Molecular Mechanism of Dahuang Tangluo (大黄糖络) Pill in Improving Insulin Resistance of Skeletal Muscle in Type 2 Diabetes Mellitus Based on RXRA/TNF-α/GLUT4 Pathway
Objective:To predict the molecular mechanism of Dahuang Tangluo (大黄糖络) Pill in improving insulin resistance in skeletal muscle of type 2 diabetes mellitus (T2DM-IRSM) based on network pharmacology and molecular docking technology and perform animal experiment verification.Methods:The network pharmacology and molecular docking technology were used to predict the possible targets of Dahuang Tan-gluo Pill in the intervention of T2DM-IRSM,and molecular biological technology was used to verify the relevant targets.A total of 30 SPF 9-week-old male ZDF (fa/fa) rats were selected for the experiment.After adaptive feeding for one week,a 5008 feed was used to induce a 3-week replication model.After modeling,rats were randomly divided into a model control group,a rosiglitazone group of 0.36 mg/kg,and Dahuang Tangluo Pill groups of 2.16,1.08,and 0.54 g/kg.Additionally,six 9-week-old SPF male ZDF (fa/+) rats were selected as the normal control group.The intervention was conducted for 12 weeks.Body mass and fasting blood glucose (FBG) were detected every two weeks.After completion,serum glucose (GLU) was measured using a fully automated biochemical analyzer.Serum fasting insulin (FINS) was measured by Elisa,and the steady-state model insulin resistance index (HOMA-IR) and insulin sensitivity index (ISI) were calculated.HE staining method was used to observe the pathological changes in skeletal muscle morphology.Result:The PPI network interaction analysis in network pharmacology prediction results revealed that tumor necrosis factor (TNF),glucose transporter type 4 (SLC2A4,also known as GLUT4),and retinoid X receptor α RXRA were key targets in the intersection of drugs and disease.Quercetin,baicalin,stigmasterol,and (2R)-7-hydroxy-5-methoxy-2-phenylbenzodihydropyran-4-one were key active ingredients of drugs.The biological processes (BP) in GO en-richment analysis results included activation of adrenergic receptor signaling pathway by adenylate cyclase,adrenergic receptor signaling path-way,and chemical signal-mediated systemic arterial blood pressure regulation.Cell composition (CC) included membrane rafts,membrane microregions,and membrane regions.Molecular functions (MFs) included G protein coupled amine receptor activity,nuclear receptor activi-ty,ligand activated transcription factor activity,etc.The key pathways involved in KEGG enrichment analysis included adipocyte cytokine sig-naling pathways,saliva secretion,adrenergic signals in myocardial cells,cGMP-PKG signaling pathway,and chemical carcinogenesis-receptor activation.The molecular docking results indicated that quercetin,baicalin,stigmasterol,and(2R)-7-hydroxy-5-met-hoxy-2-phenylbenzodihy-dropyran-4-one could bind well with TNF-α,GLUT4,and RXR-A targets,and the above targets were mainly enriched in RXRA/TNF-α/GLUT4 pathway.The results of animal experiments showed that compared with the normal control group,the body weight,GLU,FBG,FINS,and HOMA-IR of the model control group were significantly increased (P<0.05),while the ISI was significantly reduced (P<0.05).Skele-tal muscle cells underwent angular atrophy,minimal necrosis,disordered and loose arrangement of muscle cells,extensive proliferation of con-nective tissue,and infiltration of inflammatory cells,and vacuolar lipid droplets were observed.The mRNA expressions of Glut4 and Rxra,as well as the protein expressions of GLUT4 and RXRA in skeletal muscle tissue were significantly reduced (P<0.05).The mRNA and protein expressions of Tnf-α were significantly increased (P<0.05).Compared with the model control group,the body weight,GLU,FBG,FINS,and HOMA-IR of each drug treatment group were significantly reduced (P<0.05),while the ISI was significantly increased (P<0.05).The pathological damage of skeletal muscle tissue in all drug treatment groups was improved.The mRNA and protein expressions of Glut4 and Rxra in skeletal muscle tissue were significantly increased (P<0.05),while the mRNA and protein expressions of Tnf-α were significantly reduced (P<0.05).Dahuang Tangluo Pill of 2.16 g/kg showed the most significant intervention effect.Conclusion:Dahuang Tangluo Pill can obviously improve the T2DM-IRSM,and its mechanism may be related to the effective regulation of the abnormal response of key mole-cules in the RXRA/TNF-α/GLUT4 pathway in skeletal muscle tissue.
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