Objective: To observe the hypoglycemic effect of electroacupuncture (EA) at "Tianshu" (ST 25) combined with metformin on rats with type 2 diabetes mellitus (T2DM) as well as its effect on expression of adenosine monophosphate activated protein kinase (AMPK) in liver and pancreas.
Methods: Thirty-six male SD rats were randomly divided into a blank group (6 rats) and a model establishing group (30 rats). The rats in the model establishing group were fed with high-fat diet and treated with intraperitoneal injection of low-dose streptozotocin (STZ) to establish T2DM model. The rats with successful model establishment were randomly divided into a model group, a control group, a metformin group, an EA group and a combination group, 6 rats in each group. The rats in the EA group were treated with EA at "Tianshu" (ST 25), dense-disperse wave, 2 Hz/15 Hz in frequency and 2 mA in current intensity, 20 min each time. The rats in the metformin group were treated with intragastric administration of metformin (190 mg/kg) dissolved in 0.9% sodium chloride solution (2 mL/kg). The rats in the combination group were treated with EA at "Tianshu" (ST 25) and intragastric administration of metformin. The rats in the control group were treated with intragastric administration of 0.9% sodium chloride solution with the same dose. All the treatments were given once a day for 5 weeks. After the intervention, the body mass and random blood glucose were detected; the serum insulin level was detected by ELISA; the expression of AMPK and phosphorylated adenosine monophosphate activated protein kinase (p-AMPK) in liver and pancreas was detected by Western blot method; the expression of protein gene product 9.5 (PGP9.5) was detected by immunofluorescence.
Results: ①Compared with the blank group, the body mass in the model group was decreased (P<0.05); compared with the model group, the body mass in the EA group and the combination group was decreased (P<0.05); the body mass in the EA group and the combination group was lower than the metformin group (P<0.05). Compared with the blank group, the random blood glucose in the model group was increased (P<0.01); compared with the model group, the random blood glucose in the metformin group, the EA group and the combination group was decreased (P<0.01). The random blood glucose in the combination group was lower than the metformin group and the EA group (P<0.05). ②Compared with the blank group, the insulin level in the model group was decreased (P<0.05); compared with the model group, the insulin level in the metformin group, the EA group and the combination group was all increased (P<0.05). The insulin level in the combination group was higher than the metformin group and the EA group (P<0.05). ③Compared with the blank group, the protein expression of AMPK and p-AMPK in liver tissue was decreased (P<0.05), and the protein expression of AMPK and p-AMPK in pancreatic tissue was increased (P<0.05) in the model group. Compared with the model group, the protein expression of AMPK and p-AMPK in liver tissue in the metformin group, the EA group and the combination group was increased (P<0.05, P<0.01); the protein expression of AMPK in pancreatic tissue in the metformin group was increased (P<0.05); the protein expression of AMPK in pancreatic tissue in the EA group and the combination group was decreased (P<0.05); the protein expression of p-AMPK in pancreatic tissue in the metformin group, the EA group and the combination group was decreased (P<0.05). The protein expression of AMPK and p-AMPK in liver tissue in the combination group was higher than that in the metformin group and the EA group (P<0.05); the protein expression of AMPK in pancreatic tissue in the EA group and the combination group was less than that in the metformin group (P<0.05), and the expression of p-AMPK protein in pancreatic tissue in the combination group was less than that in the metformin group and the EA group (P<0.05). ④Compared with the blank group, the expression of PGP9.5 in pancreatic tissue in the model group was increased (P<0.01); compared with the model group, the expression of PGP9.5 in pancreatic tissue in the metformin group, the EA group and the combination group was decreased (P<0.05, P<0.01). The expression of PGP9.5 in pancreatic tissue in the EA group was lower than the metformin group and the combination group (P<0.05).
Conclusion: Electroacupuncture at "Tianshu" (ST 25) could promote the effect of metformin on activating AMPK in liver tissue of T2DM rats, improve the negative effect of metformin on AMPK in pancreatic tissue, and enhance the hypoglycemic effect of metformin. The mechanism may be related to the inhibition of pancreatic intrinsic nervous system.
