目的 探讨褪黑素对帕金森病大鼠转录因子NF-E2相关因子2（Nrf2）、血红素加氧酶1（HO-1）的调控作用及其对小胶质细胞活化的影响。方法 取SD大鼠72只，按照随机数字表法分为对照组、模型组、褪黑素（5 mg/kg）组、Nrf2抑制剂全反式维甲酸（ATRA，7 mg/kg）组、Nrf2抑制剂阴性对照（Nrf2-NC）（植物油，10 mL/kg）组、褪黑素＋ATRA（5 mg/kg＋7 mg/kg）组，每组12只。除对照组外，其余各组大鼠通过脑黑质区内注射6-羟多巴胺（6-OHDA）建立帕金森病模型。造模成功后各组开始给药，模型组和对照组ip等量生理盐水，各组连续给药8周，2次/d。末次给药后对大鼠进行行为学评分；免疫组化法检测黑质部多巴胺能神经元及小胶质细胞活化阳性染色细胞数目；免疫荧光共定位检测Nrf2、活化小胶质细胞（IBA1标记）重合表达的细胞数目；酶联免疫吸附法（ELISA）检测黑质部代谢产物α-突触核蛋白（α-Syn）和β淀粉样蛋白（Aβ）。采用Western blotting检测黑质部Nrf2、HO-1、肿瘤坏死因子（TNF-α）、环氧化酶2（COX-2）、小胶质细胞活化特异性标记物（OX-42）蛋白相对表达水平。结果 与对照组比较，模型组大鼠出现单侧或双侧后肢部分瘫痪及行走、进食困难现象，行为学评分显著升高（P<0.05）。与模型组相比，褪黑素组大鼠出现行走时转圈，很少有瘫痪及行走困难症状，且行为评分降低（P<0.05）。ATRA组大鼠单侧或双侧后肢部分瘫痪大鼠例数增多，行为学评分最高（P<0.05）。与对照组相比，模型组大鼠黑质区Nrf2与IBA1重合表达细胞数目、小胶质细胞活化数目、α-Syn和Aβ水平及HO-1、OX-42、TNF-α、COX-2水平和细胞核中Nrf2表达水平升高（P<0.05），多巴胺能神经元数目减少。与模型组相比，褪黑素组大鼠黑质Nrf2与IBA1重合表达细胞数目、多巴胺能神经元阳性染色数目、HO-1及细胞核中Nrf2表达升高（P<0.05），小胶质细胞活化数目、α-Syn及Aβ水平、TNF-α、COX-2、OX-42表达降低（P<0.05）；ATRA组黑质区Nrf2与IBA1重合表达细胞数目、多巴胺能神经元数目、HO-1水平及细胞核中Nrf2水平降低（P<0.05），小胶质细胞活化数目、α-Syn和Aβ水平、TNF-α、COX-2、OX-42表达水平进一步升高（P<0.05）。褪黑素＋ATRA组大鼠上述指标变化与褪黑素组相反（P<0.05）。褪黑素＋ATRA组及Nrf2-NC组上述指标与模型组相比差异无统计学意义。结论 褪黑素可能通过激活Nrf2/HO-1通路，抑制炎症反应及小胶质细胞活化，减少帕金森病大鼠黑质多巴胺能神经元丢失。

Objective To investigate the regulatory effect of melatonin on nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) in rats with Parkinson's disease, and its effect on microglia activation. Methods Seventy-two SD rats were divided into control group, model group, melatonin (5 mg/kg), Nrf2 inhibitor (ATRA, 7 mg/kg), Nrf2 inhibitor negative (Nrf2-NC) group (vegetable oil, 10 mL/kg), melatonin + ATRA (5 mg/kg + 7 mg/kg) group according to the random number table, with twelve in each group. In addition to the control group, Parkinson's disease models were established by injecting 6-hydroxydopamine (6-OHDA) into substantia nigra. After successful modeling, the drug was administered, and the model group and control group were intraperitoneally injected with the same amount of normal saline, twice daily for 8 weeks. After the last administration, the rats were scored for behavior, the positive staining numbers of dopaminergic neurons and microglia in substantia nigra were detected by immunohistochemistry, immunofluorescence co localization was used to detect the number of Nrf2 and activated microglia (Iba1 labeled) co expressing cells, the metabolites of substantia nigra, α-Syn and Aβ, were detected by enzyme-linked immunosorbent assay (ELISA). Western blotting was used to detect the relative expression levels of Nrf2, HO-1, tumor necrosis factor (TNF-α), cyclooxygenase-2 (COX-2), microglia activation specific marker (OX-42) in substantia nigra. Results Compared with the control group, rats in model group showed unilateral or bilateral partial paralysis of hind limbs and difficulty in walking and eating, and behavioral score was significantly increased (P < 0.05). Compared with the model group, the rats in the melatonin group showed little symptoms of paralysis and difficulty in walking, and the behavioral score was decreased (P < 0.05). The number of cases of unilateral or bilateral hindlimb partial paralysis was increased in ATRA group, and the behavioral score was the highest (P < 0.05). Compared with those in the control group, the number of Nrf2 and Iba1 co expressing cells, the activated number of microglia, the levels of α-Syn and Aβ, the expression of HO-1, OX-42, TNF-α, Cox2, and Nrf2 in the nucleus of rats in the model group were increased (P < 0.05), the number of dopaminergic neurons was decreased. Compared with those in the model group, the number of Nrf2 and Iba1 co expressing cells, the number of dopaminergic neurons positive staining, expression of HO-1, and Nrf2 in nucleus in melatonin group were further increased (P < 0.05), the activated number of microglia, the levels of α-Syn and Aβ, the expression of TNF-α, Cox2 and OX-42 were decreased (P < 0.05); the number of Nrf2 and Iba1 co expressing cells, the number of dopaminergic neurons positive staining, expression of HO-1, and Nrf2 in nucleus in ATRA group were decreased (P < 0.05), the activated number of microglia, the levels of α-Syn and Aβ, the expression of TNF-α, Cox2 and OX-42 were further increased (P < 0.05). The changes of the above indexes in melatonin + ATRA group were opposite to those in melatonin group (P < 0.05). There was no significant difference between melatonin + ATRA group and Nrf2-NC group. Conclusion Melatonin may inhibit inflammation and microglia activation, and reduce the loss of dopaminergic neurons in substantia nigra of Parkinson's disease rats by promoting the activation of Nrf2/HO-1 pathway.

R964

河南高等学校重点科研项目（19A320021）