电针对衰老模型大鼠睾丸AMPK/SIRT1/NF-κB信号通路的影响

Effect of electroacupuncture on the AMPK/SIRT1/NF-κB signaling pathway in testis of aging model rats

摘要:

目的 探讨电针对衰老模型大鼠睾丸腺苷酸活化蛋白激酶（adenosine monophosphate activated protein kinase，AMPK）、沉默信息调节因子1（silent information regulator of transcription 1，SIRT1）、核因子-κB（nuclear factor-κB，NF-κB）蛋白表达的影响及其延缓生殖衰老的作用机制。方法 将40只雄性SD大鼠随机分为对照组（n=10）和造模组（n=30），造模组经腹腔注射D-半乳糖复制衰老模型大鼠，从造模成功的大鼠中随机筛选24只平均分为3组：模型组、药物组和电针组，每组8只。电针组取“肾俞”“关元”予电针治疗，每日治疗1次，每次15 min，治疗5 d休息2 d，连续治疗8周。药物组予腹部皮下注射丙酸睾酮注射液（7 mg/kg，每3日1次），连续８周。对照组和模型组给予腹部皮下注射0.9%氯化钠溶液，注射剂量及疗程同药物组。治疗前后检测大鼠血清总睾酮（total testosterone，TT）和游离睾酮（free testosterone，FT）水平，免疫组化法、Western blot检测大鼠睾丸AMPK、p-AMPK、SIRT1、NF-κB p65蛋白表达。结果 治疗前，与对照组比较，模型组、药物组及电针组血清TT、FT水平降低（P=0.000，P=0.001；P=0.000，P=0.001；P=0.000，P=0.000）；治疗后，与对照组比较，模型组、药物组及电针组血清TT、FT水平降低（P=0.000，P=0.000；P=0.009，P=0.025；P=0.006，P=0.022），与模型组比较，药物组及电针组血清TT、FT明显升高（P=0.001，P=0.004；P=0.001，P=0.004）。与对照组比较，模型组、药物组及电针组p-AMPK、SIRT1蛋白表达均明显降低（P=0.000，P=0.000；P=0.000，P=0.000；P=0.000，P=0.000），NF-κB p65蛋白表达明显升高（P=0.000，P=0.000，P=0.005）。与模型组及药物组比较，电针组p-AMPK、SIRT1蛋白表达明显升高（P=0.000，P=0.000；P=0.023，P=0.002），NF-κB p65蛋白表达明显降低（P=0.000，P=0.000）。结论 电针可能通过激活睾丸间质细胞（Leydig细胞）AMPK/SIRT1/NF-κB通路，抑制Leydig细胞炎性反应，从而改善血清TT、FT水平，这可能是电针延缓生殖衰老的作用机制之一。

Abstract:

Objective To investigate the effect of electroacupuncture（EA） on the protein expression of adenosine monophosphate activated protein kinase（AMPK）， silent information regulator of transcription 1（SIRT1） and nuclear factor-κB（NF-κB） in testis of aging model rats and its mechanism of delaying reproductive aging.Methods Forty male Sprague-Dawley rats were randomly divided into the control group（n=10） and the model group（n=30）. The aging model rats were replicated by intraperitoneal injection of D-galactose in the model group. Twenty-four rats were randomly selected from the successful model rats and divided into three groups： the model group， the drug group and the EA group， with 8 rats in each group. In the EA group，“Shen yu” and “Guan yuan” were used for EA treatment， once a day，15 minutes each time，5 days of treatment and 2 days of rest，continuous treatment for 8 weeks. The drug group received abdominal subcutaneous injection of testosterone propionate（7 mg/kg once，once every 3 days） for continuous 8 weeks. The control group and model group were given abdominal subcutaneous injection of 0.9% sodium chloride solution. The injection dose and course of treatment were the same as those in the drug group. The levels of serum total testosterone（TT） and free testosterone（FT）were detected before and after treatment. The protein expression of AMPK，p-AMPK，SIRT1 and NF-κB p65 in rat testis were detected by immunohistochemistry and Western blot.Results Before treatment，compared with the control group，the levels of serum TT and FT in the model group，drug group and EA group were decreased（P=0.000，P=0.001；P=0.000，P=0.001；P=0.000，P=0.000）. After treatment，compared with the control group，the levels of serum TT and FT in the model group，drug group and EA group were decreased（P=0.000，P=0.000；P=0.009，P=0.025；P=0.006，P=0.022）. Compared with the model group，the levels of serum TT and FT in the drug group and EA group were significantly increased（P=0.001，P=0.004；P=0.001，P=0.004）. Compared with the control group， the protein expression of p-AMPK and SIRT1 in the model group，drug group and EA group was significantly decreased（P=0.000，P=0.000；P=0.000，P=0.000；P=0.000，P=0.000），and the expression of NF-κB p65 was significantly increased（P=0.000，P=0.000，P=0.005）. Compared with the model group and drug group，the protein expression of p-AMPK and SIRT1 in the EA group were significantly increased（P=0.000，P=0.000；P=0.023，P=0.002），and the protein expression of NF-κB p65 was significantly decreased（P=0.000，P=0.000）.Conclusion EA may inhibit the inflammatory response of Leydig cells by activating the AMPK/SIRT1/NF-κB pathway of Leydig cells， thereby improving the levels of serum TT and FT， which may be one of the mechanisms of EA in delaying reproductive aging.