Study On The Mechanism Of Wen Yang Jian Spleen Formula Regulating VEGF/PI3K/Aktpathway To Improve Angiogenesis At The Maternal-fetal Interface Of RSA Mice

Objective:Under the guidance of Professor Jiang Liguan’s clinical idea of "harmony and smoothness," this project explores the formula of Professor Zhang Liangying,a national famous traditional Chinese medicine,for Wen Yang Jian Spleen(WYJP,15 g Atractylodes macrocephala Koidz,15 g Dipsacus asper Wall.ex Henry,15 g Taxillus chinensis(DC.)Danse,15 g Cuscuta chinensis Lam.,15 g Epimedium brevicomu Maxim.,20 g Astragalus membranaceus(Fisch.)Bge.var.Mongholicus(Bge.)Hsiao,6g Glycyrrhiza uralensis Fisch.,20 g Codonopsis pilosula(Franch.)Nannf.,15 g Poria cocos(Schw)Wolf,15 g Eucommia ulmoides Oliv.,15 g Equus asinus L.).Based on the VEGF/PI3K/AKT signaling pathway as the entry point,to investigate the process of WYJP acting on the maternal-fetal interface vessels of RSA mice through activation of VEGF/PI3K/Akt pathway to improve the production.The expression of vascular endothelial growth factor(VEGF),hypoxia inducible factor-1(HIF-1α),and histomorphological changes at the maternal-fetal interface of RSA mice was observed,and the expression of VEGF,anti-angiogenic factor,and PI3K/AKT pathway proteins was examined.To investigate the practical improvement of vascularity at the maternal-fetal interface in RSA mice promoted by WYJP via regulation of the VEGF/PI3K/AKT signaling pathway.Furthermore,the specific mechanism of action of WYJP in protecting pregnancy will further provide theoretical support for the clinical treatment of the RSA by the MYJP.Methods:1.Reading literature and medical books to summarize the etiology and pathogenesis of RSA.Study the research and medication characteristics of the RSA by Chinese medicine practitioners in various dynasties to further understand the pathogenesis,diagnosis,and treatment of RSA in Chinese and Western medicine and to provide new ideas for the treatment of RSA.2.Experimental methods: CBA/J female mice × BALB/c pregnant mice were used as the control group(CON),and the RSA mouse model was constructed by CBA/J female mice × DBA/2pregnant mice,and they were divided into a low-dose group(WYJP-L,8.43g/kg),high-dose group(WYJP-H,16.85g/kg)and the model control group of WYJP.Ten rats in each group were given the drug for 14 days,and their meconium tissues were taken to calculate the survival rate and embryo loss rate of pregnant rats.In addition,the morphological changes of meconium were observed by H&E staining.RT-q PCR was performed to detect the expression of VEGF,HIF-α,PI3 K,and AKT m RNA in meconium tissues.The expression of VEGF,HIFA,PI3 K,and AKT in meconium was observed by immunohistochemistry,and the manifestation of VEGF,HIFA,PI3 K,and AKT in meconium was detected by Western blot.Result:1.Compared with CON group,the embryo loss rate of RSA mice in the model group was statistically significant(p<0.01);After receiving low-dose and high-dose treatment with WYJP,the rate of embryo loss was significantly reduced(p<0.01).2.Anatomy was performed for microscopic observation.Compared with normal pregnant mice,the villi of RSA mice in the model group showed varying sizes,irregular shapes,and disorderly arrangement.Mesenchymal and matrix cellulose like degeneration was very obvious,and obvious apoptotic interface vascular tissue and inflammatory interface vascular tissue could be observed;The degree of staining is relatively deep;Interstitial blood vessels also significantly decreased;In addition,there is significant tissue degeneration at the maternal fetal interface of RSA mice,with missing epithelium,wrinkled glands,and obvious blood stasis in blood vessels.After administering the WYJP,the vascular morphology of the maternal fetal interface tissue in RSA mice showed an improvement trend towards normal mouse interface blood vessels.3.RT-q PCR results showed that VEGF,HIF,PI3 K,and AKT m RNA expression was down-regulated in RSA mice compared with normal pregnant mice(p<0.05).Compared with the RSA group,the expression of VEGF,PI3 K,and AKT m RNA was upregulated in WYJP-L/H group(p<0.05),HIF m RNA expression was increased in the WYJP-L group(p<0.05),while HIF m RNA expression had an increasing trend in the WYJP-H group.4.Western blot results showed that the expression of p-PI3 k,p-AKT,VEGF,and HIF proteins was significantly down-regulated in RSA mice compared with normal pregnant mice(p< 0.05).After WYJP intervention,p-PI3 k,p-AKT,VEGF and HIF protein expression were significantly increased in WYJP-H group compared with RSA group(p<0.05).p-PI3 k and HIF protein expression were up-regulated in WYJP-L group,but the rest of the indexes were not significantly different from the model group(p>0.05).Conclusion:Wen Yang Jian Spleen Formula significantly improved the rate of embryonic loss and promoted maternal meconium tissue neogenesis in RSA mice.The high dose group of Wen Yang Jian Spleen Formula significantly improved VEGF/PI3K/AKT expression in maternal-fetal interface tissue of RSA mice to promote interface vascularization.