Complementary and Alternative Therapies for Inflammatory DiseaseView this Special Issue
The Pharmacological Mechanism of Xiyanping Injection for the Treatment of Novel Coronavirus Pneumonia (COVID-19): Based on Network Pharmacology Strategy
Purpose. The possible mechanism of Xiyanping injection treatment COVID-19 is discussed through the network pharmacology. Methods. Obtaining the chemical structure of Xiyanping injection through the patent application and obtaining control compounds I, II, III, IV, V, Yanhuning injection (VI, VII), Chuanhuning injection (VIII, IX), 10 compounds were analyzed by D3Targets-2019-nCoV. The human anti-COVID-19 gene in COVID-19 DisGeNET was intersected with the CTD Andrographolide target gene and then combined with D3Targets-2019-nCoV, resulting in 93 genes, using the Venny 2.1 platform. The PPI network was constructed by the String platform and Cytoscape 3.8.2 platform. The GO, KEGG, and tissue of the target were analyzed using the Metascape platform and DAVID platform. The gene expression in the respiratory system was analyzed using the ePlant platform. The CB-Dock is used for the docking verification and degree values of the first 20 genes. Results. Finally, 1599 GO and 291 KEGG results were obtained. GO is mostly associated with the cell stress response to chemicals, the cell response to oxidative stress, and the cell response to reactive oxygen species. In total, 218 KEGG pathway concentrations were related to infection and other diseases and 73 signaling pathways mostly related to inflammation and immune pathways, such as TNF signaling pathway and MAPK signaling pathway. The molecular docking results show that Xiyanping injection, compound III, has a good docking relationship with 20 target proteins such as HSP90AA1. Tissue has 22 genes that are pooled in the lungs. Conclusion. Xiyanping injection may inhibit the release of various inflammatory factors by inhibiting intracellular pathways such as MAPK and TNF. It acts on protein targets such as HSP90AA1 and plays a potential therapeutic role in COVID-19. Thus, compound III may be treated as a potential new drug for the treatment of COVID-19 and the Xiyanping injection may treat patients with COVID-19 infection.
According to expert consensus, Xiyanping injection belongs to Chinese medicine injections, primarily composed of sodium((1R, 2R, 4aR, 8aS)-2-hydroxy-5-((E)-2-((S)-4-hydroxy-2-oxodihydrofuran-3(2H)- ylidene) ethyl)-1,4a,6-trimethyl-1,2,3,4,4a,7,8,8a-octahydronaphthalen-1-yl)methyl sulfate (C20H29O5·SO3Na). It mainly has anti-inflammatory, antibacterial, anticough, and other effects and enhances the body’s immunity, . In the “2019 Novel Coronavirus Diagnosis and Treatment Program” (Trial 6th Edition) implemented by the State Health and Construction Commission of China, the virus infection is heavy and critical or combined with mild bacterial infection, and the treatment of Xiyanping injection is recommended with Chinese medicine injection. The recommended usage is as follows: 0.9% sodium chloride injection 250 mL plus Xiyanping injection 100 mg b.i.d. . The drug has been on the market for more than 30 years. In 2016, Xiyanping injection ranked in the top three in the market for Chinese medicine injections in the clear-heat detoxification category, with market size of about 2.6 billion yuan, often used in pediatrics .
Coronaviruses (CoVs) consist of a single positive-chain RNA virus that belongs to the coronavirus group and can develop into respiratory, intestinal, nervous, reproductive, and other diseases in infected animals and humans . In 2019, some hospitals in Wuhan City, Hubei Province, have found several cases of unexplained pneumonia with a history of exposure to the South China seafood market, which has been confirmed as a new type of coronavirus infection caused by acute respiratory infections. The World Health Organization has named the virus “COVID-19” . Similar to the pathological mechanism of Severe Acute Respiratory Syndrome (SARS), SARS-CoV-2 infections disrupt the epithelial-endothelial barrier in the bronchial and alveoli, causing alveoli-capillary oxygen transfer dysfunction and impaired oxygen diffusion. In patients with severe COVID-19, severe coagulation dysfunction and thrombosis and sepsis further lead to multiple organ failures . Zhong Nanshan’s team reported in the literature that 63.4% of COVID-19 patients had a fever and lung abnormalities in chest CT scans; experimental studies have shown that patients with COVID-19 have lymphocyte reduction and increased levels of reactive protein (CRP) .
