Ming Zhao1†，Ying Zhang2†，Jia-Yuan Cai3，Zeng Zhang3，Yan-Ming He3*，Zheng Yao3*1
Shanghai Jing ‘an District Shibei Hospital,Shanghai,200040, Shanghai, China
2The First Affiliated Hospital of Naval Medical University, Shanghai,200082, Shanghai, China
3Yueyang Hospital of Integrated Chinese and Western Medicine, Shanghai University of Traditional Chinese Medicine, Shanghai 200083,Shanghai, China
Equal contribution and first authorship:
Ming Zhao1†，Ying Zhang2†
†These authors contributed equally to this work and share first authorship
Fund：National Natural Science Foundation of China (General Program) （No.82274267）;National Natural Science Foundation of China (General Program) （No.81973818）；National Natural Science Foundation of China (Youth Science Fund Project) （No.82004263）
Corrsponding authors’ email address:
Zheng Yao：yaozheng8848@163.com
Yan-Ming He：heyanming@shyueyanghospital.com
Purpose and Background: Based on previous research, JianPi QingHua formula （JPQH）has been shown to improve insulin resistance and diabetic complications. However, the underlying mechanism is still unclear. In this study, we aim to predict the molecular mechanism of JPQH in treating Diabetic Nephropathy (DN) DN complicated with obesity using a network pharmacology approach and molecular docking.
Method: Use the Traditional Chinese Medicine Systems Pharmacology (TCMSP) database and analysis platform and related literature retrieval to obtain the chemical components of drugs, and use the SwissTargetPrediction database to search for potential effective targets based on the combination. Collect disease targets from the Genecards database, with “Diabetic Nephropathy” and “Obesity” as keywords. Use Venny2.1.0 to draw the disease component targets to obtain potential targets. Use the search tool of the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) database and Cytoscape 3.9.1 to construct the protein-protein interaction (PPI) network. Use the CytoNCA plugin in Cytoscape 3.9.1 to screen the top 20 targets based on degree, combine with the GEO dataset to screen potential hub genes, and perform GO and KEGG enrichment analysis on potential targets using R language. Use Moe to perform molecular docking and molecular dynamics verification on core targets and effective components.The impact of traditional Chinese medicine on HK2 (human renal tubular epithelial cells) and Min6 (mouse insulinoma cells) was confirmed by cell proliferation and apoptosis assays as well as oil red staining.
Result: Based on the databases and literature reports, JPQH contains 36 effective ingredients，which has been screened to identify 134 effective targets, including 100 targets related to the treatment of DN complicated with obesity. PPI network combined with cytoNAD and GEO datasets (GSE142025, GSE152991) were used to screen and identify hub genes closely related to disease treatment, such as MMP9, KDR, ESR2, PIK3CG, PTGS1, and ALOX5. GO enrichment analysis showed that the biological processes involved mainly included positive regulation of kinase activity, response to oxidative stress, and positive regulation of MAP kinase activity. The molecular functions of these proteins include transmembrane receptor protein tyrosine kinase activity, Hsp90 protein binding, oxidoreductase activity, efflux transmembrane transporter activity, and ABC-type xenobiotic transporter activity. The specific sites where genes exert regulatory effects are mainly located in lipid rafts, brush border membrane, and cell basement membrane, all of which are closely related to intercellular communication. KEGG pathway analysis further revealed that the targeted proteins of JPQH are involved in 5-hydroxytryptamine synaptic signaling pathway, ovarian steroidogenesis signaling pathway, calcium signaling pathway, and PI3K-Akt signaling pathway. Molecular docking results showed good binding abilities and stable interactions between proteins and monomeric components, including MMP9, KDR, ESR2, PIK3CG, PTGS1, ALOX5, CYP1B1, CD38, FIT3, HTR2B, INSR, MAOA, and compounds such as deflazacort, berberine, 3′,5,7-trihydroxy-4-methoxyflavone, palmatine, jatrorrhizine, oxyberberine, and baicalin. Hydrogen bonding, pi-pi stacking, and pi-H interactions are the major forms of interactions.Cell proliferation and apoptosis experiment and oil red staining experiment showed that Jianpi Qinghua could improve apoptosis and fat accumulation in model group.
Conclusion: Based on network pharmacology, this study preliminarily predicted that JPQH might treat diabetic nephropathy combined with obesity by regulating inflammation, oxidative stress, glucose and lipid metabolism and fibrotic signaling pathways. To provide reference for follow-up TCM research on the treatment of DN combined with obesity by chemical components.
Key words: Diabetes, Nephropathy, Obesity, Jianpi Qinghua prescription(JPQH), Traditional Chinese medicine,network pharmacology.
Supported by: National Natural Science Foundation of China (No.81973818); National Natural Science Foundation of China Youth Science Foundation Project (No.82004263)
1.Introduction
Diabetic nephropathy (DN) is a microvascular complication with a higher incidence rate among patients with diabetes (DM). The lesions can affect the glomeruli, renal tubules, interstitium, and renal blood vessels, among others. The main clinical features are persistent albuminuria and/or progressively declining estimated glomerular filtration rate (eGFR), which can progress to end-stage renal disease (ESRD) [1]. According to statistics, nearly 40% of DM patients will develop DN, which is the main cause of ESRD[2]. Although enhanced management focusing on simultaneous control of blood glucose, blood pressure, and blood lipid levels can slow down the progression of DN[3], a considerable proportion of patients still progress to end-stage kidney disease (ESKD) over time[4]. Therefore, it is crucial to identify and manage the underlying pathogenic factors for the prevention and treatment of DN.
