The Regulation Of Tunicamycin On The Apoptosis Of Hepatic Stellate Cell

Objectives Hepatic fibrosis(HF)is the necessary process from chronic liver disease to liver cirrhosis,the key link to prevent hepatic cirrhosis development is how to reverse the HF.Activated hepatic stellate cells(HSC)play an important role in the development of HF,and promoting the apoptosis of HSCs is the core of HF.Endoplasmic reticulum stress(ERS)is a new kind of apoptotic pathway.Tunicamycin(TM)is the inductor of endoplasmic reticulum stress,and the calpain inhibitor(ALLN)can inhibit calpain-2.Therefore in this study,TM and ALLN were used to treat HSCs which stimulated by transforming growth factor-?1(TGF-?1),to study the effect of ERS on the apoptosis of HSC and provide further laboratory evidence for the research of anti-fibrosis.Methods The rat HSCs were stored in liquid nitrogen,recovered in the water bath pot with 37? and cultured with complete medium.MTT was used to screen the dose of TM(2ug/ml),the effect time of TM(24h)and the dose of ALLN(25u M).HSCs were synchronized for 24 h and divided into four groups: blank group,TGF-?1 group,ALLN+TM group(TGF-?1+ALLN+TM)and TM group(TGF-?1+TM).The HSCs in blank group were treated by blank medium for 48 h,the rest groups were treated by TGF-?1(10ng/ml)for 24 h,TGF-?1 group replaced blank medium,ALLN+TM group pretreated by ALLN(25u M)for 30 mins,and then ALLN+TM group and TM group were treated by TM(2ug/ml)for 24 h in dark environment.Inverted phase contrast microscope was used to observe the development of HSCs after cultivated for 24 h,48h,72 h and the morphological changes after treated by TGF-?1.The proliferation rates were examined by MTT.The ultrastructural changes were watched by transmission electron microscope,the apoptosis of HSCs were examined by AO/PI double-staining method,cell cycles were evaluated by flow cytometry,the calcium were measured by laser scanning confocal microscope,the expressions of caspase-3 and calpain-2 were measured by immunohistochemistry,and the expressions of ?-SMA,GRP78,caspase-9,Bax,Bcl-2 and type I collagen protein were analyzed by Western Blot.Results 1 Morphology: Only a small quantity of HSCs adherented after 24 h,the numuber of adherented cells were increased after 48 h,and then could cover the full bottle after 72 h.The HSCs became a long spindle changed obviously treated by TGF-?1 and the intercellular space become large;2 Cell proliferation: The comparations of proliferation rates of HSCs among each groups had statistical significance(P<0.05),the proliferation rate in TGF-?1 group was higher than blank group(P<0.05),the ALLN+TM group and TM group were lower than TGF-?1 group(P<0.05);3 Cell cycle:The comparations of G1,S and G2 among each groups had statistical significance(P<0.05),the proportion of cells in G1 and G2 phases in TGF-?1 group were lower than blank group(P<0.05),while the proportion of S phase was increased significantly(P<0.05),the proportion of HSCs in G1 phase in ALLN+TM group and TM group were higher than TGF-?1 group(P<0.05),while the S phase were lower than TGF-?1 group(P<0.05)and the changes in G2 phase were not obviously(P>0.05);4 Ca2+: The comparations of Ca2+ among each groups had statistical significance(P<0.05),compared with blank group,the intensity of Ca2+ in TGF-?1 group was increased not obviously(P>0.05),compared with TGF-?1 group,the concentrations of Ca2+ in ALLN+TM group and TM group were increased significantly(P<0.05),but the ALLN+TM group was lower than TM group(P<0.05);5 AO/PI double-staining method: the most in blank group and TGF-?1 group presented green fluorescence,a small number of cells in ALLN+TM group presented orange-red or orange fluorescence,a large proportion of HSCs presented green fluorescence,the most of HSCs in TM group presented orangered or orange fluorescence;6 Transmission electron microscope: the chromatin evevly distributed in blank group and TGF-?1 group,the microvilli of HSCs in ALLN+TM group fell off,the organelles were vacuolated change.The chromatin edge accumulation and appeared “crescent region” in TM group;7 Immunohistochemical: The comparations of caspase-3 and calpain-2 among each groups had statistical significance(P<0.05),the expressions of caspase-3 and calpain-2 in ALLN+TM group and TM group were higher than TGF-?1 group(P<0.05),the caspase-3 and calpain-2 in ALLN+TM group were lower than TM group(P<0.05).8 Western Blot: The expression of ?-SMA between blank group and TGF-?1 group had statistical significance(P<0.05),the comparations of GRP78,caspase-9,Bax,Bcl-2 and type I collagen protein among each groups had statistical significance(P<0.05),compared with TGF-?1 group,the expressions of GRP78,caspase-9 and Bax in ALLN+TM group and TM group were increased significantly(P<0.05),while Bcl-2 and type I collagen were reduced obviously(P<0.05).Conclusions 1 Tunicamycin could upregulate the concentration of Ca in HSCs,induce apoptosis through ERS pathway and downregulate the expression of type I collagen to reverse HF;2 Blocking the calpain-2 in ERS pathway could inhibit the apoptosis induced by TM.