摘要
目的建立棕榈酸诱导成骨细胞凋亡的模型,探讨内质网应激在棕榈酸诱导成骨细胞凋亡中的作用。方法选取小鼠成骨细胞MC3T3-E1 14为研究对象,分组方法如下:(1)根据棕榈酸(palmitate,PA)作用浓度分为对照组(0μmol/L PA)、100μmol/L PA组、250μmol/L PA组和500μmol/L PA组,分别孵育24 h;随后选择棕榈酸作用敏感浓度500μmol/L,孵育成骨细胞不同时间(0、6、12、24 h)。(2)应用内质网应激抑制剂4-苯基丁酸(4-phenylbutyric acid,4-PBA)进一步验证棕榈酸对成骨细胞增生和凋亡的影响。选择4-PBA作用敏感浓度5 mmol/L,根据是否经4-PBA预处理分为对照组(0μmol/L PA,0 mmol/L 4-PBA)、4-PBA组(0μmol/L PA,5 mmol/L 4-PBA)、4-PBA+PA组(500μmol/L PA,5 mmol/L 4-PBA)和PA组(0 mmol/L 4-PBA,500μmol/L PA)干预24 h。培养结束后,采用CCK-8法检测细胞增生活力;Annexin V/PI双染色法流式细胞术检测细胞凋亡率;Realtime quantitative PCR检测各组细胞内质网应激相关基因GRP78、CHOP、caspase-12 m RNA的表达。结果与对照组相比,250和500μmol/L棕榈酸干预成骨细胞24 h可明显降低细胞增生活力(A值:1.015±0.068,0.816±0.108,0.479±0.050,P<0.05),细胞凋亡率则分别增高了6.70%±1.40%,15.30%±1.28%(P<0.05),CHOP和GRP78 m RNA的表达水平呈剂量依赖性增高(均P<0.05),而caspase-12 m RNA的表达水平则有所降低(P<0.05);100μmol/L PA组增生活力、凋亡率及各基因表达量差异均无统计学意义(P>0.05)。与对照组相比,用500μmol/L棕榈酸干预成骨细胞6、12、24 h后,细胞增生活力呈时间依赖性下降(A值:0.952±0.064,0.788±0.043,0.683±0.044,0.482±0.052,P<0.05);凋亡率则分别增高3.63%±1.45%,9.0%±2.31%,15.7%±0.61%(均P<0.05);CHOP m RNA表达水平呈时间依赖性增高(P<0.05),GRP78 m RNA的表达水平在12 h开始升高,24 h达到峰值(P<0.05),caspase-12 m RNA表达水平则在12 h达到峰值,24 h后开始下降(P<0.05)。与PA组相比,4-PBA+PA组成骨细胞增生活力升高(A值:0.355±0.029 vs.0.451±0.049,P<0.05),凋亡率降低8.6%±2.05%(P<0.05),GRP78、CHOP和caspase-12m RNA的变化水平则呈现与单纯棕榈酸干预组相反趋势(均P<0.05)。结论棕榈酸可能通过内质网应激途径诱导成骨细胞凋亡,4-PBA可以通过抑制内质网应激在一定程度上减少棕榈酸诱导的成骨细胞凋亡。
Objective To establish models of palmitate-induced osteoblast apoptosis and assess the role endoplasmic reticulum stress( ERS) playing in this process. Methods MC3T3-E1 14 cells were divided into different groups as follows:( 1) Groups with various concentrations of palmitate: control group( 0 μmol/L PA),100 μmol/L PA group,250 μmol/L PA group and 500 μmol/L PA group,incubated for 24 h. Then osteoblasts were pretreated with 500 μmol/L PA for different lengths of time( 0,6,12,24 h).( 2) To further characterize the role of ERS in palmitate-induced apoptosis,cells were pretreated with 500 μmol/L PA in presence or absence of 4-phenylbutyric acid( 4-PBA 5 mmol/L)for 24 h: control group( 0 μmol/L PA,0 mmol/L 4-PBA),4-PBA group( 0 μmol/L PA,5 mmol/L 4-PBA),4-PBA+ PA group,( 500 μmol/L PA,5 mmol/L 4-PBA) and PA group( 0 mmol/L 4-PB,500 μmol/L PBA). CCK-8 assay was used to determine cell viability and flow cytometry to detect cellular apoptosis,Real-time quantitative PCR was used to measure the expression of ERS markers( GRP78,CHOP,caspase-12 m RNA). Results( 1) Compared to the control group,the osteoblasts proliferation of 250 μmol/L and 500 μmol/L PA group were both decreased( A value: 1. 015 ± 0. 068,0. 816 ± 0. 108,0. 479 ± 0. 050, P < 0. 05), apoptosis rate were increased by 6. 70% ±1. 40%,15. 30% ± 1. 28%( P < 0. 05). In contrast,100 μmol/L PA had no significant effect on the osteoblasts proliferation rate,apoptosis rate and the expression levels of different gene markers( P > 0. 05); expression levels of CHOP and GRP78 m RNA were increased in a dose-dependent manner( all P < 0. 05),while the expression levels of caspase-12 m RNA was reduced( P < 0. 05). Exposure of cells to 500 μmol/L PA for 6,12 and 24 h significantly reduced the cell proliferation in a time-dependent way( A value: 0. 952 ± 0. 064,0. 788 ± 0. 043,0. 683 ± 0. 044,0. 482 ± 0. 052,P < 0. 05),and markedly increased the apoptosis rate by 3. 63% ± 1. 45%( P < 0. 05),9. 0% ±2. 31%( P < 0. 05) and 15. 7% ± 0. 61%( P < 0. 05),respectively. CHOP expression levels were increased in a time-dependent way( P < 0. 05),while GRP78 m RNA expression levels began to rise at 12 h( P < 0. 05) and peaked at 24 h( P < 0. 01). Expression levels of caspase-12 m RNA peaked at 12 h( P < 0. 05) and began to drop at 24 h( P < 0. 05).( 2) Compared to the PA group,the proliferation rate of 4-PBA + PA group was partly increased( A value: 0. 355 ± 0. 029 vs. 0. 451 ± 0. 049,P < 0. 05); apoptosis rate was partly reduce by 8. 6% ± 2. 05%( P < 0. 05),and the above results of PA on gene expression could be reversed to some extent( all P < 0. 05). Conclusion Palmitate could induce osteoblasts apoptosis through the way of endoplasmic reticulum stress. 4-PBA could partly reduce the apoptosis of osteoblasts through the inhibition of ERS.
出处
《中华骨质疏松和骨矿盐疾病杂志》
2016年第4期383-390,共8页
Chinese Journal Of Osteoporosis And Bone Mineral Research
基金
河南省医学科技攻关计划(201403028)
关键词
成骨细胞
凋亡
内质网应激
棕榈酸
osteoblasts
apoptosis
endoplasmic reticulum stress
palmitate