基于胰腺PI3K/AKT/BMAL1通路探讨针刺对2型糖尿病大鼠血糖昼夜节律的影响

Discussion about the mechanism of acupuncture in improving circadian rhythms of glucose based on the PI3K/AKT/BMAL1 pathway of the pancreas

[目的]通过观察PI3K/AKT/BMAL1通路相关蛋白的表达变化及针刺效应,探讨调理脾胃针法调节2型糖尿病大鼠血糖昼夜节律的作用机制。[方法]将36只SPF级雄性SD大鼠采用随机数字表法分为空白组(10只),造模组(26只)。造模组以高脂高糖喂养2个月,而后采用小剂量(25 mg/kg)腹腔注射链脲佐菌素的方法建立2型糖尿病大鼠针刺效应平台。并将造模成功的20只大鼠随机分为针刺组(10只),模型组(10只)。针刺组采用“调理脾胃针法”针刺,每日治疗1次,每周治疗5次,共治疗4周。空白组及模型组不进行干预。主要观察治疗前后大鼠4时相(0、6、12、18时)血糖变化及平均血糖的标准差(SDBG)、最大血糖波动幅度(LAGE);以酶联免疫吸附法(ELISA)检测治疗前后空腹血清胰岛素含量;以胰腺荧光TUNEL染色观察治疗后胰岛细胞凋亡情况,计算凋亡指数(AI);以蛋白质印迹法(Western blot)检测治疗后磷脂酰肌醇3激酶(PI3K)、丝氨酸/苏氨酸蛋白激酶(AKT)及p-AKT、脑和肌肉芳香烃受体核转位蛋白1(BMAL1)表达。[结果]干预后与空白组比较,模型组各时相血糖、SDBG、LAGE仍然显著升高,空腹血清胰岛素(Fins)含量显著降低(P<0.01),但针刺组与模型组比较,各时相血糖、SDBG、LAGE显著下降,Fins含量显著升高(P<0.01);与模型组相比,针刺可明显改善胰岛细胞的凋亡,降低AI值;干预后与模型组比较,针刺组各蛋白(PI3K、AKT、p-AKT、BMAL1)表达均显著升高(P<0.01)。[结论]调理脾胃针法可改善2型糖尿病大鼠血糖昼夜节律,机制与上调胰腺PI3K/AKT/BMAL1相关蛋白表达相关。

[Objective]To investigate the mechanism of regulating spleen-stomach acupuncture method in regulating glucose homeostasisof type 2 diabetic rats by observing the expression changes of PI3K/Akt/BMAL1 pathway.[Methods]The 36 SD rats were randomly divided into normal group(n=10)and model group(n=26).The model group was induced by a high-fat diet for 2 months,and then was injected with small dose(25 mg/kg)of streptozotocin to establish the acupuncture effect platform in type 2 diabetic rats.The 20 rats were randomly divided into acupuncture group(n=10)and model group(n=10).Acupuncture group was treated with regulating spleen-stomach acupuncture method,once a day for 4 weeks.Thenormal group and model group were not interfered.The blood glucose of rats at 4 hours(0∶00,6∶00,12∶00,18∶00)and SDBG,LAGE were observed before and after treatment.Fasting serum insulin was detected by ELISA before and after treatment.The apoptosis of pancreatic islet cells after treatment was observed by fluorescence TUNEL stainingand the apoptotic index(AI),the expressions of PI3K,Akt,p-Akt,and BMAL1 by Western blot.[Results]Compared with normal group,blood glucose in model group and SDBG and LAGE were still significantly increased and FINS were significantly decreased(P<0.01)after intervention,but the blood glucose and SDBG and LAGE were significantly decreased and FINS were increased in acupuncture group compared with model group(P<0.01);Compared with the model group,acupuncture significantly improved the apoptosis of islet cells and decreased the AI value.After intervention,compared with the model group,the expression of PI3K,Akt,p-Akt,BMAL1 in the acupuncture group were significantly increased(P<0.01).[Conclusion]Regulating spleen-stomach acupuncture method can improve glucose homeostasis in type 2 diabetic rats,enhancing of pancreatic PI3K/Akt/BMAL1 related protein expression.