RIP1/RIP3介导的程序性坏死在LPS诱导的多巴胺神经元损伤中的作用

摘要/Abstract

摘要： 目的

研究程序性坏死（necroptosis）在脂多糖（lipopolysaccharide，LPS）诱导的帕金森病大鼠模型中的作用。

方法

大鼠随机分为对照组、LPS模型组和Nec?1+LPS组。对照组动物黑质内注射生理盐水（normal saline，NS）2 μL，腹腔内连续注射NS 2 mL/kg14 d；LPS模型组动物黑质内注射LPS 2 μL （5 μg），连续14 d腹腔内注射NS 2 mL/kg；Nec?1+LPS组动物黑质内注射LPS 2 μL （5 μg），连续14 d腹腔内注射程序性坏死特异阻断剂Nec?1（2 mg/kg）。通过行为学检测PD模型是否制备成功；利用高效液相色谱（high performance liquid chromatography，HPLC）法测定大鼠纹状体中多巴胺及其代谢物含量的变化；利用免疫组织化学方法观察黑质内多巴胺能神经元损伤的数量、小胶质细胞数量与形态学变化；ELISA法检测大鼠黑质中白介素1β（interleukin?1β，IL?1β）及肿瘤坏死因子?α（tumor necrosis factor，TNF?α）表达量的变化；通过免疫印迹观察受体相互作用蛋白1（receptor-interacting protein，RIP1）与RIP3的表达量变化。

结果

皮下注射阿扑吗啡可诱导LPS组大鼠出现明显的旋转行为，与对照组比较差异有统计学意义（P< 0.001），而Nec?1+LPS组大鼠的旋转行为较LPS组有明显改善，差异有统计学意义（P< 0.01）。LPS组大鼠患侧纹状体内DA、DOPAC及HVA明显减少；黑质内小胶质细胞过度激活伴有多巴胺能神经元数量大量丢失，而应用Nec?1能显著改善LPS注射导致的上述变化，差异均具有统计学意义（P< 0.05）。LPS注射后大鼠患侧黑质内IL?1β及TNF?α、RIP1与RIP3的表达量均明显增加（P< 0.05），Nec?1干预组动物IL?1β（P< 0.01）、TNF?α（P< 0.05）、RIP1（P< 0.01）与RIP3（P< 0.01）表达量均有明显下降，差异均有统计学意义。

结论

RIP1/RIP3介导的程序性坏死与LPS诱导的多巴胺能神经元死亡密切相关，程序性坏死可作为治疗中枢炎症相关性疾病的潜在靶点。

关键词:程序性坏死,脂多糖,小胶质细胞,帕金森病,神经退行性疾病

Abstract: Objective

To explore the role of RIP1/RIP3 mediated necroptosis in lipopolysaccharide（LPS）-induced rat model of Parkinson's disease（PD）.

Methods

The SD rats were randomly divided into following three groups：（1） Control group： the sham-operated rats were administered i.p. with the normal saline （NS）（2 ml/kg/day） for 14 consecutive days；（2） LPS group： 2 μL（5 μg） LPS was stereotatically injected into unilateral substantia nigra （SNc） of rats to establish the PD models， LPS-injected rats were intraperitoneally injected with NS （2 ml/kg） for 14 consecutive days；（3） Nec-1+LPS group： Same as LPS group except that Nec-1 （2 mg/kg） was used instead of NS. Behavioral testing to observe whether the PD model is successfully established. The contents of dopamine and its metabolites in striatum were measured by high performance liquid chromatography （HPLC）.The damage of DA neurons in the substantia nigra was observed by using tyrosine-hydroxylase （TH） immunohistochemical staining.The changes in the morphology and number of microglia were detected by using immunohistochemical methods. Enzyme-linked immunoassay （ELISA） was used to detect the content of IL-1β and TNF-α in the rat substantia nigra.the changes in the expression of necroptosis-related proteins RIP1 and RIP3 were observed by western blotting.

Results

Apomorphine can induce obvious rotation behavior of rats in the LPS group， and the rotation behavior of rats in the Nec-1+LPS group was significantly improved compared with the LPS group. Nec-1 treatment obviously attenuated the depletion of DA and its metabolites in the striatum induced by LPS. As compared to the Control group， LPS injection can cause a large loss of TH immunoreactivity （TH-ir） neurons in the substantia nigra. Nec-1 treatment notably increased the number of TH-ir neurons in the substantia nigra of the LPS-injected side. Nec-1 significantly inhibited LPS-induced microglia overactivation and overexpression of pro-inflammatory factors in the substantia nigra.

Conclusions

RIP1/RIP3-mediated necroptosis is closely related to the death of dopaminergic neurons induced by LPS， and necrosis can be used as a potential target for the treatment of central inflammation-related diseases and deserves further study.

Key words:necroptosis,lipopolysaccharide,microglia,Parkinson's disease,neurodegenerative disease

郑天成, 郭俊, 孙宪昌. RIP1/RIP3介导的程序性坏死在LPS诱导的多巴胺神经元损伤中的作用[J]. betway必威登陆网址（betway.com ）学报, 2022, 43(7): 481-488.

Tiancheng ZHENG, Jun GUO, Xianchang SUN. The role of RIP1/RIP3 mediated necroptosis in LPS-induced dopamine neuron damage[J]. Journal of Shandong First Medical Unversity & Shandong Academy of Medical Sciences, 2022, 43(7): 481-488.

图/表6

图1阿扑吗啡诱导的旋转行为与对照组比较，***P < 0.001；与LPS组比较，^^P < 0.01

图2纹状体内多巴胺及其代谢产物含量的变化A、B、C分别为纹状体内DA、HVA和DOPAC的含量。与对照组比较，***P < 0.001；与LPS组比较，^P < 0.05

图3黑质内TH免疫阳性神经元数量变化A为黑质TH免疫组化染色结果（× 200），其中a为对照组，b为LPS组，c为Nec-1 + LPS组；B为损伤侧黑质内残存TH染色阳性神经元比例分析；与对照组比较，***P < 0.001；与LPS组比较，^P < 0.05

图4损伤侧黑质内CD11b免疫组化及免疫印迹结果A为损伤侧黑质CD11b免疫组化染色结果（× 200），其中a为对照组，b为LPS组，c为Nec-1+LPS组；B为损伤侧黑质CD11b免疫组化结果灰度分析；C为损伤侧黑质内CD11b免疫印迹及结果分析，与对照组比较，***P < 0.001；与LPS组比较，^P < 0.05

图5损伤侧黑质内炎症因子表达量变化A为TNF-α含量；B为IL-1β含量。与对照组比较，***P < 0.001；与LPS组比较，^P < 0.05，^^P < 0.01

图6损伤侧黑质内程序性死亡相关蛋白表达量变化A为损伤侧黑质内RIP1/β-actin表达量；B为损伤侧黑质RIP3/β-actin表达量。与对照组比较，**P < 0.01；与LPS组比较，^^P < 0.01

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