草苁蓉多糖对过氧化氢致HL-7702细胞损伤的保护作用

Protective effect of Boschniakia rossica polysaccharides on H_(2)O_(2)-induced injury of HL-7702 cells

采用草苁蓉多糖(Boschniakia rossica polysaccharides,BRP)对HL-7702细胞进行预保护,再用H 2O 2处理HL-7702细胞构建肝细胞氧化损伤模型。CCK-8法测定HL-7702细胞存活率,微板法测定HL-7702细胞培养液中乳酸脱氢酶(lactate dehydrogenase,LDH)活性,硫代巴比妥酸法检测HL-7702细胞丙二醛(malondialdehyde,MDA)的生成,Hoechst33342染色法及原位末端标记法观察HL-7702细胞的凋亡情况,蛋白印迹技术检测HL-7702细胞Caspase 3活性片段(C-casp3)、聚腺苷二磷酸核糖聚合酶活性片段[cleaved poly(ADP-ribose)polymerase,C-PARP]、细胞色素c(cytochrome c,Cyto c)、p53、B细胞淋巴瘤/白血病-2(B cell lymphoma/lewkmia-2,Bcl-2)、Bcl-2相关X蛋白(Bcl-2-associated X protein,Bax)、Bcl-2同源拮抗剂/杀伤剂(Bcl-2 homologous antagonist/killer,Bak)蛋白表达以及丝裂原活化蛋白激酶、核转录因子-κB(nuclear factor-κB,NF-κB)激活情况。结果表明,300μmol/LH 2O 2处理HL-7702细胞6 h可使细胞存活率降低58%(P<0.05),表明H 2O 2能够诱导HL-7702细胞损伤。而50和100 mg/L BRP能够减缓H 2O 2导致的HL-7702细胞损伤。与模型组比较,BRP高剂量组HL-7702细胞存活率增高30.8%(P<0.05),LDH释放率降低32.8%(P<0.05),细胞MDA生成降低81.0%(P<0.05)。此外,与模型组比较,BRP组HL-7702细胞凋亡明显减少,胞浆Cyto c水平下降(P<0.05),细胞p53、Bak、Bax蛋白表达下调(P<0.05),Bcl-2蛋白表达上调(P<0.05),C-casp3、C-PARP水平降低(P<0.05),磷酸化细胞外信号调节蛋白激酶(phosphorylated extracellular signal-regulated kinase,p-ERK)、磷酸化c-Jun氨基末端激酶(phosphorylated c-Jun N-terminal kinase,p-JNK)以及核NF-κB水平下降(P<0.05)。提示,BRP能够有效保护H 2O 2诱导的HL-7702细胞损伤,并抑制HL-7702细胞凋亡,此作用可能与BRP对ERK、JNK、NF-κB通路的调控有关。

The protective effect of Boschniakia rossica polysaccharides(BRP)on the injury of HL-7702 cells induced by hydrogen peroxide was investigated.The cellular injury model was established using hydrogen peroxide in HL-7702 cells.HL-7702 cells were pretreated with BRP,and then cell viability of HL-7702 was investigated with the cell counting kit-8(CCK-8)assay,and the lactate dehydrogenase(LDH)release was tested with the microplate method.Cellular malondialdehyde(MDA)was tested with the thiobarbituric acid method,cell apoptosis was detected by the Hoechst33342 staining and TdT-mediated dUTP nick end labeling(TUNEL)assay.The protein expression of cytochrome c(Cyto c),p53,B cell lymphoma/lewkmia-2(Bcl-2),Bcl-2-associated X protein(Bax),Bcl-2 homologous antagonist/killer(Bak),cleaved caspase 3(C-casp3),cleaved poly(ADP-ribose)polymerase(C-PARP)and the activation of mitogen-activated protein kinase(MAPK)and nuclear factor-κB(NF-κB)were determined with the western blotting method.The results showed that hydrogen peroxide at a concentration of 300μmol/L decreased the viability of HL-7702 cells by 58%(P<0.05).However,the cell viability of HL-7702 was increased by 30.8%(P<0.05),and the release of LDH was reduced by 32.8%(P<0.05).Besides,the cellular MDA was decreased by 81.0%(P<0.05)in the BRP group as compared with the model group.In addition,BRP inhibited cell apoptosis,reduced the cytoplasmic Cyto c(P<0.05),increased Bcl-2 protein expression(P<0.05),and reduced the expression of p53,Bak,Bax,C-casp3,C-PARP,phosphorylated extracellular signal-regulated kinase(p-ERK),phosphorylated c-Jun N-terminal kinase(p-JNK)as well as the nuclear translocation of NF-κB(P<0.05).It is suggested that BRP could protect HL-7702 cells from hydrogen peroxide-induced oxidative damage,and inhibit the apoptosis of HL-7702 cells through regulating ERK,JNK and NF-κB pathways.