摘要
目的:探讨寿胎丸在脂多糖(LPS)诱导的人绒毛外滋养细胞(HTR-8/SVneo)损伤中的作用及其对细胞损伤中氧化应激和焦亡的调控,为寿胎丸安胎的作用机制研究提供新的方向。方法:采用LPS(100μg·L-1)诱导HTR-8/SVneo细胞损伤建立细胞模型,设置空白组、模型组、寿胎丸组(10%寿胎丸含药血清)、抗氧化剂组(1 mmol·L-1NAC)、NOD样受体热蛋白结构域3(NLRP3)抑制剂组(50μmol·L-1MCC950)。分别采用细胞增殖与活性检测(CCK-8)试剂盒检测细胞活性情况;Hochest33342/PI双荧光染色和流式细胞术观察细胞死亡情况;酶联免疫吸附测定法(ELISA)检测细胞上清液中白细胞介素-18(IL-18)、白细胞介素-1β(IL-1β)、丙二醛(MDA)、超氧化物歧化酶(SOD)释放情况;DCFH-DA探针检测细胞内活性氧(ROS)含量;蛋白免疫印迹法(Western blot)检测NLRP3、胱天蛋白酶(Caspase)-1、消化道皮肤素D(GSDMD)、IL-1β蛋白的表达;实时荧光定量聚合酶链式反应(Real-time PCR)检测NLRP3、Caspase-1 mRNA的表达。结果:与空白组比较,模型组细胞活力显著降低(P<0.01),细胞上清液中炎症因子IL-1β、IL-18含量显著增加(P<0.01),氧化应激因子ROS、MDA含量升高,SOD活性显著降低(P<0.01),NLRP3、Caspase-1、GSDMD、IL-1β蛋白和NLRP3、Caspase-1 mRNA表达显著上调(P<0.01);与模型组比较,寿胎丸组、NAC组及MCC950组细胞活力显著升高(P<0.01),寿胎丸组和NAC组MDA、ROS活性降低,SOD活性显著升高(P<0.01),寿胎丸组和MCC950组细胞上清液中IL-1β、IL-18含量减少(P<0.01),细胞内NLRP3、Caspase-1、GSDMD、IL-1β蛋白和NLRP3、Caspase-1、IL-1β mRNA表达均明显降低(P<0.05,P<0.01)。结论:寿胎丸可通过抑制氧化应激和细胞焦亡减轻LPS诱导的HTR-8/SVneo细胞损伤,这可能是寿胎丸防治复发性流产,发挥安胎作用的机制之一。
Objective:To investigate the regulatory effect of Shoutaiwan on oxidative stress and pyroptosis in lipopolysaccharide(LPS)-induced human extravillous trophoblast(HTR-8/SVneo) cells and provide a new direction for deciphering the mechanism of action of Shoutaiwan.Method:LPS(100 μg · L-1)was used to induce the injury of HTR-8/SVneo cells(modeling).Five groups were designed in this study,including a blank group,a model group,a Shoutaiwan(10% Shoutaiwan-containing serum) group,an antioxidant(1 mmol·L-1NAC)group,and NOD like receptor thermoprotein domain 3(NLRP3)inhibitor(50 μmol·L-1MCC950)group.Cell viability was detected by cell counting kit-8(CCK-8)kit.Hochest 33342/PI double fluorescence staining and flow cytometry were employed to observe cell death.The levels of interleukin-18(IL-18),interleukin-1β(IL-1β),malondialdehyde(MDA),and superoxide dismutase(SOD)in cell supernatant was determined by enzyme-linked immunosorbent assay(ELISA).DCFH-DA probe was used to measure the level of intracellular reactive oxygen species(ROS).Western blot was employed to determine the protein levels of NLRP3,Caspase-1,gastermin D(GSDMD),and IL-1β in cells,and Real-time fluorescence quantitative polymerase chain reaction(Real-time PCR)to measure the mRNA levels of NLRP3 and Caspase-1in cells.Result:Compared with the blank group,the modeling decreased the cell viability(P<0.01),elevated the levels of IL-1β,IL-18,ROS,and MDA,and weakened the activity of SOD(P<0.01).Furthermore,it upregulated the protein levels of NLRP3,Caspase-1,GSDMD,and IL-1β and the mRNA levels of NLRP3 and Caspase-1(P<0.01).Compared with the model group,Shoutaiwan,NAC,and MCC950 increased the cell viability(P<0.01).Further,Shoutaiwan and NAC lowered the levels of MDA and ROS and increased the activity of SOD(P<0.01).Shoutaiwan and MCC950 reduced the IL-1β and IL-18 in cell supernatant(P<0.01),and down-regulated the protein levels of NLRP3,Caspase-1,GSDMD,and IL-1β and the mRNA levels of NLRP3,Caspase-1,and IL-1β(P<0.05,P<0.01).Conclusion:Shoutaiwan can regulate oxidative stress and pyroptosis to attenuate the LPS-induced damage of HTR-8/SVneo cells,which may be the mechanism of Shoutaiwan in preventing recurrent spontaneous abortion.
作者
乔宗惠
申思楠
邓敦
张舒清
雷磊
QIAO Zonghui;SHEN Sinan;DENG Dun;ZHANG Shuqing;LEI Lei(The First Affiliated Hospital of Hunan University of Chinese Medicine,Changsha 410007,China;College of Integrated Traditional Chinese and Western Medicine,Hunan University of Chinese Medicine,Changsha 410208,China)
出处
《中国实验方剂学杂志》
CAS
CSCD
北大核心
2022年第17期17-24,共8页
Chinese Journal of Experimental Traditional Medical Formulae
基金
国家自然科学基金项目(81473714)
湖南省自然科学基金项目(2021JJ30493)
湖南省教育厅重点科学研究项目(19A364)
湖南省中医药管理局重点课题项目(2021007)
湖南中医药大学校级科研基金重点项目(2020XJJJ001)。
关键词
寿胎丸
细胞损伤
氧化应激
细胞焦亡
复发性流产
Shoutaiwan
cell damage
oxidative stress
pyroptosis
recurrent spontaneous abortion
