Cadmium Exposure In Drinking Water Aggravates Liver Injury In Nonalcoholic Fatty Liver Mice Model And Its Mechanism

Background and Objective:Liver is one of the target organs of cadmium.Cadmium exposure is associated with liver damage.With the increasing prevalence of Nonalcoholic fatty liver disease(NAFLD).The effect of cadmium pollution on the liver of NAFLD patients is noteworthy.Therefore,the purpose of this study was to investigate the effects of cadmium exposure in drinking water on the liver lesions of NAFLD model mice.Methods:In this study,high fat diet combined with low dose carbon tetrachloride was used to induce NAFLD model mice.NAFLD model mice were infected with 50 mg/L cadmium chloride in drinking water for 12 weeks.(1)The effect of cadmium on lipid metabolism in the liver of NAFLD mice:The content of liver Triglyceride(TG)was determined by the kit;Liver tissue oil red O staining to observe lipid drops;qRT-PCR,Western Blot and immunohistochemistry were used to determine the expression of genes and proteins related to liver lipid metabolism.(2)The effect of cadmium on liver fibrosis in NAFLD mice:Liver tissue masson staining to observe fibrosis,qRT-PCR,Western Blot and immunohistochemistry were used to determine the expression of genes and proteins related to liver fibrosis and its formation pathway,as well as the expression of related inflammatory factors.(3)Effects of cadmium exposure on proliferation and DNA damage of liver cells in NAFLD model mice:H&E staining of liver tissue;qRT-PCR and immunohistochemistry were used to determine liver cell proliferation,DNA damage,DNA repair and apoptosis related genes and proteins.Result:(1)Cadmium exposure reduced lipid deposition in the liver of NAFLD mice.In NAFLD model mice,liver triglycerides(TG)are significantly increased,with obvious lipid accumulation and a large number of lipid vacuoles.However,cadmium exposure leads to a significant reduction in liver TG,fewer lipid droplets,and smaller lipid vacuoles in NAFLD model mice.The expression of fatty acid translocase(CD36)protein and liver fatty acidbinding protein 1(FABP1)mRNA in NAFLD model mice is significantly higher than in control mice.The expression of CD36 upstream factor,peroxisome proliferator-activated receptor y(PPARy)protein,is also significantly higher than in control mice,hepatocyte fatty acid uptake is enhanced.In cadmium-exposed NAFLD model mice,the expression of PPARy,CD36 protein,and FABP1 mRNA is significantly lower than in model mice,reducing hepatocyte fatty acid uptake.In NAFLD model mice,the expression of fatty acid synthase(FASN)protein and acetyl-CoA carboxylase(ACC)mRNA is significantly lower than in control mice,indicating suppressed hepatic fatty acid synthesis.In cadmium-exposed NAFLD model mice,FASN protein expression is higher than in model mice(P=0.063),suggesting increased hepatic fatty acid synthesis.The expression of carnitine palmitoyltransferase 1(CPT1)protein and peroxisomal acyl-coenzyme A oxidase 1(ACOX1)mRNA in NAFLD model mice is significantly higher than in control mice,indicating enhanced hepatic fatty acid β oxidation.In cadmium-exposed NAFLD model mice,CPT1 protein expression is lower than in model mice,and ACOX1 mRNA expression is significantly lower than in model mice,suggesting reduced hepatic fatty acid β oxidation.(2)Cadmium exposure aggravated liver fibrosis in NAFLD mice.In NAFLD model mice,liver collagen fibers increase,leading to obvious fibrotic lesions.Cadmium exposure causes further increase in collagen fiber formation and aggravates fibrosis in the liver of NAFLD model mice.Collagen Ⅰ(COLI)protein expression in NAFLD model mice is slightly higher than in control mice(P=0.065),while collagen Ⅳ(COLIV)and fibronectin(FN)mRNA expression is significantly higher than in control mice(P<0.05),resulting in an increase in extracellular matrix(ECM).In cadmium-exposed NAFLD model mice,COLIV mRNA expression is significantly higher than