| Objective: Based on Caspase-1-mediated pyroptosis of decidual cells,the regulatory role of pyroptosis in the pathogenesis of RSA was explored,and the mechanism of Shoutai Wan in improving the pregnancy outcome of RSA was explored.Methods: 1.SD rats were randomly divided into blank group,model group,Shoutai Wan low,medium and high dose group and dydrogesterone group,with hydroxyurea tablets and mifepristone tablets as modeling agents to establish RSA rat model,calculate embryo loss rate,HE staining to observe the morphological structure of decidual tissue,immunohistochemistry(IHC)and Western blot(WB)to detect the expression of pyroptosis-related proteins.2.The decidual cells of normal pregnant rats and RSA rats were extracted,and the cell viability was determined by CCK-8 to determine the concentration of serum and lipopolysaccharide(LPS).The pyroptosis model was established by LPS-induced decidual cell injury in the normal group.The blank group,model group,Shoutai Wan group,LPS group and LPS + Shoutai Wan group were set up.Cell viability was detected by CCK-8 method.Immunofluorescence(IF)and WB were used to detect the expression of pyroptosis-related proteins in decidual cells.Results: 1.The effect of Shoutai Wan on cell scorching of meconium t issue in RSA rats.(1)Compared with the blank group,the embryo loss rate in the model group was significantly increased(P< 0.01),suggesting that the RSA rat model was successfully established.Compared with the model group,the embryo loss rates in the low,medium and high dose groups of Shoutai Wan were significantly decreased(P< 0.01).Compared with the dydrogesterone group,the embryo loss rate of the model group and the low-dose Shoutai Wan group was significantly decreased(P< 0.01).(2)The morphological structure of decidual tissue in each group : the decidual cells in the blank group were closely arranged,the cytoplasm was rich,the interstitial blood vessels were rich,and the wall was complete;in the model group,the arrangement of decidual cells was loose and disordered,the cytoplasm was edematous,some nuclei disappeared,and the distribution of interstitial blood vessels decreased.The low and medium dose groups of Shoutai Wan were improved,but some decidual cells were still disordered,cytoplasmic edema,less interstitial blood vessels were distributed,and the wall was intact.The nucleus morphology of decidual cells in the high-dose Shoutai Wan group and the dydrogesterone group tended to be blank group,and the nucleus basically had no pyknosis,disappearance and necrosis.(3)IHC results showed that compared with the blank group,the positive expression of IL-18 and IL-1β in the model group was significantly enhanced(P< 0.05,P< 0.01).Compared with the model group,the positive expression of IL-18 and IL-1β in the low,medium and high dose groups of Shoutai Wan was significantly decreased(P< 0.05,P< 0.01).(4)WB results showed that compared with the blank group,the expression of pyroptosis-related proteins in the model group was significantly enhanced(P<0.01).Compared with the model group,the expression of Cleaved-Caspase-1,GSDMD and IL-18 protein in the low-dose group of Shoutai Wan decreased,but there was no statistical significance.The expression of Cleaved-Caspase-1protein in the middle dose group of Shoutai Wan was significantly decreased(P< 0.01),the expression of GSDMD protein was significantly decreased(P<0.05),and the expression of IL-18 protein was decreased,but there was no statistical significance.The expression of pyroptosis-related proteins in the high-dose Shoutai Wan group was significantly decreased(P< 0.05,P< 0.01).2.The effect of Shoutai Wan drug-containing serum on cell scorching of RSA rat metaphase cells.(1)The survival rate of cells in each group was detected by CCK-8.The results showed that compared with the blank group,the cell survival rate of the model group and the LPS group decreased significantly(P< 0.01).Compared with the model group,the cell survival rate of the Shoutai Wan group increased significantly(P < 0.01).Compared with the LPS group,the cell survival rate of the LPS + Shoutai Wan group was significantly increased(P<0.01).(2)The results of cellular immunofluorescence showed that compared with the blank group,the average fluorescence intensity of pyroptosis-related proteins in the model group and LPS group was significantly increased(P < 0.01).Compared with the model group,the average fluorescence intensity of pyroptosis-related protein in Shoutai Wan group was significantly decreased(P< 0.01).Compared with LPS group,the mean fluorescence intensity of pyroptosis-related protein in LPS + Shoutai Wan group was significantly decreased(P< 0.01).(3)Western blot results showed that compared with the blank group,the protein expression of pyroptosis-related proteins in the model group and LPS group was significantly increased(P< 0.01).Compared with the model group,the protein expression of pyroptosis-related proteins in Shoutai Wan group was significantly decreased(P< 0.05,P< 0.01).Compared with LPS group,the protein expression of pyroptosis-related protein in LPS + Shoutai Wan group was significantly decreased(P< 0.05,P< 0.01).Conclusion: Shoutai Wan can effectively reduce the abortion rate of RSA rats,improve the morphological structure of meconium tissues of RSA rats,reduce the levels of Cleaved-Caspase-1,GSDMD,IL-1β and IL-18 factors in meconium tissues and cells,thus reducing the scorching of uterine meconium cells and improving pregnancy outcome. |
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