1. Introduction
Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), poses a considerable public health challenge worldwide, resulting in millions of fatalities and extensive morbidity [
1], [
2]. The current small-molecule antiviral drugs utilized for COVID-19 treatment include remdesivir, nirmatrelvir/ritonavir, and molnupiravir [
3]. However, the Omicron variant strains have demonstrated heightened contagiousness and immune evasion capabilities [
4], [
5], with documented breakthrough infections postvaccination [
6]. Moreover, certain small-molecule antiviral drugs, such as monogravir and azivudine, have genotoxicity, and patients treated with these drugs can develop resistance [
7]. Additionally, potential drug-drug interactions, exemplified by those with Paxlovid, presents further challenges in the context of epidemic prevention and control.
Sanhan Huashi formula (SHHS), and the integration of three Chinese patent medicines (CPMs) and three Chinese medicine formulas including Jinhua Qinggan granules, Xuebijing injection, Lianhua Qingwen capsule, Huashi Baidu formula, Qingfei Paidu decoction, and Xuanfei Baidu formula, has been proven to be crucial for treating COVID-19 [
8], [
9]. SHHS (also known as the Hanshiyi formula) is a Chinese medicinal preparation developed by academician Xiaolin Tong for the prophylaxis and treatment of mild to moderate cases of COVID-19. SHHS was officially included in the 4th and 10th trial versions of the
Diagnosis and treatment protocol for COVID-19, and it has become the most extensively utilized formula in Wuhan, China. Subsequently, over 2.2 million doses have been distributed across Henan, Jiangsu, Jilin, and other provinces, significantly contributing to local epidemic prevention and control. The formula comprises 20 traditional Chinese medicines (TCMs) (Dataset S1(a) in Appendix A). A randomized, open-label, multicenter trial demonstrated the superiority of SHHS over nirmatrelvir/ritonavir in reducing the hospital stay duration and accelerating the clinical recovery of patients with mild to moderate COVID-19. SHSS was particularly advantageous for alleviating symptoms such as fever, cough, sore throat, and fatigue [
10].
Additionally, SHHS can prevent the progression from mild to severe COVID-19, suggesting potential antiviral and anti-inflammatory effects [
11]. Network pharmacology analyses support that the therapeutic effects of SHHS against COVID-19 might stem from its antiviral, immunomodulatory, and anti-inflammatory properties involving various components, targets, and pathways [
12]. Despite these insights, the exact mechanisms by which SHHS treats COVID-19 remain to be elucidated, necessitating further investigation.
Previous studies have shown that Lianhua Qingwen capsule, Shuanghuanglian oral liquid, and Qingfei Paidu decoction, which are used for treating COVID-19, have anti-SARS-CoV-2 properties. The primary herbal ingredient in Lianhua Qingdian capsule is
Forsythia, with “Forsythoside A” from
Forsythia suspensa recognized as an active constituent capable of inhibiting SARS-CoV-2 3C-like protease (3CL
pro) activity, thus effectively preventing viral replication and translation [
13]. In Shuanghuanglian oral liquid,
Scutellaria baicalensis is a key ingredient, and its main active constituent, baicalein, has shown potential in mitigating SARS-CoV-2-induced damage and is a promising therapeutic agent [
14]. Additionally, leucovorin in Qingfei Paidu decoction may contribute to its antiviral SARS-CoV-2 activity [
15]. These findings underscore the importance of studying herbal preparations as potential clinical therapeutic agents and identifying bioactive molecules within TCM that could contribute to disease prevention and treatment [
16]. Identifying the main active compounds in TCM can clarify the mechanism of action of TCM and promote the modernization of TCM.
In this research, we first evaluated the antiviral and anti-inflammatory properties of SHHS through both
in vivo and
in vitro experiments. Subsequent analyses involved characterizing the components of SHHS that were absorbed into the plasma, lungs, and feces via ultrahigh-performance liquid chromatography coupled with linear trap quadrupole Orbitrap mass spectrometry (UHPLC-LTQ-Orbitrap-MS). We focused on five key antiviral targets, namely, human angiotensin converting enzyme 2 (hACE2) [
17], the SARS-CoV-2 spike receptor-binding domain (RBD) [
17], 3CL
pro [
18], papain-like protease (PL
pro) [
19], and human transmembrane protease serine 2 (TMPRSS2) [
20], to elucidate the antiviral mechanisms of SHHS compounds. Moreover, the antiviral and anti-inflammatory effects of these active compounds were validated in Vero-E6 cells.
2. Materials and methods
2.1. Biosafety and ethics statement
Experiments involving SARS-CoV-2 virus-like particles (VLPs) were conducted in a biosafety level (BSL)-2 facility, while all procedures involving infection with SARS-CoV-2 were carried out in a BSL-4 facility. The animal experiments were approved by the Experimental Animal Ethics Committee of Guang’anmen Hospital, China Academy of Chinese Medical Sciences (IACUC-GAMH-2021-011), the Institute of Basic Research of the Chinese Academy of Chinese Medical Sciences (IBTCMCACMS21-2111-01), and the Ethical Review Committee for Life Science Research of Wuhan Institute of Virology, Chinese Academy of Medical Sciences (WIVA21202012). The live SARS-CoV-2 strain 2019-nCoV WIV04 was obtained from the Wuhan Institute of Virology.
2.2. Cell experiments
Vero-E6 cells (Punosai, China) were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, USA) supplemented with fetal bovine serum (FBS; Gibco) and penicillin-streptomycin, and tested negative for mycoplasma contamination. RAW264.7 cells were cultured in DMEM supplemented with 10% FBS. Vero-E6 cells (2.5 × 105 cells∙well−1) were seeded in 24-well plates and then exposed to SARS-CoV-2 at a multiplicity of infection (MOI) of 0.01 for 1 h. Following removal of the supernatant, the cells were rinsed with phosphate-buffered saline (PBS) and cultured in medium containing 2% FBS. At 24, 48, or 72 h post infection, cell supernatants and lysates were collected and analyzed using quantitative reverse transcription polymerase chain reaction (qRT-PCR) to quantify the number of viral RNA copies and the messenger RNA (mRNA) levels of cytokines.
