金松双黄酮抑制JAK2/STAT3通路对脂多糖诱导的BV2小胶质细胞炎症反应的影响

Effects of sciadopitysin on lipopolysaccharide-induced inflammatory response in BV2 cells by inhibition of JAK2/STAT3 pathway

目的探究金松双黄酮(SCI)调控酪氨酸激酶2/信号转导与转录激活因子3(JAK2/STAT3)通路,对脂多糖(LPS)诱导的BV2小胶质细胞炎症反应的影响。方法①采用格里斯(Griess)试验试剂检测不同浓度SCI(1.25~40μmol·L^(-1))干预对经LPS刺激后BV2小胶质细胞培养液中亚硝酸盐含量的影响。②采用四甲基偶氮唑盐比色法(MTT)检测不同浓度SCI(1.25~40μmol·L^(-1))干预的BV2小胶质细胞活性以此判断SCI对BV2小胶质细胞毒性作用。③将BV2小胶质细胞以2×10^(6)个/孔接种于6孔板中,并将其随机分为对照组(Ctrl组)、对照+SCI组(Ctrl+SCI组)、模型组(LPS组,100μg·L^(-1))、模型+SCI组(LPS+SCI组),各组进行相应干预。采用实时荧光定量逆转录聚合酶链式反应(RT-qPCR)法检测SCI对LPS刺激BV2细胞的促炎因子肿瘤坏死因子-α(TNF-α)和诱导一氧化氮合酶(iNOS)mRNA表达的影响;酶联免疫吸附测定(ELISA)法检测白介素-6(IL-6)、TNF-α的蛋白水平;Western blot法检测JAK2/STAT3信号通路磷酸化酪氨酸激酶2(p-JAK2)、磷酸化信号转导与转录激活因子3(p-STAT3)以及iNOS蛋白表达。结果①Greiss法结果显示,1.25~40μmol·L^(-1) SCI均显著降低LPS刺激的BV2小胶质细胞产生的亚硝酸盐(P<0.05)。②MTT法结果显示,1.25~20μmol·L^(-1)浓度的SCI对BV2小胶质细胞的活性没有影响。③RT-qPCR结果显示,与LPS组比较,LPS+SCI组中iNOS、TNF-αmRNA的表达量下降(P<0.05);ELISA法结果显示,与LPS组比较,LPS+SCI组中IL-6、TNF-α的蛋白表达量下降(P<0.05);Western blot结果显示,与LPS组比较,LPS+SCI组中p-JAK2、p-STAT3、iNOS蛋白表达降低(P<0.05)。结论SCI能够通过抑制JAK2/STAT3信号通路发挥对LPS刺激的BV2小胶质细胞的抗炎作用。

Objective This study aims to explore the anti-inflammatory effects of sciadopitysin(SCI)on lipopolysaccharide(LPS)-activated BV2 microglia based on Janus kinase 2/signal transducer and activator of transcription 3(JAK2/STAT3)signaling pathway.Methods①Griess reagent was used to detect the effects of different concentrations of SCI(1.25~40μmol·L^(-1))on the content of nitrite produced by LPS-stimulated BV2 cells.②The activity of BV2 microglia treated with different concentrations of SCI(1.25~40μmol·L^(-1))was detected by tetrazolium salt colorimetry(MTT)to determine the toxic effect of SCI on BV2 microglia.③BV2 microglia were inoculated into 6-well plates with 2×10^(6) cells per well and randomly divided into control group(Ctrl group),control+SCI group(Ctrl+SCI group),model group(LPS group,100μg·L^(-1))and model+SCI group(LPS+SCI group).Each group was intervened accordingly.Real-time fluorescence quantitative reverse transcriptase polymerase chain reaction(RT-qPCR)was used to detect the effects of SCI on pro-inflammatory factor tumor necrosis factor-α(TNF-α)and nitric oxide synthase(iNOS)mRNA expression in BV2 cells stimulated by LPS.The protein levels of interleukin-6(IL-6)and TNF-αwere detected by enzyme linked immunosorbent assay(ELISA).Western blot was used to detect the effects of SCI on the protein expression of phosphorylated tyrosine kinase 2(p-JAK2),phosphorylated signal transducer and activator of transcription 3(p-STAT3)and iNOS protein in JAK2/STAT3 signal pathway in LPS stimulated BV2 cells.Results①Griess results showed that SCI(1.25~40μmol·L^(-1))pretreatment significantly reduced the production of nitrite products in cell culture medium after LPS stimulated BV2 microglia(P<0.05).②MTT results showed that SCI(1.25~20μmol·L^(-1))had no effect on the activity of BV2 microglia.③The results of RT-qPCR showed that compared with the LPS group,the mRNA expression levels of iNOS and TNF-αdecreased in the LPS+SCI group(P<0.05).The results of ELISA showed that compared with the LPS group,the protein expression levels of IL-6 and TNF-αdecreased in the LPS+SCI group(P<0.05).Western blot results indicated that compared with the LPS group,the protein expression of phosphorylated Janus kinase 2(p-JAK2),phosphorylated signal transducer and activator of transcription 3(p-STAT3)and iNOS decreased in the LPS+SCI group(P<0.05).Conclusion SCI can inhibit the activation of BV2 cells induced by LPS through JAK2/STAT3 signaling pathway,and play an anti-inflammatory role.