钩端螺旋体脂多糖诱导J774A.1细胞凋亡及相关信号通路调控作用的研究

目的 了解问号钩端螺旋体脂多糖(L-LPS)体外诱导小鼠单核-巨噬样细胞株(J774A.1)凋亡及Toll样受体(TLR)和相关信号通路调控细胞凋亡的作用.方法 酚水法从问号钩体黄疸出血群赖型赖株中提取L-LPS.流式细胞术测定L-LPS诱导J774A.1细胞凋亡及FasL中和抗体阻断细胞凋亡的作用.实时荧光定量RT-PCR(qPCR)及流式细胞术检测L-LPS作用前后J774A.1细胞Fas/FasLmRNAs和蛋白表达水平变化.TLR2或TLR4抗体封闭试验、信号通路阻断试验及流式细胞术,检测TLR2、TLR4及p38丝裂原活化蛋白激酶(MAPK)、c-Jun氨基末端激酶(JNK)和胞外信号调节激酶(ERK)通路对L-LPS诱导细胞凋亡的调控作用.结果 100 ng/ml L-LPS作用J774A.1细胞4、12和24 h的凋亡率分别为56.50%、69.28%和24.35%,FasL中和抗体封闭后,细胞凋亡率分别下降至11.21%、21.58%和12.70%(P＜0.05).L-LPS作用4、12和24 h的J774A.1细胞FasL和Fas mRNAs水平分别为正常细胞的1.34、2.12、2.10及2.45、3.87、3.12倍(P＜0.05),FasL和Fas蛋白表达率分别从L-LPS作用前的4.82%和15.32%上调至18.61%、60.13%、42.75%(P＜0.05)和76.34%、85.70%和77.92%(P＜0.05).TLR2抗体封闭后L-LPS诱导J774A.1细胞的凋亡率(11.54%)明显低于未封闭细胞(66.56%,P＜0.05),但TLR4抗体封闭细胞的凋亡率仍高达55.27%(P＞0.05).p38MAPK与JNK通路阻断后L-LPS诱导J774A.1细胞凋亡率(20.54%和47.98%)明显低于未阻断细胞(62.17%,P＜0.05),ERK通路阻断后细胞凋亡率仍高达61.72%(P＞0.05).结论 L-LPS经TLR2识别后通过p38MAPK和JNK通路上调J774A.1细胞Fas和FasL表达,从而参与L-LPS诱导细胞凋亡过程.

J774A. 1 cell apoptosis induced by Leptospira interrogans lipopolysaccharide and apoptotic regulation of associated signaling pathways

Objective To determine the effect of Leptospira interrogans lipopolysaccharide (L-LPS) inducing apoptosis of murine mononuclear-macrophage cell line( J774A. 1 ), and apoptotic regulation of Toll-like receptor(TLR) and associated intracellular signaling pathways. Methods Lipopolysaccharide (L-LPS) of L. interrogans serogroup Icterohaemorrhagiae serovar Lai strain Lai 56601 was prepared using phenol-water method. The effects of L-LPS inducing J774A. 1 cell apoptosis and the apoptosis-blocking with FasL neutralizing antibody were detected by flow cytometry. Real-time fluorescent quantitative RT-PCR (qPCR) and flow cytometry were performed to measure the changes of Fas/FasL mRNA and protein expression levels in J774A. 1 cells before and after L-LPS treatment. The regulations in L-LPS-induced cell apoptosis by TLR2 and TLR4 as well as p38MAPK, JNK, ERK pathways were determined by either TLR2 or TLR4 antibody blocking test, signaling pathway blocking test and flow cytometry. Results 56.50%, 69.28% and 24.35% of the J774A. 1 cells after treatment with 100 ng/ml L-LPS for4, 12 and 24 h were apoptotic,while the apoptosis rates were decreased to 11.21%, 21.58% and 12.70% after the cells blocked by FasL neutralizing antibody(P ＜0.05). The levels of FasL and Fas mRNAs in J774A. 1 cells treated with L-LPS for 4, 12 and 24 h were elevated with 1.34, 2.12, 2.10 times and 2.45, 3.87, 3.12 times compared to those in the L-LPS untreated cells (P ＜ 0. 05 ), respectively, while the expression rates of FasL and Fas proteins were upregulated to 18.61%, 60.13%, 42.75% and 76.34%, 85.70%, 77.92% from 4.82% and 15.32% apoptotic rates in the L-LPS untreated cells, respectively( P ＜0.05 ). The L-LPS-induced apoptosis rate( 11.54% ) of TLR2 antibody blocked J774A. 1 cells was significantly lower than that(66.56% ) of the J774A. 1 cells without TLR2 antibody blocking( P ＜0.05 ), but L-LPS-induced apoptosis rate of TLR4 antibody blocked J774A. 1 cells was as high as 55.27% ( P ＞ 0.05 ). Compared to the apoptosis rate (62.17%) in the p38MAPK and JNK pathway-free J774A. 1 cells, the L-LPS-induced apoptosis rates in p38MAPK blocked cells(20.54% ) and JNK blocked cells(47.98% ) were significantly lower( P ＜0.05 ),and the apoptosis rate in ERK blocked cells was as high as 61.72% ( P ＞ 0.05 ). Conclusion L-LPS was recognized by TLR2 and upregulates both Fas and FasL expression via p38MAPK and JNK pathways, which involving in the process of the L-LPS-induced cell apoptosis.