1. Introduction
Porcine epidemic diarrhea (PED) is an acute, highly contagious enteric disease characterized by diarrhea, vomiting, dehydration, and high mortality, with rates reaching up to 100% in suckling piglets within their first week of life. The disease is mainly caused by infection with the porcine epidemic diarrhea virus (PEDV), a single-stranded, positive-sense RNA virus of the Alphacoronavirus genus of the Coronaviridae family [
1]. Currently, there are no effective pharmacological interventions for the prevention and control of PED. Traditional inactivated and live vaccines are considered the most effective and economical strategies for PED management. However, the emergence of PEDV mutant strains has rendered vaccine efficacy suboptimal. Furthermore, the base of pig breeding in China is large, the breeding mode is diverse, and the main body of disease prevention and control is transferred. The variability in understanding the genesis, evolution, pathogenesis, dissemination, and immunological responses of PED among various stakeholders has led to inconsistent approaches to disease management [
2]. The ongoing immune and environmental pressures have driven adaptive evolution of PEDV, resulting in the continuous emergence of new strains or subtypes through genetic variation. In the same farm, there are not only PEDV, but also other diarrheal pathogens. For example, transmissible gastroenteritis virus (TGEV), porcine colibacillosis, and porcine deltacoronavirus (PDCoV) are other pathogens that can coexist and cause mixed infections in the same field. This multi-pathogen coexistence and mixed infection contributes to the current situation of PED prevention and control in China, which remains challenging [
3].
Coptidis rhizoma is a significant component within the domain of Chinese herbal medicine. Scientific studies have demonstrated that
Coptidis rhizoma and its active ingredients play a crucial role in protecting cardiovascular and cerebrovascular systems, exhibiting hypoglycemic, anti-inflammatory, antitumor, and antimicrobial properties, and modulating intestinal flora [
4]. The existing reports indicate that berberine, the active ingredient of
Coptidis rhizoma, can inhibit PEDV infection by regulating the host signal transduction pathway or virus life cycle. Berberine has been demonstrated to significantly inhibit PEDV infection. Its mechanism of action appears to primarily involve the replication and assembly of PEDV, although the specific molecular mechanisms remain to be further elucidated [
5]. To determine whether other active ingredients of
Coptidis rhizoma can also play a role in anti-PEDV, this study employed network pharmacology–molecular docking technology to first identify the main active ingredients of
Coptidis rhizoma and then to identify the main interaction targets of the
Coptidis rhizoma active ingredients against PEDV. The study then used molecular docking technology to verify the main action sites of the identified compounds, with the aim of providing assistance for the development of anti-PEDV drugs based on
Coptidis rhizoma drugs.
2. Materials and Methods
In order to identify the principal chemical constituents of
Coptidis rhizoma, the keyword “
Coptidis rhizoma” was searched in the TCMSP pharmacology database (
https://www.tcmsp-e.com) on 24 January 2024. Subsequently, the active ingredients with oral bioavailability (OB) ≥ 30% and which were drug-like (DL) ≥ 0.18 were selected to obtain the effective active ingredients of
Coptidis rhizoma in this study. On 25 January 2024, the SwissADME tool within the SwissDrugDesign database (
https://www.molecular-modelling.ch/swiss-drug-design.html) was utilized to screen the active compounds of
Coptidis rhizoma, identifying those with superior intestinal absorption and drug-like properties. The SwissTargetPrediction tool was subsequently used to screen the effective targets of
Coptidis rhizoma active compounds. Subsequently, the GeneCards database (
https://www.genecards.org) was employed to identify disease-related gene targets associated with PED on 25 January 2024. The keyword “Porcine Epidemic Diarrhea” was utilized for retrieval on 25 January 2024. Finally, the drug targets and disease-related target genes obtained from each database were integrated and deduplicated, and their names were standardized using the UniProt database (
https://www.uniprot.org) on 25 January 2024.
The disease targets of the effective active components of
Coptidis rhizoma and PED disease targets were used to create a Venn diagram using Venny2.1 (
https://bioinfogp.cnb.csic.es/tools/venny/index.html) to explore the targets of the active components of
Coptidis rhizoma against PED disease on 26 January 2024.
The SMILES structure was constructed using the PubChem function of the NCBI database (
https://www.ncbi.nlm.nih.gov) on 26 January 2024. Subsequently, the pharmacokinetics and drug-likeness of the active components of
Coptidis rhizoma were evaluated using the SwissDrugDesign database on 26 January 2024. If the intestinal absorption capacity of pharmacokinetics is high and the drug exhibits more than two positive responses, it is deemed suitable for use.
The 69 intersection gene targets were imported into the database based on the STRING database (
https://cn.string-db.org) to obtain the interaction network of intersection gene targets on 27 January 2024. Subsequently, the Cytoscape 3.7.1 software was employed to identify the principal targets of active drugs derived from
Coptidis rhizoma that exhibit efficacy against PEDV. This was accomplished by utilizing degree centrality (degree = 7.727272727), closeness centrality (closeness = 0.005981415), and betweenness centrality (betweenness = 111.030303) to assess the interaction strength of the principal targets.