RAW264.7 cells were cultured in DMEM supplemented with 10% FBS. Cells were incubated with various concentrations of SHHS for 12 h, cells were treated with lipopolysaccharide (LPS; 1 μg∙mL−1) for 9 h, cell supernatants were collected, and the levels of inflammatory factors were assessed by enzyme-linked immunosorbent assay (ELISA).
2.3. Cell viability assay
The viability of Vero-E6 cells was assessed using a cell counting kit-8 (CCK-8) assay (Abcam, UK) following the manufacturer’s instructions. Vero-E6 cells alone were used as the control group, and the absorbance was measured at 450 nm. The cells were incubated with various concentrations of SHHS for 24 h to evaluate potential cytotoxicity.
2.4. Animal experiments
For VLP animal experiments, 6- to 8-week-old specific pathogen-free (SPF) K18-hACE2 transgenic male mice (Saiye Biologicals, China) were randomly assigned to the blank group, control group, SHHS group, or ursodeoxycholic acid (UDCA) group, with six mice in each group. In the preliminary experiment, SHHS spray dry powders were administered by gavage at a dose of 4.55 g∙kg−1∙d−1, and one animal died on the 2nd and 3rd days of administration. Therefore, the dose was adjusted to 2.275 g∙kg−1∙d−1 in the formal experiment, and no mice died. Before modeling, the mice were fasted for 12 h and had free access to water. The SHHS group was orally administered SHHS for four consecutive days at a dose of 2.275 g∙kg−1∙d−1, the UDCA group was administered UDCA by gavage for seven consecutive days at a dose of 693 mg∙kg−1∙d−1, and the control group was administered distilled water by gavage. The SARS-CoV-2 VLP lung invasion model was established in every group of hACE2 mice except for the blank group by the drip injection of VLPs 2 h after the administration of the drug on the 5th day of the experiment. The mice were anesthetized intraperitoneally, the tracheal cartilage ring was exposed, the cricothyroid membrane was punctured with an insulin needle, and 50 µL of VLPs packaged with luciferase (Luc-VLPs; 15 mg∙mL−1) was slowly injected. Mice were euthanized 6 h after VLP injection.
For the live virus animal experiments, K18-hACE2 mice (7-8 weeks) were purchased from Jiangsu GemPharmatech Co., Ltd. (China). Twelve K18-hACE2 mice were randomly assigned to a model control group or SHHS treatment group, with six mice in each group. The mice in the SHHS group were given SHHS spray dry powders daily starting seven days before the attack, at a dose of 6.825 g∙kg−1 (1.5 times the human clinical equivalent dose), and the mice in the model control group were given a daily gavage of an equal volume of PBS solution. Mice were infected with 2019-nCoV WIV04 nasal drops at a dose of 1000 50% tissue culture-infectious dose (TCID50) per 50 µL. Following infection, the body weights of the mice were observed on a daily basis, and the mice were humanely euthanized three days after infection. Tissue RNA was extracted from lung homogenates for quantitative viral and cytokine assays.
For the LPS-induced ALI model animal experiments, mice (n = 30) were randomly assigned to the control group (Control), model group (Model), SHHS low-dose group (SHHS-L; 2.275 g∙kg−1), SHHS medium-dose group (SHHS-M; 4.55 g∙kg−1; human clinical equivalent dose), SHHS high-dose group (SHHS-H; 9.1 g∙kg−1), or dexamethasone group (Dex; 5 mg∙kg−1), with five mice in each group. The SHHS group was given SHHS spray dry powders by gavage, the model group and the Dex group were given physiological saline by gavage, and each group was given a tracheal drip (LPS 5 mg∙kg−1) 2 h after gavage on the morning of the 7th day. Moreover, the Dex group was given Dex 5 mg∙kg−1 intraperitoneally 2 h prior to the administration of LPS for modeling. The mice were euthanized 24 h after modeling.
2.5. Construction and purification of SARS-CoV-2 VLPs
HEK-293T cells (ATCC, USA) were seeded in cell culture dishes, and the gene plasmids for the SARS-CoV-2 spike (S), membrane (M), envelope (E), and nucleocapsid (N) proteins with Flag tags (Genscript, USA) and an expression plasmid for luciferase were simultaneously transfected into the cells using polyethylenimine linear (PEI) transfection reagent. The supernatant was removed after 48 h, and the cells were rinsed with PBS, scraped, collected, and transferred to Eppendorf (EP) tubes. The cell lysates were obtained through a process of freeze-thaw lysis, followed by centrifugation. After removing the supernatant, 900 to 1000 μL of a 20% sucrose solution was added to the EP tube. The collected supernatant was added slowly against the wall of the tube, the tube was centrifuged at 21 690
g (
g = 9.8 m∙s
−2) for 7 h at 4 °C, and the precipitate was resuspended in PBS and filtered [
21].
2.6. Histological examination
Lung tissues taken from SARS-CoV-2-infected mice and LPS-induced ALI model mice were fixed in 4% paraformaldehyde (PFA) fix solution (Biosharp, China), embedded in paraffin (Thermo Fisher Scientific, USA), and sectioned according to standard procedures. The sections were sliced into 3 mm pieces and then stained with hematoxylin and eosin (HE) following the manufacturer’s instructions. Collagen staining was performed with Masson’s trichrome staining using a Masson’s trichrome staining kit (Wuhan Servicebio Technology Co., Ltd., China), collagen-positive areas were detected and examined microscopically using an orthogonal light microscope (Nikon, Japan), and images were obtained and processed with a Nikon DS-U3. The lung injury score was calculated by evaluating pulmonary hemorrhage, inflammatory infiltration, and pulmonary interstitial edema. Scores range from 0, indicating no significant lesions, to 4, representing critical severity. The intermediate grades were assigned as follows: 0.5 to 1 for mild cases, 2 for moderate cases, and 3 for severe cases, thus accurately reflecting the disease severity.
