| [Background]Ethylbenzene is one of the potential ototoxic chemicals,which could affect the periphery of hearing.Compared with inner hair cells,cochlear outer hair cells are more sensitive to ethylbenzene.Previous animal experiments showed that ethylbenzene could increase the hearing threshold and decrease the number of cochlear hair cells in rats,and workers with exposure of ethylbenzene combined with noise show much higher incidence of hearing loss than those exposed to noise alone.Some studies have shown that oxidative stress can up-regulate apoptosis-related genes and induce apoptosis.The activation of Nrf2/HO-1 pathway can inhibit oxidative stress and protect cells from injury.HEI-OC1 damage induced by ethylbenzene may be related to oxidative stress,but the mechanism remains to be elucidated.[Objective]This study was designed to find out the changes of mRNA and protein expression of Nrf2 and HO-1 in Nrf2/HO-1 pathway and apoptosis of HEI-OC1 cells after ethylbenzene treatment,with the purpose of building up the experimental base for the next study of molecular mechanism of ethylbenzene-induced ototoxicity and hearing loss.1.To establish an in vitro model of ethylbenzene-induced ototoxicity so that we can evaluate the effects of ethylbenzene on oxidative stress damage and apoptosis of HEI-OC1 cells.2.To explore the effects of Nrf2/HO-1 pathway on HEI-OC1 cells of ethylbenzene-induced damage and its possible mechanism.[Methods]Eleven concentration groups of 0,30,60,90,300,600,900μM,3,6,9 and 10mM were set respectively to determine the proliferation vitality of HEI-OC1 cells after 24hours of ethylbenzene exposure.After treating with the following concentration of 0,1,2,4,8,16,32 and 64mM for 24 hours,respectively,the median inhibitory concentration(IC50)of ethylbenzene on HEI-OC1 cells was calculated to determine the appropriate concentration of ethylbenzene for subsquent experiments.After HEI-OC1 cells were exposed to ethylbenzene at concentrations of 0,6,9 and 12mM for 24 hours,the total cellular protein was extracted.The content of MDA,the level of superoxide dismutase and the activity of glutathione peroxidase in HEI-OC1 cells were measured by specific kits.The apoptosis was detected by Annexin V-APC/PI double staining method using flow cytometry.After the completion of reverse transcription of cDNA from total RNA,qPCR assay was appiled to detect the mRNA expression levels of Nrf2,HO-1 after ethylbenzene exposure.Western blot assay was set to measure the protein expression levels of Nrf2 and HO-1.[Results]1.The median inhibitory concentration of ethylbenzene on HEI-OC1 cells was12.86mM(R~2=99.05).As the concentration of ethylbenzene increased,the cell proliferation vitality decreased.2.Concentration of DMF not less than 90μM could inhibit the proliferation of HEI-OC1 cells,and significant difference was found while comparing with the control group.The concentration of DMF was set to 60μM in the subsequent experiment.Survival rate of HEI-OC1 cells exposed to 60μM DMF and 9mM ethylbenzene was higher than that of 9mM ethylbenzene exposure group,and the difference was statistically significant(P<0.01).3.The contents of MDA in HEI-OC1 cells treated with ethylbenzene at 0,6,9 and12mM for 24 hours were 72.13±10.55,82.21±5.43,101.4±5.25nmol/mgprot and163.1±11.76nmol/mgprot,respectively,showing a statistical significance(P<0.05).Multiple comparisons showed that there were significant differences among all groups except 6 and 9mM(P<0.05).With a P-value less than 0.05,the activity of superoxide dismutase was 30.56±3.14,25.14±0.68,28.26±3.09,21.44±3.02U/mgprot,respectively.Multiple comparisons showed that there was significant difference between 12mM treated group and the control(P<0.05).The activity of glutathione peroxidase was740.64±25.91,527.82±7.45,661.95±118.33 and 354.30±17.50U/mgprot,separately,and the difference was statistically significant(F=22.85,P<0.01).Multiple comparisons showed that there were significant differences among treated groups named 6,9 and12mM(P<0.05).4.Compared with the control,the proportion of early apoptotic cells in 6,9 and12mM groups increased by 2.93,4.23 and 6.08 times,respectively.The statistical differences were considered significant(P<0.05).Multiple comparisons showed that there were significant differences among other groups except 6m M and 9mM concentration group.Individually,the proportion of late apoptotic cells increased by 2.18,3.38 and 5.54 times,differences were considered to be significant(P<0.01).Multiple comparisons showed that there were significant differences among other groups except the control and 9mM group while comparing with 6mM group.(P<0.05).5.Compared with the control,the mRNA expression of Nrf2 and HO-1 decreased significantly after ethylbenzene treatment of 12mM for 24 hours(P<0.05).Multiple comparisons showed that compared with the control,the mRNA expression level of Nrf2in 12mM concentration group was significantly lower(P<0.05).The mRNA expression levels of Nrf2 in 12mM concentration group was lower than that in 6mmo/L,showing a statistical significance(P<0.05).And the mRNA expression level of HO-1 in 12mM concentration group was significantly lower than that in the control(P<0.05).6.Compared with the control,the protein expression levels of Nrf2 and HO-1 in 9and 12mM groups were significantly different(P<0.05).The protein expression of Nrf2and HO-1 decreased as the increase of ethylbenzene concentration.Multiple comparisons showed that the protein expression levels of Nrf2 and HO-1 in the 9 and 12 mM showed a downward trend compared with 6 mM,and the difference was statistically significant(P<0.05).7.Compared with the control,the rate of early apoptosis in 9mM ethylbenzene-induced group,60umol/L DMF and 9mM ethylbenzene combined exposure group increased by 8.05 and 5.63times,respectively,and the difference was statistically significant(P<0.05).Multiple comparisons showed that the early apoptosis rate of cells exposed to ethylbenzene alone was higher than that in the combined exposure group of DMF and ethylbenzene,but the difference was not statistically significant(P>0.05).8.The expression levels of Nrf2 and HO-1 mRNA in DMF and ethylbenzene combined exposure group were higher than those in control and ethylbenzene exposure group,but the difference was not statistically significant(P>0.05);The protein expression levels of HO-1 showed statistical sigificances(P<0.05).[Conclusions]1.Ethylbenzene inhibits the proliferation of HEI-OC1 cells and induces damage of which might be related to oxidative stress and apoptosis.2.The activation of Nrf2/HO-1 pathway can inhibit apoptosis,up-regulate the expression of antioxidant genes,and antagonize the toxic effect of ethylbenzene on HEI-OC1 cells. |
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