The Effect Of Electroacupuncture On Autophagy Of Gastric Cajal Interstitial Cells In Diabetic Gastroparesis Rats And Its Mechanism Of Action

Objective:1.To verify the regulatory effect of electroacupuncture（EA）at"Zusanli","Sanyinjiao"and"Liangmen"on gastric motility in diabetic gastroparesis rats;2.To observe the effect of EA on autophagy flux in Interstitial cells of Cajal（ICCs）in DGP rats and explore the role of autophagy in the progress;3.To observe the effect of EA on autophagy pathway in DGP rats and explore the possible mechanism of EA on autophagy of ICCs.Methods:The DGP rat model was established by single injection of streptozotocin followed by irregular high fat and high sugar diet.After the model was successfully established,the rats in the EA group were treated with EA at"Zusanli","Sanyinjiao"and"Liangmen".The rats in the metoclopramide group（MP group）were treated with 1.7%metoclopramide solution（1ml/100g）for 3 weeks.After the intervention,blood glucose was measured by blood glucose meter;gastric emptying rate was measured by phenol red;serum insulin（INS）content was measured by radioimmunoassay;autophagy of ICC cells in gastric antrum was observed by transmission electron microscope（TEM）;the primary cells of ICC in gastric antrum were separated and extracted by Western Blot（WB）;the expression of PTEN gene was detected by fluorescence quantitative PCR.Results:1.Compared with normal control group（NC）,both the general score and blood glucose level in DGP group were remarkably increased（P<0.01）,and INS and gastric emptying rate were greatly decreased（P<0.01）;compared with DGP group,the general score and blood glucose level in EA group were significantly reduced（P<0.01）,and INS and gastric emptying rate were greatly increased（P<0.01）;compared with EA group,the general score and blood glucose level in EA group were greatly increased（P<0.05）.2.Observation under TEM:No autophagy was observed in NC group;1autophagosome was observed in DGP group,and the endoplasmic reticulum and mitochondria were obviously swollen;there were 1 autophagosome and1autophagolysosome in EA group;2 phagocytic vesicles in MP group.Observation of autophagy flux in ICCs under confocal laser:compared with the NC group,fluorescence intensity of GFP and RFP in the DGP group were significantly enhanced（P<0.01,P<0.05）;compared with the DGP group,fluorescence intensity of GFP and RFP in the EA group were significantly decreased（P<0.01,P<0.05）.Relative expression of autophagy marker protein LC3:compared with NC group,the ratio of LC3II/LC3I in the DGP group and EA group increased significantly（P<0.01,P<0.05）;compared with the DGP group,the ratio of LC3II/LC3I in EA group and MP group decreased significantly（P<0.05）.Relative expression of autophagy substrate P62protein:compared with the NC group,the relative expression of P62 in DGP,EA and MP groups increased significantly（P<0.01）;compared with DGP group,EA group and MP group decreased significantly（P<0.01）.3.The concentration of Ca²⁺in ICCs of gastric antrum:compared with the NC group,the fluorescence intensity of Ca²⁺in DGP group decreased significantly（P<0.01）;compared with DGP group,the fluorescence intensity of Ca²⁺in EA group and MP group increased significantly（P<0.01,P<0.05）.The expression of regulatory protein of autophagy pathway:compared with the NC group,the relative expression of PI3K,PIP3,Akt,p-Akt,mTOR,p-mTOR and AMPK in the DGP group increased significantly（P<0.01）,and Akt and p-Akt in the EA group and MP group also increased significantly（P<0.01,P<0.01）;compared with the DGP group,the expression of PIP3,Akt,p-mTOR and AMPK in EA group and MP group increased significantly（P<0.01）,while the expression of PI3K and mTOR in both EA group and MP group and p-Akt in MP group increased significantly（P<0.01）.The expression of PTEN gene:compared with NC group,the expression of PTEN m RNA in DGP,EA and MP groups decreased significantly（P<0.01）;compared with DGP group,the expression of PTEN m RNA in EA and MP groups decreased significantly（P<0.01,P<0.01）;compared with EA group,PTEN expression in MP group increased significantly（P<0.05）.Conclusion:1.EA could significantly improve gastrointestinal motility and general condition of DGP rats.2.EA could relive the suppression of autophagy flux of ICCs rats and improve gastric motility in DGP.3.The suppression of autophagy flux of ICCs in DGP rats might be caused by activation of PI3K-Akt-mTOR pathway.EA could relieve the blocked autophagy flux by regulating key proteins of PI3K-Akt-mTOR pathway.