甘露聚糖结合凝集素对脂多糖诱导的3T3-L1脂肪细胞炎症反应和胰岛素抵抗的调节作用及机制

目的：研究甘露聚糖结合凝集素（MBL）对脂多糖的诱导3T3-L1脂肪细胞炎症反应及胰岛素抵抗的调节作用及机制。方法：诱导3T3-L1前脂肪细胞分化,油红O染色分析细胞分化程度,使用CCK-8检测分化过程中MBL(1、10、20μg/ml)及LPS对细胞增殖能力的影响;ELISA检测细胞炎症因子IL-6、TNF-α含量,Western blot检测TNF-α、IL-6及TLR4/NF-κB信号通路中蛋白表达,并分析Akt、IRS-1 try蛋白磷酸化及GLUT4表达情况;流式细胞术检测MBL对3T3-L1细胞摄取葡萄糖的调节作用。结果：油红O染色显示,12 d时,3T3-L1前体脂肪细胞已诱导为完全成熟的脂肪细胞;CCK-8结果证实,在细胞分化全过程,不同浓度的MBL (1、10、20μg/ml)及LPS对其增殖能力无明显影响;ELISA及Western blot结果显示在脂肪细胞分化至成熟过程中,MBL处理均可抑制LPS诱导的炎症因子分泌,且其抑制作用呈浓度依赖性;Western blot结果显示MBL以浓度依赖的方式抑制LPS诱导的IκB、IKK磷酸化和TLR4以及核NF-κB表达,...     

α-亚麻酸抑制高脂诱导的脂肪细胞氧化应激和促炎因子的释放

目的通过给予3T3-L1脂肪细胞高脂处理,探讨α-亚麻酸(α-linolenic acid,ALA)抑制脂肪细胞促炎因子释放和氧化应激的作用及其机制。方法给予ALA预处理的成熟3T3-L1脂肪细胞棕榈酸酯(palmitate)以及20 mmol/L原卟啉锡(SnPP)处理,检测炎症因子TNF-α和IL-6水平;HO-1、SOD、CAT以及GCLC表达水平以及NF-κB核转位和细胞中ROS水平。结果ALA可剂量依赖性地抑制高脂诱导的脂肪细胞中促炎因子TNF-α、IL-6的产生(P0.01);ALA显著增加了高脂处理的脂肪细胞中抗氧化相关因子HO-1、CAT、SOD、GCLC的表达,减少细胞中ROS生成,抑制NF-κB的活化(P0.01);HO-1抑制剂可阻断上述ALA对脂肪细胞的抗炎和抗氧化作用。结论 ALA可以显著抑制高脂诱导的脂肪细胞促炎因子的分泌以及氧化应激反应,其机制可能与促进HO-1表达、抑制ROS产生从而抑制NF-kB介导的炎症反应有关。     

重组人生长激素对THP-1单核细胞TNF-α和IL-6分泌的影响与NF-κB活性的相关性研究

目的探讨重组人生长激素(rhGH)对THP-1单核细胞TNF-α和IL-6分泌的影响,并研究其与NF-κB活性的相关性。方法应用ELISA检测rhGH诱导THP-1细胞培养上清中TNF-α和IL-6的分泌情况,并观察LPS和NF-κB特异性抑制剂BAY11-7082对其分泌的影响;应用荧光素酶报告基因和电泳迁移率实验(EMSA)检测NF-κB在rhGH诱导的单核细胞中的活化程度。结果 rhGH单独可以促进THP-1细胞分泌TNF-α和IL-6,抑制LPS诱导的细胞因子的分泌;BAY11-7082能显著抑制rhGH和LPS诱导的TNF-α和IL-6的分泌,NF-κB活性与TNF-α和IL-6的分泌呈现明显的相关性。结论 rhGH可通过NF-κB信号通路双向调节THP-1单核细胞TNF-α和IL-6的分泌。     

基于细胞自噬探讨清血消脂片含药血清对脂肪细胞炎症模型的干预作用

<正>目的:探讨中药清血消脂片含药血清(QXXZ)对游离脂肪酸诱导的3T3-L1前脂肪细胞炎症干预作用。方法:采用3-异丁基-1-甲基黄嘌呤异丁基-3-甲基黄嘌呤、地塞米松和胰岛素诱导3T3-L1前脂肪细胞的分化。用油酸诱导细胞代谢性炎症;采用CBA法和RT-PCR法分别观察QXXZ对细胞炎症因子和NF-κB mRNA的影响;用3-甲基腺嘌呤(3-MA)抑制细胞自噬,免     