More and more studies have found that obesity is an independent risk factor for diabetic nephropathy (DN). Patients with obesity are more prone to metabolic diseases such as hyperglycemia, hypertension, and hyperlipidemia. Further research indicates that obesity is related to increased risk of DN and higher risk of occurrence. Higher Body Mass Index（BMI） levels are associated with increased risk of DN and decreased eGFR levels[5]. Ryan confirmed the independent correlation between higher BMI and higher likelihood of DN in their clinical study on Asian T2DM patients[6]. Therefore, controlling obesity is crucial for DN, and currently, this population is also increasing significantly.
The current clinical treatment principles for obesity-related diabetic nephropathy often involve weight loss, correction of lipid metabolism disorders, control of blood sugar and blood pressure, reduction of proteinuria, and kidney protection[7]. There are no clear diagnostic and therapeutic guidelines internationally, and in clinical practice, drugs such as sodium-dependent glucose transporters 2 (SGLT-2) inhibitors, a new type of glucose-lowering medication, and glucagon-like peptide-1 (GLP-1) receptor agonists, a type of insulin injection, are commonly used to control this type of disease. Domestically, SGLT-2 inhibitors can reduce proteinuria levels in patients with diabetic nephropathy[8,9]. GLP-1 receptor agonists have obvious advantages in weight reduction and are the preferred drugs for treating overweight and obese patients with diabetic nephropathy. They have a partial protective effect on the kidneys and can help with weight loss. However, these treatment methods mainly focus on improving hemodynamics and metabolic factors, and there is still a lack of effective targeted drug treatments for the important pathological process of kidney fibrosis. Furthermore, studies have shown that SGLT-2 inhibitors can reduce the risk of increased serum creatinine, progression to massive albuminuria, renal replacement therapy, and renal cause-specific mortality by up to 47% compared to placebo[10]. However, the risks of genitourinary and reproductive system infections, ketoacidosis[11], and other risks associated with these drugs also limit their widespread clinical use. Therefore, finding effective treatment methods for overweight and obese individuals with diabetic nephropathy is a hot and difficult topic in this field, and traditional Chinese medicine has certain advantages in treating this type of disease.
Compared with a single disease, there is limited research on comorbidities. Overall, obesity has a greater impact on human metabolism. Currently, the effectiveness of western medicine treatment is still not ideal. In contrast, traditional Chinese medicine’s characteristics of multi-target, multi-level, multi-axis treatment, as well as the feasibility of comprehensive intervention, make it a key research area. The JPQH is an empirical formula for the clinical treatment of diabetes at the Endocrinology Department of Shuguang Hospital, affiliated to Shanghai University of Traditional Chinese Medicine. This formula is derived from the ancient physician Li Dongyuan’s first formula “Supplement the Spleen and Stomach, Drain the Yin Fire and Elevate the Yang Decoction” in his Pi Wei Lun(《脾胃论》). The refined ingredients (Huangqin，Huanglian，Gegen) reflect his academic thought of “draining the Yin Fire”[12]. This formula mainly uses Huangqin and Huanglian as the main ingredients draining the excess, with Gegen as an adjuvant medicine, taking into account its meaning of “elevating Yang and reducing Fire”. The modified JPQH is based on this formula. Previous studies have shown that it can reduce oxidative stress, alleviate insulin resistance[13], improve endothelial function[14] and reduce blood glucose fluctuations[15], all of which are closely related to weight loss. The formula combines the medicinal group of Huangqi and Dangshen, which benefit the spleen and calm the temperament, with the medicinal group of Huangqin and Huanglian, which clear heat from the spleen and nourish the spleen yin. Additionally, it includes the medicinal group of Shanyao and Huangjing, which promote the circulation of the spleen and relieve blood stagnation. By organically combining various treatment methods, it better suits the clinical prevention and treatment of type 2 diabetes, effectively tonifying qi, nourishing yin, invigorating the spleen, clearing heat, promoting blood circulation, and lowering blood glucose. This reflects the traditional Chinese medicine principle of “treating diabetes from the perspective of the spleen” and the treatment theory of “tonifying the spleen qi and reducing yin fire”.DN complicated with obesity is closely related to oxidative stress, inflammatory factors, and lipid metabolism. Severe and persistent obesity can exacerbate DN, accelerate disease progression, and increase mortality. The purpose of this study is to preliminarily predict the mechanism of the traditional Chinese medicine formula JPQH in treating diabetes nephropathy combined with obesity from the perspective of network pharmacology. Figure 1 illustrates the overall design of this study.
Fig1 Research process of JPQH in treating DN complicated with obesity.
2.Materials and methods
- Collecting and screening disease targets
Using the keywords “diabetic nephropathy” and “obesity”, search the Therapeutic Target Database (TTD) (http://db.idrblab.net/ttd/), the Genecards database (https://www.genecards.org/), and the Ensembl database (https://uswest.ensembl.org/index.html) for information.
- Collection and screening of chemical components of JPQH
The chemical composition of the JPQH is derived from the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP) (http://tcmspw.com/tcmsp.php).
