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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 and involves many cellular functions such as calcium homeostasis, cytoskeletal dynamics, immune and inflammatory responses. This study explored the effects of different levels of canopy protein-1 on the expression of inducible nitric oxide synthase (iNOS), Toll-like receptors (TLRs) and apoptosis in macrophages Explore its molecular mechanism. According to the different level of Guanin-1 expression, experimental cells were divided into RAW264.7-Cor.Plus, RAW264.7 and RAW264.7-Cor.Minus 3 groups. The cells in each group were treated with 4μmol / L cyclosporin A (CsA) for 24 h or 2μmol / L actin polymerization inhibitor cytochalasin D (cyt D) The mRNA and protein levels of iNOS were detected by Real-time PCR and Western blot respectively. The protein levels of TLR2, TLR4, TLR9 and calcineurin (Ca N) were measured by flow cytometry Cytometry was used to detect the percentage of apoptosis. The cells were stained with tetramethyl fluorescein isothiocyanate (TRITC-phalloidin) to calculate the F-actin rearrangement rate. The results showed that the expression of iNOSmRNA and the expression of TLR2, TLR4 and TLR9 protein and apoptosis rate of canopy protein-1-overexpressing cells were significantly decreased compared with that of canonical protein-1 (P <0.05) (P <0.05). Compared with RAW264.7-Cor.Plus, the mRNA and protein levels of iNOS and TLR2, TLR4, TLR9 and RAW264.7-Cor (P <0.05). Compared with untreated control cells, the level of iNOS m RNA and the apoptosis rate of cytotoxic T lymphocytes were not significantly different (P> 0.05) ), But the expression of TLRs was significantly decreased (P <0.05). After 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 over-expression of CGRP not only promoted the rearrangement of F-actin in macrophages but also decreased the levels of iNOS and TLRs and inhibited the apoptosis of macrophages. The molecular mechanism of CGRP is related to that of CAMP-1 Ca ~ (2+) signaling pathway.