青藤碱调控NLRP3/caspase-1通路抑制BV-2小胶质细胞焦亡及炎症的机制研究

Sinomenine inhibits BV-2 microglia pyrolysis and inflammation via the NLRP3/caspase-1 pathway

目的探究青藤碱调控核苷酸结合寡聚化结构域样受体蛋白3(NLRP3)/半胱氨酸天冬氨酸蛋白酶1(caspase-1)通路抑制BV-2小胶质细胞焦亡及炎症的机制。方法以氧糖剥夺/复糖复氧(OGD/R)模型诱导BV-2细胞焦亡模型,采用CCK-8法检测不同浓度(0、10、20、50、100μmol/L)的青藤碱干预BV-2小胶质细胞24h后的细胞活性,筛选药物浓度。将BV-2细胞分为正常组、OGD/R组和OGD/R+青藤碱组(20μmol/L)进行研究。使用微量酶标法测BV-2小胶质细胞乳酸脱氢酶(LDH)活性。采用赫斯特(Hoechst)/碘化丙啶(PI)细胞染色法检测BV-2细胞中PI阳性细胞数。采用RT-PCR和Western blot方法检测青藤碱对BV-2小胶质细胞硫氧还蛋白结合蛋白(TXNIP)、NLRP3、caspase-1、白细胞介素(IL)-1β和IL-18的mRNA和蛋白表达水平的影响。结果10、20μmol/L青藤碱干预24h后,OGD/R诱导的BV-2小胶质细胞活性较对照组无显著差异[(97.69±0.51)%、(96.03±1.13)%比(100.00±0.00)%,P均>0.05)],50、100μmol/L青藤碱干预24h后,OGD诱导的BV-2小胶质细胞活性较对照组显著降低[(78.92±3.02)%、(64.12±4.55)%比(100.00±0.00)%,P均<0.05]。与正常组比较,OGD/R组BV-2小胶质细胞LDH相对释放量明显上升[(2.23±0.19)比(1.00±0.00),P<0.05],PI活性细胞比例明显升高[(46.28±4.02)%比(3.52±0.31)%,P<0.05],TXNIP、NLRP3、caspase-1、IL-1β和IL-18 mRNA[TXNIP:(1.58±0.20)比(0.69±0.08),P<0.05;NLRP3:(2.32±0.18)比(0.88±0.09),P<0.05;caspase-1:(1.61±0.11)比(0.77±0.15),P<0.05;IL-1β:(3.17±0.43)比(1.03±0.09),P<0.05;IL-18:(1.82±0.22)比(0.81±0.13),P<0.05]和蛋白表达水平明显上调[TXNIP:(1.26±0.13)比(0.72±0.09),P<0.05;NLRP3:(0.43±0.04)比(0.16±0.04),P<0.05;caspase-1:(1.30±0.09)比(0.58±0.07),P<0.05;IL-1β:(1.21±0.11)比(0.39±0.06),P<0.05;IL-18:(0.54±0.07)比(0.23±0.06),P<0.05]。与OGD/R组比较,OGD/R+青藤碱组BV-2细胞LDH相对释放量明显下降[(1.28±0.09)比(2.23±0.19),P<0.05],PI活性细胞比例显著减少[(23.08±3.46)%比(46.28±4.02)%,P<0.05],TXNIP、NLRP3、caspase-1和IL-1βmRNA[TXNIP:(0.95±0.11)比(1.58±0.20),P<0.05;NLRP3:(1.26±0.12)比(2.32±0.18),P<0.05;caspase-1:(0.95±0.05)比(1.61±0.11),P<0.05;IL-1β:(1.55±0.18)比(3.17±0.43),P<0.05]和蛋白表达水平显著下降[TXNIP:(0.78±0.04)比(1.26±0.13),P<0.05;NLRP3:(0.26±0.03)比(0.43±0.04),P<0.05;caspase-1:(0.71±0.05)比(1.30±0.09),P<0.05;IL-1β:(0.54±0.03)比(1.21±0.11),P<0.05],IL-18蛋白水平下降[(0.34±0.08)比(0.54±0.07),P<0.05]。结论青藤碱可能通过下调NLRP3/caspase-1通路磷酸化水平,抑制BV-2小胶质细胞焦亡及炎症反应。

