磷脂酰肌醇3-激酶/蛋白激酶B信号通路与高血压

综述磷脂酰肌醇3-激酶(PI3K)/蛋白激酶B(Akt)信号通路的构成、转导途径及其在高血压及相关靶器官损害中的作用机制,并阐述基于PI3K/Akt信号通路高血压的中医药治疗进展,旨在为高血压的临床诊疗提供新的思路和治疗靶点。     

替罗非班通过磷脂酰肌醇-3激酶/蛋白激酶B信号通路对氧糖剥夺心肌细胞的干预作用

目的 探讨替罗非班对氧糖剥夺(oxygen-glucose deprivation,OGD)心肌细胞的干预作用,并探索其相关机制.方法 H9C2细胞被分为正常组、模型组、替罗非班组、抑制剂组、替罗非班+抑制剂组.建立OGD细胞模型,以MTT曲线确定最终的实验浓度和作用时间为替罗非班100 nmol/L,作用时间为24 ...     

整合素连接激酶经磷脂酰肌醇3激酶/蛋白激酶B信号通路促进胃癌细胞增殖和迁移

目的探讨整合素连接激酶(ILK)对胃癌细胞增殖、侵袭的影响及相关机制。方法利用基因转染建立过表达ILK基因的HGC-27及KATOⅢ胃癌细胞株;细胞计数试剂(CCK)8和Transwell实验研究上调ILK基因表达对HGC-27及KATOⅢ细胞增殖和迁移能力的作用;Western印迹实验检测上调ILK基因表达是否可影响HGC-27及KATOⅢ细胞Akt及p-Akt蛋白的表达。结果上调ILK基因表达可显著促进HGC-27及KATOⅢ细胞的增殖和迁移能力;上调ILK基因表达可明显上调HGC-27及KATOⅢ细胞p-Akt蛋白水平。结论 ILK经磷脂酰肌醇3激酶/蛋白激酶B信号通路参与及影响胃癌细胞的致癌潜能。     

抑制PI3K/Akt/mTOR信号通路对兔原代巨噬细胞自体吞噬的影响及机制

目的 探讨抑制PI3K/Akt/mTOR信号通路对兔原代巨噬细胞自体吞噬中的影响。方法 分离培养纯种新西兰兔腹腔原代巨噬细胞并分为4组,加入磷脂酰肌醇3激酶（PI3K）抑制剂LY294002（10 μmol/L）组、哺乳动物雷帕霉素靶蛋白（mTOR）抑制剂雷帕霉素（10 nmol/L）组、蛋白激酶B（Akt）抑制剂曲西立滨组（20 μmol/L）以及空白对照组。共培养4 h、12 h后分别收集细胞,运用透射电镜观察巨噬细胞自噬体的变化,细胞免疫荧光法检测微管相关蛋白轻链3Ⅱ（LC3Ⅱ）分子的表达,Western blot检测 Akt、mTOR、磷酸化Akt（p-Akt）、磷酸化mTOR（p-mTOR）及自噬相关蛋白Beclin-1和自噬蛋白Atg5-Atg12连接体的表达,单丹酰尸胺（MDC）染色法观察自噬溶酶体的变化。结果 与空白对照组相比,透射电镜下LY294002组自噬体、自噬空泡、髓磷脂图像等自噬标记物明显减少,雷帕霉素组、曲西立滨组明显增多;激光共聚焦显微镜下LY294002组LC3Ⅱ表达显著减少,雷帕霉素组、曲西立滨组表达显著增多;Western blot结果显示LY294002组Beclin-1及Atg5-Atg12蛋白表达水平显著下降,p-mTOR、p-Akt蛋白表达显著减少;雷帕霉素组、曲西立滨组Beclin-1及Atg5-Atg12蛋白表达水平明显上调,共培养4 h后p-Akt表达增多,雷帕霉素组p-mTOR表达增多,曲西立滨组减少;共培养12 h后雷帕霉素组、曲西立滨组p-mTOR表达显著减少,雷帕霉素组p-Akt表达显著增多,曲西立滨组显著减少;MDC染色显示LY294002组自噬溶酶体明显减少,雷帕霉素组、曲西立滨组明显增多。结论 抑制PI3K/Akt/mTOR信号通路能促进兔原代巨噬细胞自体吞噬,抑制PI3K能减少兔原代巨噬细胞自体吞噬,可能是不同类型的PI3K分子通过其他通路起作用。     

