Molecular Mechanism Of Qidan Tiaozhi Capsule In The Treatment Of Metabolic Syndrome Based On Network Pharmacology And Mitophagy

ObjectiveMetabolic syndrome is a type of disease that has insulin resistance as its pathological basis and is characterized by a number of metabolic disorders such as hyperglycemia,hyperlipidemia and central obesity.According to epidemiological statistics,the prevalence rate of metabolic syndrome is about 24.0%in developed countries.And the prevalence rate of metabolic syndrome is about 21.9%in China,showing an upward trend.Therefore,metabolic syndrome has become a major health problem affecting human health.More and more evidence suggests that metabolic syndrome increases the incidence and mortality of type 2 diabetes,cardiovascular disease and cancer,and other diseases.However,due to the complex pathogenesis of metabolic syndrome,the clinical efficacy of metabolic syndrome is still unsatisfactory.Therefore,in order to prevent and treat metabolic syndrome,exploring the pathogenesis and drug treatment of metabolic syndrome is critical.The Qidan Tiaozhi capsule is composed of Astragali Radix,Salviae Miltiorrhizae Radix Et Rhizoma,Cassiae Semen and Laminariae Thallus.It has the effects of soothing the liver and strengthening the spleen,supplementing qi and activating blood circulation,removing turbidity and softening hardness,which is used to treat metabolic disorders such as hyperlipidemia and atherosclerosis.Our previous research found that Qidan Tiaozhi capsule can significantly improve lipid metabolism and insulin resistance in patients with hyperlipidemia,but it is not clear whether Qidan Tiaozhi capsule can improve insulin resistance to play a role in the treatment of metabolic syndrome and its underlying mechanism.Therefore,the present experiment is intended to investigate the effect of Qidan Tiaozhi capsule on metabolic syndrome and its underlying mechanisms through an oleic acid-induced insulin-resistant cell model and a high-fat diet-induced metabolic syndrome animal model.Methods1.Predicting the mechanism of Qidan Tiaozhi capsule in the treatment of metabolic syndrome through network pharmacologyBy searching TCMSP,TCMID,Drugbank and Swiss Target Prediction databases,we collected and collated all the chemical components and their targets of Qidan Tiaozhi capsule.By searching the Therapeutic Target Database,Online Mendelian Inheritance in Man and DisGeNET Databases,we collected the genes associated with the metabolic syndrome and selected the intersection of pharmaceutical ingredients and targets with disease targets,obtaining the target information dataset of Qidan Tiaozhi capsule in the treatment of metabolic syndrome.Thus,the "active component-target" regulation network and protein-protein interaction network of Qidan Tiaozhi capsule were constructed,and GO gene enrichment analysis and KEGG pathway analysis were also performed.Subsequently,Autodock Vina,open source software,was used to conduct molecular docking research on key components and key target proteins in the Qidan Tiaozhi capsule,and the results of molecular docking were visualized.2.Effect of Qidan Tiaozhi capsule on glucose and lipid metabolism in insulin-resistant cellsA glucose consumption experiment was conducted to examine the effect of Qidan Tiaozhi capsule on glucose metabolism in insulin-resistant HepG2 cells.The effect of Qidan Tiaozhi capsule on glucose uptake in insulin-resistant HepG2 cells was investigated by the 2-NBDG fluorescent probe.Lipid accumulation,TC and TG levels in HepG2 cells were detected by oil red O staining and biochemical assay,respectively;ROS levels in HepG2 cells were detected by confocal laser microscopy and flow cytometry.The changes in mitochondrial membrane potential in HepG2 cells were observed by confocal laser microscopy.The levels of MDA and SOD in HepG2 cells were detected by a biochemical method.Dual fluorescent mRFP-GFP-LC3 adenovirus,GFP-LC3 lentivirus and Mito-Tracker Red CMXRos mitochondrial red fluorescent probes,and transmission electron microscope were used to detect the autophagy and mitophagy activities in insulin-resistant HepG2 cells,respectively.Western Blotting was used to detect the effects of Qidan Tiaozhi capsule on the insulin signaling pathway and the mitophagy signaling pathway in insulin-resistant HepG2 cells.3.Silencing of the PINK1 gene reverses the