目的：观察电针“天枢”结合二甲双胍对2型糖尿病（T2DM）模型大鼠的降糖效应及对肝脏和胰腺组织腺苷酸活化蛋白激酶（AMPK）表达的影响。方法：将36只雄性SD大鼠随机分为空白组（6只）和造模组（30只）。造模组大鼠采用高脂饲料喂养配合小剂量链脲佐菌素（STZ）腹腔注射的方法建立T2DM模型，将造模成功的大鼠随机分为模型组、对照组、二甲双胍组、电针组和针药结合组，每组6只。电针组大鼠予电针“天枢”干预，疏密波，频率2 Hz/15 Hz，电流强度2 mA，每次20 min；二甲双胍组大鼠采用二甲双胍（190 mg/kg）溶于0.9%氯化钠溶液灌胃（2 mL/kg）；针药结合组予电针“天枢”结合二甲双胍溶液灌胃；对照组大鼠予同等剂量0.9%氯化钠溶液灌胃。均每日1次，连续干预5周。干预结束后，检测大鼠体质量和随机血糖；ELISA法检测大鼠血清胰岛素水平；Western blot法检测大鼠肝脏和胰腺组织AMPK及磷酸化腺苷酸活化蛋白激酶（p-AMPK）蛋白表达；免疫荧光法检测大鼠胰腺组织蛋白基因产物9.5（PGP9.5）表达。结果：①与空白组比较，模型组体质量降低（P<0.05）；与模型组比较，电针组及针药结合组体质量降低（P<0.05）；电针组和针药结合组体质量低于二甲双胍组（P<0.05）。与空白组比较，模型组随机血糖升高（P<0.01）；与模型组比较，二甲双胍组、电针组、针药结合组随机血糖降低（P<0.01）；针药结合组随机血糖低于二甲双胍组和电针组（P<0.05）。②与空白组比较，模型组胰岛素水平降低（P<0.05）；与模型组比较，二甲双胍组、电针组、针药结合组胰岛素水平均升高（P<0.05）；针药结合组胰岛素水平高于二甲双胍组和电针组（P<0.05）。③与空白组比较，模型组肝脏组织AMPK及p-AMPK蛋白表达均减少（P<0.05），胰腺组织AMPK和p-AMPK蛋白表达增加（P<0.05）。与模型组比较，二甲双胍组、电针组、针药结合组肝脏组织AMPK和p-AMPK蛋白表达均增加（P<0.05，P<0.01）；二甲双胍组胰腺组织AMPK蛋白表达增加（P<0.05），电针组和针药结合组胰腺组织AMPK蛋白表达减少（P<0.05），二甲双胍组、电针组、针药结合组胰腺组织p-AMPK蛋白表达减少（P<0.05）。针药结合组肝脏组织AMPK和p-AMPK蛋白表达多于二甲双胍组和电针组（P<0.05）；电针组和针药结合组胰腺组织AMPK蛋白表达少于二甲双胍组（P<0.05），针药结合组胰腺组织p-AMPK蛋白表达少于二甲双胍组和电针组（P<0.05）。④与空白组比较，模型组胰腺组织PGP9.5表达增多（P<0.01）；与模型组比较，二甲双胍组、电针组、针药结合组胰腺组织PGP9.5表达降低（P<0.05， P<0.01）；电针组胰腺组织PGP9.5表达少于二甲双胍组和针药结合组（P<0.05）。结论：电针“天枢”可促进二甲双胍激活T2DM模型大鼠肝脏组织AMPK的效应，改善二甲双胍对胰腺组织AMPK的负性影响，增强二甲双胍的降糖效应，其机制可能与抑制胰腺内在神经系统有关。.
Keywords: Point ST 25 (Tianshu); adenosine monophosphate activated protein kinase (AMPK); combination of acupuncture and medication; electroacupuncture; metformin; type 2 diabetes mellitus (T2DM).