Potential Molecular Mechanism of Yishen Capsule in the Treatment of Diabetic Nephropathy Based on Network Pharmacology and Molecular Docking

Introduction

Diabetes is a complex chronic metabolic disorder. Complications include cardiovascular and cerebrovascular diseases and microvascular diseases, such as retinopathy, peripheral neuropathy, and diabetic nephropathy.^1–3 Diabetic nephropathy (DN) is one of the most common microvascular pathologies of diabetes, and further development of it may progress to the stage of end-stage renal disease, which requires renal replacement therapy and seriously affects the patient’s health and quality of life.⁴

The pathogenesis of DN is complex. Reported evidences showed it is related to changes in glomerular hemodynamics, excessive activation of Renin-Angiotensin-Aldosterone System (RAAS), luxuriant generation of reactive oxygen species (ROS), inflammation, mitochondrial dysfunction etc. However, its exact mechanism is not yet clear.⁵

The current clinical treatments for DN mainly include blood glucose control, blood pressure control, correction of metabolic disorders, and improvement of microcirculation, while the clinical treatment has not yet achieved satisfactory results.⁶ The application of special Chinese medicine treatments for DN possesses the advantages of multi-target, multi-level treatment mechanism, fewer adverse reactions and high safety and the unique advantages for the treatment of DN have been continuously highlighted.

Diabetic nephropathy belongs to kidney deficiency. Diabetes persists for a long time, consumes qi and injures yin, damages organs, and also contains phlegm, heat, depression and blood stasis, and eventually leads to diabetic nephropathy. Diabetic nephropathy, persists for a long time, can cause deficiency of both qi and yin, deficiency of yin of liver and kidney, deficiency of spleen and kidney, internal stop of blood stasis, and dampness stagnation, resulting in a syndrome of mixed deficiency and excess. Therefore, in order to treat the symptoms of proteinuria associated with diabetic nephropathy, according to the principle of TCM syndrome differentiation and treatment, clinical treatment methods such as promoting blood circulation and removing blood stasis, clearing heat and removing dampness, strengthening spleen and nourishing qi, nourishing yin and nourishing kidney are often used.⁷

The Chinese medicine preparation Yishen Capsule is composed of Astragalus, Angelica, Gordon Euryale, Alisma, Schisandra, Rhodiola and other Chinese medicines. Yishen Capsule is a hospital preparation, the preparation number is “97 Bing Wei Preparation with Zi f01-2005”. The formula has the functions of invigorating kidney qi, diuretic and swelling, regulating and enhancing the immunity of the body. Astragalus in the prescription is the monarch drug, which has the functions of diuresis and swelling, antiperspirant, invigorating qi and nourishing yin. Alisma is a minister drug, which can eliminate dampness and diuresis, namely purging the water of the kidney meridian, and purging the dampness of the bladder meridian. Angelica is also a minister drug, which benefit blood and camp. Gorgon is an assistant drug, which has the functions of invigorating the spleen and stopping diarrhea, invigorating the kidney and strengthening the essence, removing dampness and stopping the leucorrhea.Schisandra chinensis is used as a guide medicine, which nourishing the kidney, solidifying essence, stopping diarrhea, promoting saliva and abstaining sweat. Rhodiola clears heat, detoxifies, nourishes the lungs, and regulates immunity. The combination of the whole agents has the functions of nourishing kidney qi, promoting diuresis and relieving stranguria, promoting blood circulation and removing blood stasis, regulating and enhancing the immunity of the body. It has been confirmed by acute and chronic toxicity experiments that this preparation has no toxicity and side effects.⁸ Previous studies have indicated that Yishen Capsule can improve kidney function through multiple targets and mechanisms, which effectively delays the progression of DN.^9–15

Network pharmacology is based on bioinformatics and computer technology, integrating a large amount of biological information and data. It systematically and comprehensively reflects the intervention mechanism of drugs on the disease network, which is similar to the mechanism that the complex components of traditional Chinese medicines interfere with diseases through multiple channels and multiple targets.¹⁶ In recent years, network pharmacology approaches have provided new insights into the mechanisms by which TCM treats diseases. The network pharmacology method can provide technical support for the research on the mechanism of action of traditional Chinese medicine compounds in the diagnosis and treatment of diseases. It is helpful to screen the active ingredients of drugs, predict the action targets in the ingredients, and study the mechanism of action of drugs. Thereby revealing the scientific connotation of traditional Chinese medicine compounds and guiding the research and development of new traditional Chinese medicines.^17,18 This study intends to explore the targets of Yishen Capsule in the treatment of DN and to elucidate the therapeutic mechanism of Yishen Capsule on DN from many aspects. Further, some target proteins of key signaling pathway were verified through experiment both in vitro and in vivo, which may provide evidence for the treatment of DN using Yishen Capsule.

