The Role Of Autophagy In Parenteral Nutrition-associated Liver Disease

Background and Aims Parenteral nutrition-associated liver disease(PNALD)is a serious complication of long-term use of parenteral nutrition(PN).Autophagy is an important physiological process for the body to maintain homeostasis.This study was amid to determine the changes of autophagy in hepatocytes by establishing cell model and PNALD rat model.At the same time,it was evaluated whether the pathological changes of hepatocytes could be alleviated by regulating autophagy in vivo and in vitro,and the intrinsic mechanism was explored.MethodsThree-week-old Sprague-Dawley rats were randomly divided into four groups after one week of adaptive-feeding: control group(jugular vein cather inserting combined with injection of normal saline),parenteral nutrition(PN)group(jugular vein cather inserting combined with PN injection),PN+Rapamycin(Rapa)group(jugular vein cather inserting combined with PN injection and intraperitoneally injection of Rapamycin)and the Rapamycin group(jugular vein cather inserting combined with injection of normal saline and the Rapamycin was administered intraperitoneally).Saline and PN injections were continued for 24 hours.Dose of rapamycin was 1mg/kg/qod.The rats were sacrificed one week later and liver samples and serum were collected.H&E and oil red O staining of liver tissues was proceeded.Values of serum ALT,AST,TB and DB in each group were measured.Western blot and immunofluorescence were used to detect the expression of LC3 protein in rat hepatocytes.In addition,the levels of reactive oxygen species(ROS)in rat hepatocytes were detected and the levels of endoplasmic reticulum stress(ER stress)-related markers BIP,s XBP1 and CHOP were detected by immunohistochemical(IHC)and western blot.L02 cell line was treated with 1% medium-chain and longchain triglycerides(MCT/LCT)for 48 h.Bafilomycin A1(Baf,400 n M)was used to detect the autophagy flux.Rapamycin(1 μM)and 3-Methyladenine(3-MA,5m M)were used to regulate autophagy in hepatocytes.Oil red O staining was used for detecting intracellular lipid accumulation;DAPI staining and flow cytometry(FCM)were proceeded to evaluate apoptosis;quantitative polymerase chain reaction(q PCR)and western blot were done to detect autophagy-related gene expression.Target gene was regulated to investigate its effects on autophagy and pathological changes of hepatocytes.Relusts1.In the PN group,there were lower levels of LC3Ⅱ expression combined with decreased LC3Ⅱ fluorescence intensity compared to sham group,which means inhibition of autophagy.The rats in the PN+Rapa group owned increased LC3Ⅱfluorescence intensity,higher ratios of LCⅡ to LCⅠ and lower P62 expressions than PN group,suggesting the activation of autophagy.2.Rapamycin treatment reduced intrahepatic lipid deposition and alleviated pathological changes of hepatocytes in PN rats.Compared with the sham group,the ROS of liver tissue in the PN group increased significantly,and the ER stress-related proteins BIP,s XBP1 and CHOP elevated significantly.The PN+Rapa group had lower levels of ROS and ER stress than PN group.In addition,Rapamycin treatment alleviated apoptosis in liver of PN rats,approved by lower levels of Tunel staning and cleaved-caspase3 expressons.3.Compared with control group,the L02 cells treated with MCT/LCT had increased levels of LC3Ⅱ and P62.Supplementary Baf didn’t increase the LC3Ⅱ and P62 expressions.Further induction of autophagy by rapamycin could reduce lipid deposition,apoptosis,ROS and ER stress levels in hepatocytes.Treatment with autophagy inhinitor 3-MA exacerbated the intracellular pathologic change.Cells treated with MCT/LCT presented with no decrease in the lysosomal related markers LAMP2 and CTSD,suggesting the normal function of lysosome.The expression of VPS18,which contributed to the form of autolysosome,decreased significantly.4.Up-regulating of VPS18 levels in MCT/LCT-treated L02 cells could alleviate the autophage flux blockage and reduce hepatocyte lipid deposition,apoptosis and levels of ROS and ER stress.Conclusions1.In the PNALD rat model,the levels of autophagy in liver decreased.2.Regulation of autophagy could alleviate pathological changes of hepatocytes in PN rats,which might be achieved by reducing intracellular ER stress levels.3.The blockage of autophage flux of hepatocytes caused by lipid emulsions might be caused by the dysfunction of autolysosome forming due to the down-regulation of VPS18 gene.VPS18 overexpression could improve the hepatocyte autophage flux blockage and have protective effects on hepatocytes in vitro.4.Intervention of autophagy might provide new insights for the treatment of PNALD...