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Study of the Effect and Mechanism of Protein Disulfide Isomerase in the Pathogenesis of Nonalcoholic Fatty Liver Disease in Vitro

Author: HuangJieRong
Tutor: LiYouMing
School: Zhejiang University
Course: Internal Medicine
Keywords: Nonalcoholic fatty liver disease Apoptosis Endoplasmic reticulum stress Protein disulfide isomerase
CLC: R575.5
Type: Master's thesis
Year: 2011
Downloads: 38
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Abstract

Background and Objective:Recently, with the development of economy and the modification of life style, nonalcoholic fatty liver disease(NAFLD) has been one of the most important causes of chronic liver disease in our country. NAFLD represents several overlapping clinicopathological states, ranging from simple steatosis to nonalcoholic steatohepatitis (NASH). NASH is a more serious form of liver damage, as cirrhosis and/or hepatocellular carcinoma are potential outcomes of NASH. However, cirrhosis and/or hepatocellular carcinoma rarely occurs in individuals with simple steatosis. The pathogenesis of NAFLD is still unclear. Recent data favor a model in which a pathologically increased rate of hepatocytic apoptosis and the subsequent induction and upregulation of inflammation and fibrosis in the liver. Excess endoplasmic reticulum(ER) stress can disrupt ER homeostasis and lead to unfolded proteins and misfolded proteins aggregate in the ER lumen, both of which can be detrimental to cell function and survival. Molecular chaperones in endoplasmic reticulum, such as protein disulfide isomerase(PDI), may play a role during this process. This research aims to illuminate the effect and mechanism of PDI in the pathogenesis of NAFLD in vitro.Methods:(1) Cell culture and transfection:HL-7702 cells were cultured in RPMI1640 medium supplemented with 10% fetal bovine serum at 37℃in an atmosphere containing 5% CO2. Transfection of siRNA into cells using LipofectamineTM 2000 according to the manufacturer’s instructions. Cells were incubated at 37℃for a further 72 hours and then subjected to 1 mM FFA mixture treatment for 24 hours.(2) Oil red O staining: Cell monolayers were washed twice with PBS and fixed for 15 minutes with 12% neutral formalin. Then staining in Oil Red O solution for 15 minutes and washing with 60% isopropanol. Subsequently, counterstaining with hematoxylin for 5 minutes and washing thoroughly in running tap water.(3) Western Blot:HL-7702 cells in 12-well formats were collected and proteins were extracted. Individual protein samples were separated by SDS-PAGE and then electro-transferred onto the PVDF membrane. Membranes were blocked for 1 hour and incubated with specific antibodies overnight at 4℃. After incubation with secondary antibodies, ECL detection reagents were used to detect the signals.(4) Analysis of apoptosis: Cells were washed twice with cold PBS and resuspended. Then incubated with Annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (PI) staining solution following manufacturer’s instructions. Stained cells were analyzed using a flow cytometer.Results:(1) Oil red O staining:FFA-treated groups increased intracellular lipid content compared to FFA-untreated controls. However, among all of FFA-treated groups, which contained FFA-treated group and PDI-negative siRNA+FFA-treated group and PDI-siRNA Mix+FFA-treated group,the amount of intracellular lipid content were similar.(2) Apoptosis:Compared with FFA-untreated controls, the percents of apoptosis cells in FFA-treated group and PDI-negative siRNA+FFA-treated group and PDI-siRNA Mix+FFA-treated group were risen up one by one.(3) ER-stress and apoptosis related proteins:Compared with FFA-untreated controls, the expression of protein GRP78 was risen up in FFA-treated group,PDI-negative siRNA+FFA-treated group and PDI-siRNA Mix+FFA-treated group, while the up-regulated expression was the most significance in the PDI-siRNA Mix +FFA-treated group. Moreover, the expression of protein CHOP was risen up one by one among FFA-untreated control group, FFA-treated group,PDI-negative siRNA+FFA-treated group and PDI-siRNA Mix+FFA-treated group. However, the expression of protein IRE1 was similar among all of the groups.Conclusions:(1) PDI may protect HL-7702 cells from ER stress.(2) PDI may protect HL-7702 cells from apoptosis induced by ER stress.(3) In HL-7702 cells, ER stress-mediated apoptosis may be induced by upregulation of CHOP expression for the most part.(4) PDI perhaps don’t have any affect during the course of steatosis in HL-7702 cells.

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