糖尿病患者低密度脂蛋白通过激活caspase-12途径诱导小鼠巨噬细胞凋亡

目的:研究糖尿病患者低密度脂蛋白(low density lipoprotein from diabetes mellitus patients,DM-LDL)对小鼠巨噬细胞内质网应激(endoplasmic reticulum stress,ERS)凋亡途径关键分子caspase-12的影响,以探讨其对鼠巨噬细胞凋亡的诱导作用及机制。方法:体外培养小鼠巨噬细胞RAW264.7,给予DM-LDL(25、50和100 mg/L)处理24 h;分别以正常人来源的低密度脂蛋白(normal low density lipoprotein,n-LDL;50 mg/L)和ERS诱导剂衣霉素(tunicamycin,TM;4 mg/L)处理24 h的巨噬细胞作为阴性和阳性对照组;另外以ERS抑制剂4-苯基丁酸(4-phenylbutyric acid,PBA;5 mmol/L)预处理细胞1 h,然后给予DM-LDL(100 mg/L)处理24 h。分别采用MTT法和Annexin V-FITC/PI双染法检测细胞活力和凋亡情况,乳酸脱氢酶(lactate dehydrogenase,LDH)试剂盒测定培养液中LDH的活性,Western blot法检测caspase-12蛋白的表达。结果:与TM相似,DM-LDL明显导致细胞活力下降、LDH释放和细胞凋亡率增加(P<0.05),同时显著增强caspase-12的活性,在50和100 mg/L浓度时作用更明显(P<0.01)。PBA预处理可抑制DM-LDL所诱导的巨噬细胞活力下降、LDH释放和凋亡增加(P<0.05),同时抑制DM-LDL所致的caspase-12活化(P<0.05)。结论:DM-LDL可导致小鼠巨噬细胞RAW264.7的凋亡,其机制可能与活化caspase-12途径有关。

Low density lipoprotein from patients of diabetes mellitus induces apoptosis of murine macrophages via activating caspase-12 pathway

AIM: To explore the effect of low density lipoprotein from the patients of diabetes mellitus(DM-LDL) on the activation of caspase-12 an important molecule in endoplasmic reticulum stress(ERS)-associated apoptotic pathway, in the murine macrophages, and to clarify the underlying molecular mechanisms of apoptosis. METHODS:Murine macrophage RAW264.7 was exposed to DM-LDL(25, 50 and 100 mg/L), normal low density lipoprotein(n-LDL, 50 mg/L), or tunicamycin(TM, 4 mg/L) for 24 h. Additionally, RAW264.7 macrophages were precultured with 4-phenylbutyric acid(PBA, 5 mmol/L) for 1 h and then exposed to DM-LDL(100 mg/L) for 24 h. The cell viability and apoptosis were detected by MTT assay and flow cytometry with Annexin V-FITC/propidium iodide staining, respectively. Lactate dehydrogenase(LDH) activity in the media was measured by assay kit. The protein level of caspase-12 was determined by Western blot. RESULTS:Similar to TM(an ERS inducer), treatment with DM-LDL caused significantly decrease in the viability and increase in LDH activity in the media and apoptotic rate of the RAW264.7 macrophages(P<0.05). Additionally, DM-LDL induced activation of caspase-12 especially at the dose of 50 and 100 mg/L(P<0.01). However, the ERS inhibitor PBA protected RAW264.7 macrophages from DM-LDL-induced decrease in viability and increase in LDH activity and apoptosis(P<0.05). Furthermore, PBA attenuated DM-LDL-induced activation of caspase-12(P<0.05). CONCLUSION:DM-LDL may induce apoptosis in RAW264.7 macrophages, and the mechanism may be related to the activation of caspase-12.