2.7. Immunofluorescence
For immunofluorescence staining, fresh lung tissues from K18-hACE2 mice were fixed in 4% paraformaldehyde (BioDee, China) for 48 h. The tissues were embedded in optimal cutting temperature (OCT) in a thermostatic freezer sectioning machine (Sakura, USA), sectioned at 10 μm, and equilibrated to room temperature. After washing with PBS, the cells were blocked with serum blocking solution (3% bovine serum albumin (BSA)) for 1 h. Rabbit anti-ACE2 antibody (1:200, 1% BSA; Proteintech, USA) and mouse anti-FLAG antibody (1:400, 1% BSA; Proteintech) were incubated in a wet cassette overnight at 4 °C. After washing with PBS, the sections were incubated with an anti-rabbit Alexa Fluor 488 fluorescent secondary antibody (1:1000, 1% BSA; Invitrogen), an anti-mouse Alexa Fluor 568 fluorescent secondary antibody (1:1000, 1% BSA; Invitrogen), and an Alexa Fluor 647 ghost pen cyclic peptide antibody (1:1000, 1% BSA; Cell Signaling Technology, USA) for another 2 h. Three washes of 5 min each were performed in PBS. The cells were incubated with Hoechst (1:1000, PBS) for 15 min, followed by three washes with PBS. The plates were sealed with drops of anti-fluorescence quenching sealer. Images were scanned and photographed by an Olympus fluorescence microscope (Japan) for qualitative analysis.
After the sections were slightly dried, circles were drawn around the regions of interest with a tissue pen, and the sections were sealed with BSA for 30 min. The sections were incubated with anti-SARS-CoV-2 spike RBD and nucleoprotein (NTD) antibodies (Abmart, China) overnight. The slides were treated with sulfo-cyanine3 (Cy3)-labeled goat anti-mouse IgG at a 1:300 dilution and incubated for 50 min in the dark. Following rinsing with PBS, the slides were exposed to a DAPI solution and incubated in the dark for 10 min. After washing with PBS, an autofluorescence quencher solution was added for 5 min, and the anti-fluorescence quenched tablets were sealed. A Nikon Eclipse C1 standing fluorescence microscope was used for scanning, photographing and analysis.
2.8. Immunohistochemistry
The expression of CCL-2 in paraffin-embedded sections was determined by immunohistochemical staining. The sliced samples were immersed in a solution of 3% hydrogen peroxide and incubated for 25 min in the dark. The tissue was evenly coated with a 3% BSA solution in a petri dish and sealed at room temperature for 30 min. The primary antibody of CCL-2 (MCP-1) (Servicebio, China) was applied, and the tissue was incubated overnight at 4 °C. Following a rinse with PBS (pH 7.4), the sections were then exposed to a horseradish peroxidase (HRP)-conjugated goat anti-rabbit immunoglobulin G (IgG) antibody (1:200) for 50 min. The sections were incubated with diamino-benzidine as a chromogenic substrate, counterstained with hematoxylin, and finally observed via an E100 microscope system (Nikon).
2.9. Polymerase Chain Reaction (PCR)
To quantify the number of SARS-CoV-2 RNA copies, viral RNA was extracted from infected cell supernatants or homogenized tissues using the QIAamp® Viral RNA Mini Kit (Qiagen, Germany) and utilized as the template for RT-PCR. The primer pairs RBD-qF1 (5′-CAATGGTTTAACAGGCACAGG-3′) and RBD-qR1 (5′-CTCAAGTGTCT-GTGGATCACG-3′) from our prior research were used for alignment of the S gene. The RNA quantity was verified using a HiScript® II one step qRT-PCR SYBR® green kit (Vazyme, China) and 2 μL of RNA according to the manufacturer’s instructions. The corresponding mouse gene sequence in National Center for Biotechnology Information (NCBI) was searched, the coding sequences (CDSs) region was selected, and Primer Premier 6.0 software was used to design primers across the exon region. A quantitative polymerase chain reaction (qPCR) mix kit (Tiangen Biotech (Beijing) Co., Ltd., China) was used according to the manufacturer’s instructions. The primer sequences are shown in Table S1 in Appendix A.
2.10. Luciferase activity assay
Phenylmethylsulfonyl fluoride (PMSF) (P0100; Solarbio, China) and cocktail were added to radio immunoprecipitation assay (RIPA) lysis buffer (89901; Thermo Fisher Scientific) at a 1:100 ratio. Four hundred microliters of the buffer was added to 100 mg of tissue for cell lysis, and the whole process was performed on ice. Tissue was homogenized using a homogenizer with one 4 mm bead and two 3 mm beads per EP tube at 70 Hz for 120 s. The homogenized tissue was centrifuged at 4 °C, and the resulting supernatant was obtained after the grinding and centrifugation steps were repeated. The supernatant samples were aspirated separately and stored at 4 °C. Twenty microliters of the samples were added to each well of a 96-well plate, after which 100 μL of luciferase substrate was added to each well and mixed. The luciferase activity was detected using a luciferase assay kit (E1501; Promega, USA).
2.11. ELISA
ELISA kits for the detection of interleukin (IL)-1β (KE10003), IL-6 (KE10007), and tumor necrosis factor-α (TNF-α; KE10002) in mouse lung tissue, serum, and cell culture medium were purchased from Proteintech Group, Inc. (China). The relevant tests were carried out according to the instructions.
