Yu-Ming Li, Jun-Sheng Tong, Zhi-Ya Li, Jin-Zhong Wang, Xiao-Lan Qin

Department of Emergency, The Second Affiliated Hospital of Guangzhou University of TCM, Guangzhou 510370, China

Keywords:Shenfu Decoction COVID-19 Network pharmacology Kaempferol Ginsenoside rh2 Beta-sitosterol Stigmasterol

ABSTRACT Objective: The purpose of this thesis is to explore the mechanism of ShenFu Decoction in the treatment of critically ill patients with COVID-19 based on network pharmacology. Methods: The primary active ingredients and potential targets of ShenFu Decoction were searched from the TCMSP database. The targets of COVID-19 were obtained by searching the GeneCards and OMIM databases. A ShenFu Decoction-compound-target-COVID19 network and a protein-protein interaction (PPI) network were respectively constructed through the Cytoscape 3.5.1 software and the STRING database. Gene Ontology (GO) function enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were performed via Bioconductor bioinformatics software package and R programming language. Results: ShenFu Decoction contains 255 compounds and 94 potential targets. 43 primary active ingredients were searched from the TCMSP database with oral bioavailability (OB)≥30% and drug-likeness (DL)≥0.18 as the retrieval condition. Numbers of targets of COVID-19 were 352 by searching the GeneCards and the OMIM databases. 16 key targets were acquired by intersecting the targets of drug with the targets of disease. There were 49 GO terms and 102 pathways after analyzing GO and KEGG. Conclusion: Kaempferol, ginsenoside rh2, betasitosterol, Stigmasterol and Deoxy andrographolide might be the main active ingredients which may cause the inhibition of the SARS-CoV-2 3CL hydrolase activity and regulate ACE2. As a result, the antiviral effect, immunoregulation, targeting cytokine storm of SFD may play an important role in the treatment of critically ill patients with COVID-19 through regulating multiple signaling pathways such as AGE-RAGE signaling pathway in diabetic complications, IL-17 signaling pathway, C-type lectin receptor signaling pathway, HIF-1 signaling pathway.

1. Introduction

Since December 2019, COVID-19 has spread globally. Fever, dry cough, lack of power are the main clinical manifestations of the disease. Some patients can be accompanied by a stuffy nose, runny nose, sore throat and other symptoms of respiratory tract catarrh or gastrointestinal symptoms such as diarrhea, vomiting. Some patients are asymptomatic infection. In critically ill patients, multiple organ failure occurs, including acute lung injury, acute respiratory distress syndrome, and circulatory failure. At present, the domestic epidemic has been contained, but the overseas epidemic is still on the rise. There were 2878196 confirmed cases and 198668 deaths worldwide before April 27,2020[1]. The control of COVID-19 in China is closely related to a series of preventive and medical treatment measures. The intervention of traditional Chinese medicine (TCM) has played an important role in epidemic prevention and control. Since the outbreak of COVID-19, the national health commission has released the diagnosis and treatment scheme of COVID-19(trial edition III), which has been updated to the seventh edition and involves therapeutic plans of TCM. Other provinces, autonomous regions, municipalities directly under the central government and Xinjiang production and construction corps have also released their therapeutic plans of TCM. To summarize the treatment plans of the national health commission and various regions, the current treatment of TCM for COVID-19 is mainly based on syndrome differentiation according to the disease development stage (prevention stage, clinical observation stage, clinical treatment initial stage, middle stage, critical stage and recovery stage). Statistics were made on the frequency of each syndrome type and the corresponding recommended prescription. As a result, the syndrome type with the highest frequency was the syndrome of internal closure and external separation in critical illness and the corresponding recommended prescription was ShenFu decoction and Angong Niuhuang pills, Zixuedan, Zhibaodan or Suhexiang pills [2-3]. ShenFu injection were also recommended for severe and critically ill patients with shock by the National health commission in diagnosis and treatment of severe and critical COVID-19 cases (trial version II) [4]. According to pharmacology study of ShenFu injection, which is widely used in cardiogenic shock, heart failure, septic shock and so on, it can not only strengthen the heart, dually regulate blood pressure, reduce peripheral vascular resistance, reduce cardiac afterload and myocardial oxygen consumption, but also improve the CD3+,CD4+ and the ratio of CD4+/CD8+ to enhance immune function[5].

