冠蛋白-1不同表达水平对巨噬细胞iNOS、TLRs表达以及凋亡的影响

The Effects of Coronin-1 with Different Expression Levels on iNOS and TLRs Expression and Apoptosis in RAW264.7 Cells

冠蛋白-1(Coronin-1)在真核细胞中广泛表达,涉及钙离子稳态、细胞骨架动力学、免疫及炎症反应等多种细胞功能。该研究探讨了冠蛋白-1不同表达水平对巨噬细胞诱导性一氧化氮合酶(inducible nitric oxide synthase,iNOS)、Toll样受体(Toll-like receptors,TLRs)表达和凋亡的影响并探讨其分子机制。根据冠蛋白-1表达水平的差异,实验细胞分为RAW264.7-Cor.Plus、RAW264.7、RAW264.7-Cor.Minus 3组。各组细胞分别经4μmol/L钙调磷酸酶抑制剂环孢素A(cyclosporin A,Cs A)处理24 h或2μmol/L肌动蛋白聚合抑制剂松胞菌素D(cytochalasin D,cyt D)处理48 h,同时设未处理对照,再分别用Real-time PCR法检测iNOS m RNA水平,Western blot法检测细胞TLR2、TLR4、TLR9及钙调磷酸酶(calcineurin,Ca N)的蛋白质水平,流式细胞术检测细胞凋亡百分率。各组细胞用四甲基异硫氰酸荧光素标记的鬼笔环肽(TRITC-phalloidin)染色后,计算纤维型肌动蛋白(F-actin)重排率。结果显示,冠蛋白-1过表达细胞相对于冠蛋白-1正常表达细胞,其iNOSm RNA水平以及TLR2、TLR4、TLR9蛋白表达水平和细胞凋亡率均显著降低(P<0.05);Cs A作用后,RAW264.7-Cor.Plus的iNOS m RNA水平以及TLR2、TLR4、TLR9蛋白质水平和细胞凋亡率均较未处理对照细胞显著增加(P<0.05),RAW264.7和RAW264.7-Cor.Plus细胞的Ca N水平较未处理对照细胞显著降低(P<0.05);cyt D作用后,与未处理对照细胞相比,iNOS m RNA水平及细胞凋亡率无显著性差异(P>0.05),但TLRs的表达显著降低(P<0.05);经鬼笔环肽染色后,RAW264.7-Cor.Plus细胞F-actin重排率显著高于RAW264.7细胞(P<0.05)。以上结果表明,冠蛋白-1过表达不仅促进了巨噬细胞F-actin重排,而且下调了iNOS、TLRs水平并且抑制细胞凋亡,其分子机制与冠蛋白-1促进钙调磷酸酶调控的Ca^(2+)信号通路有关。

Coronin-1 is widely expressed in eukaryotic cells, which have been reported to be involved in a wide array of cellular functions including calcium homeostasis, cytoskeletal dynamics, immune and inflammatory responses. In order to investigate the effect and molecular mechanisms of different levels of Coronin-1 on the protein levels of iNOS and TLRs and apoptosis in RAW264.7 cell line, the experiment was divided into three groups（RAW264.7-Cor.Plus, RAW264.7 and RAW264.7-Cor.Minus） according to the differences of Coronin-1 expression level. Before and after treated with the Cs A（a Calcineurin inhibitor） for 24 hours or cyt D（an actin polymerization inhibitor） for 48 hours respectively, the level of iNOS m RNA was tested by Real-time PCR. The protein levels of TLR2, TLR4, TLR9 and calcineurin were detected by Western blot, and the apoptosis percent was analyzed by flow cytometry. Three groups of cells were stained with TRITC-phalloidin, then the percentage of F-actin rearrangement was calculated. The results were showed that the m RNA level of iNOS, the protein level of TLRs and apoptosis percent were significantly decreased in cells with over-expression of Coronin-1（RAW264.7-Cor.Plus）（P〈0.05）. After treated with Cs A, the m RNA levels of iNOS, the protein levels of TLR2, TLR4 and TLR9 and apoptosis rate in RAW264.7-Cor.Plus cells were significantly increased compared with the untreated control cells（P〈0.05）, but the Ca N level of RAW264.7 and RAW264.7-Cor.Plus cells were significantly lower than that of untreated control cells（P〈0.05）. After treated with cyt D, between the treated cells and their untreated control cells, there were no significant difference in the m RNA levels of iNOS and apoptosis rate（P〉0.05）, but the m RNA level of TLRs was significantly decreased（P〈0.05）. By phalloidin staining, the F-actin rearrangement rate of RAW264.7-Cor.Plus cells was significantly higher than that of RAW264.7 cells（P〈0.05）. These results indicated that the over-expression of Coronin-1 not only promotes the F-actin rearrangement, but also down-regulates the expression of TLRs and iNOS and inhibits cell apoptosis, of which the molecular mechanism is associated with that Coronin-1 promotes Ca2＋ signaling pathway regulated by Calcineurin.