2.12. Characterization of the chemical compound profile of the SHHS spray dry powders
SHHS spray dry powders consisting of 20 Chinese herbs were obtained from Jiangsu Kanion Pharmaceutical Co., Ltd. (production lot number: Z210801; China). A rapid, sensitive, and reliable UHPLC-LTQ-Orbitrap-MS method was used to characterize the compound profile of SHHS. Approximately 1.0 g of SHHS spray dry powders were passed through a 0.45 m microporous filter membrane to gather the filtered liquid. To identify these compounds, we established a library of standards based on the Chinese Pharmacopoeia composition of each individual drug and the pharmacodynamic constituents reported in the literature, together with the retention time and precise mass number provided by high-resolution liquid chromatography. The analysis was performed on an ACQUITY UHPLC HSS T39 instrument (Waters, USA). The data were collected and analyzed using Xcalibur, Metworks, and Mass Frontier 7.0 software.
2.13. 3CL pro and PLpro purification
The genetic sequence of the SARS-CoV-2 PLpro in the pET-28 vector was kindly provided by Professor Zigang Li at the Peking University Shenzhen Graduate School. The coding sequence of SARS-CoV-2 3CLpro in the PET-28 vector was synthesized by GENEWIZ (China). Briefly, the plasmids were first transformed into the BL21 (DE3) strain of Escherichia coli. The recombinant cells were grown at 37 °C until the optical density value at 600 nm reached 0.6-0.8. Subsequently, the expression of SARS-CoV-2 PLpro or 3CLpro was induced, and the cells were further cultivated for another 20 h at 16 °C. The cells were harvested via centrifugation and sonicated. The insoluble components were then separated via centrifugation, and the supernatant was purified through a HisTrap (Cytiva, USA) followed by a Superdex 75 (Cytiva). The protein purity was confirmed to be > 95% by sodium dodecyl sulfate-polyacrylamide gel electrophocresis (SDS-PAGE).
2.14. Surface plasmon resonance (SPR)
A Biacore 8K+ instrument (Cytiva) was used to assay the binding affinities of the SHHS components to either hACE2 or the SARS-CoV-2 RBD. hACE2 (Sino Biological, China) or SARS-CoV-2 RBD (Sino Biological) were attached to a CM5 sensor chip through standard amine coupling using HEPES buffered saline with P20 (HBS-P; Cytiva). In the binding assays, the immobilized levels of both hACE2 and SARS-CoV-2 spike RBD were approximately 10 000 response units (RU). Compounds at different concentrations were injected sequentially into the channel with 5% dimethyl sulfoxide (DMSO) to assess their binding affinities. The dissociation constants (KDs) of the compounds were determined by analyzing the data with the steady-state affinity model using Biacore 8K+ Evaluation Software. In the competitive inhibition experiments, the immobilization level of hACE2 or the spike RBD was approximately 3000 RU. First, the compound with binding activity was injected for preincubation with hACE2 or spike RBD. Subsequently, 5 nmol∙L−1 spike RBD or 30 nmol∙L−1 hACE2 was injected to evaluate the association with hACE2 or spike RBD, respectively, in the presence of the active compound. The effectiveness of blocking was determined by comparing the response units obtained with and without the active compound.
2.15. Enzymatic activity assay
The activities of SARS-CoV-2 3CL
pro, PL
pro, and TMPRSS2 were measured in kinetic mode with the substrates Thr-Ser-Ala-Val-Leu-Gln-
p-nitroanilide [
22], peptide-7-amino-4-methylcoumarin (Z-Arg-Leu-Arg-Gly-Gly-AMC; Bachem Bioscience, China), and Boc-Gln-Ala-Arg-AMC (CSB-YP023924HU; CUSABIO, China), respectively. The final concentrations of 3CL
pro, PL
pro, and TMPRSS2 in the reaction were 50, 100, and 30 nmol∙L
−1, respectively. The absorbance or fluorescence signals were recorded using a BioTek Synergy Neo2 (USA).
2.16. Hydrogen-deuterium exchange mass spectrometry (HDX-MS) analysis
3 CLpro (1 mg∙mL−1) or 3CLpro (1 mg∙mL−1) with nodakenin (1 mmol∙L−1, 1% DMSO) was labeled with deuterium using D2O buffer (100 mmol∙L−1 phosphate, pH 7.0). The labeling reaction was quenched at intervals of 0.1, 0.5, 1, 10, 30, 60, and 240 min by adding quenching buffer (100 mmol∙L−1 phosphate, pH 1.8, 4 mol∙L−1 GdHCl, 0.5 mol∙L−1 trichloroethyl phosphate). Peptides were trapped, desalted for 3 min, eluted with 15% acetonitrile at 100 μL∙min−1, and separated using an ACQUITY UPLC BEH C18 column (1.0 mm × 100 mm, 1.7 μm). Mass spectrometry analyses were conducted using a Waters Xevo G2 mass spectrometer.
2.17. Molecular docking
The program Glide (Schrödinger Suite) with default parameters was used for the molecular docking studies. The protein SARS-CoV-2 3CLpro (Protein Data Bank (PDB) ID: 6LU7) was employed as the docking target for nodakenin. A grid box large enough to encompass the entire 3CL protein was created. The extra precision (XP) binding mode was utilized to examine the key interactions between nodakenin and SARS-CoV-2 3CLpro.
2.18. Statistical analysis
The statistical analysis was conducted with GraphPad Prism 9.5 software. The significance of differences between two groups was evaluated through t tests, while one-way analysis of variance (ANOVA) was used for comparing multiple groups. A p value less than 0.05 was considered to indicate statistical significance. The significance levels are denoted as * for p < 0.05, ** for p < 0.01, and *** for p < 0.001, with “ns” indicating nonsignificant results.