唾液支原体分别经NF-κB依赖性和非依赖性方式诱导HGEC产生IL-1β、IL-8和表达hBD-2

目的:研究唾液支原体感染牙龈上皮细胞(HEGC)后,人β防御素-2(hBD-2)的表达和IL-1β、IL-8的产生情况,以及这两者是否与NF-κB的激活有关。方法:HGEC经口腔支原体刺激后,RT-PCR检测hBD-2 mRNA表达,ELISA检测细胞因子的产生,Western blot分析NF-κB的激活情况。结果:HGEC经唾液支原体刺激3 h后能经NF-κB产生IL-8。同时唾液支原体也能以非NF-κB依赖性方式诱导hBD-2表达。IL-8的产生与hBD-2 mRNA的表达存在一定相关性。结论:hBD-2的非NF-κB依赖性表达可能与初始的炎症反应有关,IL-8的产生可能通过自分泌机制影响到上皮细胞表达hBD-2 mRNA的表达。     

甘氨酸对3T3-L1脂肪细胞胰岛素抵抗的影响

目的：探讨甘氨酸对3T3-L1脂肪细胞胰岛素抵抗的影响及其机制。方法:诱导分化3T3-L1脂肪前体细胞为成熟的脂肪细胞，肿瘤坏死因子α（TNF-α）诱导建立脂肪细胞的胰岛素抵抗模型，以罗格列酮为阳性对照，观察甘氨酸干预后胰岛素受体底物-1（IRS-1）和过氧化物酶体增殖物激活受体γ（PPARγ）基因的表达。结果:正常对照组脂肪细胞IRS-1mRNA和PPARγ mRNA的表达最强；TNF-α组IRS-1mRNA和PPARγ mRNA表达显著低于正常对照组；TNF-α加罗格列酮组与TNF-α加甘氨酸组相似，IRS-1mRNA和PPARγ mRNA表达显著高于TNF-α组。结论:甘氨酸对TNF-α诱导的3T3-L1脂肪细胞胰岛素抵抗具有抑制作用，其机制与其增强IRS-1、PPARγ基因的表达有关。     

白术多糖对小鼠淋巴细胞的免疫调节作用

目的:探讨白术多糖对小鼠脾淋巴细胞的免疫调节作用。方法:将白术多糖和ConA或LPS及小鼠脾淋巴细胞在体外共培养,MTT法检测淋巴细胞转化;ELISA法检测细胞培养上清中细胞因子(IL-2、IL-6、IL-10和TNF-α)和IgG含量;RT-qPCR法检测白术多糖联合ConA或LPS对转录因子T-bet、Gata3和NF-κB mRNA表达的影响;流式细胞术检测白术多糖联合LPS对CD3、CD4、CD8和CD19淋巴细胞亚群比例的影响。结果:白术多糖促进ConA诱导的脾脏T淋巴细胞转化、IL-2、IL-6、IL-10和TNF-α分泌和转录因子(T-bet和Gata3)mRNA表达,抑制LPS对脾脏B淋巴细胞的激活,减少TNF-α和IgG分泌,降低NF-κB mRNA表达水平,降低LPS诱导的CD3、CD4和CD8淋巴细胞亚群比例。结论:白术多糖可促进ConA诱导的淋巴细胞转化,但抑制LPS诱导的淋巴细胞转化。     

醛固酮对3T3-L1前脂肪细胞与脂肪细胞内脂素表达和分泌的影响

目的:探讨醛固酮对3T3-L1前脂肪细胞和脂肪细胞内脂素基因表达和蛋白分泌的影响。方法:10-8和10-6mol/L醛固酮加或不加10-6mol/L安体舒通分别干预3T3-L1前脂肪细胞和脂肪细胞24h和48h,用实时RT-PCR测定内脂素和盐皮质激素受体(MR)mRNA的表达,酶联免疫法测定培养液中内脂素的浓度。结果:醛固酮作用于3T3-L1前脂肪细胞,内脂素mRNA表达减少,培养液中蛋白浓度变化不明显,MR mRNA表达增高。醛固酮作用于脂肪细胞,内脂素mRNA表达和蛋白浓度均降低,MR mRNA表达增高。安体舒通在一定程度上可对抗醛固酮对内脂素的抑制作用。结论:醛固酮抑制3T3-L1脂肪细胞内脂素的基因表达和分泌。     

