1. Introduction
The microcirculation is defined as a branching network of small blood vessels with diameters less than 100 μm; these include arterioles, capillaries, and venules, which together account for more than 90% of the body’s vasculature. The microcirculation plays the crucial role of regulating the delivery of oxygen and nutrients to tissues and removing metabolic waste products. Disturbances of microvascular function are a common pathological basis of many acute and critical illnesses, including cardiac infarction [
1], stroke [
2], acute respiratory distress syndrome (ARDS) [
3], sepsis [
4], and trauma [
5]. As a collective, microcirculatory disturbances are a group of complex pathological processes that can manifest as abnormal vasomotor responses [
6], [
7], decreased red blood cell (RBC) velocity [
8], [
9], alterations in leukocyte and endothelial cell interactions (e.g., rolling, crawling, adhesion, and migration) [
10], [
11], [
12], [
13], perivascular mast cell degranulation [
14], [
15], plasma albumin and water leakage [
16], [
17], microvessel hemorrhage [
13], [
18], [
19], platelet aggregation and thrombosis [
20], [
21], microvessel occlusion and tissue hypoperfusion [
18], [
22], [
23], and microvascular remodeling and fibrosis [
24], [
25], [
26]. This broad group of processes involves multiple pathological mechanisms, which tipically include inflammatory storm; oxidative stress; overactivation of endothelial cells, neutrophils, monocytes/macrophages, and platelets; mitochondrial dysfunction and energy metabolism disorder; filament actin (F-actin) disassembly; stress fiber formation; microvascular hyperpermeability due to caveolae-mediated transcytosis and cell junction-mediated paracellular leakage; vascular basement membrane degradation; and collagen deposition. Currently, in clinical settings, except for the pharmacological regulation of vasomotor dysfunction and the inhibition of platelet adhesion, there is a lack of therapeutic drugs for improving the complex pathogenesis of microcirculatory disturbances and related diseases by modulating the multiple interactions and links between mechanisms.
Natural products have a long history of application in medicine. By exhibiting multi-target potential, numerous natural products have shown positive therapeutic effects in the management of microcirculatory disturbances through an ability to modulate the interacting mechanisms. In 2008 and 2017, we reviewed the effects of Salvia miltiorrhiza ingredients and compounded Chinese medicine on microcirculatory disturbances and multi-organ damage challenged by ischemia/reperfusion (I/R), respectively [
27], [
28]. In recent years, numerous publications have focused on the improvement of microcirculatory disturbances in disease via naturally derived products. In this review, we first introduce the pathophysiological mechanisms of microcirculatory disturbances, primarily focusing on I/R and lipopolysaccharide (LPS) injury. Next, we examine the ability of natural products to ameliorate the microcirculatory disturbances and tissue damage evoked by I/R and LPS. Particular attention is devoted to the aforementioned pathological processes and their underlying mechanisms and identified targets. Finally, we propose suggestions for the discovery of new drugs to improve microcirculatory function in complex diseases.
2. Natural products ameliorate I/R-induced microcirculatory disturbances and tissue injury
Restoration of blood flow to an ischemic organ is important in order to prevent irreversible damage to the tissue. However, reperfusion can worsen tissue damage beyond the damage caused by the initial ischemic insult. This process, called I/R injury, can lead to local and remote organ dysfunction. I/R injury can be caused by various factors (e.g., surgery, trauma, and organ transplantation) and can target critical organs (e.g., the heart, brain, liver, and intestine).
In the past decade, research has demonstrated that microvascular disturbances following the initial ischemia comprise a critical and early stage in the development of I/R injury [
3], [
29]. Reperfusion therapies using fibrinolytic drugs or surgical intervention do not always result in sufficient reperfusion at the microvascular level due to multiple alterations in microcirculatory function [
30]. I/R alters vasodilator and vasoconstrictor responses in arterioles and resistance arteries, reducing blood flow and impairing nutritive perfusion to the affected areas [
31]. It also disrupts the vascular barrier functions in microvessels, causing increased leakage of fluid and proteins from the vascular to extravascular compartment [
31]. Moreover, high vascular permeability initiates or promotes profibrotic remodeling in various organs, thereby playing an important role in the fibrotic long-term consequences of ischemic disease [
32]. During I/R, there is an increase in the interactions between blood cells (i.e., leukocytes and platelets) and endothelial cells. This occurs through a complex and regulated sequence of events that involves specific ligand/receptor interactions between leukocytes and the endothelium, leading to the adhesion and migration of leukocytes to the tissue [
33], [
34]. Adhesive interactions during I/R also induce a pro-thrombogenic phenotype in post-ischemic tissues, impairing perfusion during reperfusion [
31]. Thus, protection of perfusion and permeability is essential for therapies aimed at improving treatment of I/R injury and its aftereffects.
Recognition of the clinical importance of I/R injury has resulted in numerous studies investigating its causative mechanisms and mediators. Different pathological processes and mediators have been identified in each stage of I/R (
Fig. 1). These processes and mediators are interconnected and interact to result in cellular damage [
35], [
36], [
37]. Many natural products have recently been identified and reported to protect against I/R-induced tissue injury through their ability to target multiple pathological processes and signaling pathways. The structures of natural products are shown in Tables S1-S3 in Appendix A.
2.1. Cardiac microcirculatory disturbances and myocardial injury
2.1.1. Natural products ameliorate I/R-induced cardiac microcirculatory disturbances
After thrombolytic therapy, ischemic cardiac tissue suffers reperfusion damage, called cardiac I/R injury, which causes multiple disturbances in coronary microvascular function. After I/R, there is increased expression of adhesion molecules on neutrophils and endothelial cells in the coronary artery, promoting the adhesion and aggregation of leukocytes. This leads to increased vascular resistance and reduced coronary perfusion [
38]. Neutrophils that migrate to the myocardial tissue during I/R aggravate the myocardial damage [
39]. Several natural products have been reported to protect against the cardiac microcirculatory disturbance induced by I/R. For example, magnolol (Mag) can suppress neutrophil infiltration [
40], and ginsenoside Rb1 (Rb1), ginsenoside Rg1 (Rg1), notoginsenoside R1 (R1), and 3,4-dihydroxyphenyl lactic acid (DLA) can increase RBC velocity to improve myocardial blood flow [
8], [
41], [
42].
The microvascular barrier is regulated by both the transcellular and paracellular pathways [
43]. The transcellular pathway refers to caveolae-mediated vesicular transport, with caveolin-1 (Cav-1) acting as the key structural and functional factor [
44]. The paracellular pathway is controlled by endothelial cell-cell junctions, mainly including tight junctions (TJs) and adherent junctions (AJs), which are composed of several transmembrane proteins [
45]. I/R damages endothelial integrity and causes vascular hyperpermeability, which elicits protein and fluid efflux from the microvessels [
46], [
47]. Astragaloside IV (AsIV) has been shown to prevent neutrophil infiltration, albumin leakage, and myocardium edema by upregulating the expression of junction proteins and protecting cellular adenosine triphosphate (ATP) from depletion through activating insulin-like growth factor 1 receptor (IGF1R) signaling after I/R (
Table 1 [
8], [
26], [
40], [
41], [
42], [
47], [
48], [
49], [
50], [
51], [
52], [
53], [
54], [
55], [
56], [
57], [
58], [
59], [
60], [
61]) [
8], [
40], [
41], [
42], [
48], [
49].
During I/R injury, platelets are activated and form microthrombi that occlude the microcirculation and reduce tissue perfusion. This leads to a secondary ischemic attack on the cardiomyocytes, even if there has been successful revascularization of the occluded epicardial vessels [
62]. Thus far, no natural products have been reported to inhibit the aggregation and activation of platelets and thus play a role in inhibiting thrombus formation.
2.1.2. Natural products ameliorate I/R-induced myocardial injury
I/R causes sterile inflammation. Numerous prior studies have indicated that the myocardial-protective effects of natural products are achieved by inhibiting inflammation. Tanshinone IIA (Tan IIA) was found to alleviate inflammation via multiple signaling pathways, including the phosphatidyl-inositol 3-kinase (PI3K)/protein kinase B (Akt)/mechanistic target of rapamycin (mTOR)/endothelial nitric oxide synthase (eNOS), nuclear factor kappa B (NF-κB), c-Jun N-terminal kinase (JNK)/stress-activated protein kinase (SAPK)/mitogen-activated protein kinase (MAPK), and extracellular regulated protein kinase (ERK)/nuclear factor erythroid-2 related factor 2 (Nrf2) pathways [
50]. Glycyrrhizin (GL) [
51] and diosgenin (Dio) [
52] reduced myocardial I/R injury by inhibiting inflammation through regulating p38 MAPK/JNK, PI3K/Akt, and high-mobility group box 1 (HMGB1).