连接肠道炎症和肿瘤的桥梁——磷脂酰肌醇-3激酶/AKT信号转导通路的作用

磷脂酰肌醇-3激酶（phosphatidylinositol 3-kinase，PI3K）家族是一类蛋白激酶，能特异性地催化磷脂酰肌醇（phosphatidylinositol，PI）环上的3位羟基磷酸化，生成相应的肌醇脂物质，后者是细胞内新型第二信使，与胞质内含血小板-白细胞激酶C底物同源（pleckstrin homology，PH）结构域的信号分子结合后对相应分子发挥活性调节作用。丝氨酸，苏氨酸蛋白激酶AKT是PI3K下游的直接靶蛋白，含有PH结构域，主要负责由PI3K始动的生物信息转导。AKT处于这一通路的中心环节，参与了细胞生长、增殖、生存、炎症、肿瘤的发生、发展等诸多重要生理病理过程。近年来，该通路在炎症和肿瘤中的作用日益受到重视，众多学者对其进行了大量研究。本文就这方面的情况作一综述。     

通过沉默GOLPH3抑制PI3K/AKT信号通路调节食管癌氧化应激与细胞凋亡的机制研究

目的 分析GOLPH3对食管癌氧化应激与细胞凋亡的影响及其作用机制。方法 利用qRT-PCR和Western blotting实验分析食管癌组织和癌旁组织中GOLPH3 mRNA和蛋白表达水平,培养EC9706细胞,将GOLPH3 siRNA与siRNA NC、pcDNA-GOLPH3与pcDNA-3.1(+)分别转染细胞,利用丙二醛试剂盒测定MDA含量,超氧化物歧化酶试剂盒测定SOD活力,谷胱甘肽过氧化物酶试剂盒测定GSH-Px活力,过氧化氢酶试剂盒测定CAT活力,Western blotting实验分析EC9706细胞内Bax、Bcl-2、PI3K、p-PI3K、AKT和p-AKT蛋白表达,流式细胞仪分析EC9706细胞凋亡率。PI3K/AKT信号通路抑制剂LY294002作用EC9706细胞后,分析细胞氧化应激、细胞活力与细胞凋亡率。结果 食管癌组织中GOLPH3基因与蛋白表达显著上调(P<0.01)。GOLPH3表达下调后,EC9706细胞活力、SOD、CAT、GSH-Px活力、p-PI3K/PI3K比值、p-AKT/AKT比值与Bcl-2表达显著下调,细胞凋亡率、MDA...     

杨梅素通过磷脂酰肌醇3-激酶/蛋白激酶B/哺乳动物雷帕霉素靶蛋白信号通路诱导MTB感染巨噬细胞发生自噬的研究

目的对杨梅素通过磷脂酰肌醇3-激酶/蛋白激酶B/哺乳动物雷帕霉素靶蛋白(PI3K/Akt/mTOR)信号通路诱导MTB感染的巨噬细胞发生自噬进行研究,从而探讨杨梅素抗结核作用的机理。方法用CCK8法检测杨梅素对细胞增殖的影响,确定安全的用药范围;以H37Ra菌株感染的小鼠巨噬细胞Raw 264.7为模型组,并设空白组和药物处理组。按感染复数(MOI,即细菌:细胞=10:1)加入模型组、药物处理组,共孵育4 h后,磷酸盐缓冲液(PBS)洗3次以弃掉未进入胞内的MTB。药物处理组分别用不同浓度(12.5、25、50、100μmol/L)的杨梅素作用24 h,Western blot法检测自噬相关蛋白即"微管相关蛋白1轻链3-Ⅱ(LC3-Ⅱ)和p62"表达水平的变化,并以此筛选出杨梅素促进自噬的最佳作用浓度;100μmol/L杨梅素作用于感染细胞72 h后,0.1%聚乙二醇辛基苯基醚(Triton X-100)冰上裂解细胞10 min,菌落形成单位(CFU)法检测巨噬细胞胞内荷菌量;杨梅素作用感染细胞不同时间(30、60、180 min)后Western blot测定PI3K/Akt/mTO...     

