摘要
目的探讨庞氏安胎止血汤对自然流产大鼠的干预作用及机制。方法将60只雌性妊娠大鼠随机分为正常组、模型组、地屈孕酮组(2 mg·kg^(-1))和庞氏安胎止血汤低、中、高剂量组(11.025、22.05、44.1 g·kg^(-1)),每组10只。从妊娠第1天起灌胃给予相应药物,每日1次,连续12 d。妊娠第13天一次性灌胃米非司酮(5 mg·kg^(-1))复制流产大鼠模型。妊娠第14天,统计各组大鼠妊娠子宫流产胚胎数、正常胚胎数,计算流产率、子宫系数;采用ELISA法检测血清中促性腺激素释放激素(GnRH),促卵泡素(FSH),促黄体生成素(LH),雌二醇(E_(2))水平;HE染色法观察妊娠子宫组织病理形态;对妊娠子宫组织进行非标记差异定量蛋白组学分析,筛选、鉴定模型组vs正常组、庞氏安胎止血汤组vs模型组的差异蛋白。对鉴定出的差异蛋白使用STRING数据库进行蛋白互作(PPI)分析,筛选出枢纽靶点;利用DAVID数据库对枢纽靶点进行GO功能及KEGG通路富集分析。采用免疫组化法检测大鼠妊娠子宫组织中Pten、Col6a1、Cav1、Mylk、Akt3蛋白表达水平。结果(1)与正常组比较,模型组大鼠的血清GnRH、FSH、LH、E_(2)水平均明显降低(P<0.05);妊娠子宫颜色略显苍白,血供差,胚胎着床点少,分布不规则,胚胎黏附处可见明显出血点,流产率明显升高(P<0.05),子宫系数明显减小(P<0.05),妊娠子宫内膜组织血管分布减少,蜕膜细胞数量减少且有明显损伤,宫内可见大量瘀血。与模型组比较,各给药组大鼠的血清GnRH、FSH、LH、E_(2)水平均明显升高(P<0.05);妊娠子宫胚胎着床情况均有不同程度改善,流产率明显降低(P<0.05),子宫系数明显增大(P<0.05),妊娠子宫组织的蜕膜细胞损伤及充血情况均有不同程度改善。(2)正常组vs模型组共筛选出951个差异蛋白,模型组vs庞氏安胎止血汤组共筛选出1221个差异蛋白,结果共得到550个庞氏安胎止血汤干预自然流产大鼠的潜在作用靶点(交集靶点)。PPI分析得到枢纽靶点共159个,包括Pten、Polr2c、Fbn1、Polr1a、Col1a2、Dcn、Bgn、Fmod、Cd74、Col6a2。GO功能及KEGG通路分析显示,庞氏安胎止血汤干预自然流产进程与黏着斑信号途径密切相关,涉及上调Akt3、Col6a1、Cav1、Mylk,下调Pten等关键靶点。(3)与正常组比较,模型组大鼠妊娠子宫组织中Pten蛋白表达水平明显升高(P<0.05),Akt3、Col6a1、Cav1、Mylk蛋白表达水平明显降低(P<0.05)。与模型组比较,各给药组大鼠妊娠子宫组织中Pten蛋白表达水平明显降低(P<0.05),Akt3、Col6a1、Cav1、Mylk蛋白表达水平明显升高(P<0.05)。结论庞氏安胎止血汤对自然流产大鼠有明显干预作用,其机制可能与调控黏着斑途径,下调Pten蛋白表达,上调Akt3、Col6a1、Cav1、Mylk蛋白表达,进而影响滋养层细胞凋亡有关。
Objective To investigate the intervention effect and mechanism of Pang’s Antai Zhixue Decoction on spontaneous abortion in rats.Methods Sixty pregnant female rats were randomly divided into six groups:normal group,model group,Dydrogesterone group(2 mg·kg^(-1)),and low-,medium-,and high-dose Pang’s Antai Zhixue Decoction groups(11.025,22.05,44.1 g·kg^(-1)),with 10 rats in each group.From the first day of pregnancy,the rats were orally administered the corresponding drugs once daily for 12 consecutive days.On the day 13 of pregnancy,a single oral dose of Mifepristone(5 mg·kg^(-1))was administered to induce the abortion model.On the day 14 of pregnancy,the number of aborted and normal embryos in the uterine horns was counted,and the abortion rate and uterine coefficient were calculated.Serum levels of gonadotropin-releasing hormone(GnRH),follicle-stimulating hormone(FSH),luteinizing hormone(LH),and estradiol(E_(2))were measured using ELISA.Pathological changes in the pregnant uterine tissue were observed using HE staining.Label-free quantitative proteomics analysis was performed on the pregnant uterine tissue to screen and identify differentially expressed proteins between the model group versus normal group and Pang’s Antai Zhixue Decoction group versus model group.The STRING database was used for protein-protein interaction(PPI)analysis of the identified differentially expressed proteins to screen hub targets.The DAVID database was used for GO functional and KEGG pathway enrichment analysis of the hub targets.Immunohistochemistry was used to detect the protein expression levels of Pten,Col6a1,Cav1,Mylk,and Akt3 in the pregnant uterine tissue.Results(1)Compared with the normal group,the serum levels of GnRH,FSH,LH,and E_(2) in the model group were significantly decreased(P<0.05).The pregnant uterus appeared pale with poor blood supply,fewer and irregularly