- Screening and prediction of potential targets for the effects of JPQH ingredients
There are a total of 8 Chinese herbal ingredients in JPQH, including Huangqi（Astragalus）15g, Dangshen（Codonopsis）15g, Shanyao（Dioscorea） 15g, Hungjing（Polygonatum）15g,Huanglian（Coptis）3g, Huangqin（Scutellaria） 9g, Gegen（Pueraria ）15g, and Weimao（Oldenlandia 15g）. The TCMSP database (TCMSP: http://lsp.nwu.edu.cn/tcmsp.php) was used to retrieve the Chinese herbal ingredients of JPQH. In this study, bioactive compounds were retrieved with oral bioavailability (OB) ≥ 30 and drug-likeness (DL) ≥ 0.18[16], and the corresponding chemical structure was obtained from the PubChem database (https://pubchem.ncbi.nlm.nih.gov). The relevant target of the components of JPQH (≥0.05) was collected from the SwissTargetPrediction database (http://swisstargetprediction.ch/).
- common targets for screening diseases and JPQH.
Collect disease targets from the Genecards database, with “diabetic kidney disease” and “obesity” as keywords. Use Venny2.1.0 (https://bioinfogp.cnb.csic.es/tools/venenny/index.html) to plot the overlapping targets of these diseases and obtain potential targets.
- Constructing Traditional Chinese Medicine-Compound-Disease Target Network
Import information about intersecting targets into the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) database (https://string-db.org/). Import a text file consisting of herb, compound, disease, and their shared targets and logical relationships into Cytoscape software. Additionally, import a text file named “network” into Cytoscape software. The size of the resulting nodes will reflect the number (degree) of combined targets. Nodes represent drugs, compounds, diseases, and targets, while edges represent their interactions. To identify core targets for treating DN complicated with obesity using a JPQH,construct a protein-protein interaction (PPI) network of composite disease targets using STRING (version 11.0) and gather PPI information from nearly all publicly available sources (https://string-db.org). Define the species as “human” to obtain target interaction information. Import the results into Cytoscape (version 3.7.2) (https://cytoscape.org) and perform statistical calculations using R language to determine the core targets of the composite disease for further analysis. Then, plot the PPI network and a bar graph.
2.6 pathway enrichment analysis
Using R language (clusterProfiler package) to perform GO and KEGG pathway enrichment analysis on genes in order to identify potential target genes for the treatment of obesity combined with diabetic nephropathy. Using Cytoscape 3.9.2 software to visualize the relationship between the top 20 KEGG pathways and the active ingredients and potential target genes of JPQH.
2.7 molecular docking
The molecular docking software MOE is a commonly used tool for verifying the binding stability between core proteins and drug components. To carry out this work, we can use the RCSB Protein Data Bank (PDB) database (http://www.rcsb.org/) and the DrugBank database (https://www.drugbank.com/) to screen the structures of target proteins as receptors and select a series of drug ligands as positive controls based on these structures. At the same time, to obtain the 2D chemical structures of traditional Chinese medicine components, we can retrieve them from the PubChem database (https://pubchem.ncbi.nlm.nih.gov/). By using the MOE software for molecular docking experiments, we can obtain scores for the binding between different ligands and target proteins. These scores can reflect the degree of binding stability, with higher scores indicating more stable binding. Through this method, we can evaluate the performance of different drug ligands in binding with the target protein, thus finding potential drug candidates.
2.8 Cell culture and drug preparation
Human renal tubular epithelial cells (HK2) and mouse islet tumor cell lines were purchased from Cell Resource Center, Chinese Academy of Sciences (Shanghai, China). HK2 cells were cultured in MEM high-glucose medium (Gibco; Biyuntian, China) and 10% fetal bovine Serum (FBS) (Gibco; Blue sky, China), in 37 present 5% carbon dioxide °C. Jianpi Qinghua prescription purchased from. Oleic acid and palmitic acid were purchased from Sinopharm Group (Sinopharm, China). All drugs were dissolved in dimethyl sulfoxide (DMSO) and stored at -20°C.
2.9 Cell proliferation and apoptosis experiments
Cell counting Kit 8 (CCK-8) (Biyuntian, China) method was used to analyze the apoptosis and proliferation of HK-2 cells and Min6 cells. Both cells were inoculated in 96-well plates at a density of 5×103 cells/well. The cells in the model group were cultured for 48 h in a medium containing 625μmol/L oleic acid and palmitic acid (1:2). The blank control group was cultured in equivolumetric medium for 48 hours. After 24 hours of treatment with JPQH, the two kinds of cells were analyzed by CCK-8 detection according to the manufacturer’s instructions. Add 10 μL CCK-8 solution to each pore. After incubation at 37°C for 2h, the light absorption value at 450 nm was measured with an enzyme label.
2.10 Cell oil red staining experiment
Oil red staining kit (Biyuntian, China) method was used to analyze the improvement of JPQH on Min6 cell DN combined with obesity model. Min6 cells were inoculated in a 6-well plate at a density of 5×104 cells/well, and the cells in the model group were cultured in a medium containing 625μmol/L oleic acid and palmitic acid (1:2) for 48 hours. The blank control group was cultured in equivolumetric medium for 48 hours. After 24 hours of treatment with Jianpi Qinghua formula, the cells were analyzed by oil red staining kit according to the manufacturer’s instructions. Direct analysis under inverted microscope, fluoroscope (magnification, ×40, IX51; Olympus Corporation, Japan). ImageJ (ImageJ 1.53a) image analysis software was used to process the results stained by red, and prism (prism 9.5.0) statistical mapping was used to visualize the statistical analysis results.
