No association of G-protein beta polypeptide 3 polymorphism with irritable bowel syndrome: Evidence from a meta-analysis

AIM: To clarify the associations between G-protein beta polypeptide 3 (GNB3) C825T polymorphism and risk of the irritable bowel syndrome (IBS) by a meta-analysis.

MicroRNA-760 acts as a tumor suppressor in gastric cancer development via inhibiting G-protein-coupled receptor kinase interacting protein-1 transcription

BACKGROUND Gastric cancer(GC)has become a serious threat to people's health.Accumulative evidence reveals that dysregulation of numerous microRNAs(miRNAs)has been found during malignant formation.So far,the role of microRNA-760(miR-760)in the development of GC is largely unknown.AIM To measure the expression level of miR-760 in GC and investigate its role in gastric tumorigenesis.METHODS Real-time quantitative polymerase chain reaction and Western blot analysis were used to measure the expression of miR-760 and G-protein-coupled receptor kinase interacting protein-1(GIT1).Cell growth was detected by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide(MTT)and cell colony formation assays.Apoptosis was assessed by flow cytometric analysis.The relationship between miR-760 and GIT1 was verified by luciferase reporter assay.RESULTS The results showed that the expression of miR-760 was decreased in GC and associated with poor clinical outcomes in GC patients.Furthermore,miR-760 restrained cell proliferation and cell colony formation and induced apoptosis in GC cells.In addition,miR-760 directly targeted GIT1 and negatively regulated its expression in GC.GIT1 was upregulated in GC and predicted a worse prognosis in GC patients.We also found that upregulation of GIT1 weakened the inhibitory CONCLUSION In conclusion,miR-760 targets GIT1 to inhibit cell growth and promote apoptosis in GC cells.Our data demonstrate that miR-760 may be a potential target for the treatment of GC.

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.

Genetic predisposition to essential hypertension in a Mongolian population Detecting the C825T polymorphism of the G-protein beta 3 subunit gene

BACKGROUND： The prevalences of hypertension, cerebrovascular diseases, etc. are higher in Mongolian population because of the influence of various factors including genetics, geography, diet, etc. Therefore, it is helpful for prevention to develop researches on the genetics of various diseases including hypertension in Mongolian population. OBJECTIVE： To analyze the association between C825T polymorphisms of G-protein beta 3 subunit gene （GNB3）, the important candidate gene of various disease of cardiovascular system, and Mongolian patients with essential hypertension. DESIGN： A comparative observation. SETTINGS： Department of Neurology, the First Affiliated Hospital of Inner Mongolia Medical College; Wulate Houqi Red Cross Society. PARTICIPANTS： Totally 267 Mongolian residents, whose blood relations of 3 generations were all Mongolians, were selected from Wulate Houqi, Inner Mongolia. The patients were screened based on the diagnostic standard of hypertension set by WHO in 1999, and the enrolled subjects were divided into two groups according to the level of blood pressure： ① Normal blood pressure group （n =124）： 64 males and 60 females, systolic blood pressure （SBP） 〈 140 mm Hg （1 mm Hg=0.133 kPa）, diastolic blood pressure （DBP） 〈 90 mm Hg;②Essential hypertension group （n =143）： 71 males and 72 females, including 60 patients with simple high SBP （SBP ranged 145 to 195 mm Hg, whereas DBP 〈 90 mm Hg）. METHODS： Peripheral venous blood （5 mL） was drawn from all the subjects, the genome DNA was extracted, and the polymorphisms of the GNB3 C825T genotype were detected with the Sequenom system. Polyrnerase chain reaction （PCR） experiment and SNP detection were performed in Beijing Huada gene laboratory. Then the univariate analysis of variance was applied in the sample comparison among groups, and the chi-square test was used to compare the genotypes and allele frequencies. The odd ratio （OR） and 95% confidence interval （CI） were calculated. MAIN OUTCOME MEASURES： The distributions of GNB3 C825T genotypes and alleles were observed. RESULTS： All the 267 Mongolian subjects were involved in the analysis of results.① GNB3 C825T genotypes： In Mongolian population, the frequencies of CC, CT and TT genotypes at GNB3 C825T site in the essential hypertension group （48%, 41%, 11%） were not obvious different from those in the normal blood pressure group （43%, 47%, 10%, x^2 =0.162, P =0.688; OR：1.176, 95%CI： 0.533- 2.592）, whereas there were also no obvious differences between the simple high SBP group （57%, 35%, 8%） and the normal blood pressure group （x^2 =0.733, P =0.392; OR：1.957, 95%CI： 0.623- 6.143）. ②GNB3 C825T alleles： In Mongolian population, The frequencies of C and T alleles in the essential hypertension group （69%, 31%） were not obviously different from those in the normal blood pressure group （67%, 33%, x^2 =0.094, P = 0.759; OR：0.945, 95%CI：0.657 - 1.358）, whereas there were also no obvious differences between the simple high SBP group （74%, 26%） and the normal blood pressure group （ x^2 =2.133, P =0.144; OR：0.697, 95%CI： 0.428- 1.133）. CONCLUSION： GNB3 C825T site may be not a genetic marker of essential hypertension and simple high SBP in Mongolian population.

