Study On The Mechanism Of AMPK Involved In Apoptosis Of H9c2 Cells Induced By Fluoride Through Mitochondria Pathway

Objective:We explore the mechanism of AMPK involved in apoptosis through mitochondria pathway by detecting the toxic effects in H9c2 cells exposed to different concentrations of sodium fluoride.Methdos:H9c2 cells were treated with different concentrations of Na F for 48 h and 72 h, the proliferation and viability of H9c2 cells were measured by MTT assay; HE staining was used for morphological examination; The changes of mitochondrial membrane potential and the early cell apoptosis were detected by flow cytometry; The m RNA expression of genes related to cell apoptosis(caspase-3, caspase-9 and Cyt c)and AMPKα were examined by real-time PCR; The protein expressions of p-AMPKα were determined using western blot.Results:The results showed that, low dose of Na F had no significant effect on cell proliferation, with the increase of dosage of Na F, the inhibition effect increased, the morphology of H9c2 cells had a significant change, the connection between cells gradually disappeared, cell membranes became fuzzy. In the group of 20mg/L, the nucleus began to ruptures and the chromatin became agglutinates. At the dose of 40mg/L, the connection between cells was almost completely lost, cell shape contorted and the outline of cell became fuzzy; After treatment, the percentage of cells with green fluorescence increased with the increment of Na F concentration, this suggests that ΔΨm in Na F-induced H9c2 cells was down-regulated; The early apoptosis of H9c2 cells also increased, this means that Na F exerted a toxic effect on apoptosis in H9c2 cells; Compared with the control group,the m RNA expression of caspase-3、caspase-9 and Cyt c were all presented a trend of increasing with the increase of dosage of Na F,and the m RNA expression of AMPKαup-regulated; In the control group, the difference between p-AMPKα and total AMPKαwas not obvious, the grey value ratio of p-AMPKα and t-AMPKα of H9c2 cells induced with the increase of dosage of Na F.Conclusions:Cell apoptosis is an important pathological mechanism of Na F-induced myocardial cytotoxicity; The mechanism on cell apoptosis of H9c2 cells induced by fluoride may be related to activate the mitochondria pathway; AMPK is involved in the process of Na F-induced injury.