小檗碱激活AMPK抑制3T3-L1脂肪细胞分化

目的 探讨小檗碱对3T3-L1脂肪分化的作用是否与激活腺苷酸活化蛋白激酶（AMPK）有关.方法 在3T3-L脂肪细胞分化全程加入小檗碱,以油红O染色检测3T3-L1脂肪细胞胞浆中脂肪的堆积,实时定量PCR检测过氧化物酶体增殖物激活受体γ2（PPARγ2）、CCAAT增强子结合蛋白α（CEBPα）和AMPK的mRNA表达,以Western印迹法检测AMPK和乙酰辅酶A羧化酶（ACC）的磷酸化水平.结果 小檗碱剂量依赖性地抑制3T3-L1脂肪细胞分化,10 μmol/L小檗碱几乎完全抑制胞浆中脂肪的堆积.5 μmol/L小檗碱在脂肪细胞诱导分化1、3、5、7d后均显著降低CEBPα mRNA表达（P〈0.05或P〈0.01）,诱导分化3、5、7d时显著降低PPARγ2的mRNA表达（P〈0.05或P〈0.01）.AMPK的mRNA水平在分化过程中未受小檗碱的明显影响,而小檗碱明显增加其蛋白磷酸化水平,其下游靶基因ACC磷酸化水平也明显增加.结论 小檗碱抑制3T3-L1脂肪细胞的分化可能与其激活AMPK有关.     

KLF5沉默通过抑制NF-κB信号通路减少氧化应激条件下心肌细胞炎症因子分泌

目的研究沉默Krüpple样因子5(KLF5)对氧化应激条件下心肌细胞炎症因子分泌的影响及机制。方法心肌H9c2细胞用过氧化氢处理,qRT-PCR和Western blot检测细胞中KLF5 mRNA和蛋白表达水平。用KLF5 shRNA慢病毒感染H9c2细胞,给予过氧化氢处理后,qRT-PCR和Western blot检测细胞中KLF5表达水平。MTT检测沉默KLF5对过氧化氢处理的心肌细胞活性的影响,同时用qRT-PCR法检测肿瘤坏死因子-α(TNF-α)、白细胞介素-1β(IL-1β)mRNA水平,Western blot检测细胞中核因子-κBp65(NF-κBp65)蛋白水平。用NF-κB激活剂处理沉默KLF5的心肌细胞,MTT检测其在过氧化氢处理后细胞活性,qRT-PCR法检测过氧化氢处理后细胞中TNF-α、IL-1β水平。结果过氧化氢诱导H9c2细胞中KLF5 mRNA和蛋白表达。KLF5 shRNA慢病毒感染可下调氧化应激条件下H9c2细胞中KLF5的表达。过氧化氢能够下调H9c2细胞活性,提高细胞分泌的TNF-α、IL-1β水平,促进细胞中NF-κBp65蛋白表达。沉默KLF5能够提高过氧化氢条件下H9c2细胞经活性,减少细胞分泌TNF-α、IL-1β,抑制细胞中NF-κBp65蛋白表达。NF-κB激活剂可以逆转沉默KLF5对过氧化氢处理的心肌细胞增殖活性、炎症因子分泌的影响。结论氧化应激诱导心肌细胞表达KLF5,沉默其表达可以通过抑制NF-κB通路减少心肌细胞中炎症因子表达。     

生长激素调节T淋巴细胞的功能

目的：探讨生长激素（ＧＨ）对Ｔ淋巴细胞功能的影响。方法：构建ＧＨ ｃＤＮＡ表达质粒ＰｃＤＮＡ－ＧＨ ｃＤＮＡ，然后将其转染入Ｔ淋巴细胞系－Ｊｕｒｋａｔ细胞中，观察过度表达的内源ＧＨ和加入外源基因重组人ＧＨ对Ｔ淋巴细胞功能的影响。结果：过量表达的内源ＧＨ使Ｊｕｒｋａｔ细胞表达ＩＬ－２Ｒ和分泌ＩＬ－２，ＩＦＮ－γ分别增加１．４０倍，１．６０倍和１．９８倍，而在培养液中加入抗ＧＨ血清后，ＧＨ的这种作用消     