3. Results
3.1. SHHS reduces the viral load and attenuates the inflammatory response in Vero-E6 cells
Prior to conducting the cellular activity assay, we examined the cytotoxicity of SHHS on Vero-E6 cells. The viability of cells treated with SHHS was determined using the CCK-8 method. The results, depicted in
Fig. 1(a), indicated that SHHS has a cytotoxicity concentration at 50% (CC50) of 11.044 mg∙mL
−1, demonstrating minimal cytotoxicity at concentrations below 3.17 mg∙mL
−1, and cell viability remained above 85%. To study the antiviral effect of SHHS against the live SARS-CoV-2 strain 2019-nCoV WIV04 at the cellular level, SHHS at 1 mg∙mL
−1 with different drug-cell-virus pretreatments was used (Section S1.1 and Fig. S1 in Appendix A). The experimental procedure involved preincubating SHHS with the virus, followed by infecting the cells with a mixture of the virus and the drug. After 1 h, the supernatant was discarded, and the cell medium was replaced to continue cultivation. At 24 h post infection, a concentration of 1 mg∙mL
−1 SHHS significantly reduced the viral load in the supernatants of the infected cells (
Fig. 1(b)). We also investigated the effects of SHHS on the mRNA expression of
TNF-α,
IL-6,
CCL-2,
CCL-3, and
CXCL-10 in Vero-E6 cells infected with SARS-CoV-2. SHHS significantly inhibited the increase in the levels of these five cytokines in a concentration-dependent manner (
Fig. 1(c), Fig. S2 in Appendix A). These results suggested that SHHS could inhibit SARS-CoV-2 infection and proliferation at the cellular level. Moreover, SHHS may exert antiviral activity by regulating the expression of chemokines.
3.2. SHHS inhibits SARS-CoV-2 VLP invasion in mice
To determine how SHHS impacts SARS-CoV-2 invasion, we established a model in hACE2 mice in which SARS-CoV-2 VLPs invaded the respiratory tract. The luciferase activity assay results indicated that the level of VLPs in the lung tissues of the SHHS intervention group was significantly lower than that in the lung tissues of the control group (
Fig. 1(d)). The inhibition rate in the SHHS intervention group was 30% (
p < 0.05), and this rate of inhibition was consistent with previously reported outcomes for the UDCA group [
23]. The immunofluorescence results showed that the ACE2 receptor was expressed normally in the lungs of the blank group, and no SARS-CoV-2 Luc-VLPs were detected (
Fig. 1(e)). Despite the normal expression of the ACE2 receptor in the VLP group, there was significant aggregation of SARS-CoV-2 Luc-VLPs within the cell cytoplasm, particularly around the nucleus. Conversely, in the SHHS-treated group, while ACE2 expression remained normal, there was a notable reduction in the intracellular aggregation of SARS-CoV-2 Luc-VLPs. Similarly, the UDCA group exhibited decreased ACE2 receptor expression and reduced intracellular aggregation of SARS-CoV-2 Luc-VLPs. These findings suggested that SHHS effectively inhibited the invasion of SARS-CoV-2 VLPs into mouse lung tissues.
3.3. SHHS alleviates lung inflammation in K18-hACE2 mice challenged with SARS-CoV-2
To study the effect of SHHS on mice infected with SARS-CoV-2, we treated K18-hACE2 mice with 2019-nCoV WIV04 nasal drops. There were no deaths in any of the mouse groups. The study revealed that the model group had an average weight loss rate of 11.68%, whereas the SHHS group showed a reduced average weight loss rate of 10.39%, suggesting that SHHS has a tendency to protect against infection-induced body weight loss (
Fig. 2(a)). The average viral load in the lung tissue of mice in the model group was 2.9 × 10
8 copies∙g
−1, which decreased to 2.6 × 10
8 copies∙g
−1 in the SHHS-treated group. However, the difference was not significantly different, suggesting that the ability of SHHS to reduce the viral load was weak (
Fig. 2(a)). HE staining of lung tissues from the model group revealed interstitial congestion and edema, inflammatory cell infiltration, and significant widening of alveolar intervals (
Fig. 2(b), Fig. S3 in Appendix A), while lung inflammation was alleviated after SHHS treatment. Masson’s trichrome staining of the lungs showed that SHHS treatment alleviated pulmonary fibrosis and hemorrhage (
Fig. 2(c)). Furthermore, immunofluorescence analysis of lung sections revealed that SHHS treatment reduced the expression levels of SARS-CoV-2 spike RBD and NTD proteins in the anti-SARS-CoV-2 spike RBD and NTD group compared to those in the model group, suggesting an effective antiviral response (
Fig. 2(d), Fig. S4 in Appendix A).
Considering that pulmonary complications associated with SARS-CoV-2 infection are characterized by the overexpression of proinflammatory chemokines and cytokines, potentially leading to a “cytokine storm” [
24], the upregulation of inflammatory factors such as interferon-γ (IFN-γ) [
25] and various chemokines (CCL-2, CCL-3, CCL-4, CXCL-1, CXCL-6, CXCL-9, CXCL-10, CXCL-11, etc.) has been noted in patients with COVID-19 [
26], [
27]. As a result, we examined the mRNA expression of related cytokines and chemokines using PCR. SHHS notably reduced the mRNA levels of several chemokines, particularly
CCL-2,
CCL-3,
CCL-4, CXCL-1,
CXCL-6,
CXCL-9,
CXCL-10, and
CXCL-11 (
Fig. 2(e)). Subsequent immunohistochemical analysis confirmed a reduction in CCL-2 protein levels in the lung tissues of the SHHS-treated group compared to those of the Model group, highlighting the potential of SHHS to mitigate inflammatory responses in COVID-19 patients (
Fig. 2(f)).
