G-protein coupled receptors and synaptic plasticity in sleep deprivation

Insufficient sleep has been correlated to many physiological and psychoneurological disorders.Over the years,our understanding of the state of sleep has transcended from an inactive period of rest to a more active state involving important cellular and molecular processes.In addition,during sleep,electrophysiological changes also occur in pathways in specific regions of the mammalian central nervous system(CNS).Activity mediated synaptic plasticity in the CNS can lead to long-term and sometimes permanent strengthening and/or weakening synaptic strength affecting neuronal network behaviour.Memory consolidation and learning that take place during sleep cycles,can be affected by changes in synaptic plasticity during sleep disturbances.G-protein coupled receptors(GPCRs),with their versatile structural and functional attributes,can regulate synaptic plasticity in CNS and hence,may be potentially affected in sleep deprived conditions.In this review,we aim to discuss important functional changes that can take place in the CNS during sleep and sleep deprivation and how changes in GPCRs can lead to potential problems with therapeutics with pharmacological interventions.

G protein-coupled estrogen receptor in colon function, immune regulation and carcinogenesis

Estrogens play important roles in the development and progression of multiple tumor types.Accumulating evidence points to the significance of estrogen action not only in tumors of hormonally regulated tissues such as the breast,endometrium and ovary,but also in the development of colorectal cancer(CRC).The effects of estrogens in physiological and pathophysiological conditions are mediated by the nuclear estrogen receptorsαandβ,as well as the membranebound G protein-coupled estrogen receptor(GPER).The roles of GPER in CRC development and progression,however,remain poorly understood.Studies on the functions of GPER in the colon have shown that this estrogen receptor regulates colonic motility as well as immune responses in CRC-associated diseases,such as Crohn’s disease and ulcerative colitis.GPER is also involved in cell cycle regulation,endoplasmic reticulum stress,proliferation,apoptosis,vascularization,cell migration,and the regulation of fatty acid and estrogen metabolism in CRC cells.Thus,multiple lines of evidence suggest that GPER may play an important role in colorectal carcinogenesis.In this review,we present the current state of knowledge regarding the contribution of GPER to colon function and CRC.

Isoleucine, an Essential Amino Acid, Induces the Expression of Human <i>β</i>Defensin 2 through the Activation of the G-Protein Coupled Receptor-ERK Pathway in the Intestinal Epithelia

Anti-microbial peptides are essential for the intestinal innate immunity that protects the intestinal epithelia from attacks by foreign pathogens. Human β-defensin (HBD) is one of the pivotal anti-microbial peptides that are expressed in the colonic epithelia. This study investigated the effect and the signaling mechanism of inducible β-defensin HBD2 by an essential amino acid, isoleucine (Ile) in colonic epithelial cells. Here we examined the expression level of HBD2 on induction of Ile in epithelial cells, and checked this pathway. HBD2 mRNA was induced by co-incubation with IL-1α and Ile in Caco2 cells, but not by Ile alone. An inhibitor of either ERK or Gi, a subunit of G-proteins, reduced the induction of HBD2 mRNA by Ile. The treatment with Ile also increased the intracellular calcium ion concentration, thus suggesting that the GPCR and ERK signaling pathway mediate the effects of Ile. These results indicate that an essential amino acid, Ile, enhances the expression of an inducible β-defensin, namely HBD2, by IL-1α through the activation of GPCRs and ERK signaling pathway. The administration of Ile may therefore represent a possible option to safely treat intestinal inflammation.

New insights into sodium transport regulation in the distal nephron:Role of G-protein coupled receptors

The renal handling of Na^+ balance is a major determinant of the blood pressure(BP) level. The inability of the kidney to excrete the daily load of Na+ represents the primary cause of chronic hypertension. Among the different segments that constitute the nephron, those present in the distal part(i.e., the cortical thick ascending limb, the distal convoluted tubule, the connecting and collecting tubules) play a central role in the fine-tuning of renal Na^+ excretion and are the target of many different regulatory processes that modulate Na^+ retention more or less efficiently. G-protein coupled receptors(GPCRs) are crucially involved in this regulation and could represent efficient pharmacological targets to control BP levels. In this review, we describe both classical and novel GPCR-dependent regulatory systems that have been shown to modulate renal Na^+ absorption in the distal nephron. In addition to the multiplicity of the GPCR that regulate Na^+ excretion, this review also highlights the complexity of these different pathways, and the connections between them.

