The Role Of HIF-1α Mediated ROCK Signaling Pathway In The Protective Effects Of Ginsenoside Rb1 On The Lead Acetate-induced Injury In Nerve Cells

Objective Employing the treatment with lead acetate to induce PC12 cell injury, to determine the effects of ginsenoside Rb1 on cell injury and investigate the role of HIF-1α mediated ROCK signaling pathway in the protective effects.Methods Part one, in order to filter out lead acetate-induced cell damage dose, the cultured PC12 cells were treated with low, medium and high concentrations of lead acetate, respectively. The cell viability was determined by MTT reduction assay and LDH assay, the intracellular levels of oxygen species was measured by assessing SOD and MDA levels, reactive oxygen species(ROS) formation. ATP levels were measured by DCFH-DA staining and Luciferase assay. Morphologic changes of the cells were observed by inverted phase contrast microscope. Cell apoptosis was determined by Hoechst33342 staining. Moreover, the intracellular distribution of HIF-1α and ROCK-2 were examined by HIF-1α and ROCK-2 immunostaining, the expressions of HIF-1a, ROCK-1, ROCK-2, Cyto C, Caspase-9, Caspase-3, Bcl-2 and Bax were determined by Western blot analysis. By establishing the model of PC12 cells, designing and synthetizning the small interference RNA of HIF-1α(HIF-1α-si RNA), the cells transfection got. Part two, choose the appropriate lead acetate dose to deal with PC12 cell as the damage model, then dispose the PC12 cell with different doses of ginsenoside Rb1 or use the HIF-1 inhibitor(2ME2) to pretreat cell. By using the same method, detects the changes of the cell viability, LDH, SOD, MDA, ATP and ROS level. Analyse protein expression changes of HIF-1a, ROCK-1, ROCK-2, Cyto C, Caspase-9, Caspase-3, Bcl-2 and Bax by doing western blot.Results Lead acetate induced PC12 cells damage in a dose dependent manner, acclerated the oxygen radical production and cell apoptosis. Meanwhile, the intracellular ATP levels reduced. Interestingly, lead acetate enhanced HIF-1α and ROCK-2 expressions and induced the translocation of HIF-1α and ROCK-2 into nuclei. In addition, ROCK-1, Cyto C, Caspase-9, Caspase-3 expressions, Bax/Bcl-2 ratio were markedly increased. Ginsenoside Rb1 and HIF-1α inhibitor 2ME2 blocked lead acetate-induced cell damage in PC12 cells, reduced the expressions of HIF-1α,ROCK-1, ROCK-2, Cyto C, Caspase-9, Caspase-3, Bax/Bcl-2.Conclusions Lead acetate induced cell apoptosis in PC12 cells, which is associated with the activation of HIF-1α mediated ROCK signaling pathway. And the cellular oxidative stress also contributes to the injury. Ginsenoside Rb1 and HIF-1α inhibitor 2ME2 protect aganist lead acetate induced apoptosis. Taken together, ginsenoside Rb1 and HIF-1α inhibitor 2ME2 lower the degree of PC12 cells damage induced by lead acetate and reduce cell apoptosis by inhibiting HIF-1α mediated ROCK signaling pathway activation.