桑葚花色苷对PC12细胞氧化损伤的保护作用

Protective effects of mulberry anthocyanin on oxidative stress in PC12 cells

通过建立大鼠肾上腺嗜铬细胞瘤细胞(PC12细胞)氧化应激损伤模型,探讨桑葚花青素中2个矢车菊素类化合物即矢车菊素-3-O-葡萄糖苷(cyanidin-3-O-glucoside,C3G)和矢车菊素-3-O-芸香糖苷(cyanidin-3-O-rutinoside,C3R)对过氧化氢(H_(2)O_(2))诱导的细胞氧化损伤的保护作用及相关作用机制。采用噻唑蓝法检测细胞活力,DCFH-DA荧光探针检测活性氧(reactive oxygen species,ROS)的含量,试剂盒测定细胞中还原型谷胱甘肽(glutathione,GSH)、丙二醛(malondialdehyde,MDA)、超氧化物歧化酶(superoxide dismutase,SOD)以及过氧化氢酶(catalase,CAT)的水平。Western Blot检测抗氧化以及细胞凋亡蛋白的表达水平。结果表明,650μmol/L H_(2)O_(2)处理PC12细胞12 h后,细胞存活率显著降低(P<0.01),50、100μmol/L的C3G、C3R能显著升高细胞活力(P<0.05)及GSH、SOD和CAT等抗氧化酶的活性(P<0.05),降低细胞内ROS以及MDA的含量。此外50、100μmol/L的C3G、C3R处理后显著抑制PC12细胞内c-Jun氨基末端激酶和细胞外调节蛋白激酶蛋白磷酸化水平,抑制半胱氨酸天冬氨酸蛋白酶-3以及Bcl-2相关X蛋白等凋亡蛋白的表达(P<0.05),促进核因子E-2-相关因子和血红素加氧酶1抗氧化蛋白的表达水平。综上,C3G、C3R对H_(2)O_(2)所致的PC12细胞氧化损伤具有保护作用,可能与抑制细胞凋亡通路相关蛋白的表达,提高胞内抗氧化酶活性,清除胞内过量ROS,降低细胞凋亡有关。

The protective function and related mechanism against H_(2)O_(2)-induced cell injury of cyanidin compounds cyanidin-3-O-glucoside(C3G)and cyanidin-3-O-rutinoside(C3R)was investigated by establishing the model of an oxidative stress injury in PC12 cells.MTT assay and DCFH-DA fluorescent probe were employed to detect cell viability and reactive oxygen species(ROS)content,respectively.The test kits were applied to detect reduced glutathione(GSH),malondialdehyde(MDA)content,superoxide dismutase(SOD),and catalase(CAT)activity.Antioxidant and apoptotic protein expression level was detected by Western blot.Results showed that the cell survival rate was significantly decreased treated with 650μmol/L H_(2)O_(2)for 12 hours(P<0.01),50μmol/L and 100μmol/L C3G and C3R could improve the survival rate of PC12 cells,inhibit the production of ROS,decrease the content of MDA,and enhance the activities of GSH,SOD,and CAT in PC12 cells compared with H_(2)O_(2)treatment group(P<0.05).Western blot results showed that 50μmol/L and 100μmol/L C3G and C3R could significantly inhibit the phosphorylation levels of c-Jun N-terminal kinase(JNK)and extracellular regulated protein kinases(ERK1/2)proteins as well as the expression of Caspase-3 and Bcl-2-associated X protein(Bax),and promote the protein levels of nuclear factor erythroid-2-related factor 2(Nrf2)and heme oxygenase-1(HO-1)(P<0.05).In summary,C3G and C3R have protective effects on H_(2)O_(2)-induced oxidative damage in PC12 cells which may be related to inhibiting the expression of apoptosis-related proteins,improving the activity of intracellular antioxidant enzymes,clearing excess ROS in cells,and reducing the rate of apoptosis.