反义单核细胞趋化蛋白1对大鼠系膜细胞增生的影响

目的研究反义单核细胞趋化蛋白1（MCP-1）对系膜细胞MCP—1分泌及增生的影响。方法用逆转录病毒感染体外培养的系膜细胞，得到转染反义MCP—1的系膜细胞，经PCR及Southern印迹鉴定外源基因在系膜细胞基因组DNA上的整合。脂多糖（LPS）刺激反义MCP-1系膜细胞，以正常系膜细胞及转染空白载体的系膜细胞为对照，观察其增生情况。用RT-PCR法检测MCP-1、CC趋化因子受体2（CCR2）的mRNA表达。ELISA法检测细胞上清中MCP-1蛋白的分泌。结果经逆转录病毒转染得到的反义MCP-1的系膜细胞，其基因组DNA中有外源基因的整合。在正常的培养条件下，反义组与对照组系膜细胞的增生差异无统计学意义；MCP-1、CCR2的mRNA均有少量表达；MCP-1的蛋白也有微弱表达。在LPS的刺激下，反义组与对照组MCP-1的mRNA的表达均增高；MCP-1蛋白的分泌均增多。与对照组比较，反义组系膜细胞的增生受抑制[（16．83±1．16）×10^4／ml比（19．63±1．85）×10^4／ml]，MCP—1的mRNA的表达较少（0．424比0．866，P〈0．01），MCP—1蛋白的分泌减少。CCR2的mRNA表达与MCP-1的变化一致。结论（1）逆转录病毒载体pLXSN可有效地介导MCP-1 cDNA在系膜细胞的转染。（2）反义MCP-1可降低系膜细胞MCP—1的转录和翻译。（3）反义MCP—1的转染可降低系膜细胞在炎症状态时的增生。（4）大鼠系膜细胞具有CCR2的表达，在炎症状态时形成MCP-1、CCR2的自分泌环路。     

交感神经递质NE对内毒素诱导大鼠kupffer细胞TNF-α/IL-1β表达的影响

目的探讨交感神经递质去甲肾上腺素对体外培养大鼠kupffer细胞TNF-α、IL-1β炎症相关细胞因子表达的影响。方法分离大鼠kupffer细胞体外分三组培养,经体外培养48h后,给予外源性内毒素（LPS10μg／m1）刺激,同时给予不同浓度的去甲。肾上腺素（0．1μmol／L～10μmol／L）分别作用12h,运用定量逆转录多聚酶链反应检测各处理组TNF-α和IL-1β mRNA的表达,ELISA检测细胞培养上清TNF-α IL-1β蛋白的表达。结果同时给予LPS（10μg/ml）刺激和去甲肾上腺素（1μmol／L及10μmol／L）作用后,kupffer细胞培养上清TNF-α和IL-1β蛋白较LPS组显著增高（P〈0．05）;TNF-α mRNA表达分别较LPS组增加50．9％（P〈0．05）和59．1％（P〈0．05）;IL-1β mRNA较LPS组表达增加53．7％（P〈0．05）和57．8％（P〈0．05）。结论应激浓度的去甲肾上腺素能增加内毒素诱导大鼠kupffer细胞TNF-α、IL-1β表达,具有促炎效应。     

LDLr表达失衡在阿霉素肾病大鼠肾小球硬化中的作用

目的：观察阿霉素肾病大鼠肾小球硬化过程中低密度脂蛋白受体（ LDLr）的表达情况,及辛伐他汀对LDLr表达的影响,探讨LDLr在脂质导致肾病大鼠肾脏损害过程中的作用及他汀类药物肾脏保护的机制。方法：雄性SD大鼠随机分为正常对照组、阿霉素肾病组（模型组）和阿霉素肾病辛伐他汀治疗组（治疗组）,12周后收集标本,光镜观察肾小球硬化情况,酶比色法和油红染色法检测肾组织内胆固醇含量,RT-PCR和Western-blot技术分别检测肾组织LDLr、胆固醇调节元件结合蛋白-2（SREBP-2） mRNA和蛋白质表达。结果：模型组出现明显肾小球硬化（P〈0.01）,LDLr、SREBP-2 mRNA和蛋白表达明显增强（P均〈0.01）,肾组织内胆固醇含量明显升高（P〈0.01）；线性回归分析显示肾组织LDLr、SREBP-2蛋白表达上调与肾组织胆固醇酯（CE）含量呈显著正相关性（P均〈0.01）。治疗组肾小球硬化明显减轻（P〈0.01）,LDLr、SREBP-2 mRNA和蛋白表达、肾组织内胆固醇含量均较模型组低（分别P〈0.05或P〈0.01）。结论：肾病大鼠肾小球硬化过程中,肾组织内SREBP-2、LDLr表达明显上调,脂质可能通过过度表达的LDLr途径在肾组织内沉积,加重肾小球硬化；辛伐他汀可能通过下调LDLr途径,减少脂质在肾脏组织内沉积,发挥肾脏保护作用。     