Materials and Methods

Network Pharmacology Analysis

Screening of Active Chemical Components and Acquisition of Targets

Using the TCMSP database (http://tcmspw.com/) to retrieve all the chemical components of the 6 traditional Chinese medicines (Astragalus, Angelica, Gordon Euryale, Alisma, Schisandra and Rhodiola) in Yishen Capsule. According to the two ADME attribute values of oral bioavailability (≥ 30%) and drug-likeness (≥ 0.18) of these ingredients, preliminary screening was performed to obtain the active compounds, as well as their protein targets. According to the TCMID database (http://www.megabionet.org/tcmid/) and published literature reports,¹⁹ some unpredicted active compounds and reported targets have been supplemented. In the UniProt protein database (https://www.uniprot.org), human-derived targets were screened and standardized.

Target Screening of Diabetic Nephropathy

Searching the GeneCards database (https://www.genecards.org), OMIM database (http://www.omim.org), TTD database (http://bidd.nus.edu.sg/group/cjttd) and DisGeNET database (https://www.disgenet.org) for potential targets of diabetic nephropathy, by using “Diabetic nephropathy” and “Diabetic Kidney Disease” as keywords. And some target sites for disease were also supplemented through searching the DrugBank database (https://www.drugbank.ca). According to experience, the target points with Score ≥30 in the GeneCards database and Score ≥0.3 in the DisGeNET database were selected as potential targets for diabetic nephropathy.

Intersection of Chinese Medicine and Disease Target Genes and Construction of PPI Network

The intersection of the drug target of Yishen Capsule and the target of diabetic nephropathy were selected to draw the Venn diagram, using Venny 2.1.0 (https://bioinfogp.cnb.csic.es/tools/venny/index.html). Submitting the intersection target to the STRING11.0 database (https://string-db.org) to construct a protein interaction (PPI) network.²⁰ The PPI network was visually analyzed by Cytoscape 3.7.2 software, and PPI core network proteins were screened out according to the degree value≥ 2 times the median.

GO and KEGG Pathway Enrichment Analysis

Using Metascape platform (http://metascape.org/gp/index.html) for GO and KEGG pathway enrichment analysis. Setting P<0.01 is statistically significant, and the analysis results were visualized.

Construction of Components of Yishen Capsule-Targets of Diabetic Nephropathy Disease- Pathway Network Diagram

Using CytoScape 3.7.2 to construct a network diagram of Yishen Capsule components-diabetic nephropathy disease targets-pathway network. Then, using CytoScape 3.7.2 built-in tools to analyze the network topology parameters of active ingredients and targets, which helps to determine core targets and main active ingredients that exert efficacy. The core targets of Cytoscape network analysis and the core target proteins of PPI network were intersected to obtain the key targets of Yishen Capsule in the treatment of diabetic nephropathy, according to which the Venn diagram was drawn.

Verification by Molecular Docking

Downloading the crystal structure of the target protein from the PDB database (https://www.rcsb.org/), and using PyMOL and AutoDock software to remove water, remove the original ligand of the active center, and to hydrogenate the target protein. Downloading the 3D structure file of the core components of traditional Chinese medicine from the PubChem database (https://www.ncbi.nlm.nih.gov/pccompound/). And then loading it to the AutoDock Tools 1.5.6 software to add atomic charges, assign atomic types. All flexible bonds were set as rotatable Running AutoDock Vina for molecular docking. The conformation with the highest score is selected as the docking conformation, and the PyMOL program was used to visualize the results.

Experimental Verification

Reagents

Yishen Capsule was prepared at Department of Pharmacy of the First Hospital of Shanxi Medical University. Boiling the herbs in water thrice for 1 h. The decoctions were combined and filtered through a membrane, then concentrated through vacuum evaporation to a density of 1.20–1.24 at 70 °C. The concentrated decoction was further spray-dried into particles that were subsequently filled into capsule.

Streptozotocin (STZ, S8050), Penicillin,-streptomycin antibiotic (P1400), D-glucosum anhydricum (G8150) and Horseradish peroxidase-conjugated goat anti-rabbit IgG (SE134) were bought from Solarbio Company (China). PAS staining kit (BA-4114) and MASSON staining kit (BA-4079) were purchased from Baso Biotechnology (China). Fetal bovine serum (FBS, 10100147), DMEM low glucose medium (1185092) and DMEM high glucose medium (1196007) were purchased from Thermo Fisher Scientific (USA). Tris-glycine-SDS running buffer (AR1139-10) and transfer buffer (AR1149-10), SDS-PAGE protein loading buffer (AR1112) were purchased from Boster Biological Technology (China). PVDF membrane (AR0136-02), WB transfer filter paper (AR0172), hypersensitive ECL chemiluminescence solution (AR1191), and 4% paraformaldehyde fixative solution (AR1068) were purchased from Boster Biological Technology (China). Rabbit antibody of HIF-1α (ab51608), JAK2 (ab32101), STAT3 (ab68153), TGF-β (ab215715) and MCP-1 (ab7202) were purchased from Abcam (England). Both DAB chromogenic reagent (AR1022) and hematoxylin-eosin staining solution (BA-4025) were purchased from Boster Biological Technology (China).

