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Table of Contents
ORIGINAL ARTICLE
Year : 2021  |  Volume : 7  |  Issue : 3  |  Page : 339-346

Exploration of bioactive constituents and immunoregulatory mechanisms of a hanshi-yufei formulation for treating COVID-19


1 Shanghai Research Center for Modernization of Traditional Chinese Medicine, National Engineering Laboratory for TCM Standardization Technology, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai; University of Chinese Academy of Sciences, Beijing, China
2 Shanghai Research Center for Modernization of Traditional Chinese Medicine, National Engineering Laboratory for TCM Standardization Technology, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China

Date of Submission05-Feb-2021
Date of Acceptance04-Mar-2021
Date of Web Publication21-Jul-2021

Correspondence Address:
Dr. De-An Guo
Shanghai Research Center for Modernization of Traditional Chinese Medicine, National Engineering Laboratory for TCM Standardization Technology, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Haike Road 501, Shanghai 201203
China
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/wjtcm.wjtcm_45_21

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  Abstract 


Objective: The objective of this study was to characterize the chemical compounds of a Hanshi-Yufei formulation (HSYF; a modified formulation of a traditional Chinese medicine used for treating COVID-19) to elucidate the mechanism of action and to evaluate potential anti-inflammatory effects of HSYF. Materials and Methods: The chemical constituents of HSYF extract were characterized using UPLC-Q-TOF/MS. Subsequently, a set of TCM network pharmacology methods was applied to identify disease-associated genes and to predict target profiles and pharmacological actions associated with the constituents of HSYF. Then, the antiviral effects of HSYF on H1N1 were assessed in RAW264.7 cells using MTT assays. Expression levels of pro-inflammatory cytokines interleukin (IL)-6 and tumor necrosis factor (TNF)-α following infection of RAW264.7 cells with H1N1 were measured using an enzyme-linked immune sorbent assay (ELISA), and expression levels of inflammatory-related factors were detected using western blotting. Results: In total, 165 chemical constituents (including glycosides, tannins, volatile oils, amino acids, triterpenoids, polyphenols, phenylpropanoids, sesquiterpenes, alkaloids, and flavonoids, among others) were tentatively identified in HSYF. Network pharmacology demonstrated that HSYF can regulate immunomodulatory- and anti-inflammatory-related targets of multiple pathways through its active ingredients, suggesting potential anti-COVID-19 effects. Furthermore, cell viability assays and ELISA showed that HSYF significantly inhibited H1N1 replication in RAW64.7 cells and markedly reduced expression of pro-inflammatory cytokines TNF-α and IL-6 at the proteins level. Conclusions: The results of the present study help improve our understanding of the therapeutic effects of HSYF in COVID-19 treatment from multi-level perspectives.

Keywords: Anti-inflammatory, COVID-19, Hanshi-Yufei prescription, immunoregulatory


How to cite this article:
Wei WL, Wu SF, Li ZW, Li HJ, Qu H, Yao CL, Zhang JQ, Li JY, Zhang GL, Wu WY, Guo DA. Exploration of bioactive constituents and immunoregulatory mechanisms of a hanshi-yufei formulation for treating COVID-19. World J Tradit Chin Med 2021;7:339-46

How to cite this URL:
Wei WL, Wu SF, Li ZW, Li HJ, Qu H, Yao CL, Zhang JQ, Li JY, Zhang GL, Wu WY, Guo DA. Exploration of bioactive constituents and immunoregulatory mechanisms of a hanshi-yufei formulation for treating COVID-19. World J Tradit Chin Med [serial online] 2021 [cited 2022 Jan 19];7:339-46. Available from: https://www.wjtcm.net/text.asp?2021/7/3/339/323498




  Introduction Top


Acute coronavirus infections can damage the respiratory tract and have resulted in severe outbreaks in the past decades.[1],[2] The newly emerged infectious coronavirus disease COVID-19 has become a global pandemic in 2020, threatening millions of people worldwide. The cardinal symptoms of patients with COVID-19 are fever, tussiculation, and weakness and some patients also suffered from nasal obstruction, rhinorrhea, pharyngodynia, and diarrhea, among other symptoms.[3],[4] Until September 24, 2020, 31,517,087 cases of COVID-19 had been reported, and globally, 969,541 patients had died from this disease, according to the global COVID-19 tracker and interactive charts (https://coronavirus. 1point3acres.com/). The World Health Organization stated that currently no effective drugs are available to treat COVID-19.[5] Therapy of COVID-19 is presented with multiple challenges, and development of effective clinical drugs is urgently required.

