阿托伐他汀对高葡萄糖诱导的血管平滑肌细胞增殖的抑制作用

目的研究阿托伐他汀对醛糖还原酶(AR)和核因子NF-κB的表达及血管平滑肌细胞增殖的影响,探讨阿托伐他汀抗细胞增殖的可能机制.方法用高浓度葡萄糖诱导大鼠血管平滑肌细胞(VSMC)AR基因表达,然后加入不同浓度的阿托伐他汀,培养3 d后用台盼蓝染色行细胞计数.并采用RT-PCR、免疫组化、原位杂交等方法,观察阿托伐他汀对NF-κB和AR基因表达及VSMC增殖的影响.结果 1)随着阿托伐他汀浓度的提高,VSMC计数逐渐减少.2)正常浓度葡萄糖(5.6 mmol/L)时, NF-κB表达不明显,高浓度葡萄糖(22.5 mmol/L)时,NF-κB表达强阳性,阿托伐他汀则可明显抑制这一表达,0.1 μmol/L阿托伐他汀时, NF-κB表达即有降低,10 μmol/L阿托伐他汀几乎完全抑制NF-κB的表达.3)高浓度葡萄糖可明显诱导AR基因的表达,阿托伐他汀对其表达有抑制作用,且呈剂量依赖性.与高浓度葡萄糖组相比,阿托伐他汀0.1、1、10 μmol/L分别使VSMC的AR mRNA下调12%、45%和80%(P均＜0.05).结论 1)阿托伐他汀可抑制VSMC增殖.2)阿托伐他汀可抑制AR及NF-κB的表达.3)阿托伐他汀可能是通过抑制AR及NF-κB的表达继而抑制VSMC的增殖.     

阿托伐他汀对高葡萄糖诱导的血管平滑肌细胞增殖的抑制作用

目的研究阿托伐他汀对醛糖还原酶(AR)和核因子NF-κB的表达及血管平滑肌细胞增殖的影响,探讨阿托伐他汀抗细胞增殖的可能机制。方法用高浓度葡萄糖诱导大鼠血管平滑肌细胞(VSMC)AR基因表达,然后加入不同浓度的阿托伐他汀,培养3d后用台盼蓝染色行细胞计数。并采用RT-PCR、免疫组化、原位杂交等方法,观察阿托伐他汀对NF-κB和AR基因表达及VSMC增殖的影响。结果1)随着阿托伐他汀浓度的提高,VSMC计数逐渐减少。2)正常浓度葡萄糖(5·6mmol/L)时,NF-κB表达不明显,高浓度葡萄糖(22·5mmol/L)时,NF-κB表达强阳性,阿托伐他汀则可明显抑制这一表达,0·1μmol/L阿托伐他汀时,NF-κB表达即有降低,10μmol/L阿托伐他汀几乎完全抑制NF-κB的表达。3)高浓度葡萄糖可明显诱导AR基因的表达,阿托伐他汀对其表达有抑制作用,且呈剂量依赖性。与高浓度葡萄糖组相比,阿托伐他汀0·1、1、10μmol/L分别使VSMC的ARmRNA下调12%、45%和80%(P均<0·05)。结论1)阿托伐他汀可抑制VSMC增殖。2)阿托伐他汀可抑制AR及NF-κB的表达。3)阿托伐他汀可能是通过抑制AR及NF-κB的表达继而抑制VSMC的增殖。     

依折麦布和瑞舒伐他汀通过肝X受体α-三磷酸腺苷结合盒转运体A1对胆固醇的影响

目的研究瑞舒伐他汀、依折麦布及两者联合是否通过肝X受体α(LXRα)-三磷酸腺苷结合盒转运体A1(ABCA1)信号通路影响血管平滑肌细胞(VSMC)来源的泡沫细胞胆固醇水平。方法取大鼠胸主动脉3~5代VSMC进行实验,分为9组:对照组,不干预,仅单独培养VSMC 48h;模型组,用氧化型LDL(ox-LDL 50μg/ml)诱导VSMC,单独培养48h;依折麦布A组(3.0μmol/L)、依折麦布B组(10.0μmol/L)、依折麦布C组(30.0μmol/L)、瑞舒伐他汀A组(0.1μmol/L)、瑞舒伐他汀B组(1.0μmol/L)及瑞舒伐他汀C组(5.0μmol/L)在用ox-LDL 50μg/ml诱导VSMC基础上,分别加入不同剂量的依折麦布或瑞舒伐他汀培养24h;联合组,在用ox-LDL 50μg/ml诱导VSMC基础上,加入依折麦布30.0μmol/L和瑞舒伐他汀5.0μmol/L,培养24h。检测各组TC、游离胆固醇(FC)及胆固醇酯(CE)水平及各组细胞LXRα、ABCA1 mRNA及蛋白表达水平。结果与对照组比较,模型组TC、FC、CE水平显著升高,LXRα、ABCA1mRNA及蛋白表达明显降低(P0.05);与模型组比较,瑞舒伐他汀C组、依折麦布C组及联合组TC、FC、CE水平显著减少,LXRα、ABCA1mRNA及蛋白表达显著增加(P0.05);且联合组LXRα、ABCA1mRNA及蛋白表达水平明显高于依折麦布各组及瑞舒伐他汀各组(P0.05)。结论瑞舒伐他汀、依折麦布及2个最适浓度的联合均可以通过上调LXRα、ABCA1表达,促进泡沫细胞中ABCA1介导的胆固醇的逆转运,减少胆固醇在细胞中蓄积,且联合组较单药组效果更显著。     