Certain natural products, such as DLA, have been found to protect against I/R injury and attenuated apoptosis by inhibiting calcium overload via the p-JNK-NF-κB-transient receptor potential channel 6 (TRPC6) pathway [
63]. As another example, araloside C (AsC) reduced calcium overload by regulating intracellular calcium homeostasis and adenosine 5′-monophosphate (AMP)-activated protein kinase (AMPK) activation [
53].
Several natural products have been reported to protect the myocardium against I/R injury by regulating the oxidative stress- and endoplasmic reticulum (ER) stress-related signaling pathways. Examples include Dio [
52], schisandrin B (SchB) [
54], tournefolic acid B (TAB) [
47], and resveratrol (Res) [
55], all of which downregulated ER stress by regulating the expression of ER stress-related proteins to alleviate myocardial cell damage.
Natural products have exhibited therapeutic effects on myocardial I/R injury by inhibiting apoptosis. Gypenosides (GPs) attenuated apoptosis by regulating B-cell lymphoma-2 (Bcl-2)-associated X protein (Bax) and Bcl-2, inhibiting mitochondrial permeability transition pore (mPTP) opening, activating AMPK/forkhead box protein O1 (FoxO1) signaling, and decreasing miR-143-3p [
64]. Other products, such as DLA [
65], Rb1 [
8], Rg1 [
41], ginsenoside Rd (Rd) [
66], celastrol (Cel) [
56], berberine (Ber) [
57], and SchB [
54], inhibited apoptosis via the PI3K/Akt/mTOR, Akt/glycogen synthase kinase-3β (GSK-3β), Janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) pathways to protect the myocardium from I/R injury.
Mitochondrial dysfunction and energy metabolism have been proposed as critical mechanisms responsible for I/R-induced endothelial damage [
67], [
68], [
69]. Some reported evidence has demonstrated that natural products can improve mitochondrial function and energy metabolism. AsIV improved energy metabolism disorders by enhancing mitochondrial respiration [
49] and increasing ATP production via activating IGF1R signaling [
48]. R1 prevented cellular energy abnormalities by inhibiting the expression and activation of Rho-associated protein kinase (ROCK) and restoring ATP synthase subunit δ (ATP5D) expression in H9c2 cells [
70]. Both Rb1 and Rg1 inhibited the activation of the ras homolog gene family and member A (RhoA)/ROCK signaling, increased ATP5D expression and ATP synthase activity, and restored the production of ATP, thereby improving energy metabolism. Moreover, both Rb1 and Rg1 can bind to RhoA in a dose-dependent manner, as assayed by surface plasmon resonance (SPR) [
8], [
41]. Furthermore, Rg1 regulated the expression of proteins involved in glycolysis and fatty acid β-oxidation, including hypoxia inducible factor-1 (HIF-1), aldolase A (ALDOA), enoyl coenzyme A hydratase 1 (ECH1), enolase α (ENOα), and ENOβ [
41]. Molecular docking and SPR experiments indicated that Rb1 binds to the ND3 subunit, reducing nicotinamide adenine dinucleotide (NADH) dehydrogenase activity and inactivating mitochondrial Complex I during reperfusion [
71]. DLA increased NADH dehydrogenase (ubiquinone) 1 alpha subcomplex 10 (NDUFA10) expression and improved Complex I activity and mitochondrial function, possibly via binding to and activating sirtuin 1 (Sirt1) in I/R-induced myocardial injury [
72]. Curcumin (Cur) improved mitochondrial dysfunction and increased cardiac function by activating AMPK and inhibiting mTOR signaling (
Table 1) [
41], [
42], [
47], [
50], [
51], [
52], [
53], [
54], [
55], [
56], [
57], [
58], [
69].
2.1.3. Natural products ameliorate I/R-induced myocardial fibrosis
Vascular endothelial cells and cardiac myocytes damaged by I/R release monocyte chemoattractant protein-1 (MCP-1) and ribosomal protein S19 (RP S19). These proteins act on the C5a receptor to activate monocyte adhesion to endothelial cells and cause monocyte migration from microvessels. Most migrated monocytes transform into cluster of differentiation 80 (CD80)- and CD86-positive macrophages, and then into CD68-, CD163-, and CD206-positive M2 macrophages; this causes the release of pro-fibrotic factors, such as transforming growth factor-β1 (TGF-β1). In turn, TGF-β1 binds to TGF-β receptor II (TGF-βRII) to activate Smad2/3/4 in fibroblasts, driving fibroblasts to differentiate to myofibroblasts and increasing collagen deposition and myocardial fibrosis [
73]. Matrix metalloproteases (MMP) are also involved in the process of myocardial fibrosis and matrix remodeling to promote fibrosis [
74]. Natural products have been reported to regulate I/R-induced myocardial remodeling and fibrosis. AsIV, DLA, and R1 protected I/R-induced myocardial fibrosis by affecting various factors, such as RP S19, TGF-β1/TGF-βR, and the expression and phosphorylation of Smad family proteins [
26]. Tan IIA [
51], orientin (Ori) [
75], Silybum marianum (SM) [
59], and puerarin (Pue) [
60], [
61] lowered myofibroblast transdifferentiation and collagen content, suppressing myocardial fibrosis through several signaling pathways including the TGF-β1/TGF-βRs/Smad2/3, JNK/SAPK, NF-κB, and ERK/Nrf2 pathways. Protocatechualdehyde (PCA) was found to suppress collagen deposition in isoprenaline-induced myocardial fibrosis. SPR assay and cellular thermal shift assay (CETSA) confirmed the binding of PCA with collagen I [
76], indicating that collagen I may be a potential target for the treatment of myocardial fibrosis using naturally derived products (
Table 1) [
26], [
50], [
59], [
60], [
61].
Based on the collective research discussed above, it can be concluded that the signaling pathways affected by each naturally derived product are distinctly different and unique, and that different pathways can be affected by the same naturally derived product. This conclusion supports the use of natural products to therapeutically target diverse pathways.
2.2. Cerebral microcirculatory disturbances and brain injury
2.2.1. Natural products ameliorate I/R-induced leukocyte activation and cerebral hyperinflammation
Suppressing leukocyte activation and the ensuing inflammatory response is crucial in ameliorating cerebral microcirculatory disturbances such as albumin leakage, leukocyte adhesion, RBC velocity, and brain injury caused by I/R [
77]. Tetramethylpyrazine (TMP) was found to reduce the secretion of the inflammatory cytokines interleukin-6 (IL-6) and IL-1β. TMP also reduced the expression of the adhesion molecules vascular cell adhesion protein-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) in brain microvascular endothelial cells (BMECs) within an oxygen-glucose deprivation/reoxygenation (OGD/R) injury model, in part by inhibiting the p38 MAPK and NF-κB signaling pathways [
78]. Administration of AsIV was found to result in a lower mortality rate in a rat middle cerebral artery occlusion (MCAO) model and to improve ischemic brain injury (
Table 2 [
78], [
79], [
80], [
81], [
82], [
83], [
84], [
85], [
86], [
87], [
88], [
89], [
90], [
91], [
92], [
93], bold). This result was achieved by reducing the levels of the inflammatory cytokines tumor necrosis factor-α (TNF-α), IL-6, and IL-1β, the adhesion molecule ICAM-1, and lipid reactive oxygen species (ROS) such as malondialdehyde (MDA), and by preventing neuronal ferroptosis, which is related to the Nrf2/heme oxygenase-1 (HO-1) signaling pathway [
79], [
80]. Salvianolic acid A (SalA) was found to inhibit granulocyte adherence in a BMECs hypoxia/reoxygenation (H/R) model by decreasing the expression of ICAM-1(
Table 2) [
78], [
79], [
80], [
81].
2.2.2. Natural products ameliorate I/R-induced cerebral microvascular hyperpermeability and brain edema
Pretreatment with levo-tetrahydropalmatine (l-THP) was found to be effective in protecting the blood-brain barrier (BBB) and inhibiting downregulation of the TJ proteins occludin and zonula occluden-1 (ZO-1), Src kinase phosphorylation, and MMP-2/9 and Cav-1 activation in an MCAO mice model. The protective effect of l-THP was correlated with its binding to the Src kinase and inhibition of Src phosphorylation [
82]. SalA has been found to have a protective effect on cerebrovascular endothelial cells against ischemia and OGD/R injury by inhibiting the Src signaling pathway. In addition, it has been observed to reverse the increased expression of MMPs and the degradation of TJ proteins [
83], [
84]. Salvianolic acid B (SalB), when combined with Rg1, has been found to protect the BBB
in vivo. This protection is accompanied by the downregulation of MMP-2 and MMP-9, as well as the recovery of ZO-1 expression [
85]. Research has shown that Tan IIA protects the BBB against H/R injury by reducing the activation of leukocytes, inhibiting the degradation of occludin and ZO-1, and suppressing the negative effects of leukocytic products such as MMP-9, cytokines, and ROS [
86], [
87]. Both TMP and R1 were also found to have a similar effect on the protein expression of MMP-9 or TJ proteins in cerebral I/R rat models, which was achieved by regulating the JAK/STAT signaling pathway for TMP [
88] or the Cav-1/MMP-2/MMP-9 pathway for R1 [
89]. Furthermore, Rg1 and TMP reduced neurological injury and BBB disruption by decreasing the expression of aquaporin (AQP)-4 in cerebral I/R injury (
Table 2) [
82], [
83], [
84], [
85], [
86], [
87], [
88], [
89], [
90], [
91].
