基于P38 MAPK信号通路探讨艾灸督脉对APP/PS1双转基因AD小鼠自噬水平的研究

Effect of Moxibustion at Du Meridian on Autophagy Level of APP/PS1 Double Transgenic AD Mice Based on P38 MAPK Signaling Pathway

目的:观察基于p38丝裂原激活蛋白激酶(MAPK)信号通路来探讨艾灸督脉“百会”“大椎”“风府”穴对APPswe/PS1de9(APP/PS1)双转基因阿尔茨海默病(AD)小鼠脑内β-淀粉样蛋白(Aβ)清除功能的影响。方法:将48只APP/PS1双转基因AD小鼠随机分为模型组、艾灸组、西药Ⅰ组和西药Ⅱ组,每组鼠12只,同条件同龄C57BL/6J雄性小鼠为对照组。艾灸组鼠实按灸“百会”、雀啄灸“大椎”和“风府”;西药Ⅰ组每鼠腹腔注射雷帕霉素;西药Ⅱ组即艾灸组治疗加3-甲基腺嘌呤(3-MA)腹腔注射;模型组和对照组动物不给予任何干预。各组鼠于治疗结束后,用免疫组织、透射电镜、Western blot法依次分别检测小鼠皮质和海马脑区Aβ1-42表达水平、海马区自噬体形成和海马组织p38MAPK、Bcl-2、Beclin-1相关蛋白表达。结果:免疫组织化学结果显示,模型组与对照组相比小鼠大脑皮质和海马区Aβ1-42蛋白表达阳性增多(P<0.05);艾灸、西药Ⅰ、西药Ⅱ组与模型组相比小鼠脑部Aβ1-42蛋白表达阳性减少(P<0.05);西药Ⅰ组与艾灸组相比其小鼠脑部Aβ1-42蛋白表达差异无统计学意义(P>0.05),而西药Ⅱ组小鼠脑部Aβ1-42蛋白表达阳性增多(P<0.05)。透射电镜结果显示,模型组海马区见神经元变形、萎缩或不规则,自噬泡减少;艾灸组海马区部分神经元变形、萎缩及不规则,自噬泡增多;西药Ⅰ组自噬泡增多,神经元变形;西药Ⅱ组偶见少量自噬泡和变形神经元。Western blot结果显示,模型组p38MAPK、Bcl-2蛋白表达较对照组显著升高(P<0.05),而模型组Beclin-1蛋白表达显著下降(P<0.05)。艾灸组、西药Ⅰ组、西药Ⅱ组均与模型组相比小鼠p38MAPK、Bcl-2蛋白表达均明显下降(P<0.05),但Beclin-1蛋白表达上升(P<0.05)。西药Ⅰ组与艾灸组相比小鼠p38MAPK、Bcl-2蛋白表达稍微升高(P<0.05),而Beclin-1蛋白表达稍微下降(P<0.05);而西药Ⅱ组小鼠p38MAPK、Bcl-2蛋白表达显著升高(P<0.05),Beclin-1蛋白表达显著下降(P<0.05)。西药Ⅱ组与西药Ⅰ组相比其小鼠p38MAPK、Bcl-2蛋白表达显著升高(P<0.05),Beclin-1蛋白表达显著下降(P<0.05)。结论:加速自噬使艾灸督脉可以清除APP/PS1双转基因AD小鼠脑区Aβ1-42蛋白异常沉积,作用机制可能与艾灸督脉抑制P38 MAPK信号通路的异常激活有关。

Objective:To study the effect of moxibustion at Baihu(DU 20),Dazhui(DU 14)and Fengfu(DU 16)on the clearing function of amyloidβ-peptide(Aβ)in the brain of double transgenic APPswe/PS1de9(APP/PS1)mice with Alzheimer's disease(AD)by studying the p38MAPK signaling pathway.Methods:48 APP/PS1 double transgenic AD mice were randomly divided into the model group,the moxibustion group,the western medication I group and the western medication II group,with 12 mice in each group.Another 12 male C57BL/6J mice with same conditions and same age were taken as the control.The mice in the moxibustion group were treated with moxibustion at DU20,DU14 and DU16;western medication I group was treated with intraperitoneal injection of Rapamycin;western medication II group was treated with moxibustion plus intraperitoneal injection of 3-MA.The treatment duration was two weeks.The expression levels of Aβ1-42 of cortex and hippocampus,autophagy formation and relative proteins of p38MAPK,Bcl-2 and Beclin-1 in hippocampus were measured by immunohistochemical method,transmission electron microscope and Western blot respectively.Results:Immunohistochemical results showed that the positive protein expression of Aβ1-42 were increased in the cortex and hippocampus in the model group than that in the control group(P<0.05);compared to that in the model group,the positive protein expression of Aβ1-42 was decreased in the moxibustion group,the western medication I group and the western medication II group(P<0.05);there were no statistical difference in Aβ1-42 protein expression between the western medication I group and the moxibustion group(P>0.05);Aβ1-42 protein expression was increased in the western medicine II group(P<0.05).The results of transmission electron microscope showed that the deformation,atrophy or irregularity of neurons were observed in the hippocampal area of the model group,and autophagy vesicles in the cytoplasm were decreased;some neurons in the hippocampus were deformed,atrophic and irregular,and autophagy vesicles were increased in the moxibustion group;autophagy vesicles were increased and the neurons were deformed in the western medication I group;a small amount of autophagy vesicles and deformed neurons could be seen in the western medication II group.Western blot results showed that the protein expressions of p38MAPK and Bcl-2 were significantly increased(P<0.05),and the protein expression of Beclin-1 was significantly decreased in the model group compared to those in the control group(P<0.05);the protein expressions of p38MAPK and Bcl-2 were significantly decreased(P<0.05),and the protein expression of Beclin-1 was significantly increased in the moxibustion group,the western medication I group and the western medication II group compared to those in the model group(P<0.05);the protein expressions of p38MAPK and Bcl-2 were slightly increased(P<0.05),whereas the protein expression of Beclin-1 was slightly decreased in the western medication I group and the moxibustion group(P<0.05);the protein expressions of p38MAPK and Bcl-2 were greatly increased(P<0.05),whereas the protein expression of Beclin-1 was greatly decreased in the western medication II group(P<0.05);the protein expressions of p38MAPK and Bcl-2 were significantly higher(P<0.05),and the protein expression of Beclin-1 was significantly lower in the western medication II group than those in the western medication I group(P<0.05).Conclusion:By accelerating autophagy,moxibustion at Du meridian can clear abnormal deposition of Aβ1-42 protein in the brain region of APP/PS1 double transgenic AD mice,and the mechanism may be related to the abnormal activation of inhibiting the p38MAPK signaling pathway.