The related genes of the 69 intersection gene targets were analyzed using the DAVID database (
https://david.ncifcrf.gov), an online enrichment database, on 27 January 2024. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment were conducted on the ten most closely related genes. The Benjamini–Hochberg procedure was applied to control the false discovery rate (FDR), setting a threshold of FDR < 0.05. The results of the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichments are presented in the form of a bubble diagram.
Molecular docking and binding energy calculations were performed on the key effective active ingredients (e.g., epiberberine, palmatine, worenine, obacunone, berberine, and berberruine) and key targets (e.g., STAT3, ESR1, CASP3, and SRC). Some three-dimensional model structures of pig-derived proteins, including STAT3, ESR1, CASP3, and SRC proteins, were constructed based on the previous Swiss-model website. Subsequently, AutoDock1.5.7 software was utilized to modify the protein model, including the removal of pertinent water molecules and metal ions, followed by the uploading of receptors and ligands for flexible docking. Subsequently, the binding mode of small molecule drugs and proteins was identified through a process of screening and visualization, utilizing the Pymol 2.5.0a0 software. This entailed an analysis of the binding energy and binding position in order to ascertain the optimal binding mode. High-performance Computing (Sugon, I950r-G, China) was used for data processing.
4. Discussion
Network pharmacology is a discipline that employs large-scale data and computer technology to investigate the interactions between drug molecules and biological targets, pathways, genes, proteins, and other molecules in vivo. To achieve this, an interaction network between drug molecules and proteins can be constructed. By analyzing the relationships between these interactions and investigating their effects on drug efficacy, toxicity, and metabolism, this approach aids in developing efficient solutions for drug discovery, therapeutic treatments, and drug repurposing [
6]. In this study, based on network pharmacology, the potential targets of
Coptidis rhizoma against PEDV were screened using the TCMSP pharmacological database, SwissDrugDesign database, GeneCards database, and UniProt database, and 74 specific targets of
Coptidis rhizoma against PEDV were obtained. The core gene targets were screened and the interaction network diagram was made. Finally, 14 core targets were obtained. They were STAT3, ESR1, CASP3, SRC, MMP9, EGFR, PTGS2, MDM2, ICAM1, IKBKB, HSP90AA1, PTK2, SIRT1, MAPK8, among which STAT3, ESR1, CASP3, and SRC had the greatest interactions. Among the core targets, STAT3 is a member of the STAT family and is involved in a number of biological processes, including cell proliferation, survival, differentiation, and angiogenesis. In normal cells, STAT3 primarily transmits the transcriptional signals of cytokines and growth factor receptors on the plasma membrane to the nucleus through the immediate activation of phosphorylation, thereby facilitating the exchange of signals between the cytoplasm and the nucleus for a series of biological processes [
7]. It is therefore proposed that the replication of PEDV may be inhibited by targeting the STAT3 signaling pathway. This could be achieved by developing STAT3 inhibitors or degradation agents. In the data of Huang H. et al. [
8], the infection of PEDV has been observed to up-regulate the protein tyrosine phosphatase non-receptor type 14 (PTPN14), a potential tumor suppressor, and to reduce the phosphorylation of STAT3 and inhibit the activation of STAT3. The study conducted by Yang J. et al. also revealed that the inhibition of the STAT3 signaling pathway could alleviate the inflammatory response and reduce intestinal damage induced by PEDV [
9]. Additionally, the study conducted by Li X. et al. indicates that the overexpression of STAT3 may facilitate the replication of PEDV [
10]. The aforementioned studies have demonstrated that the STAT3 signaling pathway is a pivotal factor in the pathogenesis of PEDV infection. Inhibition of the STAT3 signaling pathway results in the inhibition of PEDV infection, whereas overexpression of STAT3 promotes PEDV replication. In the study of anti-PEDV infection of
Coptidis rhizoma active drugs, it was observed that STAT3 can interact with epiberberine and palmatine, which serve as important targets. It can be seen that the STAT3 signaling pathway plays an important role in the anti-PEDV infection of
Coptidis rhizoma active drugs; however, the specific molecular mechanism remains to be further elucidated.
ESR1 is a critical estrogen receptor significantly associated with the development and prognosis of estrogen-dependent malignant tumors [
11]. To date, there have been few reports on the relationship between ESR1 and PEDV. Studies have demonstrated that ESR1 is closely related to the NF-κB signaling pathway. The interaction between ESR1 and NF-κB is inhibitory; ESR1 reduces NF-κB DNA binding activity and suppresses NF-κB-mediated transcription of the IL6 promoter. In addition, ESR1 can replace RELA/p65 and associated co-regulators at the promoter, leading to the inhibition of NF-κB [
12]. Research indicates that the cooperation between ESR1 and NF-κB activates transcription through the recruitment of adjacent response elements, thereby promoting cell signal transmission [
12]. However, it remains to be determined whether the anti-PEDV effect of ESR1 is mediated by the NF-κB signaling pathway.