雌激素通过磷脂酰肌醇3-激酶/蛋白激酶B信号转导途径对钙诱导的大鼠血管平滑肌细胞钙化的影响

目的 研究雌激素对血管平滑肌细胞钙化的影响及机制.方法 体外培养大鼠胸大动脉平滑肌细胞(A7r5),按CaCl2溶液不同时间和浓度梯度进行钙化模型建立.细胞随机分为正常对照组、钙化组、雌激素干预(钙化+雌激素)组、雌激素溶剂对照(钙化+Eth)组、磷脂酰肌醇3-激酶(phosphatidylinositol3-kina...     

Induction of macrophage inflammatory cytokines by Ox-LDL is ABCA1 dependent

Objective The current study aimed to evaluate whether the induction of macrophage inflammatory cytokines by Ox-LDL is related to the expression of ABCA 1 pathway. Methods After THP 1/PMA macrophages were transfected with ABCA1 antisense oligonucleotides （100nmol/L） followed by treatment with Ox-LDL （30mg/L）, the expressions of ABCA1, ICAM-1 and MCP-1 mRNA and protein were determined by real-time fluorescent quantitative RT-PCR, Western blot or ELISA. Results Ox-LDL induced expressions of ABCA1, ICAM-1, and MCP-1 at both mRNA and protein levels from THPI/PMA macrophages. Transfection with ABCAI antisense oligonucleotides reduced ABCA1 mRNA levels after 3 and 6 hours and protein levels after 12 and 24 hours. The expression of ICAM-1 and MCP-1 induced by Ox-LDL was also decreased after inhibition of ABCA 1 protein expression by ABCA 1 antisense oligonucleotide decreased. Conclusion The induction of macrophage inflammatory cytokines by Ox-LDL is partially dependent on expression ofABCA1. Our studies disclose new functions of ABCA1 in macrophages Objective The current study aimed to evaluate whether the induction of macrophage inflammatory eytokines by Ox-LDL is related to the expression of ABCA 1 pathway. Methods After THP 1/PMA macrophages were transfected with ABCA1 antisense oligonucleotides （100nmol/L） followed by treatment with Ox-LDL （30mg/L）, the expressions of ABCA1, ICAM-1 and MCP-1 mRNA and protein were determined by real-time fluorescent quantitative RT-PCR, Western blot or ELISA. Results Ox-LDL induced expressions of ABCA1, ICAM-1, and MCP-1 at both mRNA and protein levels from THPI/PMA macrophages. Transfection with ABCAI antisense oligonucleotides reduced ABCA1 mRNA levels after 3 and 6 hours and protein levels after 12 and 24 hours. The expression of ICAM-1 and MCP-1 induced by Ox-LDL was also decreased after inhibition of ABCA 1 protein expression by ABCA 1 antisense oligonucleotide decreased. Conclusion The induction of macrophage inflammatory cytokines by Ox-LDL is partially dependent on expression ofABCA1. Our studies disclose new functions of ABCA1 in macrophages     

