Electroacupuncture Mitigates Endothelial Dysfunction Related To High-fat Diet-induced Insulin Resistance Through The PI3K/eNOS Signaling Pathway

ObjectiveOur aim was to explore the influence of electroacupuncture(EA)on endothelial dysfunction related to high-fat-diet-induced insulin resistance through the PI3K-eNOS signaling pathway.MethodsThirty-six male Sprague Dawley rats were fed high-fat diet for 12 weeks to induce the insulin resistance model and the models were divided into three groups(N=12):control group,HFD group,and HFD+EA group,respectively.Since the beginning of the eighth week,the fasting blood glucose(FPG),serum insulin(FINS)and homeostatic model assessment of insulin resistance(HOMA-IR)index were determined every two week.When the levels of HOMA-IR in the high-fat diet rats were higher than those of normal diet fed rats significantly(P<0.05),hyperinsulinemic euglycemic clamp would be operated to confirm the insulin resistance model.When compared with the blank group,the levels of GIR in HFD group and HFD+EA group were significantly lower(P<0.05).The model was sucessful after 12 weeks high-fat diet.The needles were used to the both sides of Neiguan(PC6),Zusanli(ST36),Sanyinjiao(SP6),and Shenshu(BL23).Neiguan(PC6),Zusanli(ST36),and Sanyinjiao(SP6)were oblique insertion with 8 mm,and Shenshu(BL23)straightly for 5 mm.Needles in Sanyinjiao(SP6)and Zusanli(ST36)were connected with electroacupuncture instrument,with sparse-dense wave(spares wave 2 Hz,dense wave 50 Hz,6s,respectively)for 20 min/day,lasted for four weeks.After the experiment finished,FPG,FINS,serum C-peptide(C-p),TNF-α,IL-6,NO and HOMA-IR index were determined.The pancreatic island samples and the thoracic aortas intercepted were kept for the histopathological examination.Insulin receptor substrate(IRS)-1,phosphatidylinositol 3-kinase(PI3K),protein kinase B(Akt2),and endothelial NO synthase(eNOS)in vascular endothelium were determined by Real-time PCR and Western blot analysis.ResultsDuring the study,the weights of the rats in the control group were gained,the rats were in good state with lustered hair,moving freely.Four weeks before electroacupuncture treatment,rats in both model group and electroacupuncture group became gentle,sluggish,less luster rat fur and abdominal obesity.The stool quantities of the rats was less when compared with that of the normal group.The stool were grey color and qualitative soft.Four weeks after electroacupuncture treatment,weights of the rats in electroacupuncture group were still increased.But compared to the control group,the movement and state of rats in electroacupuncture group became better.Before electroacupuncture treatment,the levels of FPG,FINS and HOMA-IR in both model group and electroacupuncture group were increased significantly but GIR decreased when compared with control group(P<0.05).After four weeks treatment,the levels of FPG,FINS,C-P,TNF-α,IL-6,ET-1 and HOMA-IR of electroacupuncture group were significantly decreased but level of GIR,C-P/INS and NO increased when compared with model group(P<0.05).Aortas and pancreatic island samples with HE staining were observed under the microscope with 20×10 times.In control group,the structure of pancreatic islet was complete,and the boundary of the cells in the islets was clear,arranged in order,the size and the distribution was uniform.Artery endothelium of control group was continuous and smooth,elastic fiber and vascular smooth muscle cells were neatly arranged without infiltrating lymphocytes.Islet structure disorder of rats in the model group,the boundary between cells was not clear,cytoplasm loose,cell fragmentation and obvious inflammatory cell infiltration.Artery endothelium of model group was discontinuous,obvious loss of membrane integrity,the layer under endothelial loose,hyperplasia and disordered arrangement of vascular smooth muscle cell.Islet in electroacupuncture group,islet cells were with clear border,form close to normal with regular arrangement.After electroacupuncture intervention,artery endothelium was almost as smooth as control group without obvious thickening.Hyperplasia and disordered arrangement of vascular smooth muscle cell of electroacupuncture group was ameliorative compared with model group.For ultrastructural changes of vascular endothelium in rats under electron microscope,there was a single layer of flat cells lining on the arterial wall in the normal group rat aortic intima.Endothelial cells were closely connected with narrow gap and no obvious abnormal mitochondria seen.Endothelial cells of the rats in the model group were under the series of loose,flat cells and arterial wall gap widened,the majority of the mitochondrial vacuolar appeared on the wall.Endothelial layer and the arterial wall gap was slightly wide in electroacupuncture group rat with a few mitochondria crista disappeared on local vascular wall.Compared with the control group,the expressions of vascular endothelial in model rats IRS-1,PI3K,Akt2 and eNOS genes and proteins were significantly reduced and phosphorylation of Akt2 and eNOS were aslo reduced(P<0.01).Compared with the model group,vascular endothelial PI3K,Akt2 and eNOS genes and proteins expression levels in electroacupuncture group was significantly increased and phosphorylation of Akt2 and eNOS were improved as well(P<0.05).ConclusionEA might improve endothelial dysfunction and insulin resistance in rats through the IRS-1/PI3K/Akt2/eNOS signaling pathway.