小胶质细胞激活在帕金森小鼠神经炎症机制中的作用

The role of microglia activation in the mechanism of neuroinflammation in Parkinson's disease mice

目的探讨不同剂量脂多糖(Lipopolysaccharide,LPS)诱导的小胶质细胞激活表型对神经炎症的不同作用,并探讨低剂量LPS预处理对帕金森(Parkinson’s disease,PD)神经损伤的保护作用。方法将小鼠随机分成四组,分别为对照组、低剂量(0.3 mg/kg)LPS组、高剂量(1 mg/kg)LPS组和低剂量LPS+高剂量LPS(0.3 mg/kg+1 mg/kg)组。不同剂量LPS分别连续4 d腹腔注射,末次注射后20 d处死取材。行为学实验选择悬挂爬行实验,观察LPS注射后的小鼠运动状态。Western blotting实验检测小鼠中脑黑质内酪氨酸羟化酶(tyrosine hydroxylase,TH)表达水平。ELISA实验检测各组小鼠促炎因子肿瘤坏死因子α(tumor necrosis factorα,TNF-α)和白介素-1β(interleukin-1β,IL-1β)蛋白含量。免疫荧光实验检测各组小鼠中脑黑质区离子钙接头蛋白抗原(Ionized calcium bindingadapter molecule 1,Iba-1)/诱导型一氧化氮合酶(inductible nitric oxide synthase,iNOS)、Iba-l/精氨酸酶-l(Arginase-l,Arg-l)双阳性细胞数量。结果与对照组相比,高剂量组小鼠的自由运动状态(通过次数、表现力)较差(均P<0.0001),低剂量组小鼠未见异常(P>0.05);与高剂量组相比,低+高剂量组小鼠自由运动障碍缓解(通过次数,表现力均P<0.001)。与对照组相比,高剂量组小鼠脑TH蛋白表达显著减少(P<0.001),低剂量组小鼠脑TH蛋白表达无明显变化(P>0.05);与高剂量组比较,低+高剂量组小鼠脑TH蛋白表达增加(P<0.001)。与对照组相比,高剂量组小鼠中脑TNF-α和IL-1β表达显著增加(均P<0.001),低剂量LPS组小鼠的TNF-α和IL-1β表达未见明显变化(均P>0.05);与高剂量组相比,低剂量+高剂量LPS组小鼠的TNF-α和IL-1β表达减少(TNF-αP<0.01,IL-1βP<0.05)。与对照组比较,高剂量组小鼠中脑黑质区Iba-l/iNOS双阳性细胞显著增加,Iba-1/Arg-1双阳性细胞显著减少(均P<0.001),低剂量组小鼠中脑黑质区Iba-l/iNOS双阳性细胞及Iba-1/Arg-1双阳性细胞数无明显变化(P>0.05);与高剂量组比较,低+高剂量组小鼠中脑黑质区Iba-l/iNOS双阳性细胞减少(P<0.05),Iba-1/Arg-1双阳性细胞增加(P<0.01)。结论小胶质细胞在不同剂量LPS刺激下呈现出不同的激活状态。高剂量LPS可诱导M1表型小胶质细胞,通过释放IL-1β、TNF-α等促炎症细胞因子,诱发PD样神经炎症反应。低剂量LPS预处理则通过增加高剂量组Iba-1/Arg-1表达,分泌抗炎症细胞因子而抑制炎症反应。低剂量LPS预处理可通过将小胶质细胞转化为神经保护表型来防止神经元损伤。

Objective To investigate the different effects of microglial activation phenotypes induced by different doses of LPS on neuroinflammation,and to investigate the protective effects of low-dose LPS pretreatment on Parkinson's nerve injury.Methods The mice were randomly divided into four groups:control,low dose(0.3 mg/kg)LPS,high dose(1 mg/kg)LPS and low dose LPS+high dose LPS(0.3 mg/kg+1 mg/kg).Different doses of LPS were injected intraperitoneally for 4 consecutive days and killed 20 days after the last injection.Suspension crawling experiment was used to observe the locomotor state of mice after LPS injection.Western blotting experiment was used to test tyrosine hydroxylase expression level in the substantia nigra.ELISA was used to test the levels of proinflammatory factor tumor necrosis factorα(TNF-α),interleukin-1β(IL-1β)content in each group of mice.Immunofluorescence experiment was used to determine the number of double-positive cells of ionic calcium adaptor protein antigen/inducible nitric-oxide synthase/arginase-l in each group of mice.Results The free movement status of high dose group was worse compared to those in the control group(both P<0.0001),no abnormalities in the low dose group(P>0.05);Mice in the low+high dose group relieved free movement impairment compared to the high dose group(Number of passes,expressive force expressive force both P<0.001).Compared with the control group,the TH expression in the high dose group decreased(P<0.001),and the TH protein expression has no significant change in the low dose group(P>0.05),the TH expression was increased in the low+high dose group compared with the high dose group(P<0.001).Significant increase of TNF-αand IL-1βin high-dose mice(both P<0.001),and no significant changes in TNF-αand IL-1β-expression were observed in low-dose LPS mice(both P>0.05);and decreased in low-dose+high-dose LPS mice compared to high-dose mice(TNF-αP<0.01,IL-1βP<0.05).When compared with the control group,Iba-l/iNOS double positive cells were significantly increased in the midbrain nigra region in the high-dose group,a significant decrease in Iba-1/Arg-1 double-positive cells(both P<0.001);The number of Iba-l/iNOS double-positive cells and Iba-1/Arg-1 double-positive cells in the low-dose group shows no significant change(P>0.05);Compared with the high-dose group,Iba-l/iNOS double positive cells in midbrain nigra region in the low+high-dose group(P<0.05),the Iba-1/Arg-1 double-positive cells were increased(P<0.01).Conclusion Microglia showed different activation states with different doses of LPS.High-dose LPS,which can induce M1 phenotype microglia,can induce PD-like neuroinflammatory response by releasing pro-inflammatory cytokines such as IL-1βand TNF-α.With low-dose LPS,pretreatment inhibited the inflammatory response by increasing the expression of Iba-1/Arg-1 and secreted anti-inflammatory cytokines.Pretreatment with low-dose LPS prevents neuronal damage by converting microglia into a neuroprotective phenotype.