2.2.3. Natural products ameliorate I/R-induced platelet aggregation and thrombus formation
The aggregation and activation of platelets induced by I/R play an important role in thrombosis formation, which leads to secondary brain injury. Therefore, suppressing platelet aggregation is an effective and important strategy for inhibiting thrombosis and protecting against post-ischemic brain injury. After cerebral ischemia, TMP has been found to suppress platelet aggregation via two different targets. The first target is inhibition of the binding of von Willebrand factor (vWF) to the glycoprotein Ib/IX complex. The second target is inhibition of the glycoprotein IIb/IIIa complex binding to adhesion proteins [
92]. Following OGD/R injury, TMP reduced platelet adhesion to BMECs by suppressing the p38 MAPK and NF-κB signaling pathways [
78]. By means of a lumi-aggregometer and turbidimetric method, it was found that xanthohumol (Xan) inhibited collagen-stimulated platelet aggregation in human platelet-rich plasma (
Table 2) [
78], [
92], [
93].
2.3. Hepatic microcirculatory disturbances and liver injury
We utilized an intravital microscope equipped with a high-speed video camera to examine the impact of caffeic acid (CA)—a compound abundant in Salvia miltiorrhiza—on microcirculatory disturbances in a rat liver I/R model. Our findings demonstrated that CA significantly decreased I/R-induced liver microcirculatory disturbance, including leukocyte rolling and adhesion in the terminal portal and hepatic venules. CA also improved the number of perfused sinusoids and RBC velocity in these areas. Furthermore, intravenous administration of CA effectively counteracted the deterioration of microvascular perfusion induced by I/R [
12]. Pretreatment with CA was found to induce the expression and activity of sirtuin 3 (Sirt3), which prevented the acetylation of NDUFA9 and succinate dehydrogenase complex subunit A. As a result, the activity of the mitochondrial respiratory chain complexes was attenuated and oxidative stress was restored, ultimately resulting in reduced liver injury after I/R [
12]. Subsequent research has indicated that the protein disulfide isomerase A3 (PDIA3) activates nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX), resulting in a burst of ROS. CA may protect a transplanted liver by inhibiting the PDIA3-NOX pathway [
94]. Likewise, R1 was discovered to significantly reduce hepatic microcirculatory disturbance induced by mouse mesenteric I/R; it acted to prevent decreased diameters of terminal portal venules and central veins, reduced RBC velocity in venules, and improved the number of perfused sinusoids. Moreover, R1 reduced the increase in leukocyte rolling and adhesion in hepatic venules and sinusoids. The mechanism behind the latter effect is related to its ability to inhibit the expression of E-selectin in the endothelium and CD18 in neutrophils [
95] (
Table 3 [
12], [
94], [
95], [
96], [
97], [
98], [
99], [
100], [
101], [
102]).
2.4. Intestinal microcirculatory disturbances and intestinal injury
Our previous study showed that DLA significantly reduces the mesenteric microcirculatory disturbances induced by intestinal I/R by inhibiting the production of hydrogen peroxide and adhesion molecule expression. Data from
in vivo experiments demonstrated that both pre- and post-administration of DLA significantly improved mesenteric microcirculatory disturbance induced by I/R without inhibiting mast cell degranulation.
In vitro experiments also demonstrated that DLA can inhibit the production of ROS induced by TNF-α and
N-formylmethionyl-leucyl-phenyl-alanine (fMLP), leading to decreased leukocyte CD11b/CD18 expression [
96]. In an animal model of intestinal I/R injury, pre- or post-treatment with R1 significantly reduced I/R-induced microvascular hyperpermeability and improved microvascular perfusion. R1 did not affect vessel diameter, which suggests that the effects on permeability and blood flow were due to the preservation of endothelial barrier integrity. Western blotting analysis provided evidence that R1 restored the degradation of TJ proteins, including claudin-5, occludin, and ZO-1, after reperfusion. R1 also inhibited the decrease of ATP5D and the increase of the ADP/ATP and AMP/ATP ratios caused by I/R, resulting in the maintenance of mucosal barrier integrity and inhibition of NF-κB activation, which prevented a subsequent inflammatory response and apoptosis [
97]. Similarly, oregonin (Ore) [
98] and anwulignan (AN) [
99] have been found to ameliorate intestinal I/R-induced microcirculatory dysfunction and reduce intestinal injury (
Table 3) [
96], [
97], [
98], [
99]. Pretreatment with Ore had a protective effect against ROS production in mesenteric microvascular walls. This was accompanied by inhibition of the adhesion of leukocytes to venules and mast cell degranulation. Post-treatment with Ore also resulted in improvement in I/R-induced insults, except for mast cell degranulation. In addition, Ore effectively inhibited the translocation of the NOX subunit p47phox from the cytoplasm to the membrane in the intestines during I/R injury [
98]. AN has been shown to significantly increase the mesenteric blood microcirculatory flow velocity and to reduce jejunal tissue injury and the production of TNF-α, IL-6, and IL-1β in rats with intestinal I/R injury. It can also improve the antioxidant capacity and reduce apoptosis in the jejunal tissue [
99].
Nrf2 is an important regulator of the expression of antioxidant enzymes (e.g., HO-1) and the enhancement of endogenous antioxidant capacity. Through microscale thermophoresis analysis, it has been shown that apigenin-7-
O-β-
D-(-6″-
p-coumaroyl)-glucopyranoside (APG) has the ability to bind specifically to two enzymes—namely, HO-1 and monoamine oxidase-B (MAO-B)—and reduce their activation, resulting in the attenuation of ROS production and Fe
2+ accumulation. Furthermore, APG preserved the normal function of mitochondria and prevented ferroptosis, which is a type of cell death caused by oxidative stress and iron accumulation [
100].
In recent studies, transcriptomics techniques were employed alongside experimental methods to investigate the potential mechanisms of natural products in mitigating intestinal I/R injury. Treatment with ethyl gallate (EG) in a mouse model of intestinal I/R injury resulted in the identification of 2592 upregulated genes and 2754 downregulated genes in the intestinal tissue. These differential genes were found to be enriched in various signaling pathways, including those involved in fat digestion and absorption, and extracellular matrix (ECM) receptor interactions. Additional investigation suggested that the protective effects of EG against intestinal I/R injury may be achieved by maintaining ECM integrity, likely through the inhibition of MMP-9 [
101]. Similarly, transcriptomics and functional experiments revealed that ellagic acid (EA) significantly improved oxidative stress by regulating the levels of superoxide dismutase (SOD), MDA, and glutathione (GSH); reduced inflammation by reversing the levels of inflammatory factors (TNF-α, IL-1β); and mitigated intestinal I/R injury by inhibiting apoptosis. Moreover, RNA-seq analysis identified Akt1 as the primary target of EA, which regulates intestinal I/R injury
in vivo by activating the PI3K/Akt pathway (
Table 3) [
100], [
101], [
102].
In summary, a growing number of natural products have demonstrated potential in reducing the microcirculatory disturbances and organ injury caused by I/R. These natural products exhibit their beneficial effects across various organs without showing any organ specificity (
Fig. 2).
3. Natural products ameliorate LPS-induced microcirculatory disturbances and tissue injury
LPS resides in the outer membrane of Gram-negative bacteria and may cause endotoxemia in the host upon the death of bacteria. When the immune response is abnormally pronounced and unregulated, endotoxemia can subsequently develop into sepsis and septic shock, which are critical illnesses. LPS-induced microcirculatory disturbances have been described extensively in the Refs. [
9], [
10], [
19], [
103], [
104], [
105]; these include decreases in RBC velocity; leukocyte rolling, adhesion, and migration; cytokines release; ROS overproduction; mast cell degranulation; albumin leakage; and microvascular hemorrhage.
3.1. LPS-induced microcirculatory disturbances
LPS evokes multiple insults in the microcirculation, among which, leukocyte activation, microvascular hyperpermeability and hemorrhage, and collagen deposition are key pathological changes during LPS-induced microcirculatory disturbances.
