This study was supported by the general project of the Natural Science Foundation of Hebei Province, China (No. H2019423037).

Jiawei Shengjiang San to Address Diabetic Nephropathy Using Network Pharmacology

The authors have nothing to disclose.

Our study employed a combination of network pharmacology, molecular docking, and in vivo animal models. A critical step was the establishment of the &#34;drug-component-target&#34; network, which was crucial for identifying the potential mechanisms of JWSJS in treating DN, focusing particularly on its interaction with the EGFR/MAPK3/1 signaling pathway.
During this study, we made several modifications, particularly in the molecular docking process, to enhance the accuracy of our predi...

Diabetes mellitus (DM) is a chronic disease that affects multiple systems and can cause various complications due to continuous hyperglycemia, such as diabetic nephropathy (DN), retinopathy, and neuropathy1. DN is a serious complication of DM, accounting for about 30%-50% of end-stage renal disease (ESRD)2. Its clinical manifestation is microalbuminuria, which can progress to ESRD characterized by increased glomerular volume, mesangial stromal hyperplasia, and thickened glomerular basement membrane3. The pathogenesis of DN is complex and has not been fully elucidated. Clinical methods such as lowering blood glucose, regulating blood pressure, and reducing proteinuria are mostly used to delay its progress, but the effect is general. 
Currently, no specific drug has been found to treat DN4. For centuries, however, Chinese herbal medicines have been widely used in treating DM and its complications5 and have improved patients' clinical symptoms and delayed disease progression. Due to the advantages of multi-component, multi-target, and multi-pathway effects, Chinese herbal medicines are expected to be an innovative drug source for the treatment of DN6.
"Shengjiang san" originated from the "Wanbing Huichun" by the Ming Dynasty medical doctor Gong Tingxian. The book "Neifu Xianfang" describes the use of Bombyx Batryticatus, Cicadae Periostracum, Curcumaelongae Rhizoma, and Radix Rhei et Rhizome. Based on this, after adding Hedysarum Multijugum Maxim, Epimrdii Herba, and Smilacis Glabrae Rhixoma, it exerts the function of shengjiang san of increasing lucidity, decreasing turbidity, releasing stagnant "heat," and harmonizing "qi" and the blood7,8. It also increases the effect of strengthening the spleen and tonifying the kidneys. Its efficacy is consistent with the pathogenesis of DN's "qi" to rise and fall out of order due to deficiency of "vital energy," excessive dryness and "heat," and stagnation of "heat" caused by a triple energizer7,8.
Previous clinical studies have shown Chinese herbal medicines have been used to treat DM and its complications, and jiawei shengjiang san (JWSJS) has been shown to regulate blood glucose and lipids, reduce proteinuria, and significantly improve the clinical efficacy of patients with early DN7. The ability of JWSJS to reduce urinary protein and blood glucose levels in DN rats has been confirmed by previous studies. This probably happens by inhibiting the TXNIP/NLRP3 and RIP1/RIP3/MLKL signaling pathways, reducing podocyte pyroptosis, and preventing necrotic apoptosis in renal tissues of DN rats, thus achieving renal protection9. JWSJS can upregulate nephrin and podocin protein expression and reduce podocyte injury in DN rats, thus suggesting that JWSJS has an inhibitory effect on podocyte injury. JWSJS has a certain anti-DN effect with good safety profiles, but there is little research on it, and this work mostly focuses on pyroptosis and necrotic apoptosis. The literature is not sufficiently deep or systematic10. Our previous findings have confirmed that JWSJS can reduce proteinuria and alleviate kidney damage in DN rats7. However, there are only a few studies on the mechanism of JWSJS for DN treatment, and these lack depth and systematization. Thus, this study aims to analyze the molecular substances and mechanisms of action of JWSJS for DN treatment using network pharmacology and provide a solid foundation for future research.
Network pharmacology is an emerging method to study the mechanism of drug action, including cheminformatics, network biology, bioinformatics, and pharmacology11,12. Network pharmacology research design is quite similar to the holistic concept of traditional Chinese medicine13,14, and it is an important method to study the mechanism of Chinese herbal medicines. Molecular docking can study interactions between molecules and predict their binding patterns and affinity. Molecular docking has emerged as a critical technique in the field of computer-aided drug research15. Therefore, this study constructed a JWSJS-DN-target interaction network through network pharmacology and molecular docking methods that offers a reliable and theoretical basis for further exploration of DN treatment with JWSJS.