CASP3 is a protease that exhibits specific cleavage activity towards poly ADP ribose polymerase (PARP1) and acetyl-DEVD-7-amino-4-methylcoumarin (Ac-DEVD-AMC), resulting in DNA cleavage and the promotion of apoptosis [
13]. In the data gathered by Zhou H.C. et al. [
14], overexpression of cell communication network factor 1 (CCN1) was observed to increase the phosphorylation level of p53, promote the expression of Bax and the cleavage of CASP9 and CASP3, and inhibit the production of Bcl-2. The knockdown of CCN1 has been demonstrated to reduce the phosphorylation level of p53, inhibit the production of Bax and the cleavage of CASP9 and CASP3, and promote the expression of Bcl-2. In the study by XU X.G. et al. [
15], it was demonstrated that the nonstructural protein Nsp9 of porcine epidemic diarrhea virus (PEDV) interacts with the H2BE protein. Overexpression of H2BE protein was found to inhibit the expression of Bax and the cleavage of CASP9 and CASP3, while promoting the expression of Bcl-2. Previous studies have demonstrated that PEDV infection can alter the CASP3 protein through various mechanisms that influence apoptosis. The primary determinant is the impact of pro-apoptotic protein Bax expression. When Bax is overexpressed, CASP3 is activated, leading to apoptosis and subsequently promoting PEDV replication.
SRC is a non-receptor tyrosine protein kinase encoded by a specific proto-oncogene, which plays a pivotal role in the growth, progression, and metastasis of cancer [
16]. In the context of infection by porcine epidemic diarrhea virus (PEDV), SRC plays a pivotal role in the coordination and promotion of cell signaling pathways. The entry of PEDV into cells is primarily dependent on the endocytosis of transferrin receptor 1 (TfR1), which necessitates the regulation of SRC [
17]. TfR1 binds to SRC and interacts with SRC, which increases the phosphorylation of TfR1 Tyr20 and promotes viral replication. Conversely, the use of SRC inhibitors has been demonstrated to reduce the phosphorylation of TfR1 Tyr20 and to inhibit viral replication [
18]. In Li X.M.’s report [
10], leflunomide active metabolite A77 1726 was also found to effectively limit PEDV replication by inhibiting JAKs and SRC. The importance of SRC in PEDV replication indicates that the study of SRC inhibitors provides a new strategy for anti-PEDV infection. Furthermore, additional potential molecular mechanisms and signaling pathways of
Coptidis rhizoma against PEDV were identified through GO enrichment and KEGG enrichment. These include protein serine/threonine/tyrosine kinase activity, zinc ion binding, and transmembrane receptor protein tyrosine kinase activity. Additionally, the following processes are positively regulated: RNA polymerase II promoter transcription, apoptosis, protein kinase B signal, and others. The transmembrane receptor protein tyrosine kinase signal pathway, protein autophosphorylation, PI3K-Akt signal pathway, fluid shear stress and atherosclerosis, chemical carcinogen-receptor activation, human cytomegalovirus infection, microRNA in cancer, and endocrine resistance and Alzheimer’s disease were also identified. The specific action sites of the active ingredients of the drug against PEDV were identified through molecular docking testing. For instance, epiberberine and palmatine demonstrated a high affinity for the STAT3 protein, worenine exhibited a high affinity for the ESR1 protein, and obacunone exhibited a high affinity for the CASP3 protein. Epiberberine, obacunone, berberine, and berberruine demonstrated high binding affinity for the SRC protein. In conclusion,
Coptidis rhizoma, as a pure natural drug of traditional Chinese medicine, contains a variety of effective active ingredients, which have the advantages of a wide effect, safety, low residue, low toxicity, and not easy to produce drug resistance. The anti-PEDV mechanism is multi-target, multi-mechanism, and multi-pathway, with multiple components working in concert. However, the precise body mechanism remains to be elucidated. Notably, this study used the GeneCards database, which focuses on human-related diseases, to research PEDV, but there are certain limitations. Significant genetic differences exist between humans and pigs; some genes critical to human diseases may exhibit different expressions or functions in pigs. Nevertheless, comparative genomics shows that over 80% of genes are homologous between pigs and humans, indicating a degree of biological similarity [
19]. This similarity renders pigs valuable models for studying human diseases, drug development, and organ transplantation [
20]. Conversely, whether human models can be utilized to study porcine diseases remains to be investigated. Additionally, PEDV, as a member of the coronavirus family, exhibits strong adaptability and extensive mutability [
3]. Research indicates that PEDV can replicate in human intestinal cells, suggesting the possibility of cross-species infection, albeit weakly. This potential highlights the promise of research into cross-species coronavirus pathogenicity or host migration [
21]. Hence, the feasibility of applying human disease targets to PEDV research warrants further investigation. Nevertheless, the use of the GeneCards database in this study has certain limitations, and subsequent research outcomes might not show significant antiviral effects in pig populations. However, these findings could still be crucial for other animals, including humans, in the future, thus bearing significance and value for public safety. Additionally, it is noteworthy that this study conducted extensive searches in the GeneCards database to obtain more target data.