紫薯花青素通过调节p53-p21^(Waf1/Cip1)信号通路对辐射致造血干/祖细胞衰老的保护作用

Protective Effect of Solanum tuberosum Anthocyanin against Radiation-Induced Hematopoietic Stem/Progenitor Cell Senescence via the p53-p21^(Waf1/Cip1) Pathway

目的:探讨紫薯花青素(Solanum tuberosum anthocyanin,STA)对辐射致造血干细胞(hematopoietic stem cell,HSC)/造血祖细胞(hematopoietic progenitor cell,HPC)衰老起保护作用的分子机制。方法:将C57BL/6小鼠随机分为对照组、模型组、STA治疗组和STA预防组,利用X射线照射构建衰老细胞模型。给药后用血细胞分析仪检测各组小鼠血液指标;免疫磁性分选法分离纯化各组小鼠Sca-1^(+)HSC/HPC,并用衰老相关β-半乳糖苷酶(senescence-associated β-galactosidase,SA-β-Gal)染色试剂盒对其进行染色实验并计算各组阳性率;利用HSC/HPC混合集落(colony forming unit-mixture,CFU-Mix)数评价各组细胞形成HSC/HPC集落能力与分化潜能;流式细胞术分析细胞周期;实时定量聚合酶链反应(quantitative real-time polymerase chain reaction,q PCR)检测p53和p21^(Waf1/Cip1)mRNA的表达;Western blot检测p53和p21^(Waf1/Cip1)蛋白的表达。结果:与对照组相比,模型组小鼠外周血指标红细胞、白细胞、血小板数均极显著降低(P<0.01),衰老细胞阳性率极显著增加(P<0.01),Sca-1^(+)HSC/HPC形成CFU-Mix的数量极显著减少(P<0.01),G0/G1期比例极显著升高(P<0.01),G2/M和S期比例极显著降低(P<0.01),p53和p21^(Waf1/Cip1)mRNA和蛋白的相对表达量极显著增加(P<0.01),STA治疗和STA预防均可分别显著和极显著恢复模型组小鼠以上指标,其中STA预防效果更好。结论:STA可以通过调节p53-p21^(Waf1/Cip1)信号通路保护受到辐射的HSC/HPC。

Objective:To investigate the molecular mechanism underlying the protective effect of Solanum tuberosum anthocyanin(STA)on radiation-induced hematopoietic stem/progenitor cell senescence.Methods:C57BL/6 mice were randomly divided into three groups:control,model,STA treatment and STA prevention,and the cell senescence model was constructed by X-ray irradiation.The hemogram of mice in each group were examined using a hematology analyzer after drug administration.Stem cell antigen 1 positive hematopoietic stem/progenitor cells(Sca-1^(+)HSC/HPCs)were isolated and purified from each group by immunomagnetic cell sorting and were stained using a senescence-associatedβ-galactosidase(SA-β-Gal)kit to calculate the proportion of SA-β-Gal positive cells.The number of mixed lineage colony forming unit(CFU-Mix)was used to evaluate the colony-forming capacity and differentiation potential of hematopoietic stem/progenitor cells.Cell cycle was analyzed by flow cytometry.The mRNA and protein expression of p53 and p21^(Waf1/Cip1) were detected by quantitative real-time polymerase chain reaction(qPCR)and Western blot.Results:The numbers of red blood cells(RBCs),white blood cells(WBCs)and platelets in the peripheral blood were significantly lower in the model group than in the control group(P<0.01).In addition,compared with the control group,the proportion of SA-β-Gal positive cells significantly increased (P < 0.01), the number of CFU-Mix significantly fell (P < 0.01), the proportion of G0/G1 phase cells significantly increased, the proportions of G2/M and S phase cells significantly dropped (P < 0.01), and the relative mRNA and protein expression levels of p53 and p21^(Waf1/Cip1) significantly increased in the model group (P < 0.01). STA could significantly restore the above parameters of mice in the model group irrespective of whether it was administered before or after X-ray radiation, with more pronounced effect being observed in the former case. Conclusion: STA can protect hematopoietic stem/progenitor cells against radiation by regulating the p53-p21^(Waf1/Cip1) pathway.