Calycosin activates the protein kinase c/NF-E2-related factor 2 pathway to attenuate the oxidative stress and apopotosis induced by streptozotocin in RIN-m5F cell

Abstract

Objective To study the effects and mechanism of calycosin on oxidative stress and β-cell apoptosis induced by streptozotocin (STZ). Methods RIN-m5F cells were divided into 5 groups: control group, STZ group, STZⅠgroup, STZⅡgroup and STZⅢgroup. The control group did not receive any treatment, while streptozotocin was added to the final concentration of 10 mmol/L in STZ group, STZⅠgroup, STZⅡgroup and STZⅢgroup. After the incubation with STZ for 6 h, calycosin was added to a final concentration of 10, 50 and 100 μmol/L in STZⅠgroup, STZⅡgroup and STZⅢgroup respectively. The cell viability and apoptosis was detected by CCK-8, LDH, caspase 3 and Tunel assay. The intracellular oxidative stress was measured using mitochondrial membrane potential, DCFH-DA, SOD activity and malondialdehyde levels assay. RIN-m5F cells were divided into control group, calycosinⅠgroup, calycosinⅡgroup and calycosinⅢgroup, which were treated with different concentrations (0, 10, 50 and 100 μmol/L, respectively) of calycosin. The expression of NF-E2-related factor 2(Nrf2) in RIN-m5F cells was detected by Western blot. The translocation of Nrf2 was detected by immunofluorescence. In RIN-m5F cells were divided intoⅢgroup and Ⅳgroup, Ⅳ group was pre-treated with protein kinase C(PKC) inhibitor. The effects of calycosin on Nrf2 translocation, oxidative stress and apoptosis were also observed. Results STZ could induce the accumulation of reactive oxygen species and apoptosis in RIN-m5F cells. Calycosin did not affect normal RIN-m5F cells, whereas it reduced the oxidative stress and apoptosis induced by STZ in a dose-dependent manner. The expression of Nrf2 in RIN-m5F cells was not affected by calycosin, whereas it promoted the translocation of Nrf2 into nucleus. The ability of calycosin promoting Nrf2 translocation was decreased after PKC inhibitor treatment, and PKC inhibitor could also significantly attenuate the anti-oxidant and anti-apoptotic ability of calycosin. Conclusions This study shows that calycosin may play an anti-oxidative and anti-apoptotic role by activating PKC to promote Nrf2 translocation, which is expected to be used as a new clinical drug for the prevention and treatment of diabetes mellitus. Key words: Calycosin; β-cell; NF-E2-related factor 2; Oxidative stress; Apoptosis