Early-life growth hormone treatment to offspring of undernourished mothers alters metabolic parameters in primary adipocytes in adulthood

Abstract

Maternal undernutrition (UN) is associated with the development of obesity and metabolic complications in adult offspring. This study investigated the impact of preweaning growth hormone (GH) treatment on adipocyte functionality in adult male offspring. Sprague-Dawley rats were assigned either standard (C) or undernourished (UN) diet (50% ad libitum) throughout gestation. Postnatal day 3–21, male C/UN pups received either saline (CS, UNS) or GH (2.5?µg/g/d; CGH, UNGH) by subcutaneous injection. Primary adipocytes were isolated following the collagenase digestion of adipose tissue. Primary adipocytes from UN offspring had significantly increased the secretion of pro-inflammatory cytokines accompanied by increased cytokine/cytokine receptor expression. This correlated with increased TLR4/NF-κB signaling. While increased inflammatory potential was not observed in adipocytes derived from UNGH offspring, there was a clear alteration in the expression of genes relating to carbohydrate and lipid metabolism along with nutrient transporters. Overall, preweaning GH treatment alters detrimental patterns of development, which predispose UN offspring to obesity and insulin resistance.     

Correlation between exon 3 polymorphism of growth hormone receptor gene and the responses to rhGH therapy

Objective: To investigate the correlation between the exon 3 polymorphism of growth hormone receptor (GHR) gene and the responses to the recombinant human growth hormone (rhGH) therapy in children with short stature. Methods: Forty-five growth hormone deficiency (GHD) children (male: 30, female: 15, aged 10.39±2.73 yrs) and twenty-five idiopathic short stature (ISS) children (male: 15, female: 10, aged 10.58±2.56 yrs) admitted to our hospital were included. The polymorphism of exon 3 of GHR gene was determined using multiple PCR amplification. Treatment duration for each subject was at least 12 months. On this basis, we evaluated the correlation between treatment efficiency of rhGH therapy and GHR exon 3 polymorphism, GHD, and treatment duration. Results: Significant difference was noted in the growth velocity (GV) of GHD children with a genotype of GHRfl compared with those with a genotype of GHRd3 (9.44±2.35 vs. 11.36±2.49, P < 0.05). Meanwhile, the GV of ISS patients with a genotype of GHRfl were remarkably decreased compared with those with a genotype of GHRd3 (8.74±2.36 vs. 11.18±2.44, P < 0.05). For the children with peak GH response of less than 5 ng/ml, statistical difference was noted in the GV of children with a genotype of GHRfl compared with those with a genotype of GHRd3 (9.55±2.76 vs. 10.84±1.53, P < 0.05). For the patients with peak GH response to clonidine or pyridostigmine bromide of > 5 ng/ml, a satisfactory response to rhGH therapy was noted in children with a genotype of GHRd3 compared with those of GHRfl (P < 0.05). Conclusions: GHRd3 was correlated with the response to rhGH therapy in children with short stature. For the patients with the same genotype, GHD caused no obvious effects on the final height. However, for the patients with peak GH response of > 5 ng/ml, a satisfactory response to rhGH therapy was noted in children with a genotype of GHRd3 compared with those of GHRfl (P < 0.05). A higher treatment efficiency was obtained in those received rhGH at an early age.     

Salicylate Inhibits Macrophage-Secreted Factors Induced Adipocyte Inflammation and Changes of Adipokines in 3T3-L1 Adipocytes

Antidiabetic effects of salicylates have been known for years, however the cellular and molecular mechanisms of the hypoglycemic activity are not well elucidated. We examined the effects of salicylate on inflammation-related changes in gene or/and protein expressions of several adipokines in 3T3-L1 adipocytes and of LPS-induced inflammatory factors in RAW 264.7 cell. Especially, we focused our attention on the cross-talk between the macrophages and adipocytes. Exposure to RAW-CM medium resulted in an increase in the gene expression or/and protein secretion of TNF-α, IL-6 and resistin, and at the same time, a decrease in the gene expression of PPARγ and adiponectin in 3T3-L1 adipocytes. Salicylate effectively reversed these changes, and up-regulated glucose consumption in adipocytes. We also found salicylate inhibited phosphorylation of NF-κB in RAW-CM-stimulated adipocytes. We conclude salicylate blocks inflammatory process in the pathogenesis of inflammation-related insulin resistance.     