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.

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.

Desensitization of G-protein-coupled receptors induces vascular hypocontractility in response to norepinephrine in the mesenteric arteries of cirrhotic patients and rats

BACKGROUND: The increased β-arrestin-2 and its combination with G-protein-coupled receptors (GPCRs) lead to GPCRs desensitization. The latter may be responsible for decreased contractile reactivity in the mesenteric arteries of cirrhotic patients and rats. The present study is to investigate the machinery changes of α-adrenergic receptors and G proteins and their roles in the contractility of mesenteric arteries of cirrhotic patients and animal models. METHODS: Patients with cirrhosis due to hepatitis B and cirrhotic rats induced by CCl 4 were studied. Mesenteric artery contractility in response to norepinephrine was determined by a vessel perfusion system. The contractile effect of G protein-coupled receptor kinase-2 (GRK-2) inhibitor on the mesenteric artery was evaluated. The protein expression of the α 1 adrenergic receptor, G proteins, β-arrestin-2, GRK-2 as well as the activity of Rho associated coiled-coil forming protein kinase-1 (ROCK-1) were measured by Western blot. In addition, the interaction of α 1 adrenergic receptor with β-arrestin-2 was assessed by co-immunoprecipitation. RESULTS: The portal vein pressure of cirrhotic patients and rats was significantly higher than that of controls. The doseresponse curve to norepinephrine in mesenteric arteriole was shifted to the right, and EC 50 was significantly increased in cirrhotic patients and rats. There were no significant differences in the expressions of the α 1 adrenergic receptor and G proteins in the cirrhotic group compared with the controls. However, the protein expressions of GRK-2 and β-arrestin-2 were significantly elevated in cirrhotic patients and rats compared with those of the controls. The interaction of the α 1 adrenergic receptor and β-arrestin-2 was significantly aggravated. This interaction was significantly reversed by GRK-2 inhibitor. Both the protein expression and activity of ROCK-1 were significantly decreased in the mesenteric artery in patients with cirrhosis compared with those of the controls, and this phenomenon was not shown in the cirrhotic rats. Norepinephrine significantly increased the activity of ROCK-1 in normal rats but not in cirrhotic ones. Norepinephrine significantly increased ROCK-1 activity in cirrhotic rats when GRK-2 inhibitor was used. CONCLUSIONS: β-arrestin-2 expression and its interaction with GPCRs are significantly upregulated in the mesenteric arteries in patients and rats with cirrhosis. These upregulations result in GPCR desensitization, G-protein dysfunction and ROCK inhibition. These may explain the decreased contractility of the mesenteric artery in response to vasoconstrictors.

