过氧化氢诱导慢性氧化应激参与小胶质细胞衰老的作用及机制

Effect and mechanism of chronic oxidative stress induced by hydrogen peroxide on microglial celluar senescence

目的探索过氧化氢(hydrogen peroxide,H_(2)O_(2))诱导慢性氧化应激在小胶质细胞衰老中的作用。方法选择购自ATCC的10代以内BV2小胶质细胞,采用0、50、100、200μmol/L不同浓度的H_(2)O_(2)处理BV2小胶质细胞。根据H_(2)O_(2)使用浓度将其分为对照组、H_(2)O_(2)-50μmol/L组、H_(2)O_(2)-100μmol/L组、H_(2)O_(2)-200μmol/L组。应用CCK8细胞增殖实验测定细胞增殖,通过衰老相关β半乳糖苷酶(senescence associatedβgalactosidase,SA-β-gal)染色实验和实时荧光定量聚合酶链反应检测衰老相关周期蛋白分子p16、p21、p53及衰老相关分泌表型基因[白细胞介素-1β(interleukin-1 beta,IL-1β)、转化生长因子β(transforming growth factor-β,TGF-β)、基质金属蛋白酶9(matrix metalloprotein 9,MMP9)]的表达变化来测定细胞衰老。结果在诱导过程中,H_(2)O_(2)-200μmol/L对BV2小胶质细胞损伤较大,因此未进行后续检测。最终收集对照组、H_(2)O_(2)-50μmol/L组和H_(2)O_(2)-100μmol/L组细胞。对照组、H_(2)O_(2)-50μmol/L组、H_(2)O_(2)-100μmol/L组的细胞存活率(F=46.176,P<0.001)、SA-β-gal染色阳性率(F=553.1,P<0.001)比较,差异均有统计学意义。H_(2)O_(2)-50μmol/L组细胞存活率无明显改变(P>0.05),H_(2)O_(2)-100μmol/L组细胞存活率明显下降(P<0.001)。H_(2)O_(2)-50μmol/L组、H_(2)O_(2)-100μmol/L组细胞SA-β-gal染色阳性率均升高(P<0.001),且H_(2)O_(2)-100μmol/L组SA-β-gal染色阳性率高于H_(2)O_(2)-50μmol/L组(P<0.001)。在50、100μmol/L H_(2)O_(2)诱导下,p16、p21、p53的mRNA水平均升高(P<0.05),IL-1B、TGF-β、MMP9 mRNA均上调(P<0.05),且H_(2)O_(2)-100μmol/L组更明显(P<0.05)。结论使用100μmol/L H_(2)O_(2)诱导8 d可成功建立BV2小胶质细胞衰老模型,其机制可能与促进p16、p21、p53、IL-1β、TGF-β、MMP9分泌有关。

Objective To explore the role of hydrogen peroxide(H_2O_2)in inducing chronic oxidative stress in microglia aging.Methods BV2 microglia purchased from ATCC in less than 10 generations were treated with 0,50,100,200μmol/L H_2O_2 at different concentrations.According to the concentration of H_2O_2 used,the BV2 microglia were divided into a control group and H_2O_2-50μmol/L Group,H_2O_2-100μmol/L Group,H_2O_2-200μmol/L Group.Cell proliferation was measured by CCK8 cell proliferation assay.Age-relatedβ-galactosidase(SA-β-gal)staining assay,and expression of age-related cyclin molecules p16,p21,p53 and senescence sssociated secretory phenotype interleukin 1 beta(IL-1β),transforming growth factor-β(TGF-β)and matrix metalloprotein 9(MMP9)detected by quantitative real-time polymerase chain reaction were used to measure celluar senescence.Results During the induction process,H_2O_2-200μmol/L caused significant damage to BV2 microglia,therefore no subsequent testing was conducted.Finally,the control group,H_2O_2-50μmol/L group and H_2O_2-100μmol/L group cells were collected.The differences in cell survival rate(F=46.176,P0.001)and positive rate of SA-β-gal staining(F=553.1,P0.001)among the three groups were statistically significant.The cell survival rate of H_2O_2-50μmol/L group had no significant change(P0.05),while the cell survival rate of H_2O_2-100μmol/L group decreased significantly(P0.001).The positive rate of SA-β-gal staining in H_2O_2-50μmol/L group and H_2O_2-100μmol/L group was increased(P0.001),and the positive rate of SA-β-gal staining in H_2O_2-100μmol/L group was higher than that in H_2O_2-50μmol/L group(P0.001).The mRNA levels of senescence related cyclin molecules p16,p21 and p53 were up-regulated under the induction of 50,100μmol/L H_2O_2(P0.05),and the expressions of IL-1β,TGF-βand MMP9 of senescence associated secretory phenotype were increased(P0.05).The increase of H_2O_2-50μmol/L group was more obvious(P0.05).Conclusion The aging model of BV2 microglia can be successfully established by inducing 8 d with 100μmol/L H_2O_2,and the mechanism may be related to promoting the secretion of p16,p21,p53,IL-1β,TGF-βand MMP9.