渥曼青霉素阻断罗格列酮对人ACAT-1表达的影响

摘 要 目的：研究过氧化体增殖物激活型受体-γ (peroxisome proliferator activated receptors-γ，PPAR-γ)配体罗格列酮对巨噬细胞酰基辅酶A：胆固醇酰基转移酶-1 (Acyl-CoA：cholesterol acyltransferases，ACAT-1) 表达的影响及可能机制。方法：在RPMI1640培养基中培养人单核细胞系（THP-1），加入佛波酯 (phorbol myristate acetate，PMA) 培养48 h，细胞贴壁呈巨噬细胞样分化。再给予PPAR-γ配体罗格列酮和磷酯酰肌醇三磷酸激酶(phosphatidylinosjtol 3-kinase，PI3K) 的特异性抑制剂渥曼青霉素 (wortmannin，WT)，运用实时定量PCR和Western Blot，观察巨噬细胞ACAT-1mRNA和蛋白表达水平的变化。结果：罗格列酮可明显抑制ACAT-1mRNA和蛋白的表达，且呈浓度依赖性；加入PI3K信号途径抑制剂wortmannin后ACAT-1表达较罗格列酮组明显增加，较巨噬细胞对照组降低。结论：PPAR-γ配体罗格列酮通过PI3K途径抑制ACAT-1表达发挥其抗动脉粥样硬化的作用。     

牛磺酸抑制THP-1细胞酰基辅酶A:胆固醇酰基转移酶-1表达

目的:观察牛磺酸对人单核细胞系THP-1细胞酰基辅酶A:胆固醇酰基转移酶-1(ACAT-1)表达的影响。方法:在有或无干扰素-γ(INF-γ)的条件下,用不同浓度的牛磺酸与THP-1持续孵育不同的时间,RT-PCR检测ACAT-1mRNA水平,WesternBlot检测其蛋白表达。结果:牛磺酸显著下调THP-1的ACAT-1mRNA和蛋白表达水平(P<0.05～0.01,n=3),并呈时间和剂量依赖性效应。INF-γ促进ACAT-1mRNA和蛋白表达(P<0.05),该作用被牛磺酸部分抑制(P<0.01)。结论:牛磺酸抑制ACAT-1的基础表达和由INF-γ诱导的表达,这可能是其抗动脉粥样硬化的机制之一。     

酰基辅酶A:胆固醇酰基转移酶与动脉粥样硬化

人酰基辅酶A：胆固醇酰基转移酶（ACAT）在生物体内催化胆固醇与长链脂肪酸连接形成胆固醇酯,在生物体内胆固醇的吸收、运输和贮存过程中起重要作用,是调节体内胆固醇代谢平衡的关键蛋白之一。遗传及细胞生物学实验研究表明,在动脉粥样硬化症的发生过程中,ACAT被认为发挥了重要作用。本文主要介绍ACAT的调控及其与动脉粥样硬化症发生关系的研究进展。     

