含miR-497野生及突变结合位点的胰岛素受体mRNA 3′UTR区报告基因载体的构建和鉴定

目的利用pmirGLO Dual-Luciferase miRNA Target Expression Vector(简称为pmirGLO报告基因载体)构建并鉴定含miR-497野生及突变结合位点的胰岛素受体mRNA 3′UTR区报告基因载体(pmir-Insr-3′UTR及pmirmutant-Insr-3′UTR)。方法以大鼠肝脏cDNA为模板,PCR获取目的片段(即含miR-497野生及突变结合位点的胰岛素受体mRNA 3′UTR区);用PmeI、XbaI双酶切pmirGLO报告基因载体和含miR-497野生及突变结合位点的胰岛素受体mRNA 3′UTR区,用T4 DNA连接酶连接纯化后的酶切产物;连接产物转化DH5α大肠杆菌感受态细胞并挑选阳性克隆,并通过PCR、双酶切、DNA测序鉴定构建的重组质粒。结果 PCR和双酶切证实pmir-Insr-3′UTR及pmir-mutant-Insr-3′UTR重组载体中均插入目的片段;DNA测序结果进一步证实pmir-Insr-3UTR重组载体中成功插入了胰岛素受体mRNA 3′UTR区,pmir-mutant-Insr-3′UTR重组载体中成功插入了在miR-497结合位点含有3个突变碱基的胰岛素受体mRNA 3′UTR片段。结论成功构建了含miR-497野生及突变结合位点的胰岛素受体mRNA 3′UTR报告基因载体pmir-Insr-3′UTR及pmir-mutant-Insr-3′UTR。

Relationship between Trp64Arg mutation in the β3-adrenergic receptor gene and metabolic syndrome： a seven-year follow-up study

Background It has been shown that the β3-adrenergic receptor （β3-AR） gene Trp64Arg mutation was closely related to obesity and insulin resistance, and may be related to the prevalence of metabolic syndrome （MS）. The aim of this study was to investigate the relationship between the 33-AR gene mutation and the prevalence of MS. Methods A seven-year follow-up study was initiated in 2000, with 496 samples of simplex obese subjects （body mass index ≥25 kg/m2） and 248 normal-weight subjects. According to the β3-AR genotypes, the subjects were classified as Trp64 homozygote group and Arg64 carrier group and after 7 years the prevalence of MS was determined. Results According to the baseline profile, there were no significant differences in the adiposity, blood pressure, lipid profile, fasting plasma glucose and fasting insulin between Trp64 homozygote group and Arg64 carrier group either in obesity or normal-weight subjects. The results of follow-up study indicated that in obese men the prevalence rate of MS was much higher in Arg64 carrier group than that in Trp64 homozygote group （54.76% vs. 40.85%, P 〈0.05）, but there was no statistical difference in women of the above groups. The prevalence rate of MS in obese men of both Trp64 homozygote group and Arg64 carrier obese group were obviously higher than that in women of the above groups （40.85% vs. 18.27% and 54.76% vs 21.28%, all P 〈0.005）. Differences were not statistically significant in the prevalence of MS for normal weight Trp64 homozygote group and normal weight Arg64 carrier group, either between men, between women, or between men and women. Comparison of populations indicated that no matter with the β3-AR gene mutation or not, the prevalence of MS in obese subjects was significantly higher than normal weight subjects （X2=28.240 and x2=15.586, all P 〈0.005）. Logistic analysis showed that the mutation of β3-AR gene was associated with the prevalence of MS in men.

DcR3在类风湿关节炎的表达及其临床意义

目的:研究诱骗受体3(DcR3)在类风湿关节炎(RA)患者体内的表达及临床意义。方法:选取80名类风湿关节炎患者作为研究组,根据病情严重程度分为活动期RA和缓解期RA,同时选取26健康者作为正常对照组。分别使用ELISA试剂盒检测血清DcR3的含量,荧光定量PCR检测DcR3在外周单核细胞中mRNA水平。结果:所有患者血清的DcR3含量和单核细胞中mRNA水平均显著高于健康者(152.5±42.3 vs 42.6±12.4,P<0.05;2.5±1.6 vs 1±0.44,P<0.05),且活动期RA的水平高于缓解期RA并高于对照组。结论:DcR3在类风湿性关节炎患者体内会表达增加,而且随着病情的加重表达量会增加。

