The Protective Effect And Mechanism Of Short-term Low-concentration H₂O₂ Treatment Involving ROS-JNK Pathway On BRL-3A Cells

Objective:To investigate the effect of ROS-mediated JNK pathway involved in short time and low concentration of H₂O₂treatment on rat normal hepatocytes BRL-3A and its mechanism.Methods:BRL-3A cells cultured in vitro were divided into DMSO group（DMSO intervention 24h）and JNK inhibitor group（10μmol/L SP600125 intervention 24h）,and treated with 12.5and 25μmol/L H₂O₂for 2,4 hours,respectively.CCK-8 method was used to detect cell viability,Annexin V-FITC/PI double-staining method by flow cytometry and m RFP-GFP-LC3 autophagy double-labeled lentivirus assay to detect apoptosis and autophagy,Real-time PCR and Western Blot was used to detect the m RNA and protein expression of pathway proteins p-JNK,JNK,autophagy-related proteins LC3,p62,Beclin-1,and apoptosis-related proteins Bcl-2 and Caspase-3.Results:1.After BRL-3A cells were treated with10μmol/L SP600125 for 24h,short time（2,4h）,low concentration（12.5,25μmol/L）H₂O₂,cell viability was significantly reduced when treated with12.5μmol/L H₂O₂for 4 hours（P<0.01）;cell viability was reduced when treated with 12.5μmol/L H₂O₂for 2 hours and 25μmol/L H₂O₂for 4 hours（P<0.05）;there was no significant change in cell viability when treated with 25μmol/L H₂O₂for 2 hours（P>0.05）.2.BRL-3A cells in the SP600125 group,compared with the DMSO group treated with H₂O₂at the corresponding concentration and time,the total apoptosis rate increased significantly when treated with 12.5μmol/L H₂O₂for 2 hours,with late apoptosis predominating and the percentage of early apoptosis remaining basically unchanged（P<0.01）;the late apoptosis rate and the total apoptosis rate increased when treated with 25μmol/L H₂O₂for2 hours（P<0.05）;the total apoptosis rate showed an increasing trend when treated with 12.5μmol/L and 25μmol/L H₂O₂for 4 hours,with late apoptosis predominating（P<0.01）and no significant difference in early apoptosis rate（P>0.05）.Compared with the DMSO group treated with the corresponding concentration and time of H₂O₂,the early autophagy,late autophagy and total autophagy levels of BRL-3A cells in the SP600125 group were all reduced,with late autophagy predominating（P<0.05）.3.In the SP600125 group of BRL-3A cells,compared with cells in the DMSO group treated with H₂O₂at the corresponding concentration and time,the relative expression of JNK m RNA was increased when treated with 25μmol/L H₂O₂for 2 hours（P<0.05）;Beclin-1 m RNA expression was reduced（P<0.05）;Bcl-2 m RNA expression was reduced when treated with 12.5μmol/L H₂O₂for 2 hours and 12.5μmol/L H₂O₂for 4 hours（P<0.05）.The cells in the SP600125 group,compared to the DMSO group treated with H₂O₂at the corresponding concentration and time,p-JNK/JNK protein expression level was reduced when treated with 12.5μmol/L H₂O₂for 2 hours（P<0.05）;LC3 protein expression level was reduced when treated with 12.5μmol/L H₂O₂for 4 hours（P<0.05）;Beclin-1 protein expression level was reduced in all cases（P<0.05）;autophagy-related protein p62 protein expression was increased in all cases（P<0.05）;the expression levels of Bcl-2 protein decreased when treated with 25μmol/L H₂O₂for 2 hours and 25μmol/L H₂O₂for 4 hours（P<0.05）;the expression levels of Caspase-3protein increased when treated with 12.5μmol/L H₂O₂for 2 hours（P<0.05）.Conclusions:1.The ROS-JNK pathway is involved in the protective effect of short-time,low-concentration H₂O₂treatment on BRL-3A in rat normal hepatocytes;2.Short-time,low-concentration H₂O₂treatment may inhibit apoptosis and induce protective autophagy by activating the ROS-JNK signaling pathway.