3.1.1. Leukocyte activation
Upon the CD14-mediated transfer of LPS to myeloid differentiation protein 2 (MD2), in collaboration with LPS-binding protein (LBP), Toll-like receptor 4 (TLR4) dimerization occurs and stimulates transcriptional responses [
106]. These transcriptional factors encode a variety of downstream pro-inflammatory mediators, among which the overexpression of adhesion molecules on endothelial cells (ICAM-1, VCAM-1) and neutrophils (L-selectin, CD11/CD18) enable leukocyte recruitment in microvessels. This recruitment is a multistep pathway involving rolling, adhesion, and migration [
107]. In studies utilizing dynamic intravital microscopy, we observed a dramatic increase in neutrophil rolling on and subsequent adhesion to the endothelium in rat mesentery venules
in vivo as early as 10-30 min after LPS infusion. This finding indicates that the interaction between neutrophils and endothelial cells may be among the first pathological steps leading to disturbed microvascular function (
Fig. 3). Excessive activation of neutrophil migration—as well as mast cell degranulation surrounding the microvessels (approximately 60 min after LPS infusion)—also damages the endothelium and basement membrane integrity, leading to multiple organ dysfunction via the release of ROS, cytokines, and other vasoactive mediators [
108]. Neutrophils also form neutrophil extracellular traps (NETs), which cause microvascular hyperpermeability by disrupting junctional proteins and rearranging the contractile cytoskeleton in endothelial cells [
109]. Moreover, intravascular NETs form clots that directly obstruct blood vessels and cause microcirculation hypoperfusion and organ damage [
110]. Accordingly, inhibiting the interaction between leukocytes and endothelial cells is thought to be a potentially useful approach to block LPS-induced tissue injury in an early phase.
3.1.2. Microvascular hyperpermeability
Microvascular hyperpermeability-induced plasma albumin leakage—which occurs within 1-2 h after leukocyte activation and subsequently results in tissue edema, blood volume reduction, and tissue hypoperfusion—is another major microcirculatory disturbance during endotoxemia (
Fig. 3). Under normal conditions, microvascular permeability is regulated by the paracellular pathway, which involves the integrity of the AJs and TJs between adjacent microvascular endothelial cells, and by the transcellular pathway via the caveolae, both of which are critical for the controlled transport of plasma albumin across the endothelium [
111]. Previous studies have shown that LPS insult induces plasma albumin leakage and concurrently increases the caveolae number in rat mesenteric venules [
9], [
17], [
105]. In addition, LPS degrades intercellular junctional proteins, including vascular endothelial (VE)-cadherin, occludin, claudin-5, and ZO-1, resulting in tissue edema and organ injury [
11], [
112]. Moreover, LPS triggers the activation of RhoA/ROCK-1, leading to impaired mitochondrial respiratory chain Complex V activity and ATP depletion, the latter of which is associated with vascular endothelial cytoskeleton depolymerization and microvascular junction disruption [
113]. Furthermore, excessive inflammatory mediators from activated leukocytes, endothelial cells, and mast cells around microvessels cause damage to microvascular endothelial integrity during LPS stimulation [
9]. LPS also intracellularly activates the nucleotide-binding domain, leucine-rich-containing family, and pyrin-domain-containing 3 (NLRP3) inflammasome, leading to endothelial pyroptosis, subsequent tissue edema, and neutrophil accumulation [
114]. Excessive LPS-evoked ROS production followed by mitochondrial dysfunction results in endothelial apoptosis and microvascular hyperpermeability [
115]. In addition, the AQPs—the transmembrane proteins that facilitate the bidirectional transport of water across cell membranes—were found to be increased after LPS insult, disturbing the normal maintenance of water homeostasis across microvessels [
116], [
117].
3.1.3. Microvascular hemorrhage
In addition to its effect on the endothelium, LPS can cause basement membrane impairment, resulting in microvascular albumin leakage and even hemorrhage as early as 2 h after LPS infusion [
19]. Basement membrane damage followed by microvascular bleeding is regarded as one of the initiating events in the pathogenesis involving platelet aggregation, fibrinogen activation, and microthrombi formation (
Fig. 3) [
118]. By interacting with platelet TLR4, LPS not only leads to platelet activation and subsequent platelet aggregation but also induces platelet secretion and thus the amplification of aggregation and thrombus formation [
119]. In addition, during endotoxemia, pyroptotic macrophages release a large amount of tissue factor (TF), causing systemic blood clotting [
120]. Furthermore, NETs released from activated neutrophils promote platelet adhesion and aggregation, resulting in microvascular coagulation and intravascular thrombus growth [
121], [
122]—all of which ultimately trigger activation of the fibrinolytic system and disseminated intravascular coagulation (DIC) after several hours of LPS stimulation (
Fig. 3).
3.1.4. Collagen deposition
In the sub-acute phase of endotoxemia (i.e., several days to weeks after LPS stimulation), chemokines released from injured microvascular endothelial cells and the surrounding tissues induce monocyte chemotaxis, followed by M2 macrophage polarization and TGF-β1 release, and this process then evokes the proliferation and accumulation of fibroblasts. Activated fibroblasts produce excessive amounts of ECM proteins such as collagen, causing perivascular tissue remodeling and fibrosis (
Fig. 3).
LPS evokes multiple insults in the microcirculation. Importantly, these include leukocyte rolling, adhesion, and migration (10-60 min after LPS stimulation), mast cell degranulation (approximately 60 min after LPS stimulation), microvascular hyperpermeability and hemorrhage (1-2 h after LPS stimulation), thrombus (several hours after LPS stimulation), and collagen deposition (several days after LPS stimulation) (
Fig. 3, dark blue background). All of these are key pathological processes that orchestrate the disturbed function of the microcirculation, ultimately resulting in multiple organ failure (MOF), DIC, fibrosis, and even death during endotoxemia and sepsis [
123].
3.2. Cardiac microcirculatory disturbances and myocardial injury
LPS-triggered myocardial damage is a multifaceted process involving various molecular and cellular mechanisms [
124]. LPS interacts with TLR4 and its associated proteins on the surface of cardiomyocytes, activating downstream signaling pathways such as NF-κB and leading to the expression of inflammatory cytokines (e.g., TNF-α, IL-1β, and IL-6) [
124]. The upregulation of inflammatory cytokines and adhesion molecules results in the infiltration of macrophages [
125]. After encountering LPS, macrophages undergo intense stimulation, and the ensuing metabolic changes redirect mitochondrial ATP synthesis to ROS production, promoting a pro-inflammatory state and exacerbating sepsis-induced cardiac injury [
126]. However, few studies have focused on the influence of LPS on cardiac microcirculation, and none (to the best of our knowledge) investigate the potential effects of natural products on LPS-induced cardiac microcirculation disturbances. Therefore, research in this field is needed in the future.
3.3. Cerebral microcirculatory disturbances and brain injury
3.3.1. Natural products ameliorate LPS-induced leukocyte activation and cerebral hyperinflammation
Accumulating evidence indicates that natural products can play a role in the amelioration of LPS-induced leukocyte activation and the subsequent overproduction of inflammatory mediators, thereby attenuating cerebral microvascular injury and brain damage (
Table 4 [
113], [
127], [
128], [
129], [
130], [
131], [
132], [
133]) [
127], [
128], [
129], [
130]. The structures of natural products are shown in Table S4 in Appendix A. A previous study showed that DLA inhibited LPS-induced cerebral microcirculatory disturbances in mice, including decreased RBC velocity, leukocyte rolling, and adhesion to the venular wall, by inhibiting CD11b/CD18 expression on neutrophils [
127]. Murrayafoline A (MA) abolished neuroinflammatory mediator production in cerebral microglial cells simulated by LPS. By performing a thermal proteome profiling strategy in combination with SPR, a CETSA, and drug affinity responsive target stability (DRATS) assays, the investigators further identified specificity protein 1 (Sp1) as a potential target of MA. By directly binding to Sp1, MA was found to block Sp1 nuclear translocation, thereby inhibiting NF-κB- and MAPK-mediated neuroinflammation, which resulted in the protection of Nissl bodies in LPS-induced mice [
128]. Other natural products, such as sulforaphane (SFN), have anti-inflammation features that have been found to be beneficial for neuroprotection by inducing receptor interacting protein 1 (RIP1) and tumor necrosis factor receptor-associated factor 6 (TRAF6) degradation [
129]. This process led to inhibition of the NF-κB pathway and prevented microglia activation. In addition, canthin-6-one (CO) was found to inhibit LPS-induced astrocyte pro-inflammatory responses, which included preventing increased TNF-α, IL-6, IL-1β, inducible nitric oxide synthase (iNOS), and MMP-9 expression, as well as decreasing the intracellular ROS level and Ca
2+ by inhibiting NF-κB and STAT3 phosphorylation. In this way, the astrocyte-mediated endothelial barrier was protected from disruption by maintaining the expression and distribution of TJ proteins (ZO-1, claudin-5) and AJ protein (VE-cadherin) [
130].