G-protein β subunit AGB1 positively regulates salt stress tolerance in Arabidopsis

The heterotrimeric GTP-binding proteins（G-proteins） in eukaryotes consisted of α, β and γ subunits and are important in molecular signaling by interacting with G-protein-coupled receptors（GPCR）, on which to transduce signaling into the cytoplast through appropriate downstream effectors. However, downstream effectors regulated by the G-proteins in plants are currently not well defined. In this study, the transcripts of AGB1, a G protein β subunit gene in Arabidopsis were found to be down-regulated by cold and heat, but up-regulated by high salt stress treatment. AGB1 mutant（agb1-2） was more sensitive to high salt stress than wild-type（WT）. Compared with WT, the cotyledon greening rates, fresh weight, root length, seedling germination rates and survival rates decreased more rapidly in agb1-2 along with increasing concentrations of Na Cl in normal（MS） medium. Physiological characteristic analysis showed that compared to WT, the contents of chlorophyll, relative proline accumulation and peroxidase（POD） were reduced, whereas the malonaldehyde（MDA） content and concentration ratio of Na＋/K＋ were increased in agb1-2 under salt stress condition. Further studies on the expression of several stress inducible genes associated with above physiological processes were investigated, and the results revealed that the expressions of genes related to proline biosynthesis, oxidative stress response, Na＋ homeostasis, stress- and ABAresponses were lower in agb1-2 than in WT, suggesting that those genes are possible downstream genes of AGB1 and that their changed expression plays an important role in determining phenotypic and physiologic traits in agb1-2. Taken together, these findings indicate that AGB1 positively regulates salt tolerance in Arabidopsis through its modulation of genes transcription related to proline biosynthesis, oxidative stress, ion homeostasis, stress- and ABA-responses.

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.

Pathogenicity Assay for <i>Cochliobolus heterostrophus</i>G-Protein and MAPK Signaling Deficiency Strains

Cochliobolus heterostrophus is an agriculturally important and emerging model pathogen for studying the signaling hierarchies' role during the maize host colonization. In particular, G-protein and MAPK-linked pathways are playing a major role during pathogenesis. Although gene disruption studies are an efficient way of identifying the role of these cascades, differentiating between the mutant strains’ virulence ability may become an intricate task. For example, in C. heterostrophus, mutants in a G-protein α subunit gene, cga1, are defective in mating and appressorium formation, but unlike mutants in homologous genes in other fungal pathogens, the cga1 mutants remained highly virulent to corn under some host physiological conditions. Here, we used the cga1 strain as a model for developing an in vivo sensitive and accurate pathogenicity assay. A detailed and well controlled analysis of wild type (WT) and cga1 pathogenic behavior revealed that detached leaves are significantly more vulnerable to the disease than intact ones. In intact leaves, cga1 mutants were less infective of maize under most conditions. This difference was maximized when the first seedling leaf was chosen for inoculation and when the infected leaves, with spores or mycelia fragments droplets, were incubated for a period of four days. This optimal condition set enabled us to classify the C. heterostrophus G-protein signaling mutants deficient in α, β or both subunits in order of decreasing virulence: WT > cga1> cgb1> cga1 cgb1. The method presented proved to be accurate and sensitive enough to identify even slight variations in virulence. Moreover, it could be modified for use in studies of other foliar phytoparasitic fungi.

Association between G-protein β3 subunit gene and isolated systolic blood pressure elevation of greater than 130 mmHg: A large-scale cross-sectional study in the Japanese population