Insights into the structural biology of G-protein coupled receptors impacts drug design for central nervous system neurodegenerative processes

In the last few years, there have been important new insights into the structural biology of G-protein coupled receptors. It is now known that allosteric binding sites are involved in the affinity and selec- tivity of ligands for G-protein coupled receptors, and that signaling by these receptors involves both G-protein dependent and independent pathways. The present review outlines the physiological and pharmacological implications of this perspective for the design of new drugs to treat disorders of the central nervous system. Specifically, new possibilities are explored in relation to allosteric and or- thosteric binding sites on dopamine receptors for the treatment of Parkinson＇s disease, and on muscarinic receptors for Alzheimer＇s disease. Future research can seek to identify ligands that can bind to more than one site on the same receptor, or simultaneously bind to two receptors and form a dimer. For example, the design of bivalent drugs that can reach homo/hetero-dimers of D2 dopa- mine receptor holds promise as a relevant therapeutic strategy for Parkinson＇s disease. Regarding the treatment of Alzheimer＇s disease, the design of dualsteric ligands for mono-oligomeric mus- carinic receptors could increase therapeutic effectiveness by generating potent compounds that could activate more than one signaling pathway.

脑小血管病患者外周血GPER30、NPAS4、FKBP5表达与认知功能障碍的相关性研究

目的探讨脑小血管病(CSVD)患者外周血G蛋白耦联雌激素受体30(GPER30)、神经元PAS结构域蛋白4(NPAS4)、FK506结合蛋白5(FKBP5)表达与认知功能障碍(CD)的相关性。方法前瞻性选取2022年1月至2023年12月郑州大学第二附属医院收治的227例CSVD患者,根据有无CD分为障碍组(n=66)与无障碍组(n=161)。比较两组患者的一般资料及外周血GPER30、NPAS4、FKBP5 m RNA表达水平,Logistic回归分析CSVD患者CD的影响因素,比较不同程度CD患者外周血GPER30、NPAS4、FKBP5 m RNA表达水平,采用Pearson法分析外周血GPER30、NPAS4、FKBP5 m RNA表达与蒙特利尔认知评估量表(Mo CA)评分的相关性。结果障碍组患者的年龄、病程分别为(72.49±5.68)岁、(2.69±0.78)年,明显高(长)于无障碍组的(67.51±7.04)岁、(2.31±0.62)年,差异均有统计学意义(P<0.05);障碍组患者的外周血GPER30 m RNA表达水平为1.02±0.17,明显低于无障碍组的1.66±0.31,NPAS4m RNA、FKBP5 m RNA表达水平分别为2.79±0.60、3.88±1.12,明显高于无障碍组的1.55±0.51、2.10±0.59,差异均有统计学意义(P<0.05);Logistic回归分析结果显示,年龄、病程、GPER30 m RNA、NPAS4 m RNA及FKBP5 m RNA均为CSVD患者CD的独立影响因素(P<0.05)。轻度组患者的外周血GPER30 m RNA表达水平为1.27±0.25,明显高于中重度组的0.70±0.12,NPAS4 m RNA、FKBP5 m RNA表达水平分别为2.31±0.58、3.19±1.07,明显低于中重度组的3.40±0.72、4.76±1.39,差异均有统计学意义(P<0.05);Pearson法分析结果显示,外周血GPER30 m RNA表达与CSVD患者Mo CA评分呈正相关(r=0.704,P<0.05),NPAS4 m RNA、FKBP5 m RNA与Mo CA评分呈负相关(r=-0.572、-0.542,P<0.05)。结论外周血GPER30、NPAS4、FKBP5是CSVD患者CD的独立相关因素,各指标表达水平与CD病情严重程度均具有一定相关性,可为临床判断CD、评估CD病情严重程度提供参考,以指导后续临床工作。

Association of hepatocyte-derived growth factor receptor/caudal type homeobox 2 co-expression with mucosal regeneration in active ulcerative colitis