Induction of macrophage inflammatory cytokines by Ox-LDL is ABCA1 dependent

Objective The current study aimed to evaluate whether the induction of macrophage inflammatory cytokines by Ox-LDL is related to the expression of ABCA1 pathway. Methods After THP1/PMA macrophages were transfected with ABCA1 antisense oligonucleotides (100nmol/L) followed by treatment with Ox-LDL (30mg/L), the expressions of ABCA1, ICAM-1 and MCP-1 mRNA and protein were determined by real-time fluorescent quantitative RT-PCR, Western blot or ELISA. Results Ox-LDL induced expressions of ABCA1, ICAM-1, and MCP-1 at both mRNA and protein levels from THP1/PMA macrophages. Transfection with ABCA1 antisense oligonucleotides reduced ABCA1 mRNA levels after 3 and 6 hours and protein levels after 12 and 24 hours. The expression of ICAM-1 and MCP-1 induced by Ox-LDL was also decreased after inhibition of ABCA1 protein expression by ABCA1 antisense oligonucleotide decreased. Conclusion The induction of macrophage inflammatory cytokines by Ox-LDL is partially dependent on expression of ABCA1. Our studies disclose new functions of ABCA1 in macrophages (J Geriatr Cardiol 2010; 7:166-170).     

Amlodipine inhibits matrix metalloproteinases expression and secretion in mouse macrophage

Objective To investigate whether the calcium channel blocker amlodipine could inhibit macrophage matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9) expression and secretion. Methods Peritoneal macrophages were isolated from BALB/C mice and incubated with low (5μg/L ), middle (15μg/L) and high (305μg/L) concentrations of amlodipine, or in the medium alone (controls) for 24 hours, and the expression and secretion of MMP-2 and MM-9 of the cells were analyzed by RT-PCR and gelatin zymography. Results Compared with controls, amlodipine at low concentration had no significant effects on the expression and secretion of either MMP-2 and MMP-9 (P>0.05) at middle concentrationit it could inhibited MMP-2 and MMP-9 expressions completely and significantly reduced the secretion of MMP-9 (P<0.05); but it had no effect on the secretion of MMP-2. At high concentration it also inhibited MMP-2 and MMP-9 expression completely. Conclusion Amlodipine at 15 ig/L inhibited the expression of MMP-2 and MMP-9 and reduced the secretion of MMP-9, suggesting that amlodipine may stabilize atherosclerotic plaque.     

ox-LDL促进树突状细胞LOX-1表达及炎症因子分泌

目的应用小鼠高脂模型及树突状细胞株研究血凝素样氧化型低密度脂蛋白受体1(LOX-1)高表达树突状细胞(DC)在动脉粥样硬化中的作用。方法氧化型低密度脂蛋白(ox-LDL)诱导原代培养的C57小鼠骨髓来源的树突状细胞(BMDC),采用Western blot检测BMDC LOX-1蛋白水平;采用流式细胞术检测高表达LOX-1和低表达LOX-1细胞亚群的比例;MACS磁珠分选LOX-1表达水平不同的两种细胞亚群,观察ox-LDL对两种细胞亚群的影响及释放炎症因子的影响;采用C57小鼠高脂模型,在高脂喂养的不同时间(4周、6周、8周)检测小鼠总胆固醇水平和主动脉中DC数量;用不同浓度的Dil标记的ox-LDL(Dil-ox-LDL)刺激DC2.4细胞,荧光显微镜观察各组细胞吞噬作用的差异,Western blot检测LOX-1的表达。结果 DC可分为高表达和低表达LOX-1两种细胞亚群,且ox-LDL能增加LOX-1~(high)DC的比例;分选后的阳性细胞被ox-LDL刺激后,TNF-α和IL-1βmRNA的表达明显上调(P0.01),且阳性细胞的上升比例高于阴性细胞。在C57小鼠高脂模型中,高脂喂养组总胆固醇水平明显升高,且随着总胆固醇水平的升高,小鼠主动脉中DC增多,且LOX-1~(high)DC比例明显增加。用不同浓度的Dil-ox-LDL刺激DC2.4细胞,随着Dil-ox-LDL浓度的升高,DC2.4细胞对ox-LDL的吞噬也增加,并且ox-LDL可以诱导DC2.4细胞表面LOX-1表达,20 mg/L和40 mg/L的ox-LDL对LOX-1表达的诱导作用明显。结论 ox-LDL能够上调DC表面受体LOX-1,增加LOX-1~(high)DC比例,促进炎症因子表达。     