Animal Experiment and Preparation of Plasma Containing Yishen Components

Forty SPF-grade healthy male SD rats (185–215g, 40) were purchased from the Animal Experimental Center of Shanxi Medical University, with the animal certificate number of SCXK (Jin) 2015-0001. Ten rats were randomly selected to give Yishen Capsule by gavage, with the frequency of once a day for 7 days. Two hours after the last gavage, the rats were anesthetized by intraperitoneal injection of 3% pentobarbital sodium. Blood was collected from the abdominal aorta. Blood was centrifugation and the upper serum was collected and stored after inactivation and sterilization.

The remaining 30 rats were randomly divided into the control group (N group, 10 rats) and the model group (20 rats). After one week of adaptive feeding, the model group was injected intraperitoneally with streptozotocin (50 mg/kg) after left nephrectomy. After 72 hours of injection, the rats with random blood glucose> 16.7 mmol/L and 24-hour urine protein quantification> 30 mg are the successful model rats. The successfully modeled DN rats were randomly divided into diabetic nephropathy group (DN) and Yishen capsule group (YS). The YS group was given Yishen capsule gavage solution at 1.25 g/kg/d by gavage (The previous study of the research has carried out different doses of intervention treatment, and it is confirmed that 1.25 g/kg/d is the effective dose of gavage treatment),⁹ and the N group and DN group were given the same amount of normal saline gavage for 8 consecutive weeks. After 8 weeks of treatment, the right kidney specimens of each group of rats were collected for subsequent immunohistochemistry and Western Blotting detection. All animal experiment protocols were reviewed and approved by the Animal Ethics Committee of Shanxi Medical University (batch number: 2019LL242), and the study was conducted in accordance with the guidelines for the care and use of laboratory animals.

Cell Culture and Treatment

Rat renal tubular epithelial cells (NRK-52E) were got from National Model and Characteristic Experimental Cell Resource Bank/Chinese Academy of Sciences Cell Bank (China) and the CSTR number is 19375.09.3101RATGNR8. And it was cultured in a standard medium (DMEM high glucose medium, containing fetal bovine serum and penicillin- streptomycin antibiotic), and in a 5% CO2 atmosphere at 37 °C. The experimental groups were as follows: normal group (5.5mmol/L glucose), high glucose group (30mmol/L glucose) and Yishen containing-serum treatment group (30 mmol/L glucose + 10% Yishen containing-serum). After each group was cultured for 24 hours, cell proteins were collected for Western Blotting detection.

Pathology Examination of Kidney

Specimens of right kidney were collected, fixed in 4% paraformaldehyde solution, embedded in paraffin, and sectioned routinely. After section, slides were stained with HE, PAS, and Masson. The pathological changes of the renal tissue were observed under light microscope.

Immunohistochemistry

Kidney tissues were embedded in paraffin, conventionally sectioned, deparaffinized, sealed, and incubated at room temperature. Slides were incubated with rabbit anti-HIF-1α, rabbit anti-JAK2, and rabbit anti-STAT3 antibody, respectively overnight at 4°C. Then Slides were incubated with biotin Labeled with goat anti-rabbit IgG (secondary antibody) at 37°C for 15 min. After that, Slides were orderly incubated with DAB reagent and hematoxylin (for 2 min). Then, slides were dehydrated and transparent, and mount with neutral gum. The tissue sections were examined under an optical microscope and quantitative analysis was performed using Image-Pro Plus 6.0 medical pathology graphic color analysis software.

Western Blotting

Extracting and collecting total protein from cell and kidney tissue and determining concentration of the total protein. Denatured protein samples (20 μL) were loaded into the wells, and the proteins were separated by electrophoresis with 8% SDS-PAGE.^21–23 Then, the protein in the gel was transferred to the PVDF membrane using a Bio-Rad electrotransmitter. Subsequently, 5% skimmed milk powder was used to seal the PVDF membrane at room temperature. After blocking, adding the primary antibody and incubating overnight at 4°C. The next day, the washed PVDF membrane was incubated with horseradish peroxidase-labeled secondary antibody at room temperature. After incubating for 1 hour, the strips on the PVDF membrane were developed with ECL chemiluminescence reagent in a dark room, and the strips were scanned and analyzed by Alpha-view software.

Data Analysis and Statistics

SPSS 22.0 statistical software was used for data analysis. If the measurement data obey a normal distribution, it is expressed as the mean ± SD. One-way analysis of variance was used for comparison between groups. LDS-t-test was used for multiple comparisons. For non-normal distribution or uneven variance, a non-parametric test of multiple independent samples is used. Extended t-test was used for multiple comparisons. P<0.05 indicates that the difference is statistically significant.