Objective To explore the underlying molecular mechanisms of sinomenium on pyroptosis and inflammation in BV-2 microglial cells,specifically the NOD-like receptor family pyrin domain containing 3(NLRP3)/caspase-1 pathway.Methods A pyroptosis model of BV-2 microglial cells was established by using oxygen and glucose deprivation/recovery(OGD/R).The cell viability was assessed using the CCK-8 assay after treatment of cells with sinomenium at different concentrations(0,10,20,50,and 100μmol/L)and an appropriate drug concentration was selected.After that,cells were divided into control,OGD/R,and OGD/R+sinomenium(20μmol/L)groups.The relative dehydrogenase(LDH)activity was determined and numbers of propidium iodide(PI)positive cells were detected using Hoechst/PI staining.The mRNA and protein expression of thioredoxin-interacting protein(TXNIP),NLRP3,caspase-1,interleukin-1β(IL-1β),and IL-18 was detected using quantitative PCR and Western blot,respectively.Results Treatment of cells with 10 and 20μmol sinomenium for 24 h showed no difference in the cell viability compared with control BV-2 cells(97.69±0.51 and 96.03±1.13 vs.100.00±0.00%;P>0.05).After 50 and100μmol sinomenium treatment for 24 h,viability of OGD/R-induced BV-2 cells was significantly reduced(78.92±3.02 and 64.12±4.55 vs.100.00±0.00%;P<0.05).Moreover,compared with control cells,in OGD/R induced pyroptosis cells,relative LDH activity was notably increased(2.23±0.19 vs.1.00±0.00;P<0.05)and percentage of the PI-positive cells was suppressed significantly(46.28±4.02 vs.3.52±0.31%;P<0.05),while levels of TXNIP(1.58±0.20 vs.0.69±0.08;P<0.05),NLRP3(2.32±0.18 vs.0.88±0.09;P<0.05),caspase-1(1.61±0.11 vs.0.77±0.15;P<0.05),IL-1β(3.17±0.43 vs.1.03±0.09;P<0.05),and IL-18(1.82±0.22 vs.0.81±0.13;P<0.05)mRNAs and TXNIP(1.26±0.13 vs.0.72±0.09;P<0.05),NLRP3(0.43±0.04 vs.0.16±0.04;P<0.05),caspase-1(1.30±0.09 vs.0.58±0.07;P<0.05),IL-1β(1.21±0.11 vs.0.39±0.06;P<0.05),and IL-18(0.54±0.07 vs.0.23±0.06;P<0.05)proteins were all increased significantly.Compared with the OGD/R cells,sinomenium treatment significantly decreased relative LDH activity(1.28±0.09 vs.2.23±0.19;P<0.05)and dramatically reduced the percentage of PI-positive cells(23.08±3.46 vs.46.28±4.02%;P<0.05),and significantly reduced levels of TXNIP(0.95±0.11 vs.1.58±0.20;P<0.05)NLRP3(1.26±0.12 vs.2.32±0.18;P<0.05),caspase-1(0.95±0.05 vs.1.61±0.11;P<0.05),and IL-1β(1.55±0.18 vs.3.17±0.43;P<0.05)mRNAs and TXNIP(0.78±0.04 vs.1.26±0.13;P<0.05),NLRP3(0.26±0.03 vs.0.43±0.04;P<0.05),caspase-1(0.71±0.05 vs.1.30±0.09;P<0.05),IL-1β(0.54±0.03 vs.1.21±0.11;P<0.05),and IL-18(0.34±0.08 vs.0.54±0.07;P<0.05)proteins.Conclusion Sinomenium was able to downregulate BV-2 microglial cell pyroptosis and inflammation by inhibiting the NLRP3/caspase-1 signaling pathway.