2.11 Retrospective analysis of clinical data
Clinical examination and test data of DN patients with obesity in the inpatient Department of Endocrinology of Yueyang Hospital from 2021 to 2023 were collected, and the correlation between the clinical data of patients was analyzed to verify the partial results of this experiment.
2.12 Statistical Methods
Values are expressed as mean ± standard deviation. SPSS 26.0 software was used for data analysis. spearman and person analysis were used to analyze the correlation between the data in each group. A P value <0.05 indicated that the difference was statistically significant.
3.Result
3.1 Screening key targets (Fig2)
Based on the TCMSP database, active compound targets and corresponding targets for JPQH were searched for various chemical components. Using OB ≥ 30% and DL ≥0.18 as screening criteria, a total of 108 compounds were collected, resulting in 8 shared components (Fig. 2A). Through SwissTargetPrediction, a total of 36 active compounds for JPQH were identified. From the collected targets in the database, 134 targets for JPQH, 6853 DN-related targets, and 4956 obesity-related targets were identified. Venny 2.1.0 software was used for network analysis of the components of JPQH, DN, and obesity targets. By cross-interaction, a total of 100 potential targets were obtained (Fig. 2), indicating that JPQH treat DN complicated with obesity through the same mechanism.
Fig.2.Intersection genes collection of DN complicated with obesity&JPQH.(A)Potential targets of JPQH in treating DN complicated with obesity;(B)Drug-component-target map of JPQH in treating DN complicated with obesity;(C)Disease-component-target-pathway map of JPQH in treating DN complicated with obesity，JPQH represents Jianpi Qinghua formula.
3.2 Analysis of cross-target PPI and co-expressed gene networks (Figure 3)
The total of 100 potential targets were imported into the STRING database and combined with Cytoscape 3.9.1 to identify 13 key targets: MMP9, KDR, ESR2, PIK3CG, PTGS1, ALOX5, CYP1B1, CD38, FIT3, HTR2B, INSR, MAOA. The PPI network is shown in Figure 3.
Fig.3. PPI network of JPQH in treating DN complicated with obesity.(A、B)Protein-protein interaction network of the intersection genes( DN complicated with obesity&JPQH) was graphed by string and cytoscape;(C)The central ring represented the core twenty genes(N&T),the darker color represented the higher degree.
3.3 GO and KEGG pathway enrichment analysis (Fig 4)
Further exploration of the mechanism of JPQH method for treating DN complicated with obesity reveals its involvement in various biological processes. These include the positive regulation of kinase activity, response to oxidative stress, and positive modulation of MAP kinase activity. The molecular functions of these proteins include transmembrane receptor protein tyrosine kinase activity, Hsp90 protein binding, oxidoreductase activity, efflux transmembrane transporter activity, and ABC-type xenobiotic transporter activity. The specific sites where genes are regulated are mainly located in lipid rafts, brush border membranes, and the basal membrane, all of which are closely related to intercellular signaling. KEGG pathway analysis further revealed that the targeted proteins of JPQH are involved in serotonergic synapse signaling pathway, ovarian steroidogenesis signaling pathway, calcium signaling pathway, and PI3K-Akt signaling pathway (Fig 4).
Comparing GO and KEGG analysis results, it is possible that the genes encoding the targeted proteins for JPQH may have functions in regulating the 5-hydroxytryptamine synaptic signaling pathway, ovarian steroid synthesis signaling pathway, calcium signaling pathway, and PI3K-Akt signaling pathway. These pathways involve the regulation of neurotransmitter transmission and metabolism-related transcription factor activity at synapses when the organism’s cells are stimulated by external and endogenous factors, including lipotoxicity, oxidative stress, and inflammatory factors.
Fig.4.(A)KEGG enrichment of intersection genes was graphed by R-studio.The top fifteen results KEGG enrichment was shown;(B)GO functional enrichment analysis of potential targets of JPQH in treating DN completed with obesity.BP represents biological process; CC represents cell composition; MF represents molecular function.
3.4 Molecular Docking Results (Fig 5)
Through molecular docking analysis, four interactions were selected that exhibited lower Ki values and strong binding affinity. These interactions demonstrated excellent bonding capabilities and stability between the proteins and monomeric components. These proteins include MMP9, KDR, ESR2, PIK3CG, PTGS1, ALOX5, CYP1B1, CD38, FIT3, HTR2B, INSR, MAOA, and dexamethasone fluoroalkane acid.Furthermore, specific compounds present in the herbal formula were identified as important contributors to the binding interactions. These compounds, namely berberine, 3′,5,7-trihydroxy-4-methoxyflavone, berberine, palmatine, hydroberberine (Corydalis yanhusuo), and wogonin, formed strong interactions with the selected proteins. In particular, they exhibited hydrogen bonding, pi-pi stacking, and pi-H interactions as the main forms of interaction.For instance, the hydroxyl group of dexamethasone fluoroalkane acid in the herbal formula’s effective ingredients was found to form a hydrogen bond with the protein target. Additionally, its aromatic and benzene rings were seen to participate in pi-pi stacking interactions, further enhancing the overall binding affinity between the compound and protein target. These findings provide a visual explanation of the molecular interactions underlying the therapeutic effects of JPQH in the context of DN complicated with obesity (Figure 5).
