活性氧和蛋白激酶B磷酸化在百草枯介导的小胶质细胞活化中的作用
Roles of reactive oxygen species and protein kinase B phosphorylation in paraquat-mediated microglia activation
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摘要:
背景 百草枯(PQ)可诱导中枢神经系统的小胶质细胞活化,但其机制尚不清楚。
目的 通过体外实验,探究PQ介导的小胶质细胞活化的分子机制,聚焦于活性氧(ROS)和蛋白激酶B(Akt)磷酸化在该过程中的作用。
方法 使用不同浓度的PQ(0、1、3.3、10、33、100 μmol·L-1)对小鼠小胶质细胞系BV-2细胞进行染毒12 h,采用细胞计数试剂盒(CCK-8)检测细胞活力。选择不引起细胞活性改变的最高染毒剂量(33 μmol·L-1)进行后续研究。用33 μmol·L-1 PQ染毒12 h,采用流式细胞技术检测小胶质细胞经典活化(M1活化)的标志一氧化氮合酶(iNOS)、选择性活化(M2活化)的标志CD206的水平以及ROS和磷酸化-Akt的水平;采用ELISA方法检测M1活化分泌的促炎细胞因子白介素6(IL-6)、肿瘤坏死因子α(TNF-α)和白介素1β(IL-1β)以及M2活化分泌的抑炎细胞因子白介素4(IL-4),胰岛素样生长因子1(IGF-1)和转化生长因子β(TGF-β)的水平。采用2 mmol·L-1乙酰半胱氨酸(NAC)在PQ染毒前处理细胞2 h以干预ROS,再更换含PQ的培养基染毒12 h后,用RT-PCR的方法检测
iNOS 、CD206 及IL-1β 的基因表达。使用Akt抑制剂(Akt inhibitor Ⅷ,5 μmol·L-1)和激活剂(SC79,20μmol·L-1)在PQ染毒前预处理细胞2h,再更换含PQ的培养基染毒12 h,检测干预后iNOS、CD206和IL-1β的水平。结果 在33 μmol·L-1 PQ染毒12 h下,小胶质细胞iNOS水平升高(
t =8.912,P < 0.001),促炎细胞因子IL-6、TNF-α、IL-1β均升高(t =2.710、3.342、5.078,P 值均 < 0.05);但CD206和抑炎因子水平改变差异均无统计学意义(P >0.05);小胶质细胞胞内ROS水平升高(t =11.907,P < 0.001),而Akt磷酸化则被抑制(t =6.152,P < 0.001)。NAC预处理后,PQ染毒引起的iNOS 、IL-1β 和CD206 基因表达水平的增加均被逆转(t =15.457、6.912、9.106,均P < 0.001)。Akt抑制剂的使用,使iNOS和IL-1β水平相较于PQ染毒组进一步升高(t =8.021、3.684,分别P < 0.001、P < 0.01),CD206的水平则降低(t =2.662,P < 0.05)。与之相反,Akt激活剂预处理抑制了PQ染毒引起的iNOS和IL-1β水平的升高(t =6.835、4.325,分别P < 0.001、P < 0.01),而CD206的水平升高(t =17.471,P < 0.001)。在ROS与Akt的关系上,使用NAC干预ROS时,PQ引起的Akt磷酸化抑制得到恢复(t =6.438,P < 0.001);而无论使用Akt抑制剂还是激活剂,均不改变ROS的水平(P >0.05)。结论 PQ可通过升高ROS,进而抑制Akt磷酸化引起小胶质细胞的促炎活化。干预ROS可同时抑制小胶质细胞的M1和M2活化;而Akt磷酸化的激活能调节PQ介导的小胶质细胞活化方向。
Abstract:Background Paraquat (PQ) can induce microglia activation in central nervous system (CNS). However, its associated mechanism is not clear yet.
Objective This study investigates the potential molecular mechanism of PQ-mediated microglia activation and focuses on the roles of reactive oxygen species (ROS) and protein kinase B (PKB, also called Akt) phosphorylation in this process through
in vitro experiments.Methods Mouse microglia cell line BV-2 cells were treated with different concentrations of PQ (0, 1, 3.3, 10, 33, and 100 μmol·L-1) for 12 h and assessed for its viability with cell counting kit 8 (CCK8). The highest dose that did not damage the cell viability (33 μmol·L-1) was selected for further experiments. Then BV-2 cells were treated with 33 μmol·L-1 PQ for 12 h to detect the levels of nitric oxide synthase (iNOS), a marker of microglia classical activation (M1 activation), CD206, a marker of microglia alternative activation (M2 activation), ROS, and phosphorylated Akt (p-Akt) by flow cytometry, as well as the levels of proinflammatory cytokines triggered via M1 activationinterleukin 6 (IL-6), tumor necrosis factor α (TNF-α), and interleukin 1β (IL-1β) and anti-inflammatory cytokines triggered via M2 activationinterleukin 4 (IL-4), insulin-like growth factor 1 (IGF-1), and transforming growth factor β (TGF-β) by ELISA. A batch of cells were pretreated with 2 mmol·L-1 N-acetylcysteine (NAC) to inhibit ROS for 2 h, and then treated with PQ for 12 h to detect the expression levels of
iNOS ,CD206 , andIL-1β by RT-PCR. Another batch of cells were pretreated with Akt inhibitor (Akt inhibitor Ⅷ, 5 μmol·L-1) and activator (SC79, 20 μmol·L-1) respectively for 2 h, and then treated with PQ for another 12 h, to detect the expression levels of iNOS, CD206, and IL-1β.Results After the cells were exposed to 33 μmol·L-1 PQ for 12 h, the levels of iNOS (
t =8.912,P < 0.001) and proinflammatory cytokines IL-6, TNF-α, and IL-1β were increased (t =2.710, 3.342, and 5.078,P < 0.05), while no significant difference was observed in the levels of CD206 and anti-inflammatory cytokines (P >0.05); in addition, intercellular ROS level was increased (t =11.907,P < 0.001), and Akt phosphorylation was inhibited (t =6.152,P < 0.001). After the NAC treatment, the increased levels ofiNOS ,IL-1β , andCD206 gene expression induced by PQ were reversed (t =15.457, 6.912, 9.106, respectively;P < 0.001). After the Akt inhibitor treatment, the levels of iNOS and IL-1β were further increased compared with the PQ group (t =8.021,P < 0.001;t =3.684,P < 0.01, respectively), and the CD206 level was reduced (t =2.662,P < 0.05). In contrast, compared with the PQ group, the levels of iNOS and IL-1β were inhibited by the Akt activator (t =6.835,P < 0.001;t =4.325,P < 0.01, respectively), while CD206 was increased (t =17.471,P < 0.001). As for the relationship between ROS and Akt, NAC restored the inhibited Akt phosphorylation induced by PQ (t =6.438,P < 0.001), while neither Akt inhibitor nor Akt activator changed the level of ROS (P < 0.05).Conclusion PQ can induce pro-inflammatory activation of microglia by increasing ROS and inhibiting Akt phosphorylation. ROS intervention can inhibit both M1 and M2 phenotypes of microglia. And Akt activation can regulate PQ-mediated microglia activation.