1. Introduction
Traditional Chinese medicine (TCM) has been widely used for thousands of years. However, there are not enough studies on the mechanisms of TCM. Inspired by the efforts to modernize TCM, the present study identified the active ingredients of
Lycium barbarum L., an important TCM used as a dietary supplement
[1].
Lycium barbarum L. strengthens bones and reinforces kidney essence, thereby improving osteoporosis
[2]. Kukoamine A and polysaccharide, bioactive compounds extracted from
Lycium barbarum L. protect against osteoporosis
[3]; however, the mechanisms by which
Lycium barbarum L. improves osteoporosis are not fully discovered.
The widespread application of new technologies led to rapid progress in RNA research and brought microRNAs (miRNAs) to the forefront of disease genomics research
[4],
[5]. Accumulating evidence shows that miRNAs are stable in biological fluids, including saliva, urine, and breast milk
[6],
[7]. These extracellular miRNAs target several genes to influence specific target cells
[8],
[9]. Different species of animals and plants can communicate through molecules such as hormone analogs
[10]. From an evolutionary perspective, miRNA transfer facilitates between-species cross-talk, communication, and signaling
[11],
[12],
[13]. A recent study reported that miRNAs play a key role in cellular communication, and miRNA transport from one species to another through feeding and other ways is involved in transboundary regulation between different species
[14],
[15],
[16],
[17].
miR168a derived from rice targets low-density lipoprotein receptor adaptor protein 1 to increase plasma cholesterol levels
[18].
One of the notable cases in TCM is the study of miRNA in honeysuckle.
miR2911 is stable in honeysuckle decoction, directly inhibits viral replication in human, and accelerates the negative conversion of patients after oral administration
[19]. Honeysuckle decoction significantly helps to treat coronavirus disease 2019 (COVID-19) patients
[19]. Moreover, the presence of herbal miRNAs in human and animal sera and tissues has been proven
[18]. Teng et al
. [13] reported that ginger-derived exosome-like nanoparticles (ELNs)-RNAs induce interleukin-22 (
IL-22) production and ameliorate colitis in mice by
mdo-miR7267-3p-mediated targeting of the
Lactobacillus rhamnosus monooxygenase ycnE. Recent findings suggest that miRNAs from food and Chinese medicine can be absorbed and delivered into cells, and food-derived miRNAs regulate gene expression and biological processes
[11],
[15],
[16],
[17],
[20],
[21],
[22],
[23],
[24]. Dietary miRNAs are a novel functional component of food and Chinese medicine. In view of the traditional application of
Lycium barbarum L. in osteoporosis, we hypothesized that miRNA from
Lycium barbarum L. may pass through the gastrointestinal (GI) tract and enter into the blood.
In the present study, we provided evidence for a functional miRNA derived from Lycium barbarum L., miR162a, which targets bone marrow to treat osteoporosis. These findings demonstrate a new mechanism of Lycium barbarum L. and support the theory that Lycium barbarum L. reinforces kidney essence and strengthens the bones. In addition, miR162a-overexpressing transgenic Nicotiana benthamiana (N. benthamiana) leaves have been developed, which protects against osteoporosis.
2. Materials and methods
2.1. Antibodies and reagents
The primary antibodies were nuclear receptor corepressor (NcoR; 20018–1-AP; ProteinTech Group, China), peroxisome proliferator-activated receptor γ (PPARγ; 16643–1-AP; ProteinTech Group), systemic RNA interference defective transmembrane family member 1 (SIDT1; 55352–1-AP; ProteinTech Group), and β-actin (4970S; Cell Signaling Technology, USA). miR162a-3p agomir was synthesized by GenePharma Co., Ltd., China, Prednisolone (batch number: N15J11Q115595; Shanghai Yuanye Biotechnology Co., Ltd., China), Alizarin red S (batch number: NO506Q031; Beijing Solaibao Technology Co., Ltd., China), 3-ethoxyaniline mesulfonate (batch number: C12590064; Shanghai Macklin Biochemical Technology Co., Ltd., China), and etidronate disodium (batch number: K22A8M34493; Shanghai Yuanye Biotechnology Co., Ltd.) were used in this study.
