Autophagy Mediated By Endoplasmic Reticulum Stress Enhances Caffeine-induced Apoptosis Of Hepatic Stellate Cells

Aims: This study was aimed to detect the directly effects of caffeine on hepatic stellate cells(HSCs)and the underlying mechanisms in vitro.Methods: Human HSCs cell line LX-2 was applied in vitro in this study and the cells were treated with caffeine at concentrations of 0m M,2.5m M,5m M,10 m M,20 m M,30 m M for the indicated times.The effects of caffeine on cell viability and apoptosis were assessed by CCK-8 assay and flow cytometry,respectively.Western blot was used to measure the marker proteins of autophagy and endoplasmic reticulum stress.Autophagosomes and enlarged endoplasmic reticulums were observed by transmission electron microscope and the number of GFP/RFP-LC3 punctain HSCs transfected with RFP-GFP-h LC3 lentivirus were explored by confocal microscope.And si RNA was applied to knock down the expression of ATG7 or IRE1-α.Results: Caffeine was found to inhibit the cell viability and increase apoptosis in dose-and time-dependent manners.ER stress was stimulated by caffeine as demonstrated by increased levels of GRP78/Bip,CHOP and IER1-α as well as many enlarged ERs detected by electron microscopy.Caffeine induced autophagy shown by increased LC3Ⅱaccumulation and decreased P62 level and the number of GFP/RFP-LC3 puncta and autophagosomes/-lysosomes.Moreover,IRE1-αknockdown decreased the level of autophagic flux,inhibition of autophagy by ATG7-si RNA and 3-MA protected LX-2 cells from apoptotic death.Conclusions: Caffeine-enhanced autophagic flux in HSCs is stimulated by ER stress via IRE1-α signaling pathway,which further weaken HSCs viability by induction of apoptosis.These findings provide a new insight into the mechanism of caffeine’s anti-fibrosis effects.