distributed embryo implantation sites,obvious bleeding points at embryo adhesion sites,a significantly increased abortion rate(P<0.05),and a significantly decreased uterine coefficient(P<0.05).The endometrial tissue showed reduced vascular distribution,fewer decidual cells with significant damage,and a large amount of intrauterine congestion.Compared with the model group,the serum levels of GnRH,FSH,LH,and E_(2) in all treatment groups were significantly increased(P<0.05).The embryo implantation status in the pregnant uterus was improved to varying degrees,the abortion rate was significantly reduced(P<0.05),the uterine coefficient was significantly increased(P<0.05),and the damage and congestion of decidual cells in the pregnant uterine tissue were improved to varying degrees.(2)A total of 951 differentially expressed proteins were screened between the normal group versus model group,and 1221 differentially expressed proteins were screened between the model group versus Pang’s Antai Zhixue Decoction group,resulting in 550 potential intervention targets of Pang’s Antai Zhixue Decoction on spontaneous abortion(intersection targets).PPI analysis identified 159 hub targets,including Pten,Polr2c,Fbn1,Polr1a,Col1a2,Dcn,Bgn,Fmod,Cd74,and Col6a2.GO functional and KEGG pathway analysis showed that the intervention of Pang’s Antai Zhixue Decoction in spontaneous abortion was closely related to the focal adhesion signaling pathway,involving the upregulation of key targets such as Akt3,Col6a1,Cav1,and Mylk,and the downregulation of Pten.(3)Compared with the normal group,the protein expression level of Pten in the pregnant uterine tissue of the model group was significantly increased(P<0.05),while the protein expression levels of Akt3,Col6a1,Cav1,and Mylk were significantly decreased(P<0.05).Compared with the model group,the protein expression level of Pten in the pregnant uterine tissue of all treatment groups was significantly decreased(P<0.05),while the protein expressions levels of Akt3,Col6a1,Cav1,and Mylk were significantly increased(P<0.05).Conclusions Pang’s Antai Zhixue Decoction has a significant intervention effect on spontaneous abortion in rats.Its mechanism may be related to the regulation of the focal adhesion pathway,downregulation of Pten protein expression,upregulation of Akt3,Col6a1,Cav1,and Mylk protein expression,and subsequent effects on trophoblast cell apoptosis.
作者
马丽亚
张童
郭申
周立汕
杜婧雯
俎兆轩
于梦
刘雨涵
张明昊
张大伟
MA Liya;ZHANG Tong;GUO Shen;ZHOU Lishan;DU Jingwen;ZU Zhaoxuan;YU Meng;LIU Yuhan;ZHANG Minghao;ZHANG Dawei(Department of Educational Administration,Henan University of Chinese Medicine,Zhengzhou 450046 Henan,China;The Fifth Clinical Medical College,Henan University of Chinese Medicine,Zhengzhou 450002 Henan,China;School of Medicine,Henan University of Chinese Medicine,Zhengzhou 450046 Henan,China;Third Affiliated Hospital,Henan University of Chinese Medicine,Zhengzhou 450008 Henan,China)
出处
《中药新药与临床药理》
北大核心
2025年第5期780-790,共11页
Traditional Chinese Drug Research and Clinical Pharmacology
基金
河南省自然科学基金面上项目(242300420168)
河南省重点研发与推广(科技攻关)项目(222102310139)
河南省中医药科学研究专项重大课题(2023ZYZD09)
河南省中医药科学研究专项重点课题(20-21ZY1053)。
