| Purpose: This research predicted the mechanism of Yitangkang and virtual screened its active components on ameliorating insulin resistance by Network Pharmacology and Molecular Docking technology.After completed the aboved work,select the corresponding indicators of important pathways and important related mechanisms,and verified them by skeletal muscle tissue of insulin resistance model rats which had been determined to be qualified by Hyperinsulinemic Euglycemic Clamp test.Finally,the Yitangkang medicated serum was prepared to interfere with C2C12 IR model cells induced by palmitic acid.The effect of the Yitangkang medicated serum was determined by the cell morphology,cell activity and glucose consumption of each group.At the same time,the corresponding mechanism of in vivo experiment was verified in vitro,and the mechanism framework of Yitangkang ameliorating insulin resistance was perfected to provide reference for clinical treatment.Material and method: Paper one: Researching and predicting the mechanism of Yitangkang ameliorating insulin resistance based on Network Pharmacology and Molecular Docking technology.Obtaining,arranging and screening the active components of Yitangkang and disease targets of insulin resistance through databases in TCMSP、Dis Ge NET、CTD、Gene Cards、OMIM.The TCMSP target prediction model was used to predict the possible targets of the active components that meet the screening criteria,and intersect with the disease targets of insulin resistance,as possible target of Yitangkang to ameliorate insulin resistance,Cluster Profile R package was used for enrichment analysis.Two insulin resistance related proteins with direct medical evidence and high influence score were selected as important protein receptors from CTD database.The sequencing data of human tissues were selected from GEO database.After analyzed the differentially expressed genes,combined with the literature,two were selected as important protein receptors.Two important targets were selected as important protein receptors from the pathway to be verified.After the binding sites of the above important protein receptors were determined,they were conducted molecular docking by Vina with the effective components of Yitangkang,virtually screened the active components that ameliorated insulin resistance,and analyzed the possible mechanism.Paper two: Animal experimental verification of Network Pharmacology based on AGE-RAGE signal pathwayA total of 96 8-week-old healthy male Wistar rats were set by blank control group,model control group,Yitangkang high-dose group,Yitangkang medium dose group,Yitangkang low-dose group and western medicine control group(pioglitazone hydrochloride)respectively,the doses were(40g/kg,20g/kg,10g/kg,1.35mg/kg),The animal model of insulin resistance was established by high glucose and high-fat diabetes model induction feed combined with low-dose STZ intraperitoneal injection,and verified by Hyperinsulinemic Euglycemic Clamp test.After the model were determined successfully,the rats in each group were given intragastric administration intervention as required.Homeostasis Insulin Resistance Index were used to evaluate the changes of insulin resistance in each group after the intervention.The core proteins of AGEs-RAGE signal pathway were verified by the technique of Immunohistochemistry and Western blotting.Paper three: Effect of Yitangkang on apoptosis of the cell of skeletal muscle tissue After a large number of literature analysis,most of the indicators of AGE-RAGE signal pathway verified in paper two were related to apoptosis.This part intended to explore whether Yitangkang can inhibit skeletal muscle cell apoptosis and its specific mechanism.The experimental animals,feeding,grouping and administration methods were the same as these in paper two.TUNEL Fluorescence Labeling method was used to observe the apoptosis of skeletal muscle cells in each group.At the same time,the expression levels of apoptotic proteins Bax,Bcl-2,P53,Caspase3 and Caspase9 were observed by Polymerase Chain Reaction and Western blotting technology.Paper four: Effect of Yitangkang medicated serum on apoptosis of C2C12 cell and verification of its mechanism 40 male SPF Wistar rats were randomly divided into Yitangkang high dose group,Yitangkang medium dose group,Yitangkang low dose group and blank control group according to body weight after adaptive feeding for one week.The high,medium and low dose