【作者】 张玲玲；

【导师】 方兰兰；

【作者基本信息】 郑州大学 ， 妇产科学（生殖医学）（专业学位）， 2024， 硕士

【摘要】 背景卵巢是女性体内重要的性腺及内分泌器官,其功能受到下丘脑-垂体-卵巢轴(Hypothalamus pituitary ovary,HPO)的复杂调控。由下丘脑合成并释放的促性腺激素释放激素(Gonadotropin-releasing hormone,GnRH)作用于垂体,垂体生成的卵泡刺激素(Follicle-stimulating hormone,FSH)和黄体生成素(Luteinizing hormone,LH)与卵巢颗粒细胞/卵泡膜细胞上的FSH受体/LH受体结合,活化下游信号通路,促进类固醇甾体类激素的生成,以维持女性正常生殖生理功能。其中,主要由卵巢颗粒细胞生成的雌激素与卵泡膜细胞/黄素化颗粒细胞分泌的孕激素是维持女性正常生殖功能的关键,二者协同调控月经周期、卵子发育、妊娠发展及其他女性生殖系统相关的生理过程。此外,雌孕激素对于维持女性骨骼健康、保护心血管系统、调节大脑功能及免疫功能等有重要作用。因此维持雌孕激素的平衡和正常分泌对于女性健康至关重要。女性类固醇甾体激素主要由胆固醇在卵巢内经一系列酶催化反应转化而成。胆固醇由线粒体外膜转运至线粒体内膜,并在位于此处的P450侧链裂解酶(P450 side-chain cleavage enzyme,P450scc)的催化作用下转化为孕烯醇酮,后者在 3β-羟基类固醇脱氢酶(Hydroxysteroid dehydrogenase,HSD)、17α-羟化酶/17,20-裂环酶(17α-hydroxylase/17,20-lyase,CYP17A1)和 17β-HSD 作用下转化为孕酮和睾酮,随后在芳香化酶(Aromatase,AROM)作用下最终生成雌激素。其中,胆固醇由细胞质转运到线粒体内部是女性甾体类激素生成的首要步骤,此转运过程是由类固醇激素合成急性调节蛋白(Steroidogenic acute regulatory protein,StAR)所介导的。因为StAR的结构域具有结合胆固醇和线粒体膜的能力,并且可以特异性地促进胆固醇转运到线粒体内,为孕激素和后续雌激素的合成提供所必需的前体物质,所以StAR是女性体内雌孕激素生成的关键限速酶。因此,探究调控StAR表达的机制对于维持女性体内雌孕激素水平正常及女性生殖系统健康具有重要意义。虫草素(Cordycepin,COR)是一种嘌呤核苷衍生物,能够识别并结合腺苷受体(Adenosine receptor,AR)、死亡受体(Death receptor,DR)和表皮生长因子受体(Epidermal growth factor receptor,EGFR),以及激活 PI3K/AKT、MAPK/ERK1/2、TGF-β/Smad和NF-κB等信号通路发挥其生物学功能,如抗氧化、免疫调节、降血脂、抗炎、抗癌、抗菌、抗病毒和降血糖等。此外,COR还可以增强两性性欲,并恢复受损的生殖功能。在女性生殖系统中,COR可以延缓卵母细胞排卵后老化,提高玻璃化卵母细胞的发育能力,还对卵巢癌、宫颈癌、子宫内膜癌和绒毛膜癌等女性生殖系统肿瘤的治疗发挥有益作用。在小鼠睾丸间质细胞中,COR通过结合AR亚型中的A1-、A2a-和A3-AR调控cAMP-PKA-StAR通路刺激睾酮生成。然而,COR调控女性生殖活动的作用机制及其是否参与调控女性生殖类固醇激素的生成及分子机制未见报道。因此,本研究首次探讨COR在人黄素化颗粒(Human granulosa-lutein cell,hGL)细胞中对StAR的调控作用及分子机制,不仅揭示COR调控甾体激素生成的作用和机制,亦为类固醇激素生成障碍相关疾病的治疗提供新的思路。目的研究COR在人颗粒细胞中对StAR的调控作用及分子机制。方法1.以人卵巢颗粒样肿瘤细胞系KGN、原代培养体外受精胚胎移植(In vitro fertilization and embryo transfer,IVF-ET)患者的人原代黄素化颗粒细胞(Human granulosa-lutein cell,hGL)作为实验模型。以 100 μM COR 在用不同时间(3h、6h、12h、24h)处理KGN细胞,关注其对StAR mRNA水平的调控作用。并以100 μM COR处理KGN细胞24 h,检测KGN细胞的细胞活力。之后采用不同时间(3 h、6 h、12 h、24 h)和(12 h、24 h)及相同浓度梯度(10-200 μM)的COR处理KGN、hGL细胞,关注COR对StAR蛋白水平的调控作用。2.确定最佳作用浓度和时间后,探究COR调控StAR表达所需要的细胞膜受体。为探究四种亚型AR是否介导此过程,分别使用使用A1-、A2a-、A2b-和 A3-AR 抑制剂 SLV-320、ZM241385、CVT-6683、VUF5574 处理 KGN、hGL细胞,观察COR处理后StAR表达的变化。3.为了进一步探究参与COR调控StAR表达过程中的信号通路,观察加药不同时间下AKT、ERK1/2蛋白表达水平,确定该过程中p-AKT、p-ERK的活化对下游的影响。分别使用能被COR结合并活化的A1-、A2a-和A3-AR的抑制剂SLV-320、ZM241385、VUF5574处理KGN细胞,再次观察加药后p-AKT、p-ERK表达的变化,进一步明确COR通过结合哪种AR活化p-AKT、p-ERK信号通路。4.分别使用p-AKT、p-ERK信号通路的抑制剂LY294002和U0126处理KGN、hGL细胞,观察加药后StAR表达的变化,进而明确COR是否通过活化p-AKT、p-ERK信号通路调控StAR。5.以不同时间(3 h、6 h、12 h)处理KGN和hGL细胞后,观察转录因子特异性蛋白1(Specificity protein 1,SP1)的蛋白质水平变化。此外,为了进一步探究SP1是否参与COR调控StAR的过程,使用A1-、A2a-和A3-AR抑制剂SLV-320、ZM241385、VUF5574 处理 KGN、hGL 细胞,观察 COR 对 SP1 表达的影响是否能被阻断。6.使用ERK1/2信号通路抑制剂U0126处理KGN、hGL细胞,观察COR对SP1表达的影响是否能被阻断。为进一步证实SP1在StAR表达调控中的作用,采用过表达SP1的方法,观察COR对StAR表达水平的影响。结果1.COR(100μM)以时间依赖的方式抑制KGN细胞中StAR mRNA的表达但并不影响KGN细胞活力,且在24 h时效果最显著;不同浓度COR(10-200μM)处理KGN细胞,结果发现100 μM COR可显著降低StAR蛋白水平,且在hGL细胞中获得相同效果。故采用100 μM COR进行后续实验。2.COR对StAR表达的抑制作用可被A1-、A2a-和A3-AR的受体抑制剂SLV-320、ZM241385、VUF5574 阻断,表明在 KGN 及 hGL 细胞中,COR 通过结合A1AR、A2aAR、A3AR抑制StAR的表达。3.在KGN及hGL细胞中,COR作用10 min可活化ERK1/2、AKT信号通路。然而在KGN细胞中,A1AR、A2aAR抑制剂SLV-320、ZM241385可以阻断ERK1/2、AKT信号通路的活化,而抑制A3AR并不影响ERK1/2、AKT信号通路的活化。4.在KGN及hGL细胞中,阻断ERK1/2信号通路可削弱COR对StAR表达的抑制作用,而阻断AKT信号通路并无显著改变。5.COR抑制KGN及hGL细胞中转录因子SP1的表达,且该作用被A1-、A2a-和A3-AR的抑制剂SLV-320、ZM241385、VUF5574及MEK特异性抑制剂U0126阻断。6.在KGN细胞中,过表达SP1可削弱COR对StAR表达的抑制作用。结论1.在人颗粒细胞中,COR通过结合A1AR、A2aAR、A3AR抑制StAR表达。2.在人颗粒细胞中,COR通过结合A1AR、A2aAR激活ERK1/2信号通路而非AKT信号通路抑制StAR表达。3.在人颗粒细胞中,COR通过结合A1AR、A2aAR、A3AR或激活ERK1/2信号通路抑制SP1表达间接抑制StAR表达,SP1可作为COR抑制StAR表达的媒介物。更多还原