AIM To investigate whether GNB3 C825 T single nucleotide polymorphism(SNP) contributes to systolic blood pressure(SBP) ≥ 130 mmH g in a large-scale cross-sectional study among the Japanese population with diastolic blood pressure(DBP) < 85 mmH g. METHODS We analyzed 11008 Japanese subjects, including 2797 cases(SBP ≥ 130 and DBP < 85 mmH g) who were not taking anti-hypertensive medication and 8211 controls(SBP < 130 and DBP < 85 mmH g), all of whom enrolled in the genome banking project of the 21 st Century COE(Center of Excellence) Program at Jichi Medical University. Subjects were divided into four groups according to gender(male and female) and age(≤ 49 years and ≥ 50 years). GNB3 gene polymorphism was determined using the TaqM an probe method. We compared the frequencies of alleles and genotypes between cases and controls by chi-squared test. The strength of the associations was estimated by odds ratios(ORs) and 95%CI by using logistic regression analysis. The ORs were adjusted for age and body mass index. RESULTS Allele and genotype distributions significantly differed between cases and controls only in males aged ≤ 49 years. Compared to the CC genotype, a significant OR was obtained in the TT genotype among males aged ≤ 49 years.CONCLUSION This study indicates that the TT genotype of the GNB3 C825 T SNP may contribute to SBP elevation of greater than 130 mmH g compared to the CC genotype in Japanese males aged ≤ 49 years.

利用CRISPR/Cas9构建敲除小鼠模型研究PPP2R3A基因对心脏功能的影响

目的 应用CRISPR/Cas9技术构建蛋白磷酸2调节亚基B″家族α亚型(PPP2R3A)基因敲除小鼠,从分子水平及组织水平上研究PPP2R3A缺失对心脏的影响。方法 将Cas9 mRNA和两个靶向PPP2R3A第3外显子翻译起始密码子附近区域的单导向RNA微注射到C57BL/6小鼠受精卵中。小鼠出生后取其基因组DNA进行聚合酶链反应(PCR)和测序以鉴定基因型,鉴定后,基因PPP2R3A缺失型小鼠为KO组,野生型C57BL/6小鼠为WT组(雄性3只,雌性2只)。小鼠心脏组织经甲醛固定并制成切片后分别进行苏木素-伊红(HE)染色和免疫组织化学染色。提取小鼠心脏组织总RNA和蛋白,应用荧光定量PCR和Western印迹验证基因敲除小鼠的有效性和检测互作蛋白表达。结果 获得F1代PPP2R3A杂合小鼠,PCR和测序结果表明突变小鼠的基因型存在113 bp的缺失突变。与WT组相比,KO组心脏组织中PPP2R3A mRNA和蛋白表达量明显下降(均P<0.05),参与心脏发育的G蛋白信号转导调控因子(RGS)19表达量明显升高(P<0.05)。PPP2R3A蛋白表达受损引起了心脏组织病理学变化。结论 PPP2R3A在体内可能通过与RGS19蛋白互作来参与心脏的发育并对心脏功能产生影响。

丁酸盐在炎症性肠病中的免疫调节机制研究进展

炎症性肠病(IBD)是一组与肠道慢性炎症相关的异质性疾病,丁酸盐是肠道微生物群产生的关键代谢产物,能够调节免疫细胞的发育和功能,调节免疫功能并防止过度免疫反应,从而延缓IBD的临床进展。本文就丁酸盐在调节免疫功能方面改善IBD作用机制的研究进展进行综述,旨在为IBD的临床治疗提供新的选择。

短链脂肪酸与糖尿病肾病关系的研究进展

糖尿病肾病是糖尿病的常见并发症之一,也是终末期肾病的常见病因。遗传因素、代谢紊乱、氧化应激、炎症反应等与糖尿病肾病的发生发展密切相关,但其发病机制尚不明确。短链脂肪酸是肠道菌群在宿主肠道内发酵膳食纤维主要的代谢产物,通过激活G蛋白偶联受体和去乙酰组蛋白受体参与调节食欲、调节糖代谢、调节脂质代谢、减轻炎症反应等一系列代谢过程,从而在糖尿病肾病的发生发展中可能起到关键作用。本文就短链脂肪酸与糖尿病肾病的关系和相关争议展开综述,旨在为了解短链脂肪酸在糖尿病的发生发展中的作用及为糖尿病肾病预防和治疗提供新的思路。

