Protective Effect Of Melatonin Against Cadmium-induced Oxidative Damage And Lipid Accumulation In The Liver Of Ducks

Cadmium is a heavy metal contaminant widely found in the environment and is a potential threat to the physiological health of the body.The liver is one of the important target organs of cadmium and is capable of enriching a large amount of cadmium entering the body.Numerous studies have demonstrated that cadmium can induce oxidative stress,abnormal lipid metabolism and blocked autophagic flow in the liver,leading to liver damage and even inducing various liver diseases such as non-alcoholic fatty liver.Melatonin is an indole hormone with important physiological effects.Melatonin was found to be an effective antioxidant to alleviate cadmium-induced hepatic oxidative stress,but its mechanism to antagonize the hepatotoxicity of cadmium has not been elucidated.In this study,Shaoxing duck was used as the research object,and a cadmium toxicity model was established by combining in vitro mouse hepatocyte line AML12 cells.The hepatotoxicity mechanism of cadmium was investigated in depth by HE staining,immunoblotting,immunofluorescence and oil red O staining,and the protective mechanism of melatonin against cadmium-induced liver injury in ducks was also investigated,which provided a new strategy for the development of targeted protective agents against cadmium-induced liver injury.1.Protective effect of melatonin on cadmium-induced liver damage in ducksTo investigate the role of melatonin in cadmium-induced liver injury in ducks,ducks were treated with 20 mg/L melatonin and 50 mg/L cadmium chloride alone or in combination for 12 weeks,and serum ALT,AST and ALP activities were measured.<0.01)and no significant changes in AST activity,disorganized hepatic cords,significantly vacuolated hepatocytes,significantly higher liver weight and liver index(P<0.05),reduced hepatocyte nuclei and dense chromatin staining.Compared with the cadmium group,the melatonin and cadmium co-treatment group showed highly significant or significantly lower serum activities of ALT,ALP and AST(P<0.01 or P<0.05),dense and intact liver tissue,clear hepatic sinusoidal structure,highly significant decrease in liver weight and liver index(P<0.01),normal nucleus morphology and homogeneous nuclear chromatin.The results indicate that cadmium can cause abnormal liver function,histological and ultrastructural damage,and melatonin has a protective effect on cadmium-induced liver damage in ducks.2.Melatonin alleviates cadmium-induced oxidative stress in duck liverIn order to investigate the mechanism of melatonin to alleviate cadmium-induced liver injury in ducks,the present study examined the indicators related to oxidative stress,and the results showed that,compared with the control group,mitochondria were damaged or even disintegrated,COX Ⅰ activity was highly significant(P<0.01),COX Ⅱ activity was significantly higher(P<0.05),COX Ⅳ and COX V activity was significantly lower(P<0.05),COX Ⅳ protein expression was significantly(P<0.05),a significant increase in superoxide anion,a highly significant decrease in GSH-Px and SOD activity(P<0.01),a highly significant increase in MDA content(P<0.01),and a highly significant decrease in Fe,Mg and Zn elements(P<0.01).Compared to the cadmium group,the melatonin and cadmium co-treated group showed clear mitochondrial cristae,intact mitochondrial structure,highly significant or significantly reduced COX Ⅰ and COX Ⅱ activity(P<0.01 or P<0.05),no significant change in COX Ⅳ activity,highly significant increase in COX V activity(P<0.01),significantly increased COX Ⅳ protein expression(P<0.05)and significantly reduced superoxide anion.GSH-Px and SOD activities were significantly increased(P<0.01),MDA content was significantly reduced(P<0.01),and Fe,Mg and Zn elements were significantly or significantly increased(P<0.05 or P<0.01).The results suggest that melatonin protects mitochondria by stabilizing their ultrastructure and restoring the activity of mitochondrial respiratory chain-related enzymes to restore cellular oxidative homeostasis and alleviate cadmium-induced oxidative damage in duck liver.3.Melatonin alleviates cadmium-induced lipid accumulation in hepatocytesIn order to investigate the protective effect of melatonin on