rehalose and carnosic acid induced LC3I, LC3II ratio, P62 down–regulation and cleaved caspase 3 expression in neural stem cells

Using neural stem cells (NSCs) in cell therapy and regenerative medicine is a growing knowledge. In this study, the protective role of carnosic acid and trehalose against H2O2-induced oxidative stress in autophagy induction and apoptosis inhibition in NSCs was investigated.The bone marrow stromal cells (BMSCs) were isolated from the femur of the rat and differentiated into NSCs using basic fibroblast and epidermal growth factors (bFGF and EGF), and B27 serum free media. To evaluate the autophagy, the P62 protein was assessed by immunocytochemistry and LC3II / LC3I ratio by Western blotting. Further, we used 3-Methyladenine (3-MA), a widely used autophagy inhibitor to study whether combined treatment of 3-MA with carnosic acid and trehalose modulates autophagy in NSCs. For studying apoptosis, the cleaved caspase-3 protein was evaluated. Carnosic acid and trehalose increased the survival of the NSCs.The H2O2 decreased the autophagy and induced apoptosis with increasing time during 24 hours, however, a pre-treatment with 2 μM carnosic acid and trehalose 3 % induced the autophagy proteins (while increasing the LC3II / LC3I ratio and decreasing the P62) and decreased the apoptosis (while decreasing the expression of the cleaved caspase-3). The results showed that the carnosic acid and trehalose increased the survival of NSCs against the oxidative stress caused by H2O2, decreased apoptosis, and induced autophagy.Due to the carnosic acid and trehalose unique properties and its low toxicity, it can be used as an agent in cellular transplantation for reducing oxidative stress and inducing autophagy (Fig. 4, Ref. 37).