瑞舒伐他汀对TPH-1巨噬细胞清道夫受体CD_（36）、SR-AⅠ表达的影响

目的观察瑞舒伐他汀对氧化型低密度脂蛋白（ox-LDL）诱导的TPH-1巨噬细胞清道夫受体CD36和SR-AⅠ表达的影响。方法体外培养TPH-1巨噬细胞,分三组置6孔培养板中。对照组为正常生长的TPH-1巨噬细胞;ox-LDL组加入100 mg/L的ox-LDL培养24 h,诱导巨噬细胞泡沫化;瑞舒伐他汀组加入100 mg/L的ox-LDL培养24 h,诱导巨噬细胞泡沫化,然后加入瑞舒伐他汀10μmol/L培养2 h。检测各组细胞内清道夫受体CD36、SR-AⅠ蛋白及其mRNA。结果与对照组相比,ox-LDL组CD36、SR-AⅠ蛋白及其mRNA表达增加（P均〈0.05）;与ox-LDL组相比,瑞舒伐他汀组CD36、SR-AⅠ蛋白及mRNA表达明显降低（P均〈0.05）。结论瑞舒伐他汀可降低ox-LDL诱导的TPH-1巨噬细胞中清道夫受体CD36、SR-AⅠ的表达。     

瑞舒伐他汀预处理对大鼠脑缺血再灌注损伤模型NF-κB表达的影响

目的探讨预防性应用瑞舒伐他汀对大鼠脑缺血再灌注损伤脑组织NF-κB表达的影响。方法 48只SD大鼠按随机化原理分成三组:假手术组、缺血再灌注组(简称对照组)、瑞舒伐他汀组。瑞舒伐他汀组大鼠术前10天予瑞舒伐他汀(5mg/kg)行灌胃治疗,每天一次;假手术组和对照组给予等容积0.9%氯化钠溶液,每天一次。采用改良的Longa氏法制备大鼠大脑中动脉缺血再灌注模型,缺血2h再灌注24h以后行神经功能缺损评分,HE染色法观察大鼠脑组织病理形态;免疫组织化学方法观察核转录因子NF-κB(nuclear factor-ΚB)的表达。结果瑞舒伐他汀组较对照组神经功能缺损评分降低(P0.05)。瑞舒伐他汀组与对照组比较,缺血区变性坏死的神经元数量,空泡化改变及组织间水肿均明显减轻。瑞舒伐他汀组较对照组NF-κB阳性表达细胞数明显减少(P0.01)。结论预防性应用瑞舒伐他汀对大鼠脑缺血再灌注损伤有神经保护作用,其机制可能与抑制炎症反应有关。     

阿托伐他汀通过抑制NF-κB对抗大鼠缺血再灌注心肌炎症反应和凋亡的机制

目的:观察阿托伐他汀预处理对大鼠心肌缺血再灌注损伤(MIRI)肿瘤坏死因子(TNF)和白细胞介素-l(IL-1)、心肌细胞内Caspases-3和NF-κB表达的影响,探讨阿托伐他汀的心肌保护作用机制。方法:将40只雄性SD大鼠随机等分为4组:假手术组(A组,0.9%氯化钠溶液5ml/d),缺血再灌注组(B组,0.9%氯化钠溶液5ml/d)、阿托伐他汀标准剂量预处理组(C组,阿托伐他汀20mg/d)和阿托伐他汀强化剂量预处理组(D组,阿托伐他汀40mg/d)。灌胃7d后,第8天制作大鼠在体心肌I/R模型,结扎左冠状动脉前降支30min,再灌注120min后,用ELISA法检测心肌中TNF-α和IL-1β水平,Western blot检测活化Caspase-3和NF-κB的表达。结果:与B组比较,C组TNF-α、IL-1β、Caspases-3、NF-κB水平均明显减少(均P0.05);与C组比较,D组TNF-α、IL-1β、Caspases-3及NF-κB表达减少更为明显(均P0.05)。结论:阿托伐他汀预处理明显抑制炎症反应,减少细胞凋亡,减轻MIRI损伤,呈现较强的心肌保护作用,其机制可能与抑制心肌NF-κB表达有关。     