Recently, network pharmacology has been widely used in the study of Chinese medicine anti-SARS-CoV-2 . The emerging network pharmacology technology can be widely used to explore the possible mechanism of Chinese medicine to treat diseases through multicomponent, multitarget, and multichannel levels and to explore potential therapeutic drugs . The Chinese medicine injections in the New Coronary Virus Pneumonia Treatment Programme (Trial 7)  are included in the recommended SARS-CoV-2 treatment options, such as Xiyanping injection, Xuebijing injection, Reduning, Tanreqing injection, Xingnaoling injection, Shenfu injection, Shenmai injection, and Shengmai injection. This has also raised awareness and acceptance of Chinese medicine injections. In Wuhan Jinyintan Hospital, Wuhan Lung Hospital, Huazhong University of Science, and Technology Tongji Medical College affiliated Concord Hospital, seriously ill patients by the Chinese and Western Medical Federation, more patients used Chinese medicine injections, achieved good results. Professor Zhangboli said “for patients with mild illness, a large number of practices have proved that Chinese medicine can improve symptoms, shorten the course of treatment, and promote healing” . Mild patients often present with fever, dry cough, and fatigue symptoms mainly. Some patients have wheezing, and lungs scattered in the oozing symptoms; Chinese medicine has detoxification, cough relief, and other good therapeutic effects. He led the clinical study of the Wuhan Fire Line project. The phase analysis shows that the first batch of 52 patients in Hubei Province combined with Western medicine hospital clinical control study: including 34 cases of Chinese and Western medicine combined treatment group, 18 cases of the simple Western medicine treatment group. The data analysis results show that compared with the Western medicine group, the clinical symptoms of the combination group of Chinese and Western medicine were shortened by 2 days. The normal temperature was reduced by 1.7 days. The average number of hospital days was shortened by 2.2 days. The CT image improvement rate was increased by 22%, and the clinical cure rate was increased by 33% .
Based on clinical data, this article uses network pharmacology to explore the possible mechanism of Xiyanping injection anti-SARS-CoV-2, while andrographolide, compound III, can be used as a potential new anti-SARS-CoV-2 drug (Figure 1).
2. Materials and Methods
2.1. Collection of Target Compounds
We obtained the chemical structures of six andrographolide salts, including Xiyanping injection and other andrographolide salts synthesized in process production, through the contents in the patent book of pharmaceutical companies. These five compounds were derived from the production process of Xiyanping injection, but they were not produced in the market as the objects of subsequent pharmacodynamic development, and there were no related clinical studies: I, II, III, IV, and V. We obtained similar andrographolide Chinese medicine injections via the DrugBank  platform (https://go.drugbank.com/): Yanhuning injection compounds: VI and VII; Chuanhuning injection compounds: VIII and IX. Ten compounds are drawn and saved in SDF format via ChemDraw 20.0 software .
2.2. Screening of Disease Targets
Based on the D3Targets-2019-nCoV platform  (https://www.d3pharma.com/D3Targets-2019-nCoV/index.php), the Xiyanping injection anti-COVID-19-related target genes were docked. We obtained human gene targets related to andrographolide based on the CTD platform  (https://ctdbase.org/). The CTD platform captures the set of genetic targets that andrographolide acts on in humans; the Human anti-COVID-19 gene dataset on the COVID-19 DisGeNET platform  (https://ctdbase.org/) takes the target intersection collection of these two gene sets through the Venny 2.1 platform (https://bioinfogp.cnb.csic.es/tools/venny/). The collection is then merged with the human anti-COVID-19 target obtained by the D3Targets-2019-nCoV platform . Finally, a target set of the andrographolide drug Xiyanping injection anti-COVID-19 was obtained [(CTD ∩ COVID-19 DisGeNET)∪D3Targets-2019-nCoV]−[(CTD ∩ COVID-19 DisGeNET)∩ D3Targets-2019-nCoV].
