Objective To study the effects and mechanism of calycosin on oxidative stress andβ-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 andⅣgroup,Ⅳgroup 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.%目的 探讨毛蕊异黄酮对链脲佐菌素(STZ)诱导的氧化应激和β细胞凋亡的作用以及机制.方法 大鼠胰岛细胞系RIN-m5F细胞分为对照组、STZ组、STZⅠ组、STZⅡ组和STZⅢ组,对照组不作任何处理,STZ组、STZⅠ组、STZⅡ组和STZⅢ组加入STZ至终浓度10 mmol/L,后3组在STZ加入6 h后再分别加入毛蕊异黄酮至终浓度为10、50和100μmol/L.24 h后通过细胞计数试剂盒8(CCK-8)、乳酸脱氢酶(LDH)和半胱氨酸天冬氨酸蛋白酶3(caspase 3)、Tunel法检测细胞活性和凋亡,通过线粒体膜电位、双氯荧光素(DCFH-DA)以及超氧化物歧化酶(SOD)活性和丙二醛(MDA)检测评价细胞内氧化应激水平.将RIN-m5F细胞分为对照组、calycosinⅠ组、calycosinⅡ组和calycosinⅢ组,分别给予0、10、50和100μmol/L毛蕊异黄酮,通过western blot检测NF-E2相关因子2(Nrf2)表达的影响,通过免疫荧光检测毛蕊异黄酮对Nrf2转核的影响.将RIN-m5F细胞分为Ⅲ组和Ⅳ组,Ⅳ组细胞经钙磷酸蛋白C(calphostin C,100 nmol/L)处理后,两组加入STZ至终浓度10 mmol/L,再分别加入100μmol/L毛蕊异黄酮,观察Nrf2转核、氧化应激和细胞凋亡的影响.结果 STZ可诱导RIN-m5F细胞内活性氧类的蓄积和细胞凋亡,而毛蕊异黄酮可减轻STZ诱导的氧化应激和细胞凋亡,并呈剂量依赖性.毛蕊异黄酮对RIN-m5F细胞中Nrf2表达无影响,但可促进Nrf2向核内转移.给予蛋白激酶C抑制剂后毛蕊异黄酮促Nrf2转核的能力下降,并且蛋白激酶C抑制剂可明显减弱毛蕊异黄酮的抗氧化和抗凋亡能力.结论 毛蕊异黄酮可能通过激活蛋白激酶C促进Nrf2转核,从而发挥抗氧化和抗凋亡能力,有望作为新型临床药物用于糖尿病的防治.
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