同型半胱氨酸对巨噬细胞CD147表达的影响及瑞舒伐他汀的干预作用

目的探讨同型半胱氨酸对U-937巨噬细胞CD147表达的影响及瑞舒伐他汀的干预作用。方法在佛波酯诱导分化的人U-937巨噬细胞中加入0、50、100及500μmol/L的同型半胱氨酸孵育48 h;半定量RT-PCR检测CD147 mRNA的表达,Western blot检测巨噬细胞CD147蛋白的表达。不同浓度的瑞舒伐他汀和500μmol/L同型半胱氨酸共同干预U-937巨噬细胞,48 h后半定量RT-PCR及Western blot检测CD147受抑制情况,细胞免疫荧光试验检测CD147表达。结果半定量RT-PCR和Western blot结果显示,随同型半胱氨酸浓度增加,U-937巨噬细胞CD147 mRNA及蛋白的表达逐渐升高,具有剂量依赖性。不同浓度瑞舒伐他汀和500μmol/L同型半胱氨酸共同处理U-937巨噬细胞,随瑞舒伐他汀浓度增加,U-937巨噬细胞CD147 mRNA及蛋白的表达逐渐受抑制,呈一定剂量依赖性。细胞免疫荧光证实同型半胱氨酸促进CD147表达增加,而瑞舒伐他汀能抑制其表达。结论同型半胱氨酸能上调U-937巨噬细胞CD147的表达,瑞舒伐他汀呈浓度依赖性抑制同型半胱氨酸诱导CD147表达。     

肝孤核受体激动剂对间充质干细胞的抗缺氧复氧损伤作用及机制

目的探讨肝孤核受体(LXR)激动剂对脂肪间充质干细胞(AD-MSCs)缺氧损伤的保护作用及可能机制。方法酶消化法分离稳定表达萤火虫荧光素酶(Fluc)报告基因的小鼠AD-MSCs,流式细胞术检测CD90、CD44、CD34、CD45细胞表面标记物。3代AD-MSCs分为7组:对照组;缺氧6 h/复氧2 h组(缺氧复氧组);缺氧/复氧+DMSO组(DMSO组);缺氧复氧+不同浓度LXR激动剂T0901317干预组(1μmol/L组、5μmol/L组、10μmol/L组、15μmol/L组)。运用Fluc报告基因生物发光成像技术对AD-MSCs细胞增殖进行定量评价,免疫印迹法检测细胞NF-κB表达水平,ELISA法检测AD-MSCs的白细胞介素6(IL-6)、TNF-α分泌水平。结果流式细胞术结果显示.AD-MSCs呈CD44(+)、CD90(+)、CD34(-)、CD45(-)。光学成像结果显示,AD-MSCs细胞数与Fluc平均生物发光信号强度呈正相关(r~2=0.97)。与对照组比较,缺氧复氧组AD-MSCs分泌TNF-α、IL-6、NF-κB明显升高;与缺氧复氧组比较,10μmol/L组AD-MSCs分泌TNF-α、IL-6、NF-κB明显减少(P<0.05,P<0.01)。结论 LXR激动剂可以促进缺氧复氧损伤后AD-MSCs的存活,并能通过NF-κB信号途径抑制炎性因子IL-6、TNF-α的释放,可为干细胞移植治疗心肌缺血再灌注损伤提供新的细胞保护方法。     

阿托伐他汀对人CD4＋T淋巴细胞张力蛋白同源第10染色体丢失的磷酸酶基因表达的影响

目的 研究阿托伐他汀在体外对人CD4＋T淋巴细胞张力蛋白同源第10染色体丢失的磷酸酶基因（PTEN）表达的影响。方法 取25例健康志愿者的新鲜外周血,免疫磁珠分选出CD4＋T淋巴细胞,随机分为空白组、植物血凝素（PHA）刺激组、PHA＋1 μmol/L阿托伐他汀组、PHA＋5 μmol/L阿托伐他汀组,PHA＋10 μmol/L阿托伐他汀组,体外培养48 h后收集各组细胞及培养基上清液,荧光定量PCR检测PTEN mRNA表达水平,Western blot检测PTEN蛋白表达,ELISA检测培养基上清液肿瘤坏死因子α（TNF-α）、白细胞介素6（IL-6）及白细胞介素10（IL-10）浓度。结果 与空白组比较,PHA刺激后,CD4＋T淋巴细胞PTEN mRNA、蛋白的表达及上清液TNF-α、IL-6浓度均升高（P<0.05）,而IL-10浓度升高无统计学差异（P>0.05）。与PHA刺激组比较,PHA＋5 μmol/L阿托伐他汀组、PHA＋10 μmol/L阿托伐他汀组CD4＋T淋巴细胞PTEN mRNA、蛋白的表达和上清液IL-10浓度增加（P<0.05）,而PHA＋1 μmol/L阿托伐他汀组具有增高趋势（P>0.05）,并随着阿托伐他汀药物浓度的增加而增加;各组上清液 TNF-α、IL-6浓度降低,PHA＋5 μmol/L阿托伐他汀组、PHA＋10 μmol/L阿托伐他汀组具有统计学差异（P<0.05）。直线相关性分析显示,TNF-α、IL-6的分泌水平与PTEN的表达量呈明显的负相关关系（r＝－0.837和r＝－0.816,P<0.01）,IL-10的分泌水平与PTEN的表达量呈明显的正相关关系（r＝0.753,P<0.05）。结论 阿托伐他汀能够通过调控人CD4＋T淋巴细胞PTEN表达发挥抗炎作用。     