2.3. Target Gene Network Construction and Analysis
The target protein is converted into the corresponding gene name through the UniProt platform  (https://www.uniprot.org/), and the target protein interaction network (PPI network) is built through the String platform  (https://www.string-db.org/). Finally, a compound-target PPI network is built using the Cytoscape 3.8.2 software platform .
2.4. GO and KEGG Pathway Enrichment Analysis
To further analyze the function of the target, the target is uploaded to the Metascape platform  (https://metascape.org/); the species is limited to “H. sapiens,” the threshold , and its GO biofunction (biological process (BP), molecular function (MF), and cell composition (CC)) enrichment analysis is carried out. At the same time, KEGG signal path enrichment analysis and tissue structural analyses are carried out through the DAVID platform  (https://david.ncifcrf.gov/), and Enrichment Plot is drawn through the image GP platform (http://www.ehbio.com/).
2.5. Drug Ingredients: Protein Targets’ Molecular Docking
Download the Human Target Protein (Table 1) in the Top 20 Degrees from the PDB platform  (https://www.rcsb.org/): 20 target macromolecules, hydrogenation, removal of water molecules, removal of ligands, etc., through Discovery Studio 2019 Client software . The target component and macromolecules are uploaded to the CB-Dock platform  (https://clab.labshare.cn/cb-dock/php/) for molecular docking to bind the energy ≤ −5.0 kcal/mol as the basis for molecular-target interactions . The docking results are mapped to heatmap via the image GP platform, and the binding sites and patterns of ligand drugs and target proteins are observed through Discovery Studio 2019 Client software .
2.6. Organizing Enrichment Analysis
We performed organizational analysis via the DAVID platform , analysis of Xiyanping injection Vina scores of higher HSP90AA1, CAT gene target and compound III Vina score of higher TNF, and FOS gene target and observed the relationship between genes and tissues, using the Human eFP Browser platform .
3.1. Screening of Active Compounds
We obtained the chemical structure of andrographolide-sulfonate: Xiyanping injection, I, II, III, IV, and V through a patent application for Jiangxi Qingfeng Pharmaceuticals . The DrugBank Platform has similar andrographolide Chinese injections: Yanhuning injection compounds: VI and VII; Chuanhuning injection compounds: VIII and IX (Figure 2).
3.2. Target Gene Network Analysis
D3Targets-2019-nCoV platform database  was used to collect 34 human genes. The 1812 human genes associated with COVID-19 in the DisGeNET platform  were intersected with 150 Homo sapiens target genes in the CTD platform . For andrographolide, 60 genes (Figure 3) were collected through the Venny 2.1 platform. The genetic results of the D3Targets-2019-nCoV platform  were combined to obtain 93 genes (Table 1, Figure 4). We standardized the 93 target genes obtained with the UniProt platform . By analyzing 93 genes through the String platform , the number of nodes of the network graph was 93, 1174 edges, and the PPI rich -value: <1.0e − 16, which is considered to have strong biological significance. The target PPI network was built using the Cytoscape 3.8.2 platform , in which 89 gene nodes were associated, average degree value of 26.38. The circle is the target gene, and the base color with the degree value less than the average value is yellow, and the larger the degree value is, the closer it is to green and the larger the shape is (Figure 5). The top 20 degree values in the PPI network diagram are used as the core targets, in which 20 targets, such as target AKT1 (Degree: 75.0), TP53 (Degree: 65.0), and TNF (Degree: 64.0), are all greater than the average of 1.5 times. These targets are considered to be the main target points for the anti-COVID-19 by the andrographolide drugs (Table 2).
