Pneumolysin Induced Huvec To Apoptosis Bymapk Signal Pathway

ObjectivePneumolysin (PLY) is one of the most important toxin of the Streptococcus pneumoniae (S.pn). The recently study suggested that PLY could induced host cell to apoptosis. But the mechanism remains poorly clarified. We obtained purified pneumolysin protein that produced by prokaryotic expression system and adopted the HUVEC as the cell model in vitro. Then, we investigated whether the PLY induced HUVEC apoptosis in vitro. And to confirmed whether it relatived the caspase pathway and MAPK pathway during PLY induced HUVEC apoptosis.Methods1.PCR amplified the Ply gene in vitro. Then the PCR fragments were then cloned into pW28 expression vector, followed by sequencing. Recombinant protein PLY was over-expressed and purified from the host of E.coli BL21, and induced by IPTG. The protein Purified by Ni-NTA resin and identified by SDS-PAGE. Purified protein PLY through hemolysis test to evaluate haemolytic activity of PLY.2. Taking the HUVEC as the experimental object, we investigated the effects of PLY on the HUVEC proliferation and apoptosis in vitro. Firstly, MTT assay was employed and evaluate the proliferative percentage after the HUVEC co-incubation with the different levels PLY for different time (24h, 48h, 72h, 96h). Secondly, the morphlolgical changes of HUVEC was observed by the optical microscope and the Annexin V/PI staining assay was perform to test the apoptosis after the HUVEC co-incubation with the different levels PLY for 24h. Thirdly, we investigated the DNA ladder after the HUVEC co-incubation with 1μg/ml PLY.3. Taking the HUVEC as the experimental object, the activities of Caspase-3, Caspase-8 and Caspase-9 were analyzed by spectrophotometry after the HUVEC co-incubation with 1μg/ml PLY. Then, Immunohistochemistry analyzed the expression of the protein ERK1/2 and p38MAPK. Finally, Western Blot analyzed the expression and phosphorylation of the protein ERK1/2 and p38MAPK.Results1. The Ply gene was successfully cloned into pW28, which was confirmed by gene sequencing. PLY protein purified and it was sucessfully over-expressed in soluble manner, which was confirmed by SDS-PAGE. The purity coefficient is 95%. Compared with control, the PLY had a high haemolytic activity in a dose-dependent manner (P<0.05). The haemolytic activity suggesting that the haemolytic activit of 100ng/ml PLY reach 100%. 2. PLY could inhibit proliferation of HUVEC in a dose-and-time-dependent manner (P<0.05). The IC50(24h) was 1.525μg/ml. The number of HUVEC was significantly reduced accompanied decreased adhesion ability after the PLY treated in a dose-dependent manner. Significantly, It showed that the PLY-treated HUVEC lost the typical morphous, a part of the HUVEC shrunk and rounded. The Annexin V/PI staining suggested that the apoptosis rate of HUVEC was 8.91%, 27.43% and 31.50% after the 0.5μg/ml, 1μg/ml and5μg/ml of PLY treated (P<0.05) respectively. The agarose gel electrophoresis showed that the DNA of PLY-treated HUVEC fragmented.3. The activities of caspase-3, caspase-8 and caspase-9 in HUVEC were not significantly change after PLY treated. Compare with the control, Immunohistochemistry indicated that the expression of ERK1/2 was decreased and the expression of p38MAPK was increased (P<0.05). Western Blot suggested that the expression of ERK1/2 and p- ERK1/2 was decreased. And the expression of p38MAPK and p- p38MAPK was increased (P<0.05).ConclusionPLY could inhibit proliferation and induce apoptosis of HUVEC. This effect partly through inhibit ERK signal pahway and activate p38MAPK signal pathway, and it induce HUVEC apoptosis not dependent active capases.