The Mechanism Underlying The Protective Effects Of Meclofenamic Acid On HEI-OC1 Cells Damage Induced By Cisplatin

ObjectiveCisplatin is a widely used anticancer drug in clinic,but it may also give rise to many side effects,for example ototoxicity,which is in many cases manifested as sensorineural deafness and vestibular dysfunction.It is of great significance to study the mechanism of cisplatin-induced deafness and screen forthe corresponding protective drugs and the study can also be used for reference in the treatment of deafness caused by other drugs.Post-transcriptional modification of RNA is one of the hotspots in epigenetics in recent years.Dynamically regulated by m6A Lethyltransferase,m6A demethylase and binding protein,N6-methyladenosine(m6A)is the most abundant modification form of RNA.Meclofenamic acid(MA),a highly selective inhibitor of RNA demethylase FTO(fatmass and obesity associated enzyme),has been discovered to increase intracellular m6A levels.In this study,the cisplatin-induced damage model of cochlear hair cell line HEI-OC1 cells is used to investigate whether the ethyl ester form of meclofenamic acid(MA2),a high-selective inhibitor of RNA demethylase FTO,has protective effects on cisplatin-induced hair cell apoptosis and what its protective mechanismis,so as to provide new englightmentfor potential therapeutic targets for the amelioration of cisplatin-induced ototoxicity.Methods1.Firstly,cisplatin was added into HEI-OC1 cells culture medium to establish hair cell loss model in vitro.CCK-8 was used to detect the relative viability of HEI-OC1 cells and flow cytometry(Annexin V-FITC/PI double staining method)was used to detect apoptosis so that the best concentration and time of cisplatintreatment were selected.Next,we determined the optimal protective concentration of MA2,and then explore the protective effect of MA2 on cisplatin-induced HEI-OC1 cells apoptosisthrough CCK-8.Westemblot analysis of Cleaved-caspase 3 protein expression and flow cytometry apoptosis detection were used to observe the protective effect of MA2 on cisplatin-induced apoptosis of HEI-OC1 cells.According to the above experimental results,different drug concentration was determined for the following groups:the control group(DMSO group),the MA2 group,the cisplatin group andthe cisplatin+MA2 group.A unified mode of action was adopted in the follow-up experiment.2.To understand the changes of reactive oxygen species(ROS)levels in mitochondria,we used CellROX(?)Deep Red to detect oxidative stress inHEI-OC1 cells,and then theflow cytometry to detect the changes.In order to eliminate theinterference of other factors,H2O2 and cisplatin were divided into 8 groups(control group,cisplatin group,H2O2 group,H2O2+ cisplatin group,MA2 group,cisplatin +MA2 group,H2O2 + MA2 group,H2O2 + cisplatin + MA2 group)in a variety of ways to determine the changes of reactive oxygen species(ROS)levels in HEI-OC1 cells after corresponding treatment.The apoptosis of HEI-OC1 cells induced by H2O2and cisplatin was detected by Annexin V-FITC/PI double staining.The protective effect of MA2 on apoptosis of HEI-OCl cells induced by H2O2 and cisplatin was analyzed to determine whether MA2 could reduce the apoptosis of HEI-OC1 cells by inhibiting the production of reactive oxygen species(ROS).3.In order to reveal the occurrence of autophagy after cisplatin injury at microcosmic level,the morphological changes of HEI-OC1 cells,including the changes of cell membrane,mitochondria,nucleolus and autophagic lysosome production,were observed by transmission electron microscopy(TEM)at 0,24 and 48 hours after cisplatin treatment.Westemblot analysis and immunofluorescence staining were used to investigate the expression of autophagy marker LC3-II and whether MA2 participated in the regulation of autophagy after cisplatin injury in HEI-OC1 cells.To further clarify the role of autophagy in the protective effect of MA2 on HEI-OC1 cells after cisplatin injury,we studied the expression of LC3-? and Cleaved-caspase 3 proteins by