2.2. Cell lines and cell culture
Human bone marrow mesenchymal stem cells (BMSCs) were purchased from Shanghai Zhong Qiao Xin Zhou Biotechnology Co., Ltd., China. BMSCs were cultured with specialized medium for BMSCs (ZQ-1318; Shanghai Zhong Qiao Xin Zhou Biotechnology Co., Ltd.).
2.3. Osteogenic induction and Alizarin red staining
BMSCs were seeded at a density of 10 000 cells per well in six-well plates. After reaching 70%–80% confluence, cells were divided into three groups: blank control group, positive control group, and experimental group. The blank control cells were cultured in specialized medium for BMSCs (ZQ-1318; Shanghai Zhong Qiao Xin Zhou Biotechnology Co., Ltd.). The positive control cells were cultured in an osteogenic induction medium consisting of specialized medium for BMSCs, osteogenesis inducer, β-glycerophosphate (10 mmol‧L–1), and ascorbic acid (50 mg·L–1). After using the osteogenesis induction solution, cells in the experimental group were treated with 30 pmol miR162a-3p agomir. The osteogenesis medium was replaced every two days for four weeks. After 28 days, cells were washed three times with phosphate-buffered saline (PBS) and fixed with 4% paraformaldehyde for 15 min. Fixed cells were washed three times with distilled water and stained with 2% Alizarin red S solution (pH 4.2) for 30 min at 37 °C. Stained cells were subsequently washed with distilled water three times and air-dried in the dark before imaging.
2.4. miRNA expression and vector construction
The precursor structure of mature
miR162a-3p was obtained by replacing 21 nucleotides (nt) of the
Arabidopsis thaliana miR319a and the partially complementary region of
miR319a with four site-directed mutant primers and two homologous recombination primers. Four replacement primers of
miR162a-3p precursor were designed by WMD
†† http://wmd3.weigelworld.org/
. Plasmid pRS300, containing the precursor sequence of At-miR319a, was used as the template for polymerase chain reaction (PCR). Primer A (5′-CACGGGGGACTCTTGACgaattcctgcagccccaaac-3′) and
miR162a* reverse primer (5′-gaACGATAATCCTCTGCATCTAGtctacatatatattcct-3′) were used to amplify the 5-terminus of miRNA162 precursor.
miR162a* forward primer (5′-gaCTAGATGCAGAGGATTATCGTtcacaggtcgtgatatg-3′) and
miR162a reverse primer (5′-gaCTGGATGCAGAGGTTTATCGAtcaaagagaatcaatga-3′) were used to amplify the middle part of precursor.
miR162a forward primer (5′-gaTCGATAAACCTCTGCATCCAGtctctcttttgtattcc-3′) and primer B (5′-TCATATggtcacctAGGggatccccccatggcgatgcc-3′) were used to amplify the 3-terminus of
miR162a precursor. The three resulting fragments were fused by overlapping PCR with primers A and B on a mixture of 0.5 μL from previous PCR as the template. The fusion product was gel purified again, then cloned into pCambin1301-heat shock protein terminator (tHSP) with restriction endonuclease Nco I and restriction endonuclease Stu I digestion via the homolog arms at both ends.
miR162a precursor expression in
N. benthamiana was driven by a 35S promoter from
Cauliflower mosaic virus. Then, pCambin1301-35S-
miR162a-tHSP and empty pCambin1301-35S-tHSP plant expression vectors were transformed into
Agrobacterium tumefaciens strains EHA105.
2.5. Agrobacterium infiltration of N. benthamiana
Agrobacterium tumefaciens containing miRNA-expressing plasmid or empty plasmid were cultured in shaking (180 r·min−1) Luria-Bertani (LB) broth supplemented with kanamycin and rifampicin at 28 °C overnight. Cells were centrifuged at 4500g for 10 min and resuspended in MMA buffer (10 mmol·L–1 2-morpholinoethanesulphonic acid, 10 mmol·L–1 MgCl2, and 200 mmol·L–1 acetosyringone). Cell suspensions with an optical density at 600 nm (OD600) = 1 were incubated for 3 h before infiltration.
