2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) Induces Hepatic Stellate Cell (HAS) Activation And Liver Fibrosis In C57BL6 Mouse Via Activating Akt And NF-?B Signaling Pathways

Objective To investigate the activation on HSC cells after exposure to TCDD,and to discuss the underlying molecular mechanism of TCDDinduced liver fibrosis.Methods(1)After adaptive feeding for one week,21 6-week-old male C57BL/6J mice of 6-week-old were randomly divided into three groups with seven mice per group: control group,and groups treated with 10?g/kg·bw·w TCDD and 25?g/kg·bw·w TCDD..On day 0,7,14,21,28 and 35,we administrated 10,25?g/kg body weight dose of TCDD or the vehicle(corn oil)by a single intraperitoneal injection per week.On the 42 nd day,the mice were executed.We randomly chose 4 mice from TCDD treated groups for clinicopathologic analysis and histomorphological observation of their livers.Livers of rest mice were taken out and frozen in liquid nitrogen for total RNA and protein extraction.(2)HSC-T6 cells were used in vitro test,and were treated with different doses(0,0.1,1,10,25,100)of TCDD for 72 hours.DMSO was used as control group.(3)Sirius red staining and H&E were used to observe fibrosis change of mouse livers and general pathological changes respectively.Realtime-PCR was used to test mRNA expressions in ?-SMA and Coll1A1 genes.Western blot was used to test,the level of protein or phosphorylated protein of ?-SMA,Coll1a1,Akt,p-Akt,p65,p-p65,TNF-? and IL-6.Enzyme-linked immunosorbent assay(ELISA)was used to test TNF-? and IL-6level in TCDD treated supernatant fluid of HSC-T6.(4)Finally,Akt inhibitor was used in HSC-T6 cell.Western blot to make a second quantitative detection on the changes of protein or phosphorylated protein level of above cells.ELISA was used to test TNF-? and IL-6level in TCDD treated supernatant fluid.Results(1)Sirius red staining showed a significant increase of collagen in mouse livers in TCDD exposed groups.Hematoxylin eosin(H&E)staining showed liver tissues exhibited inflammatory cell infiltration in the central venous area and the surrounding areas following TCDD exposure(2)TCDD exposure increased mRNA expression and protein level in ?-SMA(symbol of HSCs activation)and Coll1A1(symbol of liver fibrosis).(3)TCDD can increase phosphorylation level of Akt in C57BL/6J mice liver tissue.(4)The level of phosphorylation NF-?B p65 significantly increased.Nuclear and Cytoplasmic extraction experiment and immunocytochemistry also confirmed that TCDD exposure caused a clear nuclear translocation of p-p65 in mainly in the nucleus of HSC.(5)HSC-6T pretreated with Akt inhibitor LY294002 before exposed to TCDD showed a decrease of protein and protein phosphorylation in p-Akt,p-p65 and ?-SMA.Fluorescence test showed that TCDD exposure increased p-p65 trans-nuclear,and TNF-? and IL-6 level in supernatant fluid.While LY294002 pretreatment can reduce p-p65 trans-nuclear and inhibit the increase of TNF-? and IL-6.Conclusions(1)HSC activation and liver fibrosis can be induced by TCDD exposure.(2)TCDD exposure can stimulate inflammatory factors secretion,activate HSC and induce liver fibrosis via Akt/NF-?B signaling pathway.(3)Akt inhibitor can prevent HSC activation and liver fibrosis caused by TCDD exposure.