3.3.2. Natural products ameliorate LPS-induced cerebral microvascular hyperpermeability and brain edema
LPS evokes cerebral microvascular hyperpermeability by disrupting paracellular junctions and the upregulation of caveolae-mediated transcytosis. These combined effects lead to albumin leakage and brain edema. Pre- or post-treatment of Rb1 attenuated plasma albumin leakage and peripheral edema of cerebral microvessels, in addition to maintaining the expression of VE-cadherin, claudin-5, and ZO-1 in microvascular endothelial cells. Moreover, Rb1 reversed ATP synthase subunit beta and ATP5D underexpression. Together, these changes attenuated the decrease of mitochondrial ATP synthase activity and ATP depletion in LPS-induced rat cortex tissue and contributed to TJ maintenance [
113]. The researchers also found that, unlike Rb1, schisandrin (Sch) prevented Cav-1 phosphorylation and decreased the caveolae number in cerebral endothelium, resulting in reduced albumin leakage transcytosis [
113]. It is noteworthy that improved function was observed as the result of a synergistic effect on LPS-induced albumin leakage and tissue edema when Rb1 and Sch were used in combination—a finding that indicates the importance of both the paracellular and transcellular pathways in maintaining BBB integrity [
113]. AsIV was found to protect the BBB via the upregulation of ZO-1 and occludin, in addition to inhibiting VCAM-1 and monocyte adhesion onto brain endothelial cells upon LPS stimulation. This result was attributed to preventing ROS overproduction via activating Nrf2 [
131]. Similarly, kaempferol (KAE) has been shown to ameliorate LPS-induced striatum injury by inactivating the HMGB1/TLR4 pathway. KAE inhibited migroglia activation in the striatum tissues of LPS-injured mice, along with a decrease in the production of pro-inflammatory cytokines (IL-1β, IL-6, TNF-α, MCP-1) and adhesion molecules (ICAM-1). This resulted in increased expression of claudin-1, occludin, and connexin-43 (CX-43), all of which improved BBB integrity [
132]. In LPS-stimulated BMECs, hydroxysafflor yellow A (HSYA) restored Sirt1 induction and promoted HIF-1α degradation; in this way, it abrogated HIF-1α-dependent upregulated p47phox and NOX2 complex assembly, protecting ZO-1 against oxidative-stress-induced carbonyl modification and subsequent proteasomal degradation (
Table 4) [
113], [
131], [
132], [
133].
3.4. Pulmonary microcirculatory disturbances and lung injury
3.4.1. Natural products ameliorate LPS-induced pulmonary leukocyte-endothelial interaction and lung inflammation
Leukocyte activation and accumulation in lung tissues have widely been regarded as the initial steps in LPS-induced acute lung injury (ALI), playing a pivotal role in this process. By means of intravital fluorescence microscopy, andrographolide (AP) was demonstrated to inhibit LPS-induced rat pulmonary microcirculatory disturbances by decreasing leukocyte adhesion to pulmonary venules and inflammatory mediator release [
134]. Carnosic acid (CAA) and Res have been shown to attenuate upregulated neutrophil CD11b expression by downregulating MAPKs phosphorylation and Src family kinases pathway activation, respectively, as well as inhibiting neutrophil adherence to endothelial cells [
135], [
136]. In addition, CAA reduced neutrophil respiratory bursts and ROS-dependent NETs formation and neutrophils degranulation [
136]. Another study demonstrated that rhamnocitrin (RH) inhibited LPS-induced cytokine (IL-6, IL-8, MCP-1) and adhesion molecules (ICAM-1, VCAM-1) expression in endothelial cells, thereby preventing leukocyte adherence and migration into the surrounding tissues [
137].
Recently, using molecular docking analysis methods together with SPR imaging and protein interaction pull-down assaying, many natural products have been demonstrated to attenuate leukocyte activation. This attenuation occurs via direct binding with various key molecules that participate in the TLR4 signaling cascade, thus decreasing the release of inflammatory mediators and ROS and the infiltration of leukocytes into lung tissues. By means of an affinity assessment and the
in vivo pharmacological evaluation of compounds from the Huang-Lian-Jie-Du decoction, geniposide (Gen) was found to neutralize LPS by binding to lipid A to reduce cytokines release [
138].
Natural products have also been demonstrated to bind directly with the LPS receptor TLR4 [
139] and the adaptor protein MD2 [
140], [
141]. Through this mechanism, the natural products interfered with multiple initial steps involved in the TLR4 signaling pathway, including TLR4 dimerization, LPS-MD2 interaction, and TLR4/MD2 heterodimerization. Moreover, natural products have been found to target key upstream activators of NF-κB and attenuate lung inflammation. For example, cintelactone A (CIA) was shown to suppress LPS-induced inflammation by monocytes and macrophages by promoting K48-linked ubiquitination and subsequent TRAF6 proteasomal degradation [
142]. Naturally derived products have also been found to exert anti-inflammatory and antioxidant activities via the inhibition of NF-κB signaling by directly binding and inactivating phosphodiesterase 4 (PDE4) [
143], [
144]. In addition, a recent study identified alantolactone (ALA) as a naturally occurring inhibitor of NLRP3 inflammasomes via binding to Arg335 of the NLRP3 NACHT domain [
145]. Similarly, Xan protected LPS-induced acute lung injury (ALI) by suppressing thioredoxin interacting protein (Txnip)/NLRP3 inflammasome activation, which was related to the upregulation of the AMPK-dependent Nrf2 pathway and a decrease in oxidative stress (
Table 5 [
19], [
134], [
135], [
136], [
137], [
138], [
139], [
140], [
141], [
142], [
143], [
144], [
145], [
146], [
147], [
148], [
149], [
150], [
151], [
152]) [
134], [
135], [
136], [
137], [
138], [
139], [
140], [
141], [
142], [
143], [
144], [
145], [
146]. The structures of natural products ameliorate LPS-induced pulmonary microcirculatory disturbances and lung injury are shown in Table S5 in Appendix A.
3.4.2. Natural products ameliorate LPS-induced pulmonary hyperpermeability and hemorrhage
During LPS-induced ALI/ARDS, overactive pulmonary neutrophils and macrophages cause injuries to the microvasculature. Numerous experimental studies have verified that natural products with anti-inflammatory properties can attenuate pulmonary leukocyte accumulation and simultaneous hyperpermeability during LPS-induced ALI (
Table 5) [
19], [
134], [
147], [
148], [
149], [
150]. For example, AP protected against LPS-induced pulmonary microvessel hyperpermeability, which reduced arterial hypoxia and decreased mortality after LPS challenge in rats. The attenuation effect of AP was attributed to ameliorating the low expression of TJs junctional adhesion molecule-1 (JAM-1) and claudin-5, as well as preventing TLR4/Src-dependent Cav-1 phosphorylation [
134]. Aside from their effects on the endothelium in pulmonary microvessels, natural products have been shown to attenuate LPS-induced epithelial cell injury to maintain air-blood barrier integrity. Obacunone (OB) reduced human bronchial epithelial (BEAS-2B) cell ferroptosis due to its antioxidant capacity. This activity of OB was due to the stabilization and activation of Nrf2, which contributed to reduced LPS-induced histopathological changes. Improvements were also observed in the number of inflammatory cells in bronchoalveolar lavage fluid (BALF) and lung edema in mice [
147]. Polydatin (POD) exerted an upregulation effect on Parkin-dependent mitophagy that counteracted LPS-induced mitochondria-dependent apoptosis both in lung and in BEAS-2B cells and thereby protected against ALI [
148]. Similarly, Sch attenuated LPS-induced pulmonary hyperpermeability and tissue edema by inhibiting pulmonary epithelial cell apoptosis. Furthermore, partly due to Akt/FoxO1 signaling activation, Sch accelerated pulmonary endothelial and epithelial regeneration after LPS damages [
149].
Normalized lung epithelium function determines the effective clearance of excessive edematous liquid in pulmonary alveoli. In this respect, luteolin (Lut) has been identified as a regulator of alveolar epithelial ion transport in pulmonary edema. An
in vivo study demonstrated that Lut stimulated mouse alveolar fluid clearance in mice and decreased the lung wet/dry weight ratio. This result was attributed to increased levels of membrane α/γ-epithelial sodium channel (ENaC) protein via the activation of cyclic guanosine monophosphate (cGMP)/PI3K signaling [
150]. Pulmonary microvascular hemorrhage is another severe pathological feature in LPS-induced ALI/ARDS that can be attenuated by natural products. Zhang et al. [
19] found that catalpol (CTP) ameliorated microvessel hemorrhage in rat pulmonary tissue after LPS challenge. This treatment contributed to increasing the survival rate to 70% two days after LPS infusion, in comparison with 40% in the untreated LPS group. By binding with TLR4 and Src kinase, CTP protected against LPS-induced microvascular barrier disruption. Moreover, CTP downregulated the phosphorylation of PI3K and focal adhesion kinase (FAK) and the activation of cathepsin B, which contributed to maintaining the microvascular basement membrane in pulmonary tissue [
19].
