脂毒性应激对Bcl-2蛋白诱导胰岛β细胞凋亡的调节作用  

Regulatory effect of lipid toxicity stress on Bcl-2-induced isletβcell apoptosis

作　　者：钟丽红[1] 丘创华[2] 彭紫元[2] 佘吉佳[2] ZHONG Lihong;QIU Chuanghua;PENG Ziyuan;SHE Jijia(Department of Cadre Health,Shenzhen Second People's Hospital,Shenzhen 518000,Guangdong,China;Department of Clinical Laboratory,Shenzhen Second People's Hospital,Shenzhen 518000,Guangdong,China)

机构地区：[1]深圳市第二人民医院干部保健科,广东深圳518000 [2]深圳市第二人民医院检验科,广东深圳518000

出　　处：《检验医学》2022年第3期274-280,共7页Laboratory Medicine

基　　金：广东省自然科学基金项目(2017A030310005)。

摘　　要：目的探讨脂毒性应激对B淋巴细胞瘤(Bcl)-2蛋白诱导胰岛β细胞凋亡的调节作用。方法利用C57BL/6小鼠构建PUMA-/-小鼠,喂食高脂肪饮食72 h后处死并分离胰岛。分别用棕榈酸酯、蛋白酶体抑制剂MG132处理小鼠胰岛瘤MIN6细胞0、2、4、8、24 h,分别用萝卜硫烷(SFN)、棕榈酸酯、SFN+棕榈酸酯处理小鼠胰岛β细胞0、2、4、8、24 h。以用二甲基亚砜处理相同时间的MIN6细胞和β细胞作为对照。检测细胞蛋白酶体活性,活化转录因子4(ATF4)mRNA、重链结合蛋白(Bip)mRNA、C/EBP同源蛋白(Chop)mRNA、p53上调凋亡调控因子(PUMA)mRNA表达量,Bcl-2、Bcl-XL、Mcl-1和Akt蛋白表达量,同时评估细胞活性。结果与0 h比较,采用棕榈酸酯处理MIN6细胞不同时间的蛋白酶体活性均无明显变化(P>0.05);采用MG132处理MIN6细胞8 h的蛋白酶体活性显著降低(P<0.05);MG132处理MIN6细胞24 h的Ub水平显著升高(P<0.05)。棕榈酸酯组和MG132组处理24 h后,MIN6细胞凋亡数显著高于对照组(P<0.05),棕榈酸酯和MG132处理24 h后,MIN6细胞ATF4 mRNA、Bip mRNA、Chop mRNA、PUMA mRNA表达量高于0 h(P<0.05)。棕榈酸酯和MG132处理24 h后,MIN6细胞Bcl-2、Bcl-XL、Akt蛋白水平显著低于0 h(P<0.05);Mcl-1蛋白水平呈先升后降趋势,棕榈酸处理2 h时Mcl-1蛋白水平最高,MG132处理8 h时Mcl-1水平最高。棕榈酸酯组β细胞凋亡率均显著高于SFN组、SFN+棕榈酸酯组和对照组(P<0.05),SFN组、SFN+棕榈酸酯组及对照组之间β细胞凋亡率差异均无统计学意义(P>0.05)。棕榈酸酯组AFT4蛋白、Chop蛋白、Bip蛋白和PUMA mRNA水平均显著高于对照组(P<0.05);而SFN+棕榈酸酯组与对照组之间差异均无统计学意义(P>0.05)。结论靶向泛素-蛋白酶体系统(UPS)和Bcl-2蛋白表达能有效预防胰岛β细胞凋亡。Objective To investigate the regulatory effect of lipid toxicity stress on B-cell lymphoma(Bcl)-2-induced isletβcell 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 isletβcells were treated with sulforaphane(SFN),palmitate and SFN+palmitate for 0,2,4,8 and 24 h.MIN6 cells andβcells treated with dimethyl sulfoxide for the same time were used as controls.The cell proteasome activity was detected,and by realtime quantitative polymerase chain reaction(PCR)the activated transcription factor 4(ATF4)mRNA,heavychain 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.Theβcell apoptosis rate in palmitate

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 

分 类 号：R446.1[医药卫生—诊断学]