in model mice,and FN mRNA expression is higher than in model mice(P=0.087),leading to a further increase in hepatic ECM.Matrix metalloproteinase 9(MMP9)mRNA expression in the liver of NAFLD model mice is significantly lower than in control mice,indicating suppressed hepatic ECM degradation.In cadmium-exposed NALFD model mice,MMP9 mRNA expression is lower than in model mice(P=0.081),further inhibiting ECM degradation.Immunohistochemistry reveals that αsmooth muscle actin(α-SMA)is significantly increased in the liver of NAFLD model mice compared to control mice,myofibroblasts(MFB)increases and ECM secretion increased.Activated hepatic stellate cells(HSC)are also increased compared to control mice.In cadmium-exposed NAFLD model mice,α-SMA protein expression is significantly increased compared to model mice,MFB increases and activated HSC increased.E-cadherin(E-CAD)mRNA expression in the liver of NAFLD model mice is significantly lower than in control mice,while VIMENTIN mRNA expression is significantly higher,indicating epithelialmesenchymal transition.In cadmium-exposed NAFLD model mice,E-CAD mRNA expression is lower than in model mice,and VIMENTIN mRNA expression is higher than in model mice(P=0.079),further enhancing epithelial-mesenchymal transition.Interleukin-1β(IL1-β)mRNA and transforming growth factor-β(TGF-β)protein expression in the liver of NAFLD model mice are significantly higher than in control mice,leading to an increase in hepatic inflammatory factors.In cadmium-exposed NAFLD model mice,IL1β mRNA and TGF-β protein expression are significantly higher than in model mice,further increasing inflammatory factors.Immunohistochemistry reveals that the number of Kupffer cells and NLRP3 inflammasomes in the liver of NAFLD model mice is significantly higher than in control mice.Cadmium exposure causes a significant increase in Kupffer cells and NLRP3 inflammasomes in the liver of NAFLD model mice compared to model mice.(3)Cadmium exposure resulted in enhanced proliferation and DNA damage of hepatocytes in NAFLD model mice.Immunohistochemistry reveals that the expression of cell proliferation marker protein(Ki67)is significantly increased in the liver of NAFLD model mice compared to control mice.Cyclin D1(CCND1)mRNA expression is significantly higher than in control mice.In cadmium-exposed NAFLD model mice,Ki67 protein and CCND1 mRNA expression are significantly higher than in model mice.In the liver of NAFLD model mice,8-hydroxy-2’-deoxyguanosine(8-OHDG)and y-H2AX protein expression are significantly higher than in control mice,indicating liver DNA damage.Poly ADP ribose polymerase 1(PARP1)mRNA expression is significantly lower than in control mice,suggesting that liver DNA repair function is suppressed.In cadmium-exposed NAFLD model mice,8-OHDG and y-H2AX protein expression are significantly higher than in NAFLD model mice,further aggravating liver DNA damage.Liver PARP1 and RAD51 mRNA expression are significantly lower than in NAFLD model mice,further inhibiting DNA repair function.In NAFLD model mice,P53 and BAX mRNA expression are significantly higher than in control mice,enhancing the ability to induce apoptosis in DNAdamaged cells.In cadmium-exposed NAFLD model mice,P53 and BAX mRNA expression are significantly lower than in model mice,suppressing the ability to induce apoptosis in DNA-damaged cells.Conclusions:Cadmium exposure exacerbates liver fibrosis in NAFLD model mice,and its mechanism may be related to cadmium increase in myofibroblasts,increased secretion of ECM,and reduced ECM breakdown.At the same time,cadmium exposure reduces liver lipid accumulation in model mice,which may be related to cadmium decrease in hepatocyte fatty acid uptake.Cadmium exposure exacerbates DNA damage and enhances hepatocyte proliferation in model mice.In the context of the increasing prevalence of nonalcoholic fatty liver disease and cadmium pollution,these phenomena warrant further investigation.