SH-SY5Y细胞中过表达RIPK3对ZFP36基因转录的影响

目的探讨SH-SY5Y细胞中受体相互作用蛋白激酶-3(RIPK3)下游信号通路及其中的关键信号分子的作用。方法通过质粒转染的方式在实验组SH-SY5Y细胞内表达外源性RIPK3蛋白,将转染空载质粒载体SH-SY5Y细胞作为对照组。通过观察质粒所携带的绿色荧光蛋白(GFP)在细胞中的表达情况以验证转染结果,Westernblot对外源性RIPK3表达进行验证。MTT法检测细胞增殖活性,观察过表达RIPK3对细胞活性的影响,以确认其在细胞内是否具有生物活性。运用转录组测序技术(RNAseq)分别检测2组细胞内基因转录丰度并应用IngenuityPathway Analysis(IPA)数据库进行分析,获得RIPK3下游信号通路及关键分子。通过微滴式数字化PCR(dd PCR)对部分RNAseq结果进行验证。结果外源性RIPK3在细胞内具有生物活性,能够抑制SH-SY5Y细胞的增殖。RNAseq数据经IPA分析后得出锌指蛋白36(ZFP36)为RIPK3下游效应分子,其相关效应分子包括血管内皮生长因子(VEGF)、人脱帽酶2(DCP2)、脑源性神经营养因子(BDNF)、肿瘤坏死因子(TNF)的转录丰度也发生了相应的变化。结论 RIPK3能够参与对ZFP36以及与其相关效应分子的转录调控,进而在神经系统的发育、炎症和肿瘤发生等生理、病理过程中发挥重要作用。

Molecular mechanism of the effects of 1,25-dihydroxyvitaminn D_3 and its novel analogues on proliferation and differentiation of a human megakaryoblastic leukemia cell line

Our previous studies revealed that 1, 25-dihydroxyvrtamin D_3[1, 25 (OH)_2, D_3] and its two novel analogues (MC903 and EB1089) play an important role in the modulation of proliferation and differentiation of a newly established human megakaryoblastic leu

Galectin-3-centered paracrine network mediates cardiac inflammation and fibrosis upon β-adrenergic insult

Rapid over-activation of β-adrenergic receptors (β-AR) following acute stress initiates cardiac inflammation and injury by activating interleukin-18 (IL-18),however,the process of inflammation cascades has not been fully illustrated.The present study aimed to determine the mechanisms of cardiac inflammatory amplification following acute sympathetic activation.With bioinformatics analysis,galectin-3 was identified as a potential key downstream effector of β-AR and IL-18 activation.The serum level of galectin-3 was positively correlated with norepinephrine or IL-18 in patients with chest pain.In the heart of mice treated with β-AR agonist isoproterenol (ISO,5 mg kg^(-1)),galectin-3 expression was upregulated markedly later than IL-18 activation,and Nlrp3^(-/-)and Il18^(-/-)mice did not show ISO-induced galectin-3 upregulation.It was further revealed that cardiomyocyte-derived IL-18 induced galectin-3 expression in macrophages following ISO treatment.Moreover,galectin-3deficiency suppressed ISO-induced cardiac inflammation and fibrosis without blocking ISO-induced IL-18 increase.Treatment with a galectin-3 inhibitor,but not a β-blocker,one day after ISO treatment effectively attenuated cardiac inflammation and injury.In conclusion,galectin-3 is upregulated to exaggerate cardiac inflammation and injury following acute β-AR activation,a galectin-3 inhibitor effectively blocks cardiac injury one day after β-AR insult.

Ginsenoside F1 administration promotes UCP1-dependent fat browning and ameliorates obesity-associated insulin resistance

Obesity-induced type 2 diabetes is mainly due to excessive free fatty acids leading to insulin resistance.Increasing thermogenesis is regarded as an effective strategy for hypolipidemia and hypoglycemia.Ginsenoside is a natural active component in Panax ginseng C.A.Meyer,and some of them enhance thermogenesis.However,there are few studies on the mechanism and target of ginsenosides enhancing thermogenesis.Using thermogenic protein uncoupling protein 1(UCP1)-luciferase reporter assay,we identifi ed ginsenoside F1 as a novel UCP1 activator in the ginsenosides library.Using pull down assay and inhibitor interference,we found F1 binds toβ3-adrenergic receptors(β3-AR)to enhance UCP1 expression via cAMP/PKA/CREB pathway.We also investigated the ability of F1 on energy metabolism in obesity-induced diabetic mice,including body weight,body composition and energy expenditure.The results of proteomics showed that F1 signifi cantly up-regulated thermogenesis proteins and lipolytic proteins,but down-regulated fatty acid synthesis proteins.Ginsenoside F1 increased thermogenesis and ameliorated insulin resistance specifi cally by promoting the browning of white adipose tissue in obese mice.Additionally,ginsenoside F1 improves norepinephrine-induced insulin resistance in adipocytes and hepatocytes,and shows a stronger mitochondria respiration ability than norepinephrine.These fi ndings suggest that ginsenoside F1 is a promising lead compound in the improvement of insulin resistance.

