| Diabitic Gastroparesis is a common gastrointestinal complication of diabetes,manifested by delayed gastric emptying caused by gastric motility disorder.Diabitic Gastroparesis has the characteristics of making hypoglycemic drugs unstable,difficult to control blood sugar concentration,and increased fluctuations,thereby accelerating the occurrence of other related acute and chronic complications of diabetes,which seriously affects the quality of life of patients.Because the clinical treatment of Diabitic Gastroparesis is relatively single,and the relative side effects or trauma are relatively large.Therefore,exploring and determining the related mechanism of Diabitic Gastroparesis is essential for clinical exploration of new treatment options and drug development.At present,there are relatively few studies on gastric smooth muscle cells themselves in the pathogenesis of Diabitic Gastroparesis.Previous studies have confirmed that there are gastric motility disorders and cell apoptosis in the gastric smooth muscle of Diabitic Gastroparesis rats,and cell apoptosis is an important cause of gastric motility disorders.If a certain mechanism can be determined and regulated by drugs to inhibit gastric smooth muscle cell apoptosis,then it will have a positive effect on the treatment of Diabitic Gastroparesis and other complications of diabetes.Therefore,this study uses multidisciplinary experimental techniques such as flow cytometry,ELISA,RNA interference,Western Blot,antibody protein chip,etc.,using diabetic rat models and in vitro cultured gastric smooth muscle cells as the research objects to explore IGF-1 under high glucose conditions.Regulating the related mechanisms of gastric smooth muscle cell apoptosis through the ROS-AMPK pathway is aimed at providing scientific theoretical and experimental basis for clinical exploration of new treatment options,and has potential application value for the clinical prevention and treatment of DGP and drug development.Objective:To investigate the effect of IGF-1 on the apoptosis of gastric smooth muscle cells induced by high glucose based on the ROS-AMPK pathway and to clarify its underlying mechanism.Methods:Prepare diabetic rat models,intraperitoneally inject IGF-1（1.5μg.kg-1.d-1）,the experiment is divided into NC group,DM4w group,DM6w group,DM8w group,DM4w+IGF-1 group,DM6w+IGF-1 group,DM8w+IGF-1 group,observe the general state,blood sugar,weight and gastric emptying changes of diabetic rats in each group before and after IGF-1 intervention;observe IGF-1 in vitro Changes in the spontaneous contraction movement of the gastric smooth muscle of each group of diabetic rats before and after intervention;the Biochemical test and western blot method was used to observe the changes of oxidative stress and AMPK activity of the gastric smooth muscle of each group of diabetic rats before and after IGF-1 intervention;The changes in the apoptosis of gastric smooth muscle cells of diabetic rats before and after IGF-1intervention were determined.To culture rat gastric smooth muscle cells in vitro,firstly use in situ probe technology to observe the effect of IGF-1 on the activity and activation mode of ROS and AMPK of rat gastric smooth muscle cells under high glucose conditions in the high glucose 24h group and high glucose 48 h group and high glucose 24 h+IGF-1 group and high glucose 48 h+IGF-1 group;then using antioxidantα-thiozinc acid,western blot and Elisa method were used to observe IGF-1 under high glucose conditions,Whether and how to regulate the AMPK activity of rat gastric smooth muscle cells through ROS in the high glucose 24 h group and high glucose 48 h group and high glucose 48 h+α-thiozinc acid group.Then AMPK using Small interfering RNA（si RNA）was silenced.The experiment was divided into high glucose group,high glucose+IGF-1 group,high glucose+si RNA group and high glucose+si RNA+IGF-1 group.Flow cytometry observed and confirms the conditions of high glucose Whether IGF-1regulates rat gastric smooth muscle cell apoptosis through AMPK at 24h and 48h;protein chip was used to observe the changes of AMPK phosphorylation pathway of rat gastric smooth muscle cells under high glucose conditions.The experimental group was high Determine the relevant functional proteins in the high