梓醇对MPTP介导的亚急性帕金森病小鼠氧化应激水平的影响

Effects of Catalpol on MPTP-Mediated Oxidative Stress in Mice with Subacute Parkinson’s Disease

为了研究梓醇(Catalpol)对甲基-4-苯基1,2,3,6-四氢吡啶(1-methyl-4-phenyl-1,2,3,6-tetmhydropyridine,MPTP)所诱导的亚急性帕金森病模型小鼠氧化应激反应的影响,选取33只野生型小鼠随机分为对照组(n=11),给予生理盐水腹腔注射14d;MPTP模型组(n=11),给予生理盐水腹腔注射3d,第4天开始连续腹腔注射MPTP(30mg·kg^(-1),溶于生理盐水)5d,最后腹腔注射生理盐水6d;梓醇治疗组(n=11),给予腹腔注射梓醇(溶于生理盐水)3d,第4天腹腔注射MPTP(30mg·kg^(-1),溶于生理盐水)连续注射5d,最后腹腔注射梓醇6d。应用旷场实验、旋转杆实验和悬尾实验分别检测3组小鼠的运动能力和焦虑行为;利用免疫荧光染色技术检测小鼠黑质多巴胺能神经元的凋亡情况;运用Western blot方法考察小鼠黑质区TH、Bcl2、BAX、SOD1、SOD2、HO1、NQO1和Nrf2的蛋白表达情况;使用试剂盒检测小鼠黑质区SOD活性、ROS含量和GSH的活性。结果表明:MPTP模型组小鼠在旷场中心区域的移动距离和在旋转杆上的运动时间明显短于对照组,悬尾实验小鼠的不动时间明显长于对照组(p<0.05),与模型组相比,梓醇处理后,小鼠在旷场中心区域移动距离和旋转时间明显延长,悬尾实验小鼠的不动时间明显缩短(p<0.05);免疫荧光染色结果显示,与对照组相比,MPTP模型组小鼠的黑质区多巴胺能神经元死亡率明显升高,与模型组小鼠相比,梓醇处理后,黑质区多巴胺能神经元死亡率明显降低;Western blot和ELISA结果显示,与对照组相比,SOD1、SOD2、HO1、NQO1和Nrf2表达降低(p<0.05),Bcl2/BAX比例降低(p<0.05),与MPTP模型组小鼠相比,梓醇处理后,黑质区SOD1、SOD2、HO1、NQO1和Nrf2表达增加(p<0.05),Bcl2/BAX比例升高(p<0.05)。梓醇可缓解由MPTP介导的亚急性帕金森模型鼠脑内多巴胺能神经元凋亡和氧化应激反应,进而提高小鼠的运动能力,其作用机制可能是通过调控Nrf2/HO1/NQO1信号通路发挥神经保护作用。

To investigate the effect of oxidative stress response by the catalpol on the mouse with 1-methyl-4-phenyl-1,2,3,6-tetmhydropyridine(MPTP)-induced Parkinson disease(PD).The C57BL/6 mice were divided into three groups,namely the vehicle group,the MPTP model mice group and the catalpol administration group.The mice were intraperitoneally injected with catalpol dissolved in saline or vehicle for 3 days,followed by MPTP(30mg·kg^(-1)·d^(-1)),MPTP+catalpol,or vehicle starting on day 4 for 5 days.Mice in the MPTP+catalpol treatment group were continually administered catalpol for 6 days,and those primed with MPTP received vehicle for 6 days.Mice in the control group were given the vehicle saline.The open field,rotating rod tests test and tail suspension test was used to detect the motor ability and depression of the three groups,and the immunofluorescent staining method was investigated to the expression of tyrosinehydroxylase(TH)positive cells in substantia nigra(SN).Western blot was used to determine the expression of TH,Bcl2,BAX,SOD1,SOD2,HO1,NQO1and Nrf2 in SN.The kits were applied to detect the contents of ROS,the activities of SOD and GSH in SN.The results of behavior experiment showed that compared with the control group,the MPTPinduced mice reduced the distance of walking,the rotating time and activity time(p<0.05)compared with the MPTP administration group,the catalpol treatment group improved the motor ability and anxious emotion of the mice.Compared with the control group,the results of immunofluorescent staining and Western blot showed that the number of TH positive cells in SN in MPTP group decreased,while the expression level of TH protein decreased in SN.After the model mice were treated with catalpol,the above indexes were inhibited or restored.The Western blot results showed that compared with the vehicle,MPTP administration decreased the expression of SOD1,SOD2,HO1,NQO1,Nrf2 and decrease the ratio of Bcl2/BAX.Compared with the MPTP group,catalpol treatment obviously promoted the expressions of SOD1,SOD2,HO1,NQO1,Nrf2 and increase the ratio of Bcl2/BAX.The kits results showed that activities of SOD and GSH were inhibited in SN of MPTP group with the vehicle group,after treatment with catalpol,SOD and GSH activities obviously increased.Additionally,the content ROS were significantly decreased after the treatment of catalpol.Catalpol could significantly improve the motor ability and anxious emotion of the MPTP-induced mice by inhibiting the dopaminergic neuron death,and the mechanism may be related to the antioxidant stress levels through the Nrf2/HO1/NQO1 signal pathway.