The Roles And Mechanisms Of Different Types Of Cell Death In Microcystin-LR-induced Hepatotoxicity In Rats

Objective:Microcystins(MCs)are cyanobacterial toxins produced by eutrophic water bodies and are highly hepatotoxic.Acute exposure to MCs can induce hepatomegaly,hemorrhage,hepatocyte necrosis,and even individual death in human or animals.Chronic exposure to MCs can cause liver injury,nonalcoholic steatohepatitis disease and hepatocyte carcinoma.However,the exact mechanisms of hepatotoxicity induced by MCs have not been fully elucidated.In this study,Wistar rats were used to investigate the effects of MC-LR on oxidative damage in rat liver and the effect of MC-LR on the expression of genes related to different types of cell death in rat liver tissues.This study will strengthen the understanding for mechanisms of hepatotoxicity caused by MCs exposure.Methods:Healthy male rats were and acclimatized for one week and randomly divided into 4 groups:control group,low-dose group(0.25 LD50,25μg MC-LR/kg),medium-dose group(0.5 LD50,50 μg MC-LR/kg),and high-dose group(0.75 LD50,75 μg MC-LR/kg).After 24 h of intravenous injection,serum and liver tissues were collected.The effects of histopathology,blood biochemistry and antioxidant system were detected in rats.qRT-PCR and Western blotting were used to determine the transcription and protein expression levels of genes related to autophagy,apoptosis,necroptosis,pyroptosis,ferroptosis and cuproptosis in the liver of rats.Results:1.Inflammatory cell infiltration,hepatocytes necrosis and hemorrhage were observed after exposure to MC-LR.2.MC-LR exposure can affect serum biochemical parameters in rats.3.75 μg MC-LR/kg exposure caused oxidative damage in the liver of rats.4.MC-LR exposure could alter thetranscription and protein expressions of genes involved in multiple types of celldeath including autophagy,apoptosis,necroptosis,pyroptosis,ferroptosis and cuprotosis.And a dose relationship appeared.(1)Autophagy.The transcriptions or protein expressions of genes associated with initiation and elongation of autophagy were significantly up-regulated in rats administrated 25 μg MC-LR/kg,bm,but down-regulated in rats administrated 75 μg MC-LR/kg,bm.The ratio of LC3II/I was up-regulated in all MC-LR treatment groups.In addition,the transcriptions of mitochondrial autophagy-related genes were up-regulated in 25 μg MC-LR/kg group,while there were down-regulated in 50 or 75 μg MC-LR/kg group.(2)Apoptosis.MC-LR exposure could induce rather the intrinsic pathway apoptosis than extrinsic apoptosis pathway regulated by transcriptions or proteins levels of related genes.(3)Necroptosis.the transcriptions or proteins levels of genes related necroptosis were significantly up-regulated in rats administrated 50 or 75μg MC-LR/kg,bm.(4)Pyroptosis.The transcriptions or proteins levels of genes involved in pyroptosiswere up-regulated in the 25 or 50 μg MC-LR/kg group;The transcriptions of IL-1β and IL-18 were up-regulated in each group.(5)Ferroptosis.The transcriptions or proteins levels of genes related to amino acid metabolism were altered in rats administrated 50 or 75 μg MC-LR/kg,bm;the transcriptions or proteins levels of genes related to lipid metabolism were altered in rats administrated MC-LR;the transcriptions or proteins levels of genes related to iron metabolism were altered in rats administrated MC-LR.(6)Cuprotosis.The transcriptions or proteins levels of genes related to copper ion metabolism were significantly down-regulated in rats administrated 50 or 75 μg MC-LR/kg,bm;the transcriptions or proteins levels of genes involved in protein lipid acylation were significantly down-regulated in rats administrated 50 or 75 μg MC-LR/kg,bm.Conclusion:1.MC-LR exposure induced a dose-dependent manner of pathological damage and liver function impairment in rat in.2.The balance of antioxidant systems in liver tissue of rat was disrupted after MC-LR exposure.3.Autophagy,mitophagy,apoptosis,necroptosis,pyroptosis,ferroptosis,and cuprotosis are involved in MC-LR-induced liver injury,suggesting that different types of cell death might cooperate to mediate the hepatotoxicity of Wistar rats induced by acute MC-LR exposure.This was manifested by the occurrence of mainly autophagy,mitophagy,pyroptosis and ferroptosis at low and medium doses groups;autophagy,apoptosis,necroptosis,pyroptosis,ferroptosis,mainly occurred in the medium-dose MC-LR group;apoptosis,necroptosis,ferroptosis,cuprotosis mainly occurred in the high-dose MC-LR group.