Effects of Actin Depolymerization on TLRs Expression and Apoptosis in Macrophages

Abstract: 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 an 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.

CSTR: 32200.14.cjcb.2018.12.0003