Pang Tao, Sun Li-Xin, Wang Tao, Jiang Zhen-Zhou, Liao Hong, Zhang Lu-Yong
1] New Drug Screening Center, China Pharmaceutical University, Nanjing 210009, China [2] State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing 210009, China [3] Key Laboratory of Drug Quality Control and Pharmacovigilance (China Pharmaceutical University), Ministry of Education, Nanjing 210009, China.
New Drug Screening Center, China Pharmaceutical University, Nanjing 210009, China.
Acta Pharmacol Sin. 2014 Jun;35(6):727-37. doi: 10.1038/aps.2013.199. Epub 2014 May 5.
To determine whether angiotensin II receptor blockers (ARBs) could protect central neurons against nutrient deprivation-induced apoptosis in vitro and to elucidate the underlying mechanisms.
Primary rat cerebellar granule cells (CGCs) underwent B27 (a serum substitute) deprivation for 24 h to induce neurotoxicity, and cell viability was analyzed using LDH assay and WST-1 assay. DNA laddering assay and TUNEL assay were used to detect cell apoptosis. The expression of caspase-3 and Bcl-2, and the phosphorylation of Akt and GSK-3β were detected using Western blot analysis. AT1a mRNA expression was determined using RT-PCR analysis.
B27 deprivation significantly increased the apoptosis of CGCs, as demonstrated by LDH release, DNA laddering, caspase-3 activation and positive TUNEL staining. Pretreatment with 10 μmol/L ARBs (telmisartan, candesartan or losartan) partially blocked B27 deprivation-induced apoptosis of CGCs with telmisartan being the most effective one. B27 deprivation markedly increased the expression of AT1a receptor in CGCs, inhibited Akt and GSK-3β activation, decreased Bcl-2 level, and activated caspase-3, which were reversed by pretreatment with 1 μmol/L telmisartan. In addition, pretreatment with 10 μmol/L PPARγ agonist pioglitazone was more effective in protecting CGCs against B27 deprivation-induced apoptosis, whereas pretreatment with 20 μmol/L PPARγ antagonist GW9662 abolished all the effects of telmisartan in CGCs deprived of B27.
ARBs, in particular telmisartan, can protect the nutrient deprivation-induced apoptosis of CGCs in vitro through activation of PPARγ and the Akt/GSK-3β pathway.
确定血管紧张素II受体阻滞剂（ARBs）能否在体外保护中枢神经元免受营养剥夺诱导的凋亡，并阐明其潜在机制。
原代大鼠小脑颗粒细胞（CGCs）在无B27（一种血清替代物）的条件下培养24小时以诱导神经毒性，使用乳酸脱氢酶（LDH）测定法和WST-1测定法分析细胞活力。采用DNA梯状条带分析和TUNEL测定法检测细胞凋亡。使用蛋白质免疫印迹分析检测半胱天冬酶-3（caspase-3）和Bcl-2的表达，以及Akt和糖原合成酶激酶-3β（GSK-3β）的磷酸化。使用逆转录-聚合酶链反应（RT-PCR）分析测定AT1a mRNA表达。
如LDH释放、DNA梯状条带、caspase-3激活和TUNEL阳性染色所示，无B27培养显著增加了CGCs的凋亡。用10μmol/L的ARBs（替米沙坦、坎地沙坦或氯沙坦）预处理可部分阻断无B27培养诱导的CGCs凋亡，其中替米沙坦最为有效。无B27培养显著增加了CGCs中AT1a受体的表达，抑制了Akt和GSK-3β的激活，降低了Bcl-2水平，并激活了caspase-3，而用1μmol/L替米沙坦预处理可逆转这些变化。此外，用10μmol/L过氧化物酶体增殖物激活受体γ（PPARγ）激动剂吡格列酮预处理在保护CGCs免受无B27培养诱导的凋亡方面更有效，而用20μmol/L PPARγ拮抗剂GW9662预处理则消除了替米沙坦对无B27培养的CGCs的所有作用。
ARBs，尤其是替米沙坦，可通过激活PPARγ和Akt/GSK-3β途径保护体外营养剥夺诱导的CGCs凋亡。