3.4. SHHS ameliorates LPS-induced inflammatory injury in ALI model mice and RAW264.7 cells
To assess the anti-inflammatory effects of SHHS, a mouse model of ALI was created by administering 5 mg∙kg
−1 LPS directly into the trachea. Six hours post-LPS administration, notable structural alterations were observed in the lung tissues of the model group, including disrupted alveolar architecture, thickened alveolar walls, expanded alveoli, and dense interstitial spaces. Histiocyte hyperplasia, vascular congestion, and edema were observed. Different doses of SHHS effectively improve pathological damage in pulmonary tissue, as reflected in the corresponding lung injury scores. (
Fig. 3(a), Fig. S5 in Appendix A). After SHHS treatment, ELISA analysis revealed a notable decrease in the levels of proinflammatory factors such as IL-1β and TNF-α in the serum and lung tissue samples (
Fig. 3(b)). However, the treatment did not exhibit a clear dose-response relationship. In a parallel
in vitro study, LPS was administered to RAW264.7 cells to simulate an inflammatory environment, and the impact of SHHS on cell viability was assessed using an CCK-8 assay. There was basically no cytotoxicity (cell viability > 85%) when the concentration of SHHS was lower than 1 mg∙mL
−1 (Fig. S6 in Appendix A). Moreover, the ELISA results demonstrated that SHHS decreased the levels of IL-6 and IL-1β in the supernatant of RAW264.7 cells (
Fig. 3(c)). Collectively, these findings confirm the potent anti-inflammatory effects of SHHS both
in vivo and in
vitro.
3.5. Characterization of the chemical compound profile of SHHS
To clarify the material composition of SHHS, UHPLC-LTQ-Orbitrap-MS technology was used to analyze and identify the components in SHHS. An initial screening identified 308 compounds, with 224 detected in positive ion mode and 84 in negative ion mode (Dataset S1(a)). As shown in
Fig. 4(a), compounds in SHHS that were absorbed into plasma, lungs, and feces were detected in positive and/or negative ion modes. The structures of the compounds identified in the SHHS were subsequently classified and compared. Each compound was assigned to the relevant herb by applying the same extraction method and chromatographic conditions to each herb in the SHHS compound and extracted accordingly (
Fig. 4(b)). To increase the accuracy of this identification process, 60 standard references were employed to examine the fragmentation patterns of these compounds. This rigorous approach significantly improved the reliability of the compound identification, ensuring a robust analytical outcome.
Detailed information on the chemical identification of SHHS was provided in Section S1.2 in Appendix A. Compounds that were absorbed into plasma, distributed into the lungs, and transferred into feces were also identified in normal rats after seven days of gavage with SHHS (Section S1.3 in Appendix A). The identified compounds within SHHS, delineated based on their presence in plasma, lungs, and feces, were systematically categorized in
Fig. 4(c). Detailed information such as the retention times,
m/
z values, mass errors, molecular formulas, fragment ions, identifications, and structural classifications of these compounds was provided in Section S1.3 and was listed in Dataset S1 in Appendix A.
3.6. Research on the antiviral mechanism of SHHS in treating COVID-19
To further study the antiviral mechanism of SHHS, we studied 80 simple components (released into the lungs, plasma, and feces) from SHHS and successfully sourced 28 of these components from the market. These compounds predominantly originated from the primary (monarch) and secondary (minister) herbs within the formula, comprising 16 components present in the plasma, 11 in the lung, and 20 in the feces. Structurally, these compounds included three terpenoids, seven coumarins, six flavonoids, two lignans, two alkaloids, and eight other types of compounds (Dataset S1(e)). These compound structure types basically represented the full spectrum of compound types found in SHHS, serving as representative monomers for investigating the mechanism of action of the formula. To this end, we focused on five key antiviral targets, hACE2, RBD, 3CLpro, PLpro, and TMPRSS2, to study the antiviral mechanisms of these SHHS compounds.
For the hACE2 and SARS-CoV-2 RBDs, preliminary binding screenings revealed that gallic acid, aegeline, and apigenin all had suitable binding activities to both hACE2 and the RBD in the micromolar range (Fig. S7 in Appendix A). In addition, marmesin showed suitable binding to hACE2 (Fig. S7), while imperatorin, atractylenolide I, atractylon, wogonin, scoparone, aegeline, isorhamnetin, propyl gallate, diosmetin, quercetin, and luteolin exhibited suitable RBD-binding capacities (Fig. S8 in Appendix A). Considering that SARS-CoV-2 mainly achieves viral invasion through its own spike protein binding to the ACE2 receptor on the surface of cells in humans [
16], we then tested whether these hACE2- or RBD-active compounds could block the binding of the RBD to hACE2. As shown in
Fig. 5(a) and Fig. S9 in Appendix A, compared with the injection of 5 nmol∙L
−1 hACE2 alone, the addition of gallic acid and apigenin notably reduced the binding of hACE2 to the SARS-CoV-2 RBD immobilized on the sensor chip in a dose-dependent manner. Furthermore, compared with injection of 30 nmol∙L
−1 SARS-CoV-2 RBD alone, the presence of gallic acid, apigenin, atractylon, and quercetin effectively reduced the binding of the SARS-CoV-2 RBD to hACE2 in a dose-dependent manner. The maximum binding inhibition rate was approximately 90% (
Fig. 5(a), Fig. S9).