According to the treatment experience of epidemics for thousands of years and recordation in medical books, traditional Chinese medicines (TCMs) have been used as first-line drugs to treat patients with COVID-19 in China, which showed promising outcomes.[6],[7] TCMs have long been used to treat common disorders such as influenza, even though the underlying mechanisms are frequently unclear. Currently, as TCMs have been clinically used to treat COVID-19, elucidating the respective molecular mechanisms has become more significant. The National Health Commission (Pay Revision Commission) updated and released the Guideline (diagnosis and treatment of novel coronavirus (2019-nCoV)) Pneumonia, and TCMs were recommended for the prevention and treatment of COVID-19. HSYF formulation consists of Magnoliae Officinalis Cortex, Citri Reticulatae Pericarpium, Atractylodis Rhizoma, Agastache Rugosus, Tsaoko Fructus, Ephedrae Herba, Notopterygii Rhizoma et Radix, Arecae Semen, and Zingiberis Rhizoma Recens. It is easily available and is commonly used for patients with COVID-19 or suspected patients during the early phase of the COVID-19 outbreaks. The effects of HSYF are believed to alleviate the symptoms, decelerate the progression of severe inflammatory responses, and improve the clinical outcome. However, data on clinical characteristics associated with HSYF treatments of COVID-19 are limited, partly due to the emergency situation of the pandemic.

In this study, we examined the chemical constituents of HSYF using Ultra-Performance Liquid Chromatography-tandem Quadrupole Time-of Flight Mass Spectrometer (UPLC-Q-TOF/MS) to identify effective compounds and secondary metabolites. Network pharmacology analysis was applied to further explore the potential therapeutic targets and pathways involved in the pharmacodynamic effects of HSYF. Subsequently, biochemical in vitro experiments were employed to assess potential antivirus and immunomodulatory effects of HSYF using HIN1-infected RAW264.7 cells. The results suggested that HSYF can inhibit virus replication in a dose-dependent manner and significantly decrease pro-inflammatory cytokine expression, which may be help prevent or mitigate damage caused by COVID-19.


  Materials and Methods Top


Reagents

Acetonitrile and formic acid were purchased from Merck KGaA (Merck, Darmstadt, Germany) and Sigma-Aldrich (Sigma-Aldrich, St. Louis, MO, USA), respectively. Deionized water (18.2 MΩ cm at 25°C) was prepared using a Millipore Alpha-Q water purification system (Millipore, Bedford, USA). Herbal raw materials including Magnoliae Officinalis Cortex, Citri Reticulatae Pericarpium, Atractylodis Rhizoma, Agastache Rugosus, Tsaoko Fructus, Ephedrae Herba, Arecae Semen, Zingiberis Rhizoma Recens, and Notopterygii Rhizoma et Radix were purchased from Shanghai Dehua National Pharmaceutical Products Co., Ltd. (Shanghai, China), Shanghai Huaying Pharmaceutical Co., Ltd. (Shanghai, China), Shanghai Qingpu Traditional Chinese Medicine Yinpian Co., Ltd. (Shanghai, China), and Shanghai Yutiancheng Traditional Chinese Medicine Yinpian Co., Ltd. (Shanghai, China). Four reference standards (ephedrine, pseudoephedrine, magnolol, and honokiol) were purchased from the China National Institute of Pharmaceutical and Biological Products (Beijing, China) and the Shanghai Nature Standard Technical Service Co., Ltd. (Shanghai, China) at >98% purity.