Results

Network Pharmacology Analysis

Screening of Active Chemical Components and Acquisition of Targets

Through searching the TCMSP and TCMID database and published literature, a total of 53 active ingredients of traditional Chinese medicine were obtained. Among them, there were 20, 7, 1, 7, 8 and 10 ingredients belonging to Astragalus, Angelica, Gordon Euryale, Alisma, Schisandra, and Rhodiola respectively (see Table 1). There are 3 repeated active ingredients in each traditional Chinese medicine. 571, 106, 21, 9, 27, and 562 targets of effective active compounds of Astragalus, Angelica, Gordon Euryale, Alisma, Schisandra, and Rhodiola were obtained respectively. After merging and deleting duplicate values, a total of 516 targets were obtained.

Table 1 Main Ingredients of Yishen Capsule

Acquisition of Target Genes in Diabetic Nephropathy

Diabetic nephropathy disease targets were collected through GeneCards, OMIM, TTD, DisGeNET and DrugBank databases. After merging and deleting duplicate values, a total of 1324 disease targets were obtained.

PPI Network

Taking the intersection of the diabetic nephropathy targets and the drug action targets of the Yishen capsule, and obtaining a total of 150 intersection targets. And a Venn diagram was drawn based on the above data (see Figure 1A). The intersection targets were submitted to the STRING11.0 platform to obtain the PPI network of the components of the Yishen capsule-diabetic nephropathy disease targets (see Figure 1B). The PPI network was visually analyzed by Cytoscape software (see Figure 1C). Setting the degree value ≥ 2 times the median, and 26 core network proteins were obtained using this as a threshold value to screen out. The core network proteins screened out include STAT3, AGT, TP53, SRC, CAV1, EGFR, CTNNB1, ESR1, STAT1, PTEN, JAK2, AKT1, MAPK1, JUN, TNF, AR, CCND1, FGF2, TLR4, CDH1, MYC, VEGFA, MAPK8, EGF, FOS, HSPB1, and the above target proteins are the core nodes in the PPI network.

Figure 1 Common targets of Yishen capsule ingredients and diabetic nephropathy. (A) Venn diagram. Venn diagram shows the intersection of Yishen Capsule target genes and DN disease target genes, and the overlapping part is the number of intersection target genes. (B) PPI network of the common targets of Yishen Capsule and diabetic nephropathy. (C) PPI protein interaction network diagram. The nodes in the picture represent the common target proteins, and the color depth is positively correlated with the degree value of the node.

GO and KEGG Pathway Enrichment Analysis

GO and KEGG enrichment analysis were performed on the targets of Yishen Capsule in the treatment of diabetic nephropathy. A total of 255 significantly related biological processes were screened, including MAPK cascade regulation, DNA binding transcription factor activity regulation, lipopolysaccharide response, reactive oxygen metabolism and apoptosis signaling pathway (see Figure 2A). A total of 74 significant related cell components were screened, mainly including various cell structures such as membrane rafts, nucleoli, myelin sheaths, endoplasmic reticulum lumen, and extracellular matrix (see Figure 2B). 96 significant related molecular functions were obtained, mainly related to protein tyrosine kinase activity, receptor regulatory activity, kinase binding, protein homodimerization activity, transcription factor binding and integrin binding (see Figure 2C). KEGG enrichment screening resulted in 159 main signal pathways, mainly including AGE-RAGE signal pathway, PI3K-Akt signal pathway, HIF-1 signal pathway, JAK-STAT signal pathway, insulin signal pathway and other signal pathways. Pathways ranking the top 20 was visualized (see Figure 2D).

Figure 2 GO and KEGG pathway enrichment analysis. (A–C) The BP, CC and MF of GO enrichment analysis, respectively. (D) The KEGG signal pathway analysis. The size of the dot indicates the number of enriched genes. The color from blue to red indicates significance. The smaller the p.value, the more significant the difference.

Construction of Components of Yishen Capsule-Targets of Diabetic Nephropathy Disease- Pathway Network Diagram

CytoScape 3.7.2 was used to construct a network diagram of Yishen Capsule components-diabetic nephropathy disease target-pathway network. The network has a total of 586 nodes and 1748 component-target protein-pathway relationships (see Figure 3A). Analyzing network topology parameters through CytoScape’s built-in Network Analyzer to obtain core components and core targets. According to analysis and prediction, Quercetin was the main component of Yishen Capsule in the treatment of diabetic nephropathy. Kaempferol (Kaempferol), gallic acid (Epigallocatechin gallate), and astragaloside IV (Astragaloside IV) were minor components (see Table 2). It was predicted that PTGS2 was the main target of Yishen Capsule in the treatment of diabetic nephropathy, and PTGS1, AR, AKT1, NOS2, and PPARG were the secondary targets (see Table 3). Taking the intersection of the above targets and the core targets of the PPI network and drawing the Venn diagram (see Figure 3B). Ten intersection targets were obtained, namely AR, AKT1, TP53, ESR1, JUN, TNF, MAPK1, EGFR, CCND1, VEGFA.