【Abstract】 BackgroundThe ovary is a crucial gonadal and endocrine organ in the female body,with its function intricately regulated by the hypothalamus-pituitary-ovary(HPO)axis.Gonadotropin-releasing hormone(GnRH),synthesized and released by the hypothalamus,acts on the pituitary gland to stimulate the production of follicle-stimulating hormone(FSH).FSH,along with luteinizing hormone(LH),binds to their respective receptors on ovarian granulosa cells/theca cells,activating downstream signaling pathways that promote steroid synthesis.These steroids are essential for maintaining normal reproductive physiological functions in women.Notably,estrogen primarily produced by ovarian granulosa cells and progesterone secreted by theca cells/luteinized granulosa cells play pivotal roles in regulating the menstrual cycle,oocyte development,pregnancy progression,and other physiological processes related to the female reproductive system.Moreover,estrogen and progesterone significantly contribute to bone health maintenance,cardiovascular protection as well as regulation of brain function and immune response in women.Therefore,it is imperative to maintain a balanced secretion of estrogen and progesterone for optimal women’s health.The female steroid hormones are mainly converted from cholesterol through a series of enzymatic reactions in the ovary.Cholesterol is transported from the outer mitochondrial membrane to the inner mitochondrial membrane,where it is converted to pregnenolone by the P450 side-chain cleavage enzyme(P450scc).Under the action of 3β-hydroxy steroid dehydrogenase(3β-HSD),17α-hydroxylase/17,20-lyase(CYP17A1),and 17β-hydroxysteroid dehydrogenase(17β-HSD),pregnenolone can be further converted to progesterone and testosterone,and then to estrogen under the action of Aromatase(AROM).Cholesterol transport from cytoplasm to mitochondria,which is mediated by Steroidogenic acute regulatory protein(StAR),is the primary step in female steroidogenesis.Because the StAR domain can bind cholesterol and mitochondrial membrane,and specifically promotes cholesterol transport into mitochondria,providing essential precursors for progesterone and subsequent estrogen synthesis.Thus StAR is a key rate-limiting enzyme in the production of estrogen and progesterone in females.Therefore,exploring the regulatory mechanism of StAR expression is of great significance for maintaining the normal level of estrogen and progesterone in females and the health of the female reproductive system.Cordycepin(COR)is a purine nucleoside derivative that exhibits diverse biological activities,including antioxidant,immune-modulating,hypolipidemic,anti-inflammatory,anticancer,antibacterial,antiviral