Mechanistic Study Of Endoplasmic Reticulum Stress-Mitochondrial Pathways In Fluorosis-induced Apoptosis In HepG2Cells

Objective To explore the pathological roles of endoplasmicreticulum stress and mitochondrial apoptotic pathways in fluorosis-induced apoptosisin HepG2cells, we measured the levels of endoplasmic reticulum stress responseproteins at both mRNA and protein, e.g., GRP78, GRP94, CHOP in HepG2liver cellswere exposed to a series concentrations of fluoride, the expression of proteins relatedto mitochondrial apoptosis pathway cells such as AIF, Bcl-2, Bax, Cytochrome C,Caspase9and Caspase3, we tried to reveal the possible mechanism of thepathological roles of endoplasmic reticulum stress and mitochondrial apoptoticpathways in fluorosis-induced apoptosis. Methods HepG2cells were cultured in a5%CO2humidi ed atmosphere culture. Under the stimulation of differentconcentrations of NaF in24h,48h or72h, the cells density and morphology wereobserved under light microscope. The apoptosis of HepG2cells were measured byflow cytometry, and the mitochondrial function was detected by MTT. Under thestimulation of different concentrations of NaF, those factors related to theendoplasmic reticulum stress and mitochondrial apoptotic pathways, such as GRP78,GRP94, CHOP, AIF were measured at both mRNA and protein levels. Bcl-2, Bax,Cytochrome C, Caspase9and Caspase3were measured at protein level. Results Asignificant decrease of cells density was observed in HepG2cells treated withdifferent concentrations of NaF for24hours. We observed that the cells shapebecomes gradually irregular, which changes from spindle into round. To measure theapoptosis rate, HepG2cells were treated with a series concentration. The results showed that the apoptosis rate of HepG2cells was positive related to theconcentration of NaF. The apoptotic cells significantly increased after treated with2mM NaF for24hours. Under the same condition MTT assay showed that the cellstoxicity was significantly increased after treated with2mM NaF for24hours and thecells toxicity was positive related to the concentration of NaF. After treated with3mM NaF for24h, the mRNA and protein level of GRP78and CHOP increasedsignificantly compared to control. At the same time, the mRNA level of GRP94significantly increased after treated with3mM NaF for24h. Meanwhile, the proteinlevel of GRP94significantly decreased compared to control. In addition, the mRNAand protein levels of mitochondrial apoptosis protein AIF increased sharply comparedto control. The expression of Bcl-2, Cytochrome C, Caspase9and Caspase3whichare related to mitochondrion apoptosis pathway significantly increased while theprotein level of Bax significantly decreased. Conclusion In the apoptosis model ofHepG2liver cells exposed to NaF stimulation, we measured the mRNA and proteinexpressions of GRP78, GRP94and CHOP which are the endoplasmic reticulum stressresponse proteins. We detected significant changes of those proteins that HepG2cellshave endoplasmic reticulum stress after exposed to NaF. By measuring the expressionof proteins like AIF, Bcl-2, Bax, Cytochrome C, Caspase9and, Caspase3which arerelated to mitochondrion apoptosis pathway, we detected significant changes of thosefactors above in the apoptosis model of HepG2liver cells which indicated thatmitochondrial apoptotic pathway was activated in the apoptosis model of HepG2livercells exposed to NaF. Our study suggests that the endoplasmic reticulum stress andmitochondrial apoptotic pathways are involved in NaF-induced cells death in HepG2cells.