Secretion of growth hormone and thyroid-stimulating hormone in patients with dementia

We studied the growth hormone (GH) response to GH-releasing hormone (GHRH) and the thyroid-stimulating hormone (TSH) response to thyrotropin-releasing hormone (TRH) in four groups of patients with dementia and examined whether GH and TSH secretion is altered in patients with Alzheimer's disease. The four groups included those with Alzheimer's disease (n=28), parkinsonism with dementia (n=10), progressive supranuclear palsy with dementia (n=10), and dementia of vascular origin (n=28). The results showed no differences among the four groups in GH response to GHRH (12.2 ± 2, 10.7 ± 2, 8.9 ±1.1, and 9.9 ± 1.9 g/ml, respectively); there was no correlation between GH response to GHRH and sex, stage of the disease, or cerebral atrophy. The proportion of patients with exaggerated, normal, or lower GH response was similar in the four groups. There were also no differences among the groups in terms of TSH response to TRH (9.2 ±0.9, 11.1 ± 1, 11.1 ± 1, and 10.3 ± 1 mU/ml, respectively), nor was there a correlation between TSH response to TRH and sex, stage of the disease, cerebral atrophy, or GH response to GHRH. The proportion of those with exaggerated, normal, or lower TSH response was similar in the four groups. Cerebrospinal somatostatin levels were similar in Alzheimer's disease and vascular dementia patients. These findings indicate that neither GH response to GHRH nor TSH response to TRH provides a useful diagnostic adjunt in Alzheimer's disease patients.Abbreviations AD Alzheimer's disease - PD parkinsonism with dementia - PSP progressive supranuclear palsy - VD dementia of vascular origin - GH growth hormone - GHRH growth hormone releasing hormone - TRH thyrotropin releasing hormone - TSH thyroid stimulating hormone Correspondence to: J.M. Gomez     

GRK5对大鼠星形胶质细胞活化的作用及其机制研究

目的:探讨培养新生大鼠皮层星形胶质细胞G蛋白偶联受体激酶5(GRK5)基因沉默后核因子κB(NF-κB)表达的变化及其与胶质细胞活化、炎症反应和氧化应激的关系。方法:采用分别或联合RNA干扰沉默GRK5基因表达与NF-κB抑制剂N-乙酰半胱氨酸干预进行实验分组。利用免疫荧光、实时定量PCR和Western blotting观察GFAP与活性caspase-3表达,细胞分泌TNF-α与NO的浓度,p65、TNF-α、IL-1β和iNOS的表达情况等。结果:GRK5 siRNA刺激星形胶质细胞活化,并检测到NF-κB表达增多(P0.01),TNF-α、IL-1β和iNOS mRNA表达水平增高(P0.01),细胞分泌TNF-α和NO增多(P0.01),活性caspase-3表达增多(P0.01)。采用GRK5 siRNA+NF-κB抑制剂联合干预可部分逆转GRK5 siRNA引起的上述变化(P0.05)。结论:GRK5基因沉默可能通过刺激NF-κB表达增多引起星形胶质细胞活化。GRK5的正常表达可能具有抑制星形胶质细胞活化相关炎症反应及氧化应激的作用。     