2.6. RNA extraction and quantitative PCR (qPCR)
Total RNA was extracted from
N. benthamiana leaves and
Lycium barbarum L. as previously described
[19].
miR162a-3p-specific stem-loop reverse transcription (RT) primer was 5′-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACCTGGAT-3′. The RT product was subjected to qPCR analysis using the ChamQ SYBR qPCR Master Mix (Vazyme Biotech Co., Ltd., China) with a
miR162a-3p-specific forward primer (5′-GCGGCGGTCGATAAACCTCTGC-3′) and stem-loop RT primer.
2.7. Animals and treatments
All C57BL/6J mice were housed in a room with 12 h light/dark cycles at Nanjing University of Chinese Medicine, China. Female C57BL/6J mice were removed from both ovaries and then kept continuously for 90 days to induce a mouse model of osteoporosis.
Wild-type AB strain zebrafish embryo (1dpf) was provided by Nanjing Yishu Lihua Biotechnology Co., Ltd., China. The culture system of zebrafish was provided by ESEN Water-Purification Tech. The breeding conditions of zebrafish were 26 °C, 14 h light/10 h dark. The pH (7.0–7.4) and conductivity (500–550 μs·cm−1) of culture water were maintained by NaCl and NaHCO3 solutions. Zebrafish embryos were collected in 12-well plates and divided into blank group, prednisolone model group (20 μmol·L–1 prednisolone), positive group (30 μg·mL−1 etidronate disodium), and agomir group (100, 300, and 900 pmol·L–1) after fertilization. Drugs were all dissolved in distilled water except for prednisolone, which was dissolved in dimethyl sulfoxide (DMSO). During the experiment, the final concentration of DMSO in the control group was 0.1%. Starting from the 3rd day of fertilization, the model and drug groups were treated with 20 μmol·L–1 prednisolone, and the blank group was given 0.1% DMSO. From the 5th day of fertilization, different concentrations of agomir were administered. The experiment ended on the 9th day and was repeated three times.
All animal studies were performed in accordance with the governmental recommendations for the Care and Use of Laboratory Animals and approved by the Institutional Ethics Review Boards of Nanjing University of Chinese Medicine (ethics registration No.: 201905A003).
2.8. Immunofluorescence staining and confocal imaging
Immunofluorescent staining was conducted according to previously described methods
[25]. A confocal microscope (TCS SP8; Leica, Germany) was utilized to capture images.
2.9. Statistical analyses
Statistical analyses were performed using SPSS version 22.0, and all values were expressed as mean ± standard deviation (SD) unless otherwise specified. A two-tailed Student’s t-test (2 groups) or one-way analysis of variance (ANOVA) (≥ 3 groups) was utilized to evaluate statistical significance. P < 0.05 was considered statistically significant.
3. Results
3.1. Herbal miR162a is absorbed and transferred into the blood of mice following administration of Lycium barbarum L.
C57BL/6J mice were gavaged with
Lycium barbarum L. 10 mL·kg
−1 to verify whether herbal miRNA can be absorbed into the blood. After 14 days, blood samples were collected and subjected to Illumina deep-sequencing (
Fig. 1(a), GSE232626). Base preference analysis of miRNAs in
Lycium barbarum L. showed that the first base preference significantly differed between miRNAs with different lengths. For example, the first base of miRNAs with a length of 18–24 nt was mostly U, while the first base of miRNAs with a length of 25–29 nt was mostly A (
Fig. 1(b)). Sequence length distribution analysis demonstrated that the length of
Lycium barbarum L.
-derived miRNAs was mostly 22 nt (
Fig. 1(c)). The copy numbers of the majority of miRNAs with a high expression in
Lycium barbarum L. were reduced in blood samples of mice, while the copy number of
miR162a remained high (
Fig. 1(d)). In addition, stem-loop PCR assay was performed to detect
miR162a (
Fig. 1(e)).
