摘要
目的探究滋肾育胎(ZSYT)丸对人卵巢颗粒细胞焦亡的调控作用及其机制。方法将30只8周龄雌性SD大鼠随机分为对照组和给药组,每组各15只,分别灌胃等量蒸馏水或ZSYT混悬液,重复7 d后腹主动脉采血,制备对照组血清和含药血清。将人卵巢颗粒细胞(KGN细胞)分为A组(对照)、B组(诱导焦亡)、C组(诱导焦亡+5%含药血清)、D组(诱导焦亡+10%含药血清)、E组(诱导焦亡+15%含药血清)、F组(诱导焦亡+20%含药血清)和G组(诱导焦亡+25%含药血清)。B~G组细胞孔中加入LPS联合尼日利亚菌素处理,A组加入等体积不含药培养基;而后C~G组加入相应浓度的ZSYT含药血清,A、B组加入20%对照组血清,置于培养箱中培养。通过CCK-8法检测A~G组细胞的细胞活力,检测A、B、F组细胞培养基上清液中的乳酸脱氢酶(LDH)水平和雌二醇(E2)合成能力,通过RT-qPCR和蛋白免疫印迹(WB)实验检测A、B、F组细胞中焦亡相关分子以及雌激素合成关键酶CYP19a1 mRNA和蛋白的表达水平。结果CCK-8实验结果显示,与B组相比,E~G组细胞活力显著升高(F=80.08,t_(LSD)=7.35~12.57,P<0.05)。与B组相比,F组细胞培养基上清液中LDH水平以及细胞中NLRP3、GSDMD、IL-1β、IL-18、Caspase-1 mRNA的相对表达水平和NLRP3、GSDMD、GSDMD-N、Caspase-1蛋白的相对表达水平显著降低(F=21.66~319.40,t_(LSD)=3.88~13.65,P<0.05),E2合成能力以及细胞中CYP19a1 mRNA和蛋白的相对表达水平显著升高(F=72.26~151.01,t_(LSD)=2.60~8.38,P<0.05)。结论ZSYT丸可能通过抑制NLRP3/GSDMD通路降低卵巢颗粒细胞焦亡水平,并恢复其E2合成能力。
Objective To investigate the regulatory role and mechanism of action of Zishen Yutai(ZSYT)pills on the pyroptosis of human ovarian granulosa cells.Methods A total of 30 female Sprague-Dawley rats,aged 8 weeks,were randomly divided into control group and treatment group,with 15 rats in each group.The rats in the treatment group were given ZSYT suspension by gavage,while those in the control group were given an equal volume of distilled water by gavage.After treatment for 7 d,blood samples were collected from the abdominal aorta to prepare control serum and serum containing ZSYT.Human ovarian granulosa cells(KGN cells)were divided into group A(control),group B(inducing pyroptosis),group C(inducing pyroptosis+5%drug-containing serum),group D(inducing pyroptosis+10%drug-containing serum),group E(inducing pyroptosis+15%drug-containing serum),group F(inducing pyroptosis+20%drug-containing serum),group G(inducing pyroptosis+25%drug-containing serum).The cells in groups B-G were treated with LPS combined with nigericin in the culture wells,while those in group A were cultured in an equal volume of drug-free medium;subsequently,the cells in groups C-G were added with serum containing ZSYT at the corresponding concentrations,and those in groups A and B were added with 20%control serum;all groups were cultured in an incubator.CCK-8 assay was used to measure cell viability in groups A-G;groups A,B,and F were measured in terms of the level of lactate dehydrogenase(LDH)in supernatant and estradiol(E2)synthesis capacity;RT-qPCR and Western blotting were used to measure the mRNA and protein expression levels of pyroptosis-related molecules and the key estrogen synthesis enzyme CYP19a1 in groups A,B,and F.Results CCK-8 assay showed that compared with group B,groups E-G had a significant increase in cell viability(F=80.08,t_(LSD)=7.35-12.57,P<0.05).Compared with group B,group F had significant reductions in the level of LDH in supernatant,the relative mRNA expression levels of NLRP3,GSDMD,IL-1β,IL-18,and caspase-1 in cells,and the relative protein expression levels of NLRP3,GSDMD,GSDMD-N,and caspase-1(F=21.66-319.40,t_(LSD)=3.88-13.65,P<0.05),as well as significant increases in E2 synthesis capacity and the relative mRNA and protein expression levels of CYP19a1 in cells(F=72.26-151.01,t_(LSD)=2.60-8.38,P<0.05).Conclusion ZSYT pills reduce the level of pyroptosis in ovarian granulosa cells and restores their E2 synthesis capacity by inhibiting the NLRP3/GSDMD pathway.
作者
李娟娥
赵峻
杜鑫
LI Juan′e;ZHAO Jun;DU Xin(Qingdao Medical College of Qingdao University,Qingdao 266003,China)
出处
《精准医学杂志》
2025年第6期516-521,共6页
Journal of Precision Medicine
基金
国家自然科学基金青年项目(81904244)
山东省“十二五”中医药科技发展计划项目(2011-218)
北京大学人民医院学术新星项目(RS2021-15)。