Next, the inhibitory effects of these 28 compounds on the activity of SARS-CoV-2 3CL
pro, PL
pro, and human TMPRSS2 were detected sequentially at a concentration of 50 μmol∙L
−1. Dose-response curves of compounds with inhibition rates greater than 50% at 50 μmol∙L
−1 were generated. As shown in
Fig. 5(b) and Fig. S10 in Appendix A, gallic acid, nodakenin, marmesin, epicatechin, and quercetin effectively reduced the enzyme activity of 3CL
pro, with the half maximal inhibitory concentration (IC
50) values of (8.2 ± 1.3), (10.2 ± 1.6), (17.8 ± 1.8), (30.2 ± 7.8), and > 100.0 μmol∙L
−1, respectively. Additionally, marmesin, scoparone, and luteolin inhibited PL
pro activity with IC
50 values of (20.2 ± 5.8), (29.3 ± 7.8), and (65.0 ± 30.0) μmol∙L
−1, respectively (Fig. S11 in Appendix A). The IC
50 values of human TMPRSS2 inhibition by scoparone, nodakenin, and marmesin were (29.0 ± 3.5), (47.0 ± 10.0), and (40.0 ± 44.0) μmol∙L
−1, respectively (Fig. S12 in Appendix A). Marmesin, despite its inhibitory action on multiple targets, was classified as a potential pan assay interference compound (PAINS) due to its broad spectrum of activity and was consequently excluded from subsequent antiviral assays.
3.7. Antiviral and anti-inflammatory activity of SHHS compounds against SARS-CoV-2 in vitro
The screening and validation results of the five primary target proteins (ACE2, RBD, 3CL
pro, PL
pro, and TMPRSS2) screened for COVID-19 in the previous stage were obtained. We then selected five compounds for advanced antiviral activity assessments, namely, gallic acid, apigenin, atractylon, quercetin, and nodakenin (
Fig. 5(c)). Gallic acid, apigenin, atractylon, and quercetin share a binding epitope with hACE2 or the SARS-CoV-2 RBD, which can directly reduce the binding of SARS-CoV-2 RBD and hACE2, thus exerting antiviral activity. Nodakenin had suitable SARS-CoV-2 3CL
pro inhibitory activity, with an IC
50 value near 10 μmol∙L
−1, suggesting the potential to reduce the replication rate of the virus. The antiviral effects of the five compounds were further verified by infection of Vero-E6 cells with WIV04 (
Fig. 5(d), Fig. S13 in Appendix A). Compared with the control, apigenin and nodakenin significantly reduced the viral load in the cell supernatant, with inhibition rates exceeding 50%. Notably, nodakenin had a significant antiviral effect, with an inhibition rate surpassing 66%, underscoring its efficacy in hindering SARS-CoV-2 infection and replication at the cellular level (
Fig. 5(d)). Additionally, nodakenin reduced the expression of TNF-α, CCL-2, CCL-3, and CXCL-10, revealing an antiviral phenotype akin to that of SHHS. The anti-inflammatory activities of these compounds against SARS-CoV-2 were shown in Section S1.4 and Fig. S13 in Appendix A. The method for determining the content of nodakenin in plasma was shown in Section S1.5 and Tables S2 and S3 in Appendix A.
3.8. Mechanism by which nodakenin inhibits 3CLpro enzyme activity
We used HDX-MS and molecular docking to investigate the compound with the best antiviral activity, nodakenin, and its ability to inhibit 3CLpro enzyme activity and exert antiviral function. Moreover, considering that nodakenin has a weak inhibitory effect on TMPRSS2, no further binding mechanism studies were conducted.
The HDX-MS results revealed that the addition of nodakenin led to significant differences in the molecular weights of the peptides 35-44, 37-44, 133-148, 139-156, 155-172, and 192-209, with changes ranging from approximately 0.5-2.0 Da (
Fig. 5(e), Fig. S14 in Appendix A). According to the molecular docking results, nodakenin formed hydrogen bonds with L141, N142, G143, and Q192 and engaged in hydrophobic interactions with H41, M165, L167, and P168 (
Figs. 5(f) and (g)). These findings align well with the HDX-MS results, offering a comprehensive understanding of the mechanism of action of nodakenin at the molecular level.
4. Discussion
TCM has been used in China to fight epidemics for thousands of years, and many effective TCM prescriptions and drugs have been developed. At the onset of the COVID-19 epidemic, when faced with a novel infectious disease and a dearth of available antiviral medications, TCM was rapidly deployed to deduce the etiology of the conditions of patients using a holistic observational approach, encompassing visual, auditory, olfactory, tactile, and interrogative assessments, alongside treatment analysis. One substantial advantage of TCM is its ability to provide early and symptomatic treatment. In clinical practice, SHHS and the “three medicines and three prescriptions” [
28] have been identified as effective therapeutic agents for the prevention and treatment of COVID-19 in China. These prescriptions have been found to improve patient prognosis, lower mortality rates, and promote patient recovery [
29]. Nevertheless, the complex compositions of TCM formulations pose challenges in identifying their active constituents and elucidating their mechanisms of action, which, in turn, hinders the global adoption and application of TCM.
The Vero-E6 cell model and K18-hACE2 mouse model are commonly utilized to evaluate the virulence of viruses such as SARS-CoV-2 and to explore potential treatment strategies [
30], [
31]. The CCL-2/C-C motif chemokine receptor 2(CCR-2) axis plays a crucial role in recruiting inflammatory monocytes/macrophages to the lung, and its dysregulated activation may lead to hyperinflammation in patients with COVID-19 [
32]. Our study revealed that SHHS can reduce viral load and downregulate the expression of CCL-2 and CCL-3 in Vero-E6 cells. SHHS has been shown to inhibit the invasion of SARS-CoV-2 VLPs into lung tissues. Here, SHHS treatment tended to reduce the viral load in the lung tissue of mice infected with the 2019-nCoV WIV04 virus. Concurrently, SHHS mitigated lung inflammation and reduced the mRNA expression of chemokines such as
CCL-2,
CXCL-1,
CXCL-6, and
CXCL-10. Due to experimental constraints related to the virus, it was not possible to acquire protein samples for analysis. Therefore, we were limited to examining paraffin-embedded lung tissue samples using immunofluorescence and immunohistochemistry techniques. The immunofluorescence results indicated that SHHS treatment effectively reduced the expression levels of SARS-CoV-2 spike RBD and NTD proteins. Notably, CCL-2 protein expression in the lung tissue of the SHHS group was reduced. These
in vivo and
in vitro experiments demonstrated that SHHS has the potential to inhibit viral invasion. Moreover, these results also suggested that SHHS may regulate immune responses and exert anti-inflammatory effects on COVID-19 through the CCL-2-CXCL axis. We obtained similar results for the effect of SHHS on SARS-CoV-2 (Omicron) infection in K18-hACE2 mice (Fig. S15 in Appendix A). Relevant studies have indicated that during the initiation stage of SARS-CoV-2 infection and viral sepsis, chemokines are predominantly released. Treatment at this initial stage with agents that block chemokines and inflammatory cytokines is crucial for preventing COVID-19 patient death [
33]. This mechanism may explain why SHHS is effective in preventing the progression of COVID-19 from mild to severe stages.
