大定风珠对帕金森病模型小鼠脑黑质小胶质细胞活化及TLR4/MyD88/NF-κB信号通路的影响

Effect and Dadingfengzhu Pill(大定风珠)on the Activation of Microglial Cells in the Substantia Nigra and TLR4/MyD88/NF-κB Signaling Pathway in Parkinson's Disease Model Mice

目的探讨大定风珠对帕金森病神经损伤的影响及可能作用机制。方法84只C57BL雄性小鼠随机分为正常组、模型组、多巴丝肼组及大定风珠低、中、高剂量组,每组14只。除正常组外,其余各组小鼠每天腹腔注射鱼藤酮2.5 mg/kg连续28天复制帕金森病小鼠模型。造模同时大定风珠低、中、高剂量组分别给予大定风珠混悬液5、10、20g/(kg·d)灌胃,多巴丝肼组给予多巴丝肼混悬液0.034 g/(kg·d)灌胃,正常组和模型组小鼠给予生理盐水0.08 ml灌胃,均每日1次,共28天。灌胃结束后对各组小鼠进行旷场实验(包括水平运动总距离、中央区域运动总距离、静止时间)、爬杆实验(包括转身时间及爬杆总时间)评价小鼠行为学改变;高效液相色谱法检测小鼠纹状体多巴胺(DA)、5羟色胺(5-TH)水平;HE染色法观察小鼠中脑黑质致密部多巴胺能神经细胞病理改变;免疫组化法检测黑质致密部、纹状体酪氨酸羟化酶(TH)表达;免疫荧光法检测黑质致密部α-突触核蛋白(α-synuclein)、离子钙结合衔接分子1(Iba-1)表达;蛋白免疫印迹法检测纹状体Toll样受体4(TLR4)、髓样分化因子88(MyD88)、核转录因子κB p65(NF-κB p65)、磷酸化核转录因子κB p65(p-NF-κB p65)蛋白表达。结果与正常组比较,模型组小鼠水平运动总距离和中央区域运动总距离明显减少、静止时间延长(P<0.01),爬杆实验转身时间及爬杆运动总时间显著延长(P<0.01);小鼠纹状体DA、5-HT含量减少,TLR4、MyD88、NF-κB p65、p-NF-κB p65蛋白表达明显上调(P<0.05或P<0.01);黑质DA能神经细胞数量明显减少,排列紊乱且胞体皱缩;纹状体和黑质致密部TH阳性表达显著减少,α-synuclein、Iba-1荧光强度显著增强(P<0.05或P<0.01)。与模型组比较,大定风珠中、高剂量组小鼠水平运动总距离增加、静止时间减少,爬杆实验转身时间及爬杆运动总时间显著缩短(P<0.05或P<0.01);纹状体DA、5-HT含量增加,TLR4、MyD88、NF-κB p65、p-NF-κB p65蛋白表达明显减少;黑质DA能神经细胞数量增加,排列有序,TH阳性表达显著增加,α-synuclein、Iba-1荧光强度减弱(P<0.05或P<0.01)。与多巴丝肼组比较,大定风珠中、高剂量组α-synuclein、Iba-1荧光强度及MyD88蛋白表达降低(P<0.05或P<0.01)。结论大定风珠可缓解鱼藤酮对DA能神经元的神经毒性,抑制小胶质细胞活化,其机制可能与TLR4/MyD88/NF-κB信号通路有关。

Objective To investigate the effect of Dadingfengzhu Pill(大定风珠,DDFP)on nerve damage in Parkinson's disease(PD)and the possible mechanism.Methods Eighty-four C57BL male mice were randomly divided into normal group,model group,dopaserazide group,DDFP low-,medium-and high-dose groups,with 14 mice in each group.Except for the normal group,the mice were injected with rotenone(2.5 mg·kg-1)intraperitoneally every day for 28 days to replicate the PD model.Simultaneously,the DDFP low-,medium-and high-dose groups were given 5,10,and 20 g/(kg·d)of DDFP suspension by gavage,while the dopaserazide group was given 0.034 g/(kg·d)of dopaserazide suspension,and the normal group and model group were given 0.08 ml of normal saline by gavage,once daily for a total of 28 days.The behavioral changes of the mice were evaluated by the open field test measuring the total horizontal locomotor distance,total central region locomotor distance and resting time,and pole climbing test measuring time to turn and total time.The levels of DA and 5-hydroxytryptamine(5-TH)in the striatum of mice were measured by high performance liquid chromatography(HPLC).The pathological changes of dopaminergic neurons in the midbrain substantia nigra compacta were observed by hematoxylin-eosin(HE)staining.Immunohistochemical method was used to detect the expression of tyrosine hydroxylase(TH)in the substantia nigra compacta and striatum.The expression ofα-synuclein and ionized calcium street protein(Iba-1)in the substantia nigra compacta were detected by immunofluorescence(IF).The protein expression of striatal Toll-like receptor 4(TLR4),myeloid differentiation factor 88(MyD88),nuclear transcription factor-κB p65(NF-κB p65)and phosphorylated nuclear transcription factor-κB p65(p-NF-κB p65)were detected by Western blotting.Results Compared to those in the normal group,the total horizontal locomotor distance and central region locomotor distance of mice in the model group were significantly reduced,while the resting time was prolonged(P<0.01),and the time to turn and total time of pole climbing were significantly prolonged(P<0.01);striatal DA and 5-TH contents were significantly reduced,while protein expression of TLR4,MyD88,NF-κB p65,and p-NF-κB p65 increased(P<0.05 or P<0.01);number of dopaminergic neurons in the substantia nigra significantly decreased with disorganized and wrinkled cytosol,and positive expression of TH in the striatum and substantia nigra compacta decreased as well,while fluorescence intensity ofα-synuclein and Iba-1 were significantly enhanced(P<0.05 or P<0.01).Compared to those in the model group,the total horizontal locomotor distance in the DDZP medium-and high-dose groups significantly increased,resting time shortened,while the time to turn and total time of pole climbing reduced(P<0.05 or P<0.01);striatal DA and 5-TH contents significantly increased,while protein expression of TLR4,MyD88,NF-κB p65,and p-NF-κB p65decreased;number of dopaminergic neurons in the substantia nigra significantly increased with ordered arrangement,and positive expression of TH increased as well,while fluorescence intensity ofα-synuclein and Iba-1 were significantly reduced(P<0.05 or P<0.01).Compared to those in the dopaserazide group,the fluorescence intensity ofα-synuclein and Iba-1 and the MyD88 protein expression in the DDZP medium-and high-dose group significantly decreased(P<0.05 or P<0.01).Conclusion DDFP can alleviate the neurotoxicity of rotenone on dopaminergic neurons and inhibit microglia activation in PD mice,and the mechanism may be related to TLR4/MyD88/NF-κB signaling pathway.