Western blot analysis using cisplatin and 10 or 20 ?M of the autophagy inhibitorsLY294002,which inhibited PI3K/Akt signaling.4.To further determine the role of RNA epigenetic regulation,we investigated the changes of m6A in HEI-OC1 cells at 0,3,6,12,24,and 48 hours after cisplatin injury,and the changes of m6Aafter MA2 ptotecting HEI-OC1 cells from cisplatin-induced damage.We extracted the total RNA from the corresponding samples,then extracted the mRNA with the Dynabeads(?)mRNA purification kit and determined the ratio of m6A to A by LC-MS/MS after passing the Agilent 2100 bioanalyzer.The results would verifywhether the function of FTO demethylase inhibitor in cisplatin-induced HEI-OC1 cell injury was exerted.Results1.Through CCK-8 assay,the cellviabilities of HEI-OC1 cells were 100%,70.3 ±2.69%,56.79 ± 2.97%,41.21 ± 4.71%,20.72 ± 3.67%,7,14 ± 1.48%,and 5.88 ± 1.42%,respectively,after 48 hours of treatmentwith different cisplatin concentrations(0,5,10,15,20,30,and 40?M).Next,we treated HEI-OC1 cells with cisplatin at 15?M concentration for 0,3,6,12,24 and 48 hours.The proportion of apoptotic cells were 5.29± 0.77%,6.55 ± 0.65%,7.85 ± 0.52%,10.13 ± 0.65%,20.1 ±0.43%,and 27.47 ± 0.5%respectively by flow cytometry.Therefore,we chose 15?M cisplatin for 48 hours to establish HEI-OC1 cells injury model.2.According to the concentration range provided in the literature,the cells were treated with different MA2 concentrations(0 ?M,70 ?M,80 ?M,and 90 ?M)for 48 h,and the CCK-8 assay showed cell viabilities of 100%,104.26 ± 9.33%,95.22 ±5.67%,and 81.73 ± 7.08%,respectively.Therefore,we chose 80?M as the working concentration of MA2(no significant difference compared with the control group).To clarify the protective effect of 80?M MA2 on HEI-OC1 cells with 15?M cisplatin for 48 hours,CCK-8 assaywas carried out and the cell viabilities of the control group(DMSO group),MA2 group,cisplatin group and cisplatin+MA2 group were 100%,95.13± 2.20%,43.67 ± 4.74%,and 78.66 ± 8.89%,respectively.The difference between cisplatin group and cisplatin+MA2 group was statistically significant.The results suggested that MA2 play a protective role in cisplatin-induced cytotoxicity in HEI-OC1 cells.3.Cleaved-caspase 3 was an activated form of protease caspase 3,which indicated apoptosis.Westernblot analysis showed that the expression of Cleaved-caspase 3 increased gradually at 0,3,6,12,24 and 48 hafter cisplatin injuryand reached a peak at 48 h.Westernblot analysis also showed that the expression of Cleaved-caspase 3 in cisplatin+MA2 group was significantly lower than that in cisplatin group.The difference was statistically significant,suggesting that MA2 inhibited the apoptosis of HEI-OC1 cells induced by cisplatin.Flow cytometryexperiments showed that the proportion of apoptotic cells was significantly increased after cisplatin treatment(29.05 ± 9.50%apoptotic cellscompared to the undamaged control 4.44 ± 1.68%apoptotic cells),while theproportion in cisplatin + MA2 group decreased significantly(17.51 + 0.81%apoptotic cells).There was a significant difference between the two groups,suggesting that MA2 significantly reduced the apoptosis of HEI-OC1 cells induced by cisplatin.4.There was a close relationship between reactive oxygen species(ROS)and apoptosis of hair cells induced by ototoxic drugs.By flow cytometry,we found that the ROS levels induced by cisplatin,H2O2 and cisplatin+H2O2(2.16±0.07,2.01±0.18,4.73±1.21 fold changes,respectively)significantly decreased with MA2 co-treatment(the fold change of cisplatin+MA2 group,H2O2+MA2group and cisplatin+H2O2+MA2group were 1.45±0.07,1.37±0.1,1.77±0.16,respectively),the difference was statistically significant.Similarly,CCK-8 assay showed that the relative cell viability of the cisplatin+MA2 group was significantly higher than that of the cisplatin+H2O2+MA2 group(80.54+3.44%vs 59.22+2.29%).The apoptotic cell ratio of the cisplatin+MA2 group was significantly lower than that of the cisplatin+H2O2+MA2 group(17.56+0.49%vs 24.7+1.4%)by flow cytometry.All these