Berberine Inhibits Chlamydia Pneumoniae Infection-induced Vascular Smooth Muscle Cell Migration Through Regulating Expression Of MMP3and MMP9via PI3K

ObjectiveUsing the model of rat primary vascular smooth muscle cell (VSMC) infected with Chlamydia pneumoniae (C.pn) in vitro, to observe the roles of MMPs in VSMC migration induced by C.pn infection; and to observe the antagonistic effects of berberine on C.pn infection-induced VSMC migration, and to explore its related molecular mechanisms.Methods1. Rat primary VSMCs were infected with C.pn in vitro after the culture and propagation of C.pn.2. After the MMPs were inhibited by a broad-spectrum MMP inhibitor GM6001, wound-healing assay and invasion assay were performed to observe the roles of MMPs in VSMC migration induced by C.pn infection.3. Reverse transcription-polymerase chain reaction (RT-PCR) was used to detect themRNA expression of MMPs in C.pn-infected VSMCs.4. Gelatin zymography was detected the change of MMP9enzymatic activity in the infected VSMCs.5. ELISA was performed to determine the protein expression levels of MMP3and MMP9in Cpn-infected VSMCs.6. After PI3K activity was inhibited by a PI3K specific inhibitor LY294002, gelatin zymography and ELISA were used to determine the changes in MMP9enzymatic activity and the protein expression levers of MMP3and MMP9, respectively.7. CCK-8assay was determined the effects of berberine at the differet concentrations on VSMCs viability..8. After adminstration with berberine at the different concentrations, wound-healing assay and invasion assay were performed to investigate the effects of berberine on VSMCs migration induced by C.pn infection; gelatin zymography and ELISA were used to detect the changes in MMP9enzymatic activity and the protein expression levers of MMP3and MMP9, respectively. 9. After VSMCs were administrated with berberine at the indicated concentrations, western blot was performed to detect the p-Akt expression level in the infected VSMCs.Results1. Wound-healing assay results showed that the migration area of C.pn-infected VSMCs was significantly larger than that of normal control group (P<0.05); the migration area of C.pn-infected VSMCs pretreated with GM6001was found to migrate less than that of C.pn infection group (P<0.05). In the invasion assay, the number of VSMCs infected with C.pn that invased through Matrigel was much larger than that of the normal (P<0.05); the number of Cpn-infected VSMCs pretreated with GM6001through Matrigel was less than that of C.pn infection group (P<0.05).2. RT-PCR results showed that there was no significant difference in the mRNA expressions of MMP1and MMP2in the VSMCs between the different time points after C.pn infection (P>0.05), whereas the mRNA expression of MMP3was up-regulated at4h and reached the peak value at18h post-infection in the infected VSMCs, and remained until72h post-infection (P<0.05); MMP9mRNA expression started to go up at18h and reached the peak value at36h post-infection in the infected VSMCs, remained until72h post-infection (P<0.05).3. The result of gelatin zymography showed that the MMP9activity began to increase at6h post-infection, and finally reached a peak at72h. The MMP9activity was positively correlated with the time of C.pn-infected VSMCs in C.pn infection period (r=0.9829, P<0.05). When the infected VSMCs were pretreated with LY294002, the MMP9activity was significantly inhibited compared to C.pn infection group (P<0.05).4. ELISA results showed that the protein expression of MMP3began to rise at12h post-infection, and increased obviously at24h (P<0.05) and reached a peak72h after C.pn infection. The protein expression of MMP9began to rise at24h post-infection, and increased obviously at72h postinfection (P<0.05). The protein expression of MMP3and MMP9in Cpn-infected VSMCs pretreated with LY294002were both decreased compared to C.pn infection group (P<0.05).5. The results from CCK-8assay showed that berberine at the concentrations of less than100μM did not reduce the viability of VSMCs compared with normal control group, whereas berberine at the concentrations of higher than100μM resulted in a reduction of viability with the increase in berberine concertrations compared with normal control group (P<0.05). Berberine at150μM decreased the VSMC viability to63%of normal.200μM and300μM of berberine resulted in53%and81%reduction of normal in the viability of VSMCs, respectively.6. After VSMCs were adminstrated with berberine at the indicated concentrations, the migration area of VSMCs induced by C.pn infection reduced gradually with the increase in berberine concertrations in a wound-healing assay compared to C.pn infection group (P<0.05). The resusts from the invasion assay showed that the number of VSMCs through Matrigel decreased gradually with the increases in berberine concertrations (P<0.05), when C.pn-infected VSMCs were adminstrated with berberine at the indicated concentrations.7. After C.pn-infected VSMCs were adminstrated with berberine at the different concentrations, MMP9activity and the protein expression of MMP3and MMP9induced by C.pn infection were significantly inhibited with the increase in berberine concertration (P<0.05).8. Western blot analysis showed that berberine significantly decreased the expression of p-Akt induced by C.pn infection(P<0.05). There were no significant changes in total Akt and β-actin protein expressions during the infection.ConclusionC.pn infection promotes VSMC migration possibly through up-regulating of MMP3and MMP9via PI3K; berberine may inbihit VSMC migration through negatively regulating the PI3K/Akt related enzymatic activities and protein expression of MMP3and MMP9.