电针诱导沉默信息调节因子1依赖性自噬对小鼠脑卒中后中枢性痛的影响

Effect of electroacupuncture in alleviating central post-stroke pain in mice by inducing silent information regulator 1-dependent autophagy

目的探讨电针对小鼠脑卒中后中枢性痛(CPSP)的影响及其与沉默信息调节因子1(SIRT1)和自噬的关系。方法选取无特定病原体(SPF)级健康雄性ICR小鼠40只,随机分为假手术组(Sham组)、CPSP组、CPSP+电针组(EA组)和CPSP+电针+SIRT1抑制剂EX527组(EA+EX527组),每组10只。采用Ⅳ型胶原酶注射入丘脑腹后外侧核建立小鼠CPSP模型。模型建立24 h后,EA组和EA+EX527组采用电针(2 Hz/15 Hz频率疏密波)刺激小鼠人中、三阴交及内关穴(30 min/d,持续14 d),且EA+EX527组每次电针刺激前30 min腹腔注射5 mg/kgEX527。于模型建立前1 d(T_(0))和模型建立后3、7、14 d(T_(1)、T_(2)、T_(3))时测定各组小鼠的机械缩足频率(PWF)、热缩足潜伏期(TWL)和冷缩足潜伏期(CWL)。疼痛行为学测试完成后,处死小鼠取脑组织。采用干湿质量法检测脑组织含水量,采用蛋白质印迹法(Western blot)检测SIRT1、Beclin-1、p62表达水平。结果与Sham组比较,CPSP组、EA组和EA+EX527组小鼠T_(1)、T_(2)、T_(3)时PWF升高,TWL、CWL缩短,脑组织含水量升高,SIRT1、Beclin-1表达下调,p62表达上调,差异有统计学意义(P<0.05)。EA组小鼠T_(1)时PWF、TWL、CWL与CPSP组比较,差异无统计学意义(P>0.05);与CPSP组比较,EA组小鼠T_(2)、T_(3)时PWF降低,TWL、CWL延长,脑组织含水量降低,SIRT1、Beclin-1表达上调,p62表达下调,差异有统计学意义(P<0.05)。EA+EX527组小鼠T_(1)时PWF、TWL、CWL与EA组比较,差异无统计学意义(P>0.05);与EA组比较,EA+EX527组小鼠T_(2)、T_(3)时PWF升高,TWL、CWL缩短,脑组织含水量升高,SIRT1、Beclin-1表达下调,p62表达上调,差异有统计学意义(P<0.05)。结论电针可能通过激活SIRT1的表达促进自噬,从而减轻小鼠脑卒中后中枢性痛。

Objective To explore the effect of electroacupuncture on central post-stroke pain(CPSP)and its relationships with silent information regulator 1(SIRT1)and autophagy in mice.Methods Forty male specific pathogen free(SPF)grade healthy male ICR mice were selected,and were randomly divided into Sham group,CPSP group,CPSP+electric acupuncture group(EA group)and CPSP+electroacupuncture+SIRT1 inhibitor EX527 group(EA+EX527group),with 10 mice in each group.CPSP model was induced in mice by injection of type VI collagenase in the nucleus ventralis posterolateralis thalami.The mice in the EA group and the EX527+EA group were stimulated at Neiguan,Renzhong and Sanyinjiao acupoints by frequency of condensation and rarefaction wave of 2/15 Hz at 24 h after modelingfor 30 min per day,lasting for 14 d and the EA+EX527 group was intraperitoneally injected 5 mg/kg EX527 at 30 min before each stimulation.Before the modeling(T_(0))and 3,7 and 14 days(T_(1),T_(2) and T_(3),respectively)after the establishment of the model,the paw withdrawal frequency(PWF),thermal withdrawal latency(TWL)and cold withdrawal latency(CWL)of each group were measured.The mice were sacrificed and brains were collected after pain behavioral tests.The brain water content was measured using dry-humid weight method,and the expressions of SIRT1,Beclin-1 and p62 were analyzed by western blotting.Results Compared with the Sham group,the PWF was significantly increased,the TWL and CWL were significantly shortened,the brain water content increased,the expressions of SIRT1 and Beclin-1 in brain tissue were down-regulated,and the expressions of p62 in brain at T_(1),T_(2) and T_(3)were up-regulated in the CPSP group,EA group and EA+EX527 group(P<0.05).Compared with the CPSP group,the PWF,TWL and CWL in the EA group showed no significant change at T_(1)(P>0.05).The values of PWF were significantly increased at T_(2) and T_(3) in the EA group,and the TWL and CWL were significantly prolonged,water content in the brain decreased,the expressions of SIRT1 and Beclin-1 were up-regulated,and the expression of p62 in brain was down-regulated in the EA group compared with the CPSP group(P<0.05).Compared with the EA group,the PWF,TWL and CWL in the EA+EX527 group showed no significant changes at T_(1)(P>0.05).The values of PWF were significantly increased at T_(2) and T_(3) in the EA+EX527 group,and the TWL and CWL were significantly shortened,water contentin the brain increased,the expressions of SIRT1 and Beclin-1 were down-regulated,and the expression of p62 in brain was up-regulated compared with the EA group(P<0.05).Conclusion Electroacupuncture can promote autophagy via increasing the expression of SIRT1,thereby alleviating central post-stroke pain in mice.