AIM:To characterize the regeneration-associated stem cell-related phenotype of hepatocyte-derived growth factor receptor(HGFR)-expressing cells in active ulcerative colitis(UC).METHODS:On the whole 38 peripheral blood samples and 38 colonic biopsy samples from 18 patients with histologically proven active UC and 20 healthy control subjects were collected.After preparing tissue microarrays and blood smears HGFR,caudal type homeobox 2(CDX2),prominin-1(CD133) and Musashi-1conventional and double fluorescent immunolabelings were performed.Immunostained samples were digitalized using high-resolution Mirax Desk instrument,and analyzed with the Mirax TMA Module software.For semiquantitative counting of immunopositive lamina propria(LP) cells 5 fields of view were counted at magnification x 200 in each sample core,then mean ± SD were determined.In case of peripheral blood smears,30 fields of view with 100 μm diameter were evaluated in every sample and the number of immunopositive cells(mean ± SD) was determined.Using 337 nm UVA Laser MicroDissection system at least 5000 subepithelial cells from the lamina propria were collected.Gene expression analysis of HGFR,CDX2,CD133,leucine-rich repeat-containing G-protein coupled receptor 5(Lgr5),Musashi-1 and cytokeratin20(CK20) were performed in both laser-microdisscted samples and blood samples by using real time reverse transcription polymerase chain reaction(RT-PCR).RESULTS:By performing conventional and double fluorescent immunolabelings confirmed by RT-PCR,higher number of HGFR(blood:6.7 ± 1.22 vs 38.5 ±3.18;LP:2.25 ± 0.85 vs 9.22 ± 0.65;P < 0.05),CDX2(blood:0 vs 0.94 ± 0.64;LP:0.75 ± 0.55 vs 2.11± 0.75;P < 0.05),CD133(blood:1.1 ± 0.72 vs 8.3± 1.08;LP:11.1 ± 0.85 vs 26.28 ± 1.71;P < 0.05)and Musashi-1(blood and LP:0 vs scattered) positive cells were detected in blood and lamina propria of UC samples as compared to controls.HGFR/CDX2(blood:0 vs 1± 0.59;LP:0.8 ± 0.69 vs 2.06 ± 0.72,P < 0.05)and Musashi-1/CDX2(blood and LP:0 vs scattered) coexpressions were found in blood and lamina propria of UC samples.HGFR/CD133 and CD133/CDX2 coexpressions appeared only in UC lamina propria samples.CDX2,Lgr5 and Musashi-1 expressions in UC blood samples were not accompanied by CK20 mRNA expression.CONCLUSION:In active UC,a portion of circulating HGFR-expressing cells are committed to the epithelial lineage,and may participate in mucosal regeneration by undergoing mesenchymal-to-epithelial transition.

Identification of a key G-protein coupled receptor in mediating appressorium formation and fungal virulence against insects

Fungal G-protein coupled receptors(GPCRs)play essential roles in sensing environmental cues including host signals.The study of GPCR in mediating fungus-insect interactions is still limited.Here we report the evolution of GPCR genes encoded in the entomopathogenic Metarhizium species and found the expansion of Pth11-like GPCRs in the generalist species with a wide host range.By deletion of ten candidate genes MrGpr1–MrGpr10 selected from the six obtained subfamilies in the generalist M.robertsii,we found that each of them played a varied level of roles in mediating appressorium formation.In particular,deletion of MrGpr8 resulted in the failure of appressorium formation on different substrates and the loss of virulence during topical infection of insects but not during injection assays when compared with the wild-type(WT)strain.Further analysis revealed that disruption of MrGpr8 substantially impaired the nucleus translocation of the mitogen-activated protein kinase(MAPK)Mero-Fus3 but not the MAPK Mero-Slt2 during appressorium formation.We also found that the defect ofΔMrGpr8 could not be rescued with the addition of cyclic AMP for appressorium formation.Relative to the WT,differential expression of the selected genes have also been detected inΔMrGpr8.The results of this study may benefit the understanding of fungus-interactions mediated by GPCRs.

