银杏总黄酮对汞中毒致人胚肾细胞毒性的干预作用及对SIRT1/NLRP3通路的影响

摘要/Abstract

摘要： 目的探讨银杏总黄酮对汞中毒致人胚肾细胞毒性的干预作用及对沉默信息调节因子1（silent information regulator 1,SIRT1）/Nod样受体3（nod-like receptors-3,NLRP3）通路的影响。方法体外培养HEK293细胞为对照组、汞中毒模型组（1 000 μmol/L HgCl₂）、银杏总黄酮低剂量组、中剂量组和高剂量组（1 000 μmol/L HgCl₂+100、200、400 mg/L 银杏总黄酮）,孵育24 h后,四甲基偶氮唑蓝法（tetramethylazolyl blue,MTT）检测细胞存活情况,流式细胞仪测定细胞凋亡水平,微管相关蛋白1轻链3B-Ⅱ抗体（microtubule-associated protein 1 light chain 3B-Ⅱ,LC3B-Ⅱ）染色检测细胞自噬水平,测定细胞谷胱甘肽（glutathione,r-glutamyl cysteingl+glycine,GSH）、丙二醛（Malondialdehyde,MDA）水平,实时荧光PCR（real-time fluorescent reverse transcription,RT-PCR）法及蛋白印迹法测定SIRT1、NLRP3水平。结果与对照组比较,汞中毒模型组细胞存活率[（93.63±2.51）% vs（48.63±3.25）%]、细胞LC3B-Ⅱ阳性率[（58.25±12.15）% vs（10.19±3.20）%]、GSH含量[（115.82±15.91） vs （52.74±6.40）U/mg]、SIRT1 mRNA蛋白表达水平（2.93±0.80 vs 0.48±0.14,0.30±0.05 vs 0.09±0.03）均降低,差异均有统计学意义（均P<0.05）。与汞中毒模型组比较,银杏总黄酮治疗后细胞存活率[（48.63±3.25）% vs（59.28±3.18）%、（74.21±3.63）%、（82.29±3.59）%]、细胞LC3B-Ⅱ 阳性率[（10.19±3.20）% vs（15.20±5.33）%、（22.02±8.65）%、（36.22±11.35）%]、GSH含量[（52.74±6.40）vs（67.36±9.71）、（84.52±7.46）、（92.48±5.29）U/mg]、SIRT1mRNA蛋白表达水平（0.48±0.14、0.78±0.23、1.52±0.30 vs 2.20±0.72,0.09±0.03 vs 0.23±0.06、0.19±0.08、0.14±0.06）升高,且呈剂量反应关系,差异均有统计学意义（均P<0.05）。与对照组比较,汞中毒模型组细胞凋亡率[（0.16±0.08）% vs（1.14±0.19）%]、MDA含量[（5.42±0.68）vs（18.60±3.21）nmol/mg]、NLRP3 mRNA蛋白表达水平（0.35±0.09 vs 3.28±1.02,0.24±0.04 vs 0.73±0.12）升高,差异均有统计学意义（均P<0.05）。与汞中毒模型组比较,银杏总黄酮治疗后细胞凋亡率[（1.14±0.19）% vs（0.83±0.12）%、（0.32±0.07）%、（0.21±0.07）%]、MDA含量[（18.60±3.21）vs（15.79±4.26）、（10.21±2.47）、（8.16±1.05）nmol/mg]、NLRP3 mRNA蛋白表达水平（3.28±1.02 vs 2.45±0.86、1.53±0.46、0.83±0.32,0.73±0.12 vs 0.60±0.09、0.41±0.06、0.37±0.07）均降低,且呈剂量反应关系,差异均有统计学意义（均P<0.05）。结论银杏总黄酮对汞中毒致人胚肾细胞毒性具有保护作用,能降低细胞凋亡及氧化损伤,增强细胞自噬。其机制可能与银杏总黄酮增加SIRT1表达而降低NLRP3表达,进而抑制SIRT1/NLRP3通路有关。