游泳训练对载脂蛋白E基因敲除小鼠过氧化物酶体增殖物激活受体-γ及脂代谢酶肉碱棕榈酰转移酶-1、中链酰基辅酶A脱氢酶的影响

目的:观察游泳训练对ApoE基因敲除小鼠胰岛素抵抗模型血清游离脂肪酸(FFA)、肝脏组织过氧化物酶体增殖物激活受体-γ(PPAR-γ)及肉碱棕榈酰转移酶-1(CPT-1)、中链酰基辅酶A脱氢酶(MCAD)mRNA表达的影响,初步探讨游泳训练改善ApoE基因敲除小鼠胰岛素抵抗(IR)的可能机制.方法:选取8周雄性ApoE基因敲除小鼠26只,随机分为:高脂运动组(n=13)和高脂静止组(n=13).高脂运动组小鼠给予高脂饮食加游泳训练12周,高脂静止组除不进行游泳训练外,余同高脂运动组.另以健康雄性C57BL/6J (n=10)小鼠为正常对照组,普通饲料喂养12周.干预12周后,测各组小鼠空腹胰岛素(FIN)、空腹血糖(FPG)、并以HOMA法计算胰岛素抵抗指数(IRI),确定胰岛素抵抗模型建立;全自动生化分析仪测定血清中总胆固醇(TC)、甘油三酯(TG)、高密度胆固醇脂蛋白(HDL)、低密度胆固醇脂蛋白(LDL)、游离脂肪酸(FFA)含量;RT-PCR法测肝脏组织中PPAR-γ、CPT-1、MCAD mRNA表达水平.结果:运动训练干预12周以后:①与正常对照组相比较,高脂静止组体重显著增加(P＜0.05);与高脂静止组比较,高脂运动组体重显著下降(P＜0.05).②与正常对照组相比较,高脂静止组FIN、FPG、Homa-IRI水平明显升高(P均＜0.01);与高脂静止组相比较,高脂运动组FIN、FPG、Homa-IRI明显降低(P分别＜0.05、0.01、0.01).③与正常对照组相比较,高脂静止组TC、LDL、FFA水平明显升高(P均＜0.01);与高脂静止组相比较,高脂运动组TC、LDL、FFA水平明显降低(P分别＜ 0.05、0.05、0.01),HDL水平明显升高(P＜0.05).④与正常对照组相比较,高脂静止组PPAR-γ、CPT-1、MCAD mRNA表达明显降低(P均＜0.01);与高脂静止组相比较,高脂运动组PPAR-γ、CPT-1、MCADmRNA表达明显增加(P均＜0.01).结论:游泳训练可能通过上调肝脏组织PPAR-γ表达、进而上调CPT-1、MCAD的表达,改善小鼠脂代谢,从而改善ApoE基因敲除小鼠胰岛素抵抗.     

同型半胱氨酸对THP-1单核细胞源性泡沫细胞形成中ABCA1、ACAT1表达的影响

目的探讨同型半胱氨酸(Hcy)对THP-1单核细胞源性泡沫细胞形成中三磷酸腺苷-结合转运子A1(ABCA1)和酰基辅酶A:胆固醇酰基转移酶(ACAT1)表达的影响。方法将THP-1单核细胞与佛波酯(PMA)、氧化低密度脂蛋白(ox-LDL)共同培养,复制泡沫细胞模型,并分别用50、100、200、500μmol/L Hcy和100μmol/L Hcy+叶酸+维生素B12(Vit B12)干预72 h,并设对照组(不加入Hcy)。采用油红O染色检测泡沫细胞的形成。采用酶终点法测定细胞内总胆固醇(TC)、游离胆固醇(FC)和胆固醇酯(CE)含量的变化,观察Hcy对泡沫细胞CE流出的影响。采用荧光定量逆转录-聚合酶链反应(RT-PCR)测定ABCA1、ACAT1 mRNA表达;免疫印迹法检测ABCA1、ACAT1蛋白表达。结果油红O染色显示Hcy加剧了泡沫细胞的形成,但不呈量效关系。实验组(加入50、100、200、500μmol/L Hcy和100μmol/L Hcy+叶酸+Vit B12)泡沫细胞阳性百分率均高于对照组(P<0.05、P<0.01),以100μmol/L Hcy组效应最为明显(P<0.01)。在Hcy的干预下,泡沫细胞胞内TC、FC、CE流出减少,与对照组比较差异有统计学意义(P<0.05),以100μmol/L Hcy组效应最为明显(P<0.01)。100μmol/L Hcy+叶酸+VitB12组与100μmol/L Hcy组比较,前者泡沫细胞形成减少,胆固醇流出增多。RT-PCR结果显示ABCA1 mRNA表达下调,ACAT1 mRNA表达上调,均以100μmol/L Hcy组效应最为明显(P<0.01);免疫印迹法检测ABCA1、ACAT1蛋白的表达与其mRNA表达一致。结论 Hcy下调了ABCA1的表达,上调了ACAT1的表达,促使了泡沫细胞形成。     