3.2. SIDT1-mediated absorption of dietary miRNA occurs in the stomach
Fluorescein amidites-labeled synthetic
miR162a was administrated by gavage. As depicted in
Fig. 2(a), obvious fluorescence was observed in whole blood and bone marrow, indicating that
miR162a entered the blood and bone marrow. Furthermore, significant fluorescence was observed in the kidney and liver of mice (
Fig. 2(b)). As SIDT1 localizes on the plasma membrane
[26] and mediates intercellular miRNA transport and extracellular small interfering RNA (siRNA) uptake
[27], we assumed that SIDT1 might mediate
miR162a absorption in mice. SIDT1 expression was analyzed in the GI tract 0.5 h after gavage to detect miRNA absorption through SIDT1. SIDT1 was mainly detected in the stomach and large intestine, including the cecum, colon, and rectum (
Fig. 2(c)), but not in the small intestine. Intriguingly, SIDT1 was colocalized with fluorescein amidites-labeled
miR162a. Primary gastric epithelial cells (PGECs) were collected, and it was found that fluorescent-labeled
miR162a entered the cytoplasm and nucleus of PGECs (
Fig. 2(d)). Of note, no miRNA was observed 3 h after gavage, suggesting that miRNA was absorbed in the stomach (
Fig. 2(e)). The schematic diagram shows that the stomach is the main organ for absorbing
miR162a through gastric SIDT1 (
Fig. 2(f)).
3.3. Herbal miR162a directly binds to NcoR and promotes osteogenic differentiation of BMSCs
Bioinformatics analysis was conducted for structural analysis (
Fig. 3(a)) and downstream targets of
miR162a were screened (
Fig. 3(b)) to verify whether
miR162a is a bioactive ingredient of
Lycium barbarum L. Using a human reference genome and predictions made by Mireap online tool, we found that
NcoR was a downstream target gene of
miR162a (
Fig. 3(b)). It has been reported that targeting
NcoR increases insulin sensitivity, reduces blood sugar, and improves osteoporosis in diabetic patients
[28].
NcoR knockdown can induce osteogenic differentiation in rat BMSCs
[29]. Alizarin red staining was performed to confirm the bioactivity of
miR162a. It was found that
miR162a promotes osteogenic differentiation (
Fig. 3(c)). Furthermore, luciferase assay identified
NcoR as the downstream target of
miR162a (
Fig. 3(d)). Western blotting (WB) confirmed that
NcoR and its client protein PPARγ were decreased after treatment with
miR162a (
Fig. 3(e)). In addition, we found that
miR162a significantly decreased PPARγ expression, which was reversed by rosiglitazone, a PPARγ agonist (
Fig. 3(f)). Moreover, qPCR assay confirmed that classical osteogenic markers, runt-related transcription factor 2 (RUNX2)
, bone sialoprotein (BSP), osteocalcin (OCN), and osterix were significantly upregulated after treatment with
miR162a, and rosiglitazone substantially attenuated this effect (
Fig. 3(g)).
3.4. miR162a promotes osteogenic differentiation in zebrafish
A zebrafish osteoporosis model was established to validate the anti-osteoporosis effect of
miR162a in vivo [30]. Fluorescence localization analysis showed that synthetic
miR162a entered the body of the zebrafish and mainly accumulated in the GI tract of zebrafish (
Fig. 4(a)). Compared with the blank group, prednisolone treatment at 20 μmmol·L
–1 significantly inhibited the formation of the first vertebrae of juvenile zebrafish, evidenced by decreased alizarin red staining area (
Figs. 4(b) and
(c)) and reduced optical density in staining (
P < 0.001;
Fig. 4(d)). These findings indicate that the juvenile zebrafish model of osteoporosis was successfully established. Etidronate disodium, used for treating osteoporosis as a positive drug, significantly increased the mineralized area and optical density of the first vertebrae of juvenile zebrafish. Different concentrations of synthetic
miR162a significantly improved the stained area and optical density of the first vertebrae of juvenile zebrafish (
Figs. 4(c) and
(d)). Synthetic
miR162a at 100 and 300 pmol·L
–1 evidently promoted vertebral node formation in juvenile zebrafish (
Fig. 4(e)). These data suggest that
miR162a relieves the inhibitory effect of prednisone on bone development in juvenile zebrafish.
3.5. Transgenic N. benthamiana leaves overexpressing miR162a vigorously protect against osteoporosis in mice
Transgenic technology was used to overexpress
miR162a in
N. benthamiana leaves and examine whether transgenic
N. benthamiana leaves with
miR162a protect against osteoporosis (
Fig. 5(a)).