基于Kisspeptin/GPR54系统探讨新加二甲地黄汤对PCOS模型大鼠卵泡发育的影响

目的探讨经新加二甲地黄汤干预后的多囊卵巢综合征(polycystic ovary syndrome,PCOS)模型大鼠的卵泡发育情况及其可能存在的效应机制。方法筛选28只拥有规律动情周期的雌性SD大鼠,随机分为正常对照组、模型组、中药组及西药组,每组7只。除正常对照组外,其余三组均连续予来曲唑-羧甲基纤维素混悬液0.1mg/(kg·d)灌胃,以构建PCOS大鼠模型。自第22天起,中药组以新加二甲地黄汤5.268g/(kg·d)灌胃,西药组以炔雌醇环丙孕酮片0.286mg/(kg·d)灌胃,正常对照组及模型组均以10ml/(kg·d)蒸馏水灌胃。3周后比较各组大鼠卵巢系数,苏木精-伊红染色观察各组大鼠卵巢组织形态学改变,对各组大鼠血清激素均采用酶联免疫吸附试验进行检测,蛋白质印迹法检测卵巢亲吻素(kisspeptin,Kp)、G蛋白偶联受体54(G-protein-coupled receptor 54,GPR54)蛋白表达水平。结果与正常对照组比较,模型组大鼠卵巢内呈现为囊状扩张和闭锁的卵泡增多,其颗粒细胞层数变少,卵巢系数增大;血清睾酮(testosterone,T)、黄体生成素(luteinizing hormone,LH)、Kp水平升高;血清卵泡刺激素(follicle stimulating hormone,FSH)、雌二醇(estradiol,E2)水平及大鼠卵巢组织中Kp、GPR54蛋白表达均显著降低(P<0.05)。予中药干预后,与模型组比较,中药组大鼠卵巢囊样扩张卵泡数量变少,颗粒细胞的层数增多,存在近成熟的卵泡,并见少量黄体存在;大鼠血清LH、T、Kp水平下降,血清FSH、E2水平及卵巢Kp、GPR54蛋白表达显著升高(P<0.05)。结论PCOS模型大鼠的卵泡发育情况经新加二甲地黄汤干预后得以改善,该治疗机制可能为通过调控Kisspeptin/GPR54系统影响FSH和LH的释放,以此影响激素含量,调整卵巢功能,使卵泡发育和排卵能力得以改善。

Kiss-1/GPR54系统在PCOS模型大鼠卵巢颗粒细胞中的表达及对卵泡发育障碍的作用机制

目的初步探讨Kiss-1/GPR54系统在多囊卵巢综合征(PCOS)模型大鼠卵巢颗粒细胞中的表达,并分析其与PCOS之间的关联。方法筛选出14只动情规律的雌性SD大鼠,按照随机数字表法分为正常对照组和模型组,每组7只。其中模型组大鼠予来曲唑-羧甲基纤维素混悬液0.1mg/(kg·d)连续灌胃21 d以构建PCOS大鼠模型。收集大鼠血清、卵巢组织、卵巢颗粒细胞。然后计算大鼠卵巢指数,对卵巢组织进行苏木精-伊红染色法,采用酶联免疫吸附测定法检测血清激素水平,免疫荧光染色检测颗粒细胞中亲吻素(Kp)、G蛋白偶联受体54(GPR54)荧光强度,实时定量反转录聚合酶链式反应检测颗粒细胞中Kiss-1、GPR54基因表达。结果与正常对照组比较,模型组大鼠卵巢囊状扩张卵泡及闭锁卵泡增加,卵泡颗粒细胞层变薄;卵巢指数增大[(1.22±0.10)mg/g比(0.82±0.13)mg/g,P<0.05];血清黄体生成素[(66.32±3.98)IU/L比(11.87±5.54)IU/L]、睾酮[(33.72±3.57)ng/mL比(8.40±2.94)ng/mL]、Kp水平[(1506.79±154.82)pg/mL比(289.57±89.20)pg/mL]均升高(P<0.05);卵巢颗粒细胞中Kp荧光强度[(1601852.67±378567.82)比(3685156.00±359825.63)]、GPR54荧光强度[(1298372.25±297701.61)比(2961456.58±309119.01)]及Kiss-1基因表达[(0.10±0.03)比(1.11±0.14)]、GPR54基因表达[(0.10±0.05)比(1.00±0.13)]均降低(P<0.05)。结论卵巢表达的Kiss-1/GPR54系统可能通过调节颗粒细胞从而影响PCOS大鼠卵泡发育。