An MTT kit was purchased from BSD Biotechnology Co., Ltd. (Wuhan, China). Tumor necrosis factor (TNF)-α and interleukin (IL)-6 enzyme-linked immunosorbent assay (ELISA) kits were purchased from Shanghai QIYA Co., Ltd. (Shanghai, China). p-NF-Κb and NF-κB antibodies were purchased from Abcam (Cambridge, UK). p-MAPK, MAPK, p-ERK, ERK, and β-actin antibodies were purchased from Santa Cruz Biotechnology (CA, USA). Immunoglobulin G (IgG) antibodies and anti-GAPDH were purchased from Boster Biological Engineering Co., Ltd. (Wuhan, China). Oseltamivir phosphate capsules (lot SH0037) were purchased from Roche Pharmaceutical Co., Ltd. (Shanghai, China).

Sample preparation

Sample preparation for characterization of chemical constituents of HSYF:

The amount of each herbal raw material was accurately weighed according to the respective prescription dose. Herbal materials (Magnoliae Officinalis Cortex, Citri Reticulatae Pericarpium, Atractylodis Rhizoma, Agastache Rugosus, Tsaoko Fructus, Ephedrae Herba, Arecae Semen, Zingiberis Rhizoma Recens, and Notopterygii Rhizoma et Radix) were placed in a gallipot and were soaked for 10 min using the tenfold amount of water (v/w). Then, the formulation was decocted twice for 30 min, each. The extracts were then mixed and freeze-dried.

UPLC-Q-TOF/MS analysis

A Waters ACQUITY I-Class UPLC® system (Waters, Manchester, UK) equipped with an ACQUITY UPLC® HSS T3 column (1.8 μm; 2.1 mm × 100 mm) was used for chromatographic separation of HSYF. A binary mobile phase (acetonitrile; B) and 0.1% formic acid (v/v; A) were used to elute HSYF with the following gradient elution program: 0–20 min: 0%–60% B; 20–22 min: 60%–90% B; and 22–25 min: 90%–90% B. The following parameters were used: column temperature, 30°C; flow rate, 0.3 mL/min; and sample injection, 5 μL.

Raw data were acquired using a Waters Xevo® G2-S QTOF mass spectrometer (Waters) equipped with a ZSpray™ ESI source. The parameters were as follows: mass range, m/z 100–1500; collision energy ramp, 15–25 V and 35–45 V; cone voltage, 40 V; capillary voltages, 2 kV; source temperature, 140°C; cone gas flow, 30 L/h; and desolvation gas flow, 700 L/h at 500°C. Leucine-enkephalin was used as a lock mass for data calibration. MassLynx V4.1 software (waters company, Milford, USA) was used for data acquisition and processing.

Cell lines, virus, cell viability assay, and western blotting

Mouse-adapted influenza virus A/PR/8/34 (H1N1) was kindly donated by Professor Zhi-Kun from the Southwest Medical University and was conducted as described previously.[8] The Reed–Muench method was used to calculate virus titers, and the results were expressed as median tissue infectious doses. RAW264.7 cells were obtained from the American-Type Culture Collection (Rockville, MD, USA). The virus growth medium (a serum-free Dulbecco's modified Eagle's medium [DMEM]) was supplemented with 2 μg/mL L-1-(tosyl-amido-2-phenyl) ethyl chloromethyl ketone-treated trypsin after infecting RAW264.7 cells with influenza virus (multiplicity of infection [MOI] = 20).

RAW264.7 cells were cultured at 37°C in high-glucose DMEM containing 10% (v/v) fetal bovine serum (Gibco, Waltham, MA, USA) and 1% penicillin–streptomycin. Cell viability was measured during the logarithmic growth stage using the MTT method. Cells seeded in 96-well culture plates (2 × 104 cells/well) were treated with 100 μL HSYF at different concentrations or with oseltamivir for 24 h. MTT solution at 20 μL/well (5 mg/mL in phosphate buffer solution (PBS)) was added to further incubation for an additional 4 h at 24 h. Formazan precipitates were dissolved in dimethyl sulfoxide (150 μL/well), and absorbance at 490 nm was measured using a microplate reader (Model 680, O-524; Bio-Rad, Hercules, CA, USA). A microplate reader (Shenzhen Sante Electronics Co., Ltd., Shenzhen, China) was used to measure optical density at 450 nm wavelength and 600 nm reference wavelength.