SARS-CoV-2 enters human cells through ACE2, which serves as a cellular receptor that is expressed on ciliated cells and alveolar epithelial type II cells of the lung and is highly expressed in the intestines [
34]. The role of ACE2 in amino acid transport is related to gastrointestinal tract microbial ecology [
35]. COVID-19 manifests with respiratory, gastrointestinal [
36], and systemic symptoms, with viral RNA persisting in the respiratory tract [
37] and feces [
38], [
39]. Studies have shown that SARS-CoV-2 is retained in fecal samples significantly longer than in respiratory and serum samples [
40]. TCM exerts therapeutic effects on COVID-19 through multiple pharmacological pathways, targets, and sites [
41]. Given the expression of ACE2 receptors in both the respiratory and intestinal tracts and the prolonged presence of novel coronavirus RNA in these locations, it is hypothesized that SHHS may exert its influence initially via the small intestine, subsequently entering the bloodstream and impacting the lungs. To explore this hypothesis, we characterized the components in SHHS and identified the components present in the plasma, lungs, and feces by UHPLC-LTQ-Orbitrap-MS conditions.
Research on the active ingredients of TCM has facilitated the discovery of effective compounds in TCM preparations. Of the 80 identified compounds, 28 were commercially available. By focusing on five antiviral targets (hACE2, RBD, TMPRSS2, 3CLpro, and PLpro), five potential antiviral compounds (gallic acid, apigenin, atractylon, quercetin, and nodakenin) were finally identified based on the results of the SPR and enzymatic assays. Among them, gallic acid had strong effects on ACE2, RBD, and 3CLpro; apigenin on ACE2 and RBD; atractylon on RBD; quercetin on RBD and 3CLpro; and nodakenin on 3CLpro. The spike protein of SARS-CoV-2 comprises two subunits: S1, which recognizes cell receptors, and S2, which facilitates membrane fusion. We utilized SPR technology to investigate the binding affinities of five potential antiviral compounds—gallic acid, apigenin, atractylon, quercetin, and nodakenin—to the S2 protein. Notably, gallic acid, apigenin, quercetin, and atractylon bound to the S2 protein with KD values of 3.53, 2.81, 8.75, and 7.46 µmol∙L−1, respectively (Fig. S16 in Appendix A). The antiviral activity of these compounds may be due to their interference with the binding of the spike protein to ACE2 or their binding to the S2 subunit, preventing necessary conformational changes in the S2 protein. However, the specific mechanisms underlying these interactions require further investigation. The antiviral effects of these monomers were further verified by infecting Vero-E6 cells with WIV04. Nodakenin had the most obvious inhibitory effect on viral load and could reduce the expression of CCL-2, CCL-3, and TNF-α. Therefore, nodakenin is considered an effective antiviral and anti-inflammatory compound of SHHS for treating COVID-19.
Previous studies have demonstrated that this coumarin glycoside has anti-inflammatory, antioxidant, and antiapoptotic effects [
42], [
43], However, our study suggested that nodakenin could exert antiviral effects by inhibiting 3CL
pro bioactivity, although it has not been reported to have antiviral effects. Nodakenin is a chemical component of the TCM Qiang Huo, which is the rhizome and root of
Notopterygium incisum Ting. ex H. T. Chang or
Notopterygium forbesii Boiss. In TCM, Qiang Huo is known to alleviate cold and dampness, a concept that aligns with the effects of SHHS. Notably, nodakenin was detected in the plasma, lungs, and feces of rats, indicating its systemic distribution. Further investigation using HDX-MS revealed that nodakenin interacted with 3CL
pro via noncovalent binding, in contrast to the majority of previously reported 3CL
pro inhibitors that function via covalent binding to cysteine, thus revealing a distinct pathway for its inhibitory action.
In conclusion, we demonstrated through in vivo and in vitro experiments that SHHS has the ability to inhibit viral invasion and proliferation, and exert a notable anti-inflammatory effect. Nodakenin, a compound found in SHHS, exerts antiviral effects through the inhibition of 3CLpro enzyme activity. Our findings offer valuable insights into the mechanisms through which SHHS combats COVID-19, contributing to a broader understanding of its therapeutic potential.
Acknowledgments
This work was supported by the National Key Research and Development Program of China (2021YFE0201100 and 2022YFC0868300) and the National Natural Science Foundation of China (22107004).
Compliance with ethics guidelines
Chuanxi Tian, Hang Liu, Qian Wang, Jinyue Zhao, Chensi Yao, Yanfeng Yao, Xu Zhang, Qinhai Ma, Weihao Wang, Yanyan Zhou, Mengxiao Wang, Xiaomeng Shi, Xiangyan Li, Shan Wang, Yingying Yang, Xiaowen Gou, Lijuan Zhou, Jingyi Zhao, Li Wan, Jiarui Li, Stefanie Tiefenbacher, Juntao Gao, Rudolf Bauer, Min Li, and Xiaolin Tong declare that they have no conflict of interest or financial conflicts to disclose.
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