阿托发他汀对糖尿病大鼠外周血和肾组织核因子κB的影响

目的 探讨阿托伐他汀对链脲佐菌素诱导的糖尿病大鼠肾组织与外周血单个核细胞(PBMC)中核因子κB(NF-κB)活性的影响.方法 30只雄性SD大鼠分成对照组、糖尿病组和阿托伐他汀治疗组;酶联免疫吸附法(ELISA)测定各组大鼠PBMC中NF κB活性;免疫组化检测各组大鼠肾组织NF-κB、单核细胞趋化蛋白1(MCP-1).结果 糖尿病大鼠PBMC和肾小球中NF κB显著高于对照组(P＜0.01).与糖尿病大鼠相比,阿托伐他汀明显抑制NF-κB活化及MCP-1和纤黏连蛋白(FN)表达(P＜0.05),减少24 h尿蛋白排泄,改善肾功能及肾病理学损害.结论 NF-κB在糖尿病肾病发病中具有重要作用,抑制NF-κB的活化可能是他汀类药物发挥肾脏保护作用的机制之一.     

过氧化物酶体增殖物激活受体γ激动剂对大鼠胰腺腺泡细胞信号传导途径的影响

目的 了解经雨蛙肽处理的胰腺腺泡细胞AR42J中过氧化物酶体增殖因子活化受体γ(PPARγ)与核因子(NF)-κB活性间的关系.方法 将胰腺腺泡AR42J细胞分为对照组(常规培养)、吡咯列酮组(40 μmol/L吡咯列酮)、吡咯列酮+雨蛙肽组(40 μmol/L吡咯列酮+10~(-8) mol/L雨蛙肽)、雨蛙肽组(40 μmol/L二甲基哑砜+10~(-8)mol/L雨蛙肽)和吡咯列酮+GW9662+雨蛙肽组(40 μmol/L吡咯列酮+5 μmol/L GW9662+10~(-8)mol/L雨蛙肽),各组培养30 min后检测PPARγ和NF-κB活性.Western印迹法检测NF-κB、PPARγ蛋白和磷酸化NF-κB抑制物(IκB)α抗体、IκB激酶(IKK)β和IκBα的表达差异、IKKβ活性和IκBα磷酸化的变化.免疫荧光法和Western印迹法检测NF-κB(p65和p50)的核移位.免疫沉淀法检测IκBα与NF-κB的变化.结果 吡咯列酮不仅抑制IKKβ活性(吡咯列酮+雨蛙肽组:雨蛙肽组为1.6;3.7)及IκBα的磷酸化(吡咯列酮+雨蚌肽组:雨蛙肽组为0.9:1.5),还能加强IκBα与NF-κB的结合(吡咯列酮+雨蛙肽组:雨蛙肽组为0.8:0.3),抑制NF-κB的核移位及NF-κB的转录活性(P<0.01),而PPARγ拮抗剂GW9662则逆转了吡咯列酮对NF-κB活性的抑制作用(P<0.05).结论 在雨蛙肽处理的AR42J细胞中PPARγ通过干扰NF-κB的活化产生抗炎作用.     

Prolongation of liver allograft survival by dendritic cells modified with NF-κB decoy oligodeoxynucleotides

