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  

Interventional effect of total flavonoids of Ginkgo biloba on human embryonic kidney cytotoxicity induced by mercury poisoning and its effect on SIRT1/NLRP3 pathway

作　　者：张建新 何清 张剑彬[1] 刘杰[1] ZHANG Jian-xin;HE Qing;ZHANG Jian-bin;LIU Jie(Department of Nephrology and Hematology,Ba'nan Hospital Affiliated to Chongqing Medical University,Chongqing,401320,China)

机构地区：[1]重庆医科大学附属巴南医院肾病血液内科,重庆401320

出　　处：《职业与健康》2023年第9期1158-1164,共7页Occupation and Health

基　　金：重庆市永川区2019年公益类科技计划项目(Ycstc,2019nb0201)。

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 regulator1,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 000μmol/L HgCl_(2))、银杏总黄酮低剂量组、中剂量组和高剂量组(1 000μmol/L HgCl_(2)+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(1000μmol/L HgCl_(2)),low-dose,middle-dose and high-dose ginkgo total flavonoids groups(1000μmol/L HgCl_(2)+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 3BII(LC3B-I)antibody staining,the levels of glutathione,r-glutamyl cysteingl+glycine(CGSH)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)%us(48.63±3.25)%],the positive rate of cell LC3B-I[(58.25±12.15)%vs(10.19±3.20)%],GSH content[(115.82±15.91)U/mg vs(52.74±6.40)U/mgl,SIRT1 mRNA protein expression(2.93+0.80 us 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)%us(59.28±3.18)%,(74.21±3.63)%,(82.29±3.59)%),the positive rate of cell LC3B-I[(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/mgl,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 us 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(

关 键 词：汞中毒 银杏总黄酮 SIRT1 NLRP3 人胚肾细胞 

分 类 号：R114[医药卫生—卫生毒理学]