Shoutaiwan Regulate Lipopolysaccharide-induced Oxidative Stress and Pyroptosis in Human Extravillous Trophoblast Cells / 中国实验方剂学杂志

ABSTRACT

ObjectiveTo 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. MethodLPS （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-1 NAC） group， and NOD like receptor thermoprotein domain 3 （NLRP3） inhibitor （50 μmol·L-1 MCC950） 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-1 in cells. ResultCompared 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 up-regulated 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）. ConclusionShoutaiwan 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.