内质网应激介导的凋亡在糖尿病ApoE^(-/-)小鼠内膜钙化中的作用

Effects of Endoplasmic Reticulum Stress-mediated Apoptosis in Intimal Calcification of Diabetic ApoE^(-/-) Mice

目的观察内质网应激(ERS)介导的凋亡在糖尿病动脉粥样硬化(As)性载脂蛋白E基因敲除(ApoE^(-/-))小鼠内膜钙化启动中的作用。方法 6周龄雄性Apo E-/-小鼠,给予腹腔注射链脲佐菌素(STZ)40mg/(kg·d),连续5天。2周后血糖水平>3000 mg/L的小鼠纳入本研究,并由普通饮食转为半合成型高脂饮食(HFD),同时给予尾静脉注射羧甲基赖氨酸(CML)10 mg/(kg·d),连续4个月。在转换高脂饮食和给予CML注射后的0个月(0月组,n=10)、2个月(2月组,n=10)和4个月(4月组,n=10)时对小鼠实施安乐死,进行相关检测与分析。结果病理形态学研究证实STZ-CML-HFD联合处理2个月后,糖尿病ApoE^(-/-)小鼠可形成早期的As斑块,4个月后形成典型的晚期As内膜钙化。主动脉壁平滑肌细胞固有表型SM22α逐渐丢失,而成骨表型骨形成蛋白2、核心结合因子α1、碱性磷酸酶的表达与活性则增加。主动脉壁CML沉积信号和糖基化终产物受体(RAGE)表达主要局限于粥样斑块内。Western blot检测显示,随着糖尿病Apo E-/-小鼠病程的延长,主动脉壁CML、RAGE、CD36表达显著上调,而胆固醇外流调控子三磷酸腺苷结合盒转运体A1先出现代偿性增加继而又减少至基线附近。TUNEL染色与Cleaved Caspase-3免疫组织化学染色发现,随着糖尿病As的演进,斑块内细胞凋亡率明显增加。定位分析显示ERS伴侣分子葡萄糖调节蛋白78(GRP78)、C/EPB同源蛋白(CHOP)主要分布在As病变区的脂质池内,而且相对于CHOP,4月组GRP78的分布位置似乎更倾向于脂质池的基底部。Western blot半定量分析发现,ERS相关指标GRP78、磷酸化蛋白激酶样内质网激酶、磷酸化eIF2α、活化转录因子4和CHOP的表达随着动物实验时间的延长均呈上调趋势。结论 STZ-CML-HFD联合干预4个月可成功诱导ApoE^(-/-)小鼠形成糖尿病As钙化。CML/RAGE可能首先启动了斑块ERS介导的凋亡,继而诱发了钙化级联信号。

Aim To investigate the role of endoplasmic reticulum stress( ERS)-mediated apoptosis in atherosclerotic calcification of diabetic apolipoprotein E gene knocked-out( ApoE^(-/-)) mice. Methods 6 weeks old male ApoE^(-/-)mice were first rendered diabetic by intraperitoneal injection of streptozotocin( STZ) for 5 days [40 mg /( kg·d) ]. After 2 weeks,the blood glucose level > 3000 mg / L mice were included in this study,and then were given a semisynthetic high-fat diet( HFD),plus daily tail vein injection of carboxy methyl lysine( CML) [10 mg /( kg·d) ]. The mice were euthanized at 0 month( group 0M,n = 10),2 months( group 2M,n = 10),and 4 months( group 4M,n = 10)after the triple administration of STZ-CML-HFD. Related detection and analysis were carried out for each group of mice.Results Morphological analysis showed that early atherosclerotic plaques appeared at 2 months after the triple administrations of STZ-CML-HFD,and that typically advanced plaques with extensive calcification lesions,abundant cholesterol crystals,and proliferative collagen were formed at 4 months after the triple administrations of STZ-CML-HFD. The intrinsic phenotype( SM22α) of aortic smooth muscle cells was gradually lost,and osteoblast-like phenotypes( bone morphogenetic protein-2,core binding factor α1,alkaline phosphatase) were increased. CML deposition signal and the expression of receptor for advanced glycosylation end product( RAGE) in the aortic wall were mainly restricted in the atherosclerotic plaques. Western blot assay showed the expressions of CML,RAGE and CD36 in aortic wall of diabetic Apo E- /-mice were significantly up-regulated,but the expression of ATP binding cassette transporter A1 firstly displayed a compensated increase and then reduced near the baseline. Experiment of TUNEL staining and cleaved caspase-3 immunohistochemical staining found intra-plaque cells apoptotic rate rised with the progression of diabetic atherosclerosis. Immunohistochemical location analysis showed glucose regulated protein 78( GRP78),a molecular chaperone of ERS and C / EPB homologous protein( CHOP) were mainly restricted in the lipid poor of atherosclerosis. Furthermore,compared with CHOP,the distribution signal of GRP78 in group 4M appeared more basal in lipid pool. Western blot semi-quantitative analysis found that the related indexes of ERS( GRP78,phosphorylated protein kinase RNA-like endoplasmic reticulum kinase,phosphorylated e IF2α,activating transcription factor 4,CHOP) were up-regulated with the extension of diabetic course in ApoE^(-/-)mice. Conclusions STZ-CML-HFD combined intervention for 4 months can induce atherosclerotic calcification of diabetes in ApoE^(-/-)mice. CML / RAGE may firstly start-up ERS-mediated apoptosis,and then cause to happen the calcification cascade signal.