Miaoting Yang1, Zhijuan Chen1, Chunxin Xiao2, Waijiao Tang1, Beijie Zhou1,3. 1. Department of Pharmacy, Zhujiang Hospital, Southern Medical University, Guangzhou 510282, China. 2. Shenzhen Beimei Pharmaceutical Co., Ltd, Shenzhen 518057, China. 3. Department of Pharmacy, Seventh Affiliated Hospital of Sun Yat-sen University, Shenzhen 518017, China.
Abstract
OBJECTIVE: To investigate the effects of sera from rats fed with Huganqingzhi tablets (HGT) on endoplasmic reticulum (ER) stress in a steatotic hepatocyte model of free fatty acids (FFAs)-induced nonalcoholic fatty liver disease (NAFLD) and explore the possible mechanism. METHODS: FFAs prepared by mixing oleic acid and palmitic acid at the ratio of 2:1. HepG2 cells were treated with the sera from rats fed with low-, moderate-or high-dose HGT (HGT sera) or sera of rats fed with fenofibrate (fenofibrate sera), followed by treatment with 1 mmol/L FFAs for 24 h to induce hepatic steatosis. Oil red O staining was used to observe the distribution of lipid droplets in the cells. The biochemical parameters including triglyceride (TG), lactated hydrogenase (LDH), aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were measured using a commercial kit. The morphological changes of the ER in the cells were observed using transmission electron microscopy. The protein/mRNA expressions of ER stress-related signal molecules including GRP78, PERK, p-PERK, ATF6, ATF4, CASPASE-12, CHOP, XBP-1, PKC, and p-PKC-δ were detected using Western blotting and/or quantitative real-time PCR (qRT-PCR). The changes in the protein expressions of GRP78, p-PERK, CASPASE-12 and CHOP were also detected in cells with transient transfection of PKC-δ siRNA for PKC-δ knockdown. RESULTS: Compared with the control cells, the cells treated with FFAs showed significantly increased levels of TG, AST, and ALT (P < 0.05). Compared with FFAs-treated cells, the cells pretreated with HGT sera or fenofibrate sera all showed significantly decreased TG, AST and ALT levels (P < 0.05), reduced accumulation of the lipid droplets (P < 0.05), and lowered protein or mRNA expression levels of GRP78, p-PERK, ATF6, ATF4, CHOP, CASPASE-12, XBP-1 and p-PKC-δ (P < 0.05). PKC-δ knockdown caused significantly reduced protein expressions of GRP78, p-PERK, CASPASE-12 and CHOP in the cells with FFA-induced hepatic steatosis (P < 0.001); treatment with high-dose HGT serum more significantly reduced the expressions of GRP78 (P < 0.001) and P-PERK (P < 0.01) in FFAs-induced cells with PKC-δ knockdown. CONCLUSIONS: HGT serum can effectively prevent FFAs-induced steatosis in HepG2 cells by alleviating ER stress, in which PKC-δ may act as an important target.
OBJECTIVE: To investigate the effects of sera from rats fed with Huganqingzhi tablets (HGT) on endoplasmic reticulum (ER) stress in a steatotic hepatocyte model of free fatty acids (FFAs)-induced nonalcoholic fatty liver disease (NAFLD) and explore the possible mechanism. METHODS: FFAs prepared by mixing oleic acid and palmitic acid at the ratio of 2:1. HepG2 cells were treated with the sera from rats fed with low-, moderate-or high-dose HGT (HGT sera) or sera of rats fed with fenofibrate (fenofibrate sera), followed by treatment with 1 mmol/L FFAs for 24 h to induce hepatic steatosis. Oil red O staining was used to observe the distribution of lipid droplets in the cells. The biochemical parameters including triglyceride (TG), lactated hydrogenase (LDH), aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were measured using a commercial kit. The morphological changes of the ER in the cells were observed using transmission electron microscopy. The protein/mRNA expressions of ER stress-related signal molecules including GRP78, PERK, p-PERK, ATF6, ATF4, CASPASE-12, CHOP, XBP-1, PKC, and p-PKC-δ were detected using Western blotting and/or quantitative real-time PCR (qRT-PCR). The changes in the protein expressions of GRP78, p-PERK, CASPASE-12 and CHOP were also detected in cells with transient transfection of PKC-δ siRNA for PKC-δ knockdown. RESULTS: Compared with the control cells, the cells treated with FFAs showed significantly increased levels of TG, AST, and ALT (P &lt; 0.05). Compared with FFAs-treated cells, the cells pretreated with HGT sera or fenofibrate sera all showed significantly decreased TG, AST and ALT levels (P &lt; 0.05), reduced accumulation of the lipid droplets (P &lt; 0.05), and lowered protein or mRNA expression levels of GRP78, p-PERK, ATF6, ATF4, CHOP, CASPASE-12, XBP-1 and p-PKC-δ (P &lt; 0.05). PKC-δ knockdown caused significantly reduced protein expressions of GRP78, p-PERK, CASPASE-12 and CHOP in the cells with FFA-induced hepatic steatosis (P &lt; 0.001); treatment with high-dose HGT serum more significantly reduced the expressions of GRP78 (P &lt; 0.001) and P-PERK (P &lt; 0.01) in FFAs-induced cells with PKC-δ knockdown. CONCLUSIONS: HGT serum can effectively prevent FFAs-induced steatosis in HepG2 cells by alleviating ER stress, in which PKC-δ may act as an important target.
Authors: Ekaterina Bobrovnikova-Marjon; Georgia Hatzivassiliou; Christina Grigoriadou; Margarita Romero; Douglas R Cavener; Craig B Thompson; J Alan Diehl Journal: Proc Natl Acad Sci U S A Date: 2008-10-13 Impact factor: 11.205