COVID-19 was thought to be one kind of epidemic disease in traditional Chinese Medicine and characterized by dampness as the core pathogenesis accompanied with cold or heat. Patients in critical stage of the disease present with hyperthermia, dyspnea, cyanosis and other signs of internal closure and shock, cool of limb, sweating, oliguria, dizziness, large and floating pulse. In this phase, ShenFu decoction is applied to restore the effect of Yang-qi. However, what is the mechanism of ShenFu decoction in the treatment of critical COVID-19 patients? Many studies suggested that cytokine storms are closely associated with critical transformation in COVID-19 patients. But there are currently no specific drugs for cytokine storms. Therefore, the mechanism of ShenFu decoction in the treatment of critical COVID-19 is worth discussing.

2. Research method

2.1 Screening of main active compounds of ShenFu decoction and prediction of corresponding targets

Two traditional Chinese medicines(Renshen, Fuzi) in ShenFu decoction were respectively input into Traditional Chinese Medicine Systems Pharmacology and Analysis Platform(TCMSP, http://tcmspw.com/tcmsp.php). Combined with literature reports[6], the active ingredients of ShenFu decoction were screened by the two conditions of oral bioavailability(OB) value ≥30% and drug similarity(DL) value ≥0.18.

2.2 Screening of targets of COVID-19

Through the search with “Novel Coronavirus” as a key word in the GeneCards database (https://www.genecards.org/) and the OMIM database (http://omim.org), the targets of COVID-19 retrieved form the above two databases were incorporated and duplicated targets were eliminated.

2.3 Selection of core targets of ShenFu decoction for COVID-19

The core targets of ShenFu decoction in the treatment of COVID-19 were obtained by intersecting the corresponding targets of the effective components of ShenFu decoction with the genes related to COVID-19. And the Venn diagram was drawn by Venny2.1 software.

2.4 Constructing the network of ShenFu decoction-active compounds-targets-disease and PPI

The article constructed the network of ShenFu decoction-active compounds-targets-disease by using Cytoscape3.5.1 software(http://www.cytoscape.org/). The targets of ShenFu decoction for COVID-19 were input into the multiple proteins of the STRING databases(http://string-db.org/) and the research species was set as “Homo sapiens”. As a result, PPI was acquired.

2.5 Analysis of GO function enrichment and KEGG signaling pathway enrichment

Analysis of GO function enrichment and KEGG signaling pathway enrichment was conducted using the Bioconductor(http://www.bioconductor.org) and R language with p＜0.05 and q＜0.05 as thresholds.

3. Results

3.1 Main active compounds and corresponding targets of ShenFu decoction

Through the search in the TCMSP database, 190 active compounds of Renshen, 65 of Fuzi were obtained. 22 active chemical constituents of Renshen, 21 of Fuzi were screened by the two conditions OB value≥30% and DL value≥0.18. After retrieval and removal of duplicates, 94 corresponding targets of ShenFu decoction were achieved.

3.2 Result of screening of targets of COVID-19

Through the search with “Novel Coronavirus” as a key word in the GeneCards database and the OMIM database, 352 genes associated with COVID-19 were obtained.

3.3 Result of selection of core targets of ShenFu decoction for COVID-19

The Venn diagram (Fig.1) was obtained by Venny2.1 software after intersecting targets of ShenFu decoction with the genes related to COVID-19.

3.4 The network of ShenFu decoction-active compoundstargets-disease and PPI network.

The network of ShenFu decoction-active compounds-targetsdisease was constructed by Cytoscape 3.5.1 software (Fig.2). In this figure, yellow nodes represent ShenFu decoction, green nodes represent disease, light green represents main active compounds of ShenFu decoction, blue represents targets, and lines represent interaction.

According to the PPI network (Fig.3), it contains 16 proteins and 79 interacting wires. The proteins with degree values ranging from large to small are CASP3、MAPK8、IL1B、PPARG、PTGS2、CASP8、HMOX1、ICAM1、IFNG、NOS2、RELA、STAT1、BAX、BCL2、PRKCA、PTGS1. The degree values of CASP3、MAPK8、IL1B、PPARG、PTGS2、CASP8、HMOX1、ICAM1、IFNG、NOS2、RELA、STAT1, which may be the main targets of ShenFu decoction for COVID-19, are higher than others.

