Study On The Mechanism Of Liver Fibrosis Induced By DEHP In Mice

Phthalates（PAEs）are a kind of plastic additives with increasing usage.Di（2-ethylhexyl）phthalate（DEHP）is the most abundant type in the environment and has been found in food,soil,and water.DEHP can cause liver cancer and abnormal hyperplasia.Fibrosis or cirrhosis can be seen in more than 90%of hepatocellular carcinoma（HCC）cases,with fibrosis preceding the development of HCC.Hepatic stellate cells（HSCs）are the main cause of liver fibrosis.Whether DEHP-induced hepatotoxicity is related to HSCs has not been studied.This study aims to reveal the key role of HSCs in DEHP-induced hepatic fibrosis.The 100 mice were divided into five groups:Con group（control group）,Vcon group（vehicle control group）,D50 group（50 mg/kg DEHP group）,D200 group（200 mg/kg DEHP group）and D500 group（500 mg/kg DEHP group）were administered by gavage,and liver samples were collected after 28 consecutive days.The degree of hepatic fibrosis was evaluated by histological staining.The accumulation and metabolome changes of DEHP and its metabolites in liver were analyzed by liquid chromatography-mass spectrometry.Using real-time fluorescent quantitative PCR,Western Bloting and immunofluorescence co-localization and other experimental techniques to detect liver mitochondrial damage-related indicators,apoptosis and pyroptosis-related factors in the liver,and explore the role of hepatic stellate cells in liver fibrosis.The results showed that:（1）DEHP exposure can cause the accumulation of its metabolites in the liver,change the composition of liver metabolites,and lead to liver fibrosis injury,type I collagen deposition,liver cell arrangement disorder,cellular vacuolar degeneration,nuclear condensation,mitochondrial ridge breakage and other pathological injuries,and up-regulated protein expression of fibrosis markers（α-SMA,TGF-β1,TGF-β2）;（2）DEHP exposure can damage the structure and function of mitochondria,cause damage to mt DNA,up-regulate the expression of 8-OH and decrease the content of ATP,inhibit the activities of CAT and T-SOD,and increase the content of H₂O₂ and ROS.（3）DEHP exposure up-regulated the content of TNF-αand the expression of protein level,and activated Caspase-1-mediated pyroptosis through NLRP3 inflammatosome,and up-regulated the expression of genes and proteins of the classical pathway related factors（ASC,IL-1β,IL-18,GSDMD-n/GSDMD）.（4）DEHP exposure induced pyroptosis of hepatocytes,released inflammatory bodies into the extracellular,up-regulated the expression and aggregation of F4/80 positive cells,inhibited the expression of GFAP,altered the status of static HSCs,and led to hepatic fibrosis injury;（5）DEHP exposure up-regulated the expression of apoptosis suppressor XIAP in activated HSCs,led to increased expressions of pro-apoptotic factors Caspase-3,Caspase-8 and Caspase-9,inhibited the apoptosis of activated HSCs,and accelerated the process of liver fibrosis.Conclusion:The accumulation of DEHP and its metabolites in liver increases the deposition of type I collagen in liver,resulting in liver fibrosis.DEHP exposure results in mitochondrial function injury,abnormal accumulation of reactive oxygen species induced pyroptosis of hepatocytes,and then activates HSCs.DEHP also inhibits activated HSCs apoptosis and further accelerates the progression of fibrosis.