and hypoglycemic effects.Moreover,COR has been shown to enhance sexual desire in both males and females and improve impaired reproductive function.In the female reproductive system,COR regulates post-ovulation aging and enhances the developmental potential of vitrified oocytes.It also demonstrates beneficial effects on female reproductive system tumors such as ovarian cancer,cervical cancer,endometrial cancer and choriocarcinoma.In the male reproductive system,COR promotes the biosynthesis of male reproductive steroid hormones in vivo and in vitro,thereby promoting testosterone production to ameliorate age-related testicular dysfunction and enhance sexual function.Additionally,it exhibits significant efficacy in treating male reproductive system tumors like testicular stromal cell tumors.However,there have been no studies conducted thus far investigating the regulatory mechanisms of COR on female reproductive steroid hormone production.Therefore,this study delves into the regulatory effect and molecular mechanism of COR on StAR expression in human granulosa cells,aiming to provide new ideas for the treatment of steroid hormone production disorder-related diseases.Cordycepin(COR)is a purine nucleoside derivative that recognizes and binds to the Adenosine receptor(AR),Death receptor(DR),and Epidermal growth factor receptor(EGFR).COR can activate PI3K/AKT,MAPK/ERK1/2,TGF-β/Smad,and NF-κB signaling pathways to exert its biological functions including antioxidant activity,immune regulation,lipid-lowering effects,anti-inflammatory properties,anticancer potential,antibacterial and antiviral activities as well as hypoglycemic effects.Additionally,COR has been found to enhance sexual desire and restore impaired reproductive function.In the female reproductive system context specifically,COR has demonstrated the ability to delay post-ovulatory aging and improve the developmental capacity of vitrified oocytes.Furthermore,it exhibits beneficial effects on female reproductive system tumors such as ovarian cancer,cervical cancer,endometrial cancer,and choriocarcinoma.In mouse Leydig cells research studies have shown that COR stimulates testosterone production by regulating the cAMP-PKA-StAR pathway through binding with A1-,A2a-,and A3-AR isoforms of AR.However,the mechanism underlying how COR regulates female reproductive activity remains unclear.Moreover,it is yet unknown whether COR plays a role in regulating steroid hormone production in females or what molecular mechanisms are involved.Therefore,this study aims to investigate for the first time both the regulatory effect of COR on StAR in human granulosa-lutein cells(hGL)and its associated molecular mechanism.This investigation will not only shed light on the role played by COR in steroidogenesis regulation but also provide novel insights for treating disorders related to steroidogenesis.ObjectiveTo investigate the role and molecular mechanism of COR on StAR expression and in human granulosa cells.MethodsKGN cells and primary human granulosa cell line hGL were used as study objects.Firstly,KGN cells were treated