植物异三聚体G蛋白研究进展

植物细胞中的异三聚体G蛋白信号转导系统包括异三聚体G蛋白、G蛋白偶联受体、类受体激酶、G蛋白信号调节因子等,这些信号转导组分在感受物理及化学刺激、启动细胞内信号级联反应、调控细胞内代谢和基因表达过程中发挥重要作用.异三聚体G蛋白参与调控植物生长发育(如胚胎形成、营养器官生长、有性生殖等)、植物对生物及非生物胁迫的响应、根瘤形成等过程.因此,异三聚体G蛋白信号系统组分参与调控多种农作物的农艺性状,并最终影响农产品的产量和品质.对植物异三聚体G蛋白的结构、活化机制和生理功能等方面近年来取得的研究进展进行了回顾和总结.

基于G蛋白信号调节因子2敲低探讨血管紧张素Ⅱ诱导的主动脉夹层形成的机制

目的探讨G蛋白信号调节因子2(RGS2)在调节血管紧张素Ⅱ(AngⅡ)诱导的主动脉夹层形成中的作用。方法将C57BL/6小鼠分为3组:Control组(n=10)、AngⅡ组(n=20)、AngⅡ+sh-RGS2组(n=20)。AngⅡ组和AngⅡ+sh-RGS2组小鼠建立了主动脉夹层模型。在体内评估主动脉夹层的发生率,在体外和体内评估血管平滑肌细胞(VSMC)表型转化。结果RGS2的敲低逆转了AngⅡ导致的αSMA、ACTA2和MYH11的表达下调,并抑制了AngⅡ诱导的SPP1和Vimentin蛋白表达。AngⅡ组和AngⅡ+sh-RGS2组的主动脉夹层发生率分别为45%(9/20)和10%(2/20)。与AngⅡ组小鼠比较,AngⅡ+sh-RGS2组小鼠中观察到更少的弹性层增厚、主动脉破裂和主动脉壁胶原纤维含量。此外,与AngⅡ组比较,AngⅡ+sh-RGS2组主动脉的最大直径减小(P<0.05),ACTA2、MYH11蛋白增加(P<0.01),RGS2、SPP1、Vimentin蛋白降低(P<0.01)。结论RGS2敲低抑制AngⅡ诱导的VSMC从可收缩表型转变为合成表型,降低了主动脉夹层形成的发生率。

