Protective effects of Qinglongyi-Buguzhi herb pair against H 2 O 2 ‑induced oxidative injury in B16F10 mouse melanoma cells via PI3K/Akt/Nrf2 signaling pathway

Abstract

Abstract The herbal pair of Qinglongyi (Q, the exocarp of Juglans regia L.)-Buguzhi (B, the fruit of Psoralea corylifolia L.) (QB) is commonly used for treating vitiligo in traditional Chinese medicine (TCM). However, the relevant mechanism of QB in the treatment of vitiligo is still unclear. This study aimed to investigate the protective role and mechanism of QB on B16F10 mouse melanoma cells after H2O2-induced oxidative stress injury. Firstly, 17 experimental groups were designed as follows: normal control group, H2O2 group, Q, B, and QB (mass ratio 1:1, 1:2 and 2:1 ) low, middle and high concentration groups. MTT was used to detect cell survival rate and flow cytometry was used to determine the content of reactive oxygen species (ROS). Superoxide dismutase (SOD), catalase (CAT) and melanin levels were evaluated using corresponding commercial kits. Based on all the results, the drug group with the best effect was selected for follow-up mechanism study. Then, six experimental groups were designed as follows: normal control group, H2O2 group, LY294002 (PI3K inhibitor) group, QB་LY294002 group, positive control VE group. Flow cytometry was used to detect cell apoptosis and ROS content. The nuclear translocation of Nrf2 was analyzed using immunofluorescence. RT‑qPCR was used to determine the changes in the expression of Akt, Nrf2 and HO-1 genes. Western blot was used to detect the expression of Akt, phospho-Akt (p-Akt), total nuclear factor erythroid 2‑related factor 2 (Nrf2), nuclear Nrf2 and heme oxygenase-1 (HO-1) proteins. From our findings, Q, B and QB (1:1, 1:2, 2:1) groups all increased cell survival rate, decreased ROS level and cell apoptosis, upregulated SOD and CAT activity, and increased melanin content of B16F10 cells with H2O2 injury. It was worth noting that the QB (1:2) 0.08 mg/mL group had the most prominent effect among all drug groups, so we chosed this group to study the mechanism of action. From the results, when compared with H2O2, LY294002, QB་LY294002 groups, QB pretreatment of cells in QB group significantly decreased ROS level and apoptosis rate of B16F10 cells with H2O2 injury. Moreover, QB up-regulated Akt, Nrf2, HO-1 mRNA expression level and increased p-Akt, HO-1, nuclear Nrf2 proteins expression level, and promoted the nuclear translocation of Nrf2. Notably, LY294002 could largely block the effect of QB. This study demonstrated that QB reversed H2O2-induced oxidative stress injury of melanocytes may be related to the activation of PI3K/Akt/Nrf2 signaling pathway.