四君子丸对SAMP8小鼠海马CA3区神经元Lon蛋白表达的影响

Effect of Si Junziwan on Expression of Lon Protein in Hippocampal CA3 Region of SAMP8 Mice

目的:研究SAMP8小鼠海马Lon蛋白及线粒体动力学相关蛋白表达变化,为健脾益气法治疗阿尔茨海默病提供理论依据。方法:3月龄SAMR1小鼠8只作为正常组,3月龄SAPM8小鼠32只分为模型组、多奈哌齐组(0.013 g·kg^(-1))、四君子丸低、高剂量组(3.24、12.56 g·kg^(-1)),每组8只,多奈哌齐组灌胃多奈哌齐,四君子丸低、高剂量组灌胃四君子丸溶液,灌胃30 d;第25天开始水迷宫定位航行实验,第30天开始水迷宫空间探索实验;第30天取材后,免疫组化检测线粒体融合蛋白2(MFN2)蛋白表达变化,酶联免疫吸附测定法(ELISA)检测腺苷酸活化蛋白激酶(AMPK)表达,比色法检测三磷酸腺苷(ATP)含量,电镜检测神经元线粒体微观结构变化,蛋白免疫印迹法(Western blot)检测β淀粉样蛋白(Aβ)、Lon蛋白、线粒体动力相关蛋白1(DRP1)蛋白、MFN1蛋白表达变化。结果:与正常组比较,模型组小鼠逃避潜伏期时间增加、穿越次数减少、AMPK表达上调,ATP含量降低,Aβ蛋白表达升高(P<0.01),DRP1蛋白表达显著升高(P<0.01),Lon、MFN1蛋白表达降低(P<0.05,P<0.01),MFN2蛋白减少,线粒体空泡化增加,嵴断裂;与模型组比较,四君子丸低、高剂量组逃避潜伏期时间减少(P<0.01)、穿越次数明显增加(P<0.05),AMPK表达下调(P<0.01);四君子丸高剂量组ATP含量显著升高(P<0.01),Aβ、DRP1蛋白表达下调(P<0.05,P<0.01);MFN1蛋白表达上调(P<0.05),四君子丸低剂量组空泡化较为明显,四君子丸高剂量组空泡化有所恢复,嵴较为清晰。结论:四君子丸可以通过上调Lon蛋白表达变化、纠正线粒体分裂融合蛋白紊乱,改变SAMP8小鼠的记忆功能,达到治疗阿尔茨海默病的目的。

Objective:To study the expression changes of Lon protein and mitochondrial dynamicsrelated protein in the hippocampus of SAMP8 mice and provide a theoretical basis for the treatment of Alzheimer’s disease by invigorating the spleen and supplementing Qi.Method:Eight 3-month-old SAMR1mice were used as the normal group,and 323-month-old SAPM8 mice were divided into model group,western medicine group(0.013 g·kg^(-1)),low-dose Si Junziwan group(3.24 g·kg^(-1)),and high-dose Si Junziwan group(12.56 g·kg^(-1)),with 8 mice in each group.The western medicine group was gavaged with donepezil,and the S Junziwan low-and high-dose groups were gavaged with Si Junziwan for 30 days.The positioning navigation experiment of the water maze was started on the 25day,and the space exploration experiment of the water maze was started on the 30day.On the 30day,the protein expression of mitofusin 2(MFN2)was detected by immunohistochemistry,the expression of adenosine 5’-monophosphate(AMP)-activated protein kinase(AMPK)was detected by enzyme-linked immunosorbent assay(ELISA),the content of ATP was detected by colorimetry,the microstructure of neuron mitochondria was detected by electron microscope,and the expressions of Aβ protein,Lon protein,dynamin-related protein 1(DRP1)protein,and MFN1 protein were detected by Western blot.Result:As compared with the normal group,the latency escape period increased,the number of crossings decreased,the expression of AMPK increased,and the content of ATP decreased in the model group.The expressions of Aβ protein and DRP1 protein increased(P<0.01),whereas the expressions of Lon protein,MFN1 protein decreased in the model group(P<0.05,P<0.01),and MFN2 protein decreased.The vacuolation of mitochondria increased and the cristae broke in the model group.As compared with model group,the time of the latent escape period decreased(P<0.01),and the number of crossings increased in the low-dose and high-dose Si Junziwan groups(P<0.05).The expression of AMPK(P<0.01)decreased,the content of ATP increased(P<0.01),the expression of Aβ and DRP1 protein decreased(P<0.05,P<0.01),and the expression of MFN1 protein was up-regulated(P<0.05)in high-dose Si Junziwan groups.The vacuolation was more obvious in the low-dose Si Junziwan group,whereas the vacuolation was restored and the ridge was clear in the high-dose Si Junziwan group.Conclusion:Si Junziwan treats Alzheimer’s disease by up-regulating the protein expression of Lon,correcting the disorder of mitochondrial division and fusion protein,and changing the memory function of SAMP8 mice.