Investigation On The Mechanism Of High-Fat-Diet-Induced Fatty Liver Disease Aggravated By Early Low-Dose Cadmium Exposure

Cadmium（Cd）is a heavy metal pollutant widely present in ecological environment.Recent years,advanced environmental protection technology and industry standards have decreased the incidence of acute Cd poisoning year by year,but the adverse effects of low-dose Cd pollution are increasing in the ecological environment.Previous studies have confirmed that lipid metabolism disorders are involved in high-dose Cd-induced metabolic diseases.However,the effects of environmental Cd level on lipid metabolism have not been well known.Liver is the main organ of lipid metabolism,and the incidence rate of fatty liver diseases has increased year by year with the consumption of high-fat-diet（HFD）.Recent epidemiological evidences have demonstrated that environmental pollutants（such as PM2.5）can exacerbate the progression of HFD-induced fatty liver diseases,but the effects of environmental Cd pollution on HFD-triggered fatty liver diseases remain unclear.The experimental design of this study was conducted based on the Cd pollution level in the ecological environment and the current incidence rate of fatty liver diseases.Rats were first poisoned with environmental dose of Cd Cl₂for 4 weeks,and then fed HFD for 6 weeks,so as to build an in vivo model of fatty liver injury aggravated by early Cd exposure.In this study,the molecular mechanism of HFD-induced hepatic steatosis aggravated by early low-dose Cd exposure was explored through in vivo and in vitro experiments from the perspective of mitochondrial homeostasis.1.Effects of early low-dose Cd exposure on HFD-induced hepatic steatosisTwenty-four 4-week-old healthy male SD rats（body weight 110-120 g）were randomly assigned to four treatments after one week of adaptation period:Control group（Cont）was fed the basal diet for 10 weeks;High fat diet（HFD）group was raised with basal diet for 4 weeks and then raised with HFD for 6 weeks;Early low-dose Cd combined with HFD（Cd+HFD）group was raised with diet contained 2 mg Cd²⁺/kg diet for 4 weeks,and then fed with HFD for6 weeks;Cadmium（Cd）group was raised with diet contained 2 mg Cd²⁺/kg diet for 4 weeks,and then fed with base diet for 6 weeks.At last,serum and liver were collected for the detection of various biological indicators.The results showed that no significant pathological injury was observed in the liver of rats in the Cd group,and there were no evident differences in the concentrations of liver TG and TC,the activities of serum enzymes（AST and ALT）,and the contents of blood lipid compared with the control rats.Hepatocytes in the HFD-fed rats showed severe steatosis,but no obvious inflammatory cell infiltration was observed in hepatocytes.The concentrations of liver TG and TC and the activities of serum enzymes（AST and ALT）were evidently increased in the HFD group,and the metabolism of blood lipid was abnormal,suggesting that hepatic steatosis was induced in rats fed with HFD for 6 weeks.Hepatic steatosis with inflammatory cell infiltration in Cd+HFD-fed rats was further aggravated compared with HFD-fed rats,contributing to increased pathological score of NAFLD.Compared with HFD-fed rats,the indexes of lipid metabolism in liver and serum of Cd+HFD group were evidently different from those of HFD-fed rats.These results show that early low-dose Cd exposure significantly enhances the degree of HFD-evoked hepatic steatosis and promotes the development of NAFLD to NASH,suggesting that environmental Cd exposure is a risk factor for the development of fatty liver disease.2.Early low-dose Cd exposure aggravates HFD-induced hepatocyte mitochondrial dysfunctionRat liver cell lines were exposed to 2.5μM Cd for 4 h and/or 200μM palmitic acid（PA）for 24 h to establish an in vitro model of HFD-induced fatty liver aggravated by low-dose Cd exposure.In this study,an in vivo model combined with an in vitro model was established to further investigate the mechanism of HFD-induced fatty liver aggravated by low-dose Cd exposure from the perspective of mitochondrial function.The transcriptomic results of rat liver tissue showed that 236 up-regulated and 473 down-regulated DEGs were identified in HFD and Cd+HFD groups.KEGG pathway analysis demonstrated that DEGs were primarily concentrated in pathways involved in mitochondrial function.Mitochondrial ultrastructure was observed by transmission electron microscopy in vitro.Compared with the control group,mitochondrial morphology was abnormal in the Cd,PA and Cd+PA groups,and the mitochondrial damage degree in the Cd+PA group was further aggravated compared with PA group,as evidenced by the reduction or disappearance of mitochondrial crest and the rupture of mitochondrial outer membrane.Then,results found that the ATP levels of Cd,HFD and Cd+HFD groups were evidently lower than that of the control group,and the ATP level of the Cd+HFD group was further reduced compared with HFD group,suggesting that Cd and HFD treatment caused mitochondrial dysfunction of hepatocytes.Further in vitro studies showed that the mitochondrial function of hepatocytes in Cd,PA and Cd+PA groups was evidently lower than that in the Cont group,and the mitochondrial function