glucose（48 h）group and the high glucose（48 h）+IGF-1 group,then they were verified the determined functional protein by western blot.The experimental group was divided into high glucose（48 h）group and the high glucose（48 h）+IGF-1 group.Results:In animal experiments,IGF-1 could alleviate the general state of diabetic rats,reduce blood sugar in diabetic rats and increase body weight,reduce the residual rate of gastric pigment,increase the intestinal transit rate and the recommended gastrointestinal rate.IGF-1could increase the frequency and amplitude of voluntary contraction of gastric smooth muscle in diabetic rats.IGF-1 could reduce AMPK activity in gastric smooth muscle tissue of diabetic rats.IGF-1 could reduce the content of MDA in gastric smooth muscle tissue of diabetic rats,increase the content of T-SOD,and increase the content of CAT.IGF-1 could up-regulate gastric smooth muscle cell proliferation in diabetic rats IGF-1 can reduce gastric smooth muscle cell apoptosis in diabetic rats.In vitro cell experiments,under high glucose conditions IGF-1 could down-regulated rat gastric smooth muscle ROS,while up-regulating AMPK activity at 24 h and down-regulated AMPK activity at 48 h,while down-regulated the AMP/ATP ratio,LKB1 activity,TAK1 activity and Ca MKKβexpression at 48 h.After the intervention ofα-thiozinc acid,AMPK activity was decreased,AMP/ATP ratio was decreased,LKB1 activity was decreased,TAK1 activity was decreased and the expression of Ca MKKβwas decreased.When AMPK was silenced for 24 h under high glucose,the apoptosis rate of rat gastric smooth muscle cells decreased;after IGF-1was added,the apoptosis rate of rat gastric smooth muscle cells also decreased;while AMPK was silenced and IGF-1 was added,the apoptosis rate of was not statistically significant;the apoptosis rate of IGF-1 after adding IGF-1 and silencing AMPK was not statistically significant.After AMPK was silenced for 24 h under high glucose conditions,the apoptosis rate of rat gastric smooth muscle cells decreased;after IGF-1 was added,the apoptosis rate of rat gastric smooth muscle cells also decreased;while the apoptosis rate of AMPK and IGF-1 was not statistically significant;the apoptotic rate of adding IGF-1 and silencing AMPK was not statistically significant.After silencing AMPK for 48 hours under high glucose conditions,the apoptotic rate of gastric smooth muscle cells in rats decreased;after adding IGF-1,the apoptotic rate of gastric smooth muscle cells in rats also decreased;while the apoptotic rate of AMPK was higher than that of adding IGF-1;the apoptotic rate of adding IGF-1 after adding IGF-1 and silencing AMPK was not statistically significant.Protein chip results found that IGF-1 had an effect on the phosphorylation of 48 sites in the AMPK phosphorylation pathway of rat gastric smooth muscle cells under high glucose conditions,involving p53,PI3K,Akt,e NOS,TSC-2,m TOR,4E-BP1,p70S6K,p21,Ca MKⅡ,PLC-β3,Rap GEF1 and other apoptosis-related proteins.IGF-1 could down-regulate the expression of Ca MKⅡin rat gastric smooth muscle cells under high glucose,down-regulate the expression of e NOS,down-regulate the expression of p53,up-regulate the expression of p21,and up-regulate the expression of Rap GEF1,up-regulate the expression of PLC-β3,up-regulate the expression of PI3K p110 Ser1070,up-regulate the activity of Akt,up-regulation the activity of p70S6K,up-regulate the expression of 4E-BP1,up-regulate the activity of m TORC1,up-regulation the activity of m TORC2;at the same time,IGF-1 could up-regulated the expression of Bcl-2 and down-regulate the expression of BAX,down-regulate the expression of Caspase-3.Conclusion:（1）IGF-1 could increase the gastric emptying capacity of diabetic gastroparesis rats by improving gastric motility,which is related to IGF-1 promotion of gastric smooth muscle cell proliferation and inhibition of cell apoptosis and increase the number of effective smooth muscle cells.（2）IGF-1 could inhibit apoptosis of rat gastric smooth muscle cells,it is one of the mechanisms that IGF-1 could regulate the expression and activity of p53,PI3K,TSC-2,Akt,m TOR,4E-BP1,p70S6K,p21,Ca MKⅡ,PLC-β3 through Oxidative Stress-AMPK pathway,under high glucose conditions. |
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