3,5-二羟基苯甘氨酸对体外氨基核苷嘌呤霉素诱导的足细胞凋亡的影响

Effect of (S)-3,5-dihydroxyphenylglycine on puromycin aminonucleosideinduced apoptosis of podocytes in vitro

目的研究代谢型谷氨酸受体1(mGluR1)和mGluR5的激动剂3,5-二羟基苯甘氨酸(DHPG)在体外对由氨基核苷嘌呤霉素(PAN)诱导的足细胞凋亡的影响,探讨可能的作用途径。方法体外培养小鼠条件永生型足细胞株5P12,根据添加干预药物的不同进行分组。采用CCK-8法、Western blotting、EIA法、JC-1单标法流式细胞术、免疫荧光法和TUNEL染色激光共聚焦显微镜观察等方法,对足细胞的细胞活力、mGluR1/5和活化的胱冬肽酶-3(cleaved caspase-3)的表达、环磷酸腺苷(cAMP)水平、线粒体膜电位变化、磷酸化cAMP反应元件结合蛋白(CREB)及细胞凋亡等指标进行检测。结果 DHPG诱导足细胞使cAMP生成和CREB表达增加,DHPG可防止PAN诱导的足细胞活力降低、cleaved caspase-3表达升高、线粒体膜电位下降以及晚期凋亡,而这些作用可被mGluR1和mGluR5的选择性拮抗剂(AIDA)、腺苷酸环化酶抑制剂(SQ22536)及mGluR1和mGluR5的RNA干扰加以抑制。结论 mGluR1和mGluR5的选择性激动剂DHPG可防止体外PAN诱导的足细胞凋亡,其作用可能与mGluR/cAMP信号途径有关。

Objective To investigate the effect of （ S）-3, 5-dihydroxyphenylglycine （DHPG）, a selective metabotropic glutamate receptors （mGluR） 1 and 5 agonist, on aminonucleoside puromycin （PAN）-induced podocyte apoptosis in vitro, and explore the possible mechanism. Methods Conditional immortalized mouse podocytes 5P12 were cultured in vitro, and were grouped based on different interventions. CCK-8, Western blotting, EIA, JC-1 staining and flow cytometry, immunofluorescence staining, TUNEL staining and confocal laser scanning microscopy were employed to examine the cell viability, expression of mGluR1/5 and activated cleaved caspase-3, cyclic adenosine monophosphate （cAMP） level, mitochondrial membrane potential, cAMP responsive element binding protein （CREB） and apoptosis of podocytes. Results Podocytes treated with DHPG generated cAMP and activated expression of CREB. DHPG inhibited PAN-induced podocyte loss, cleaved caspase-3 apregulation, decrease of mitochondrial membrane potential and advanced apoptosis, which could be suppressed by selective antagonist of mGluR1/5 （AIDA）, adenylate cyclase inhibitor （SQ22536） and RNA interference- mediated knockdown of mGluR1 or mGluRS. Conclusion DHPG can protect against PAN-induced apoptosis of podocytes, which may be associated with mGluR/cAMP signalling pathway.