LPA_(1) antagonist-derived LNPs deliver A20 mRNA and promote anti-fibrotic activities

Activated fibroblasts are major mediators of pulmonary fibrosis.Fibroblasts are generally found in the connective tissue but upon activation can generate excess extracellular matrix(ECM)in the lung interstitial section.Therefore,fibroblasts are one of the most targeted cells for treating idiopathic pulmonary fibrosis(IPF).Here,we develop an anti-fibrotic platform that can modulate both the lysophosphatidic acid receptor 1(LPA_(1))and the inflammatory pathway through tumor necrosis factorα-induced protein 3(TNFAIP3,also known as A20)in fibroblasts.First,we synthesized a series of LPA_(1) antagonists,AM095 and AM966,derived amino lipids(LA lipids)which were formulated into LA-lipid nanoparticles(LA-LNPs)encapsulating mRNA.Specifically,LA5-LNPs,with AM966 head group and biodegradable acetal lipid tails,showed efficient A20 mRNA delivery to lung fibroblasts in vitro(80.2%±1.5%)and ex vivo(17.2%±0.4%).When treated to primary mouse lung fibroblasts(MLF),this formulation inhibited fibroblast migration and collagen production,thereby slowing the progression of IPF.Overall,LA5-LNPs encapsulated with A20 mRNA is a novel platform offering a potential approach to regulate fibroblast activation for the treatment of IPF.

Activating Connexin43 gap junctions primes adipose tissue for therapeutic intervention

Adipose tissue is a promising target for treating obesity and metabolic diseases.However,pharmacological agents usually fail to effectively engage adipocytes due to their extraordinarily large size and insufficient vascularization,especially in obese subjects.We have previously shown that during cold exposure,connexin43(Cx43)gap junctions are induced and activated to connect neighboring adipocytes to share limited sympathetic neuronal input amongst multiple cells.We reason the same mechanism may be leveraged to improve the efficacy of various pharmacological agents that target adipose tissue.Using an adipose tissue-specific Cx43 overexpression mouse model,we demonstrate effectiveness in connecting adipocytes to augment metabolic efficacy of theβ_(3)-adrenergic receptor agonist Mirabegron and FGF21.Additionally,combing those molecules with the Cx43 gap junction channel activator danegaptide shows a similar enhanced efficacy.In light of these findings,we propose a model in which connecting adipocytes via Cx43 gap junction channels primes adipose tissue to pharmacological agents designed to engage it.Thus,Cx43 gap junction activators hold great potential for combination with additional agents targeting adipose tissue.

FOLR1-induced folate deficiency reduces viral replication via modulating APOBEC3 family expression

Folate receptor alpha(FOLR1)is vital for cells ingesting folate(FA).FA plays an indispensable role in cell pro-liferation and survival.However,it is not clear whether the axis of FOLR1/FA has a similar function in viral replication.In this study,we used vesicular stomatitis virus(VSV)to investigate the relationship between FOLR1-mediated FA deficiency and viral replication,as well as the underlying mechanisms.We discovered that FOLR1 upregulation led to the deficiency of FA in HeLa cells and mice.Meanwhile,VSV replication was notably sup-pressed by FOLR1 overexpression,and this antiviral activity was related to FA deficiency.Mechanistically,FA deficiency mainly upregulated apolipoprotein B mRNA editing enzyme catalytic subunit 3B(APOBEC3B)expression,which suppressed VSV replication in vitro and in vivo.In addition,methotrexate(MTX),an FA metabolism inhibitor,effectively inhibited VSV replication by enhancing the expression of APOBEC3B in vitro and in vivo.Overall,our present study provided a new perspective for the role of FA metabolism in viral infections and highlights the potential of MTX as a broad-spectrum antiviral agent against RNA viruses.

