寿胎丸对脂多糖诱导的人绒毛外滋养细胞氧化应激及焦亡的调节作用

Shoutaiwan Regulate Lipopolysaccharide-induced Oxidative Stress and Pyroptosis in Human Extravillous Trophoblast Cells

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.