The complexity of G-protein coupled receptor-ligand interactions

The G-protein coupled receptors(GPCRs)play fundamental roles in the human biololgy and drug discovery.GPCRs function as signalling molecules that transduce extracellular signals into cells.The signalling transduction is generally triggered by interacting with ligands,including photons,ions,small organic compounds,peptides,proteins and lipids.In this review,we focus on interactions with diffusible ligands such as hormones and neurotransmitters.We discuss three aspects of the complexity of the GPCR-ligand interactions:functional selectivity of ligands,receptor subtype selectivity of ligands and orphan GPCRs.

Role of opioid receptor heterodimerization in pain modulation and tolerance development

Protein to protein interactions leading to homo/heteromerization of receptor is well documented in literature. These interactions leading to dimeric/oligomers formation of receptors are known to modulate their function, particularly in case of G-protein coupled receptors. The opioid receptor heteromers having changed pharmacological properties than the constituent protomers provides preferences for novel drug targets that could lead to potential analgesicactivity devoid of tolerance and physical dependence. Heterodimerization of opioid receptors appears to generate novel binding properties with improved specificity and lack of side effects. Further the molecules which can interact simultaneously to both the protomers of the heteromer, or to both the binding sites(orthosteric and allosteric) of a receptor protein could be potential therapeutic molecules. This review highlights the recent advancements in exploring the plausible role of heteromerization of opioid receptors in induction of tolerance free antinociception.

G protein-coupled receptors in energy homeostasis

G-protein coupled receptors(GPCRs)compromise the largest membrane protein superfamily which play vital roles in physiological and pathophysiological processes including energy homeostasis.Moreover,they also represent the up-to-date most successful drug target.The gut hormone GPCRs,such as glucagon receptor and GLP-1 receptor,have been intensively studied for their roles in metabolism and respective drugs have developed for the treatment of metabolic diseases such as type 2 diabetes(T2D).Along with the advances of biomedical research,more GPCRs have been found to play important roles in the regulation of energy homeostasis from nutrient sensing,appetite control to glucose and fatty acid metabolism with various mechanisms.The investigation of their biological functions will not only improve our understanding of how our body keeps the balance of energy intake and expenditure,but also highlight the possible drug targets for the treatment of metabolic diseases.The present review summarizes GPCRs involved in the energy control with special emphasis on their pathophysiological roles in metabolic diseases and hopefully triggers more intensive and systematic investigations in the field so that a comprehensive network control of energy homeostasis will be revealed,and better drugs will be developed in the foreseeable future.

Molecular mechanism of endocrine-disruptive effects induced by Bisphenol A:The role of transmembrane G-protein estrogen receptor 1 and integrin αvβ3

Bisphenol A(BPA) is one of the highest volume industrial products worldwide and has been widely used to make various products as the intermediates of polycarbonate plastics and epoxy resins.Inevitably, general population has been widely exposed to BPA due to extensive use of BPAcontaining products. BPA has similar chemical structure with the natural estrogen and has been shown to induce a variety of estrogen-like endocrine effects on organism in vivo or in vitro. High doses of BPA tend to act as antagonist of estrogen receptors(ERs) by directly regulating the genomic transcription. However, BPA at environmentally relevant low-dose always disrupt the biological function via a non-genomic manner mediated by membrane receptors, rather than ERs. Although some studies had investigated the non-genomic effects of low-dose BPA, the exact molecular mechanism still remains unclear. Recently, we found that membrane G protein-coupled estrogen receptor 1 and integrin αvβ3 and its relative signal pathways participate in the induction of male germ cell proliferation and thyroid transcription disruption by the low-dose BPA. A profound understanding for the mechanism of action of the environmentally relevant BPA exposure not only contributes to objectively evaluate and predict the potential influence to human health, but also provides theoretical basis and methodological support for assessing health effects trigged by other estrogen-like environmental endocrine disruptors. Based mainly on our recent findings, this review outlines the research progress of molecular mechanism on endocrine disrupting effects of environmental low-dose BPA, existing problems and some consideration for future studies.

