竹节参乙酸乙酯提取物对脂多糖诱导的BV2小胶质细胞炎症反应的影响及其机制研究

Ethyl Acetate Extract of Panax japonicus Alleviates LPS-induced Inflammation in BV2 Cells

目的探讨竹节参乙酸乙酯提取物(EtOAc-PJ)对脂多糖(LPS)诱导的BV2小胶质细胞炎症损伤的保护作用及其机制。方法采用LPS(1μg·mL^-1)对BV2小胶质细胞进行诱导炎症反应,并以EtOAc-PJ 3.125、6.25、12.5、25μg·mL^-1进行干预。采用噻唑蓝法(MTT)检测EtOAc-PJ对BV2细胞增殖的影响;Griess法检测BV2细胞上清液中一氧化氮(NO)的释放量;逆转录聚合酶链反应法(RT-PCR)检测炎症因子诱导型一氧化氮合酶(iNOS)、白细胞介素1β(IL-1β)及肿瘤坏死因子α(TNF-α)mRNA表达水平;免疫荧光法(IF)检测BV2细胞中核转录因子κB(NF-κB)的表达及定位;免疫印迹法(Western Blot)检测炎症小体3(NLRP3)、凋亡相关斑点样蛋白(ASC)、半胱氨酸蛋白酶-1(Caspase-1)及IL-1β的蛋白表达情况。结果 3.125、6.25、12.5、25μg·mL^-1EtOAc-PJ单独作用或与1μg·mL^-1LPS共同培养24 h,对BV2细胞存活率均无明显影响(P>0.05)。与正常对照组比较,LPS模型组的BV2细胞上清液中NO含量显著增加(P<0.01);BV2细胞iNOS、IL-1β及TNF-αmRNA表达水平均明显升高(P<0.05,P<0.01);BV2细胞NF-κB p65核移位的水平明显增加;BV2细胞NLRP3、ASC、Caspase-1及IL-1β蛋白表达水平均显著升高(P<0.05,P<0.01)。与LPS模型组比较,EtOAc-PJ 6.25、12.5、25μg·mL^-1剂量组的BV2细胞NO释放水平明显降低(P<0.05,P<0.01);EtOAc-PJ 12.5、25μg·mL^-1剂量组的iNOS、TNF-αmRNA表达水平均明显降低(P<0.05,P<0.01),EtOAc-PJ 25μg·mL^-1剂量组的IL-1βmRNA表达水平明显降低(P<0.01);EtOAc-PJ 12.5、25μg·mL^-1预处理能明显抑制LPS诱导的BV2细胞NF-κB p65核转位;EtOAc-PJ各剂量组的BV2细胞NLRP3、ASC、IL-1β蛋白表达水平明显降低(P<0.01),EtOAc-PJ 6.25、12.5、25μg·mL^-1剂量组的Caspase-1蛋白表达水平均明显降低(P<0.01)。结论 EtOAc-PJ可以通过抑制BV2小胶质细胞激活,减少炎症介质释放和促炎细胞因子表达,从而减轻LPS诱导的BV2小胶质细胞炎症反应,其作用机制可能与抑制NF-κB活化及NLRP3炎症小体激活有关。

Objective To investigate the protective effects of ethyl acetate extract of Panax japonicas(EtOAc-PJ)on lipopolysaccharide(LPS)-induced inflammation in BV2 cells and its underlying mechanisms.Methods LPS(1 μg·mL^-1) was used to induce inflammation in BV2 cells,and intervention was performed with EtOAc-PJ 3.125,6.25,12.5,25 μg·mL^-1.The cytotoxicity of EtOAC-PJ on BV2 cells was assessed by MTT method.The level of nitric oxide(NO) was measured using Griess method.RT-PCR was used to detect the mRNA levels of iNOS,IL-1βand TNF-α.The expression and localization of NF-κB were observed by immunofluorescence(IF).Additionally,Western Blot(WB) was used to evaluate the protein levels of NLRP3,ASC,Caspase-1 and IL-1β.Results EtOAc-PJ at doses of 3.125,6.25,12.5,25 μg·mL^-1 alone or co-cultured with 1 μg·mL^-1 LPS for 24 h had no significant effect on the survival rate of BV2 cells(P>0.05).Compared with the normal control group,the NO content in BV2 cell supernatant of the LPS model group increased significantly(P<0.01);the mRNA expression of iNOS,IL-1β and TNF-α rose notably(P<0.05,P<0.01).The level of NF-κB p65 nuclear translocation escalated remarkably;the protein levels of NLRP3,ASC,Caspase-1 and IL-1β were up-regulated obviously(P<0.05,P<0.01).Compared with the LPS group,EtOAc-PJ 6.25,12.5,25 μg·mL^-1 could significantly reduce NO levels in LPS-induced BV2 cells(P<0.05,P<0.01);the mRNA levels of iNOS and TNF-α in EtOAc-PJ 12.5 and 25 μg·mL^-1 groups were appreciably alleviated(P<0.05,P<0.01),and IL-1β mRNA expression in EtOAcPJ 25 μg·mL^-1 was considerably decreased(P<0.01);EtOAc-PJ 12.5,25 μg·mL^-1 pretreatment could inhibit LPS-induced nuclear translocation of NF-κB p65;NLRP3,ASC,IL-1β protein levels in each dose group of EtOAc-PJ were markedly lowered(P<0.01),the Caspase-1 protein expression in EtOAc-PJ 6.25,12.5,25 μg·mL^-1dose groups were apparently dropped(P<0.01).Conclusion EtOAc-PJ can attenuate LPS-induced inflammation,which is mainly manifested as reducing the release of inflammatory mediators and inhibiting the expression of pro-inflammatory cytokines.Its underlying mechanism may be related to inhibition of NF-κB pathway and NLRP3 inflammasome activation.