3.5 The result of analysis of GO function enrichment and KEGG signaling pathway enrichment

Through GO function enrichment analysis, 49 related items were achieved. The top 20 items were drawn as a bubble diagram (Fig.4), including Cytokine receptor binding, heme binding, tetrapyrrole binding, tumor necrosis factor receptor superfamily binding, oxidoreductase activity acting on paired donors with incorporation or reduction of molecular oxygen, repressing transcription factor binding, BH domain binding, death domain binding, cysteinetype endopeptidase activity involved in apoptotic process, death receptor binding, ubiquitin-like protein ligase binding, integrin binding, protein phosphatase binding, tumor necrosis factor receptor binding, protein phosphatase 2A binding, phosphatase binding, actinin binding, cell adhesion molecule binding, peroxidase activity, oxidoreductase activity acting on peroxide as acceptor. The result of analysis of GO function enrichment suggests that ShenFu decoction acts on COVID-19 through regulating a variety of biological pathways [7].

KEGG signaling pathway enrichment analysis revealed 102 signaling pathways. The top 20 signaling pathways were drawn as histogram (Fig.5).

Fig.1 Venn diagram of shenfu decoction for COVID-19 targets

Fig.2 SFD-compound-target-COVID19 network

Fig.3 PPI network of SFD and COVID-19 (A) and the common genes with the top 16 degree values(B)

Fig.4 The result of GO function enrichment analysis

Fig.5 The result of KEGG pathway analysis

4. Discussion

The COVID-19 belongs to the category of "epidemic disease" in TCM. Treatment for COVID-19 based on stage and syndrome differentiation is possible because the clinical course of the disease presents a periodical rule [8]. Critically ill patients often present with fever, asthma, cyanosis, fatigue, irritability, even coma, chest and abdomen heat and limbs cold, pulse count, etc. In the current treatment of TCM for COVID-19 of the national health commission and various regions, the corresponding recommended prescription was ShenFu decoction and Angong Niuhuang pills, Zixuedan, Zhibaodan or Suhexiang pills. ShenFu injection were also recommended for severe and critically ill patients[9]. The medicine of ShenFu decoction consists of Renshen and Fuzi. The prescription has the function of restoring Yang-qi and nourishing qi to stop collapse. It is mainly used for treatment of qi-deficiency which may present limb cooling, cold sweat, pale complexion, shortness of breath, and weak pulse, etc[10].

At present, many studies suggest that inflammatory reaction, immune imbalance and cytokine storm are the key pathogenesis of COVID-19. Some scholars found that the serum levels of inflammatory factors such as IL-1β and γ-IFN increased, which was more obvious in ICU patients with critical illness [7,11-12]. The SARS-CoV-2 enters human cells through binding the S protein of the virus and angiotensin converting enzyme II (ACE2) and then promotes the imbalance between ACE2 and ACE1 in the lung, which may lead to increase pulmonary capillary permeability and patients develop pulmonary edema [13]. The genome of SARSCoV-2 is a single chain RNA and SARS-CoV-2 3CL hydrolase is the core protease of polymeric proteolysis of precursor of single plus strand RNA virus [14], which is the breakthrough of anti-SARS-CoV-2 drug research and development [15]. The network of ShenFu decoction-active compounds-targets-disease in this article showed that kaempferol, ginsenoside rh2, arachidonic acid, betasitosterol, stigmasterol, Deoxy andrographolide might be the main active compounds. Kaempferol may be a replication inhibitor of SARS-CoV, which can bind to SARS-CoV-2 3CL hydrolase and then effectively inhibit viral precursor protein cleavage and block viral replication [14], thus interfering with the life cycle of the virus [15]. Ginsenoside rh2 and deoxy andrographolide can inhibit the inflammatory reaction of sepsis and have obvious anti-inflammatory effect [13]. Ginsenoside Rh2 also has a selective immunomodulatory effect under different disease backgrounds [16]. Beta-sitosterol and stigmasterol play an antiviral role because they have the ability to bind to ACE2 which is the key receptor when SARS-CoV-2 infect target cells and then inhibit the binding of virus S protein to ACE2 [6]. Therefore, we speculate that ShenFu decoction may have antiviral effect in the treatment of critically ill COVID-19 patients. This effect may be through the following mechanisms: The active compounds combine with ACE2 and block the Virus from entering human cells through the pathway that binds the S protein to ACE2. It may suppress the virus-induced inflammatory cascade response. The active compounds may inhibit the activity of SARS-CoV-2 3CL hydrolase and block its regulation of virus replication. In addition, decreasing the level of inflammatory factors and regulating immune function and suppressing cytokine storms may be the antiviral mechanism of the drug.

