Electroacupuncture Protects Enteric Neurons By Regulating GDNF Secretion Of Enteric Glial Cells

Objective : Colon motility disorders are pretty common among patients with diabetes.And colon motility is controlled by local enteric neuronal networks.In recent years,electroacupuncture(EA)has become an alternative treatment of Colon motility disorders,but specific mechanisms of EA’s effects on enteric neurons remain incompletely understood.Glia cell line-derived neurotrophic factor(GDNF),mainly secreted by enteric glial cells(EGCs),is vital to the survival of enteric neurons.Our aim in this study is to investigate whether EA could accelerate colon transit and protect enteric neurons by activating EGCs to play a neuroprotective effect via GDNF/GFRα1/Ret,PTEN/PI3K/Akt pathways.Methods: Phase one: Wild C57BL/6 male mice were randomly divided into two groups: normal control group(N group),diabetic mellitus group(DM group).Diabetes was induced by a single intraperitoneal injection of streptozotocin(STZ).At the end of 4 weeks,6 weeks,8 weeks,and 12 weeks,the expression of PGP9.5(pan-marker of enteric neurons),GFAP(pan-marker of EGCs),and GDNF in colon tissues were detected by western blot and quantitative PCR.Phase two: Based on results of phase one,8 weeks of diabetes and EA treatment were selected in this phase.Wild C57BL/6 male mice were randomly divided into five groups: normal control group(N group),diabetic mellitus group(DM group),diabetic mellitus +sham EA group(DM+SEA group),diabetic mellitus + low-frequency EA group(DM+LEA group),and diabetic mellitus + high-frequency EA group(DM+HEA group).After the diabetes model was successfully established,EA treatment started and continued for 8 weeks.Then,colon transits were assessed by defecation frequency and fecal water content after whole process of EA treatment finished.Western Blot,real-time PCR and immunofluorescence double-staining were used to detect the expression of PGP9.5,n NOS,GFAP and GDNF in colon tissues,and Western Blot and real-time PCR were used to detect the expression differences ofGDNF/GFR level 1 / Ret and PTEN/PI3K/Akt pathway molecules in the colon of each group of mice.Results:(1)PGP9.5,GFAP and GDNF all declined at 8 weeks of diabetes;(2)EA could effectively promote colon transit of diabetic mice,restore enteric neurons in the colon of diabatic mice and increased the number but also enhanced the function of EGCs,especially HEA.(3)Compared with the N group,the expression of GDNF,GFRα1 in the DM group decreased,which could be partially corrected by EA and the effect of HEA was more significant.There was no significant difference in the expression levels of Ret,PTEN and Akt in each group.The expression level of p-Akt protein was decreased in the DM group,and increased EA,and the effect of HEA was significant.There was no significant difference in p-PTEN protein expression between the DM group and the N group,but it increased in the EA groups.Conclusion: There were damage of enteric neurons,EGCs and decreased colon transmission in diabetic mice.EA increased the expression of EGCs in the colon of diabetic mice and protected enteric neurons through the GDNF/GFRα1 / Ret and PTEN/PI3K/Akt signaling pathways,thereby improving colon motility.