胰岛素样生长因子1通过AMP蛋白活化激酶调节糖尿病胃轻瘫大鼠胃平滑肌细胞缝隙连接重构OA

AIM:To investigate the mechanism of high glucose-induced gap junction(GJ)remodeling in gas-tric smooth muscle cells of diabetic rats,and the interventional effect of insulin-like growth factor-1(IGF-1)through the AMP-activated protein kinase(AMPK)pathway.METHODS:A total of 70 specific pathogen-free male Sprague-Dawley rats were used.Diabetes was induced in 40 randomly selected rats by intraperitoneal injection of streptozotocin,while the remaining 30 served as normal controls.At 8 weeks post-induction,10 diabetic rats were randomly selected for assessment of gastric dye retention and small intestinal transit,and another 10 for gastrointestinal propulsion.These parameters were compared with those of 20 normal rats to confirm successful establishment of the diabetic gastroparesis(DGP)model.The animals were then re-grouped:10 normal rats served as the control group,and the remaining DGP model rats were random-ly assigned to a DGP group(n=10)or an IGF-1+DGP group(n=10).The rats in IGF-1+DGP group received daily intra-peritoneal injections of IGF-1(1.5 µg/kg),while those in control and DGP groups received equal volume of normal sa-line.All treatments were continued for 8 weeks.Gastric smooth muscle contractility was evaluated ex vivo by measuring spontaneous contraction frequency and amplitude.Immunohistochemistry was used to examine connexin 43(Cx43)distri-bution.Western blot was performed to quantify protein expression of AMPK,phosphorylated AMPK,membrane and cyto-solic Cx43,and phosphorylated Cx43.In parallel,gastric smooth muscle cells were exposed to high glucose with or with-out IGF-1.Protein microarray analysis was used to screen AMPK phosphorylation pathway changes under high glucose,and Western blot was applied to validate AMPK-related functional protein expression.RESULTS:Compared with con-trols,DGP rats showed significant weight loss,elevated blood glucose,delayed gastric emptying,reduced gastrointestinal propulsion,and impaired antral smooth muscle contractility(P<0.05).Immunohistochemistry and Western blot revealed increased Cx43 expression,elevated membrane-to-cytoplasm ratio,and enhanced phosphorylation at Ser368 in gastric smooth muscle of DGP rats(P<0.05).IGF-1 intervention improved general health and restored spontaneous contractile activity in DGP rats,while significantly reducing Cx43 membrane localization and phosphorylation(P<0.05).In high glu-cose-treated cells,AMPK activity was enhanced(P<0.01),accompanied by an increased AMP/ATP ratio,upregulated activity of upstream kinases transforming growth factor-β-activated kinase 1(TAK1)and liver kinase B1(LKB1),and ele-vated calcium/calmodulin-dependent protein kinase kinase β(CaMKKβ)expression.IGF-1 treatment reversed these changes,significantly reducing the AMP/ATP ratio,TAK1/LKB1 activity,and CaMKKβ expression(P<0.05),thereby attenuating GJ remodeling.CONCLUSION:The GJ remodeling occurs in gastric smooth muscle of DGP rats,a process mediated in part by AMPK signaling.IGF-1 ameliorates high glucose-induced GJ remodeling via the AMPK pathway,con-tributing to the recovery of spontaneous contractile function in gastric smooth muscle.