Telmisartan protects central neurons against nutrient deprivation-induced apoptosis in vitro through activation of PPARγ and the Akt/GSK-3β pathway

Abstract

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.

Figure 1

Telmisartan prevents nutrient deprivation-induced neuronal death in CGCs. (A, B) CGCs were pretreated with 10 μmol/L telmisartan (Telm), 10 μmol/L candesartan (Cand), or 10 μmol/L losartan (Los) for 24 h, followed by B27 deprivation for an additional 24 h. LDH release (A) in the culture supernatant was detected using the LDH Activity Assay kit, and cell viability was measured using the WST-1 assay (B) as described in Materials and Methods. (C) CGCs were pretreated with different concentrations of telmisartan (Telm) for 24 h, followed by incubation with or without B27. LDH release in the culture supernatant was measured using the LDH Activity Assay kit, as described in Materials and Methods. (D) Morphology of the neurons. The cells were pretreated with 1 μmol/L telmisartan (Telm) for 24 h, followed by the B27 deprivation for an additional 24 h. Photomicrographs were taken using an inverted light microscope. Scale bar, 50 μm. The results represent the mean±SEM of at least three independent experiments. ^bP<0.05, ^cP<0.01.

Figure 2

Telmisartan protects CGCs from nutrient deprivation-induced apoptosis. CGCs were pretreated with 1 μmol/L telmisartan (Telm) for 24 h, followed by B27 deprivation for an additional 24 h. Apoptosis was determined by the cell morphology using the In Situ Cell Death Detection Kit (A) and DNA laddering using the DNA fragmentation assay (B). The figure represents a typical experiment that was repeated three times. (C) CGCs were pretreated with or without 1 μmol/L Telm for 24 h, followed by B27 deprivation for the indicated time points. The levels of the apoptotic protein caspase-3 and the anti-apoptotic protein Bcl-2 were determined using Western blot analysis. All results represent the mean±SEM of three independent experiments. ^cP<0.01.

Figure 3

Telmisartan inhibits the nutrient deprivation-induced alterations in Akt and GSK-3β phosphorylations. CGCs were pretreated with 1 μmol/L telmisartan (Telm) for 24 h, followed by nutrient deprivation (-B27) for the indicated times. The levels of phosphorylated Akt (Ser473) (A, B) and GSK-3β (Ser9) (A, C) were determined using Western blot analysis. The results represent the mean±SEM of the groups from three independent experiments. ^cP<0.01.

Figure 4

The AT₁ receptor is involved in telmisartan neuroprotection. (A) CGCs were pretreated with 1 μmol/L telmisartan (Telm) for 24 h, followed by B27 deprivation, and AT_1a gene expression was determined 24 h later. The data are presented as the mean±SEM of three independent experiments. ^cP<0.01. (B) Nutrient deprivation-induced neuronal death is reduced in CGCs isolated from AT_1a receptor knock-out mice (KO) when compared to the wild-type mice (WT). Primary CGCs from wild-type or AT_1a knock-out mice were pretreated with 1 μmol/L Telm or 10 μmol/L losartan (Los) for 24 h, followed by B27 deprivation for an additional 24 h. The maximum nutrient deprivation-induced LDH release was significantly lower in the CGCs obtained from the AT_1a knock-out mice compared to that from the wild-type mice. (C) Primary CGCs from the AT_1a knock-out mice were pretreated with 20 μmol/L GW9662 for 2 h, followed by 24 h exposure to 1 μmol/L Telm or 10 μmol/L Los and B27 deprivation for an additional 24 h to determine the LDH release. The results represent the mean±SEM of the groups from three independent experiments. ^cP<0.01.

Figure 5

PPARγ activation is partly involved in the neuroprotective effects of telmisartan in CGCs. (A) CGCs were pretreated with 1 μmol/L telmisartan (Telm) or 10 μmol/L PPARγ agonist pioglitazone (PGZ) for 24 h, followed by B27 deprivation for an additional 24 h. (B) CGCs were pretreated with 1 μmol/L Telm for 24 h, followed by B27 deprivation for an additional 24 h. The PPARγ antagonist GW9662 (GW, 20 μmol/L) was added 2 h prior to Telm treatment. (C, D) CGCs were pretreated with 20 μmol/L GW9662 for 2 h, followed by 24 h exposure to 1 μmol/L Telm and B27 deprivation for an additional 24 h to determine the caspase-3 activation, as indicated by caspase-3 cleavage, and Bcl-2 expression using Western blot analysis (C). Alternatively, the cells were starved for 4 h to determine the phosphorylation levels of Akt and GSK-3β using Western blot analysis (D). One representative picture is presented here. The data are presented as the mean±SEM of three independent experiments. ^cP<0.01.

Figure 6

Proposed mechanisms of telmisartan neuroprotection. Nutrient depletion induces neuronal injury by stimulating apoptosis. The involved mechanisms include increasing the expression of the AT₁ receptor and inhibiting the anti-apoptotic Akt/GSK-3β pathway. Telmisartan reduces nutrient depletion-induced neuronal apoptosis in CGCs. Telmisartan neuroprotection is due to both AT₁ receptor blockade and PPARγ activation, which decreases apoptosis by preventing the nutrient deprivation-induced alterations in the Akt/GSK-3β pathway.