关键词: B淋巴细胞瘤-2蛋白, β细胞, 细胞凋亡, 脂毒性应激, 泛素化蛋白

Abstract:

Objective To investigate the regulatory effect of lipid toxicity stress on B-cell lymphoma（Bcl）-2-induced islet β cell apoptosis. Methods C57BL/6 mice were used to establish PUMA-/- mice,and pancreatic islets were isolated after 72 h of feeding a high-fat diet. The mouse pancreatic tumor cells MIN6 were treated with palmitate and proteasome inhibitor MG132 for 0,2,4,8 and 24 h. The mouse pancreatic islet β cells were treated with sulforaphane（SFN）,palmitate and SFN + palmitate for 0,2,4,8 and 24 h. MIN6 cells and β cells treated with dimethyl sulfoxide for the same time were used as controls. The cell proteasome activity was detected,and by real-time quantitative polymerase chain reaction（PCR） the activated transcription factor 4（ATF4） mRNA,heavy-chain binding protein（Bip） mRNA,C/EBP homologous protein（Chop） mRNA,p53 up-regulated modulator of apoptosis（PUMA） mRNA expression were determined. Western blotting was used to determine the expressions of Bcl-2,Bcl-XL,Mcl-1 and Akt protein,and the cell viability at the same time was evaluated. Results Compared with 0 h,the cell proteasome activity of MIN6 cells treated with palmitate for different times did not change（P>0.05）. The proteasome activity of MIN6 cells treated with MG132 for 8 h was reduced（P<0.05）. After MG132 treated MIN6,the level of ubiquitinated protein in the cells at 24 h was increased（P<0.05）. After 24 h of treatment in palmitate group and MG132 group,the number of cell death in MIN6 was higher than that in control group（P<0.05）. The expressions of ATF4 mRNA,Bip mRNA,Chop mRNA and PUMA mRNA in MIN6 cells were higher than those of 0 h after palmitate and MG132 treatment for 24 h（P<0.05）. The protein levels of Bcl-2,Bcl-XL and Akt in MIN6 cells were lower than those of 0 h after treatment with palmitate and MG132 for 24 h（P<0.05）. The level of Mcl-1 protein increased firstly and then decreased. The level of Mcl-1 protein was the highest when treated with palmitate for 2 h,and the level of Mcl-1 was the highest when treated with MG132 for 8 h. The β cell apoptosis rate in palmitate group was higher than those in SFN group,SFN+palmitate group and control group（P<0.05）. There was no statistical significance in the β cell apoptosis rate among SFN group,SFN+palmitate group and control group（P>0.05）. AFT4 protein,Chop protein,Bip protein and PUMA mRNA in palmitate group were higher than those in control group（P<0.05）. There was no statistical significance between SFN+palmitate group and control group（P>0.05）. Conclusions Targeting ubiquitin-proteasome system （UPS） and Bcl-2 may prevent β-cell apoptosis in islet β cell effectively.

Key words: B-cell lymphoma-2, β cell, Cell apoptosis, Lipid toxicity stress, Ubiquitinated protein