3.3. GO and KEGG Signal Path Enrichment Analysis
GO Enrichment 2343 items through the Metascape platform, which can be clustered into five items by their target properties (Figure 6). The most significant category of items is the mitogen-activated protein kinase MAPK family (MAPK1, MAPK2, MAPK8, etc.), which has the function of regulating cell growth, differentiation, stress adaptation to the environment, inflammatory response, and other important cellular physiological and pathological processes . Studies have also shown an unexpected mechanical link between MAPK signals and inflammatory network control during reprogramming caused by tumor therapy . As can be seen from (Table 3), Cytokine Signaling in Immune system, Nanomaterial induced apoptosis, and PID HIV NEF PATHWAY may be Xiyanping injection anti-SARS-CoV-2 important biologic processes. In total, there are1599 GO biofunctions, of which 1447 are biological processes (BP), 61 are cell compositions (CC), and 91 are molecular functions (MF) (Figures 7(a), 7(b)). Most of the top 20 GO features are related to the reaction of response to an inorganic substance, cellular response to chemical stress, response to oxidative stress, and response to reactive oxygen species. We obtained 291 KEGG path enrichment features (Figures 8(a), 8(b)): 218 were related to infection and other diseases and 73 signaling pathways are mostly related to inflammation and immune pathways. Enriched by the DAVID platform  to 107 KEGG results, 47 correspond to a variety of diseases such as Hepatitis B, Chagas disease (American trypanosomiasis), colorectal cancer, and influenza A, tubeless. There are 60 pathways, the first three of which are TNF signaling pathways, the Toll-like receptor signaling pathway, and the HIF-1 signaling pathway, most of which are related to immune and inflammatory pathways, and we concluded that injection may be anti-SARS-CoV-2 through inflammation and immune pathways. Showing the TNF pathway (Figure 9), the sunflower represents Xiyanping injection, and the three arrows represent inhibition, dual-directional regulation, and active.
3.4. Analysis of Molecular Docking Results
It is generally believed that the smaller the binding energy of the ligand to the receptor, the greater the likelihood of its interaction and the easier the binding . Andrographolide sulfonates include the following: Xiyanping injection, compound I, compound II, compound III, compound IV, and compound V; Yanhuning injection: VI and VII; Chuanhuning injection: VIII and IX. The degree value of the top 20 target proteins (Table 2) was selected for molecular docking with the above compounds. As can be seen from the molecular docking results, the binding energy of the target compound with the 20 target protein macromolecules is less than −5.0 k·J/mol . Studies suggest that these ingredients may have anti-SARS-CoV-2 effects , and the results indicate that compounds III and Xiyanping injection have a good binding ability with target proteins. See heatmap for molecular docking binding energy (Figure 10). Colors range from red to blue. The bluer the color, the smaller the binding energy, the stronger the binding ability, and the better the docking results. Xiyanping injection and gene: HSP90AA1 (target protein: 3U93) has the highest Vina score of −9.7, Xiyanping injection, and HSP90AA1 (target protein: 3U93) docking mode (Figure 11).
3.5. Organizing Enrichment Analysis
The differential expression of 93 gene targets in human tissue was analyzed using DAVID platform  Tissue Enrichment. As can be seen from (Figure 12), the redder the -value circle, the greater the enrichment value, and the larger the count circle shape, the more enriched the gene. It can be seen that in tissues, placenta, liver, epithelial, lung, and other common target expressions are significant, of which the number of lung targets is 22. Among them, the results of molecular docking show that the high scores of Xiyanping injection Vina are HSP90AA1 and CAT gene target, and TNF and FOS gene target have higher compound III Vina scores. The expression of the above four genes in the respiratory and circulatory system was analyzed by the Human eFP Browser platform , and the distribution and absoluteness of the genes in the human body were analyzed (Figures 13–16). The higher the absolute, the redder the color, and the higher the gene expression. Human HSP90AA1 and other targets are believed to be effective potential therapeutics.
According to the clinical trials of Zhang XY, Lv L, and Zhou YL, it can be proved that Xiyanping has the effect of treating COVID-19 . Zhang XY, Lv L, Zhou YL, and others recruited 130 consecutive COVID-19 patients with mild-to-moderate symptoms from five research sites and randomly assigned them to receive Xiyanping injection combined with standard treatment or standard support treatment alone at a 1 : 1 ratio. The results showed that Xiyanping injection significantly reduced the time of cough relief, fever regression, and virus clearance. No serious adverse events were reported during the study. The above clinical trials can prove that Xiyanping has the effect of treating COVID-19, which is worthy of clinical promotion.