N. benthamiana leaves were selected to express
miR162a for its easy planting, short life cycle, and large-scale production. A modified precursor of mature
miR162a was obtained by replacing the 21 nt of
Arabidopsis thaliana miR319a, and the partially complementary region of
miR319a (
Fig. 5(b)).
Agrobacterium tumefaciens containing miRNA-expressing plasmid was used to overexpress
miR162a in transgenic
N. benthamiana leaves (
Fig. 5(c)). The results of stem-loop real time quantitative polymerase chain reaction (RT-qPCR) (
Fig. 5(d)) and semiquantitative reverse transcription and polymerase chain reaction (RT-PCR) assays (
Fig. 5(e)) confirmed high expression of
miR162a-3p in
miR162a-overexpressed (OE)
N. benthamiana leaves, but not in empty vector (EV)
N. benthamiana leaves.
Both ovaries were removed from female C57BL/6J mice. Mice were observed for 90 days to establish an osteoporosis model. Hematoxylin and eosin (H&E) and tartrate-resistant acid phosphatase (TRAP) staining of the left femur showed that bone loss occurred in the model group, and bone density of the epiphysis significantly decreased (
Fig. 5(f)). Transgenic
N. benthamiana leaves,
Lycium barbarum L.
, and etidronate disodium all significantly prevented bone loss in mice (
Fig. 5(f)). As shown in
Fig. 5(g), it was found that biliary glycoprotein (BGP) levels in the model group remarkably decreased, and TRAP and alkaline phosphatase (ALP) levels significantly increased, indicating that mice in the model group suffered from severe osteoporosis (
Figs. 5(h) and
(i)). In addition, transgenic
N. benthamiana leaves,
Lycium barbarum L.
, and etidronate disodium all maintained bone homeostasis, inhibited bone loss, and promoted bone formation in mice (
Figs. 5(g)–(j)). Changes in
BGP level indicated that the effect of
Lycium barbarum L. on bone formation was greater than transgenic N.
benthamiana leaves and disodium etidronate (
Fig. 5(g)). Changes in
TRAP and
ALP levels revealed that the effects of transgenic
N. benthamiana leaves and disodium etidronate on the prevention of bone loss and maintenance of bone metabolism were greater than
Lycium barbarum L. (
Figs. 5(h) and
(i)). The results of micro-computed tomography (microCT) showed that the application of transgenic
N. benthamiana leaves,
Lycium barbarum L.
, and etidronate disodium significantly prevented bone loss in mice (
Fig. 5(k)).
4. Discussion
The modernization of TCM is a major problem for many pharmaceutical experts. Despite the clear efficacy of TCM, international pharmaceutical communities hardly recognize TCM due to its unclear mechanisms and active compounds, which seriously restricts its modernization and marketing.
Lycium barbarum L. has been a functional food and medicine for thousands of years. It has been reported that
Lycium barbarum L. increases the osteogenic differentiation and mineralization of BMSCs
[31],
[32]. In addition,
Lycium barbarum L. increases type II muscle fiber types by activating estrogen-related receptor γ (ERRγ) and promoting the effect of exercise
[33]. These findings indicate that
Lycium barbarum L. regulates bone metabolism through direct or indirect means. Some studies have been conducted to explore the role of polysaccharides in improving osteoporosis; however, few studies focused on the anti-osteoporosis activity of other bioactive ingredients of
Lycium barbarum L.
Currently, calcium, active vitamin D, recombinant parathyroid hormone, calcitonin, and bisphosphonate are used for treating osteoporosis in clinics; however, the effect of these western medicines is not promising. According to the TCM theory, the kidney stores essence, which can produce marrow, thus nourishing the bone. Western medicine believes that stemness and pluripotent differentiation make stem cells the most primitive cell type. Stem cells, including BMSCs and hematopoietic stem cells, have many functions, such as blood transformation, repair, and development
[34]. It is in agreement with TCM theory, which claims that kidney essence, including congenital and acquired essence, is the source of life. Kidney essence distributes and regulates the function of the whole body. Particularly, kidney essence plays a significant regulatory role in the function of bone marrow
[35]. Based on TCM theory, the present study identified a new mechanism of
Lycium barbarum L. against osteoporosis and explained its mechanism in reinforcing kidney essence and strengthening bone from miRNA perspective.