3.4.3. Natural products ameliorate LPS-induced pulmonary fibrosis
LPS was found to induce pulmonary fibrosis in a mouse model, which manifested as increased collagen deposition in mouse pulmonary tissues with a time course that paralleled the production of TGF-β1 and the activation of Smad signaling. Treatment with Res attenuated LPS-treated pulmonary fibrosis by directly regulating TGF-β1-Smad signaling in mice. In addition, Res reversed LPS-induced epithelial-mesenchymal transition through the downregulation of oxidative stress [
151]. Moreover, pterostilbene (Pts)—a structural analog of Res—ameliorated LPS-induced mouse pulmonary fibrosis by affecting multiple targets. Pts attenuated neutrophil and macrophage infiltration and cytokine overproduction by inhibiting the tumor necrosis factor alpha-induced protein 3 (TNFAIP3)/NF-κB and NLRP3 signaling pathways. Also, Pts inhibited NOX4 and Kelch-like ECH-associated protein 1 (Keap1) expression and activated Nrf2 and downstream genes, all of which contributed to a decrease in LPS-induced pulmonary apoptosis, lung injury, and early fibrosis. Furthermore, LPS activated tissue inhibitor of metalloproteinase-1 (TIMP-1) and inhibited MMP-1, which were reversed by Pts, indicating that Pts can promote collagen degradation and attenuate pulmonary fibrosis induced by LPS (
Table 5) [
151], [
152].
3.5. Hepatic microcirculatory disturbances and liver injury
3.5.1. Natural products ameliorate LPS-induced leukocyte activation and hepatic inflammation
Leukocyte activation and the subsequent overexpression of inflammatory mediators participate in various pathological processes in LPS-induced hepatic microcirculatory disturbances. Intravital microscopy revealed that, 2 h after LPS injection, the numbers of leukocytes adhering to the sinusoidal wall and swollen endothelial cells increased significantly, accompanied by a reduction in the number of sinusoids containing flow. All of these were inhibited by pretreatment with curcuminoids, which mainly included three active metabolites: Cur, desmethoxycurcumin (DMC), and bidesmethoxycurcumin (BDMC) [
153]. Rb1, one of the main bioactive components in ginseng, protected mice against LPS/
D-galactosamine (LPS/Gal)-induced acute liver injury by decreasing hepatic ROS, myeloperoxidase (MPO), and MDA, while increasing the activity of SOD and GSH peroxidase (GSH-Px). This reduced the levels of inflammatory cytokines (e.g., IL-6, TNF-α, IL-1β, and IL-18) in serum and liver tissue by inhibiting the TLR4/NF-κB signaling pathway and NLRP3 inflammasome activation in Kupffer cells (KCs), which contributed to improving the survival rate of mice to 80%, in comparison with 20% for the LPS/Gal group [
154]. Similarly, AsIV downregulated NF-κB-mediated adhesion molecules and pro-inflammatory mediators, including ICAM-1, TNF-α, IL-6, and TLR4 [
155]. Asperosaponin VI (AVI) reduced the expression of inflammatory factors (i.e., TNF-α, IL-6, and IL-10) and liver organ damage in LPS-induced sepsis mice by inhibiting the Hippo and Rho signaling pathways (
Table 6 [
153], [
154], [
155], [
156], [
157], [
158], [
159]). The structures of natural products ameliorate LPS-induced hepatic microcirculatory and liver injury are shown in Table S6 in Appendix A.
3.5.2. Natural products ameliorate LPS-induced liver injury and fibrosis
In addition to leukocytes, liver sinusoidal endothelial cells (LSECs), hepatic stellate cells (HSCs), and other cell components are components of the hepatic microcirculation that can be affected by LPS intervention. In LSECs, LPS-induced Cav-1 upregulation and Rho/ROCK signaling pathway activation eliminated endothelin-1 (ET-1)-induced eNOS translocation, reduced ET-1-mediated eNOS phosphorylation activation and nitric oxide (NO) production, and consequently disrupted the hepatic microcirculation [
160], [
161]. Lethal-7a (Let-7a) and Let-7b were found to be reduced in LPS/TGF-β-treated HSCs, activating these cells. The activated HSCs moved to the damaged site of the liver to secrete ECM, providing a basis for cell division and adhesion. If the liver is unable to replace these damaged cells successfully, HSCs deposit excessive ECMs to induce fibrosis and scarring [
162]. Isoliquiritigenin (ISL) is a natural flavonoid that alleviates liver fibrosis through Cav-1-mediated hepatic stellate cells ferroptosis in zebrafish and mice [
157]. Tetrandrine (Tet) plays an antifibrotic role in fibrotic rat models via the concentration-dependent inhibition of TNF-α-induced NF-κB transcription, TGF-β1-induced α-smooth muscle actin (α-SMA) secretion, and collagen deposition [
158].
Astragalus decoction is composed of astragalus and licorice. Total astragalus saponin (TAS) and glycyrrhizic acid (GA) are the main components of astragalus and licorice, respectively. Studies have shown that LPS-induced macrophage exosomes promoted HSC activation, and that TAS in combination with GA preconditioning exerts an anti-fibrosis effect by decreasing the expression levels of p-Smad2/3, collagen I, and α-SMA (
Table 6) [
157], [
158], [
159].
3.6. Intestinal microcirculatory disturbances and intestinal injury
3.6.1. Natural products ameliorate LPS-induced leukocyte activation and intestinal inflammation
LPS can trigger a series of mesenteric venular microcirculatory disorders that cause damage to the intestinal endothelium and basement membrane, accompanied by leukocyte adhesion and albumin exudation. Many natural products have been demonstrated to inhibit LPS-induced mesenteric microcirculatory disturbances
in vivo. For example, DLA and SalB from
Salvia miltiorrhiza and Rb1, Rg1, and R1 from
Panax notoginseng have been found to inhibit LPS-induced microcirculatory disturbances in rat mesenteric venules. This included the prevention of a decrease in RBC velocity, leukocyte adhesion to and emigration out of the venular wall by inhibiting CD11b/CD18 expression, and mast cell degranulation [
10], [
103]. Similarly, through intravital fluorescence microscopy, emodin (Emo) was shown to inhibit leukocyte rolling, adhesion to and emigration out of mesenteric venules induced by LPS. This inhibition was correlated with the expression of the TLR4-NF-κB-dependent adhesion molecules L-selectin, CD11b, and ICAM-1 [
163]. Natural products have demonstrated good biological activity in their ability to reduce LPS-induced leukocyte activation. This is one of the most important mechanisms in regulating inflammatory pathway activation, production of inflammatory cytokines, and inflammatory cell infiltration. For example, enhancer of zeste homolog 2 (EZH2) is a clear therapeutic target for lonicerin (LNC). This result was confirmed using a series of techniques, such as molecular docking, molecular dynamics simulation, chromatin immunoprecipitation assay, CETSA, solvent-induced protein precipitation assay, site-directed mutagenesis, and lentiviral transduction. By targeting EZH2, LNC disturbs the NLRP3-apoptosis-associated speck-like protein containing a caspase-recruitment domain (ASC)-pro-caspase-1 complex assembly [
164]. Also, alpinetin (APT) significantly attenuated colon inflammation in mice. APT decreased inflammatory infiltration by suppressing the NF-κB inflammatory pathway and pro-inflammatory mediators; it was found to bind pregnane X receptor (PXR), which was confirmed via competitive ligand binding assay [
165]. Similarly, convallatoxin (CNT) potentially controls colon inflammation by inhibiting the NF-κB pathway through targeting peroxisome proliferator-activated receptor γ (PPARγ) [
166]. Moreover, Ber exhibited a significant protective effect against LPS-evoked intestinal injury; it reduced enterocyte apoptosis, decreased neutrophil infiltration, and deactivated the TLR4-NF-κB-macrophage inflammatory protein-2 (MIP-2) pathway (
Table 7 [
10], [
17], [
19], [
103], [
163], [
164], [
165], [
166], [
167], [
168], [
169], [
170], [
171]) [
10], [
103], [
163], [
164], [
165], [
166], [
167]. The structures of natural products are shown in Table S7 in Appendix A.