cadmium-induced lipid accumulation in hepatocytes,this study examined lipid metabolism-related indicators by Western blot,oil red O staining and BODIPY staining.The results showed that in the in vivo model,the number of lipid droplets in hepatocytes was increased,TG and T-CHO contents were significantly or very significantly increased(P<0.05 or P<0.01),PPARa and CPT 1 protein expression was down-regulated(P<0.05)and ACC and FAS protein expression was up-regulated(P<0.05 or P<0.01)in the cadmium group compared with the control group.In an in vitro model,AML 12 cells were treated with different concentrations(0,2.5,5 and 10μM)of cadmium chloride for 12 h.It was confirmed that cadmium increased HDL-C and T-CHO concentration-dependently(P<0.05 or P<0.01).5 μM cadmium treatment of AML12 cells for 12 h resulted in a significant increase in lipid accumulation in the cadmium group compared to the control group,and both PPARa and CPT 1 protein expression were highly The expression of PPARa and CPT 1 proteins were significantly reduced(P<0.01),while the expression of ACC and FAS proteins were significantly increased(P<0.01).The expression of PPARa and CPT 1 was significantly or very significantly increased(P<0.05 or P<0.01)and the expression of ACC and FAS was significantly or very significantly decreased(P<0.05 or P<0.01)in the melatonin and cadmium co-treatment group compared to the cadmium group.The above results suggest that cadmium can cause lipid accumulation in hepatocytes by disrupting lipid metabolism,while melatonin may alleviate cadmium-induced lipid accumulation in the liver by reducing fatty acid production and increasing beta-oxidation of fatty acids in the liver.4.Role of melatonin in alleviating cadmium-induced blockade of autophagic flowTo investigate the relationship between autophagy and abnormal lipid metabolism in hepatocytes caused by cadmium and the protective effect of melatonin,this study observed the autophagic vesicles in hepatocytes by transmission electron microscopy and detected LC3 and P62 protein expression by Western blot.The cadmium co-treatment significantly reduced the number of autophagic vesicles and down-regulated LC3 and P62 protein expression(P<0.05 or P<0.01).This suggests that cadmium promotes autophagy and causes autophagic flow block in hepatocytes,and melatonin can alleviate autophagic flow block.To further investigate the role of autophagy in cadmium-induced lipid accumulation in hepatocytes,this study used 5 μM cadmium chloride,10 μM melatonin and 5 μM RAPA and 1 μM toxic carotene(TG)to treat AML 12 cells alone or in combination,and detected the lipid distribution in hepatocytes by oil red O staining.The results showed that the RAPA and toxic carotene groups,compared with the control group,showed no significant changes,and the RAPA and cadmium co-treatment group,significantly reduced lipids compared to the cadmium group,while toxic carotene(TG)and cadmium co-treatment group had significantly increased lipids compared to the cadmium group.This suggests that autophagy has a positive effect on cellular alleviation of cadmium-induced lipid accumulation.To further clarify the protective mechanism of melatonin against cadmium-induced hepatic lipid accumulation,hepatocyte lipid accumulation was detected by knockdown of PPARα,Western blot for LC3 and P62 protein expression,and BODIPY staining,and the results showed that PPARα siRNA showed highly significant increase in LC3 and P62 protein expression compared with the cadmium co-treatment group and the cadmium alone group(P<0.01)and the lipid droplets were significantly increased.The above results suggest that PPARa may be a bridge between autophagy and cadmium-induced lipid accumulation in hepatocytes.It is suggested that cadmium may cause the blockage of autophagic flow through the down-regulation of PPARa protein expression,which in turn causes lipid accumulation in hepatocytes.In conclusion,cadmium causes liver injury through oxidative stress,lipid accumulation and autophagic flow block in duck liver.Melatonin alleviates the hepatotoxicity of cadmium by enhancing mitochondrial function,reducing fatty acid production,up-regulating PPARa expression and alleviating autophagic flow block,promoting lipid degradation and reducing lipid accumulation in hepatocytes.