Telmisartan protects central neurons against nutrient deprivation-induced apoptosis in vitro through activation of PPARγ and the Akt/GSK-3β pathway
Abstract
Tao PANG1, 2, 3, #, Li-xin SUN1, #, Tao WANG1, Zhen-zhou JIANG1, Hong LIAO1, 2, *, Lu-yong ZHANG1, 2, *
1New Drug Screening Center, China Pharmaceutical University, Nanjing 210009, China; 2State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing 210009, China; 3Key Laboratory of Drug Quality Control and Pharmacovigilance (China Pharmaceutical University), Ministry of Education, Nanjing 210009, China
Aim: 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.
Methods: 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.
Results: 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.
Conclusion: 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.
Keywords: cerebellar granule cell; nutrient deprivation; apoptosis; angiotensin II receptor blocker; telmisartan; AT1a receptor; Akt; GSK-3β; PPARγ; neurotoxicity
This study was supported by the Natural Science Foundation of Jiangsu Province (BK20130653, BK2010433), the Fundamental Research Funds for the Central Universities (JKZD2013006), the National Natural Science Foundation of China (81070967, 81271338), the Specialized Research Fund for the Doctoral Program of Higher Education of China (20130096110011) and the Initial Fund of China Pharmaceutical University to Dr Tao PANG.
# These authors contributed equally to this work.
* To whom correspondence should be addressed.
E-mail lyzhang@cpu.edu.cn (Lu-yong ZHANG); liaohong56@hotmail.com (Hong LIAO)
Received 2013-10-04 Accepted 2013-12-30
Keywords:
1New Drug Screening Center, China Pharmaceutical University, Nanjing 210009, China; 2State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing 210009, China; 3Key Laboratory of Drug Quality Control and Pharmacovigilance (China Pharmaceutical University), Ministry of Education, Nanjing 210009, China
Aim: 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.
Methods: 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.
Results: 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.
Conclusion: 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.
Keywords: cerebellar granule cell; nutrient deprivation; apoptosis; angiotensin II receptor blocker; telmisartan; AT1a receptor; Akt; GSK-3β; PPARγ; neurotoxicity
This study was supported by the Natural Science Foundation of Jiangsu Province (BK20130653, BK2010433), the Fundamental Research Funds for the Central Universities (JKZD2013006), the National Natural Science Foundation of China (81070967, 81271338), the Specialized Research Fund for the Doctoral Program of Higher Education of China (20130096110011) and the Initial Fund of China Pharmaceutical University to Dr Tao PANG.
# These authors contributed equally to this work.
* To whom correspondence should be addressed.
E-mail lyzhang@cpu.edu.cn (Lu-yong ZHANG); liaohong56@hotmail.com (Hong LIAO)
Received 2013-10-04 Accepted 2013-12-30