罗格列酮对糖尿病大鼠血中细胞间粘附分子-1的影响

目的观察罗格列酮对糖尿病大鼠血中ICAM-1水平的影响,以了解其对糖尿病大血管病变的保护作用。方法建立糖尿病大鼠模型,24只糖尿病大鼠随机分为正常对照(A组)、糖尿病组(B组)、罗格列酮治疗组(C组)。糖尿病大鼠模型建立1周后,C组以罗格列酮(5 mg.kg-1.d-1)灌胃,A组和B组灌以相应量的生理盐水,检测各组每周血糖、检测第8周血清中ICAM-1水平。结果血糖水平B组和C组相近,均显著高于A组(P<0.01),B组和C组血中ICAM-1水平均显著高于A组(P<0.01),其中C组ICAM-1水平明显低于B组(P<0.01)。结论罗格列酮具有抗炎作用,对糖尿病大血管病变有一定的保护作用。     

食管鳞癌组织中磷脂酰肌醇3-激酶和血管内皮生长因子的表达及其临床意义

目的研究磷脂酰肌醇3-激酶(PI3K)和血管内皮生长因子(VEGF)在食管鳞癌组织(esophageal squamous cell carcinoma,ESCC)中的表达及其与食管鳞癌细胞发生、发展与转移的关系以及两者之间的相关性。方法应用免疫组织化学链菌素亲生物素-过氧化酶连接法(SP)法检测24例正常食管黏膜、94例食管鳞癌组织中PI3K和VEGF的表达情况。结果食管癌组织中PI3K和VEGF的阳性表达率与正常食管黏膜组织相比差异均有统计学意义,分别为71.3%(67/94)vs 4.2%(1/24)、61.7%(58/94)vs 0(均P<0.01);在淋巴结转移阳性组两者的表达显著高于淋巴结转移阴性组,分别为92.0%(46/50)vs 47.7%(21/44)、76.0%(38/50)vs 45.4%(20/44)(均P<0.01)。VEGF的表达与肿瘤组织类型、浸润深度等因素有关(P<0.01);PI3K与VEGF表达有关联性(C=0.311,P<0.05)。结论 PI3K、VEGF表达与食管癌发生发展及侵袭转移有关,可作为评价食管鳞癌生物学行为的指标。     

电针对APP/PS1小鼠皮质区磷脂酰肌醇-3激酶/糖原合成酶激酶-3α通路相关蛋白表达和老年斑沉积的影响

目的 观察电针对APP/PS1双转基因小鼠皮质区磷脂酰肌醇-3激酶/糖原合成酶激酶-3α (PI3K/GSK3α)信号通路上相关蛋白的分布表达,以及老年斑沉积的影响。 方法 基因鉴定后,3月龄雄性转基因小鼠随机分为模型组和电针组,野生型C57小鼠为对照组,每组6只。电针组电针百会(GV20)和双侧肾俞(BL23)14 d。治疗后,Western blotting和免疫组化法检测各组小鼠皮质区老年斑数量,PI3K亚单位P85α和P110α,以及GSK3α和pS^21-GSK3α等蛋白的分布和表达。 结果 模型组皮质区老年斑数较对照组显著升高(P < 0.001);电针组老年斑数较模型组显著降低( P < 0.001)。各相关蛋白主要表达于皮质神经元胞质。与对照组相比,模型组pS ^21-GSK3α、P110α和P85α蛋白表达明显降低(P < 0.01),GSK3α蛋白表达升高( P < 0.05);与模型组相比,电针组pS ^21-GSK3α、P110α和P85α的蛋白表达明显升高(P < 0.01),GSK3α蛋白表达降低( P < 0.05)。 结论 电针能调节APP/PS1双转基因小鼠皮质区PI3K/GSK3α通路相关蛋白表达,减少老年斑沉积。     

