Dihydromyricetin Inhibits The Activetion Of Hepatic Stellate Cell Induced By Iron Overload Through The Inhibition Of Ferritinophagy

OBJECTIVE:To investigate the effect of dihydromyricetin(DMY)on the activation of hepatic stellate cell induced by ferric ammonium citrate(FAC)in rat hepatic stellate cell line HSC-T6 cells,to explore the possible mechanism and to provide new ideas and strategies for prevention and treatment of hepatic fibrosis.METHODS:Rat hepatic stellate cell line HSC-T6 cells were cultured in DMEM culture medium.After HSC-T6 cells were treated with the inducer of autophagy rapamycin(100 nmol/L)for 0.5 h,HSC-T6 cells were treated with FAC(400 ?mol/L)and DMY(10,20,40,80,160 and 320?g/mL)for 24 h.The experiment was divided into the following groups: blank control group,FAC group,DMY intervention group with different concentrations,FAC+DMY group,and ra-pamycin preconditioning group.Cell viability was tested by MTT assay.The levels of hyaluronic acid(HA),laminin(LN),procollagen ?(PC?)and collagen IV(IV-C)in supernatant of cell culture liquid were tested by ELISA assay.Iron deposit in HSC-T6 cells was observed by prussian blue staining.The levels of total iron in HSC-T6 cells by colorimetry assay.The ultrastructural of HSC-T6 cells was observated by transmission electron microscope.The expressions of autophagy related gene microtubule associated protein 1 light chain 3(LC3)and p62/SQSTM1 in HSC-T6 cells were measured by Western Blot.RESULTS:1.Compared with control group,the protein expression of ?-SMA in HSC-T6 cells and the levels of HA,LN,PC ?and IV-C in supernatant of cell culture liquid were significantly increased in FAC group(P<0.05).Compared with FAC group,the protein expression of ?-SMA in HSC-T6 cells were significantly decreased in DMY(20,40,80,160 and 320 ?g/mL)+FAC group(P<0.05).2.Compared with control group,the iron deposit,the levels of total iron and ferrous iron in HSC-T6 cells were significantly increased in FAC group(P<0.05).Compared with FAC group,the iron deposit and the levels of total iron in HSC-T6 cells were significantly decreased in DMY(20,40,80,160 and 320 ?g/mL)+FAC group(P<0.05).3.Compared with control group,the number of autophagosome,the protein expression of NCOA4,LC3-? and the ratio of LC3-?/LC3-?in HSC-T6 cells were significantly increased and the protein expression of p62 in HSC-T6 cells was significantly decreased in FAC group(P<0.05).Compared with FAC group,the protein expression of NCOA4,FTH1 and LC3-? and the ratio of LC3-?/LC3-? in HSC-T6 cells were significantly decreased and the protein expression of FTH1 and p62 in HSC-T6 cells was significantly increased in DMY(40 ?g/mL)+FAC(P<0.05).4.Compared with FAC+DMY(40 ?g/mL)group,the protein expression of ?-SMA,the levels of HA,LN,PC ?and IV-C in supernatant of cell culture liquid,the iron deposit,the levels of total iron and ferrous iron in HSC-T6 cells were significantly increased in FAC+DMY(40 ?g/mL)+ rapamycin group(P<0.05).CONCLUSIONS:Dihydromyricetin inhibits the activation of hepatic stellate cell induced by iron overload through the inhibition of Ferritinophagy.