人参皂苷Rg1基于SIRT6/NF-κB信号通路对辐射致造血干/祖细胞衰老的保护作用

Effect of ginsenoside Rg_1 on delaying radiation-induced senescence of hematopoietic stem cell and progenitor cell based on SIRT6/NF-κB signal pathway

目的探讨去乙酰化酶SIRT6/核转录因子-κB(NF-κB)信号通路在人参皂苷Rg_1抗辐射致造血干/祖细胞(Sca-1+HSC/HPC)衰老中的作用。方法 C57BL/6小鼠随机分为对照组、模型组和人参皂苷Rg_1组。给予模型组和人参皂苷Rg_1组小鼠全身一次性6.5 Gy ^(60)Coγ线照射,人参皂苷Rg_1组于照射前ip人参皂苷Rg_1 20 mg/kg,每天1次,连续给药7 d,照射后继续ip等剂量人参皂苷Rg_1 7 d。给药结束后第2天,外周血血象指标检测确定人参皂苷Rg_1促进造血恢复情况;免疫磁性分选法分离纯化各组Sca-1+HSC/HPC,造血祖细胞混合性集落(CFU-Mix)培养、细胞周期分析和衰老相关β-半乳糖苷酶(SA-β-Gal)染色分析人参皂苷Rg_1抗辐射损伤致Sca-1+HSC/HPC衰老的生物学作用;荧光定量PCR及Western blotting法检测衰老调控分子SIRT6、NF-κB mRNA及蛋白的表达。结果辐射后,与对照组相比,模型组小鼠外周血象恢复缓慢,Sca-1+HSC/HPC出现细胞衰老特征,G0/G1期细胞比例及SA-β-Gal染色阳性率增高,CFU-Mix数量下降,SIRT6 mRNA及蛋白表达下调,NF-κB mRNA及蛋白表达上调。与模型组比较,人参皂苷Rg_1组小鼠白细胞(WBC)、红细胞(RBC)、血小板(PLT)数量增高,Sca-1+HSC/HPC G_0/G_1期细胞比例及SA-β-Gal染色阳性率下降,CFU-Mix数量升高,SIRT6 mRNA及蛋白表达上调,NF-κB mRNA及蛋白表达下调。结论人参皂苷Rg_1可通过调控SIRT6/NF-κB信号通路发挥抗辐射致Sca-1+HSC/HPC衰老的作用。

Objective To investigate the effect of SIRT6/NF-κB signal axis on delaying radiation-induced senescence of hematopoietic stem cell and progenitor cell with ginsenoside Rg1.Methods C57 BL/6 mice were randomly divided into control group,model group and ginsenoside Rg1 group.The mice in model and ginsenoside Rg1 groups were exposed to total body irradiation with 6.5 Gy 60 Coγ,the mice in ginsenoside Rg1 group were intraperitoneal injected with ginsenoside Rg1（20 mg/kg） for 7 d before and after radiation.On day after taking medicine,the indicators in peripheral blood were observed to definite the information of hematopoietic reconstruction and the effect of ginsenoside Rg1 on it.Sca-1＋ HSCs/HPCs were isolated and purified by magnetic activated cell sorting（MACS）.The effect of ginsenoside Rg1 on delaying radiation-induced senescence of hematopoietic stem cell and progenitor cell was evaluated by mixed hematopoietic progenitor cell culture,cell cycle assay and senescence-associated β-galactosidase（SA-β-gal） staining.The expression of senescence associated SIRT6 and NF-κB mRNA and protein was examined by realtime fluorescence quantitative PCR（q RT-PCR） and Western blotting.Results After radiation,the indicator in peripheral blood of model group was decreased compared with control group.Sca-1＋ HSC/HPC appeared aging character：The number of cells entered G0/G1 phase,the percentage of SA-β-gal positive cells was increased,the expressing of SIRT6 mRNA and protein were down regulated and NF-κB mRNA and protein were up regulated,and the number of CFU-Mix was decreased.Compared with the model group,the number of white blood cells（RBC）,red blood cells（RBC）,and platelet（PLT） in mice were increased,the number of cells entered G0/G1 phase and the percentage of SA-β-gal positive cells were decreased,and the number of CFU-Mix was increased in Sca-1＋ HSC/HPC from the ginsenoside Rg1 group.Moreover,the expression of SIRT6 mRNA and protein were up regulated,and NF-κB mRNA and protein were down regulated in the ginsenoside Rg1 group.Conclusion SIRT6/NF-κB signal axis may play a key role in the antiaging effect of ginsenoside Rg1 to Sca-1＋ HSC/HPC senescence induced by radiation.