GPER抑制星形胶质细胞活性减少OIR小鼠视网膜新生血管生成的机制研究

目的探究在氧诱导下的新生小鼠视网膜缺血模型(oxygen-induced retinopathy,OIR)中,G蛋白偶联雌激素受体(G proteincoupled estrogen receptor,GPER)减少OIR小鼠视网膜新生血管生成的机制。方法42只新生小鼠采用随机数字表法分为4组:常氧对照组(n=11)、OIR组(n=11)、G-1(GPER激动剂)组(n=10)和溶剂对照组(n=10)。出生后17 d(P17)时,眼球冰冻切片免疫荧光染色观察常氧对照组小鼠GPER在视网膜分布情况。G-1组和溶剂对照组在P12~P15时分别腹腔注射给予G-1[50μg/(kg·d)]或玉米油溶剂,P17时视网膜铺片免疫荧光染色观察视网膜血管标志物(IB4)、星形胶质细胞标志物胶质纤维酸性蛋白(glia fibrilary acidic protein,GFAP)表达,蛋白免疫印迹法定量检测视网膜血管内皮生长因子A(vascular endothelial growth factor A,VEGFA)、GFAP、炎症因子TNF-α、IGF-1、IL1-β蛋白表达情况。结果GPER存在于小鼠视网膜全层,在视网膜神经节细胞层与星形胶质细胞标志物GFAP共染。与常氧对照组相比,OIR组小鼠视网膜出现新生血管与无血管区,且GPER、VEGFA和GFAP表达增多,差异具有统计学意义(P<0.05);与溶剂对照组相比,G-1组视网膜新生血管生成减少,VEGFA、GFAP、TNF-α、IGF-1、IL1-β蛋白表达下降,差异具有统计学意义(P<0.05)。结论GPER能抑制星形胶质细胞活性,减少VEGFA、炎症因子释放,减少OIR小鼠视网膜新生血管生成。

GPER在TNBC中介导的相关通路分析

三阴性乳腺癌(TNBC)是乳腺肿瘤中最具侵袭性的分子亚型,占所有乳腺癌的10%~15%,且预后最差。其特点是缺乏雌激素受体(ER)和孕激素受体(PR)表达,缺乏人表皮生长因子受体2(ERBB2/HER2)过表达或基因扩增,故早期TNBC被认为是一种不依赖雌激素的乳腺癌。乳腺癌的治疗主要包括激素治疗、抗HER2治疗和化疗,故TNBC长期以来缺乏有效的靶向治疗和精确的治疗选择,直到G蛋白偶联雌激素受体(GPER)的发现才揭示出TNBC的雌激素介导通路。有研究表明GPER与TNBC促转移途径有关,这些结果为研究GPER介导雌激素在TNBC中的致癌作用提供了新的见解,为TNBC的治疗提供了新思路。综述了与GPER相关的各通路及其作为TNBC潜在治疗靶点的合理性,为探究治疗TNBC的有效作用通路提供理论基础。

2型糖尿病患者血清GPER1、SESN2水平与糖尿病视网膜病变的关系研究

目的研究2型糖尿病(T2DM)患者血清G蛋白偶联雌激素受体1(GPER1)、应激诱导蛋白2(SESN2)水平与糖尿病视网膜病变(DR)的关系。方法回顾性选取2020年2月—2022年2月联勤保障部队第九四〇医院眼科诊治的T2DM患者162例为T2DM组。根据眼部检查结果,将T2DM患者分为DR亚组(n=68)和非DR亚组(n=94);根据DR病情程度,将DR患者分为增生性亚组(n=28)和非增生性亚组(n=40)。另选取同期医院健康体检者60例为健康对照组。采用酶联免疫吸附试验检测血清GPER1、SESN2水平;多因素Logistic回归分析DR的影响因素;受试者工作特征(ROC)曲线评价血清GPER1、SESN2水平对DR的预测价值。结果T2DM组血清GPER1水平高于健康对照组,血清SESN2水平低于健康对照组(t/P=36.850/<0.001,28.112/<0.001);DR亚组糖尿病病程、胰岛素抵抗指数(HOMA-IR)、血清GPER1高于非DR亚组,肾小球滤过率(eGFR)、血清SESN2低于非DR亚组(t/P=11.552/<0.001,3.078/0.009,17.226/<0.001,21.915/<0.001,11.100/<0.001);增生性DR亚组血清GPER1水平高于非增生性DR亚组,血清SESN2水平低于非增生性DR亚组(t/P=12.151/<0.001,3.534/<0.001);多因素Logistic回归结果示,糖尿病病程长、HOMA-IR高、GPER1高是DR的独立危险因素[OR(95%CI)=1.237(1.096~1.397),1.265(1.079~1.483),1.347(1.054~1.721)],eGFR高、SESN2高是DR的独立保护因素[OR(95%CI)=0.736(0.598~0.906),0.759(0.596~0.967)];血清GPER1、SESN2水平及二者联合预测DR的曲线下面积(AUC)分别为0.731、0.710、0.820,二者联合的AUC大于血清GPER1、SESN2水平单独预测(Z=4.711、4.816,P均<0.001)。结论DR患者血清GPER1升高,SESN2降低,两者均参与DR的发生发展,两者联合检测对DR的发生具有较高的预测价值。

