Objective: To observe the effect of electroacupuncture (EA) of "Zusanli" (ST36), "Sanyinjiao" (SP6) and "Liangmen" (ST21) on gastrointestinal motility, blood glucose content and expression of autophagy-related proteins 1 light chain 3 (LC3), p62, phosphatidyli-nositol-3 kinase (PI3K), protein kinase B (Akt), p-Akt and mammalian target protein of rapamycin (mTOR) of interstitial cells of Cajal (ICCs) in the cultured gastric antrum cells in diabetic gastroparesis (DGP) rats, so as to reveal its mechanisms underlying improvement of DGP.
Methods: A total of 45 Sprague Dawley (SD) rats were randomly divided into blank control, model, EA, medication (3-methyladenine, 3-MA) and EA+3-MA groups, with 9 rats in each group. The DGP model was established by intraperitoneal injection of 2% streptozotocin (STZ) combined with high-fat and high sugar diet for 8 weeks. The gastric emptying rate was measured by using gavage of phenol red (to measure the propelling length of the phenol red/total length of small intestine ×100%). The symptom score (mental state, coat color and luster, behavior and activity, stool traits) of rats was observed every week and the blood glucose content was measured by using a glucometer. EA (20 Hz/100 Hz, 2 mA) was applied to unilateral ST36, SP6 and ST21 alternatively for 15 min, once daily, 5 days a week for 3 weeks. Rats of the 3-MA and 3-MA+EA groups received intraperitoneal injection of 3-MA (30 mg·kg-1·d-1, 10 mg/mL), once daily, 5 days a week for 3 weeks. After 15 days' intervention, the rats were operated for gastric emptying rate test, specimen collection, isolation, and culture of primary ICCs. The expression levels of microtubule associated protein LC3, p62, PI3K, Akt, p-Akt and mTOR of ICCs of cultured gastric antrum cells were detected using Western blot, and the number of autophagosomes in ICC of gastric antrum was observed under transmission electron microscope.
Results: Compared with the blank control group, the symptom score, blood glucose, and the expression levels of p62, class Ⅰ PI3K, Akt, p-Akt and mTOR proteins were increased significantly (P<0.01), while the gastric emptying rate and ratio of LC3Ⅱ/LC3Ⅰ and the expression level of class Ⅲ PI3K protein were significantly decreased (P<0.05, P<0.01) in the model group. In comparison with the model group, the increase of symptom score, blood glucose, and expression levels of p62, class Ⅰ PI3K, Akt, p-Akt and mTOR proteins and the decrease of gastric empty rate and LC3Ⅱ/LC3Ⅰ ratio and the expression level of class Ⅲ PI3K protein were all reversed in both EA and EA+3-MA groups (P<0.05, P<0.01), rather than in the 3-MA group. In addition, 3-MA also reversed modeling-induced increase of class Ⅰ PI3K, Akt, p-Akt and mTOR proteins expression (P<0.01). No significant differences were found between the EA and EA+3-MA in downregulating the levels of symptom score and blood glucose content, and in upregulating gastric empty rate(P>0.05). The effect of EA was notably superior to that of EA+3-MA in upregulating the ratio of LC3Ⅱ/LC3Ⅰ and the expression level of class Ⅲ PI3K protein, and in downregulating the expression of p62, class Ⅰ PI3K, Akt, p-Akt and mTOR proteins (P<0.05, P<0.01). The findings of transmission electron microscopy showed obvious swelling, breakage of some mitochondrial cristae in the ICC cells of antrum and no autophagosomes in the model group and 3-MA group, which was milder in the damage of mitochondrial cristae and marked increase in the autophagosomes in both EA and EA+3-MA groups.
Conclusion: EA can improve the gastrointestinal motility and symptoms in DGP rats, which may be related to its functions in downregulating PI3K/Akt/mTOR signaling to promote autophagy level of ICC.
目的：观察电针“足三里”“三阴交”“梁门”对糖尿病胃轻瘫(DGP)大鼠血糖、胃排空及胃窦部Cajal间质细胞(ICC)自噬及磷酯酰肌醇-3 激酶(PI3K)/蛋白激酶B(Akt)/哺乳动物雷帕霉素靶蛋白(mTOR)信号通路的影响，探讨电针改善DGP胃肠动力功能障碍的作用机制。方法： SD大鼠随机分为空白组、模型组、电针组、3-MA组和3-MA+电针组，每组9只。采用腹腔注射链脲佐菌素结合高脂高糖饲料喂养制备DGP大鼠模型。电针组予单侧“足三里”“三阴交”“梁门”电针治疗，每日1次；3-MA组予3-甲基腺嘌呤(3-MA)腹腔注射，浓度为10 mg/mL，剂量为30 mg·kg-1·d-1；3-MA+电针组予3-MA腹腔注射联合电针治疗；每个疗程5 d，共治疗3个疗程。评定大鼠症状积分，测量血糖；酚红灌胃法检测大鼠胃排空率；Western blot法检测胃窦部ICC中微管相关蛋白1轻链3(LC3)、p62、PI3K、Akt、磷酸化Akt(p-Akt)、mTOR的蛋白表达水平；透射电镜观察胃窦部ICC内自噬体数量。结果：造模后，与空白组比较，模型组、电针组、3-MA组和3-MA+电针组症状积分及血糖水平均升高(P<0.01)。治疗后，与空白组比较，模型组症状积分、血糖水平仍升高(P<0.01)，胃排空率降低(P<0.01)，LC3Ⅱ/LC3Ⅰ比值和Ⅲ型PI3K蛋白表达降低(P<0.05)，p62、Ⅰ型PI3K、Akt、p-Akt、mTOR蛋白表达升高(P<0.01)；与模型组比较，电针组和3-MA+电针组症状积分、血糖水平均降低(P<0.01)，胃排空率升高(P<0.01)，LC3Ⅱ/LC3Ⅰ比值和Ⅲ型PI3K蛋白表达升高(P<0.01)，p62、Ⅰ型PI3K、Akt、p-Akt、mTOR蛋白表达降低(P<0.01，P<0.05)，3-MA组LC3Ⅱ/LC3Ⅰ比值及Ⅲ型PI3K、Ⅰ型PI3K、Akt、p-Akt、mTOR蛋白表达降低(P<0.05,P<0.01)；与3-MA组比较，电针组、3-MA+电针组症状积分、血糖水平均降低(P<0.01)，胃排空率升高(P<0.01)，LC3Ⅱ/LC3Ⅰ比值和Ⅲ型PI3K蛋白表达升高(P<0.01)，p62、Ⅰ型PI3K、Akt、p-Akt、mTOR蛋白表达均降低(P<0.01，P<0.05)；与电针组比较，3-MA+电针组LC3Ⅱ/LC3Ⅰ比值和Ⅲ型PI3K蛋白表达降低(P<0.01)，p62、Ⅰ型PI3K、Akt、p-Akt、mTOR蛋白表达均升高(P<0.01)。透射电镜结果显示，模型组、3-MA组大鼠胃窦部ICC细胞内受损较重，未见明显自噬结构；电针组、3-MA+电针组胃窦部ICC细胞内自噬小体数量明显增多，ICC受损状况有所改善。结论： 电针可能通过调控PI3K/Akt/mTOR通路调节胃窦部ICC自噬水平，从而改善DGP胃肠动力功能障碍。.
Keywords: Autophagy; Cajal interstitial cells; Diabetic gastroparesis; Electroacupuncture; PI3K/Akt/mTOR signaling pathway.