differenceshad statistical significance.These results demonstrated that MA2 reduced apoptosis by inhibiting the increase in intracellular ROS levels in HEI-OC-1 cells after cisplatin exposure.5.Autophagy was a normal physiological phenomenon of cells,but excessive autophagy could lead to cell death and pathological changes.The morphology of the cells and the occurrence of autophagy of HEI-OC1 cells was observed by transmission electron microscopy(TEM)after cisplatin treatment for 0,24 and 48 h.We found that,in undamaged cells,the cytoplasmic membrane was intact,the microvilli were clearly visible,the mitochondria exhibited an oval or round shape with almost no cavitation,and the nuclei were round and clear.After cisplatin exposure for 24 h,the plasma membrane was still intact,the microvilli were still visible,the mitochondria were almost normal but showed a small amount of mitochondrial cavitation,a small number of autophagic lysosomes can be observed in the cytoplasm,and nuclear atypia was obvious.After cisplatin exposure for 48 h,chrysanthemum-like bodies were visible throughout the cells,and a large number of autophagic lysosomes appeared in the cytoplasm.There was nuclear and cytoplasmic condensation.The electron density increased,and there was significant heterochromatin aggregationLC3-? is a classical index reflecting the number of autophages in cells.Westernblot analysis showed that the expression of LC3-? increased gradually after cisplatin exposure for 0,3,6,12,24,48 h,and reached a peak at 48 h.All these results suggested that thecisplatin-induced excessive autophagy might be involved in HEI-OC1 apoptosis.6.In order to determine the role of MA2 in the autophagy regulation of HEI-OC1 cells after cisplatin exposure,immunofluorescence staining with anti-LC3B antibodies was performed.We found that a large number of autophagosome were formed in the cytoplasm of the HEI-OC1 cells in the cisplatin group,and LC3-? was strongly positive.In the cisplatin+MA2 group,autophagosome formation was significantly reduced.Similarly,Westernblot analysis also confirmed that the expression of LC3-? in cisplatin+MA2 group,compared with cisplatin group,decreased significantly.Bothsuggested that MA2 inhibiteautophagy.LY294002 could inhibit the signaling pathway of PI3K/Akt and was recognized as an autophagy inhibitor.Westernblot analysis showed that both 10?Mand 20?Mof LY294002 inhibited cisplatin-induced excessive autophagy,and the expression of LC3-II was significantly decreased while the expression of Cleaved-caspase 3 was also decreased.These results suggested that MA2 treatment could inhibite the cisplatin-induced activation of autophagy,which in turn inhibited apoptosis after cisplatin exposure.7.The mRNA,extracted and purifiedwith multi-step methodsfrom samples,was first analyzed by NanoDrop and Agilent 2100 bioanalyzer.The reports showed thatpurity and quality of the mRNA met the experimental requirements.Then the ratio of m6A to A was determined by LC-MS/MS.We found that the level of m6A in HEI-OC1 cells did not change regularly after cisplatin treatment for 0,3,6,12,24,and 48 h respectively.There was no significant difference in the ratio of m6 A to A at each time point,suggesting that the cisplatin-induced damage on HEI-OC1 cells is not directly related to the RNA epigenetical regulation of m6A.On the other hand,there was no significant difference in the ratio of m6A to A among the treatment groups(control group,MA2 group,cisplatin group and cisplatin+MA2 group).Together,these results showed that the inhibition of cisplatin-induced apoptosis in HEI-OC-1 cells by MA2 might not be caused by directly targeting the level of m6A.ConclusionsMeclofenamic acidattenuated cisplatin-induced apoptosis in cochlear hair cell line HEI-OC1 cells by reducing the accumulation of reactive oxygen species and inhibiting excessive autophagy.The protective effects of meclofenamic acidfrom cisplatin-induced HEI-OC1 cellsdamagemight notdirectly relate to the function of its own RNA demethylase inhibitor.