siRNA 技术沉默SOCS3 对缺氧复氧心肌细胞增殖、凋亡的影响以及作用机制

目的：研究小干扰RNA（siRNA）靶向沉默细胞因子信号转导抑制因子3（SOCS3）基因对心肌细胞增殖、凋亡的影响以及可能作用机制。方法：采用脂质体Lipofectamine 2000转染大鼠H9C2心肌细胞,分为对照组、缺氧/复氧组、缺氧/复氧+siRNA NC组、缺氧/复氧+siRNA SOCS3组;免疫印迹试验（Western blot）检测细胞的转染效果;噻唑蓝（MTT）观察细胞的增殖活性,观察细胞中乳酸脱氢酶（LDH）、超氧化物歧化酶（SOD）、丙二醛（MDA）水平的变化;膜联蛋白 V-FITC（Annexin V-FITC）/碘化丙啶(PI)染色法检测细胞凋亡率,Western blot检测核因子-κB（NF-κB）、细胞周期蛋白D1（Cyclin D1）、c-Myc蛋白水平。结果：与对照组相比,缺氧/复氧组心肌细胞SOCS3蛋白水平显著增加（P<0.05）,细胞的增殖活性、SOD显著下降（P<0.05）,LDH、MDA、凋亡率显著升高（P<0.05）;靶向沉默缺氧复氧心肌细胞SOCS3基因表达较缺氧/复氧组细胞的增殖活性和SOD显著增加（P<0.05）,LDH、MDA、凋亡率显著降低（P<0.05）;缺氧/复氧干预心肌细胞显著抑制NF-κB、Cyclin D1、c-Myc蛋白表达（P<0.05）,下调SOCS3基因表达显著增加NF-κB、Cyclin D1、c-Myc蛋白表达（P<0.05）。结论：沉默SOCS3表达促进缺氧复氧心肌细胞增殖,抑制其凋亡,增强机体氧自由基的清除能力,其作用机制与NF-κB信号通路的激活有关。     

脂联素siRNA对3T3-L1细胞基础葡萄糖转运的影响

目的：构建携带小鼠脂联素（Acrp30）siRNA腺病毒载体，并检测其对小鼠脂肪细胞Acrp30表达以及对3T3-L1脂肪细胞基础葡萄糖转运的影响。方法:设计并化学合成小鼠脂肪细胞Acrp30 siRNA片段，将其亚克隆入AdEaxy XL 腺病毒载体系统，在293细胞内包装扩增为重组腺病毒。用此重组腺病毒感染3T3-L1脂肪细胞，用RT-PCR和ELISA检测其Acrp30 mRNA和蛋白表达。采用2-Deoxy-［3H］D-glucose掺入法测定脂肪细胞葡萄糖转运。结果:设计并构建了小鼠Acrp30 基因特异性siRNA腺病毒载体，该载体感染脂肪细胞后，能显著抑制Acrp30 mRNA和蛋白表达，影响3T3-L1脂肪细胞基础葡萄糖的转运，与对照组相比，差异显著(P<0.05)。结论:构建的Acrp30 基因特异性siRNA腺病毒载体能有效地抑制脂联素在3T3-L1脂肪细胞中的表达，从而影响3T3-L1脂肪细胞基础葡萄糖转运。     

丝胶对2型糖尿病大鼠睾丸生长激素/胰岛素样生长因子-1轴的作用

目的 探讨丝胶对2型糖尿病大鼠睾丸生长激素（GH）/胰岛素样生长因子-1（IGF-1）轴的作用。 方法 40只雄性SD大鼠随机分为正常对照组、糖尿病模型组、丝胶治疗组和阳性对照组,每组均为10只。链脲佐菌素连续腹腔注射制作2型糖尿病大鼠模型,1周后以血糖≥16.7mmol/L作为成模标准。待模型成功建立后,模型组大鼠不做任何处理,丝胶治疗组大鼠给予丝胶灌胃［2.4g/(kg&#8226;d)］,阳性对照组大鼠给予二甲双胍［55.33mg/(kg&#8226;d)］灌胃,均为35d。采用酶联免疫吸附测定（ELISA）方法检测大鼠血清睾酮、GH和IGF-1水平;分别采用免疫组织化学染色、免疫印迹法和RT-PCR法检测睾丸GH、GH受体（GHR）和IGF-1的表达。 结果 丝胶可明显降低糖尿病大鼠血GH水平、下调睾丸GH的表达,升高血IGF-1和睾酮水平,上调睾丸IGF-1和GHR的表达（P＜0.05,P＜0.01）。并且丝胶治疗组各项指标与阳性对照组比较无明显差别（P＞0.05）。 结论 丝胶可通过调节糖尿病时GH/IGF-1轴紊乱改善生精功能,发挥对糖尿病生殖功能损害的保护作用,其作用与二甲双胍相当。