Table 2 Network Node Characteristic Parameters of the Main Active Ingredients of Yishen Capsule

Table 3 The Characteristic Parameters of the Target Network Node of the Main Active Ingredients of Yishen Capsule

Figure 3 Network diagram and Venn diagram. (A) The inverted triangle node represents the name of the pathway, the octagonal node represents the active ingredient of traditional Chinese medicine, and the diamond node represents the gene. The larger the node area of the traditional Chinese medicine component, the more the number of genes enriched by this component, or the more this component is enriched in the pathway. (B) Venn diagram shows the intersection of the key target genes screened by the drug-disease-pathway network diagram and the key target genes screened by the PPI network.

Verification with Molecular Docking

Selecting the key genes AR, AKT1, TP53 obtained in the previous steps, and the core targets HIF-1α, JAK2, and STAT3 in the key signal pathway, and carrying out molecular docking with the main active ingredients of the Yishen capsule. The docking results were shown in Figure 4A. Among the 60 docking results, 57 suggested a better docking activity (binding energy less than −1.2kcal/mol). Among them, β-sitosterol binded best to the core target protein, and TP53 was the target protein with the best binding activity to the key chemical components in the Yishen capsule.

Figure 4 The molecule docking heat map of the key active compounds in the Yishen capsule and the core target proteins. (A) The vertical axis represents the effective active ingredient of Yishen capsule and the horizontal axis represents the core target protein receptor. Each value represents the total score of molecular docking, and the darker the color, the higher the score. (B) Docking analysis of formononetin with AKT1 receptor. (C) Docking analysis of formononetin with STAT3 receptor. (D) Docking analysis of formononetin with TP53 receptor. (E) Docking analysis of β-sitosterol with AR receptor. (F) Docking analysis of β-sitosterol with TP53 receptor. (G) Docking analysis of Calycosin with HIF-1α receptor. (H) Docking analysis of Calycosin with JAK2 receptor. For all the docking analysis results, the right panel shows the global diagram of the simulated docking of each ligand molecule and the receptor protein. The colored ribbon model, the protein structure. The yellow grid structure, the ligand molecule. The left panel is a partial three-dimensional image of simulated docking. Wheat, small ligand molecules. Green, amino acid residues that have interaction with active molecules. The yellow dashed line, interacting hydrogen bonds. The blue dashed line, Pi-Pi Stacked. The magentas dashed line, Pi-Alkyl interaction.

The docking results with better binding energy was shown in Figure 4. Formononetin formed a hydrogen bond with the AKT1 receptor target protein through the amino acid residue ASN-231 and TYR-229 and formed Pi-Alkyl interaction through the amino acid residue ARG-174 (see Figure 4B). Formononetin formed two hydrogen bonds with the STAT3 receptor target through the amino acid residues GLU-444 and THR-443, formed Pi-Alkyl interaction through the amino acid residue LYS-363 (see Figure 4C). Formononetin formed two hydrogen bonds with the TP53 receptor target through the amino acid residues GLU-221 and THR-224 (see Figure 4D). β-sitosterol formed a hydrogen bond with the AR receptor target protein through the amino acid residue PRO-913, formed Pi-Alkyl interaction through the amino acid residue LYS-912 (see Figure 4E). β-sitosterol binded to the TP53 receptor target protein formed hydrogen bonds through the amino acid residue GLN-100, formed Pi-Alkyl interaction through the amino acid ALA-138 (see Figure 4F). Calycosin binded to the HIF-1α receptor target protein through amino acid residue GLN-96 to form hydrogen bond and through amino acid residue TYR-98 to form Pi-Pi Stacked (see Figure 4G). Calycosin formed two hydrogen bonds through amino acid residues ASP-994 and LEU-855 with the JAK2 receptor target protein, formed Pi-Pi Stacked through amino acid residue PHE-995, and formed Pi-Alkyl interaction through amino acid residue ARG-980 (see Figure 4H). The above-mentioned ligand compounds could be well embedded in the active pocket of the receptor target protein.

