TLR2-PI3K/Akt Signaling Pathway Regulates Chlamydia Pneumoniae Infection-induced Vascular Smooth Muscle Cell Migration

Objecti veUsing the model of Rat primary vascular smooth muscle cell (VSMC) infected with Chlamydia pneumoniae (C.pn) in vitro, to observe the effects of C.pn infection on VSMC migration, to investigate the roles of phosphoinositide3-kinase (PI3K)/Serine/threonine kinase (Akt) signaling pathway and Toll like receptor2(TLR2) in VSMC migration induced by C.pn infection, to explore the related molecular mechanisms.Methods1. VSMCs were infected with C.pn in vitro after the culture and propagation of C.pn.2. Successful infection of VSMCs with C.pn was identified by the observation of C.pn inclusions under Transmission electron microscope (TEM).3. Wound-healing assay and Transwell assay were performed to investigate the effect of C.pn infection on VSMC migration.4. Reverse transcription-polymerase chain reaction (RT-PCR) was used to determine the mRNA expression level of PI3K after C.pn infected VSMCs.5. Western blot was performed to detect the p-Akt expression level in the VSMCs at different time potins after C.pn infection.6. After the PI3K activity was inhibited by PI3K specific inhibitors, LY294002. Western blot was performed to detect the p-Akt expression level in the infected VSMCs:Wound-healing assay and Transwell assay were performed to explore the changes in VSMC migration induced by C.pn infection.7. The activity of TLR2was blocked by a TLR2neutral antibody, and then Western blot was performed to detect the p-Akt expression level in order to explore the roles of PI3K/Akt signaling pathway and TLR2in VSMC migration induced by C.pn infection. Results1. The typical C.pn elementary body, were observed in C.pn inclusions in the cytoplasm of the infected VSMCs. indicating the successful infection of VSMCs with C.pn.2. The migration area of C.pn-infected VSMCs was significantly larger than that of control group in a wound-healing assay (P<0.05). In a Transwell assay, VSMCs infected with C.pn were found to migrate more than the control group (P <0.05).3. RT-PCR results showed that there was no significant difference in the mRNA expression of PI3K in the VSMCs between C.pn infection group pretreated with LY294002and the control group. Also no significant difference of the PI3K mRNA expression in the VSMCs between C.pn infection group pretreated with LY294002and C.pn infection group4. Western blot results showed that the significant activation of Akt was observed in the C.pn-infected VSMCs. C.pn stimulated phosphorylation of Akt occurred as early as30min and increased gradually to reach a peak at1h postinfection and lasted up to6h. and then decreased until24h after infection. No significant changes in total Akt protein expression were detected during the infection.5. Western blot results showed that the expression of p-Akt of C.pn-infected VSMCs pretreated with LY294002was decreased compared to C.pn infection group (P<0.05). No significant changes in total Akt protein expression were detected during the infection.6. Wound-healing assay results showed that the migration area of C.pn-infected VSMCs pretreated with LY294002was significantly lower than that of C.pn infection group (P<0.05). In the Transwell assay, the infected VSMCs pretreated with LY294002were found to migrate less than C.pn infection group (P <0.05).7. Western blot resultes showed that the expression of p-Akt induced by C.pn infection significantly decreased after the activity of TLR2was blocked by TLR2neutral antibody (P<0.05). No significant changes in total Akt protein expression were detected during the infection.ConclusionC.pn infection promotes VSMC migration through PI3K/Akt signaling pathway via TLR2.