电针"足三里"恢复糖尿病前期血糖稳态的效应机制研究OA

Objective To observe the effect of electroacupuncture(EA)at"Zusanli"(ST36)on blood glucose in acute hyperglycemia(AH),low-risk prediabetes(LRP)and high-risk prediabetes(HRP)mice,and to explore its underlying mechanisms in regulating the function of pancreatic β-cells in LRP.Methods This study included 3(AH,LRP and HRP)experiments.In each part of the experiments,C57BL/6J mice were randomly divided into normal,model and EA groups,with 6 mice in each group in AH and HRP experiments,and 18 mice in LRP experiment.The AH model was established by intraperitoneal injection of 2 g/kg glucose solution.After injection,EA(1 mA,10 Hz)was applied to bilateral"Zusanli"(ST36)for 20 min.The LRP model was established by feeding the mice with high-fat diet for 1 week.From the 4th day when the impaired glucose tolerance criteria of prediabetes were reached,EA was applied to bilateral ST36,with the parameters being the same as above,once a day for a total of 4 times.In the HRP experiment,the animal model was established by feeding the mice with a high-fat diet for 8 weeks.Four weeks after beginning of the modeling,EA intervention(1 mA,10 Hz)was performed at ST36 for 20 min,3 times a week for a total of 12 times.Blood glucose and glucose tolerance were measured using a glucose meter,and serum glycated hemoglobin A1c(HbA1c),insulin,and glucagon levels were detected by enzyme-linked immunosorbent assay(ELISA).Changes of the pancreatic morphology were examined by HE staining.The expression levels of activating transcription factor 6(ATF6),endoplasmic reticulum to nucleus signaling 1(ERN1),eukaryotic translation initiation factor 2 alpha kinase 3(EIF2AK3),p21(a cell cycle factor in cellular senescence),muscarinic acetylcholine receptor M2(M2-AchR)and M3-AchR in the pancreatic β-cells were detected using Western blot,and the expression levels of pancreatic and duodenal homeobox factor-1(PDX-1),beta-cell-specific transcription factor(MAFA)and forkhead box O1(FOXO1)mRNAs in the pancreatic β-cells detected using quantitative real-time PCR.The insulin+/P21+co-positive cells,and the number of ChAT+/c-fos+co-labeled neurons in the pancreatic ganglia and CTB647/ChAT+/c-fos+co-labeled neurons in the dorsal motor nucleus of the vagus(DMV)innervating the pancreas were determined using fluorescent immunohistochemistry.Results 1)In the AH experiment,compared to the normal group,the blood glucose level in the model group was significantly increased 15 min after modeling(P<0.001),without pathological damage in the pancreas,and the serum insulin level was significantly increased after modeling(P<0.05).In comparison with the model group,the blood glucose levels at 30 and 60 min were significantly reduced(all P<0.01),while the serum insulin content was considerably increased(P<0.05)in the EA group,suggesting that EA lowered blood glucose by increasing stress-induced insulin level.2)In the LRP experiment,compared to the normal group,the blood glucose levels from the 3rd day to the 7th day,and the glucose tolerance from 15 to 120 min were significantly elevated in the model group(P<0.001,P<0.05,P<0.01).No significant changes were found in the serum HbA1c,serum insulin and glucagon levels and in the morphological result of the pancreas tissue in the model group.In comparison with the model group,the blood glucose levels on day 4,6 and 7,the glucose tolerance at 15,30,60 and 120 min were strikingly decreased(P<0.001,P<0.01,P<0.05),while the serum insulin content was notably increased(P<0.05)in the EA group.No significant changes were found in the serum HbA1c and glucagon levels after EA intervention.3)In the HRP experiment,compared to the normal group,the blood glucose levels from 1 to 8 weeks,the glucose tolerance levels at 15,30,60 and 120 min,HbA1c and insulin levels were significantly increased(P<0.001,P<0.05,P<0.01),whereas the serum glucagon level was notably decreased(P<0.05)in the model group,accompanied with an increase of the pancreatic islet volume.Following EA intervention,the blood glucose at the 5th and 6th week,insulin and glucagon levels were considerably down-regulated in the EA group(P<0.01,P<0.05),accompanied with a slight decrease of the pancreatic islet volume.4)In LRP mice,in comparison with the normal group,the expression levels of M2-AchR and M3-AchR protein were significantly decreased(P<0.05),while the expression levels of ATF6,ERN1,EIF2AK3,P21 and the number of insulin+/P21+positive cells in the pancreas tissue were significantly increased(P<0.01,P<0.05)in the model group.In contrast to the model group,the EA group had an obvious increase in the number of CTB647/ChAT+/c-fos+co-labeled neurons in the DMV and ChAT+/c-fos+co-labeled neurons in the pancreatic ganglia,and the expression of M2-AchR and M3-AchR protein(P<0.05,P<0.01),and a notable down-regulation in the expression levels of ATF6,ERN1,EIF2AK3 and P21 proteins and in the number of pancreatic insulin+/P21+positive cells(P<0.01,P<0.05).No significant changes were found in the number of CTB647/ChAT+/c-fos+co-labeled neurons in the DMV and ChAT+/c-fos+co-labeled neurons in the pancreatic ganglia in the model group,and in the mRNA expression levels of pancreatic PDX1,MAFA and FOXO1 in both model and EA groups.Conclusion EA at ST36 can promote the restoration of blood glucose balance in the AH,LRP and HRP stages in mice,with the most effective phase being the LRP.The underlying mechanism may be related to its functions in activating vagus nerve to inhibit the endoplasmic reticulum stress in pancreatic β-cells.