关键词: 汞中毒, 银杏总黄酮, SIRT1, NLRP3, 人胚肾细胞

Abstract: Objective To investigate the intervention effect of total flavonoids of Ginkgo biloba on the cytotoxicity of human embryonic kidney induced by mercury poisoning and its effect on silent information regulator 1（SIRT1）/nod-like receptors-3（NLRP3） pathway. Methods HEK293 cells were cultured in vitro and included the control group,mercury poisoning model group（1 000 μmol/L HgCl₂）,low-dose,middle-dose and high-dose ginkgo total flavonoids groups（1 000 μmol/L HgCl₂+100,200,400 mg/L ginkgo total flavonoids）. After incubation for 24 hours,the cell survival was detected by tetramethylazolyl blue（MTT） method,the level of apoptosis was detected by flow cytometry,the level of autophagy was detected by microtubule-associated protein 1 light chain 3B-Ⅱ（LC3B-Ⅱ） antibody staining,the levels of glutathione,r-glutamyl cysteingl+glycine（GSH） and malondialdehyde（MDA） were measured,and levels of SIRT1 and NLRP3 were detected by real-time fluorescent reverse transcription（RT-PCR） and Western blotting. Results Compared with the control group,the survival rate of cells in the mercury poisoning model group[（93.63±2.51）% vs（48.63±3.25）%],the positive rate of cell LC3B-Ⅱ[（58.25±12.15）% vs（10.19±3.20）%],GSH content[（115.82±15.91）U/mg vs（52.74±6.40）U/mg],SIRT1 mRNA protein expression（2.93±0.80 vs 0.48±0.14,0.30±0.05 vs 0.09±0.03） decreased,and the difference were statistically significant（all P<0.05）. Compared with the mercury poisoning model group,the cell survival rate after ginkgo total flavonoids treatment[（48.63±3.25）% vs（59.28±3.18）%,（74.21±3.63）%,（82.29±3.59）%],the positive rate of cell LC3B-Ⅱ[（10.19±3.20）% vs（15.20±5.33）%,（22.02±8.65）%,（36.22±11.35）%],GSH content[（52.74±6.40）U/mg vs（67.36±9.71）U/mg,（84.52±7.46）U/mg,（92.48±5.29）U/mg],SIRT1 mRNA protein expression（0.48±0.14,0.78±0.23,1.52±0.30 vs 2.20±0.72, 0.09±0.03 vs 0.23±0.06,0.19±0.08,0.14±0.06） increased,and the difference were statistically significant（all P<0.05）. Compared with the control group,the apoptosis rate of mercury poisoning model group[（0.16±0.08）% vs（1.14±0.19）%],MDA content[（5.42±0.68）nmol/mg vs（18.60±3.21）nmol/mg],NLRP3 mRNA protein expression levels（0.35±0.09 vs 3.28±1.02,0.24±0.04 vs 0.73±0.12） significantly increased,and the difference were statistically significant（all P<0.05）. Compared with the mercury poisoning model group,the cell apoptosis rate after ginkgo total flavonoids treatment[（1.14±0.19）% vs（0.83±0.12）%,（0.32±0.07）%,（0.21±0.07）%],MDA content[（18.60±3.21）nmol/mg vs（15.79±4.26）nmol/mg,（10.21±2.47）nmol/mg,（8.16±1.05）nmol/mg],NLRP3 mRNA protein expression levels（3.28±1.02 vs 2.45±0.86,1.53±0.46,0.83±0.32,0.73±0.12 vs 0.60±0.09,0.41±0.06,0.37±0.07） decreased,with the dose-response relationship,and the differences were statistically significant（all P<0.05）. Conclusions The total flavonoids of Ginkgo biloba have a protective effect on the cytotoxicity of human embryonic kidney caused by mercury poisoning,can reduce cell apoptosis and oxidative damage,and enhance cell autophagy. The mechanism may be related to total flavonoids of Ginkgo biloba increasing the expression of SIRT1,reducing the expression of NLRP3,and then inhibiting SIRT1/NLRP3 pathway.

Key words: Mercury poisoning, Total flavonoids of Ginkgo biloba, SIRT1, NLRP3, Human embryonic kidney cells

中图分类号:

R114

张建新, 何清, 张剑彬, 刘杰. 银杏总黄酮对汞中毒致人胚肾细胞毒性的干预作用及对SIRT1/NLRP3通路的影响. [J]职业与健康, 2023, 39(9): 1158-1164.

ZHANG Jian-xin, HE Qing, ZHANG Jian-bin, LIU Jie. Interventional effect of total flavonoids of Ginkgo biloba on human embryonic kidney cytotoxicity induced by mercury poisoning and its effect on SIRT1/NLRP3 pathway. [J]OCCUPATION AND HEALTH, 2023, 39(9): 1158-1164.

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