Fig.5.Docking results of key targets and core components of traditional Chinese medicine.(A)Docking results of MMP9 with diflucortolone valerate；(B)Docking results of MMP9 with (Rac)-Hesperetin;(C)Docking results of MMP9 with wogonin;(D)Docking results of MMP9 with Epiberberine;(E)Docking results of INSR with Epiberberine;(F)Docking results of INSR with Palmatine;(G)Docking results of CYP1BI with Palmatine;(H)Docking results of CYP1BI with diflucortolone valerate;((I)Docking results of ALOX5 with diflucortolone valerate.
3.5 Apoptosis and proliferation experiments
After treatment with JPQH, the apoptosis and proliferation of HK2 cells and Min6 cells were detected by CCK-8 method. As shown in Figure 6, after 24 h of intervention, compared with the model group and the blank control group, JPQH significantly improved the apoptosis of the two kinds of cells and promoted cell proliferation.
3.6 Cell oil red staining experiment
After 24h treatment with JPQH, compared with the control group and the model group, JPQH significantly improved the steatosis of Min6 cells (Fig. 6)
Fig.6.(A) Effects of JPQH on proliferation and apoptosis of HK2 and Min6. *, P < 0.05;(B)The effects of JPQH traditional Chinese medicine components on the min6 cells of DN complicated with obesity model.*, P < 0.05.
3.7 Retrospective analysis of clinical data
In clinical practice, we collected endothelial function and related metabolic indexes of 46 patients with diabetic nephropathy combined with obesity from inpatients of Yueyang Hospital of Integrated Traditional Chinese and Western Medicine Affiliated to Shanghai University of Traditional Chinese Medicine, and found that endothelial function of these patients was significantly correlated with glycated hemoglobin and postmeal 2hC peptide (P < 0.05). Table 1-2 shows the results.
Table 1
Table2 Clinical correlation between endothelial index and HbA1C and C-Peptide 2h parameters in 46 diabetic nephropathy patients complicated with obesity
|
|
|
R |
P |
|
endothelial index |
HbA1C（person） |
.348* |
.024 |
|
|
C-Peptide 2h（spearman） |
-.309* |
.047 |
*, P < 0.05.
4.Discussion
Diabetic nephropathy is a modern medical term, which was not used by the ancients. It can be referred to as traditional Chinese medicine’s secondary edema, Guange (a condition characterized by lower limb swelling), and xulao (general fatigue). Huangdi Neijing （《黄帝内经》）already discussed “water,” “wind-water,” “water swelling,” and “stone water.” The Suwen Shuirre Xue Lun（《素问–水热穴论》） states: “The kidney is the gate of the stomach, and when the gate is obstructed, water accumulates and causes similar symptoms.” “The injury of the five viscera will ultimately lead to kidney damage.” With prolonged polyuria and thirst, the kidney Qi becomes deficient and unable to steam and transform fluid. As a result, fluid retention occurs and eventually leads to edema. Guange is considered a critical condition in traditional Chinese medicine, and it is often seen in the late stage of diseases such as edema, urinary retention, and urinary disorders. Zhengzhi Huibu（《证治汇补》） describes Guange as follows: “If the pulse is obstructed and bound, there will be urine retention. Within a day or night, sudden increase in vomiting and nausea will occur. This is due to the stagnation and obstruction of turbid pathogens, which inhibits the ascending and descending movement of Triple Burner Qi…” Chronic renal function decline caused by the urinary system is also related to this condition, and the treatment of DN can learn from this. Obesity is a chronic metabolic disease characterized by excessive accumulation or abnormal distribution of body fat and weight gain. In Chinese medicine, it is referred to as “fei ren” (fat person) or “fei gui ren” . It usually falls under the category of “fei ren” .
With the rapid development of the current economy and society, the improvement of people’s living standards, the global prevalence of obesity combined with DN is gradually increasing. Traditional Chinese medicine believes that the pathogenesis of DN complicated with obesity is excessive consumption of fatty and sweet foods, inadequate spleen transportation and transformation, accumulation of dampness and phlegm, the formation of greasy and turbid phlegm in the body, leading to stagnation of qi, blood stasis, and internal heat. This further damages the spleen and stomach, causing deficiency of spleen yang and impaired spleen function. The disease changes from excess to deficiency, and in the long run, it affects the spleen and kidneys. Therefore, the fundamental treatment principle is to invigorate the spleen and replenish qi. JPQH is the experience formula for the clinical treatment of diabetes in the Endocrinology Department of Shuguang Hospital Affiliated to Shanghai University of Traditional Chinese Medicine. After using JPQH granules clinically, patients experienced varying degrees of decreased appetite. Previous studies confirmed that this formula can effectively improve insulin resistance in diabetic rats and diabetic patients. Chronic inflammation is the fundamental cause of insulin resistance, and its mechanism may be related to the regulation of intestinal immune cells, reducing the ratio of Th1, Th2, and Th17 cells in the intestinal tissue and increasing the ratio of Treg cells[17].Huangqi contains astragalosides, astragalus polysaccharides, astragalus saponins, amino acids, and various trace elements. This study explores the potential therapeutic targets of JPQH in the treatment of obesity combined with diabetes mellitus.
In clinical studies, it has been found that the visceral adiposity index is closely related to insulin resistance[18]. Insulin resistance further leads to endothelial dysfunction[19], oxidative stress[20], and inflammatory state, promoting the development of microvascular lesions. Podocytes are insulin-sensitive renal cells, so insulin resistance is more likely to cause kidney damage[21]. Research has shown that the release of free fatty acids from visceral adipose tissue can increase the secretion of pro-inflammatory factors such as TNF-α and IL-6, leading to endothelial dysfunction in the glomerulus and increased urinary albumin excretion[22].