In the diagram of PPI network, we obtained 16 core targets. The CASP3 is the main effector of apoptosis. BAX and BCL2 are the major proteins regulating apoptosis. BAX is pro-apoptotic protein and BCL2 is an anti-apoptotic protein. The level of both is directly related to the regulation of apoptosis. The expression of BAX is upregulated and the expression of BCL2 is down-regulated, which promote the apoptosis of alveolar epithelial cells by activating the expression of CASP3 [17]. The MAPK is a kind of serine/threonine kinases and signal transduction mediators [18], which has a regulatory effect on cells. As a member of MAPK family, MAPK8, also known as C-jun amino-terminal kinase 1, plays a role in regulating cell growth, proliferation, differentiation and apoptosis through threestage enzymatic cascade reaction stimulated by pro-inflammatory cytokines and other factors [19]. In the current studies of the mechanism of antiviral action of TCM, it is thought that regulating the expression of certain genes in the process of cell apoptosis may play an antiviral role [20]. As a member of the interleukin family, IL1B can aggravate the body's inflammatory response by activating the terminal kinase pathway of c-JUN, which is another protooncogene in the MAPK signaling pathway [6]. PTGS, also known as cyclooxygenase (COX), mainly includes PTGS1 and PTGS2, and is a key rate-limiting enzyme for synthesis of prostaglandins by arachidonic acid [21]. By inhibiting PTGS and reducing prostaglandin production, it can reduce the secretion of inflammatory factors and play an anti-inflammatory role. Researchers such as Ren-yue [12] found that ginseng can treat COVID-19 by inhibiting the metabolic pathway of arachidonic acid, reducing the release of inflammatory cytokines and suppressing the cytokine storm. At the same time, PTGS2 can be activated by the MAPK pathway to participate in SARS-CoV-2 injury of epithelial cells in tissues and organs by regulating inflammation and immunity [20]. Interferon can activate macrophages and play a role in suppressing the virus and regulating immunity [7]. In summary, according to literatures, the inflammatory cascade, immune imbalance and cytokine storm may be the key mechanisms of critical COVID-19 patients and the above targets play an important role in inflammatory response, apoptosis, immune regulation. This article revealed that the main ingredients of ShenFu decoction such as kaempferol, ginsenoside rh2, arachidonic acid, beta-sitosterol, stigmasterol, deoxy andrographolide may take the therapeutic effects in the treatment of critical COVID-19 through the above targets.

In terms of KEGG pathway enrichment analysis, the AGEs-RAGE signaling pathway had the most gene target enrichment. The AGEs-RAGE signaling pathway stimulates the expression and release of various inflammatory factors by activating multiple signaling pathways such as MAPK, ERK1/2 and NF-κB [23]. This signal pathway is closely related with renal injury because it can also induce the expression of monocyte chemoattractant protein-1 and activate the renin-angiotensin system [23]. Members of the IL-17 family bind to various IL-17 receptors and play an important role in acute and chronic inflammatory responses by activating the NF-κB and Janus kinase-signaling and transcriptional activator signaling pathways, regulating the secretion of various inflammatory factors, inducing the expression of antimicrobial peptides, promoting neutrophils aggregation, and recruiting TH17 and ILC3s cells[24]. Tumor necrosis factor signaling pathway, C type lectin receptor signaling pathway and hypoxia-induced factor-1 signaling pathway also play important biological effects in inflammatory response and immune regulation [25-27]. Therefore, according to the function of 16 targets and the enrichment analysis of KEGG pathway, the main active components of ShenFu decoction may act in critical COVID-19 patients through AGEs-RAGE signaling pathway, IL-17 signaling pathway, c-type lectin receptor signaling pathway, HIF-1 signaling pathway and so on. Moreover, among the top 20 pathways with gene target enrichment, 10 pathways are related to infectious diseases, which suggests that ShenFu decoction may play an important role in the treatment of infectious disease.

To sum up, based on the network pharmacology, this article revealed that ShenFu decoction exerts antiviral, anti-inflammatory and immunomodulatory effects through multiple components, multiple targets and multiple pathways. However, many factors such as the complexity of TCM, unelucidated pathogenesis of COVID-19 and comprehensive treatment of critically ill patients should be considered. Although the conclusion of this essay can provide a theoretical basis for ShenFu decoction to treat critical patients with COVID-19, it needs to be verified by further experiments.