肌动蛋白解聚对巨噬细胞TLRs的表达及细胞凋亡的影响

Effects of Actin Depolymerization on TLRs Expression and Apoptosis in Macrophages

在肺癌等的癌细胞中,脂多糖(lipopolysaccharide, LPS)等致炎因子介导的促瘤效应涉及Toll样受体(Toll like receptors, TLRs)/纤维型肌动蛋白(fibrous actin, F-actin)通路。该研究探讨了外源性松胞菌素D(cytochalasin D, cytD)和细胞内源过表达的重组肌动蛋白解聚因子丝切蛋白(Cofilin-1)所诱导的肌动蛋白解聚对巨噬细胞TLR2、TLR4和TLR9的表达和细胞凋亡水平的影响。分别用6种不同浓度的cytD处理RAW264.7小鼠巨噬细胞48 h,用Western blot检测6组细胞TLR2、TLR4、TLR9的表达水平。构建重组pEGFP-N1-Cofilin-1质粒并转染RAW264.7细胞,设置空质粒对照和空白细胞对照组。用Real-time PCR法和Western blot检测细胞转染前后Cofilin-1表达水平;Western blot检测巨噬细胞TLR2、TLR4、TLR9的表达情况;用流式细胞术检测3组细胞的凋亡水平。结果显示,终浓度≥1.5μmol/L的cytD处理细胞后, RAW264.7细胞表达TLR2明显降低(P<0.05);终浓度≥1.0μmol/L的cytD处理细胞后,巨噬细胞表达TLR4、TLR9明显降低(P<0.05)。重组质粒转染组细胞的cofilin-1 mRNA及Cofilin-1蛋白水平均高于空质粒对照组和空白细胞对照组(P<0.05),且其TLR2、TLR4、TLR9蛋白表达水平和细胞凋亡水平均显著低于空质粒对照组和空白细胞对照组(P<0.05)。结果表明,外源性cytD和巨噬细胞内源性高表达的Cofilin-1蛋白均可下调巨噬细胞TLR2、TLR4、TLR9的表达,其中,细胞高表达的Cofilin-1蛋白还显著降低了细胞凋亡率。该文通过揭示肌动蛋白解聚对巨噬细胞TLRs的表达及细胞凋亡的抑制,为进一步研究炎症与肿瘤的关系及分子机制奠定了基础。

In lung cancer and other tumor cells, the tumor-promoting effect mediated by lipopolysaccharide(LPS) involves the TLRs/F-actin pathway. This study investigated the effect of actin depolymerization induced by exogenous cytochalasin D and over-expressed recombinant Cofilin-1 in cells on the expression of TLR2, TLR4 and TLR9 and the apoptosis level in macrophages. After the RAW264.7 cells were treated with 6 different concentrations of cytD for 48 h, the expression levels of TLR2, TLR4 and TLR9 in each group were detected by Western blot respectively. The pEGFP-N1-Cofilin-1 recombinant plasmids were constructed and then transfected into RAW264.7 cells, and the blank plasmid control group and the blank cell control group were set up. The mRNA and protein expression level of Cofilin-1 were detected by Real-time PCR and Western blot respectively. The TLRs expression level were detected by Western blot, and the cell apoptosis level of the three groups was detected by flow cytometry. The results showed that the expression of TLR2 in RAW264.7 cells significantly decreased after cytD treatment with a concentration no less than 1.5 μmol/L and the expression of TLR4 and TLR9 was significantly decreased after cytD treatment with a concentration no less than 1.0 μmol/L(P<0.05). The level of cofilin-1 mRNA and Cofilin-1 protein in the cells transfected with recombinant plasmid were higher than that in the negative control group and blank control group(P<0.05), and the protein level of TLR2, TLR4, TLR9 and the apoptosis level in the cells transfected with recombinant plasmid were significantly lower than those in the control groups(P<0.05). Above all, cytD could reduce the expression of TLR2, TLR4 and TLR9 in macrophages. cofilin-1 mRNA and Cofilin-1 protein expression in macrophage transfected with the recombinant plasmid significantly increased. High expression of Cofilin-1 protein inhibited the expression of TLR2, TLR4 and TLR9 in macrophages, and reduced apoptosis rate. All results showed that the exogenous cytD and the endogenous over-expression of Cofilin-1 protein both could down regulate the expression of TLR2, TLR4 and TLR9 in macrophages, and the over-expression of Cofilin-1 protein also significantly reduced the apoptosis rate. The inhibition effect of actin depolymerization on TLRs expression and apoptosis of macrophages could lay a foundation for further study on the relationship between inflammation and tumor and its molecular mechanism.