groups of Yitangkang administration according to the converted drug dose(40g/kg,20g/kg and 10g/kg),and the blank control group was given the same volume of normal saline for 1 week.One hour after the last administration,the rats were anesthetized,the blood was taken from the abdominal aorta and centrifuged to prepare drug medicated serum of Yitangkang.C2C12 cells were cultured and the its insulin resistance model was induced by palmitic acid.They were divided into model control group,Yitangkang medicated serum high dose group,Yitangkang medicated serum medium dose group,Yitangkang medicated serum low dose group and RAGE inhibitor group.Normal C2C12 cells were set as blank control group,The corresponding Yitangkang medicated serum and RAGE inhibitor were used forintervention.The morphological changes of cells in each group were observed by Inverted Microscope,the cell viability was detected by CCK-8 method,and the cell glucose consumption was measured.The proportion of apoptosis was observed by Flow Cytometry,the expression levels of Bax,Bcl-2,P53,Caspase3 and Caspase9 were observed by Western blotting,and the m RNA expression level of the above important apoptotic proteins was tested by Polymerase Chain Reaction,to verify whether Yitangkang can ameliorate insulin resistance by inhibiting AGE-RAGE signal pathway and then inhibiting apoptosis.Results: Paper one: 1.A total of 202 potential targets of Yitangkang in the treatment of insulin resistance were obtained by analysing corresponding websites and databases.Involving multiple Biological Processes such as the regulation of nutrients,vascular diseases and oxidant metabolism,which were related to the joint action of multiple organelles and the activities of neurotransmitters and adrenoceptors,and many related to nerves,inflammation pathways related to metabolic processes.2.INSR and NOS3 were selected as important protein receptors from CTD Database.GSE73034 Chip was found to be related to this research from GEO database,it was the skeletal muscle tissue sequencing data of patients with insulin resistance and normal healthy volunteers.14 significantly difference proteins were screened out.Combined with the literature analysis,LPL and GRB14 are of research significance.Therefore,LPL and GRB14 were selected as important protein receptors,RAGE and JAK2 were selected as important protein receptors from the AGE-RAGE pathway to be verified.3.Through molecular docking with all the effective components of Yitangkang,we found that diosgenin,Xambioona,isotanshinone II,lantadene A,Glycyrrhizin E,astragaloside A,glycyrrhizin H2,isotanshinone I,neoflavanone,glycyrrhizin C2,Physalin A and Longikaurin A were the potential active components of Yitangkang to ameliorate insulin resistance,The mechanism of action mainly involves anti-inflammatory and antioxidant stress.Paper two: 1.Molding situation of Type 2 diabetes and insulin resistance in experimental rats: After injection of STZ,70 rats were in accord with Type 2 diabetes mellitus,except fordead and unmodeled rats.After the Hyperinsulinemic Euglycemic Clamp test,all the rats showed insulin resistance.2.Effects of Yitangkang on HOMA-IR levels in rats in each group HOMA-IR in the model group increased significantly.After use of various doses of Yitangkang and western medicine,the HOMA-IR decreased.The above differences were statistical significance(P<0.05 or 0.01).3.Immunohistochemical test results: Compared with the blank control group,the expression level of AGEs,RAGE,PKCβII,Egr-1,JNK,JAK2,STAT1,STAT3 in skeletal muscle tissue of rats in model control group,Yitangkang high,medium and low dose group,western medicine control group increased significantly(P<0.05 or 0.01);Compared with model control group,the expression level of AGEs,RAGE,PKCβII,Egr-1,JNK,JAK2,STAT1,STAT3 in skeletal muscle tissue of rats in Yitangkang high,medium and low dose group,western medicine control group decreased significantly(P<0.05 or 0.01);Compared with Yitangkang medium dose group,the expression level of AGEs,RAGE,Egr-1,JNK,STAT1,STAT3 in Yitangkang high dose group decreased significantly(P<0.05 or 0.01),there was no significant difference in expression level of PKCβII and JAK2(P>0.05),the expression level of AGEs,RAGE,PKCβII,Egr-1,JNK,JAK2,STAT1,STAT3 in Yitangkang low dose group increased significantly(P<0.01);Compared with Yitangkang medium dose group,the expression level of RAGE in western medicine control group