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河北医学  2021, Vol. 27 Issue (10): 1585-1591    DOI: 10.3969/j.issn.1006-6233.2021.10.001
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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
李静1, 黄娅芬1, 夏晓枫2, 唐家国2
1.湖北省武汉市第三医院骨科, 湖北 武汉 430000;
2.长江航运总医院骨科, 湖北 武汉 430010
Effects of Total Flavonoids of Ginkgo Biloba on Apoptosis of Nucleus Pulposus Cells Induced by IL-1β and Nrf2/ARE Signaling Pathway
LI Jing, HUANG Yafen, et al
Wuhan Third Hospital, Hubei Wuhan 430000, China
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摘要 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。
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关键词 银杏总黄酮髓核细胞核因子E2相关因子2/抗氧化反应元件信号通路白细胞介素-1β凋 亡    
AbstractObjective: 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-1β induced 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-1β induced group, TFG low (20 mg/mL), high (80 mg/mL) dose groups, Nrf2 pathway inhibitor group (Brusatol group, 0.2 μg/mL) group, TFG + Brusatol group (80 mg/mL + 0.2 μg/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-1β induced group was lower (P<0.05). Compared with the IL-1β induced 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-1β induced group was decreased (P<0.05), and the apoptosis rate, ROS content, Nrf2 number, Nrf2, ARE, cleaved caspase-3, HO-1 and TNF-α protein expression levels were increased (P<0.05). Compared with IL-1β induced 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-α protein 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-α protein 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-α protein 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-α protein 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.
Key wordsTotal flavonoids of Ginkgo biloba    Nucleus pulposus cells    Nuclear factor E2 related factor 2/antioxidant response element signaling pathway    Interleukin-1β    Apoptosis
    
基金资助:湖北省卫生健康委员会联合基金立项项目,(编号:WJ2019H388)
通讯作者: 夏晓枫   
引用本文:   
李静, 黄娅芬, 夏晓枫, 唐家国. 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[J]. 河北医学, 2021, 27(10): 1585-1591.
LI Jing, HUANG Yafen, et al. Effects of Total Flavonoids of Ginkgo Biloba on Apoptosis of Nucleus Pulposus Cells Induced by IL-1β and Nrf2/ARE Signaling Pathway. HeBei Med, 2021, 27(10): 1585-1591.
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