The Role of GPER in Sepsis-Induced Myocardial Cell Damage and 28-Day Mortality Risk

Purpose: The role of GPER in sepsis-induced myocardial cell injury and its potential impact on the risk of death within 28 days in sepsis. Methods: An in vitro experiment was conducted to establish a sepsis-induced myocardial cell model. H9C2 myocardial cells were treated with 10 μg/ml lipopolysaccharide (LPS) for 24 hours. The effects of different concentrations of the GPER agonist G1 (1, 3, and 10 μmol/L) on cell viability, expression of inflammatory markers, cell apoptosis, and the NF-κB pathway were evaluated. A Mendelian randomization analysis was conducted using Single Nucleotide Polymorphism (SNPs) related to the GPER gene as instrumental variables to investigate the causal relationship between the GPER gene variations and sepsis (28-day death). Results: The results indicate that the group treated with LPS showed a significant decrease in myocardial cell viability, an increase in concentrations of tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6), higher apoptosis rates, and increased phosphorylation levels of NF-κB p65 (p-P65/P65) and IκB-α (p-IκB-α/IκB-α) compared to the control group (P κB pathway. However, genetic evidence did not show a causal relationship between GPER gene variations and sepsis (28-day death) (P κB pathway. However, genetic evidence did not show a causal relationship between GPER gene variations and sepsis (28-day death).

G蛋白偶联雌激素受体在雌激素相关肿瘤发生中的作用

在经典的雌激素核受体α和β之外,雌激素或雌激素样物质也可以经过膜受体,即G蛋白偶联雌激素受体(GPER)发挥功能。G蛋白偶联雌激素受体是雌激素非基因通路信号转导过程的重要介导因子,在雌激素相关肿瘤细胞的发生和治疗中具有重要的意义。现针对G蛋白偶联雌激素受体在雌激素相关肿瘤细胞中介导的效应及有关机制的研究进展进行综述。

丹参酮ⅡA抗乳腺癌T47D细胞增殖的GPER途径研究

目的利用经典雌激素受体（estrogenic receptor,ER）和G蛋白偶联雌激素受体（G protein-coupled estrogen recep-tor,GPER）阳性乳腺癌T47D细胞,探索丹参酮IIA（TanshinoneIIA）对细胞增殖活性的影响及其GPER介导与调节功能.方法以GPER激动剂G1和GPER拮抗剂G15为工具药干预,并应用GPERSiRNA转染构建GPER基因沉默的T47D细胞,利用MTT细胞增殖实验观察丹参酮IIA对T47D细胞增殖速率的影响及GPER的介导作用.利用Westernblot方法检测丹参酮IIA对T47D细胞GPER表达情况的影响.结果1×10^-5 mol·L^-1 ～1×10^-7 mol·L^-1丹参酮IIA能够明显抑制T47D细胞增殖,且该抑制作用可被G1拮抗,可被G15增强.丹参酮IIA作用于GPER基因沉默的T47D细胞,该细胞表现出更为明显的生长抑制效应.West-ernblot测定结果表明,1×10^-5 mol·L^-1和1×10^-6 mol·L^-1丹参酮IIA可使T47D细胞GPER蛋白表达明显降低.结论丹参酮IIA具有抑制乳腺癌T47D细胞增殖的作用,该抑制作用可经GPER途径介导;且丹参酮IIA具有对靶细胞GPER表达的调节功能.