for 3,6,12 and 24 h to observe the regulation of StAR mRNA and the effect of 24 h treatment on KGN cell viability.The regulation effect of COR on the level of StAR protein was studied by treating COR with different times and concentrations.The expression of adenosine receptors in KGN cells and hGL cells was inhibited and the protein levels of StAR were detected by Western blot.Selective receptor inhibitors were used to block the activation of the receptor,and the phosphorylation level of the signaling pathway was detected.Specific chemical inhibitors were further applied to block the signaling pathway and detect the changes in StAR expression.The regulation effect of COR on the expression of transcription factor SP1 was observed by treating KGN cells and hGL cells at different times.Then,selective receptor inhibitors and specific inhibitors were used to block the signaling pathway,and the protein level of SP1 was detected by Western blot.Finally,the transcription factor SP1 was overexpressed in KGN cells to further determine whether COR affected the protein level of StAR by regulating the expression of SP1.Results1.COR(100 μM)inhibited the expression of StAR mRNA in KGN cells in a time-dependent manner but did not affect the viability of KGN cells,and the effect was most significant at 24 h.When KGN cells were treated with different concentrations of COR(10-200 μM),100 μM COR significantly decreased the level of StAR protein.The same effect was obtained in hGL cells.Therefore,100 μM COR was used for subsequent experiments.2.The inhibitory effect of COR on the expression of StAR could be blocked by A1-,A2a-,and A3-AR receptor inhibitors SLV-320,ZM241385,and VUF5574,indicating that COR inhibits the expression of StAR through A1-,A2a-and A3-AR in KGN and hGL cells.3.ERK1/2 and AKT signaling pathways were activated by COR for 10 min in KGN and hGL cells.However,A1AR and A2aAR inhibitors SLV-320 and ZM241385 blocked the activation of ERK1/2 and AKT signaling pathways in KGN cells,while A3AR inhibition did not affect the activation of ERK1/2 and AKT signaling pathways.4.Blocking the ERK1/2 signaling pathway attenuated the inhibitory effect of COR on StAR expression in KGN and hGL cells while blocking the AKT signaling pathway had no significant change.5.COR inhibited the expression of transcription factor SP1 in KGN and hGL cells,and this effect was blocked by A1-,A2a-,and A3-AR inhibitors SLV-320,ZM241385,VUF5574,and MEK-specific inhibitor U0126.6.Overexpression of SP1 attenuated the inhibitory effect of COR on StAR expression in KGN cells.Conclusions1.In human granulosa cells,COR downregulates StAR expression by binding to A1AR、A2aAR and A3AR.2.In human granulosa cells,COR inhibits StAR expression by activating the ERK1/2 signaling pathway rather than the AKT signaling pathway through binding A1 AR and A2aAR.3.COR indirectly inhibits StAR expression in human granulosa cells by binding to A1 AR,A2aAR,and A3AR or inhibiting SP1 expression by activating the ERK1/2 signaling pathway,and SP1 can be used as a mediator for COR to inhibit StAR expression.更多还原