of hepatocytes in Cd+PA group was further reduced compared with PA group,which was mainly manifested by the decrease of mitochondrial membrane potential,mitochondrial dynamics disorder and the increase of mitochondrial ROS（mt ROS）level.The results show that severe hepatocyte mitochondrial dysfunction is implicated in the development of HFD-induced fatty liver aggravated by early low-dose Cd exposure.3.Reduced TFEB transcription activity due to early Cd exposure aggravates HFD-induced fatty liver by inhibiting mitophagyMitophagy is an important mechanism for removing damaged mitochondria.In this study,plasmid transfection and drug intervention were performed on the model in vitro.Western blotting method,immunofluorescent-laser confocal technology,and chromatin immunoprecipitation test were used to determine whether mitophagy was involved in the HFD-induced mitochondrial dysfunction aggravated by low-dose Cd exposure.The results showed that Cd+PA group significantly decreased the mitochondrial LC3-II（mito-LC3II）protein level and the colocalization of autophagosome marker protein LC3 and mitochondrial marker protein Tom20,and significantly enhanced the protein expression of mitochondrial marker molecules COX IV and HSP60 compared with PA group.These results suggested that PA-inhibited mitophagy was further downregulated by early low-dose Cd exposure.To identify the receptors involved in mitophagy,protein levels of Parkin,BNIP3 and FUNDC1 were detected in vitro.Results found that Cd+PA only inhibited the protein level of mitophagy receptor FUNDC1.In this study,it was found that overexpression of FUNDC1 improved mitophagy inhibition;mitochondrial function impairment and hepatic steatosis induced by Cd+PA,confirming that FUNDC1-mediated mitochondrial autophagy is involved in Cd+PA-induced hepatic steatosis.Then,primers were designed for the FUNDC1 promoter region,and Ch IP test was used to detect transcription factors regulating FUNDC1.Results demonstrated that transcription factor EB（TFEB）could bind FUNDC1 promoter and regulated FUNDC1 transcription,contributing to FUNDC1-mediated mitophagy inhibition.In addition,TFEB overexpression improved mitochondrial function impairment and hepatic steatosis induced by Cd+PA,but FUNDC1knockdown significantly inhibited the beneficial effects of TFEB on hepatic steatosis.These results suggest that reduced TFEB transcription activity due to early Cd exposure aggravates HFD-induced fatty liver by inhibiting FUNDC1-mediated mitophagy.4.Early low-dose Cd aggravates HFD-inhibited mitophagy by targeting the SIRT1/TFEB pathwayTFEB exerts a crucial role in the regulation of mitophagy.However,the mechanism by which TFEB regulates mitophagy in HFD-induced fatty liver aggravated by early low-dose Cd is not yet understood.Therefore,this study comprehensively explored the possible mechanism by which TFEB was involved in HFD-triggered fatty liver aggravated by early low-dose Cd exposure.The results of RNA-seq sequencing showed that the level of histone deacetylase SIRT1 in Cd+HFD group was significantly down-regulated compared with that in HFD group,and these results were consistent with the verification results.The PPI network demonstrated that SIRT1 could activate the transcription factor EB.Then,SIRT1 expression was regulated by plasmid transfection and activator.Results found that SIRT1 could regulate the nuclear translocation and acetylation status of TFEB and affected the transcriptional activity of TFEB.In addition,overexpression SIRT1 significantly improved Cd+PA-induced mitophagy inhibition,mitochondrial function impairment and hepatic steatosis,but TFEB knockdown significantly inhibited the beneficial effects of SIRT1 on hepatic steatosis.At the same time,this study also confirmed that SIRT1 regulated FUNDC1-mediated mitophagy inhibited by Cd+PA.Moreover,this process was strictly dependent on TFEB,and SIRT1-TFEB-FUNDC1pathway was implicated in HFD-induced fatty liver aggravated by early low-dose Cd exposure.Finally,in view of the important effects of mitochondria in hepatic steatosis,it was confirmed that Cd+PA-triggered SIRT1-TFEB-FUNDC1 axis aggravated the development of fatty liver by excess mt ROS production.These data suggest that Cd-inhibited SIRT1 regulates transcriptional activity of TFEB and inhibits FUNDC1-mediated mitophagy,thereby leading to accumulation of mt ROS and aggravating HFD-induced fatty liver.To sum up,this study draws the following conclusions:Early low-dose Cd exposure aggravated HFD-evoked fatty liver,in which mitochondrial dysfunction played an important role in this process;reduced TFEB transcription activity due to early Cd exposure aggravated HFD-induced fatty liver by inhibiting FUNDC1-mediated mitophagy;early low-dose Cd exposure aggravated HFD-inhibited mitophagy by targeting SIRT1/TFEB pathway,leading to mt ROS accumulation and promoting the development of fatty liver.This study enriched and supplemented the new understanding that environmental pollution was an important risk factor to accelerate the development of HFD-induced fatty liver,and provided a theoretical basis for prevention and treatment of HFD-evoked fatty liver aggravated by early low-dose Cd exposure from the perspective of mitochondrial protection.