过表达乙酰肝素酶对胆囊癌细胞增殖及PI3K/AKT信号通路的影响

目的 探讨过表达乙酰肝素酶(HPSE)对胆囊癌细胞系GBC-SD细胞增殖及磷脂酰肌醇3-激酶/蛋白质丝氨酸苏氨酸激酶(PI3K/AKT)信号通路的影响。方法 构建HPSE真核表达载体,然后获得转基因过表达HPSE的胆囊癌细胞株GBD-SD。通过实时荧光定量聚合酶链反应(qRT-PCR)和Western blot分析GBC-SD和GBD-SD中HPSE mRNA和蛋白表达。通过集落形成实验、MTT来评估各组细胞的增殖情况。采用Western blot分析PI3K/AKT信号通路相关蛋白(p-PI3K、PI3K、p-AKT和AKT)的水平。结果 成功转染后的GBD-SD中HPSE mRNA水平和蛋白水平升高,差异有统计学意义(P<0.01)。与正常表达HPSE的GBC-SD相比,过表达HPSE的GBD-SD细胞光密度(OD)值和集落形成能力提高,差异有统计学意义(P<0.05)。Western blot检测结果显示,与GBC-SD细胞相比,过表达HPSE的GBD-SD组细胞p-AKT、p-PI3K蛋白表达水平、p-AKT/AKT值和p-PI3K/PI3K值增加,差异有统计学意义...     

PI3K/Akt信号通路对转化生长因子β1诱导的人肺上皮-间质细胞转分化的影响

目的:探讨磷脂酰肌醇3激酶(PI3K)/丝氨酸/苏氨酸蛋白激酶(Akt)通路在转化生长因子β1(TGF-β1)体外诱导的人肺上皮细胞A549间充质化过程中的作用。方法:将体外培养的A549细胞随机分成3组:正常对照组、TGF-β1组及抑制剂组,正常对照组不加入TGF-β1,TGF-β1组加入10ng/mLTGF-β1,抑制剂组加入TGF-β1(10ng/mL)和PI3K的抑制剂Ly294002(10nmol/L),72h后通过RT-PCR检测各组A549细胞上皮标志物E-钙黏蛋白(E-cad)及间充质细胞标志物α-平滑肌肌动蛋白(α-SMA)表达的变化,ELISA方法检测间充质细胞标志物纤连蛋白(Fn)表达的变化,Westernblot检测Akt磷酸化(p-Akt)水平的变化。所有实验至少重复3次。结果:正常体外培养的A549细胞有E-cad表达及微量的α-SMA、Fn、p-Akt表达;TGF-β1组E-cad的表达下调,α-SMA、Fn、p-Akt的表达上调;抑制剂组与TGF-β1组比较E-cad的表达上调,α-SMA、Fn、p-Akt的表达明显抑制。结论:PI3K/Akt途径参与TGF-β1介导的肺上皮-间质细胞转分化过程,PI3K特异性抑制剂Ly294002可有效抑制TGF-β1介导的肺上皮-间质细胞转分化过程。     

Esterification of cholesterol in human small intestine: the importance of acyl-CoA:cholesterol acyltransferase

Human intestinal mucosa contains acyl-CoA:cholesterol acyltransferase activity. The enzyme has been studied by using oleylcarnitine, CoA and carnitine palmitoyltransferase as an oleyl-CoA regenerating system. The enzyme was found in the particulate fraction of the cells, it had a pH optimum between 7.2 and 8.2, and was inhibited by taurocholate. The specific enzymic activity in biopsies from intestinal mucosa of normal men was found to be 3.6 +/- 1.37 nmol cholesteryl ester formed mg protein-1 h-1, an activity which can account for all cholesteryl esters in intestinal lymph. Low enzymic activity was found in biopsies from patients with small intestinal disorders. Two pancreatectomized patients had values within the normal range.     

The effect of feeding and fasting on the activity of acyl-CoA:cholesterol acyltransferase in rat small intestine

The purpose of the present study was to test if the microsomal acyl-CoA:cholesterol acyltransferase (ACAT) in rat small intestine is regulated under physiological conditions. Two previously described in vitro assays were used, both based on the esterification of endogenous cholesterol with exogenous acyl-CoA, performed or generated during the incubation. The important and consistent finding with rats on normal diet was an increase in ACAT activity with fasting and a decrease with feeding. Independent of the assay used, the ratio between ACAT activity in night-fasted and night-fed animals was about 2 (P less than 0.005). When the fasting period was extended to 36 h a corresponding difference was found whether the ACAT assay was based on preformed [1-14C]oleoyl- or [1-14C]palmitoyl-CoA as acyl-donor (P less than 0.05). The microsomal content of unesterified cholesterol was higher in fasted than fed animals, suggesting that availability of this substrate might be a factor in the regulation of rat intestinal ACAT activity.     

