银杏总黄酮对IL-1β诱导的髓核细胞凋亡及Nrf2/ARE信号通路的影响

Effects of Total Flavonoids of Ginkgo Biloba on Apoptosis of Nucleus Pulposus Cells Induced by IL-1βand Nrf2/ARE Signaling Pathway

目的:探讨银杏总黄酮(TFG)对白细胞介素-1β(IL-1β)诱导的髓核细胞凋亡的影响,及对核因子E2相关因子2(Nrf2)/抗氧化反应元件(ARE)信号通路的调控作用。方法:将人椎间盘髓核细胞分为正常组、IL-1β诱导组、二甲基亚砜(DMSO)组、TFG不同浓度组(5、10、20、40、80、160mg/mL),除正常组外,各组均用浓度为10ng/mL的IL-1β诱导培养24h后,CCK-8法检测细胞增殖活性。将髓核细胞分为正常对照组、IL-1β诱导组、TFG低(20mg/mL)、高(80mg/mL)剂量组、Nrf2通路抑制剂组(Brusatol组,0.2μg/mL)、TFG+Brusatol组(80mg/mL+0.2μg/mL);用CCK-8法检测细胞活性;hochest 33258染色检测细胞凋亡情况;免疫荧光染色法检测活性氧(ROS)含量;免疫荧光共聚焦法检测Nrf2入核情况;蛋白免疫印迹法检测各组细胞Nrf2、ARE、活化的半胱氨酸天冬氨酸蛋白酶3(cleaved caspase-3)、肿瘤坏死因子-α(TNF-α)、血红素加氧酶1(HO-1)蛋白表达水平。结果:与正常组比较,IL-1β诱导组细胞增殖活性降低(P<0.05);与IL-1β诱导组比较,随着TFG浓度升高,细胞增殖活性逐渐升高,在80~160mg/mL时保持在稳定水平,后续选用20mg/mL、80mg/mL为TFG低、高剂量组进行后续试验。与正常对照组比较,IL-1β诱导组细胞增殖活性降低(P<0.05),细胞凋亡率、ROS含量、Nrf2入核数目、Nrf2、ARE、cleaved caspase-3、HO-1、TNF-α蛋白表达水平升高(P<0.05)。与IL-1β诱导组比较,TFG低、高剂量组细胞增殖活性、Nrf2入核数目、Nrf2、ARE、HO-1蛋白表达水平升高(P<0.05),细胞凋亡率、ROS含量、cleaved caspase-3、TNF-α蛋白表达水平降低(P<0.05);且TFG高剂量组细胞增殖活性、Nrf2入核数目、Nrf2、ARE、HO-1蛋白表达水平高于TFG低剂量组(P<0.05),细胞凋亡率、ROS含量、cleaved caspase-3、TNF-α蛋白表达水平低于TFG低剂量组(P<0.05);Brusatol组细胞增殖活性、Nrf2入核数目、Nrf2、ARE、HO-1蛋白表达水平降低(P<0.05),细胞凋亡率、ROS含量、cleaved caspase-3、TNF-α蛋白表达水平升高(P<0.05)。与TFG高剂量组比较,TFG+Brusatol组细胞增殖活性、Nrf2入核数目、Nrf2、ARE、HO-1蛋白表达水平降低(P<0.05),细胞凋亡率、ROS含量、cleaved caspase-3、TNF-α蛋白表达水平升高(P<0.05)。结论:TFG可通过激活Nrf2/ARE通路,发挥抗炎、抗氧化、抗髓核细胞凋亡作用。

Objective:To investigate the effect of total flavonoids of Ginkgo biloba(TFG)on apoptosis of nucleus pulposus cells induced by interleukin-1β(IL-1β),and its regulation on nuclear factor E2 related factor 2(Nrf2)/antioxidant response element(ARE)signaling pathway.Methods:Human nucleus pulposus cells were divided into normal group,IL-1βinduced group,dimethyl sulfoxide(DMSO)group and TFG groups with different concentrations(5,10,20,40,80,160 mg/mL),in addition to the normal group,all groups were induced with IL-1β(10 ng/mL)for 24 hours,and CCK-8 method was used to detect the cell proliferation activity.The nucleus pulposus cells were divided into normal control group,IL-1βinduced group,TFG low(20 mg/mL),high(80 mg/mL)dose groups,Nrf2 pathway inhibitor group(Brusatol group,0.2μg/mL)group,TFG+Brusatol group(80 mg/mL+0.2μg/mL).CCK-8 assay was used to detect cell viability;hochest 33258 staining was used to detect apoptosis;immunofluorescence was used to detect the content of reactive oxygen species(ROS);detection of Nrf2 entry by immunofluorescence cofocal method;Western blot was used to detect the expression level of Nrf2,ARE,cleaved caspase-3,tumor necrosis factor-α(TNF-α),heme oxygenase 1(HO-1)protein.Results:Compared with that in the normal group,the proliferation activity of IL-1βinduced group was lower(P<0.05).Compared with the IL-1βinduced group,the proliferation activity increased with the increase of TFG concentration,it was stable at 80 mg/mL-160 mg/mL,and 20 mg/mL and 80 mg/mL were selected as TFG low and high dose groups for follow-up test.Compared with the normal control group,the cell proliferation activity of IL-1βinduced group was decreased(P<0.05),and the apoptosis rate,ROS content,Nrf2 number,Nrf2,ARE,cleaved caspase-3,HO-1 and TNF-αprotein expression levels were increased(P<0.05).Compared with IL-1βinduced group,cell proliferation activity,Nrf2 number,Nrf2,ARE and HO-1 protein expression levels of TFG low dose and high dose groups were increased(P<0.05),cell apoptosis rate,ROS content,cleaved caspase-3 and TNF-αprotein expression levels were decreased(P<0.05);and the cell proliferation activity,Nrf2 number,Nrf2,ARE and HO-1 protein expression levels in TFG high dose group were higher than those in TFG low dose group(P<0.05),and the apoptosis rate,ROS content,cleaved caspase-3 and TNF-αprotein expression levels in TFG high dose group were lower than those in TFG low dose group(P<0.05);the cell proliferation activity,Nrf2 number,Nrf2,ARE and HO-1 protein expression levels of Brusatol group were decreased(P<0.05),while the apoptosis rate,ROS content,cleaved caspase-3 and TNF-αprotein expression levels were increased(P<0.05).Compared with the TFG high dose group,the cell proliferation activity,Nrf2 number,Nrf2,ARE and HO-1 protein expression levels of TFG+Brusatol group were decreased(P<0.05),and the apoptosis rate,ROS content,cleaved caspase-3 and TNF-αprotein expression levels were increased(P<0.05).Conclusion:TFG can play an anti-inflammatory,anti-oxidation and anti-apoptosis of nucleus pulposus cells effect by activating Nrf2/ARE pathway.