Protective Effect Of S-allyl Cysteine On Liver Cell Damage Induced By Indomethacin And Its Molecular Mechanism

Non-steroidal anti-inflammatory drugs(NSAIDs)are the most commonly used antipyretic,anti-inflammatory and analgesic drugs at present,and their drug varieties include paracetamol,indomethacin,diclofenac,aspirin,celecoxib,etc.As well as the therapeutic effects of these drugs,their side effects are increasingly apparent due to their frequent use,and in many cases they are discontinued due to their side effects.Of drug-induced liver injury caused by indomethacin in clinic is one of the most common side effects,this experiment in normal rat hepatocyte cell line BRL-3 a as experimental object,explore the mechanism of indomethacin for liver damage,and the garlic extract S-allyl cysteine(S-allylcysteine,SAC)for the protective effect and mechanism of the injury.Purpose:The effect and molecular mechanism of indomethacin induced hepatocyte injury were studied experimentally.And to study the protective effect of SAC on indomethacin induced hepatocyte apoptosis and its molecular mechanism.Methods:1.Study on the effect of indomethacin and SAC on the proliferation of hepatocytes:CCK8 experiment and inverted phase contrast microscope were used to confirm the effect of indomethacin and SAC on the proliferation of hepatocytes;2.Effects on apoptosis:Tunel staining was used to detect the possible effects of indomethacin and SAC on the promotion or inhibition of apoptosis in liver cells;3.Western blot assay was used to detect the changes in the expression levels of p-eif2,CHOP and Cleaved caspase-3 in the ER stress pathway,so as to confirm that the mechanism of indolethacin and SAC action on hepatocytes occurred in the ER molecular pathway.results:1.To confirm that indomethacin might induce apoptosis in rat BRL-3A cells:indomethacin treated with different concentrations(12.5 M;25 microns;50 microns.After treatment with 100 M for 24 hours,(1)the results of cck-8 kit showed that the inhibitory rates of indomethacin on brl-3a proliferation were 10.28%,23.63%,36.52%and 61.50%,respectively,with statistically significant differences(p<0.05 for pair-to-pair comparison),while the inhibitory rates of cell proliferation between the blank group and the solvent group were not statistically significant(p>0.05).(2)The morphology of BRL-3A was observed under inverted phase contrast microscope:the mononuclear cells were moderately polyhorned in size.After 24 hours of culture,the inhibition of cell number decreased,indicating that indomethine could induce the inhibition of proliferation of BRL-3A cells.(3)TUNEL staining:apoptosis rate of apoptotic cells was determined by TUNEL staining to be 22.43%,37.54%,49.71%and 72.4%(p<0.05 for pair-based comparison),while the difference between the apoptosis rate of the blank group and the solvent group(p>0.05)was not statistically significant.(4)Western blot results:p-eIF2 alpha,CHOP and Cleaved Caspase 3 three kinds of protein expression level tends to increase as time,showed that indomethacin could be on BRL-3 a cell of p-eIF2 alpha,CHOP and Cleaved Caspase 3 three proteins promote the expression of the function,and the promoting effect been time dependence(as the effect of indomethacin and the longer the promoting effects express,the more obvious).2.In the study of SAC protective hepatocyte apoptosis,the indomethacin group and each group were treated for 24 hours,with SAC concentration of 50 M and indomethacin concentration of 100 M,divided into five groups(blank group,solvent group,SAC group,indomethacin group,SAC+indomethacin group).Results show that(1)the CCK 8 kit determination results show:the indomethacin group,SAC+indomethacin groups BRL-3 a proliferation inhibition rate:67.35%,26.50%,with statistical significance(p<0.05),between the blank group and solvent,and cell proliferation inhibition rate between groups of SAC there was no statistically significant difference(p>0.05)shows that after adding SAC can alleviate indomethacin inhibit proliferation and induce apoptosis of liver cells.(2)The morphology of BRL-3A was observed by inverted phase contrast microscope:based on the indomethacin induced apoptosis of hepatocytes,the number of hepatocytes increased significantly after SAC addition,the number of apoptotic cells decreased,the number of adherent cells increased,and the morphology of the cells partially restored polygonia.(3)TUNEL staining:Apoptosis rate of cells in each group was determined by TUNEL staining to be 63.7%,33.9%(P<0.05),and the difference was statistically significant.The blank and solvent groups and the SAC group(p>0.05 for pairwise comparisons).(4)Western blot results showed that p-eif2,CHOP,and Cleaved Caspase 3 showed no significant changes in bands in the first three groups,and the expression level of indomethacin in SAC+indomethacin group was higher than that in the first three groups,but lower than that in the indomethacin intervention group alone.conclusion:1.Indomethacin induces apoptosis and inhibits proliferation of hepatocytes,and the increased expression of p-eif2 PCR,CHOP,and Cleaved Caspase 3 explains that indomethacin may induce apoptosis of hepatocytes through the unfolded protein response of endoplasmic reticulum to promote apoptosis signaling pathway.2.In the S-allyl cysteine(S-allylcysteine,SAC)to join the apoptosis induced by indomethacin liver cells on the basis of the experimental phenomenon of promote the proliferation and induce the apoptosis of the inhibition of SAC,and p-eIF2 alpha,CHOP and Cleaved Caspase 3 three kinds of protein expression to decrease illustrates the SAC in liver cells may also be not fold proteins by endoplasmic reticulum stress reaction,its role is to inhibit indomethacin for the apoptosis of liver cells.