补肾活血颗粒对亚急性帕金森病模型小鼠脑黑质多巴胺神经元铁死亡的影响

Effect of Bushen Huoxue Granule (补肾活血颗粒) on Ferroptosis of Dopamine Neurons in Substantia Nigra of Subacute Parkinson's Disease Model Mice

目的 探讨补肾活血颗粒治疗帕金森病的作用及可能机制。方法 将48只C57/BL6小鼠随机分为空白组(12只)和造模组(36只),造模组小鼠连续1周腹腔注射1-甲基-4-苯基-1,2,3,6四氢吡啶(MPTP)30 mg/(kg·d)制备亚急性PD小鼠模型,将造模成功36只小鼠再随机分为模型组、补肾活血颗粒组、铁死亡抑制剂组,各12只。补肾活血颗粒组给予补肾活血颗粒药液3 g/(kg·d)灌胃,铁死亡抑制剂组予铁死亡抑制剂Liproxstatin-1溶液10 mg/(kg·d)腹腔注射,空白组和模型组给予蒸馏水10 ml/(kg·d)灌胃,均每日1次,连续2周。给药结束后的第2天运用旷场实验和步态行为分析进行小鼠行为学测试,免疫组化法观察脑黑质酪氨酸羟化酶(TH)阳性表达,检测脑黑质谷胱甘肽(GSH)、4-羟基异壬烯醛(4-HNE)、铁离子(Fe^(2+))含量变化,Western-blot和RT-qPCR法检测脑黑质铁死亡相关因子溶质载体家族7成员11(SLC7A11)、谷胱甘肽过氧化物酶4 (GPX4)、酰基辅酶A合成酶长链家族成员4 (ACSL4)蛋白及mRNA的表达。结果 与空白组比较,模型组小鼠在旷场实验中运动总路程缩短、平均速度明显降低,静止时间明显延长,步态分析实验中步宽增大、步长减小、步态变异率增大,脑黑质TH表达、GSH含量及SLC7A11、GPX4 mRNA及蛋白表达降低,4-HNE、Fe^(2+)含量、ACSL4 mRNA及蛋白表达升高(P<0.01)。与模型组比较,补肾活血颗粒组小鼠运动总路程和平均速度增加,静止时间缩短,步宽缩小、步长增加、步态变异率减小,脑黑质TH阳性表达、GSH含量及SLC7A11、GPX4 mRNA及蛋白表达升高,4-HNE、Fe^(2+)含量、ACSL4mRNA及蛋白表达降低(P<0.05或P<0.01);铁死亡抑制剂组运动总路程延长、平均速度增加,步长增加,脑黑质TH阳性表达、GSH含量及SLC7A11、GPX4 mRNA及蛋白表达升高,4-HNE、Fe^(2+)含量、ACSL4mRNA及蛋白表达降低(P<0.05或P<0.01)。铁死亡抑制剂组与补肾活血颗粒组比较,GPX4mRNA表达升高(P<0.01),其余各指标差异均无统计学意义(P>0.05)。结论 补肾活血颗粒治疗帕金森病可能机制是通过抑制多巴胺神经元铁死亡,改善氧化应激,从而改善运动能力。

Objective To investigate the effects of Bushen Huoxue Granule(补肾活血颗粒,BSHXG)on Parkinson's disease(PD)and the possible mechanism.Methods Thirty-six successful modeling subacute PD mice prepared by 7-day continuous intraperitoneal injection of 30 mg(/kg·d)1-Methyl-4-phenyl-1,2,3,6tetrahydro⁃pyridine(MPTP)were randomly divided into model group,BSHXG group,Liproxstatin-1(Lip-1)group with 12 mice in each group,and another 12 C57/BL6 mice were taken as blank group.The BSHXR group was given 3 g/(kg·d)BSHXG solution by gavage,while the Lip-1 group was administered with intraperitoneal injection of 10 mg/(kg·d)Lip-1 solution,and the blank and model group were given 10 ml(/kg·d)distilled water by gavage,once daily for two weeks.The behavioral performance of mice was detected by open field experiment and gait analysis,and the positive expression of tyrosine hydroxylase(TH)in substantia nigra was observed by immunohistochemistry.The con⁃tents of glutathione(GSH),4-hydroxyisononenal(4-HNE)and ferric ion(Fe^(2+))in substantia nigra were assessed,and the protein and mRNA expressions of ferroptosis related factors solute including carrier family 7 member 11(SLC7A11),glutathione peroxidase 4(GPX4)and acyl-CoA synthase long-chain family member 4(ACSL4)were de⁃tected by Western-blot and RT-qPCR.Results Compared to those in the blank group,the total distance and average speed of mice in the model group decreased,and the resting time increased in the open field experiment;the gait analysis showed the step width in the model group increased,while the step length decreased,and the gait variation rate increased;the TH expression,the GSH content and the expression of SLC7A11 and GPX4 mRNA and protein decreased,while the contents of 4-HNE and Fe^(2+)and expression of ACSl4 mRNA and protein increased(P<0.01).Compared to those in the model group,the total distance and average speed of mice in BSHXG group increased,while the resting time decreased significantly;the step width was shortened,while the step length increased,and the gait variation rate decreased;the TH positive expression,the GSH content and the mRNA and protein expression of SLC7A11 and GPX4 increased,while 4-HNE and Fe^(2+)contents and ACSl4 mRNA and protein expression decreased(P<0.05 or P<0.01).Compared to the model group,the Lip-1 group increased the total distance,average speed and step length,increased the TH positive expression,GSH content and the SLC7A11 and GPX4 mRNA and protein expression,and decreased 4-HNE and Fe^(2+)contents and ACSl4 mRNA and protein expression(P<0.05 or P<0.01).Compared to that in the BSHXG group,the expression of GPX4 mRNA in Lip-1 group increased(P<0.01),while there were no statistical significant differences in other outcomes between groups(P>0.05).Conclusion BSHXG in the treatment of PD may improve athletic ability by inhibiting ferroptosis of dopamine neurons and improvimprovingoxidative stress.