Total protein was extracted from RAW264.7 cells using RIPA buffer containing 2% proteinase inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA). A 10% polyacrylamide SDS-PAGE gel was used to separate a 30-μg protein sample under denaturing conditions, which was then transferred to a polyvinylidene fluoride membrane (0.22 μm, Invitrogen, Thermo Fisher Scientific). The membranes were blocked at room temperature for 1 h and were then incubated overnight at 4°C in Tris-buffered saline Tween-20 which contained 5% bovine serum albumin and antibodies including anti-p-NF-κb (1:1,000), anti-NF-κb (1:5,000), anti-p-MAPK (1:1,000), anti-MAPK (1:5,000), anti-p-ERK (1:1,000), anti-ERK (1:5,000), and anti-GAPDH (1:10,000). Subsequently, the membrane was exposed to secondary horseradish peroxidase -conjugated anti-rabbit or anti-mouse IgG antibody (Sigma-Aldrich) for 2 h at room temperature. Blots were visualized using ECL reagent (GE Healthcare, Piscataway, NJ, USA), and gel images were analyzed using ChemiDoc XRS + analysis software (Bio-rad, California, USA).

Network pharmacology analysis

The identified chemical constituents of HSYF were used for network pharmacology analysis. Network construction was executed according to the following main steps: (1) potential targets of the chemical constituents were predicted using the online resources DrugBank database (http://www.drugbank.ca/, version 4.3) or PharmGKB database (https://www.pharmgkb. org/). (2) Relevant compound-target and target-pathway networks were established for further network pharmacology analysis. Active HSYF compounds and their corresponding targets were used to establish compound-target-function-disease networks for systematical network pharmacology analysis. (3) Potential biological mechanisms of action of HSYF were assessed using GO and GOplot enrichment analysis. Detailed information on network construction is provided.

Enzyme-linked immune sorbent assay

RAW264.7 cells (4 × 106 cells/well) were cultured in a 6-well tissue culture plate (Corning Inc., Corning, NY, USA). Cells were exposed to H1N1 at an MOI of 20 at 37°C with 1 h under 5% CO2 for incubation within 24 h. After infection, the supernatant was removed, and cells were treated with HSYF or oseltamivir for 24 h. The supernatant was harvested after 4 h or 12 h, was centrifuged at 1500 × g and 4°C for 5 min, and was stored at −20°C until analysis. Concentrations of IL-6 and TNF-α were measured using an ELISA kit.

Statistical analyses

A one-way analysis of variance was used to test differences between multiple groups, and means ± standard deviation were calculated. Statistical significance was reported at P < 0.05 (*P < 0.05, **P < 0.001).


  Results Top


Characterization of Hanshi-Yufei formulation chemical constituents

UPLC-Q-TOF/MS in negative and positive ion mode was applied to systematically characterize chemical constituents of HSYF [Figure 1]. The compounds were identified according to the elemental composition and characteristic fragments summarized from relevant references and standards information. For example, alkaloids in Ephedrae Herba showed higher abundance in positive mode, and the main adduct form produced an [M + H] + peak. As an example, ephedrine presented an [M + H] + ion peak at m/z 166 and was fragmented in three products at m/z 148, 133, and 117. Ephedrine generated bond fission of hydroxyl with loss of an H2O (18 Da); then, dehydroxylated-ephedrine was further fragmented to the product ion at m/z 133 and 117 through losing a CH3 or NH2-CH3. In total, 165 chemical constituents (including glycosides, tannins, volatile oils, amino acids, triterpenoids, polyphenols, phenylpropanoids, sesquiterpenes, alkaloids, and flavonoids, among others) were tentatively identified according to fragmentation patterns and references, and 163 and 52 chemical constituents were characterized in negative and positive ion modes, respectively [Figure 2]a. NO. 16, 17, 128, and 129 were identified as ephedrine, pseudoephedrine, magnolol, and honokiol, respectively, using reference standards. The results suggested that phenylpropanoids and flavonoids are the predominant chemical compounds in HSYF extract, accounting for 16% and 47%, respectively [Figure 2]b.
Figure 1: Base peak chromatogram of HSYF by UPLC-Q-TOF/MS in negative and positive ion mode