To further explore the specificity binding sites of JPQH in exerting the above effects, we conducted molecular docking verification. Generally, the structure of molecular docking is mainly evaluated by the minimum binding free energy[23]. The lower the binding energy, the less energy is required for the drug molecule to bind to the target, indicating a higher possibility and stability of binding[24]. Through molecular docking, we found JPQH can act on the gene expressions of MMP9, KDR, ESR2, PIK3CG, PTGS1, ALOX5, CYP1B1, CD38, FIT3, HTR2B, INSR, MAOA in DN complicated with obesity.
4.1 Potential targets
4.1.1 MMP9 regulates fibrosis and improves DN
MMP9 is a member of the matrix metalloproteinase (MMP) family. Studies have shown significant elevation of MMP9 in the urine, serum, and renal tissues of patients with DN. Moreover, the upregulation of urinary MMP9 concentration occurs earlier than the occurrence of microalbuminuria, and the serum levels of MMP9 are negatively correlated with estimated glomerular filtration rate (eGFR) [25]. Research has confirmed that diabetes can induce the expression and activity of MMP9 in diabetic mice, and knocking out MMP9 can alleviate proteinuria, reduce glomerular filtration rate, and decrease glomerular basement membrane thickness associated with DN[26]. Furthermore, MMP9 is a member of the zinc-dependent enzyme family MMPs[27]. Among MMPs, MMP9 has been proven to be associated with DN, and certain drugs can reduce the inflammatory response caused by DN by attenuating the expression of high glucose-induced MMP9 and relieving extracellular matrix accumulation[28,29], as well as inhibiting the synthesis of fibrogenic enzymes to alleviate renal fibrosis caused by DN[30,31]. Zhang revealed that long non-coding RNA growth arrest-specific transcript 5 (GAS5) downregulates the expression of MMP9 by recruiting EZH2 to the MMP9 promoter region, thus alleviating the progression of renal fibrosis in DN rats[32]. In addition, Fan found through experiments that the renal protective effect against diabetic nephropathy in mice may occur through the MMP-9/NF-κB signaling pathway[33], and MMP9 has also been identified as a potential therapeutic target for treating DN-related renal cell carcinoma[34]. On the other hand, abnormal expression of MMP9 is associated with epithelial-mesenchymal transformation (EMT) in podocytes, a process related to DN. High glucose induces podocyte EMT through demethylation-mediated MMP9 expression[35].
4.1.2 MMP and ALOX5 improve DN by intervening in oxidative stress and inflammation
The concentration of MMP9 in the saliva of obese women is significantly higher than that of women with normal weight[36]. Furthermore, it further induces insulin resistance. Related research results also indicate that antacid drugs can also change the oxidative and vascular remodeling responses of overweight and obese men to psychological stress by reducing MMP9 activity[37]. 5-lipoxygenase (5-LOX, ALOX5) is an iron-containing non-heme dioxygenase that catalyzes the peroxidation of polyunsaturated fatty acids such as arachidonic acid. ALOX5 is the rate-limiting enzyme in leukotriene biosynthesis. Leukotrienes are a family of proinflammatory lipid mediators derived from arachidonic acid. ALOX5 can regulate cell death in two ways: inflammation and lipid peroxidation. Furthermore, it has been discovered through pharmacological research on JPQH that ALOX5 plays a key role in iron death in renal tubulointerstitial injury in DN[38]. ALOX5 may be involved in the molecular mechanism of iron death in DN patients through metabolic pathways and immune-inflammatory response mechanisms. Studies have found that increased expression of ALOX5 can have an antioxidant effect on cardiovascular and cardiac tissues[39]. Recent research has found that interfering with the 5-LOX gene family can alleviate high-glucose-induced glomerular mesangial cell inflammation and fibrosis[40].
4.1.3 ESR2, INSR, CYP1B1 and KDR regulate the body’s glucose and lipid metabolism
Another estrogen-related gene is ESR2. In a mouse study, it was found that the addition of estrogen receptors ESR1 and ESR2 in a single extract can effectively improve the abnormal lipid metabolism caused by obesity[41]. Krz et al found that the visceral fat of obese study participants was significantly lower than that of other participants. The level of ESR2 gene was significantly lower than that of normal body weight[42], and the related gene dependent down-regulation of ESR2 expression in visceral fat promoted metabolic inflammation and further aggravated obesity[43].
Insulin is involved in the regulation of energy and lipid metabolism by activating the intracellular signaling cascade of insulin receptor (INSR), insulin receptor substrate (IRS) protein, phosphoinositol-3-kinase (PI3K) and protein kinase B (AKT). Insulin regulates adipose tissue development and function and stimulates the differentiation process of adipose cells. Recent studies have found that different distributions of INSR and changes in the InSR-A: InSR-B ratio may lead to insulin resistance and metabolic abnormalities in the development of type 2 diabetes[44]. Blood glucose control is crucial for the prevention and treatment of diabetic nephropathy. High extracellular glucose induces intracellular reactive oxygen species to initiate podocyte apoptosis[45], which is a key feature of diabetic glomerular injury in vitro and in vivo. The change of insulin receptor is the key to insulin action, an important intermediary between extracellular and intracellular insulin signaling pathways, and is crucial for insulin action[46], which is involved in adipogenesis and β-cell insulin secretion, and may also be involved in type 2 diabetes[47]. In addition, the expression of INSR in the kidney changes during diabetes, suggesting that INSR is a factor promoting the occurrence and development of DN[48].