In vitro and in vivo Experimental Verification

Pathological Changes of Rat Kidney Tissues

The results of HE staining of kidney tissue specimens (see Figure 5A) showed that the glomerulus in group N was normal, and the structure of mesangium and basement membrane was normal. In the DN group, the glomerular capillary loops were hypertrophy, the basement membrane was thickened, the mesangial cells and mesangial matrix were proliferated, and the renal tubular epithelial cells were hypertrophy. However, there was no glomerular sclerosis or interstitial fibrosis. Compared with the DN group, the YS group had increased glomerular volume, mesangial hyperplasia, basement membrane thickening, and renal tubular epithelial cell hypertrophy to varying degrees. The results of PAS staining (see Figure 5B) showed that the glomeruli and tubules of rats in group N were normal in structure and morphology. PAS-positive protein deposits were seen in the glomerular mesangial area and renal tubular epithelial cells of rats in the DN group. Compared with the DN group, the YS group had less PAS-positive protein deposition. The results of Masson staining (see Figure 5C) showed no abnormal changes in the morphology and structure of the kidney tissue in the normal group, and blue staining of collagen tissue was rarely seen in Masson staining. The glomerulus volume of rats in the DN group increased, the renal tubular epithelial cells were obviously edema, and the lumen became narrow. Masson staining showed blue-stained collagen tissue. Compared with the DN group, the YS group had increased glomerular volume, reduced renal tubular epithelial cell edema, and decreased blue-stained collagen tissue.

Figure 5 Pathological analysis of rat kidney tissues in each group (×100,×200,×400). (A) HE staining of rat kidney tissue. In the DN group, the glomerular capillary loops were hypertrophy, the basement membrane was thickened, the mesangial cells and mesangial matrix were proliferated, and the renal tubular epithelial cells were hypertrophy, while there was no glomerular sclerosis or interstitial fibrosis. Compared with the DN group, the above-mentioned lesions in the YS group were reduced to varying degrees. (B) PAS staining. PAS-positive protein deposits can be seen in the glomerular mesangial area and renal tubular epithelial cells of rats in the DN group (Arrows in the figure). Compared with the DN group, the YS group had less PAS-positive protein deposition. (C) Masson staining. There were no abnormal changes in the morphology and structure of the kidney tissue in the normal group. Masson staining rarely showed blue staining of collagen tissue. The glomerulus volume of rats in the DN group increased, the renal tubular epithelial cells were obviously edema, and the lumen became narrow. Masson staining showed blue-stained collagen tissue (Arrow in the figure). Compared with the DN group, the YS group had increased glomerular volume, reduced renal tubular epithelial cell edema, and decreased blue-stained collagen tissue.

Immunohistochemical Detection of Key Proteins in the Signal Pathway in Rat Kidney Tissue

After 8 weeks of rearing, the kidney tissues of rats in each group were collected for immunohistochemical examination. The results showed that HIF-1α was expressed in the nucleus and cytoplasm of renal tubular cells. There was a certain amount of HIF-1α expression in the kidney tissue of rats in the N group. The expression of HIF-1α in the DN group was higher than that in the N group. Compared with the DN group, the expression of HIF-1α in the YS group was further increased (P<0.05, see Figure 6A). JAK-2 and STAT3 were expressed in the nucleus and cytoplasm of renal tubules. There was a small amount of JAK-2 and STAT3 expression in the kidney tissue of rats in the N group. The expression of JAK-2 and STAT3 in the DN group was higher than that in the N group, while the expression in the YS group was lower than that in the DN group (P<0.05, see Figure 6B and C). The above results indicated that the expressions of HIF-1α, JAK-2 and STAT3 in diabetic nephropathy were elevated. Yishen Capsule could interfere with HIF-1α and JAK/STAT signaling pathways and improved the glomerulus and renal tubular interstitial damage in rats with diabetic nephropathy.

Figure 6 Immunohistochemical detection and quantitative analysis of HIF-1α (A), STAT3 (B) and JAK2 (C) in kidney tissues of rats in each group. The expression of HIF-1α, STAT3 and JAK-2 in the DN group was higher than that in the N group. The expression of HIF-1α was further up-regulated in the YS group (P<0.05). The expression of STAT3 and JAK-2 in the YS group were decreased compared with the DN group (P<0.05). *Compared with the N group P<0.05, **Compared with the DN group P<0.05.

Effects of High Glucose and Yishen Capsule Intervention on Renal Signaling Pathway, Inflammation and Fibrosis Index Expression

Western Blotting indicates that, there was a trace amount of HIF-1α expression in kidney tissue and NRK-52E cells in the normal group. After 24 hours of high glucose stimulation, results showed that the expression of HIF-1α, JAK-2, STAT3, TGF-β and MCP-1 protein in NRK-52E cells and renal tissues all increased compared with the N group (P<0.05). Compared with the DN group, the expression of HIF-1α in kidney tissue and NRK-52E cells increased after the treatment of Yishen capsule (P<0.05), while the expression of JAK2, STAT3, MCP-1 and TGF-β were all decreased (P<0.05). The above results were shown in Figure 7A–D.