G-蛋白偶联雌激素受体在肾阴虚和肾阳虚小鼠睾丸中的表达及其与生殖功能的关系

目的:探讨肾阴虚与肾阳虚雄性小鼠睾丸G-蛋白偶联雌激素受体(GPER)的定位与表达,及其对肾阴虚与肾阳虚雄性小鼠生殖功能的影响。方法:将6周龄雄性昆明小鼠随机分为正常组、肾阳虚组和肾阴虚组。采用矿场实验、高架十字迷宫、游泳力竭等评价小鼠精神萎靡程度和自主活动次数等行为学改变;电化学发光法检测睾酮(T)和雌二醇(E2),并计算T/E2;全自动精子分析仪检测精液质量;与雌鼠合笼后记录雌鼠产仔数,计算雄鼠平均育仔数;免疫组织化学和免疫荧光染色检测GPER在小鼠睾丸中的定位和表达。结果:与肾阴虚组比较,肾阳虚组小鼠开臂进入次数百分率和中央区进入次数明显减少,同时,游泳力竭时间缩短更为明显(P<0.05),但开臂滞留和中央区滞留时间百分率无明显差异(P>0.05)。与肾阴虚组比较,肾阳虚组小鼠附睾精子计数和活动精子百分率明显减少,且育仔数显著下降(P<0.05);精子畸形率略有升高(P>0.05);血清T水平明显降低(P<0.05),E2水平略有下降(P>0.05),T/E2明显下降(P<0.05)。肾阴虚和肾阳虚小鼠睾丸GPER均表达于睾丸间质细胞的细胞质内,细胞核和细胞膜表达为阴性,肾阳虚小鼠睾丸GPER的表达显著高于肾阴虚组(P<0.05)。结论:肾阳虚与肾阴虚小鼠均出现精子数量与育仔数下降,精子畸形率增加,但以肾阳虚小鼠更为明显,这种改变可能与肾阳虚小鼠睾丸GPER表达增加有关,从而造成血清T水平及T/E2比值下降。

G蛋白偶联受体在雌激素诱导人甲状腺未分化癌FRO细胞增殖中的作用及其机制

目的探讨G蛋白偶联受体(G-protein coupled receptor,GPER)在雌激素促进人甲状腺未分化癌FRO细胞增殖中的作用及其机制。方法不同浓度(0、10-10、10-9、10-8mol/L)的17β-雌二醇(17β-Estradiol,E2)处理FRO细胞不同时间(0、12、24h),MTT法检测细胞增殖率;10-8mol/L的E2处理FRO细胞不同时间(0～24h),流式细胞术检测E2对FRO细胞周期的影响;设计合成针对GPER的小干扰RNA(GPER-siRNA)并转染FRO细胞;Western blot检测FRO细胞中Akt与ERK1/2磷酸化水平与GPER蛋白的表达。结果不同浓度(0、10-10、10-9、10-8mol/L)的E2处理FRO细胞不同时间(0、12、24h),细胞增殖率随浓度和时间的增加而增加,且10-8mol/L的E2处理24h后,细胞增殖率为(16.5±2.1)%;10-8mol/L的E2处理FRO细胞不同时间(0、12、24h),细胞周期S/G2/M期细胞的比例随时间的增加而增加;E2(10-8mol/L)处理FRO细胞不同时间(0、5、10、15、30min),ERK1/2与Akt的磷酸化水平分别在15min与10min达到最大;将GPER-siRNA转染FRO细胞48h,GPER的蛋白表达显著减少;E2作用于分别加入PD98059(30μmol/L)、LY294002(50μmol/L)以及转染GPER-siRNA的FRO细胞15min和10min,与E2处理组相比,ERK1/2和Akt的磷酸化水平降低;E2作用于分别加入PD98059、LY294002及转染GPER-siRNA的FRO细胞24h,与E2处理组相比,增殖率由(16.5±2.1)%降至(11.2±1.3)%、(9.6±1.5)%、(7.2±1.3)%(P<0.05)。结论 E2通过GPER激活ERK1/2、PI3K-Akt途径,促进FRO细胞的增殖。