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Figure 2: The chemical constituents characterized in HSYF. The numbers of chemical constituents in negative and positive ion modes (a). The proportion of chemical constituents in HSYF (b)

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Network pharmacology and target-pathway analysis

Network pharmacology helps elucidate relationships between drugs and targets on a molecular level and provides a global perspective for the investigation of drug efficacy.[9] Emerging evidence suggested a variety of inflammation-related biomarkers in patients with severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) pneumonia, including IL-6 and IL-1 β, among others.[10],[11] Thus, a network pharmacology method was developed to explore potential multicomponent, multi-target, and multi-pathway modes of action of HSYF for treating COVID-19. A C-T-P-D network was produced, including 59 compounds of HSYF, 52 potential target nodes, and 17 related pathways. As shown in [Figure 3]a, active compounds and corresponding targets were strongly associated with immunization-related proteins including PTGS2, TNF, MAPK14, and AKT1, among others. PTGS2 matched with the majority of candidate compounds, which indicated that PTGS2 plays a crucial role in regulating immune responses. PTGS2 encodes an inducible isozyme and may be regulated by specific stimulatory events, which suggests that it is important for prostanoid biosynthesis referred to as inflammation.[12],[13] MAPK14 was directly correlated with the ability to inhibit the expression of IL-1 and TNF, suggesting that regulation of MAPK14 was critical for cytokine production.[14] Collectively, the C-T-P-D network results may help reveal the potential pharmacological effects of HSYF on SARS-CoV-2 infection.
Figure 3: Network pharmacology and target-pathway analysis of HSYF. The active compounds - corresponding targets – pathway disease analysis (a). The significantly enriched GO terms of pathways correlated with HSYF treatment during SARS-CoV-2 pneumonia (b). The signaling pathways analysis of HSYF (c)

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To further explore the major signaling pathways which may be affected by treatment of SARS-CoV-2 pneumonia with HSYF, biological pathways were extracted from the KEGG database. The significantly enriched GO terms of pathways correlated with HSYF treatment during SARS-CoV-2 pneumonia are shown in [Figure 3]b. To further explore the synergistic effects of HSYF for treating SARS-CoV-2, an integrated pathway including the five signaling pathways such as PI3K-Akt, NF-kappa B signaling, TNF signaling, T-cell receptor signaling, and MAPK signaling was assembled based on the current knowledge of the disease pathology [Figure 3]c. The pathways were regulated through multiple target proteins, and the majority of protein targets have been reported as potential therapeutic targets for inflammatory and viral-related diseases. Activation of PI3K-Akt signaling is beneficial for promoting the production of pro-inflammatory cytokines such as TNF-α and IL-6 and inhibiting the production of anti-inflammatory cytokines such as IL-10.[15],[16] The TNF signaling pathway has been implicated in the pathogenesis of various human diseases and may be used as a therapeutic pathway target in the prevention and treatment of sepsis, inflammatory bowel disease, or rheumatoid arthritis.[17],[18] TNF-α and IL-6 were also considered robust indicators of COVID-19, and the associated pathways were interdependent from each other through the potential targets and compounds, indicating that HSYF may exert synergistic effects through these different pathways.

Hanshi-Yufei formulation inhibits expression of pro-inflammatory cytokines in H1N1-infected RAW264.7 cells

