Effects of sera of rats fed with tablets on endoplasmic reticulum stress in a HepG2 cell model of nonalcoholic fatty liver disease / 南方医科大学学报

OBJECTIVE@#To investigate the effects of sera from rats fed with 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 ( < 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 ( < 0.05), reduced accumulation of the lipid droplets ( < 0.05), and lowered protein or mRNA expression levels of GRP78, p-PERK, ATF6, ATF4, CHOP, CASPASE-12, XBP-1 and p-PKC-δ ( < 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 ( < 0.001); treatment with high-dose HGT serum more significantly reduced the expressions of GRP78 ( < 0.001) and P-PERK ( < 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.

Role of protein kinase C-δ in Dectin-1-Src-Syk-mediated killing of Candida albicans by macrophages / 中国免疫学杂志

Objective:To study the role of protein kinase C-δin the Dectin-1-Src-Syk-mediated killing of Candida albicans by macrophages and investigate the molecular mechanism of antifungal innate immunity .Methods:Cell surface receptors were accessed by Flow Cytometry.Mouse bone marrow derived macrophages were pre-incubated with different protein kinase inhibitors and then stimulated with C.albicans.The phosphorylation of related proteins was determined by Western blot.The ROS production,phagocytosis and killing of C.albicans by macrophages were measured.Results:Either Src or Syk inhibitor reduced C.albicans induced PKC-δphos-phorylation.PKC-δinhibitor Rottlerin reduced p40phox phosphorylation,ROS production and killing of C.albicans but had no effect on the phagocytosis of C.albicans by macrophages.Conclusion:PKC-δinhibitor Rottlerin reduced the killing of Candida albicans by mac-rophages through the inhibition of NADPH complex activation and ROS production ,suggesting that PKC-δplays an important role in an-tifungal innate immunity.

Effects of different dialysates on apoptosis and expression of PKCδ of U937 cell line / 上海交通大学学报（医学版）

Objective To investigate the effects of different dialysates on expression of protein kinase C-δ (PKCδ) and apoptosis of U937 cell line. Methods Different dialysates were added into culture fluid with U937 cell line at exponential phase of growth, and groups were divided: fluid A+fluid B group (dialysate A+dialysate B), fluid A+fluid B+rottlerin (PKCδ specific inhibitor)group, fluid A+powder B group (dialysate A+powder B) and fluid A+powder B + rottlerin group. Besides, blank control group and normal control group were established. Cells were harvested 24 h and 48 h after treatment, morphological changes were observed by Hoechst33258 fluorescence staining, cell apoptosis was measured by Annexin-V-FITC/PI double staining, and expression of PKCδ mRNA and protein was detected by RT-PCR and Western blotting, respectively. Results Cell apoptosis significantly increased in fluid A+powder B group, with typical morphology of apoptosis. After treatment for 24 h and 48 h, cell apoptosis rates in fluid A+powder B group were significantly higher than those at corresponding time points in blank control group , normal control group and fluid A+powder B+rottlerin group (P<0.05). Compared with normal control group, blank control group and fluid A+powder B+rottlerin group, the expression of PKCδ mRNA and protein of U937 cells in fluid A+powder B group were significantly increased (P<0.05). There was no significant difference in cell apoptosis rates and expression of PKCδ mRNA and protein between fluid A+fluid B group and blank control group, normal control group and fluid A+fluid B+rottlerin group (P＞0.05). Conclusion Fluid A+powder B can significantly increase apoptosis of U937 cell line, the mechanism of which may be associated with the up-regulation of expression of PKCδ. Compared with fluid A+powder B, fluid A+fluid B is superior in reducing apoptosis of peripheral blood monouclear cells.