Figure 7 Western blotting and quantitative analysis. (A and B) The expression and quantitative analysis of key proteins in NRK-52E cells. After HG stimulation, the expression of HIF-1α, JAK-2, STAT3, TGF-β and MCP-1 protein increased in NRK-52E cells. After Yishen Capsule treatment, compared with the HG group, the expression of HIF-1α in NRK-52E further increased, while the expression of JAK2, STAT3, MCP-1 and TGF-β decreased. (C and D) The expression and quantitative analysis of key proteins in kidney tissue. Compared with the normal group (N), the expression of HIF-1α, JAK-2, STAT3, TGF-β and MCP-1 protein in the kidney tissue of the DN group increased. After treatment with Yishen Capsule, the expression of HIF-1α increased compared with the DN group. Compared with the DN group, the expressions of JAK2, STAT3, MCP-1 and TGF-β all decreased after treatment with Yishen capsule. *Compared with the N group P<0.05, **Compared with the DN group P<0.05.

Discussion

DN has become the leading cause of end-stage renal disease worldwide. At present, the treatment of DN mainly involves reducing cardiovascular risk, controlling blood glucose, controlling blood pressure and inhibiting the renin-angiotensin system (RAS).^24,25 With the in-depth exploration of the pathogenesis of DN, many studies are devoted to finding new drugs to treat DN.²⁶ Over the past few decades, Chinese herbal medicine is emerging as an effective drug candidate for the treatment of diabetes and its complications.⁴ Traditional Chinese medicine has certain advantages in treating DN. Yishen Capsule is composed of Astragalus, Angelica, Gordon Euryale, Alisma, Schisandra, Rhodiola and other Chinese medicines. It possesses the effect of improving the pathological changes of DN kidney tissue and delaying the progression of the disease, while its specific mechanism of action is not completely clear.

This study applied network pharmacology methods to discover the key active ingredients of Yishen Capsule in the treatment of DN. Our results showed that Quercetin, Kaempferol, Epigallocatechin gallate, Astragaloside IV, Luteolin and Calycosin were important components in the treatment of DN, which may act on multiple pharmacology targets of DN treatment. Lei et al found that quercetin inhibits the proliferation of glomerular mesangial cells induced by high glucose through activating the Hippo pathway.²⁷ The study of Alshehri confirmed that kaempferol exerts an antioxidant effect by activating the Nrf-2/Ho-1 axis to reduce DN damage.²⁸ Clinical and animal experiments have confirmed that gallic acid targets the activation of Notch signal by inhibiting the TGFβ/Smad3 pathway in diabetic mice, leading to the improvement of renal fibrosis.²⁹ Astragaloside IV is a saponin extracted from Astragalus. Astragaloside IV reduces endoplasmic reticulum stress, inhibits mitochondrial division and increases autophagy activity through mechanisms of anti-fibrosis, anti-oxidation, and anti-apoptosis, which is benefit to the improvement of DN symptoms.³⁰ Recent studies have found that astragaloside IV may inhibit oxidative stress and reduce DN podocyte apoptosis by activating the PPARγ-Klotho-FoxO1 signaling pathway.³¹ Luteolin, a natural flavonoid compound, may inhibit the activation of STAT3, inhibit inflammation and oxidative stress, and improve glomerular sclerosis and renal interstitial fibrosis in DN mice.³² In addition, mullein, β-sitosterol, formononetin may also act on DN disease targets and signal pathways through a variety of biological processes.

The potential target genes and signal pathways of Yishen capsule in the treatment of DN were predicted based on network pharmacology. Our results showed that AR, AKT1, TP53, ESR1, JUN, TNF, MAPK1, EGFR, CCND1 and VEGFA were the potential key targets for the treatment of DN. AGE-RAGE, PI3K-Akt, HIF-1, JAK-STAT may be the key signaling pathways for the treatment of DN. Some of the above results have been confirmed in the preliminary research of our group. Yishen capsule gavage treatment can reduce urinary protein excretion in DN rats, improve renal function, reduce foot process fusion, improve podocyte Podocin expression, and reduce DN podocyte damage.¹⁰ Treated with Yishen capsule-containing serum, the expression of p-JAK2 and p-STAT3 in high glucose cultured podocytes were decreased.¹¹ These results initially suggest that Yishen Capsule can alleviate the pathological damage of the kidney of DN and improve the podocyte damage induced by high glucose. Cytokine suppressor (SOCS) is a classic JAK/STAT signaling pathway inhibitor.³³ Yishen Capsule may reduce the degree of glomerular sclerosis and tubular interstitial fibrosis in DN rats by up-regulating the expression of SOCS-3.^12,13 In addition, Yishen Capsule can also inhibit the overexpression of TLR4, NF-κB¹⁴ and other inflammatory factors and Vascular Endothelial Growth Factor (VEGF)¹⁵ in the kidney tissue of DN rats. The reduction of inflammatory response can inhibit the damage of vascular epithelial cells caused by proliferative inflammatory response, which leads to the delay of DN progression. Recent research team found that Yishen Capsule has the function of improving diabetic nephropathy by promoting autophagy and inhibiting inflammation.⁹

Based on the preliminary research foundation of our group and the results of this study, HIF-1α and JAK2/STAT3 signaling pathway were selected for further experimental verification. JAK/STAT signaling pathway is involved in the signal transduction of IL-10, IL-6, TNF-α and other cytokines and activates the expression of genes related to inflammation and cell proliferation,³⁴ which promotes the progression of DN. This study suggests that Yishen Capsule may inhibit the downstream inflammation cascade by inhibiting the JAK/STAT signaling pathway, and improve DN kidney injury.