Despite their relatively low abundance in recipient organisms, dietary miRNAs are functional. In addition to the well-known honeysuckle
miR2911 that can treat COVID-19 patients, other dietary miRNAs play important roles in metabolism and immune response
[36],
[37],
[38],
[39],
[40].
miR168a derived from rice increases plasma cholesterol levels
[18]. Milk-derived
miR-155 regulates B cell maturation and immune response, while milk-derived
miR181a and
miR181b mediate B cell differentiation
[41]. Moreover, milk exosome-derived
miR-155 regulates forkhead box P3 (FoxP3) expression and IL-4 signaling and controls immunoglobulin E (IgE) binding to high-affinity IgE receptor to modulate regulatory T cell response
[40]. Dietary herbal miRNAs can affect consumers’ organisms in a cross-kingdom manner
[42]. Strawberry fruit-derived
miR168 induced and exacerbated experimental autoimmune encephalomyelitis by limiting dendritic cell migration, which is associated with toll-like receptor 3 (TLR3) and Toll/interleukin-1 receptor-like protein (TIR)-domain-containing adapter-inducing interferon-β (IFN-β) signaling
[39]. Oral administration of tumor suppressor miRNAs reduced colon cancer growth in a mouse model. The combination of plant and tumor suppressor miRNAs further reduced tumor growth
[43]. Detectable concentrations of
miR159 inhibited breast cancer growth by directly targeting transcription factor 7 (TCF7), which encodes a Wnt signaling-related transcription factor
[44].
Dietary miRNAs are effective after being processed and absorbed by the GI tract. Previous studies have found that miRNAs are stable during food processing. The serum concentrations of corn-derived miRNAs (
zma-miR164a-5p and
zma-miR319a-3p) in pigs fed with fresh corn peaked at 6–12 h
[45], and miRNAs remained stable in the GI tract for more than 1 h
[20],
[46]. Serum miRNA concentrations maintained stable over the next 7 days after random feeding with fresh corn
[47]. It is worth noting that half of exogenous lettuce
miR156a was detected in circulating human exosomes 3 h after feeding
[48]. However, only a small fraction of food miRNAs is absorbed, indicating that miRNAs in food are selectively absorbed
[16],
[49]. The specificity of miRNA is an important factor affecting their absorption
[49].
miR2911 is stable in honeysuckle decoction and can be absorbed into the circulation and delivered to tissues following oral administration. The stable properties of
miR2911 mainly depend on its special structure and GC content
[19]. In the present study, by searching
miR162a-3p sequences of different species from the microRNA database and analyzing the conservation of their mature sequences, we found that
miR162a-3p was highly conserved among different species. In addition, the RNAFold web server† was used to verify the precursor characteristics, and it was found that the
miR162a-3p precursor sequence had typical stem-ring structure characteristics. The mature sequence was located at the 3′ end of the precursor sequence, whose length and GC content were 228 bp and 36.84%, respectively. The minimum folding free energy (MFE) was –68.30 kcal·mol
−1, and the MFE index (MFEI) was 0.81, indicating that the miRNA precursor was relatively stable. Using a mouse model, it was confirmed that
miR162a-3p entered the blood and bone marrow and passed through the mouse GI tract via SIDT1-dependent absorption. SIDT1 was initially proposed as a double strain RNA (dsRNA) transporter in mammalian cells
[26]. The absorption efficiency of
miR156a,
miR168a, and
miR2911 is largely different, suggesting that SIDT1 may selectively mediate the uptake of exogenous miRNAs
[18],
[19].
miR162a is the first dietary miRNA identified from TCM that can target BMSCs. However, the direct absorption and stability of food-derived miRNAs in human GI tract have not yet been demonstrated, and many studies have failed to detect dietary miRNAs in animal models
[50],
[51]. Thus, the absorption of dietary miRNAs requires further investigation, especially in mammals.