IL-37 inhibits the production of pro-inflammatory cytokines in MSU crystal-induced inflammatory response

Acute gouty arthritis (AGA) is an auto-inflammatory disease characterized by resolving spontaneously, which suggests that negative feedback loops control inflammatory and immunological responses to monosodium urate (MSU) crystals. By now, the molecular mechanism for spontaneous resolution of acute GA remains unclear; this study was undertaken to evaluate whether IL-37 is involved in spontaneous resolution of AGA. A total of 45 acute GA (AGA),29 non-acute GA (NAGA) male patients and 82 male health control (HC) were involved in this study, we measured IL-7 expression in the peripheral blood mononuclear cells (PBMCs), together with levels of IL-1β, IL-6, IL-10, TNF-α and TGF-β1 in the serum. Further, we either inhibited IL-37 expression in human PBMCs with siRNA or over-expressed the cytokine in human macrophages. Pro-inflammatory cytokine IL-1β, IL-6, and TNF-α expressions were significantly higher in the AGA group than in the NAGA or HC group (P?<?0.05, respectively). However, anti-inflammatory IL-37, TGF-β1, and IL-10 were greater in the NAGA group than in the AGA and HC groups (P?<?0.05, respectively). Expression of IL-37 in MSU crystal-treated macrophages inhibited the expression of pro-inflammatory cytokines, whereas the abundance of these cytokines increased with silencing of endogenous IL-37 in human blood cells. However, anti-inflammatory TGF-β1 and IL-10 expressions in these supernatants were unaffected by over-expression or knockdown of IL-37. Our study indicates that IL-37 is an important anti-inflammatory cytokine in AGA by suppressing the production of pro-inflammatory cytokines. Thus, IL-37 may provide a novel research target for the pathogenesis and therapy of GA.     

PPARα配体对泡沫细胞炎性介质分泌的影响

目的在证实泡沫细胞有过氧化物酶体增殖物激活受体α（PPARα）表达的基础上,探讨其配体对泡沫细胞炎性介质分泌的影响。方法体外诱导THP-1单核细胞分化为巨噬细胞,给予oxLDL进一步诱导其转变为泡沫细胞,应用RT-PCR检测PPARα基因表达;PPARα配体氯贝丁酯（50μmol/L）干预后用ELISA法测定泡沫细胞培养上清液中IL-6、TNF-α、MMP-2、MMP-9浓度,Gelatin Zymography测定MMPs活性。结果巨噬细胞转化为泡沫细胞后其PPARα基因表达无显著变化;氯贝丁酯可显著抑制泡沫细胞IL-6、TNF-α（P<0.05）、MMP-9（P<0.01）的分泌,抑制MMP-9的活性,对MMP-2的分泌和活性无影响。结论PPARα配体抑制致动脉粥样硬化炎性因子的分泌,减少基质金属蛋白酶的释放并抑制其活性,对防治粥样硬化有利。     

Old drug, new tricks: haloperidol inhibits secretion of proinflammatory cytokines

OBJECTIVES: It was noted that treatment of a patient with acute mania by haloperidol was associated with marked improvement in activity of rheumatoid arthritis. The objective of this study was to examine the effects of haloperidol on inflammatory cytokine release in vitro, as a potential mechanism to explain the in vivo anti-inflammatory effects of haloperidol. METHODS: The effect of haloperidol on the production of inflammatory cytokines interleukin 1beta (IL1beta) and tumour necrosis factor alpha (TNFalpha) was measured in bacterial lipopolysaccharide stimulated whole blood cultures and on the promonocyte cell line THP-1, using commercial and in house enzyme linked immunosorbent assays to measure cytokine concentrations. RESULTS: Haloperidol inhibited lipopolysaccharide stimulated production of both IL1beta and TNFalpha in vitro in a dose dependent manner and over a prolonged time period. Marked inhibition was seen over a range of concentrations of haloperidol from 0.5 microgram/ml to 50 microgram/ml, including those predicted to occur in the patient's blood. CONCLUSIONS: Haloperidol treatment seemed to alleviate inflammation in rheumatoid arthritis. In vitro experiments would suggest that the mechanism is by direct inhibition of proinflammatory cytokine release. This phenomenon requires further investigation and may potentially lead to the development of novel treatment.     