AIM: To induce the tolerance of rat liver allograft by dendritic cells (DCs) modified with NF-κB decoy oligodeoxynucleotides (ODNs).METHODS: Bone marrow (BM)-derived DCs from SD rats were propagated in the presence of GM-CSF or GM-CSF+IL-4to obtain immature DCs or mature DCs. GM-CSF+IL-4-propagated DCs were treated with double-strand NF-κB decoy ODNs containing two NF-κB binding sites or scrambled ODNs to ascertain whether NF-κB decoy ODNs might prevent DC maturation. GM-CSF-propagated DCs, GMCSF+NF-κB decoy ODNs or scrambled ODNs-propagated DCs were treated with LPS for 18 h to determine whether NF-κB decoy ODNs could prevent LPS-induced IL-12production in DCs. NF-κB binding activities, costimulatory molecule (CD40, CD80, CD86) surface expression, IL-12protein expression and allostimulatory capacity of DCs were measured with electrophoretic mobility shift assay (EMSA),flow cytometry, Western blotting, and mixed lymphocyte reaction (MLR), respectively. GM-CSF-propagated DCs, GMCSF+IL-4 -propagated DCs, and GM-CSF+NF-κB decoy ODNs or scrambled ODNs-propagated DCs were injected intravenously into recipient LEW rats 7 d prior to liver transplantation and immediately after liver transplantation.Histological grading of liver graft rejection was determined 7 d after liver transplantation. Expression of IL-2, IL-4 and IFN-γ mRNA in liver graft and in recipient spleen was analyzed by semiquantitative RT-PCR. Apoptosis of liver allograft-infiltrating cells was measured with TUNEL staining.RESULTS: GM-CSF-propagated DCs, GM-CSF+NF-κB decoy ODNs-propagated DCs and GM-CSF+ scrambled ODNspropagated DCs exhibited features of immature DCs, with similar low level of costimulatory molecule(CD40, CD80,CD86) surface expression, absence of NF-κB activation,and few allocostimulatory activities. GM-CSF+IL-4-propagated DCs displayed features of mature DCs, with high levels of costimulatory molecule (CD40, CD80, CD86) surface expression, marked NF-κB activation, and significant allocostimulatory activity. NF-κB decoy ODNs completely abrogated IL-4-induced DC maturation and allocostimulatory activity as well as LPS-induced NF-κB activation and IL-12protein expression in DCs. GM-CSF+NF-κB decoy ODNspropagated DCs promoted apoptosis of liver allograftinfiltrating cells within portal areas, and significantly decreased the expression of IL-2 and IFN-γ mRNA but markedly elevated IL-4 mRNA expression both in liver allograft and in recipient spleen, and consequently suppressed liver allograft rejection, and promoted liver allograft survival.CONCLUSION: NF-κB decoy ODNs-modified DCs can prolong liver allograft survival by promoting apoptosis of graft-infiltrating cells within portal areas as well as downregulating IL-2 and IFN-γ mRNA and up-regulating IL-4 mRNA expression both in liver graft and in recipient spleen.     

p38丝裂原活化蛋白激酶对HBZY-1系膜细胞株表达NF-κB和MCP-1的调控

目的 探讨p38丝裂原活化蛋白激酶(p38 mitogen-activated protein kinase, p38MAPK)与NF-κB、单核细胞趋化蛋白1(monocyte chemoattractant protein-1, MCP-1)之间的关系,从而研究p38MAPK和NF-κB、MCP-1在糖尿病肾病中的作用机制.方法 分别以高葡萄糖、高胰岛素、H2O2和糖基化终产物孵育大鼠肾小球系膜细胞株HBZY-1;先以p38MAPK特异抑制剂SB203580预处理细胞株HBZY-1,再给予上述4种因素孵育细胞株HBZY-1,观察其p38MAPK和NF-κB、MCP-1的表达.结果 高葡萄糖、高胰岛素、H2O2和糖基化终产物均可独立激活p38MAPK,使其磷酸化表达量增加,NF-κB、MCP-1表达也明显增加;SB203580预处理后,NF-κB、MCP-1表达被显著抑制.结论 p38MAPK可能通过激活NF-κB、MCP-1而诱导糖尿病时肾脏的损害,p38MAPK和NF-κB、MCP-1在糖尿病肾病的发生发展过程中可能起重要作用.     

