The extract of Gnaphalium affine D. Don protects against H ₂ O ₂-induced apoptosis by targeting PI3K/AKT/GSK-3β signaling pathway in cardiomyocytes

Ethnopharmacological relevance: Gnaphalium affine D. Don is an important Traditional Chinese herbal Medicine (TCM) used to treat hyperuricemia, asthma, rheumatic arthritis, antitussive, expectorant and cardiovascular in folk medicine because of anti-inflammatory and anti-oxidant activity. The aim of this study was to investigate the potential beneficial effect of G. affine extract (GAE) on hydrogen peroxide (H₂O₂)-induced apoptosis and explore the possible underlying mechanism in cardiomyocyte.

Materials and methods: The ingredients of GAE were isolated and tentatively identified by HPLC-ESI-Q-Qribatrip-MS/MS. The cardioprotective and anti-oxidant effects of GAE were evaluated in the experimental model with H₂O₂ induced apoptosis in H9c2 cells. H9c2 cells were pretreated for 3 h with or without GAE or with GAE plus PX866 (PI3K inhibitor), then exposed to H₂O₂ for 6 h, H9c2 cells viability were detected by CCK8 kit, the content of intracellular reactive oxygen species (ROS) and malondialdehyde (MDA) and intracellular superoxide dismutase (SOD) activity were measured by the commercial biochemical kits, western blotting, immunohistochemical (IHC), immunofluorescence (IF) and reverse transcription-polymerase chain reaction (RT-PCR) assays were performed to evaluate the proteins and mRNA expression, propidium iodide (PI) staining was adopted to indicate H9c2 cells apoptosis.

Results: Firstly, seventeen polyphenols and flavonoids compounds with the characteristics of anti-inflammatory and anti-oxidant in GAE were tentatively identified by HPLC-ESI-Q-Qribatrip-MS/MS. In the experimental model, GAE not only significantly improved cells viability, but also showed anti-oxidant effects through improving SOD activity, up-regulating nuclear factor E2-related factor 2 (Nrf2), and decreasing intracellular concentration of ROS and MDA and the proteins expression of p47^phox, p67^phox and gp91^phox. On the other hand, GAE revealed anti-apoptotic effect through up-regulating the expression of B-cell lymphoma-2 (Bcl-2), down-regulating Bcl2-associated X (BAX) and cleaved-caspase 3. Furthermore, GAE significantly facilitated phosphorylation of AKT and glycogen synthase kinase-3 beta (GSK-3β) but not AMPK, while the effects were blocked by PX866 (PI3K inhibitor).

Conclusions: Our data suggested that GAE showed strong anti-oxidant effect to ameliorate oxidative stress and attenuate apoptosis induced by H₂O₂ in H9c2 cells by targeting PI3K/AKT/GSK-3β signaling pathway.

Keywords: AMPK; Gnaphalium affine D. Don; PI3K/AKT/GSK-3β signaling pathway; anti-apoptosis; anti-oxidant; oxidative stress.