Effect of lipoprotein concentration and lecithin: cholesterol acyltransferase activity on cholesterol esterification in human plasma after plasma exchange

The rate of cholesterol esterification in plasma, plasma lecithin cholesterol acyltransferase (LCAT) activity and plasma lipoprotein levels have been measured in five subjects who underwent therapeutic plasma exchange to reduce their plasma cholesterol concentration. In the week following the exchange the cholesterol esterification rate and the plasma triglyceride concentration returned rapidly in parallel to pre-exchange levels, while high density lipoprotein (HDL) cholesterol and LCAT activity returned to normal more slowly but also in parallel. The data suggest that the rate-limiting factor for cholesterol esterification in plasma is unlikely to be solely the enzyme levels, but is probably a combination of factors, including the enzyme level and either substrate availabiltiy or product removal. Plasma very low density lipoprotein (VLDL) may either provide substrates for the reaction or provide a means of removing one of the products from the site of reaction.     

Decreased hepatic expression of PPAR-gamma coactivator-1 in cholesterol cholelithiasis

BACKGROUND: Cholesterol cholelithiasis (gallstone disease) is a common disease in the Western world. The aim of the present study was to analyze the hepatic expression of a number of nuclear receptors involved in bile acid metabolism in human cholesterol gallstone disease. MATERIALS AND METHODS: Surgical liver biopsies were obtained from 11 patients with untreated cholesterol cholelithiasis and nine gallstone-free subjects; mRNA levels of cholesterol 7alpha-hydroxylase (CYP7A1) and related nuclear receptors and coactivators were assayed by quantitative real-time RT-PCR. RESULTS: No differences between the two groups were detected in mRNA levels of CYP7A1 and related nuclear receptors, with the exception of peroxysome proliferator-activated receptor-gamma coactivator 1 (PGC-1), which was significantly (P < 0.01) less expressed in gallstone subjects. Expression of PGC-1 was linearly correlated with farnesoid X receptor (FXR) in gallstone patients (r = 0.87 on a log scale, P < 0.01), but not in control subjects; in gallstone patients PGC-1 expression was also correlated with hepatocyte nuclear factor 4 (HNF-4) (r = 0.78, P < 0.01). CONCLUSION: These findings suggest that PGC-1 can play a role in the prevention of cholesterol gallstone disease in humans; this might take place via interaction with the bile acid receptor FXR, whose protective role in cholelithiasis has been suggested by recent evidence in animal models and other coactivators. The present data might help to understand the pathophysiology and possibly focus on new therapeutical targets in cholesterol gallstone disease.     

罗格列酮对人腹膜间皮细胞和脐静脉内皮细胞水孔蛋白-1的影响

目的探讨体外条件下核受体Peroxisome proliferator-activated receptor gamma（PPAR-gamma）激动剂罗格列酮（RosiglitazoneRGZ）对人腹膜间皮细胞（human peritoneal mesothelial cells HPMCs）、人脐静脉内皮细胞（Human Umbilical Vein Endothelial Cells HUVECs）水孔蛋白-1（aquaporin 1 AQP1）增殖以及基因水平的影响。方法以HPMCs和HUVECs为研究对象,将实验分成四组：对照组、RGZ组（10uMRGZ）、GW9662组（peroxisome proliferator-activated receptor gamma antagonist 20uMGW9662）以及GW9662＋R组（20uMGW9662提前作用1H后加入10uMRGZ）。应用比色法（CCK-8）观察RGZ等对HPMCs和HUVECs增殖的影响。应用Realtime-PCR方法检测罗格列酮等对水孔蛋白-1基因水平表达的影响。结果①GW9662＋R组与对照组相比,可以明显抑制HPMCs增殖（P〈0.01）,其他组对HPMCs增殖没有明显影响（P〉0.05）。而对HUVECs,RGZ组、GW9662组以及GW9662＋R组都可以促进其增殖（P〈0.01）;②罗格列酮上调这两种细胞AQP1基因水平的表达。结论在体外,罗格列酮可影响HPMCs和HUVECs水孔蛋白质1基因水平的表达。其作用机制可能是通过激活PPAR-gamma从而影响上述两种细胞的AQP1的表达。     

