Study On The Involvement Of Ferroptosis In Liver Injury In Cadmium-exposed Chickens

Cadmium（Cd）is one of the most toxic heavy metal pollutants in the environment.It is easy to accumulate in the body and cause serious tissue and organ damage because of long half-life and bioaccumulation.Liver is the main target organ for Cd accumulation and toxic damage,and oxidative stress is considered to be an important mechanism of Cd-induced hepatotoxicity.The previous study in our lab found that the accumulation of peroxides was accompanied by severe inflammatory damage in the liver of Cd-exposed chickens;at the same time,the expression of glutathione peroxidase 4（GPX4）was significantly reduced in the liver.GPX4 is a key regulatory protein of ferroptosis,suggesting that ferroptosis is involved in the process of liver injury in Cd-exposed chickens.But so far,there is no research report on the mechanism of ferroptosis involved in Cd-induced hepatotoxicity.In order to study the role of ferroptosis in Cd-induced liver damage in chickens,this study adopted a research strategy by conducting in-vivo and in-vitro experiments.30 1-day-old Hylan white egg-laying chickens were randomly divided into control group（group C）and Cd exposure group（group Cd）after pre-rearing for 7 days.The chickens in group C were fed standard mixed diet,and the chickens in Cd group were fed diet mixed with 140 mg·kg^-1 CdCl₂.Samples were taken at 20 d,40 d and 60 d after Cd exposure.Using ELISA,qRT-PCR,Western Blot and histopathology and ultra-pathological techniques to detect and observe the liver function enzymes ALT and AST activities in the serum,liver morphological structure,the expression of inflammatory factors lipoxygenase（LOX）,nuclear factor kappa-B（NF-κB）,tumor necrosis factor alpha（TNF-α）,interleukin-6（IL-6）and interleukin-1β（IL-1β）,antioxidant enzymes glutathione peroxidase（GSH-Px）and superoxide dismutase（SOD）activity,and superoxide malondialdehyde（MDA）content;the expression of iron metabolism-related factors ferritin heavy chain 1（FTH1）and transferrin receptor（TRE）;ferroptosis markers GPX4,acyl-Co A synthetase long-chain family member 4（ACSL4）and prostaglandin endoperoxide synthase 2（PTGS2）;the change of mitochondrial fusion-related factors Optic atrophy 1（OPA1）,Mitofusin 1/2（Mfn1/2）expression in Cd-exposed liver of chicken.At the same time,based on the establishment of Cd-exposed model of liver cancer cell line（LMH）cells,the LMH cells were divided into control group（group C）,Cd-exposed group（group I）,ferroptosis activator RSL3 group（group II）,Ferrostatin-1 group（group III）,Cd+RSL3 group（group IV）and Cd+Ferrostatin-1 group（group V）,Cd exposure for 24 h.Using CCK-8,qRT-PCR and Western Blot to detect the LMH cell activity,the expression of inflammatory factors LOX,NF-κB,TNF-α,IL-6 and IL-1β;iron metabolism-related factors FTH1 and TRE;ferroptosis markers GPX4,ASCL4and PTGS2;mitochondrial fusion-related factors OPA1 and Mfn1/2.The results showed that the activity of liver functional enzymes in the Cd group was significantly increased in the serum of chickens;the disordered arrangement of liver cells,vacuolar degeneration and even necrosis,liver congestion,serous-fibrinous exudation and inflammatory cell infiltration;Volume shrinks,cristae rupture,and endoplasmic reticulum expands.The mRNA expression of the inflammatory factors LOX,NF-κB,TNF-α,IL-6,and IL-1βwere upregulated in the liver;contents of TNF-α,IL-6,and IL-1βwere significantly increased;the content of MDA,and the levels of GSH-Px and SOD were significantly increased;and mRNA expression of FTH1 and TRE were significantly increased;the expression of ferroptosis marker GPX4 was significantly decreased,however,the expression of ACSL4 and PTGS2 were extremely increased;mRNA expression of OPA1 and Mfn1/2 were significantly decreased.Both Cd exposure and ferroptosis activators can trigger ferroptosis in LMH cells,further promoting the release of inflammatory factors and mitochondrial dysfunction which lead to cell damage;ferroptosis inhibitors significantly inhibited the release of inflammatory factors and mitochondrial damage in LMH cells exposed to Cd.Cell viability was decreased caused by Cd.The results showed that Cd exposure resulted in morphological and functional impairment of chicken livers,and Cd promoted hepatic damage by inducing ferroptosis in hepatocytes.