The hallmark of COVID-19 pathogenesis is a cytokine storm which results in increased levels of TNF-α, IL-1 β, IL-6, chemokine (C-C-motif) ligand 2, and granulocyte-macrophage colony-stimulating factor.[19] Effects of HSYF on cell viability were evaluated in vitro using an MTT assay to identify a reasonable concentration for experimental application; the respective results. As shown in the figures, the 50% cytotoxic concentration of HSYF was 858 μg/mL and the maximum nontoxic concentration of HSYF to RAW264.7 cells was approximately 100–200 μg/mL. Pretreatment of H1N1-infected RAW264.7 cells with HSYF improved cell viability in a dose-dependent manner. Thus, a concentration series was established to assess a suitable concentration for application in the subsequent experiment. In the present study, the expression of COVID-19-related cytokines in H1N1-infected RAW264.7 cells was measured with or without HSYF treatment. As shown in [Figure 4]a and [Figure 4]b, IL-6 and TNF-α expression levels in virus-infected cells were significantly upregulated, compared with those in noninfected controls, and treatment with HSYF substantially reduced IL-6 and TNF-α protein expression in H1N1-infected cells in a dose-dependent manner. The results suggested that HSYF may prevent or ameliorate the cytokine storm due to inhibition of pro-inflammatory cytokines.
Figure 4: (a,b) IL-6 and TNF-α expression levels in virus-infected cells were significantly upregulated, compared with those in noninfected controls, and treatment with HSYF substantially reduced IL-6 and TNF-α protein expression in H1N1 infected cells in a dose dependent manner

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Hanshi-Yufei formulation suppresses the nuclear factor kappa B/MAPK signaling pathway in H1N1-infected cells

To further elucidate potential antiviral mechanisms of HSYF, expression of type-I IFN-related protein phosphorylation was assessed. Activation of MAPK and NF-κB was measured by screening phosphorylation levels of MAPK p38, ERK1/2, and NF-κB p65 using western blotting. As shown in [Figure 5], expression levels of p-MAPK, p-NF-κB, and p-ERK were substantially increased in H1N1-infected cells, compared with noninfected controls. However, expression levels of p-MAPK, p-NF-κB, and p-ERK were significantly reduced after treatment with HSYF (100 and 500 μg/mL) or oseltamivir (100 μM). Thus, reducing the levels of MAPK p38 and NF-κB p65 phosphorylation, which are essential regulators of MAPK- and NF-κB-related inflammation responses, may help ameliorate the cytokine storm induced by viral infection.
Figure 5: Compared with noninfected controls, the expression levels of p-MAPK, p-NF-κB, and p-ERK were substantially increased in H1N1-infected cells. The expression levels of p-MAPK, p-NF-κB, and p-ERK were significantly reduced after treatment with HSYF (100 and 500 μg/mL) or oseltamivir (100 μM).

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  Discussion Top


TCMs have been commonly used in clinical treatments of SARS-CoV-2 infection. At the onset of the COVID-19 outbreak, HSYF was recommended for treating patients infected with SARS-CoV-2, especially for patients with early symptoms. Thus, HSYF was also considered an “early-stage prescription” for COVID-19 patients. Clinical observation reports showed that HSYF treatment helped improve immunity and ameliorate the symptoms of mild COVID-19 cases such as fever, cough, and fatigue, among others.[20],[21],[22]

Identifying the bioactive constituents of TCMs has become one of the key issues for modernization of TCMs. The chemical constituents of HSYF were examined in the present study using UPLC-Q-TOF/MS, and 165 chemical constituents (6 glycosides, 3 tannins, 1 volatile oil, 3 amino acids, 3 triterpenoids, 14 polyphenols, 27 phenylpropanoids, 9 sesquiterpenes, 15 alkaloids, 78 flavonoids, and 6 other compounds) were tentatively identified. The results revealed that the number of phenylpropanoids and flavonoids was higher than those of other compounds, accounting for 47% and 16%, respectively. Furthermore, an incorporated pathway was developed to further explore synergistic mechanisms of potential ways by which the active HSYF compounds and corresponding compound targets may act through modulating multiple metabolism pathways against COVID-19. To improve our understanding of relatively complete immunity- and inflammation-related pathways, they were analyzed to further elucidate compound-synergy mechanisms. C-T-P-D networks and integrated pathway analysis provided deep insights to explain correlations between functions and synergistic effects of each herb, indicating that the potential therapeutic effect of HSYF on COVID-19 may be by regulating these proteins, and HSYF may exert multi-target regulations.