The main component of Xiyanping injection is andrographolide, which is the main effective component of the natural plant Andrographis paniculata. It has the effects of dispelling heat and detoxification, anti-inflammatory, and analgesic. It has special effects on bacterial and viral upper respiratory tract infections and dysentery and is known as a natural antibiotic drug . In clinical application of the product, its side effects are small. The median lethal dose of alcohol extracts of Andrographis paniculata in mice was 13.19 g/kg (calculated by andrographolide). The modern injection has adverse reactions because it directly enters the internal circulation without passing through the liver metabolism of the digestive system. The adverse reactions of andrographolide injections—Yanhuning injection and Chuanhuning injections—are more serious; therefore, they should be used with caution in children. China’s National Medical Products Administration has reported security issues with Yanhuning injection/Chuanhuning injection and noted that Yanhuning injection/Chuanhuning injection was used cautiously for children . In the consensus of experts on the clinical application of Xiyanping injection (Child Edition) in 2019, experts believed that Xiyanping injection can be used in children and has good clinical efficacy . Xiyanping injection was recommended by multiple pediatric diseases guidelines/consensuses, and the adverse reactions were much smaller than those of similar drugs. In 2010, Xiyanping injection was recommended in the new editions of Hand, Foot and Mouth Disease Diagnosis and Treatment Guidelines, Influenza A (H1N1) Diagnosis and Treatment Programme, and Fever with Thrombocytopenia Prevention and Treatment Guidelines issued by the Ministry of Health (Table 4).
Excessive TNF can cause tissue damage, which is one of the core cytokines of an “inflammatory storm” caused by infection. TNF can induce cells to release cytokines, but in some cases, it can also induce apoptosis or programmed cell necrosis . After Xiyanping injection enters the patient, its anti-inflammatory effect may be related to the TNF pathway, triggering many TNF pathway reactions: inhibiting TNFR1 and TNFR2 in the TNF pathway. At the same time, Xiyanping injection can regulate Caspase-3 and Caspase-8 in the Caspase family involved in the TNF pathway and mediate cell apoptosis. The activation of NFB JNK protein kinase-mediated by TRAF is inhibited by Xiyanping injection. Xiyanping injection inhibits the extracellular MAPK inflammatory pathway (inhibition of MAPK1, MAPK3, inhibition of MAP production), inhibits NF-κB and other pathways in the nucleus through various pathways, inhibits the release of various inflammatory factors, and plays a role in the treatment of COVID-19.
Target gene HSP90AA1: HSP90AA1 is an important regulator of inflammation because HSP90AA1 is currently understood as an extracellular agent secreted in wound healing and inflammation. Extracellular HSP90 + induces inflammation by activating NF-κB (RELA) and STAT3 transcription procedures, including proinflammatory cytokines IL-6 and IL-8 . In addition, it has an intracellular anticancer effect and induces autophagy. The anti-inflammatory mechanism of Xiyanping injection may be highly related to this target.
Target gene CAT: CAT gene occurs in almost all aerobic breathing organisms, which helps protect cells from the toxic effects of hydrogen peroxide. It promotes cell growth, including T cells, B cells, bone marrow leukemia cells, melanoma cells, breast cells, and normal and transformed fibroblasts ; Xiyanping injection may act on this target, anti-inflammatory effect.
Target gene TNF: TNF gene induces VEGF production by cooperating with IL1B and IL6 , plays an anti-inflammatory role, and plays a role in angiogenesis.
Target gene FOS: FOS is considered to play an important role in signal transduction, cell proliferation, and cell differentiation. In growth cells, phospholipid synthesis may be activated by CDS1 and PI4K2A . This activity requires Tyr dephosphorization and association with the endoplasmic retina .
From the molecular docking Vina data, it was found that the overall score of compound III was better than that of Xiyanping injection. This also indicates the possibility of a potential new drug, with compound III having better water solubility than Xiyanping injection.