大鼠内毒素性急性肝损伤后肝细胞凋亡与炎性因子的表达

目的 探讨脂多糖诱导D-氨基半乳糖胺致敏大鼠急性肝损伤肝细胞凋亡、炎性因子表达情况及其发生机制.方法 56只大鼠分为0 h对照组与1、2、4、6、24和48 h脂多糖+D-氨基半乳糖胺处理组.在相应时间点处死大鼠后收集肝组织及血清,肝组织苏木精-伊红染色后光学显微镜下观察ELISA法检测血清细胞因子表达;反转录(RT)-PCR法检测TNF-α、IL-β、诱导型一氧化氮合酶(iNOS)和p53基因表达;收集24 h肝组织用底物显色法检测Caspase-3、8、9,12活性.组间比较用方差分析.结果 经药物处理后,肝组织出现碎片状坏死、大量炎性细胞浸润等表现,从6 h开始,24 h和48 h显著加重.血清TNF-α浓度在1 h处理组为(727.8±261.3)ng/L,显著高于对照组及其他处理组(F=49.82,P<0.01),2 h处理组为(156.4±52.2)ng/L,显著低于1 h组,但高于对照组(F:30.23,P<0.01);血清IL-β浓度逐渐上升,24 h处理组最高,为(360.5±121.6)ng/L(F=18.61,P<0.01).24 h处理组肝组织Caspase-3、8、9、12活性明显高于对照组(F=84.96,P<0.01).iNOS基因在对照组无表达,药物作用后6 h达最高,24 h和48 h则显著下降(F=34.07,P<0.01);p53基因在24 h和48 h处理组表达明显增高(F=37.43,P<0.01);TNF-α和IL-1β基因表达均较对照组升高(F=2.94,P<0.05),其峰值均出现在1 h处理组.结论 小剂量脂多糖可诱导D-氨基半乳糖胺致敏大鼠发生急性肝损伤;Caspase-3、8、9、12活性明显增强是其特征性改变之一;肝损伤的发生与TNF-α、iNOS和p53基因早期高水平表达有密切关系.     

Insulin inhibits apoptosis of macrophage cell line, THP-1 cells, via phosphatidylinositol-3-kinase-dependent pathway

Hyperinsulinemia has recently been reported as a risk factor for atherosclerotic diseases such as coronary heart disease; however, its precise mechanism is not well understood. To elucidate the role of insulin in the development of atherogenesis, we have investigated the effect of insulin on cell survival in macrophages, which are known to be important in the atherosclerotic process. Apoptosis was induced in macrophage cell lines derived from human monocytes or murine macrophages by serum starvation. Insulin administration retarded macrophage apoptosis by means of DNA laddering, dimethylthiazol diphenyltetrazolium bromide assay, and annexin V binding assay. Insulin also enhanced mRNA expression and protein production of the antiapoptotic Bcl-XL gene in a dose-dependent manner within the range of physiological concentrations. In the exploration of the signaling pathway involved in these antiapoptotic effects of insulin, pretreatment of cells with a specific inhibitor of phosphatidylinositol-3-kinase significantly suppressed insulin-mediated cell survival and insulin-induced Bcl-XL expression in macrophages. These data indicate that the survival effect of insulin on the apoptosis of macrophages is associated with the upregulation of Bcl-XL expression, and it may be mediated through the phosphatidylinositol-3-kinase signaling pathway. These mechanisms could be involved in the possible role of insulin in the development of atherosclerosis.