decreased,and the difference was statistically significant(P<0.05),there were no significant difference in AGEs、PKCβII、Egr-1、JNK、JAK2、STAT1、STAT3(P>0.05).4.Western blotting results: Compared with blank control group,the expression level of AGEs,RAGE,PKCβII,Egr-1,JNK,JAK2,STAT1,STAT3 in other groups increased significantly(P<0.01);Compared with model control group,the expression level of AGEs,RAGE,PKCβII,JNK,JAK2,STAT1,STAT3 in high,medium and low dose group,western medicine control group decreased significantly(P<0.05 or 0.01),there was no significant difference in expression level of Egr-1(P>0.05);Compared with Yitangkang medium dose group,the expression level of AGEs,RAGE,PKCβII,JNK,JAK2,STAT3 decreased significantly(P<0.05 或 0.01),there was no significant difference in expression level ofEgr-1 and STAT1(P>0.05).The expression level of aboved index proteins in Yitangkang low dose group increased significantly(P<0.01);The expression level of AGEs in western medicine control group was significantly lower than that in Yitangkang medium dose group(P<0.01),and there were no significant difference in the expression level of RAGE,PKCβII,Egr-1,JNK,JAK2,STAT1,STAT3(P>0.05).Paper three: 1.Results of the apoptosis of skeletal muscle cells by TUNEL technology in each group: Compared with blank control group,the number of positive apoptotic cells of skeletal muscle tissue in model control group was significantly increased(P<0.01).compared with model control group,the number of positive apoptotic cells in skeletal muscle tissue in Yitangkang high,medium,low dose group and western medicine control group decreased significantly(P<0.01).Compared with Yitangkang medium dose group,the number of positive apoptotic cells in Yitangkang high dose group decreased and the number of positive apoptotic cells in Yitangkang low dose group increased(P<0.01).There was no significant difference between Yitangkang medium dose group and western medicine control group(P>0.05).2.Polymerase Chain Reaction results: Compared with blank control group,the m RNA expression levels of Bax,P53,Caspase3 and Caspase9 in model control group increased significantly,and the m RNA expression level of Bcl-2 decreased significantly(P<0.01),Compared with model control group,the m RNA expression level of Bax,P53,Caspase3,Caspase9 in Yitangkang high dose group,medium dose group,low dose group and western medicine control group decreased significantly,Bcl-2 increased significantly(P<0.05 or 0.01).Compared with Yitangkang medium dose group,the m RNA expression levels of Bax,P53 and Caspase3 in Yitangkang high dose group decreased significantly,and the m RNA expression level of Bcl-2 increased significantly(P<0.01),and there was no significant difference in m RNA expression level of Caspase9(P>0.05),the m RNA expression levels of Bax,P53,Caspase3 and Caspase9 increased significantly,Bcl-2 decreased significantly(P<0.05 or 0.01);There were no significant difference in Bax,Bcl-2,P53,Caspase3,Caspase9 m RNA expression between Yitangkang medium dose group and western medicine control group(P>0.05).3.Western blotting results: Compared with blank control group,the expression level of Bax,P53,Caspase3 and Caspase9 in model control group increased significantly,and the expression level of Bcl-2 decreased significantly(P<0.01);Compared with model controlgroup,the above results of Yitangkang high dose group,medium dose group,low dose group and western medicine control group were on the contrary(P<0.01);Compared with Yitangkang medium dose group,the expression level of Bcl-2 in Yitangkang high dose group increased significantly,Bax、Caspase9 decreased significantly(P<0.05 or 0.01),there was no significant difference in the expression level of P53 and Caspase3(P>0.05),the expression level of Bax,P53,Caspase3 and Caspase9 in Yitangkang low dose group increased significantly(P<0.05 or 0.01),and the difference of Bcl-2 expression level was no statistically significant(P>0.05).There were no significant difference in the expression level of Bax,Bcl-2,P53,Caspase3,Caspase9 between Yitangkang medium dose group and western medicine control group(P>0.05).Paper four: 1.Cell culture and induction of insulin resistance model: After C2C12 was induced by palmitic acid,they were spherical changes,cell activity and glucose consumption decreased,the insulin resistance model was successfully established.After intervention with various doses of Yitangkang medicated serum