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			<td>2&#215;SYBR Green qPCR Master Mix&#160;</td>
			<td>Servicebio, Wuhan, China</td>
			<td>G3320-05</td>
			<td></td>
		</tr>
		<tr>
			<td>24-h urine protein quantification (UTP)</td>
			<td>Nanjing Jiancheng Institute of Biological Engineering</td>
			<td>N/A</td>
			<td></td>
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			<td>3,3&#39;-Diaminobenzidine</td>
			<td>Shanghai Huzheng Biotech, China</td>
			<td>91-95-2</td>
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			<td>Automatic biochemical analysis instrument</td>
			<td>Hitachi, Japan</td>
			<td>7170A</td>
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			<td>Anhydrous Ethanol</td>
			<td>Biosharp, Tianjin, China</td>
			<td>N/A</td>
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			<td>BAX Primary antibodies&#160;</td>
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			<td>AF0120</td>
			<td>Rat</td>
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			<td>BCL-2 Primary antibodies&#160;</td>
			<td>Affinity, USA</td>
			<td>AF6139</td>
			<td>Rat</td>
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			<td>BX53 microscope</td>
			<td>Olympus, Japan</td>
			<td>BX53</td>
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			<td>Chloroform Substitute</td>
			<td>ECOTOP, Guangzhou, China</td>
			<td>ES-8522</td>
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			<td>Desmond software&#160;</td>
			<td>New York, NY, USA</td>
			<td>Release 2019-1</td>
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			<td>Digital Constant Temperature Water Bath</td>
			<td>Changzhou Jintan Liangyou Instrument, China</td>
			<td>DK-8D</td>
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			<td>EGFR Primary antibodies&#160;</td>
			<td>Affinity, USA</td>
			<td>AF6043</td>
			<td>Rat</td>
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			<td>Embed-812 RESIN</td>
			<td>Shell Chemical, USA</td>
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			<td>Fasting blood glucose (FBG)</td>
			<td>Nanjing Jiancheng Institute of Biological Engineering</td>
			<td>N/A</td>
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			<td>FC-type full-wavelength enzyme label analyser</td>
			<td>Multiskan; Thermo, USA</td>
			<td>N/A</td>
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			<td>GAPDH&#160; Primary antibodies&#160;</td>
			<td>Affinity, USA</td>
			<td>AF7021</td>
			<td>Rat</td>
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			<td>Glycated serum protein (GSP)</td>
			<td>Nanjing Jiancheng Institute of Biological Engineering</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Transmission electron microscope</td>
			<td>Hitachi, Japan</td>
			<td>H-7650</td>
			<td></td>
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			<td>Haematoxylin/eosin (HE) staining solution</td>
			<td>Servicebio, USA</td>
			<td>G1003</td>
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			<td>Image-Pro Plus</td>
			<td>MEDIA CYBERNETICS, USA</td>
			<td>N/A</td>
			<td></td>
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			<td>Real-Time PCR Amplification Instrument</td>
			<td>Applied Biosystems, USA</td>
			<td>iQ5&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Irbesartan tablets</td>
			<td>Hangzhou Sanofi Pharmaceuticals</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Isopropanol</td>
			<td>Biosharp, Tianjin, China</td>
			<td>N/A</td>
			<td></td>
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			<td>&#160;JWSJS granules</td>
			<td>Guangdong Yifang Pharmaceutical</td>
			<td>N/A</td>
			<td></td>
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			<td>Kodak Image Station 2000 MM imaging system</td>
			<td>Kodak, USA</td>
			<td>IS2000</td>
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			<td>Low-density cholesterol (LDL-C)</td>
			<td>Nanjing Jiancheng Institute of Biological Engineering</td>
			<td>N/A</td>
			<td></td>
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		<tr>
			<td>MAPK3/1Primary antibodies&#160;</td>
			<td>Affinity, USA</td>
			<td>AF0155</td>
			<td>Rat</td>
		</tr>
		<tr>
			<td>Medical Centrifuge</td>
			<td>Hunan Xiangyi Laboratory Instrument Development, China&#160;</td>
			<td>TGL-16K</td>
			<td></td>
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		<tr>
			<td>Mini trans-blot transfer system</td>
			<td>Bio-Rad, USA</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Mini-PROTEAN electrophoresis system</td>
			<td>Bio-Rad, USA</td>
			<td>N/A</td>
			<td></td>
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			<td>NanoVue Plus Spectrophotometer</td>
			<td>Healthcare Bio-Sciences AB, Sweden</td>
			<td>111765</td>
			<td></td>
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		<tr>
			<td>p-EGFR Primary antibodies&#160;</td>
			<td>Affinity, USA</td>
			<td>AF3044</td>
			<td>Rat</td>
		</tr>
		<tr>
			<td>Periodic acid-Schiff (PAS) staining solution</td>
			<td>Servicebio, USA</td>
			<td>G1008</td>
			<td></td>
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			<td>p-MAPK3/1 Primary antibodies&#160;</td>
			<td>Affinity, USA</td>
			<td>AF1015</td>
			<td>Rat</td>
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			<td>Secondary antibodies</td>
			<td>&#160;Santa Cruz, USA</td>
			<td>sc-2357</td>
			<td>Rabbit</td>
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			<td>Streptozotocin</td>
			<td>Sigma, USA</td>
			<td>S0130</td>
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			<td>SureScript First-Strand cDNA Synthesis Kit</td>
			<td>GeneCopeia, USA</td>
			<td>QP056T</td>
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			<td>TriQuick Reagent</td>
			<td>Solarbio, Beijing, China</td>
			<td>R1100</td>
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			<td>Ultra-Clean Workbench</td>
			<td>Suzhou Purification Equipment, China</td>
			<td>SW-CJ-1F&#160;</td>
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antagonistic effect of jiawei shengjiang san on a rat model of diabetic nephropathy: related to egfr/mapk3/1 signaling pathway

