Antagonistic Effects And Molecular Mechanisms Of Berberine On Chlamydia Pneumoniae Induced-Vascular Endothelial Cell Migration And Angiogenesis

Objectives:To establish the model of Chlamydia pneumoniae (C.pn)-infected vascular endothelial cell (VEC) in vitro, to observe the development cycle of C.pn and ultrastructural alteration of infected VEC, to observe the effects of C.pn infection on migration of VEC and angiogenesis, to investigate the antagonistic effects of berberine on C.pn induced-VEC migration and angiogenesis, then to explore its related molecular mechanisms.Methods:1. C.pn was proliferated in HEp-2 cells and purified with gradient centrifugation; the model of C.pn-infected VEC in vitro was established with the purified C.pn.2. At 24 h,48 h and 72 h post-infection, the morphological changes of C.pn inclusions and infected VECs were observed under fluorescence microscope by acridine orange staining (AO).3. At 24 h,48 h and 72 h post-infection, the ultrastructure characteristics of C.pn inclusions and infected VECs were observed under transmission electron microscope (TEM).4. Wound healing assay was performed to observe the changes of C.pn-infected VEC migration, and the effects of berberine on the cell migration.5. Transwell assay was performed to observe the changes of C.pn-infected VEC migration, and the effects of berberine on the cell migration.6. ELISA was performed to detect the levels of MMP-1 and MMP-9 secreted by C.pn-infected VECs, and the effects of berberine on the levels of MMPs.7. Capillary tube formation assay was performed to observe the changes of angiogenesic ability of C.pn-infected VECs, and the effects of berberine on angiogenesis.8. RT-PCR was performed to detect the PI3K mRNA expression in C.pn-infected VECs, and the effects of berberine on the PI3K mRNA expression.9. ELISA was performed to detect the PI3K enzymatic activity of C.pn-infected VECs, and the effects of berberine on the PI3K enzymatic activity. Results:1. Under light microscope, the C.pn inclusions were observed in the infected VECs; the model of C.pn-infected VEC in vitro was successfully established, which was identified by amplification products of C.pn specific DNA sequences.2. Under fluorescence microscope, the typical C.pn inclusions appeared in VECs at 24 h post-C.pn infection; more C.pn inclusions can be observed after 48 h infection; and the inclusions began to release from the disintegrated VEC at 72 h after infection.3. Under TEM, C.pn inclusions proliferated in VEC at 24 h post-C.pn infection; the rough endoplasmic reticulum (RER) dilated in VECs, C.pn elementary bodies (EBs) containing electron-dense deposits and bubble-like C.pn reticulate bodies (RBs) appeared after 48h infection; C.pn inclusions began to release from the disintegrated VEC at 72 h infection.4. At 24h,48 h and 72 h post-C.pn infection, the two-dimensional migration of VECs was accelerated (P<0.01)while inhibited by LY294002(P<0.01), and it decreased markedly after administration of the middle and high-dose berberine (P<0.01)5. At 72 h post-C.pn infection, the three-dimensional migration of VECs significantly increased (P<0.01) while inhibited by LY294002 (P<0.01); VEC migration decreased gradually after administration of the low, middle and high-dose berberine (low dose P<0.05, middle and high dose P<0.01), and the inhibitory effects of berberine were enhanced with the increase in its concertrations.6. At 24,48 and 72 h post-C.pn infection, the levels of MMP-1 and MMP-9 secreted by infected VECs showed no difference, compared with the nomal group (P> 0.05), and there is no statistic difference between LY294002 group, berberine groups and infected group on the levels of MMPs (P> 0.05)7. At 72 h post-Cpn infection, the ability of VECs to form capillary tube increased (P<0.01) while blocked by LY294002 (P<0.01), and the angiogenesic ability of VECs decreased gradually after administration of the low, middle and high-dose berberine (P<0.01), and the inhibitory effects of berberine were enhanced with the increase in its concertrations.8. At 72 h post-C.pn infection, the mRNA expression and enzymatic activity of PI3K increased significantly (P<0.01), while inhibited by LY294002 (P<0.01), and they both decreased markedly after administration of the middle-dose and high-dose berberine (P<0.01). The migration and angiogenesis of VECs was positively correlated with the mRNA expression and enzymatic activity of PI3K (r1=0.841, r1'=0.656, P<0.01; r2=0.832, r2'=0.813, P<0.01).Conclusions:C.pn may induce VEC migration and angiogenesis via the activation of PI3K, but not via stimulating the secretion of MMP-1 and MMP-9. Berberine can antagonize the effect of C.pn infection on VEC migration and angiogenesis possibly through down-regulating the PI3K mRNA expression and inhibiting the activation of PI3K, but not changing the levels of MMP-1 and MMP-9.