石菖蒲挥发油对帕金森病模型小鼠黑质多巴胺能神经元自噬的影响及其机制

Mechanisms of Acorus calamus volatile oil on autophagy of substantia nigra dopaminergic neurons in Parkinson’s disease model mice

目的探讨石菖蒲挥发油对帕金森病模型小鼠黑质多巴胺能(Dopamine,DA)神经元自噬的影响及其可能机制。方法取45只C57BL/6小鼠,随机选取10只设为正常组,其余小鼠建立帕金森病模型,成功30只,随机分为帕金森病组、石菖蒲组、石菖蒲+激活剂组,每组各10只;石菖蒲+激活剂组灌胃石菖蒲挥发油90 mg/kg,腹腔注射蛋白激酶B(Protein kinase B,AKT)/哺乳动物雷帕霉素靶蛋白(Mammalian target of rapamycin,mTOR)信号通路激活剂MHY1485(10 mg/kg);石菖蒲组灌胃石菖蒲挥发油90 mg/kg,腹腔注射等体积二甲基亚砜(Dimethylsulfoxide,DMSO);正常组、帕金森病组灌胃等体积生理盐水,腹腔注射等体积DMSO;转棒实验检测运动协调能力;Western Blotting法检测黑质微管相关蛋白轻链3B(Light chain 3B,LC3B)、Bcl-2同源结构域蛋白(Bcl-2 homeodomain protein,Beclin1)蛋白表达水平;免疫组织化学染色检测黑质酪氨酸羟化酶(Tyrosine hydroxylase,TH)阳性表达水平;Western Blotting法检测黑质AKT、磷酸化蛋白激酶B(Phosphorylation protein kinase B,p-AKT)、mTOR、磷酸化哺乳动物雷帕霉素靶蛋白(Phosphorylation mammalian target of rapamycin,p-mTOR)蛋白表达水平。结果与帕金森病组比较,石菖蒲组小鼠坚持运动时间延长,LC3B,Beclin1蛋白表达水平、TH阳性细胞占比升高(P<0.05);与石菖蒲组比较,石菖蒲+激活剂组小鼠坚持运动时间缩短,LC3B、Beclin1蛋白表达水平、TH阳性细胞占比降低(P<0.05);与帕金森病组比较,石菖蒲组黑质p-AKT/AKT,p-mTOR/mTOR降低(P<0.05);与石菖蒲组比较,石菖蒲+激活剂组黑质p-AKT/AKT,p-mTOR/mTOR升高(P<0.05)。结论石菖蒲挥发油可提升帕金森病(Parkison’s disease,PD)模型小鼠运动协调能力,促进黑质多巴胺能神经元自噬,提升多巴胺水平,抑制AKT/mTOR通路可能是其发挥作用的机制之一。

Objective To study the effect of calamus volatile oil on autophagy of dopaminergic(DA) neurons in the substantia nigra of Parkinson’s disease model mice and its underlying mechanisms. Methods Ten out of 45 C57 BL/6 mice were randomly taken as the normal group. and the others were taken to establish the Parkinson’s disease model. Finally thirty mice were successfully established and were randomly divided into 3 groups. They were the Parkinson’s disease group(n=10), Acori Graminei Rhizoma group(n=10), and Acori Graminei Rhizoma with activator group(n=10). The Acori Graminei Rhizoma with activator group, was given Acori Graminei Rhizoma volatile oil 90 mg/kg and intraperitoneally injected with protein kinase B(AKT)/mammalian target of rapamycin(mTOR) signaling pathway activator MHY1485(10 mg/kg). Meanwhile the Acori Graminei Rhizoma group was gavaged daily with Acori Graminei Rhizoma volatile oil 90 mg/kg and intraperitoneal injected with equal volume of DMSO. The control group and the Parkinson’s disease group were intragastrically administered with equal volume of saline and intraperitoneally injected with equal volume of DMSO separately. The rotating rod experiment was used to test the ability of motor coordination. Western Blotting method was used to detect the expression of LC3 B, Beclin1, AKT, p-AKT, mTOR, and p-mTOR protein in substantia nigra. Immunohistochemical staining was applied to detect the positive expression of substantia nigra tyrosine hydroxylase(TH). Results Compared with the Parkinson’s disease group, mice in Acori Graminei Rhizoma group had longer exercise duration, and the expression of LC3 B and Beclin1 protein and the proportion of TH-positive cells were significantly increased(P<0.05). Compared with the Acori Graminei Rhizoma group, mice in Acori Graminei Rhizoma with activator group had shorter exercise duration and the expression of LC3 B, Beclin1 protein and the proportion of TH-positive cells were substantially decreased(P<0.05). Compared with the Parkinson group, the substantia nigra p-AKT/AKT and p-mTOR/mTOR of Acori Graminei Rhizoma group were decreased(P<0.05). Compared with Acori Graminei Rhizoma group, the substantia nigra p-AKT/AKT and p-mTOR/mTOR of Acori Graminei Rhizoma+activator group were increased(P<0.05). Conclusion Volatile oil of Acori Graminei Rhizoma may improve the motor coordinative ability of PD mice, promote the autophagy of dopaminergic neurons in the substantia nigra, increase the content of dopamine via inhibiting the AKT/mTOR pathway.