The expression of the solute carriers NTCP and OCT-1 is regulated by cholesterol in HepG2 cells

Drug disposition and response are greatly determined by the activities of drug-metabolizing enzymes and transporters. While the knowledge in terms of CYP enzymes and efflux ABC transporters (such as MDR1, P-glycoprotein) is quite extensive, influx transporters are increasingly being unveiled as key contributors to the process of drug disposition. There is little information on the regulation of these proteins in human cells, especially as regards the effect of endogenous compounds. In this study, we analysed the expression of CYP3A4 and three uptake transporters NTCP (SLC10A1), OATP-A/OATP1A2 (SLCO1A2) and OCT-1 (SLC22A1) in HepG2 cells following treatment with cholesterol. While CYP3A4 and OATP1A2 expression was unaffected, cholesterol treatment led to increased levels of NTCP and OCT-1 mRNAs. Alterations in the functional characteristics and/or expression levels of drug transporters in the liver may conceivably contribute to the variability in drug oral bioavailability often observed in the clinical settings.     

脂质前列腺素E1对急性肺损伤T细胞特异性转录因子T-bet与Gata-3的作用

目的 探讨脂质体前列腺素E1(Lipo-PGE1)对脂多糖(lipopolysaccharide,LPS)诱导的小鼠急性肺损伤中表达失衡的转录因子T-bet/Gata-3的影响及其对Th1/Th2平衡的调节机制.方法 60只雌性BALB/C小鼠随机(随机数字法)分成3组(每组20只):尾静脉注射生理盐水10 ml/kg为对照组,尾静脉注射LPS 5 mg/kg(稀释至0.5 mg/ml)为LPS模型组,给予LPS 1 h后给予脂质前列腺素E1 15 μg/kg尾静脉注射为治疗组(LPS+ PGE1组).各组于6h后观察肺组织变化,并测定肺湿/干质量比(W/D).采用流式细胞仪分别检测Th1、Th2细胞的阳性率,SYBRGreen Ⅰ实时荧光定量PCR技术检测T-bet和Gata-3 mRNA的表达.应用SPSS 13.0统计软件行单因素方差分析.结果 模型组肺组织出现病理学改变,W/D为(5.74±0.31)较对照组(4.79±0.27)升高(P＜0.01),而PGE1治疗组W/D(4.92±0.27)较模型组明显降低(P＜0.01),肺组织病理学改变也明显减轻.模型组Th1、Th2细胞的阳性率及两者比值[(16.65±1.70)％,(9.40±1.25)％,(1.77 ±0.03)]较对照组[(23.67±2.10)％,(12.17±1.80)％,(1.95±0.04)]均显著降低,差异具有统计学意义(P＜0.01);与模型组比较,PGE1治疗组上述3个指 标[(20.31±2.20)％,(10.50±0.80)％,(1.93 ±0.05)]均升高,差异具有统计学意义(P＜0.01).模型组转录因子T-bet与Gata-3基因表达量及两者比值[(1.183 ±0.495),(0.693±0.285),(1.713 ±0.131)]较对照组[(3.439 ±0.557),(1.203±0.238),(2.857±0.290)]均降低,差异具有统计学意义(P＜0.01);与模型组比较,PGE1治疗组T-bet基因的表达量(1.827 ±0.705),T-bet与Gata-3表达量比值(2.502±0.352)均明显增高,差异有统计学意义(P＜0.01),而Gata-3的表达量(0.719 ±0.186)增高不明显,差异无统计学意义(P＞0.05).结论 脂质前列腺素E1可能通过增加T-bet基因的表达,纠正Th1/Th2平衡失调,从而改善相应的炎性症状.