黏附性G蛋白偶联受体F1在胰腺导管腺癌中的表达及其促进癌症进展的机制研究

目的·分析黏附性G蛋白偶联受体F1(adhesion G protein-coupled receptor F1,ADGRF1)在胰腺导管腺癌(pancreatic ductal adenocarcinoma,PDAC)发生及发展过程中的表达变化,探究ADGRF1对PDAC细胞增殖的影响以及促进PDAC进展的潜在分子机制。方法·基于癌症基因组图谱(The Cancer Genome Atlas,TCGA)数据库和基因表达综合(Gene Expression Omnibus,GEO)数据库分析ADGRF1在正常胰腺组织及PDAC组织中的mRNA水平表达。利用实时荧光定量PCR(quantitative real-time PCR,qPCR)和蛋白质印迹法(Western blotting)检测ADGRF1在正常胰腺导管上皮细胞hTERT-HPNE及多种PDAC细胞中的表达情况。利用免疫组织化学染色(immunohistochemistry staining,IHC)检测PDAC患者的癌组织及癌旁组织中ADGRF1的表达差异。转染小干扰RNA(small interfering RNA,siRNA)敲低ADGRF1后,通过CCK8和平板克隆形成实验检测PDAC细胞AsPC-1、SW1990增殖能力的变化。构建稳定过表达ADGRF1的Patu8988细胞,通过CCK8实验检测过表达ADGRF1引起的PDAC细胞增殖变化。利用RNA测序(RNA-sequence,RNA-seq)、基因集富集分析(gene set enrichment analysis,GSEA)和免疫浸润分析预测与ADGRF1促进PDAC癌症进展相关的信号通路。结果·TCGA数据库和GEO数据库的分析结果显示ADGRF1 mRNA在PDAC组织中的表达高于正常胰腺组织(均P=0.000)。qPCR和Western blotting结果显示,与hTERT-HPNE细胞相比,多种PDAC细胞中ADGRF1的mRNA和蛋白水平均有所上调(均P<0.05)。IHC结果显示ADGRF1在PDAC患者癌组织中的表达也高于癌旁组织。此外,下调ADGRF1能够抑制PDAC细胞AsPC-1、SW1990的增殖能力;而过表达ADGRF1则促进Patu8988细胞的增殖能力(均P<0.05)。RNAseq、GSEA富集分析和免疫浸润的结果显示,ADGRF1的表达与干扰素α(interferon-α,IFN-α)、肿瘤坏死因子α(tumor necrosis factor-α,TNF-α)和核因子κB(nuclear factorκB,NF-κB)等信号通路有关。结论·ADGRF1在PDAC细胞和组织中高表达,促进PDAC细胞的增殖,其机制可能与多个免疫相关信号通路有关。

机械应力调控血管平滑肌细胞的凋亡

背景:随着生物力学的发展,其在心血管疾病中的研究也日益广泛,通过研究血管的力学特性可以有效地揭示动脉粥样硬化、再狭窄等心血管疾病的发病机制,开发心血管疾病治疗的新思路和新方法。目的:综述机械应力刺激对血管平滑肌细胞凋亡的研究现状,寻找临床治疗潜在靶分子和信号通路,以期改善临床动脉粥样硬化及再狭窄等心血管疾病的临床治疗效果。方法:检索1992年1月至2023年5月在中国知网、PubMed及ScienceDirect数据库收录的文献。中文检索词为“血管平滑肌细胞,机械应力,剪切力,牵张力,凋亡”;英文检索词为“vascular smooth muscle cell,mechanical stress,shear stress,stretch stress,apoptosis”,最终纳入63篇文献进行综述分析。结果与结论:①血管平滑肌细胞的生理性和病理性凋亡都是为了适应血管力学变化而发生的适应性重构。不同部位的平滑肌细胞承受不同的力学刺激,其发病机制也不同。②低剪切力、生理剪切力和高剪切力可通过直接与平滑肌细胞表面分子、受体及蛋白等作用调控凋亡信号分子和抑制增殖实现对血管平滑肌细胞凋亡的调控,该部分未对促进增殖的研究进行综述。③低牵张力、生理牵张力和高牵张力可导致平滑肌细胞凋亡,但仍存在争议。平滑肌表面存在多种力学感受分子(如整合素及受体络氨酸激酶等)可以将力学信号转导为细胞内的化学信号(如Hippo通路),启动平滑肌细胞的凋亡信号,调控平滑肌细胞的凋亡。④总之,不同力学刺激通过多种信号分子,启动多个信号通路共同作用,调控平滑肌细胞的凋亡,例如剪切力主要通过刺激分泌前列腺素、转化生长因子等影响Fas/FasL、Akt通路;而牵张力主要通过Yes相关蛋白等调控YAP通路和Notch通路等。这些分子在不同的作用时间,或不同作用强度下可能发挥相反的双向作用,比如,小鼠血管平滑肌细胞受到10%生理牵张1 h时,其增殖增加;然而,人的血管平滑肌细胞牵张12 h后可以减少其增殖。临床可以通过寻找其关键作用时间点干扰力学转导关键分子,达到预防和治疗临床心血管疾病的目的。