目的 探討褪黑素對(duì)帕金森病大鼠轉(zhuǎn)錄因子NF-E2相關(guān)因子2（Nrf2）、血紅素加氧酶1（HO-1）的調(diào)控作用及其對(duì)小膠質(zhì)細(xì)胞活化的影響。方法 取SD大鼠72只，按照隨機(jī)數(shù)字表法分為對(duì)照組、模型組、褪黑素（5 mg/kg）組、Nrf2抑制劑全反式維甲酸（ATRA，7 mg/kg）組、Nrf2抑制劑陰性對(duì)照（Nrf2-NC）（植物油，10 mL/kg）組、褪黑素＋ATRA（5 mg/kg＋7 mg/kg）組，每組12只。除對(duì)照組外，其余各組大鼠通過腦黑質(zhì)區(qū)內(nèi)注射6-羥多巴胺（6-OHDA）建立帕金森病模型。造模成功后各組開始給藥，模型組和對(duì)照組ip等量生理鹽水，各組連續(xù)給藥8周，2次/d。末次給藥后對(duì)大鼠進(jìn)行行為學(xué)評(píng)分；免疫組化法檢測(cè)黑質(zhì)部多巴胺能神經(jīng)元及小膠質(zhì)細(xì)胞活化陽性染色細(xì)胞數(shù)目；免疫熒光共定位檢測(cè)Nrf2、活化小膠質(zhì)細(xì)胞（IBA1標(biāo)記）重合表達(dá)的細(xì)胞數(shù)目；酶聯(lián)免疫吸附法（ELISA）檢測(cè)黑質(zhì)部代謝產(chǎn)物α-突觸核蛋白（α-Syn）和β淀粉樣蛋白（Aβ）。采用Western blotting檢測(cè)黑質(zhì)部Nrf2、HO-1、腫瘤壞死因子（TNF-α）、環(huán)氧化酶2（COX-2）、小膠質(zhì)細(xì)胞活化特異性標(biāo)記物（OX-42）蛋白相對(duì)表達(dá)水平。結(jié)果 與對(duì)照組比較，模型組大鼠出現(xiàn)單側(cè)或雙側(cè)后肢部分癱瘓及行走、進(jìn)食困難現(xiàn)象，行為學(xué)評(píng)分顯著升高（P<0.05）。與模型組相比，褪黑素組大鼠出現(xiàn)行走時(shí)轉(zhuǎn)圈，很少有癱瘓及行走困難癥狀，且行為評(píng)分降低（P<0.05）。ATRA組大鼠單側(cè)或雙側(cè)后肢部分癱瘓大鼠例數(shù)增多，行為學(xué)評(píng)分最高（P<0.05）。與對(duì)照組相比，模型組大鼠黑質(zhì)區(qū)Nrf2與IBA1重合表達(dá)細(xì)胞數(shù)目、小膠質(zhì)細(xì)胞活化數(shù)目、α-Syn和Aβ水平及HO-1、OX-42、TNF-α、COX-2水平和細(xì)胞核中Nrf2表達(dá)水平升高（P<0.05），多巴胺能神經(jīng)元數(shù)目減少。與模型組相比，褪黑素組大鼠黑質(zhì)Nrf2與IBA1重合表達(dá)細(xì)胞數(shù)目、多巴胺能神經(jīng)元陽性染色數(shù)目、HO-1及細(xì)胞核中Nrf2表達(dá)升高（P<0.05），小膠質(zhì)細(xì)胞活化數(shù)目、α-Syn及Aβ水平、TNF-α、COX-2、OX-42表達(dá)降低（P<0.05）；ATRA組黑質(zhì)區(qū)Nrf2與IBA1重合表達(dá)細(xì)胞數(shù)目、多巴胺能神經(jīng)元數(shù)目、HO-1水平及細(xì)胞核中Nrf2水平降低（P<0.05），小膠質(zhì)細(xì)胞活化數(shù)目、α-Syn和Aβ水平、TNF-α、COX-2、OX-42表達(dá)水平進(jìn)一步升高（P<0.05）。褪黑素＋ATRA組大鼠上述指標(biāo)變化與褪黑素組相反（P<0.05）。褪黑素＋ATRA組及Nrf2-NC組上述指標(biāo)與模型組相比差異無統(tǒng)計(jì)學(xué)意義。結(jié)論 褪黑素可能通過激活Nrf2/HO-1通路，抑制炎癥反應(yīng)及小膠質(zhì)細(xì)胞活化，減少帕金森病大鼠黑質(zhì)多巴胺能神經(jīng)元丟失。

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

河南高等學(xué)校重點(diǎn)科研項(xiàng)目（19A320021）