We aimed to delve into the mechanisms underpinning Jiawei Shengjiang San's (JWSJS) action in treating diabetic nephropathy and deploying network pharmacology. Employing network pharmacology and molecular docking techniques, we predicted the active components and targets of JWSJS and constructed a meticulous "drug-component-target" network. Gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) enrichment analyses were utilized to discern the therapeutic pathways and targets of JWSJS. Autodock Vina 1.2.0 was deployed for molecular docking verification, and a 100-ns molecular dynamics simulation was conducted to affirm the docking results, followed by in vivo animal verification. The findings revealed that JWSJS shared 227 intersecting targets with diabetic nephropathy, constructing a protein-protein interaction network topology. KEGG enrichment analysis denoted that JWSJS mitigates diabetic nephropathy by modulating lipids and atherosclerosis, the PI3K-Akt signaling pathway, apoptosis, and the HIF-1 signaling pathway, with mitogen-activated protein kinase 1 (MAPK1), MAPK3, epidermal growth factor receptor (EGFR), and serine/threonine-protein kinase 1 (AKT1) identified as collective targets of multiple pathways. Molecular docking asserted that the core components of JWSJS (quercetin, palmitoleic acid, and luteolin) could stabilize conformation with three pivotal targets (MAPK1, MAPK3, and EGFR) through hydrogen bonding. In vivo examinations indicated notable augmentation in body weight and reductions in glycated serum protein (GSP), low-density lipoprotein cholesterol (LDL-C), uridine triphosphate (UTP), and fasting blood glucose (FBG) levels due to JWSJS. Electron microscopy coupled with hematoxylin and eosin (HE) and Periodic acid-Schiff (PAS) staining highlighted the potential of each treatment group in alleviating kidney damage to diverse extents, exhibiting varied declines in p-EGFR, p-MAPK3/1, and BAX, and increments in BCL-2 expression in the kidney tissues of the treated rats. Conclusively, these insights suggest that the protective efficacy of JWSJS on diabetic nephropathy might be associated with suppressing the activation of the EGFR/MAPK3/1 signaling pathway and alleviating renal cell apoptosis.

Author Spotlight: Network Pharmacology and Molecular Docking to Decipher the Action of Jiawei Shengjiang San Against Diabetic Kidney Disease

Antagonistic Effect of Jiawei Shengjiang San on a Rat Model of Diabetic Nephropathy: Related to EGFR/MAPK3/1 Signaling Pathway

Following the protocol, 90 active ingredients of JWSJS were finally obtained from the analysis after screening and deduplication according to the set standards of OB and DL. These included 20 kinds of Hedysarum Multijugum Maxim, 23 kinds of Epimrdii Herba, 15 kinds of Smilacis Glabrae Rhixoma, 16 kinds of Radix Rhei et Rhizome, four kinds of Curcumaelongae Rhizoma, 15 kinds of Cicadae Periostracum, and six kinds of Bombyx Batryticatus components. Because ther...

Read this Scientific Article Protocol about Antagonistic Effect of Jiawei Shengjiang San on a Rat Model of Diabetic Nephropathy: Related to EGFR/MAPK3/1 Signaling Pathway at JoVE.com

Here, we present a protocol describing network pharmacology and molecular docking techniques to explore the mechanism of action of Jiawei Shengjiang San (JWSJS) in treating diabetic nephropathy.

We aimed to delve into the mechanisms underpinning Jiawei Shengjiang San's (JWSJS) action in treating diabetic nephropathy and deploying network ...

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390 Views.  Hebei University of Chinese Medicine. Here, we present a protocol describing network pharmacology and molecular docking techniques to explore the mechanism of action of Jiawei Shengjiang San (JWSJS) in treating diabetic nephropathy.