Cytotoxicity and protective effects of APMCG in vitro
Based on the strong scavenging activity of the four fractions, APMCG, APMCG-1, APMCG-2, APMCG-3, and APMCG-4 were selected for furtherin vitro assay. Initially, different concentrations of PA were screened, and the PA concentration of 0.2 mM was selected because its cell viability was 60% (Fig. 2A). The treatment concentration of samples was set from 6.25 μg/mL to 100 μg/mL. All concentrations of the samples showed low toxic except concentration of 100 μg/mL (Fig. 2B). Fractions ranging from 6.25 μg/mL to 50 μg/mL significantly increased cell viabilities in PA-induced H9c2 cells, implicating a strong cardioprotective effect on cardiomyocytes (Fig. 2C). Especially, the concentrations of APMCG-1 markedly down-regulated intracellular ROS production and LDH content in PA-induced H9c2 cells dose-dependently, revealing strong protection against oxidative caused injury (Fig. 2D, 2E). Oil Red O staining revealed that PA resulted in shrinking, rounding, and shedding of H9c2 cells, as observed via an inverted microscope. After APMCG-1 treatment, the intracellular lipid accumulation of PA-induced H9c2 cells decreased (Fig. 2F). Compared with that in the untreated group, no significant difference was observed in the AKT level in each group of H9c2 cells, whereas p-AKT significantly increased in PA-induced H9c2 cells after APMCG-1 treatment (Fig. 2G). Thus, APMCG-1exhibits a cardioprotective effect by regulation of AKT/p-AKT signaling protein. We employed PI3K/AKT inhibitors to determine whether the inhibitory impact of APMCG-1 on PA-induced apoptosis in H9c2 cells was due to the PI3K/AKT signaling pathway (Wei et al., 2012). The cells were pretreated with 10 μM LY294002 for 1 h, followed by 1 h of treatment with 50 μg/mL APMCG-1 and 24 h of treatment with PA. The outcomes demonstrate that the reduction in p-AKT level caused by 50 μg/mL APMCG-1. According to the aforementioned findings, APMCG-1 can partially prevent PA from inducing the death of H9c2 cells through activating the PI3K/AKT signaling pathway (Fig. 2H).
Similarly, a 0.2 mM PA was selected for the simulating C2C12 cell damage (Fig. 2I). As shown in Fig. 2J and Fig. 2K, the indicated concentrations of active fraction showed low cytotoxicity, and APMCG-1 significantly increased cell viability in damaged PA-induced C2C12 cells. Meanwhile, APMCG-1 reduced the CK content compared with that in the PA-treated group in a dose-dependent manner, implicating the alleviation of insulin resistance and microcirculation disorders in the skeletal muscle (Fig. 2L). In addition, APMCG-1 significantly increased cellular glucose consumption at 8, 16, 24, and 48 h in time- and dose-dependent manners compared with the model group, suggesting that it attenuated high glucose-induced damage (Fig. 2M). Fig. 2N shows that different concentrations of APMCG-1 decreased intracellular lipid accumulation in PA-induced C2C12 cells. Western blotting showed that the expressions of IRS1, PI3K, AKT and GLUT4 in PA-induced C2C12 cells were up-regulated by APMCG-1 treatment (Fig. 2O). These results revealed that APMCG-1 activates the PI3K/AKT signaling pathway to promote the survival and function of cardiomyocytes and skeletal myoblasts.