and RAGE inhibitor,the situation was improved significantly(P<0.01).2.Results of Flow Cytometry: Compared with blank control group,the apoptosis rate of the model control group was significantly increased(P<0.01).Compared with the model control group,the apoptosis rate of Yitangkang medicated serum high,medium,low dose group and RAGE inhibitor group decreased significantly(P<0.01).Compared with Yitangkang medicated serum medium dose group,the apoptosis rate of Yitangkang medicated serum low dose group increased,and the apoptosis rate of Yitangkang medicated serum high dose group and RAGE inhibitor group decreased,there were significant statistical significance between the above groups(P<0.01).3.Western blotting results: Compared with normal control group,the expression level of Bcl-2 in model control group decreased significantly,and the expression level of Bax、P53、Caspase3、Caspase9 increased significantly(P<0.01);Compared with model control group,the aboved results were on the contrary in Yitangkang medicated serum high,medium,low dose groups and RAGE inhibitor group(P<0.01);Compared with Yitangkang medicated serum medium dose group,the expression level of Bcl-2 increased,Bax,Caspase 3,Caspase 9 decreased in Yitangkang medicated serum high dose group(P<0.05 or 0.01),there was no significant difference in the expression levels of P53(P>0.05),the expressions of Bax,P53 and Caspase3 in Yitangkang medicated serum low dose group increased(P<0.01),there were nosignificant difference in the expression level of Bcl-2 and Caspase 9(P>0.05);There were no significant difference between Yitangkang medicated serum medium dose group and RAGE inhibitor group(P>0.05).3.Polymerase Chain Reaction results: Compared with normal control group,the m RNA expression level of Bax,P53,Caspase3 and Caspase9 in model control group increased significantly,the m RNA expression level of Bcl-2 decreased significantly(P<0.01).Compared with model control group,the m RNA expression level of Bax,P53,Caspase3 and Caspase9 in Yitangkang medicated serum high,medium,low dose group and RAGE inhibitor group decreased significantly,the m RNA expression level of Bcl-2 increased significantly(P<0.01);Compared with Yitangkang medicated serum medium dose group,the m RNA expression level of Bcl-2 in Yitangkang medicated serum high dose group increased(P<0.05),Bax,P53,Caspase3 and Caspase9 decreased(P<0.05 or 0.01),the m RNA expression level of Bcl-2 in Yitangkang medicated serum low dose group decreased significantly,and P53,Caspase3 and Caspase9 increased significantly(P<0.01),there was no significant difference in m RNA expression level of Bax(P>0.05).There were no significant difference in Bax,Bcl-2,P53,Caspase3,Caspase9 m RNA expression level between Yitangkang medicated serum medium dose group and RAGE inhibitor group(P>0.05).Conclusion: 1.The results of Network Pharmacology suggested that the improvement of insulin resistance by Yitangkang through many Biological Processes,such as the regulation of nutrients,vascular diseases and oxidant metabolism,were related to the joint action of multiple organelles,activities of neurotransmitters and adrenoceptors,involved many pathways related to nerve,inflammation and metabolic processes,the predicted AGE-RAGE signal pathway was the object of further research.2.The potential active components of Yitangkang which can ameliorate insulin resistance were successfully screened by Molecular Docking technology,such as diosgenin,Xambioona,isotanshinone II,lantadene A,Glycyrrhizin E,astragaloside A,glycyrrhizin H2,isotanshinone I,neoflavanone,glycyrrhizin C2,Physalin A and Longikaurin A,the binding ability of all important protein receptors to most of the effective active components in Yitangkang were better than Pioglitazone.3.The results showed that Yitangkang can ameliorate insulin resistance.From the perspective of skeletal muscle,the specific process may involved reducing the levels of AGEs,RAGE and their downstream targets to inhibit inflammation and ameliorate oxidative stress,mainly through PKCβII-Egr-1、JAK2/STAT1/STAT3.4.AGE-RAGE signaling pathway was closely related to apoptosis.Yitangkang can inhibit excessive apoptosis of skeletal muscle cells by inhibiting AGE-RAGE signaling pathway through increasing the expression levels of anti apoptotic protein Bcl-2 and inhibiting pro apoptotic proteins Bax,P53,Caspase3 and Caspase9. |
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