3.6.2. Natural products ameliorate LPS-induced microvascular hyperpermeability
Some active molecules also play a role in protecting the mucosal barrier while controlling the production of ROS and inflammation by preventing microvascular albumin leakage (
Table 7) [
17], [
168], [
169], [
170], [
171]. For example, studies using SPR and bio-layer interferometry (BLI) demonstrated that SalB can target Src, leading to the inhibition of albumin leakage from rat mesenteric venules and a decrease in vesicle number in venular endothelial cells by preventing the degradation of ZO-1, VE-cadherin, and Cav-1 [
168]. Rb1 also ameliorates LPS-induced microvascular hyperpermeability by suppressing caveolae formation, junction disruption, NF-κB phosphorylation, and Src activation. In addition, post-treatment with Rb1 after LPS infusion increased the rat survival rate to 90% four days after stimulation, which was significantly higher than that of the LPS group [
17]. Atractylenolide III (AT III) improved mitochondrial function and protected mice against colitis via AMPK/Sirt1/proliferator-activated receptor-γ coactivator-1α (PGC-1α) signaling [
169]. Similarly, KAE protects the intestinal barrier by increasing the levels of ZO-1, claudin-1, and occludin, while decreasing the expression of TNF-α, IL-1β, and IL-6 and downregulating TLR4-NF-κB signaling [
170]. Moreover, AsIV reduced intestinal barrier dysfunction by regulating the RhoA/NLRP3 inflammasome pathway [
171].
3.6.3. Natural products ameliorate LPS-induced microvascular hemorrhage
Certain natural products have exhibited pharmacological activity against LPS-induced platelet aggregation, hemorrhage, and even microthrombi formation in an inflamed and bleeding colon (
Table 7) [
19]. For example, CTP alleviated not only mesenteric venular hyperpermeability but also hemorrhage by ameliorating the degradation of JAM-1, claudin-5, collagen IV, and laminin. This result was attributed to a reduction in TLR4 levels, prevention of cathepsin B activation, and inhibition of the phosphorylation of Src, PI3K, and FAK by directly binding with TLR-4 and Src [
19].
In summary, defining the temporal sequence of microcirculatory disturbances is the first step in addressing them and can serve to establish a common pathological process of LPS-induced multi-organ injury (
Fig. 4, solid black arrow). Through their attenuation effects on various stages of microcirculatory disturbances, including numerous processes such as leukocyte-endothelial interaction, mast cell degranulation, microvascular hyperpermeability, hemorrhage, and ECM deposition (
Fig. 4, solid red line) caused by a diverse array of underlying mechanisms (
Fig. 4, solid green line), many natural products have the ability to limit LPS-induced organ injury in the brain, lung, liver, and intestine (
Fig. 4, solid black line).
4. Clinical application of natural products
4.1. Application of natural products in I/R-related clinical disorders
Cardiovascular diseases are the leading cause of death worldwide and are associated with a high disability rate, mortality rate, recurrence rate, and complications. The prevalence of cardiovascular diseases in China is on an upward trend, and it is estimated that there are currently 330 million people with cardiovascular diseases in China. In the composition of disease-related deaths among urban and rural residents in China, cardiovascular diseases rank first. In 2020, cardiovascular diseases accounted for 48% and 46% of the causes of death in rural and urban areas, respectively; thus, two out of every five deaths in China are caused by cardiovascular diseases. Ischemic heart disease, ischemic stroke, and hemorrhagic stroke are the three main causes of cardiovascular-disease-related deaths in China.
While double-blind, controlled, and randomized studies are not commonly used to evaluate the efficacy of natural products in cardiovascular diseases therapy, some frequently prescribed preparations based on natural products—such as Qishen Yiqi dripping pill, Compound Danshen dripping pill, Qili Qiangxin capsule, Tong-Xin-Luo capsule, and Xinyue capsule—have already undergone significant clinical evaluations (
Table 8 [
172], [
173], [
174], [
175], [
176], [
177], [
178], [
179], [
180], [
181], [
182], [
183], [
184], [
185]) [
172], [
173], [
174], [
175], [
176], [
177], [
178], [
179], [
180], [
181]. The available clinical trial evidence suggests that certain natural products may effectively treat conditions, such as coronary heart disease and chronic heart failure. However, the inconsistent use of outcome measures in the treatment of cardiovascular diseases hampers accurate evaluation, the secondary use of clinical trial data, and the development of clinical guidelines. Moreover, there is a need for further rigorously designed randomized controlled trials to evaluate the impact of natural products on overall mortality in patients with cardiovascular diseases or major adverse cardiovascular events.
4.2. Application of natural products in LPS-related clinical disorders
LPS-induced endotoxemia, which can subsequently develop into sepsis and septic shock, is a critical problem in the clinic, with a mortality rate of approximately 27% worldwide [
186]. According to a recent global analysis, about 48.9 million people are estimated to be affected by sepsis, and 11 million deaths occur each year [
187], [
188]. Current management of endotoxemia and sepsis only includes infection control via broad-spectrum antibiotics and/or fluid administration with vasoactive agents. However, a recent randomized double-blind and placebo-controlled trial showed that curcumin nanomicelles supplementation resulted in decreased sequential organ failure. The scores assessed for multiple organ dysfunction syndromes were also reduced, as were the serum levels of inflammatory biomarkers in sepsis patients [
182]. Until recently, very few individual natural products have been approved or have undergone clinical trials for endotoxemia and sepsis therapy. Considering the complexity of microcirculatory disturbances during the pathogenesis of endotoxemia or sepsis, the combined utilization of various natural products that act on different key targets and phases of disturbed microcirculatory functions—rather than a single active ingredient—may prove to be more advantageous for sepsis therapy. Encouragingly, several preparations composed of multiple natural products have recently been shown to be effective in sepsis management through clinical trials. For example, a multicenter, randomized, double-blind, placebo-controlled trial demonstrated that, among patients with sepsis, the administration of Xuebijing injection—an herbal-based intravenous preparation containing multiple natural products—significantly reduced 28-day mortality (
Table 8, bold) [
183]. Two other clinical trials showed that the JinHong formula and Shenfu injection significantly reduced all causes of mortality on 28 and 60 days in sepsis patients (
Table 8, bold) [
185] and improved sublingual microcirculatory perfusion in patients with septic shock [
184], respectively. It seems prudent to suggest that the effectiveness and clinical translation of natural products deserve increased attention and further investigation. There is also a need for many more clinical trials on the use of natural products in these contexts. The present data provide a foundation and demonstrate the potential of natural products as an alternative and adjuvant therapy for LPS-induced endotoxemia and sepsis. Clinical trials of the use of natural products and naturally derived product-based preparations to address I/R- or LPS-related diseases are summarized in
Table 8 [
182], [
183], [
184], [
185], [
186], [
187], [
188].
5. Conclusions and future perspectives
Microcirculatory disturbances are the common pathological basis of I/R- and LPS-induced multi-organ injury. These clinically important injuries involve complex pathological processes mediated by multiple signaling pathways. It has been proposed that multi-target management is required to regulate the complex links of these microcirculatory disturbances. In this review, we systematically summarized and showed that natural products can play amazing ameliorative roles in improving I/R- and LPS-induced microcirculatory disturbances and organ damage, which are mainly attributed to multi-pathway modes of action. More importantly, multiple specific targets of natural products that are involved in disturbed microvascular function have been identified, all of which highlight natural products as a potential therapeutic option for microcirculatory disturbance-related diseases in the clinic.
5.1. Natural products improve I/R-induced dysfunction of organ microcirculation
5.1.1. Experimental studies provide insights into mechanisms by which natural products mitigate microcirculatory disturbances and organ injury induced by I/R
In I/R injury, the main problem arises from the interruption of blood flow (ischemia), followed by the restoration of blood flow (reperfusion) to an organ or tissue. This process can result in several dysfunctions and forms of damage, including oxidative stress, inflammation, and cellular damage. The resolution of I/R injury with the help of natural products involves reducing oxidative stress, dampening inflammation, regulating energy metabolism, alleviating endothelial dysfunction, and promoting cell survival and tissue repair. By targeting these mechanisms, natural products can help mitigate the damage caused by I/R injury and enhance the recovery process (
Fig. 5). It is important to note that, while natural products show therapeutic potential in mitigating I/R injury, further research is needed to understand their optimal dosages, delivery methods, and efficacy in clinical settings.
5.1.2. Studies offer insights into the structural basis underlying the effects of certain natural products
In the context of I/R injury, various mechanisms and relationships contribute to the overall pathology, including mitochondrial dysfunction, oxidative stress, inflammatory response, Ca
2+ overload, endothelial dysfunction, ER stress, apoptosis, and autophagy. While some mechanisms may be considered to be stronger or more prominent than others, it is important to note that the interplay between these mechanisms and their relationships can vary depending on the specific context. In addition, the relative importance of these mechanisms may vary based on the organ or tissue affected. Further research is needed to fully understand the intricate relationships and relative significance of these mechanisms in order to develop effective therapeutic approaches (
Fig. 6).
