寿胎丸抑制滋养细胞miR-29c-3p/Caspase-8/GSDME焦亡信号轴治疗URSA的作用与机制研究  

Effect and mechanism of Shoutai Pill on inhibiting trophocyte miR-29c-3p/Caspase-8/GSDME pyroptosis signaling axis to treat URSA

作　　者：许珂 张振[2] 魏然 褚楚 李莉华 刘泳琳 高淑凤 范楠楠 周苗苗 石飞飞 李霞(指导)[2] XU Ke;ZHANG Zhen;WEI Ran;CHU Chu;LI Lihua;LIU Yonglin;GAO Shufeng;FAN Nannan;ZHOU Miaomiao;SHI Feifei;LI Xia(Innovative Research Institute of Traditional Chinese Medicine,Shandong University of Traditional Chinese Medicine,Jinan 250355,China)

机构地区：[1]山东第一医科大学(山东省医学科学院)基础医学院,济南250062 [2]山东中医药大学中医药创新研究院,济南250355

出　　处：《中国免疫学杂志》2023年第4期750-758,共9页Chinese Journal of Immunology

基　　金：国家自然科学基金面上项目(82274575,81873337);山东省自然科学基金面上项目(ZR2019MH039);山东省中央引导地方科技发展资金项目(YDZX20203700001407);泰山学者青年专家项目(Tsqn201812125);济南市科技局科研带头人工作室项目(2020GXRC050)资助。

摘　　要：目的:探讨寿胎丸通过靶向miR-29c-3p/Caspase-8/GSDME信号通路减轻滋养细胞焦亡治疗不明原因复发性自然流产(URSA)的作用与关键机制。方法:全转录组高通量测序分析正常早孕妇女(NP组)和URSA患者绒毛组织中的差异mRNAs;Western blot检测Caspase-8、Caspase-3和GSDME表达及活化水平;qRT-PCR检测miR-29c-3p和CASP8 mRNA表达。体外调控HTR-8/SVneo细胞miR-29c-3p表达,分析其对Caspase-8和细胞焦亡相关分子表达及细胞培养上清中IL-1β、IL-18蛋白水平的影响。补肾固冲经典方剂寿胎丸含药血清作用于HTR-8/SVneo细胞后,观察miR-29c-3p、CASP8 mRNA表达和Caspase-8、Caspase-3及GSDME蛋白表达及活性,并检测培养上清中IL-1β与IL-18蛋白水平。CBA/J雌鼠和DBA/2雄鼠交配构建URSA小鼠模型,随机分为溶剂对照组、寿胎丸组、寿胎丸+INC组及寿胎丸+miR-29c-3p inhibitor组,观察各组胚胎吸收情况;检测各组胎盘组织中miR-29c-3p、Caspase-8、Caspase-3和GSDME表达及血清中IL-1β、IL-18蛋白水平。结果:与NP组比较,URSA患者绒毛组织Caspase-8、Caspase-3及GSDME表达及活化水平显著升高,而miR-29c-3p表达显著降低,且与CASP8 mRNA表达呈显著负相关。上调HTR-8/SVneo细胞miR-29c-3p表达后,CASP8 mRNA表达显著降低,Caspase-8、Cleaved Caspase-8、Cleaved Caspase-3、GSDME-N及细胞培养上清中IL-1β、IL-18蛋白水平明显下降,而抑制miR-29c-3p表达作用相反。双荧光素酶报告基因系统显示,miR-29c-3p能够与CASP8 mRNA 3’UTR直接结合。与对照组血清相比,寿胎丸含药血清能够上调HTR-8/SVneo细胞miR-29c-3p表达,而Caspase-8、Caspase-3、GSDME活性及IL-1β、IL-18表达均显著下调。与溶剂对照组相比,寿胎丸治疗组小鼠胚胎吸收减少,胚胎吸收率显著降低,小鼠胎盘组织中miR-29c-3p显著升高,Caspase-8、Caspase-3、GSDME活性及血清中IL-1β、IL-18表达显著下降;而miR-29c-3p inhibitor能够抑制寿胎丸的妊娠保护作用。结论:miR-29Objective:To investigate role and key mechanism of Shoutai Pill in treating unexplained recurrent spontaneous abortion(URSA)by targeting miR-29c-3p/Caspase-8/GSDME signaling pathway to alleviate trophocyte pyroptosis.Methods:Differen‐tial mRNAs in villous tissue of normal pregnant women(NP group)and URSA patients were analyzed by full transcription high-throughput sequencing.Expressions and activation of Caspase-8,Caspase-3 and GSDME were analyzed by Western blot.miR-29c-3p and CASP8 mRNA expressioins were detected by qRT-PCR.miR-29c-3p expression in HTR-8/SVneo cell was regulated in vitro,and effects on Caspase-8 and pyroptosis-related molecules and protein levels of IL-1βand IL-18 in cell culture supernatant were analyzed.After Bushenguchong's classic prescription Shoutai Pill containing serum acted on HTR-8/SVneo cells,miR-29c-3p,CASP8 mRNA,protein expressions and activity of Caspase-8,Caspase-3 and GSDME were observed,and expressions of IL-1βand IL-18 proteins in culture supernatant were detected.CBA/J female mice and DBA/2 male mice were mated to construct URSA mouse model,and ran‐domly divided into vehicle control group,Shoutai Pill group,Shoutai Pill+INC group,Shoutai Pill+miR-29c-3p inhibitor group,embryo resorption in each group were observed;expressions of miR-29c-3p,Caspase-8,Caspase-3 and GSDME in placental tissue and serum IL-1βand IL-18 protein levels in each group were detected.Results:Compared with NP group,expression and activation levels of Caspase-8,Caspase-3 and GSDME in villous tissues of URSA patients were significantly increased,while miR-29c-3p was signifi‐cantly decreased,and negatively correlated with CASP8 mRNA.After up-regulating miR-29c-3p in HTR-8/SVneo cells,CASP8 mRNA was significantly decreased,and protein levels of Caspase-8,Cleaved Caspase-8,Cleaved Caspase-3,GSDME-N;and IL-1β,IL-18 in cell culture supernatant were significantly decreased.However,inhibition of miR-29c-3p expression had opposite effect.Double luciferase reporter gene system showed that miR-29c-3p could

关 键 词：原因不明复发性自然流产 miR-29c-3p 寿胎丸 滋养细胞 细胞焦亡 

分 类 号：R285.5[医药卫生—中药学]