effect of Qidan Tiaozhi capsule on glucose and lipid metabolism in insulin-resistant cellsAfter silencing the PINK1 gene by RNA interference technology,the effect of Qidan Tiaozhi capsule on the glucose metabolism of insulin-resistant HepG2 cells was detected by a glucose consumption assay;the lipid accumulation,TC and TG levels in HepG2 cells were detected by oil red O staining and a biochemical method,respectively;the levels of ROS in HepG2 cells were detected by laser confocal microscopy and flow cytometry;the Laser confocal microscopy to observe the changes in mitochondrial membrane potential in HepG2 cells;a biochemical method to detect the levels of MDA and SOD in HepG2 cells;dual fluorescent mRFP-GFP-LC3 adenovirus,and GFP-LC3 lentivirus and Mito-Tracker Red CMXRos mitochondrial red fluorescent probes,and transmission electron microscope were used to detect the autophagy and mitoophagy activities in insulin-resistant HepG2 cell,respectively;Western Blotting detected the effect of Qidan Tiaozhi capsule on insulin signaling pathway and mitophagy signaling pathway in insulin-resistant HepG2 cells with PINK1 gene silencing.4.The effect of Qidan Tiaozhi capsule on glucose and lipid metabolism in mice with metabolic syndromeThe animal model of metabolic syndrome was prepared by feeding a high-fat diet for 24 weeks.At the 16th week.the mice were treated with Qidan Tiaozhi capsule for 8 weeks,and the body weight and wet weight of liver and abdominal fat were collected,and the coefficients of liver and abdominal fat were calculated;the insulin sensitivity of mice with metabolic syndrome was detected by the insulin tolerance test and oral glucose tolerance test;the serum and hepatic TC,TG,LDL-C and HDL-C levels,and the serum MDA and SOD levels were detected;HE staining was used to observe the effects of Qidan Tiaozhi capsule on hepatic steatosis in mice with metabolic syndrome;subsequently,the expression of AMPK-PINK1/Parkin pathway-related proteins in liver tissues was detected by Western Blotting.Results1.Network pharmacology predicts the mechanism of Qidan Tiaozhi capsule in the treatment of metabolic syndromeAccording to network pharmacology,173 possible key targets of Qidan Tiaozhi capsule in the treatment of metabolic syndrome were obtained.And then we performed GO function enrichment analysis and KEGG signaling pathway enrichment analysis on these 173 targets.The results showed that Qidan Tiaozhi capsule treats metabolic syndrome mainly through the following mechanisms:steroid metabolic process,response to decreased oxygen levels,response to oxygen levels,response to hypoxia,regulation of reactive oxygen species metabolic process,reactive oxygen species biosynthetic process,response to oxidative stress,reactive oxygen species metabolic process,cellular response to oxidative stress,regulation of inflammatory response,response to nutrient levels.Relevant signaling pathways include AGE-RAGE signaling pathway,TNF signaling pathway,PI3K-AKT signaling pathway,reactive oxygen species,and AMPK signaling pathway.The results of molecular docking indicated that the binding free energies between most of the key active components in Qidan Tiaozhi capsule and the key target proteins of metabolic syndrome were less than-5.0 kcal/mol,suggesting an interaction between the active component and the target protein.Only a small number of compounds have binding energies greater than-1.20 kcal/mol with the target protein,suggesting that there is no interaction between the active components and the target proteins.2.Qidan Tiaozhi capsule improves glucose and lipid metabolism in insulin-resistant cellsWe found that Qidan Tiaozhi capsule had no significant effect on the cell viability of normal HepG2 cells and insulin-resistant HepG2 cells in the concentration range of 50-800μg/ml.And 50、100、200、400、600 and 800 μg/ml Qidan Tiaozhi capsule can promote glucose consumption in insulin-resistant HepG2 cells and effectively inhibit the accumulation of lipid droplets in HepG2 cells.Compared with the control group,the glucose uptake and consumption capacity of HepG2 cells administrated with oleic acid were significantly reduced.However,Qidan Tiaozhi capsule treatment significantly increased the glucose uptake and consumption capacity of insulin-resistant HepG2 cells.Compared with the control group,the intracellular cholesterol and triglyceride