阿托伐他汀对晚期糖基化终末产物诱导的人内皮细胞单核细胞趋化蛋白-1mRNA表达影响及其机制的实验研究

目的 观察阿托伐他汀对晚期糖基化终末产物(advanced glycation end products,AGE)诱导的人脐静脉内皮细胞表达单核细胞趋化蛋白-1(MCP-1)的影响,并探讨其作用机制.方法 实验分组:(1)空白对照组.(2)牛血清白蛋白(BSA)对照组.(3)AGE诱导组:不同作用浓度的AGE(10-4、10-3、10-2及10-1g/L)与细胞共同培养24 h.(4) AGE+阿托伐他汀组:用不同作用浓度的阿托伐他汀(0.1、1、10 μmol/L)分别与细胞培养1 h,而后加入10-1 g/L AGE[根据(3)实验结果选取最佳浓度]与细胞共孵育24 h.(5)PPAR-γ激动剂(15 d-PGJ2)组:15 d-PGJ2( 10 μmol/L)与细胞孵育1 h后加入10-1 g/L AGE再与细胞共孵育24 h.(6)PPAR-γ抑制剂(GW9662)组:GW9662(5000 g/L)与细胞孵育1 h后加入AGE(10-1 g/L)和阿托伐他汀(1 μmol/L)[根据(4)实验结果选取最佳浓度]再与细胞共孵育24 h.胶原酶消化法获取人脐静脉内皮细胞.逆转录聚合酶链反应法分析细胞MCP-1和过氧化物酶增殖物活化受体γ(PPAR-γ)基因的表达.蛋白免疫印迹法测定细胞核因子-κB(NF-κB )p65表达水平.结果 (1)AGE(10-4、10-3、10-2及10-1 g/L)呈浓度依赖性提高人脐静脉内皮细胞MCP-1 mRNA表达水平,分别是空白对照组的1.53倍、2.12倍、2.56倍及4.71倍;AGE浓度为10-4 g/L时,细胞MCP-1 mRNA表达明显高于空白对照组(0.26±0.02比0.17±0.04,P<0.01).(2)与AGE组比,阿托伐他汀(0.1、1、10 μmol/L)呈浓度依赖性抑制AGE诱导的人内皮细胞MCP-1 mRNA的表达;阿托伐他汀浓度为1 μmol/L时,细胞MCP-1 mRNA表达水平显著低于AGE(10-1g/L)组(0.63±0.11比1.03±0.07,P<0.01).(3)AGE(10-1 g/L)组人脐静脉内皮细胞PPAR-γ mRNA的表达水平显著低于空白对照组(0.22±0.08比0.69±0.09,P<0.01),磷酸化和非磷酸化NF-κB p65蛋白表达水平显著高于空白对照组(0.78±0.06比0.31±0.01和1.61±0.16 比0.59±0.14,P均<0.01).(4)阿托伐他汀(1、10 μmol/L)组人脐静脉内皮细胞PPAR-γ mRNA表达水平显著高于AGE(10-1 g/L)组(0.59±0.02和0.61±0.06比0.22±0.08,P均<0.01);阿托伐他汀(1 μmol/L)组磷酸化和非磷酸化NF-κB p65蛋白表达水平显著低于AGE(10-1g/L)组(0.40±0.03比0.78±0.06和0.65±0.12比1.61±0.16,P均<0.01).(5)PPAR-γ激动剂(15 d-PGJ2)组细胞磷酸化和非磷酸化NF-κB p65蛋白表达水平显著低于AGE(10-1g/L)组 (0.21±0.01比0.78±0.06和0.67±0.14比1.61±0.16,P均<0.01),MCP-1 mRNA表达水平显著低于AGE(10-1g/L)组(0.17±0.02比0.93±0.12,P<0.01).(6)PPAR-γ抑制剂(GW9662)组细胞磷酸化和非磷酸化NF-κB p65蛋白表达水平显著高于AGE(10-1g/L)+阿托伐他汀(1 μmol/L)组(0.53±0.02比0.40±0.03和1.38±0.18比0.65±0.12,P均<0.01),MCP-1 mRNA表达水平显著高于AGE(10-1g/L)+阿托伐他汀(1 μmol/L)组(0.62±0.05比0.30±0.07,P<0.01).结论 阿托伐他汀可通过提高AGE诱导的人脐静脉内皮细胞对PPAR-γ表达抑制细胞NF-κB信号途径,进而抑制AGE诱导的人脐静脉内皮细胞的炎性反应.

Abstract：

Objective To investigate the effects of atorvastatin on advanced glycation end products (AGE) induced monocyte chemoattractant protein-1(MCP-1) expression in human umbilical vein endothelial cells(HUVECs)and whether this effect could be linked to peroxisome proliferator-activated receptor-γ (PPAR-γ) and nuclear factor-κB(NF-κB).Methods Grouping: (1)Blank control group;(2)BSA group;(3)AGE group:cells were incubated with different concentrations of AGE(10-4,10-3, 10-2 and 10-1g/L)for 24 hours; (4)AGE+Atorvastatin group: cells were incubated with different concentrations of atorvastatin(0.1,1,10 μmol/L)for 1 hour,then incubated with AGE (10-1 g/L) for 24 hours; (5)PPAR-γ agonist(15 d-PGJ2)group: cells were incubated with 15 d-PGJ2(10 μmol/L)for 1 hour,then incubated with AGE (10-1g/L) for 24 hours;(6)PPAR-γ inhibitor(GW9662)group:cells were incubated with GW9662(5000 nmol/L)for 1 hour,then incubated with atorvastatin (1 μmol/L)and AGE (10-1g/L) for 24 hours. Collagenase was used to isolate the endothelial cell from human umbilical vein;RT-PCR was performed to examine the mRNA expression of MCP-1 and PPAR-γ;Western blot was performed to detect NF-κB p65 protein.Results (1) The expression of MCP-1 mRNA was increased in proportion with increasing concentrations of AGEs which could be blocked by atorvastatin in a dose-dependent manner. (2) AGE(10-1g/L)significantly downregulated the expression of PPAR-γ mRNA(0.22±0.08 vs. 0.69±0.09, P<0.01) while upregulated the expression of phospho-NF-κB p65 protein (0.78±0.06 vs. 0.31±0.01,P<0.01) and nonphospho-NF-κB p65 protein (1.61±0.16 vs. 0.59±0.14,P<0.01) comparaed with the control group which could be significantly attenuated by atorvastatin. (3) PPAR-γ agonist decreased the expression of phospho-NF-κB p65 protein (0.21±0.01 vs. 0.78±0.06, P<0.01),nonphospho-NF-κB p65 protein (0.67±0.14 vs. 1.61±0.16,P<0.01)and MCP-1 mRNA (0.17±0.02 vs. 0.93±0.12, P<0.01)compared with AGE(10-1g/L)group. (4) PPAR-γ inhibitor antagonized the effect of atorvastatin on the expression of phospho-NF-κB p65 protein, nonphospho-NF-κB p65 protein and MCP-1 mRNA stimulated by AGE in HUVECs(P<0.01).Conclusion The anti-inflammatory properties of atorvastatin in AGE stimulated HUVECs may partly be attributed to the effect on upregulation of PPAR-γ and downregulation of NF-κB signaling pathway.     