电针足三里对严重烫伤致大鼠急性肺损伤的影响

目的 探讨电针足三里对严重烫伤致大鼠急性肺损伤的影响.方法 雄性SD大鼠40只,体重200～250 g,随机分为5组（n=8）：对照组、烫伤组、足三里组、非经非穴组和α-银环蛇毒素组（α-BGT组）.对照组尾静脉注射生理盐水1 ml.烫伤组、足三里组、非经非穴组和α-BGT组先制备30%总体表面积Ⅲ度烫伤模型,然后烫伤组尾静脉注射生理盐水1 ml;足三里组于双侧足三里穴垂直进针7 mm,给予脉冲电流（电压3V,电流2ms,频率3 Hz）持续刺激12 mim,间隔8 h刺激1次,持续2 d;非经非穴组于双侧足三里穴旁5mm处给予脉冲刺激,方法同足三里组;α-BGT组尾静脉注射α-BGT 1.0 μg/kg,再于双侧足三里穴给予脉冲刺激,方法同足三里组.各组处理结束后,处死大鼠,取肺组织,光镜下观察病理学结果,电镜下观察超微结构,采用ELISA法测定肺组织高迁移率族蛋白B1（HMGBl）含量,采用免疫组化法测定HMGBl蛋白表达,采用RT-PCR法测定HMGBl mRNA表达.结果 烫伤组肺组织光镜下可见肺泡壁崩解,泡内大量渗出液,间质水肿、肥厚和增生,伴大量炎性细胞浸润;电镜下可见细胞核形态不规则,核膜僵硬,部分凸凹不平和核溶解,胞质内板层小体明显减少,肺组织病理损伤程度较对照组减轻.与对照组比较,烫伤组、非经非穴组和α-BGT组肺组织HMGBl含量升高,HMGBl蛋白及其mRNA的表达上调（P〈0.05）,足三里组各指标差异无统计学意义（P〉0.05）;与烫伤组比较,足三里组肺组织HMGBl含量降低,HMGBl蛋白及其mRNA的表达下调,非经非穴组和α-BGT组肺组织HMGBl mRNA表达下调（P〈0.05）;与足三里组比较,非经非穴组和α-BGT组肺组织HMGBl含量升高,HMGBl蛋白及其mRNA的表达上调（P〈0.05）.结论 电针足三里可减轻严重烫伤致大鼠急性肺损伤,其机制与激活含α7亚基N型胆碱能受体介导的胆碱能抗炎通路,抑制肺组织HMGBl的表达有关.

Effects of catecholamine-β-adrenoceptor-cAMP system on severe patients with heart failure

Objective To investigate the association between catecholamine-β-adrenoceptor (β-AR)-adenosine 3’,5’-monophosphate (cAMP) system and long-term prognosis in patients with chronic heart failure (CHF).Methods The study population comprised 73 patients with CHF (EF: 23%±10%) with a mean follow-up of 3.8±1.9 years. Plasma levels of norepinephrine (NE) were measured using high performance lipid chromatography,β-adrenergic receptor density (Bmax) and the content of cAMP in peripheral lymphocytes were calculated using 3H-dihydroalpneolo as ligand and competitive immunoassay,respectively. Deaths due to cardiovascular events within the follow-up period were registered.Results The total mortality was 64.7%,57.4% of which was for cardiogenic (worsening heart failure: 32.4%; sudden death: 25.0%). In the cardiogenic death group,plasma levels of NE and epinephrine (E) (3.74 nmol/L±0.09 nmol/L and 3.17 nmol/L±1.0nmol/L) and the contents of peripheral lymphocyte cAMP (3.64 pmol/mg protein±1.4 pmol/mg protein) were significantly increased as compared with the survival group (2.68 nmol/L±0.07 nmol/L,2.41 nmol/L±0.24 nmol/L and 2.73 pmol/mg protein±0.9 pmol/mg protein,respectively,all P <0.01). In the sudden death group,plasma levels of NE and E (5.01 nmol/L±0.06 nmol/L and 4.13 nmol/L±0.08 nmol/L) were significantly increased as compared with the worsening heart failure group (2.49 nmol/L±0.07 nmol/L and 2.33 nmol/L±0.8 nmol/L,all P <0.001) and to the survival group (2.68 nmol/L±0.07 nmol/L and 2.41 nmol/L±0.14 nmol/L,all P <0.01). The incidences of sudden death were 0%,75%,and 100% (χ 2=16.018, P <0.01) in patients with plasma NE<2.5 nmol/L,NE 2.5 nmol/L-4.5 nmol/L,and NE>4.5 nmol/L,respectively. In the worsening heart failure group,the content of peripheral lymphocyte cAMP (4.46 pmol/mg protein±0.18 pmol/mg protein) was significantly increased compared with the sudden death group (2.39 pmol/mg protein±0.9 pmol/mg protein,P <0.001) and to the survival group (2.73 pmol/mg protein±1.1 pmol/mg protein,P <0.001). The worsening heart failure death occurences were 5.0%,72.2%,and 100% (χ 2=14.26,P <0.01) in patients with a content of peripheral lymphocyte cAMP <2.5 nmol/L,cAMP 2.5 nmol/L-4.5 nmol/L,and cAMP>4.5nmol/L,respectively. B max in peripheral lymphocyte was not significantly different ( P >0.05) among the sudden death,worsening heart failure,and survival groups in CHF patients.Conclusions Plasma levels of catecholamine increase significantly,and B max and the contents of cAMP in peripheral lymphocytes decrease significantly in patients with CHF. High plasma catecholamine levels may be associated with sudden death,and high intralymphocyte cAMP content may be associated with worsening heart failure in CHF patients.