The miRNA transporters and surface proteins of exosomes also play a key role in the endocytosis of miRNA. Endocytosis contributes to the absorption and delivery of dietary miRNAs
[52],
[53]. Previous studies reported that miRNAs are encapsulated by plant-derived exosome-like nanoparticles
[54],
[55]. The protease K-dependent destruction of milk exosome surface proteins significantly reduces the transport rate of human endothelial cells, suggesting that exosome surface proteins are involved in miRNA transport
[56]. Modification of β-galactoside and
n-acetylglucosamine on the surface of milk-associated exosomes and intestinal cells plays a vital role in endocytosis
[57]. In addition, miRNAs are transported to the intestines of infants and young children through lactation. Under the protection of exosomes and other substances, they remain intact in the digestive system and are absorbed by intestinal epithelial cells
[38],
[46],
[57]. Thus, it is necessary to study the absorption mechanism of miRNAs available in TCM to provide a better foundation for treating diseases and accelerate the modernization of TCM.
The modernization of TCM inspires the use of TCM theory as a guide. It analyzes effective substances through modern technologies. For example, arsenic trioxide was shown to treat leukemia, and artemisinin was shown to treat malaria by TCM records. Recently, the applications of transgenic technology and nanomedicine significantly improved the modernization of TCM
[58]. Transgenic technology has empowered plants to produce therapeutic molecules and revolutionize miRNA generation. In a previous study, Zhang et al.
[59] generated small silencing RNAs in edible lettuce, which could repress
HBsAg gene expression and reduce liver damage in a mice model. Fu et al.
[60] developed a controllable siRNA self-assembly and delivery, regulating gene expression in a purpose-driven model. In this study, we found that
miR162a is a new active compound of
Lycium barbarum L.
, which can be absorbed into the blood to target
NcoR and promote osteogenic differentiation (
Fig. 6). This finding supports the TCM theory, in which
Lycium barbarum L. reinforces the kidney and strengthens the bone. Furthermore, it was shown that SIDT1 in the stomach mediates
miR162a absorption. Due to the relatively low abundance of
miR162a in
Lycium barbarum L., we developed transgenic
N. benthamiana leaves to overexpress
miR162a, one of the most widely used experimental models in plant science. Transgenic
N. benthamiana leaves not only produced
miR162a in large quantities but also effectively protected against osteoporosis in mice. Because of high yield, low cost, and strong environmental adaptability, using transgenic plants to produce miRNAs for treating diseases may have broad application prospects and marketing value. However, there are still some limitations to the present study. Although
miR162a absorption into blood has been confirmed, the pharmacokinetics and biodistribution of
miR162a in vivo still need to be investigated. Importantly, we need to extend the basic research on
miR162a to clinical application.
Collectively, the present study demonstrated that Lycium barbarum L.-encoded miR162a can be absorbed into the blood to target NcoR and promote osteogenic differentiation to improve osteoporosis. In the future, we will focus on the development of genetically engineered plants and multi-functional nano-platforms for oral delivery of miR162a, which are effective, safe, economical, and feasible.
Acknowledgments
This work was supported by Key Project of Jiangsu Province’s Administration of Traditional Chinese Medicine (ZD202203), Jiangsu Province’s Innovation Program (JSSCTD202142), and a project funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (traditional Chinese medicine).
Authors’ contribution
Chunyan Gu, Ye Yang, Xiaosong Gu, Jinao Duan, and Kai Xu designed and supervised the projects. Chunyan Gu and Xiaozhu Tang drafted the manuscript and drew the figures. Xichao Yu, Xiaozhu Tang, Leilei Gong, Jingquan Tan, Yuanjiao Zhang, Huili Zheng, Ze Wang, and Chenqian Zhang performed the experiments and analyzed the data. Yejin Zhu, Zuojian Zhou, and Heming Yu provided technical and theoretical supports. All authors have read and approved the final version of the manuscript.
Compliance with ethics guidelines
Chunyan Gu, Xichao Yu, Xiaozhu Tang, Leilei Gong, Jingquan Tan, Yuanjiao Zhang, Huili Zheng, Ze Wang, Chenqian Zhang, Yejin Zhu, Zuojian Zhou, Heming Yu, Kai Xu, Jinao Duan, Xiaosong Gu, and Ye Yang declare that they have no conflict of interest or financial conflicts.
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