NF-κB activation and zinc finger protein A20 expression in mature dendritic cells derived from liver allografts undergoing acute rejection

AIM: To investigate the role of NF-κB activation and zinc finger protein A20 expression in the regulation of maturation of dendritic cells (DCs) derived from liver allografts undergoing acute rejection. METHODS: Sixty donor male SD rats and sixty recipient male LEW rats weighing 220-300 g were randomly divided into whole liver transplantation group and partial liver transplantation group. Allogeneic (SD rat to LEW rat) whole and 50 % partial liver transplantation were performed. DCs from liver grafts 0 hour and 4 days after transplantation were isolated and propagated in the presence of GM-CSF in vitro. Morphological characteristics and phenotypical features of DCs propagated for 10 days were analyzed by electron microscopy and flow cytometry, respectively. NF-κB binding activity, IL-12p70 protein and zinc finger protein A20expression in these DCs were measured by EMSA and Western blotting, respectively. Histological grading of rejection was determined. RESULTS: Allogeneic whole liver grafts showed no signs of rejection on day 4 after the transplantation. In contrast,allogeneic partial liver grafts demonstrated moderate to severe rejection on day 4 after the transplantation. After propagation for 10 days in the presence of GM-CSF in vitro,DCs from allogeneic whole liver grafts exhibited features of immature DC with absence of CD40 surface expression,these DCs were found to exhibit detectable but very low level of NF-κB activity, IL-12 p70 protein and zinc finger protein A20 expression. Whereas, DCs from allogeneic partial liver graft 4 days after transplantation displayed features of mature DC, with high level of CD40 surface expression, and as a consequence, higher expression of IL-12p70 protein, higher activities of NF-κB and higher expression of zinc finger protein A20 compared with those of DCs from whole liver grafts (P＜0.001). CONCLUSION: These results suggest that A20expression is up-regulated in response to NF-κB activation in mature DCs derived from allogeneic liver grafts undergoing acute rejection. Given the NF-κB inhibition function of this gene, it is suggested that their expression survives to limit NF-κB activation and maturation of DCs,and consequently inhibits the acute rejection and induces acceptance of liver graft.     

塞来昔布对人胶质瘤U251细胞增殖及NF-κB蛋白表达的影响

目的观察塞来昔布对人恶性胶质瘤U251细胞增殖的影响及分子机制。方法将对数生长期人胶质瘤U251细胞随机分为塞来昔布组、TNF-α组,两组均分别加入0、10、25、50、100μmol/L的塞来昔布,其中TNF-α组于1h后加50ng/mlTNF-α。其后采用甲基噻唑蓝（MTT）法检测两组U251细胞增殖水平及抑制率,采用免疫组化及Western blot检测NF-κB细胞内分布及核内表达情况。结果塞来昔布浓度为10～100μmol/L时两组细胞增殖抑制率均显著高于浓度为0者且呈浓度依赖性,增殖水平则相反,组间比较无显著差异;塞来昔布浓度为50、100μmol/L时NF-κB蛋白在细胞核内的表达显著低于浓度为0者,且呈浓度依赖性。结论塞来昔布可抑制人胶质瘤U251细胞增殖,可能机制为间接抑制NF-κB向核内移位。     

