针刺对SAMP8小鼠海马线粒体SIRT3调控因子GATA-2表达的影响

Effect of Acupuncture on the Expression of Mitochondria SIRT3 Regulator GATA-2 in Hippocampus of SAMP8 Mice

目的观察针刺对SAMP8小鼠学习记忆能力、海马线粒体SIRT3调控因子GATA-2表达的影响,探讨其防治阿尔茨海默病的可能机制。方法将27只6月龄SAMP8小鼠按随机数字表法分为模型组、针刺组、抑制剂组,每组9只,另取9只6月龄SAMR1小鼠作为空白组。针刺组和抑制剂组针刺"百会""涌泉",每日1次,每次20 min,5 d为1个疗程,连续干预4个疗程。抑制剂组在治疗结束后水迷宫实验测试中,第1、3、5、7日提前2h腹腔注射SIRT3抑制剂。Morris水迷宫实验观察SAMP8小鼠学习记忆能力;RT-PCR检测GATA-2的mRNA表达;免疫荧光观察海马区GATA-2阳性细胞平均表达面积;Western blot检测小鼠海马线粒体GATA-2蛋白的表达。结果 Morris水迷宫实验:与空白组比较,模型组小鼠平均逃避潜伏期明显延长,第4、5、7日差异有统计学意义(P<0.05,P<0.01),穿越平台次数显著减少(P<0.01);与模型组比较,针刺组小鼠第4、7日平均逃避潜伏期明显缩短(P<0.05),穿越平台次数显著增加(P<0.01);与针刺组比较,抑制剂组第6日小鼠平均逃避潜伏期明显延长(P<0.05),穿越平台次数无明显变化(P>0.05)。RT-PCR检测:与空白组比较,模型组小鼠GATA-2 mRNA表达明显升高(P<0.05);与模型组比较,针刺组小鼠GATA-2mRNA表达明显降低(P<0.05);免疫荧光检测:与空白组比较,模型组小鼠GATA-2阳性细胞平均表达面积明显减少(P<0.05);与模型组比较,针刺组小鼠GATA-2阳性细胞平均表达面积明显增加(P<0.05);与针刺组比较,抑制剂组小鼠GATA-2阳性细胞平均表达面积明显减少(P<0.05);Westernblot检测:与空白组比较,模型组小鼠GATA-2蛋白表达明显降低(P<0.05);与模型组比较,针刺组小鼠GATA-2蛋白表达明显升高(P<0.05);与针刺组比较,抑制剂组小鼠GATA-2蛋白表达明显降低(P<0.05)。结论针刺可提高SAMP8小鼠学习记忆能力,其机制可能与促进SIRT3转录因子GATA-2蛋白的表达、改善线粒体能量代谢有关。

Objective To observe the effect of acupuncture on the SAMP8 mice learning and memory ability and regulatory factor GATA-2 of mitochondria SIRT3 in hippocampus;To explore its possible mechanism to prevent AD.Methods Totally twenty-seven 6-month-old SAMP8 mice were divided into model group,acupuncture group and inhibitor group according to random number table method,and nine 6-month-old SAMR1 mice were set as blank group.The acupuncture group and the inhibitor group were treated with acupuncture at“Baihui”and“Yongquan”acupoints,once a day for 20 minutes each time,5 days as a course of treatment,and 4 consecutive courses of treatment.In the inhibitor group,during the water maze test after treatment,the inhibitor(3-TYP)was injected intraperitoneally two hours earlier on days 1,3,5,and 7.Morris water maze was used to observe the effects of acupuncture on learning and memory ability of SAMP8 mice;RT-PCR was used to detect GATA-2 mRNA expression;immunofluorescence was used to observe the average expression of GATA-2 positive cells in the hippocampus;Western blot was used to detect GATA-2 protein expression in hippocampus mitochondria.Results Morris water maze experiment results showed that compared with the blank group,the average escape latency of the model group was significantly prolonged,of which the difference was significant on the 4th,5th(P<0.05),and 7th(P<0.01)day.The number of animal crossings on the platform decreased significantly(P<0.01).Compared with the model group,the average escape latency of the acupuncture group on the 4th and 7th days was significantly shortened(P<0.05),and the number of crossing platforms increased significantly(P<0.01).Compared with the acupuncture group,the average escape latency on the 6th day of the inhibitor group was significantly prolonged(P<0.05),and the number of crossing platforms was not statistically significant(P>0.05).RT-PCR results showed that compared with the blank group,the GATA-2 mRNA content in the model group significantly increased(P<0.05);compared with the model group,the GATA-2 mRNA content in the acupuncture group was significantly reduced(P<0.05).Immunofluorescence results showed that compared with the blank group,the average expression area of GATA-2 positive cells in the model group was significantly reduced(P<0.05);compared with the model group,the average expression area of GATA-2 positive cells in the acupuncture group increased significantly(P<0.05);compared with the acupuncture group,the average expression area of GATA-2 positive cells in the inhibitor group was significantly reduced(P<0.05).Western blot detection showed that the relative expression level of GATA-2 protein in the model group was significantly reduced compared with the blank group(P<0.05);compared with the model group,the expression of GATA-2 protein in the acupuncture group significantly increased(P<0.05);compared with the acupuncture group,the expression of GATA-2 protein in the inhibitor group significantly decreased(P<0.05).Conclusion Acupuncture can improve the learning and memory ability of SAMP8 mice,which may be related to promoting the expression of SIRT3 transcription factor GATA-2 protein and improving mitochondrial energy metabolism.