In this study, through network pharmacology, molecular docking, target analysis, and tissue analysis, it can be concluded that Xiyanping injection and andrographolide sulfonates such as compound III in the treatment of COVID-19 mainly act on HSP90AA1, CAT, TNF, FOS, and other targets. At the same time, Xiyanping injection inhibits the release of a variety of inflammatory factors by inhibiting intracellular MAPK, TNF, and other pathways and inhibiting NF-κB and other pathways in the nucleus, thus playing a role in the treatment of COVID-19. Compound III is a potential new drug for the treatment of COVID-19. Xiyanping injection can achieve a therapeutic effect in patients with infectious pneumonia, which is in line with the “Chinese Association of Traditional Chinese Medicine Expert Consensus (literature reports)”  conclusion. In summary, Xiyanping injection is effective and safe in treating COVID-19, which is an important method for the treatment of COVID-19 in integrative medicine. The clinical practice and popularization and application are of far-reaching significance.
All data used to support the findings of the study are included within the article.
Conflicts of Interest
No potential conflicts of interest were reported by the authors.
All the authors of the manuscript are immensely grateful to their respective universities and institutes for their technical assistance and valuable support in the completion of this research project. This research was partially supported by grants from the National Natural Science Foundation of China (81760698), the Seventh Batch of Yunnan Specialty Plant Polysaccharide Engineering Research Center Construction Plan (-57), the General Project of the Department of Science and Technology of Yunnan Province (202001BA070001-041), and the Administration of Traditional Chinese Medicine of Sichuan Province (2020JC0086).
National Health Commission National Administration of Traditional Chinese Medicine, “Diagnosis and treatment of new coronavirus pneumonia (trial sixth edition),” Chinese Journal of Viral Diseases, vol. 10, pp. 81–85, 2020.View at: Google Scholar
Z. F. Wang, X. Chen, W. Zhang, and Y. M. Xie, “Literature review on premarketing and postmarketing evidence of xiyanping injection,” Zhongguo Zhongyao Zazhi, vol. 39, no. 18, pp. 3637–3640, 2014.View at: Google Scholar
X. W. Ye, Y. L. Deng, X. Zhang et al., “Study on the mechanism of treating COVID-19 with shenqi wan based on network pharmacology,” Drug Development and Industrial Pharmacy, vol. 4, pp. 1–22, 2021.View at: Google Scholar
“Diagnosis and treatment protocol for novel coronavirus pneumonia (trial version 7),” Chinese Medical Journal, vol. 133, no. 9, pp. 1087–1095, 2020.View at: Google Scholar
Z. Li, H. Wan, Y. Shi, and P. Ouyang, “Personal experience with four kinds of chemical structure drawing software: review on ChemDraw, ChemWindow, ISIS/Draw, and ChemSketch,” Journal of Chemical Information and Computer Sciences, vol. 44, no. 5, pp. 1886–1890, 2004.View at: Publisher Site | Google Scholar
C. Tang, W. Ye, Y. Hu, and X. Yang, “Sulfonated derivative of andrographolide and combination of medication,” China Unexamined Application. Open to Public Inspection CN20051038561, 2005.View at: Google Scholar
National Medical Products Administration, “Adverse drug reaction information circular (No. 23) concerned about the safety of Chuanhuning injection and Yanhuning injection,” https://www.nmpa.gov.cn/xxgk/yjjsh/ypblfytb/20090901120001886.html.View at: Google Scholar
J. E. Bohonowych, M. W. Hance, K. D. Nolan, M. Defee, C. Parsons, and J. Isaacs, “Extracellular Hsp90 mediates an NF-κB dependent inflammatory stromal program: implications for the prostate tumor microenvironment,” The Prostate, vol. 74, no. 4, pp. 395–407, 2014.View at: Publisher Site | Google Scholar
A. Takeuchi, T. Miyamoto, K. Yamaji et al., “A human erythrocyte-derived growth-promoting factor with a wide target cell spectrum: identification as catalase,” Cancer Research, vol. 55, no. 7, pp. 1586–1589, 1995.View at: Google Scholar