Currently, acute viral infections may cause severe complications related to hyper induction of pro-inflammatory cytokines, typically termed “cytokine storm” and first presented in severe influenza. There were high concentrations of cytokines (such as MCP1, MIP1A, and TNF-α) during SARS and Middle East respiratory syndrome, and a similar phenomenon was observed in plasma of patients with COVID-19 who required intensive care unit (ICU) admission, suggesting that the cytokine storm was closely correlated with disease severity. However, the mechanisms underlying the cytokine storm during COVID-19 are not yet clearly understood. Xu et al. analyzed the immune characteristics of patients with COVID-19 and suggested that pathogenic T-cells and inflammatory monocytes which secreted large amounts of IL-6 may elicit the “inflammatory storm”. A clinical study reported that 123 patients with COVID-19, including 102 mild and 21 severe cases, showed low levels of CD4+T and CD8+ T which are common indicators of severe Novel coronavirus pneumonia (NCP) (2019-nCoV pneumonia), while IL-10 and IL-6 levels were significantly higher in severe cases. It was reported that expression levels of IL-10, IL-7, TNF-α, IL-2, granulocyte-colony-stimulating factor , and IP-10 in patients infected with COVID-19 in ICUs were significantly higher than those in non-ICU patients, and the incidence of acute respiratory distress syndrome (ARDS), acute heart, secondary infection, shock, and kidney injury was significantly higher in patients in ICUs than in non-ICU patients.[23] Thus, emergence of a cytokine storm may be a major clinical concern to monitor changes in cytokines during the treatment process to optimize treatment plans and to predict the outcome of COVID-19. It may be speculated that the benefits of HSYF treatment were partly due to the immunomodulatory effects of pro-inflammatory cytokines. In our study, HSYF effectively decreased expression levels of IL-6 and TNF-α 4 and 12 h after H1N1 infection of RAW263.7 cells. Regulation of inflammatory cytokines production may be one of the aspects of antiviral protective effects of HSYF. Western blot analysis was also used to analyze the expression levels of phosphorylated forms of ERK, MAPK p38, and NF-κB p65, which were the key signaling molecules of antiviral responses like transcription of type-I IFNs and inflammatory cytokines. Compared with H1N1-infected RAW264.7 cells, HSYF (100 μg/mL and 500 μg/mL) significantly downregulated the phosphorylation levels of ERK, MAPK p38, and NF-κB p65, indicating that HSYF can mediate phosphorylation levels of proteins related to IFNs and inflammatory cytokines.


  Conclusion Top


In this study, the chemical constituents of HSYF were characterized by UPLC-Q-TOF/MS, and a total of 165 chemical constituents were tentatively identified. Network pharmacology analysis was applied to further explore the potential therapeutic targets and pathways involved in the pharmacodynamic effects of HSYF. Furthermore, biochemical in vitro experiments were employed to assess potential antivirus and immunomodulatory effects of HSYF using HIN1-infected RAW264.7 cells. The results showed that HSYF could regulate immunomodulatory- and anti-inflammatory-related targets of multiple pathways through its active ingredients, and HSYF could markedly reduce the expression of pro-inflammatory cytokines TNF-α and IL-6 at the proteins level. The results implied that HSYF could inhibit virus replication in a dose-dependent manner and significantly decrease pro-inflammatory cytokine expression, which may be help prevent or mitigate damage caused by COVID-19.

Acknowledgments

This work was supported by the National Key R&D Program of China (NO. 2019YFC1711000), the National Natural Science Foundation of China (NO. 81530095, 81673591), the Strategic Priority Research Program of the Chinese Academy of Sciences (NO. XDA12020348), the National Standardization of Traditional Chinese Medicine Project (NO. ZYBZH K LN 01), the Science and Technology Commission Foundation of Shanghai (NO.15DZ0502800), and the Projects of Research and Develop Plan in the Key Field of Guangdong (No 2020B1111110007).

Financial support and sponsorship

Nil.

Conflicts of interest

Prof. De-An Guo is the editor in cheif of World Journal of Traditional Chinese Medicine. The article was subject to the journal's standard procedures, with peer review handled independently of this editorial board member and their research groups.



 
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