Visceral adipose tissue is the main site of estrogen production, and CYP1B1 is highly expressed in adipose tissue. Studies have shown that inhibiting the expression of CYP1B1 in adipose tissue causes changes in estrogen metabolism, which may be beneficial to obese patients[49]. CYP1B1 has not only been found to participate in the metabolism of endogenous compounds, such as steroid hormones and lipids, and regulate the metabolism, accumulation and distribution of fats[50], but also the arachidonic acid metabolized by CYP1B1 is the main source of fatty acids that constitute glycerophospholipids in cell membranes, which can eventually lead to obesity and insulin resistance[51]. Related to this, compared with wild-type animals, high-fat diet is glucose intolerant to transgenic CYP1B1 deficient mice and can ameliorate obesity to a certain extent[52].
The activity of MAOA in visceral adipose tissue of obese patients is higher than that of non-obese patients, and it can catalyze oxidative deamination of norepinephrine, and is expected to affect thermogenesis by inhibiting adrenergic tension[53,54], that is, inhibition of MAOA may induce Browning of human adipocytes to a certain extent[55], and another study found that, Electroacupuncture stimulation can reduce the expression of MAOA and inhibit the degradation of norepinephrine to achieve weight loss and chronic inflammatory damage caused by obesity[56]. Moreover, studies[57] found that baroreceptors can prevent heart remodeling in obese rats by inhibiting MAOA-mediated oxidative stress.
KDR is an endothelial cell marker vascular endothelial growth factor receptor 2(VEGFR2)[58], which has a high diagnostic certainty in the diagnosis of DN[59]. Narisa[60] analyzed the failure of renal function recovery during microalbuminuria or macroalbuminuria treatment in late DN by simulating the mechanism of vascular homeostasis, and found that it may be related to the increase of anti-angiogenic factors such as angiopoietin-2 and KDR. Moreover, studies have found that one of the key roles in the process of fat accumulation caused by obesity is the expression and release of angiogenic factor VEGF by fat cells, while KDR and VEGF-R1 are expressed in the vascular endothelium[61].
ABCG2 is an effector uric acid transporter. Studies have shown that ABCG2 is related to uric acid and has the strongest correlation among most genes, and it is expressed in the proximal tubule and intestine of the kidney[62,63]. Meanwhile, the interaction between the variation of ABCG2 gene and BMI will increase the risk of renal dysfunction in patients[64]. The Breast Cancer Resistance Protein (BCRP) encoded by ABCG2 gene is involved in the transport of endogenous substances including sugars, amino acids, lipids and steroids such as cortisol[65]. Studies have shown that the expression level of ABCG2 gene in mice with a high-fat diet is significantly higher than that in mice with a low-fat diet[66].
In addition, for some genes, relevant studies have found that upregulation of PTGS1 and related genes can enhance antioxidant effects to protect pancreatic dysfunction[67]. Won[68]demonstrated through studies that HTR2B(serotonin receptor 2B) inhibits serotonin signaling and reduces lipolysis in visceral fat cells to improve insulin resistance.
4.2 Signal path analysis
Enrichment analysis suggests that the main role of JPQH is involved in regulating signaling pathways related to ovarian steroidogenesis, PI3K-Akt signaling pathway, calcium signaling pathway, and 5-hydroxytryptamine synaptic signaling pathway. The ovarian steroidogenesis signaling pathway mainly involves the genes CYP1B1, ALOX5, and INSR. ALOX5 plays a crucial role in regulating the conversion of cholesterol to androgenone and testosterone in granulosa cells, while CYP1B1 plays a key role in regulating the conversion of estradiol to 4-OHE2 in ovarian granulosa cells. INSR is distributed on the surface of follicular membrane cells and ovarian granulosa cells, and INSR is regulated by insulin, thereby regulating the biological processes involving ALOX5 and CYP1B1. Obesity can lead to disruption of steroidogenesis in the ovaries, causing problems in the generation and secretion of sex hormones. The estrogen action in pancreatic cells also regulates insulin secretion, nutrient homeostasis, and survival[69]. Lack of estrogen promotes metabolic disorders and can protect the kidneys by regulating the renin-angiotensin system (RAS), nitric oxide (NO) levels, antioxidant effects[70]. If estrogen conversion is affected, it will further impact the kidneys. Changes in estrogen directly or indirectly affect the action of insulin and endocrine homeostasis, thus affecting the development of DN with obesity. Our study shows that some monomeric components of JPQH can effectively bind to key target proteins involved in this pathway, thereby effectively regulating metabolic disorders. Therefore, the regulation of DN and obesity by JPQH is closely related.