HIF-1α is a regulatory protein that monitors the body’s sensitivity to oxygen.³⁵ Hypoxia exists in the subclinical stage of DN, and hypoxic environment may induce the expression of HIF-1α.³⁶ In addition to hypoxia, factors such as inflammation and stress can also up-regulate the expression of HIF-1α.^37,38 Studies have suggested that enhanced HIF-1α activity is one of the reasons for the dysfunction of podocytes, glomerular mesangial cells, and tubular epithelial cells (RTECs) and for the activation of pro-inflammatory pathways.^39,40 Thus, HIF-1α may be related to glomerulosclerosis and renal interstitial fibrosis. Diabetes hyperglycemia environment and AGEs directly affect the transcription of HIF-1α and lead to HIF-1α activation.⁴¹ Inhibiting the HIF-1α/VEGF signaling pathway in RTECs can reduce the expression of extracellular matrix (ECM) markers in DN.^41,42 However, studies have also found that the diabetic environment activates the atypical proteasome-dependent pathway of HIF-1α degradation in human renal tubular epithelial cells (HK-2), and reduces the basic expression of HIF-1α.⁴³ HIF-1α can promote autophagy, which reduces kidney damage caused by oxidative stress, ischemia and diabetes.^44,45 Conditional knockout of HIF-1α aggravates DN renal tubular damage, promotes mitochondrial rupture in HK-2 cells cultured under hypoxia, promotes ROS generation and promotes mitochondrial membrane potential loss and apoptosis. However, overexpression of HIF-1α or HO- 1 agonist treatment can reverse the above changes.⁴⁶ The hypoglycemic drug SGLT2 inhibitor empagliflozin can reduce the proximal tubular epithelial cell damage induced by high glucose, through up-regulation of HIF-1α.⁴⁷ Under physiological conditions, when the oxygen concentration of the body’s tissues or cells decreases, the expression of HIF-1α and HIF-2α increases, thereby mediating the adaptive response of cells to hypoxia to maintain the body’s homeostasis.⁴⁸ HIF-1α and HIF-2α are important regulatory proteins in the body’s adaptive regulation of hypoxia. Studies have found that both of them can promote autophagy, thereby reducing diabetic kidney damage.⁴⁹ HIF-1α promotes clearance of damaged mitochondria,⁵⁰ whereas HIF-2α promotes lysosomal dysfunction-mediated peroxisomal clearance.⁵¹ The controversial results may be due to the intensity of the HIF response in DN that regulates the progression of kidney injury in a different subtype and time-dependent manner. The results of this study showed that the expression of HIF-1α increased after the intervention of high glucose. And the expression of HIF-1α further increased after the intervention of Yishen Capsule. HIF-1α activation under high glucose conditions may have a protective effect on the kidneys. After the intervention of Yishen Capsule, the expression of HIF-1α was further increased, which may improve the renal injury of diabetic nephropathy by activating autophagy and other mechanisms.⁴⁹

Molecular docking is mainly used to study the interaction between molecules.^52–55 The more stable the binding conformation of the ligand and the receptor, the stronger the binding and the lower the energy.⁵⁶ The screened active ingredients and key targets were verified by molecular docking technology.β-sitosterol binds best to the core target protein, suggesting that it plays the greatest role in the treatment of DN by Yishen capsule. According to the optimal composite structure of key targets and active ingredients, it is found that formononetin has better binding ability with AKT1, STAT3 and TP53. In addition, β-sitosterol has good binding ability with AR and TP53, and mullein has good binding ability with HIF-1α and JAK2. The above data suggest that the key chemical components of Yishen Capsule have good binding activity with key targets of diseases.

Conclusion

This study used bioinformatics to initially analyze the mechanism of action of Yishen capsule in the treatment of DN, and selected key pathways for experimental verification both in vitro and in vivo. Results showed that the identical compound of Yishen Capsule may regulate different targets, and the identical target may interfere with different biological processes and signal pathways. It reflects the combined effect characteristics of multi-pathway and multi-target of Yishen Capsule. This study provides a scientific basis for Yishen Capsule in the treatment of DN, and also provides a new direction for exploring the potential mechanism of Yishen Capsule. Based on the network pharmacology analysis, the key components and core targets of Yishen Capsule were obtained. In the future, the effect of the key monomer components in Yishen Capsule on the core targets and the internal mechanism of the compatibility of traditional Chinese medicine components can be further verified.