Microcystin-LR-induced Endoplasmic Reticulum Stress Interferes With Zebrafish Liver Lipid Metabolism

Microcystin(MCs)is a family of cyanotoxins that are widely distributed,highly toxic and completely studied so far.Recent studies have shown that MCs can cause liver endoplasmic reticulum stress(ERS),whereas the ERS response plays a very important role in lipid metabolism.In our study,MC-LR were selected as the research object.Combined with in vivo zebrafish experiments and in vitro zebrafish liver cells(ZFL cells)experiments,the effects and mechanisms of MC-LR on liver lipid metabolism base on the ERS pathway were investigated by determining changes in histotology,lipid metabolic parameters and the expression of related genes.In in vivo experiments,adult male zebrafish were exposed at long-term low-level MC-LR(0,1,5 and 25 ?g/L)exposure,and hepatic histopathology,lipid metabolism parameters as well as m RNA levels of ERS signal molecules and genes associated with lipid metabolism were measured in zebrafish liver.The results revealed that prolonged exposure to MC-LR remarkably altered the levels of hepatic total cholesterol and triglyceride and led to hepatic steatosis,which were also confirmed by hepatocellular vacuolization in HE stain and lipid droplet accumulation in oil red-O stain.MC-LR exposure upregulated significantly transcriptional levels of ERS markers including hspa5,mapk8 and chop,indicating that MC-LR induced the occurrence of ERS in the liver of zebrafish.Concurrently,MC-LR significantly improved m RNA expression of unfolded protein response(UPR)pathways related genes including atf6,eif2ak3,ern1 and xbp1 s,suggesting that all of three UPR branches were activated by MC-LR.MC-LR also induced significant upregulation of downstream lipid metabolism-related factors and genes including srebf1,fasn,acaca,scd,srebf2,hmgcra and hmgcs1,but downregulation of genes associated with lipolysis such as atgl,hsla and cpt1 aa.The above results indicated that the cause of hepatic lipid accumulation by MC-LR was mainly by upregulating lipogenic and cholesterol genes but downregulating the expression of lipolytic genes through the induction of srebf1 and srebf2,which were involved with the activation of ERS signal pathways.In in vitro experiments,zebrafish liver cells(ZFL)were exposed to MC-LR(0,10,20,40,80,160 ?g/m L)for 24 h.The cell viability was measured with CCK8 and the concentration was 70.503 ?g/m L.10 ?g/m L MC-LR was selected as the subsequent exposure concentration.Also,tauroursodeoxycholic acid(TUDCA)was used alone-and co-exposed with MC-LR.The results showed that the intracellular TC and TG contentswere increased significantly in the MC-LR(10 ?g/m L)treatment group,and UPR pathways related genes including atf6,eif2ak3,ern1,and xbp1 s and downstream lipid metabolism-related genes including srebf1,fasn,acaca,scd,srebf2,hmgcra and hmgcs1 m RNA expression were significantly up-regulated.In contrast,the contents of TC and TG were decreased significantly in the TUDCA treatment group,and the expression levels of related genes were significantly downregulated.In the TUDCA pretreatment group,the TC and TG contents showed a downward trend compared with the MC-LR treatment group,and the expression levels of related genes decreased conrespondly.This findind indicates that MC-LR mainly affects lipid metabolism of in vitro ZFL by interfering with the expression of liver lipid synthesis-related genes.The mechanism is that the ERS caused by MC-LR further destroys the expression of the steroid regulatory element protein factor srebf1 and genes related to lipid synthesis.The exposure of TUDCA and the recovery of corresponding test indicators further verified the role of ERS in the abnormal lipid metabolism of zebrafish liver caused by MC-LR.