The related component targets of JPQH are involved in the regulation of the core pathogenic genes and pathways of DN complicated with obesity. The PI3K-Akt pathway is closely related to the occurrence and development of DN. PI3K and AKT are extremely critical insulin signaling molecules in the body, and play a significant role in the regulation of glucose and lipid metabolism in the body. Studies have shown that insulin mainly achieves its physiological functions by activating the insulin receptor substrate 2(IRS-2)/PI3K/Akt signal transduction pathway. After insulin binds to its receptor in the body, insulin receptor tyrosine kinase is activated and causes IRS-2 phosphorylation, and downstream PI3K can be activated by phosphorylated IRS-2 to start the PI3K/Akt signal transduction pathway[71]. Studies have shown that Akt activation, to adjust the glucose transporters – 4 (glucose from 4, GLUT – 4), GSK – 3 beta, fork frame protein O1 (forkhead box protein O1, The activities of FoxO1) and mTOR regulate the body’s glucose and lipid metabolism, autophagy and apoptosis, inflammation and oxidative stress levels, and thus participate in the occurrence and development of DN and drug therapy. The core targets of these pathways are related to the active ingredients of JPQH, and play an important role in the treatment of DN with obesity.
4.3 Molecular docking
In this study, 134 potential drug targets, 100 overlapping targets and 7 core compounds (epimerberine, diflumethasone valerate, 3 ‘,5, 7-trihydroxy-4-methoxy-flavonoids, berberine, palmitine, hydrogenated berberine (fumarate), and baicalin were identified.)
4.3.1 Epiberberine
Epiberberine (EPI), extracted from wheat roots, has been shown to reduce symptoms of hyperlipidemia in vivo, and in HepG2 cells and the livers of McD-fed mice, EPI increased SHP levels, accompanied by a reduction in sterol-regulatory element binding protein-1C (SREBP-1c) and FASN. Therefore, EPI reduces the accumulation of triglycerides (TG) in steatogenic hepatocytes and even improves the microbiome in HepG2 cells treated with siRNA-SHP[72]. Berberine (BBR) is an alkaloid in Coptis chinensis that has been reported to improve non-alcoholic fatty liver disease (NAFLD). In fact, BBR may regulate NAFLD/NASH by activating the AMPK pathway, enhancing phosphorylation of AMPK(Thr172)[73,74], and may reduce lipid accumulation and bile acids through the gut microbiome[75]. BBR is also thought to regulate insulin sensitivity and glucose metabolism or mitochondrial function, and reduce inflammation and oxidative stress[76–83]. Our study also found that monomers such as EPI and BBR can closely bind to key protein gene targets of DN associated with obesity, such as ALOX5, CD38, CYP1B1, FIT3, INSR, KDR, MMP9, PIK3CG, and play a certain regulatory role. Existing studies have shown that epimerberine is a low-toxic multi-target small molecule, which exerts various activities such as anti-lipogenesis through Akt and ERK pathways, anti-cancer and anti-bacterial effects through p53/Bax apoptosis pathway, and ameliorates diabetes mellitus and Alzheimer’s disease[84].
4.3.2 Hesperetin
Hesperetin(Hst) is a citrus flavonoid that has gained increasing interest in scientific research in recent years due to its properties in fighting diabetes and its complications, Naila Rabbani et al. found through HATFF study that the treatment of overweight and obese subjects with tRES-HESP(trans-resveratrol combined with 3 ‘,5, 7-trihydroxy-4-methoxy-flavonoids) reversed insulin resistance and reduced abnormal blood sugar, blood pressure, vascular inflammation and lipid disorders[85]. In clinical evaluation[86,87], tRES and HESP did not reverse insulin resistance through Tres-HESP alone, suggesting that Tres-HESP has a pharmacological synergistic effect. Doses of HESP may be sufficient to inhibit intestinal glucuronidase and promote uptake of uncoupled tRES and HESP[88]. The results of the HATFF study may provide a pointer to effective synergistic agents for dietary polyphenol combinations to combat insulin resistance. Alemeh Taheri et al. found that HST could reduce the expression of adipose tissue fibrosis markers in preadipocytes[89]. Other studies have shown that HST improves diabetes-related anxiety and depression in rats by activating the Nrf2/ARE pathway[90].
4.3.3 Palmitine antioxidant
It has been reported that palmitine has antioxidant, anti-diabetic and hypoglycemic properties both in vitro and in vivo[91], which may enable palmitine to up-regulate IRS1 gene[92]. Antioxidants interact with active free radicals such as oxygen and nitrogen to neutralize their oxidative damage. Neutralization of antioxidants involves disrupting the oxidative cascade reactions in cell membranes to fine-tune the levels of free radicals.
4.3.4 wogonin
Xue-qi Liu[93] demonstrated that wogonin reduced the damage of MPC5 cells induced by high glucose (HG) in a dose-dependent manner, while increasing the expression of WT-1, renin, and podoprotein, and decreasing the expression of TNF-α, MCP-1, IL-1β, and phosphorylated p65. In addition, wogonin treatment significantly inhibited HG-induced apoptosis of MPC5 cells, reversed HG-inhibited autophagy in MPC5 cells, significantly alleviated proteinuria, histopathological lesions and p65 NF-κB mediated nephritis inflammatory response, and dose-dependent inhibited STZ-induced podocytic apoptosis in diabetic mice, promoting podocytic autophagy. This study is the first to demonstrate a novel effect of wogonin in alleviating glomerular disease and podocyte damage by modulating the crosstalk between bcl-2 mediated autophagy and apoptosis. However, Lei Lei[94] found that wogonin alleviated renal tubule interstitial fibrosis and tubular cell injury by regulating autophagy and inflammation mediated by PI3K/Akt/NF-κB signaling pathway.
4.conclusion
Based on the complex mechanism of action of JPQH, the author will conduct the follow-up study of experimental verification from the aspects of material basis of the JianPi QingHua formula-pharmacodynamic evaluation-metabolomics pathway verification.
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