levels of insulin-resistant HepG2 cells in the model group increased significantly.However,the intracellular cholesterol and triglyceride levels of HepG2 cells in the Qidan Tiaozhi capsule group were significantly decreased.Compared with the control group,the ROS levels in insulin-resistant HepG2 cells increased significantly,the mitochondrial membrane potential,autophagy and mitophagy were also significantly decreased.After the intervention of Qidan Tiaozhi capsule,the ROS levels in HepG2 cells were significantly reduced,mitochondrial membrane potential,autophagy and mitophagy were significantly increased.Compared with the model group,Qidan Tiaozhi capsule effectively activated the insulin signaling pathway IRS2 and PI3K proteins,mitophagy-related proteins LC3-Ⅱ/Ⅰ,p-AMPK,PINK1 and Parkin expression,and inhibited p62 expression.3.Silencing the PINK1 gene reverses the effect of Qidan Tiaozhi capsule on glucose and lipid metabolism in insulin-resistant cellsAfter silencing the PINK1 gene by RNA interference technology,we found that compared with the model group,Qidan Tiaozhi capsule could significantly increase the glucose uptake and consumption capacity of insulin-resistant HepG2 cells.However,after the PINK1 gene was silenced,the improving effect of Qidan Tiaozhi capsule on the glucose uptake and consumption capacity of insulin-resistant HepG2 cells was significantly inhibited.Compared with the model group,the intracellular cholesterol and triglyceride levels of HepG2 cells in the Qidan Tiaozhi capsule groups were significantly reduced.After the PINK1 gene was silenced,the intracellular cholesterol and triglyceride levels in HepG2 cells increased significantly.Compared with the model group,after the intervention of Qidan Tiaozhi capsule,the ROS levels in HepG2 cells was significantly decreased,and the mitochondrial membrane potential,autophagy and mitophagy were significantly increased.However,after the PINK1 gene was silenced,the improvement of mitochondrial function and mitophagy by Qidan Tiaozhi capsule was reversed.The results of protein expression were consistent with the previous findings,when the PINK1 gene was silenced,the IRS2,PI3K,LC3-Ⅱ/Ⅰ,PINK1,and Parkin proteins activated by Qidan Tiaozhi capsule were significantly inhibited.and the repressed p62 protein expression showed marked enhancement.4.Qidan Tiaozhi capsule improves glucose and lipid metabolism in mice with metabolic syndromeThe experimental results of the metabolic syndrome animal model showed that after 24 weeks of high-fat diet feeding,compared with the normal group,the mice with metabolic syndrome showed significant weight gain,insulin resistance,increased serum and hepatic TC,TG and LDL-C levels,inhibited serum and hepatic HDL-C levels,increased serum MDA level,inhibited serum SOD content,hepatic steatosis,and the expression of AMPK-PINK1/Parkin pathway is inhibited.However,compared with the model group,after 8 weeks of treatment with Qidan Tiaozhi capsule,the body weight of the mice with metabolic syndrome decreased to a certain extent,and the insulin sensitivity was significantly increased.The serum and hepatic TC,TG and LDL-C levels were significantly decreased,the serum and hepatic HDL-C levels were significantly increased,the content of MDA in serum was significantly decreased,the level of serum SOD significantly increased,the steatosis of liver tissue was significantly improved,and the AMPK-PINK1/Parkin pathway was significantly activated:insulin signaling pathway IRS2 and PI3K proteins were activated,mitophagy-related proteins LC3-Ⅱ/Ⅰ,p-AMPK,PINK1 and Parkin were activated,and p62 protein expression was inhibited.Conclusions1.Network pharmacology studies have found that Qidan Tiaozhi capsule may play a key role in the treatment of metabolic syndrome by regulating oxidative stress.2.Qidan Tiaozhi capsule can improve the insulin sensitivity of insulin-resistant cells by activating AMPK-PINK1/Parkin-mediated mitophagy,thereby improving disordered glucose and lipid metabolism.3.Silencing the PINK1 gene can reverse the therapeutic effects of Qidan Tiaozhi capsule on insulin sensitivity,glucose and lipid metabolism in insulin-resistant cells.4.Qidan Tiaozhi capsule can increase insulin sensitivity in mice with metabolic syndrome by activating AMPK-PINK1/Parkin-mediated mitophagy,thereby exerting a therapeutic effect on metabolic syndrome.