阿托伐他汀钙对扩张型心肌病患者促炎性细胞因子表达和NF-κB/p65活化的影响

目的探讨不同浓度的阿托伐他汀钙对脂多糖(LSP)诱导的扩张型心肌病(DCM)心力衰竭患者外周血单个核细胞(PBMCs)促炎性细胞因子表达以及核转录因子κB/p65(NF-κB/p65)活化的影响.方法选择心功能Ⅱ、Ⅲ、Ⅳ级的稳定期DCM患者25例,采清晨外周静脉血并分离出单个核细胞,用细胞因子刺激剂LPS刺激单个核细胞并分别加入终浓度为0、10-7、10-6、10-5mol/L的阿托伐他汀钙培养24 h,离心后提取上清液并用放射免疫法测白细胞介素-1β(IL-1β)、白细胞介素-6(IL-6)、肿瘤坏死因子-α(TNF-α)水平;细胞悬液用免疫组化染色检测NF-кB/p65,显微镜下计算其阳性细胞率,并分析二者的相关性.结果随着阿托伐他汀钙浓度的增加,IL-1β、IL-6、TNF-α和NF-κB/p65水平呈进行性下降(P<0.01);10-7、10-6、10-5mol/L阿托伐他汀钙组IL-1β、IL-6、TNF-α、NF-κB/p65水平均显著低于0 mol/L组(P<0.05或P<0.01);10-5mol/L阿托伐他汀钙组IL-1β、IL-6、TNF-α、NF-κB/p65水平均显著低于10-6、10-7mol/L组(P<0.05或P<0.01),而10-6mol/L组和10-7mol/L组IL-1β、IL-6、TNF-α、NF-κB/p65水平差异无统计学意义(P>0.05);且在不同浓度的阿托伐他汀钙组NF-κB/p65和IL-1β、IL-6、TNF-α的水平均有明显的正相关性(r值分别为0.647、0.527、0.459,P<0.001,P<0.05).结论阿托伐他汀钙呈剂量依赖性抑制LPS诱导的DCM患者PBMCs促炎性细胞因子表达增加,可能是通过下调NF-κB/p65的水平来实现的.这可能是他汀类药物对DCM有益的原因之一.     

高糖通过激活人脐静脉内皮细胞ⅠκB-α/NF-κB途径诱导MCP-1的表达

目的观察高糖诱导人脐静脉内皮细胞NF-κB的活性和单核细胞趋化蛋白1（MCP-1）的表达,探讨糖尿病并发动脉粥样硬化的发病机制。方法在培养的人脐静脉内皮细胞中加入不同浓度葡萄糖,检测内皮细胞中MCP-1mRNA、MCP-1蛋白的表达,NF-κB的活性及IκB-α的磷酸化水平。结果高糖明显地诱导了血管内皮细胞NF-κB的活性、MCP-1的表达及IκB-α的磷酸化;NF-κB活性抑制剂明显地降低了高糖所诱导的MCP-1的表达。结论高糖通过诱导血管内皮细胞IκB-α的磷酸化激活NF-κB,从而诱导了MCP-1的表达。提示在糖尿病并发动脉粥样硬化的发病过程中,高血糖通过激活血管内皮细胞IκB-α/NF-κB途径诱导MCP-1的表达,进而发挥了重要的作用。     

Effect of Tetrandrine on LPS-induced NF-κB activation in isolated pancreatic acinar cells of rat

AIM: To investigate the effect of Tetrandrine (Tet) on LPS-induced NF-κB activation and cell injury in pancreatic acinar cells and to explore the mechanism of Tetrandrine preventing LPS-induced acinar cell injury.METHODS: Male rat pancreatic acinar cells were isolated by collagenase digestion, then exposed to LPS (10mg/L), Tet (50 μmol/L, 100 μmol/L) or normal media. At different time point (30 min, 1 h, 4 h, 10 h) after treatment with the agents, cell viability was determined by MTT, the product and nuclear translocation of subunit p65 of NF-κB was visualized by immunofluorescence staining and nuclear protein was extracted to perform EMSA which was used to assay the NF-κB binding activity.RESULTS: LPS induced cell damage directly in a time dependent manner and Tet attenuated LPS-induced cell damage (50 μmol/L, P ＜ 0.05; 100 μmol/L, P ＜ 0.01).NF-κB p65 immunofluorescence staining in cytoplasm increased and began showing its nuclear translocation within 30 min and the peak was shown at 1 h of LPS 10 mg/L treatment. NF-κB DNA binding activity showed the same alteration pattern as p65 immunofluorescence staining. In Tet group, the immunofluorescence staining in cytoplasm and nuclear translocation of NF-κB were inhibited significantly.CONCLUSION: NF-κB activation is an important early event that may contribute to inflammatory responses and cell injury in pancreatic acinar cells. Tet possesses the protective effect on LPS-induced acinar cell injury by inhibiting NF-κB activation.     

CCDC80对巨噬细胞源性泡沫细胞炎症因子表达的影响及机制

目的 探讨卷曲螺旋结合域蛋白80(CCDC80)对THP-1 巨噬细胞源性泡沫细胞炎症因子表达的影响及相关分子机制.方法 体外培养的THP-1 细胞用佛波酯(160 nmol/L)处理,诱导分化为巨噬细胞,然后使用氧化型低密度脂蛋白(50 mg/L)处理使其荷脂形成泡沫细胞,并进行常规细胞体外培养.ELISA 检测细胞...