Studies have provided insights into the structural basis of the effects of certain natural products. In this regard, natural saponin products such as AsIV from Astragalus membranaceus and R1 from
Panax notoginseng have been observed to enhance the expression of ATP5D in the mitochondrial respiratory chain, leading to improved energy metabolism. Moreover, CA, DLA, SalA, and SalB—derived from
Salvia miltiorrhiza—have the ability to inhibit peroxide production. It is noteworthy that all of these compounds possess phenolic hydroxyl groups in their chemical structures (
Fig. 6). In addition, studies have identified specific targets and binding sites of natural products that affect key molecules involved in the development of microcirculatory disturbances and organ injury caused by I/R (
Fig. 7). These findings provide valuable insights for the discovery of additional natural products with similar therapeutic potential and the development of new drugs targeting similar molecular pathways. It is important to note that the effects of natural compounds on specific dysfunction in I/R injury can vary depending on dosage, formulation, method of administration, and experimental models used. Further research is needed to fully understand the specific effects and underlying mechanisms of these natural compounds in the different dysfunctions associated with I/R injury in different organs.
5.1.3. Potential translation of basic findings into clinical interventions
Previous clinical studies have shown that microvascular I/R injury is an independent risk factor for increased mortality and rehospitalization rates in patients who undergo reperfusion therapy [
64]. The basic findings on the mechanisms and treatment of microcirculatory disturbances using natural products can potentially lead to the development of clinical interventions. These interventions may include: ① early identification and monitoring of microcirculatory disturbances and microvascular I/R injury; ② optimization of reperfusion therapy to minimize microvascular I/R injury; ③ development of targeted therapies specifically aimed at mitigating microcirculatory disturbances and microvascular I/R injury; and ④ development of novel pharmacological agents that can protect or repair the microvasculature. It is important to note that the translation of basic findings into clinical interventions requires further research, validation, and collaboration among researchers, clinicians, and healthcare professionals.
5.1.4. Future perspectives
There are still some aspects of this field of research that need to be studied in depth, and it is necessary to continue the investigation of the ability of natural products to combat I/R injury. Firstly, increasing evidence suggests that microcirculatory disturbances are a common and decisive factor in organ injury in ischemic diseases. These disturbances significantly contribute to the high mortality rate observed in patients. Nevertheless, studies on the effects of natural products on I/R-induced microcirculatory disturbances are relatively scarce. The insights gained from experimental studies on the mechanisms and treatment of microcirculatory disturbances using natural products should be translated into clinical practice, which would enable the early initiation of treatment to prevent the manifestation of organ injury. Secondly, although current research has found that various natural products can exert effects on I/R-induced tissue injury, our knowledge of the mechanisms of action is incomplete and requires more in-depth study. The scope of existing research is relatively limited, and it is unclear how many of these natural products exhibit similarities and differences in the ways they act on their target. In addition, the combined use of various natural products that act on different targets or phases of microcirculatory disturbances should be investigated both in the lab and in clinical trials. Also, the effects and mechanisms of naturally derived products on microvascular dysfunction and organ injury in other pathological conditions should be fruitful and important areas of future investigation.
5.2. Natural products improve LPS-induced dysfunction of organ microcirculation
5.2.1. Insights into mechanisms by which natural products mitigate microcirculatory disturbances and organ injury induced by LPS
LPS cause multiple insults on end-organ microcirculations, among which the activation of TLR4-dependent inflammatory cells (i.e., neutrophils, monocytes, and mast cells) and microvascular endothelial cells is regarded as the initial and key step. The expression of excessive adhesion molecules on leukocytes and endothelial cells enables leukocyte-endothelial cell interaction and activation. Subsequently, by releasing a large amount of ROS, cytokines, NETs, MMPs, elastase, and other vasoactive mediators, inflammatory cells damage the endothelium and basement membrane integrity. They also cause many kinds of cell death in microvessels and parenchyma, which contributes to multiple facets of organ dysfunction during the acute phase of LPS-induced endotoxemia (
Fig. 8, solid red arrow). In addition, LPS directly induces TLR4-Src-caveolae-dependent albumin transcytosis. Meanwhile, LPS evokes RhoA/ROCK-dependent mitochondrial dysfunction, followed by ATP depletion and F-actin depolymerization; all of this disrupts the endothelial junctions, causing microvascular hyperpermeability, which leads to tissue edema and hypoperfusion, further aggravating the tissue injury (
Fig. 8, solid green arrow). Moreover, LPS degrades the basement membrane of microvessels, which results in microvascular hemorrhage—an early pathological event that activates a coagulation cascade and causes subsequent thrombosis formation and even DIC, accompanied by platelet activation (
Fig. 8, solid blue arrow). In the sub-acute phase of endotoxemia, monocyte chemotaxis followed by M2 macrophage polarization and TGF-β1 release evoke fibroblasts activation and collagen deposition, causing perivascular tissue remodeling and fibrosis (
Fig. 8, solid black arrow). By targeting the multiple aforementioned signaling pathways (e.g., TLR-4-NF-κB, TLR4-Src-caveolae, endothelial junction disruption, and basement membrane damage) and subsequent microcirculatory disturbance processes (e.g., leukocyte-endothelial cell interaction, microvascular hyperpermeability, hemorrhage, and fibrosis), natural products mitigate the multi-organ damage caused by LPS and enhance the recovery process (
Fig. 8, solid black line).
5.2.2. Identification of multiple targets of natural products in LPS-induced microcirculatory disturbance
It is clear that LPS-induced microcirculatory disturbances comprise a multi-linked and complex pathological process involving multiple signaling pathways that interact with each other (
Fig. 9, solid red arrow). Among them, leukocyte-endothelial interaction, inflammation, and oxidative stress play the initial and causative role in this pathological process (
Fig. 9, thick solid red arrow). The present review illustrates how natural products ameliorate LPS-induced microcirculatory disturbances and organ injury by means of a multi-target mode (
Fig. 9, solid green arrow). The major targets of natural products in the process of LPS-induced microcirculatory disturbances are the expression of adhesion molecules and subsequent leukocyte-endothelial interaction, TLR4-NF-κB and NLRP3 signaling activation and hyperinflammation, and endothelial junction disruption followed by microvascular hyperpermeability (
Fig. 9, thick solid green line). Direct ameliorative effects of natural products on other microcirculatory disturbances involved in endotoxemia, such as oxidative stress, caveolae-mediated albumin leakage, mitochondrial dysfunction, basement membrane damage, and so forth, have also been reported (
Fig. 9, fine solid green line). Through molecular docking methods, in combination with SPR, BLI, CETSA, and DRATS assays, protein interaction pull-down assay, and other advanced structural pharmacological techniques, numerous specific targets and binding sites of natural products have been identified for multiple key molecules involved in TLR4-NF-κB signaling and NLRP3 signaling during the pathogenesis of LPS-induced microcirculatory disturbances and organ injury (
Fig. 10).
5.2.3. Future perspectives
Many of the current studies investigating the effects of natural products focus only on LPS-induced tissue damage or certain types of cell injury, without examining the causative effects of microcirculatory disturbances. Thus, it is important to underscore the necessity of defining the entire time course of the changes that occur within the microcirculation, particularly in the heart (
Fig. 4, black dotted arrow). Moreover, further investigation of the ameliorative effects of natural products and their specific targets in other LPS-induced injuries is required, especially in energy-metabolism-disorder-related intercellular junction disruption, basement-membrane-degradation-related hemorrhage, platelet activation and thrombus, monocyte chemotaxis, and macrophage-polarization-related fibroblast activation.
Acknowledgments
This work was partially supported by the National Natural Science Foundation of China (81873217 and 82074310) and the State Key Laboratory of Core Technology in Innovative Chinese Medicine (20221108). We thank Yin Li, Li Yan, Lu-Lu Yan, Gui-Zi-Meng Hu, An-Qing Li, and De-Xin Li for their help with data collection.
Authors’ contribution
Jingyan Han designed this study; Jingyan Han, Quan Li, Kai Sun, Chunshui Pan, Jian Liu, Ping Huang, Juan Feng, and Yanchen Liu searched literature, wrote the first draft of this manuscript, created tables, and drew figures; Gerald A. Meininger and Jingyan Han participated in discussion and improvement related to the manuscript; and Gerald A. Meininger and Jingyan Han critically revised and approved the final manuscript.
Compliance with ethics guidelines
Jingyan Han, Quan Li, Kai Sun, Chunshui Pan, Jian Liu, Ping Huang, Juan Feng, Yanchen Liu, and Gerald A. Meininger declare that they have no conflict of interest or financial conflicts to disclose.
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.eng.2023.11.016.
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