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-29c-3p低表达促进Caspase-8/GSDME介导的滋养细胞焦亡,参与URSA发生;寿胎丸靶向miR-29c-3p/Caspase-8/GSDME信号通路,减轻滋养细胞焦亡,进而保护妊娠,治疗URSA。
Objective: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 be directly combined with CASP8 mRNA 3'UTR.Compared with control serum,Shoutai Pill containing serum up-regulated miR-29c-3p in HTR-8/SVneo cells,while activity levels of Caspase-8,Caspase-3,GSDME,IL-1βand IL-18 were obviously down-regulated.Compared with vehicle control group,embryo resorption of mice was reduced,embryo resorption rate was decreased significantly in Shoutai Pill treatment group;miR-29c-3p in placental tissue was significantly increased,activities of Caspase-8,Caspase-3 and GSDME in placental tissue as well as expressions of IL-1βand IL-18 in serum were markedly decreased;but miR-29c-3p inhibitor was able to inhibit protective effect of pregnancy by Shoutai Pill.Conclusion:Low-expression of miR-29c-3p promotes Caspase-8/GSDME-mediated trophocyte pyroptosis and participates in occur‐rence of URSA;Shoutai Pill targets miR-29c-3p/Caspase-8/GSDME signaling pathway,which reduces trophocyte pyroptosis,thus pro‐tecting and treating URSA.
作者
许珂
张振
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)
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)
出处
《中国免疫学杂志》
CAS
CSCD
北大核心
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。
作者简介
许珂,女,在读硕士,主要从事中药生殖表观遗传免疫调控研究,E-mail:1165208529@qq.com。;通信作者:李霞,女,博士,教授,博士生导师,主要从事中药生殖表观遗传免疫调控研究,E-mail:60230033@sdutcm.edu.cn。
