Heat stress induced hepatocyte apoptosis in largemouth bass Micropterus salmoides via IRE1α/TRAF2/ASK1/JNK pathway

Heat stress (HS) has been shown to adversely affect fish livers and can lead to extensive apoptosis.To investigate the relationship between endoplasmic reticulum (ER) stress and HS-induced apoptosis in fish livers,we isolated and cultured primary hepatocytes of largemouth bass,Micropterus salmoides by trypsin method,then established an in-vitro model of liver cells under HS (35℃).The contents of lactic dehydrogenase (LDH) and hydrogen peroxide (H2O2) were determined to evaluate the effects of HS on hepatocyte injury and oxidative stress.RT-qPCR was performed to discover the key genes in unfolded protein response (UPR) pathways involved at different HS duration.ERS inhibitor 4-PBA and IRE1α inhibitor 4μ8C were used to further investigate the effects of HS on IRE1α apoptosis pathway in hepatocytes.Results show that HS led to significant increases in the release of LDH,the content of H2O2,and the expressions of oxidative protein folding genes (ero1α and pdi) under HS,suggesting severe hepatocyte injury and oxidative stress happened in heat-stressed largemouth bass hepatocytes.The continuous activation of IRE1α pathway genes (grp78,grp94,atf6,perk,eif2a,atf4,chop,ire1α,traf2,ask1,jnk1,and jnk2) indicated that HS led significantly to ER stress.In particular,the mRNA expression levels of ER stress-related genes (grp78,grp94,atf6,perk,ire1α,chop,jnk1,and jnk2)in the high temperature (HT)+4-PBA group and the HT+4μ8C group were significantly down-regulated under HS.After 4μ8C treatment,the expression levels of apoptosis-related genes (caspase-2,caspase-3,caspase-6,caspase-7,caspase-8,caspase-9,and caspase-10) and LDH content were significantly decreased,whereas the cell survival rate was significantly increased when given 4-PBA or 4μ8C treatment.These findings demonstrate that HS could induce liver apoptosis of largemouth bass through the IRE1α pathway,which may act as a key switch mediating liver apoptosis of largemouth bass under HS.