地塞米松对脂多糖刺激THP-1释放炎性因子的影响及其机制

目的探讨地塞米松（Dexamethasone,DEX）对脂多糖（lipopolysaccharide,LPS）诱导的人外周血单核细胞（human peripheral mononuclear cell,THP-1）炎症因子释放的影响及其机制。方法体外培养人THP-1单核细胞,随机分为对照组（Control）、脂多糖组（LPS）和地塞米松组（DEX）。地塞米松组（1microg/ml）孵育地塞米松2 h后与脂多糖组（0.1μg/ml）均加入脂多糖刺激24 h。应用免疫蛋白印迹电泳法（Western blotting）检测各组细胞总蛋白、糖皮质激素受体（Glucocorticoid receptor ,GR）、磷酸化的糖皮质激素受体（p-GR）,IκB-α,磷酸化 IκB-α（p-IκB-α）,核因子κB （NF-κB）,磷酸化核因子κB （p-NF-ΚB）,巨噬细胞炎症趋化因子（MCP-1）的水平。用 ELISA 检测各组细胞培养上清中 MCP-1,肿瘤坏死因子-α（TNF-α）和白介素-6（IL-6）的表达水平,用实时定量 PCR（RT-PCR）检测各组细胞MCP-1,TNF-α和IL-6mRNA表达水平。结果 Westernblotting检测显示脂多糖组与对照组相比,脂多糖组 p-GR、p-NF-κB、p-IKB-α和 MCP-1蛋白表达水平明显升高（P〈0.05）,IκB-α表达明显下降（P〈0.05）,GR和 NF-KB表达水平无明显差异（P〉0.05）;RT-PCR 和 ELASA 检测显示MCP-1,TNF-α和IL-6分泌水平和mRNA 表达水平均明显增高（P〈0.05）。与脂多糖组相比,地塞米松组p-NF-κB,p-IKB-α和MCP-1蛋白水平表达明显下降,以及 MCP-1,TNF-α和 IL-6分泌和mRNA水平也均表达明显降低,但p-GR和IκB-α蛋白表达水平明显增加（P〈0.05）,但在 NF-κB和 GR表达水平依然无明显差异（P〉0.05）。结论地塞米松预处理可通过激活糖皮质激素受体而下调 NF-κB活化从而抑制脂多糖诱导单核细胞THP-1产生的炎症反应。     

尿足细胞及其相关分子在肾小球疾病中的表达

目的：探讨尿液检测局灶节段性肾小球硬化足细胞损伤与其他足细胞病之间的特点和差异。方法：入选原发性局灶节段性肾小球硬化（KSGS）患者54例,膜性肾病（MN）23例及微小病变（MCD）12例,正常对照20例。免疫荧光法计数尿足细胞,荧光实时定量PCR法定量尿沉渣足细胞相关分子nephrin、podocin、synaptopodin mRNA的表达水平,Western印迹法检测尿液WilmsTumor1（WT1）蛋白水平,免疫荧光法检测肾脏组织podocalyxin的表达及分布。结果：（1）FSGS组、MN组、MCD组和对照组尿足细胞阳性率分别是63％、34．8％、33．3％和0,FSGS组与其余各组相比差异均有统计学意义（P〈0．05）。FSGS组足细胞脱落数目显著高于MCD组、MN组和对照组（P〈0．05）,伴足细胞尿FSGS患者与不伴足细胞尿FS—GS患者相比,24h尿蛋白和血清白蛋白（Alb）差异均有统计学意义（P〈0．05）。（2）FSGS组尿沉渣足细胞nephrin mRNA表达水平显著高于MCD和MN组（P〈0．05）;FSGS组尿沉渣足细胞podocinmRNA表达显著高于MCD组（P〈0．05）,与MN组相比有升高趋势但差异无统计学意义;尿沉渣足细胞synaptopodin mRNA表达各组间差异无统计学意义。尿沉渣足细胞nephrin、podocin．synaptopodin mRNA的表达与24h蛋白尿无相关性。（3）FSGS组尿WT1蛋白量显著高于MCD和MN组。部分足细胞阴性患者尿液检测到WT1分子。（4）FSC-S患者肾组织podocalyxin较对照组、MCD和MN有明显的节段缺失。结论：局灶节段性肾小球硬化病患者足细胞损伤严重,尿足细胞与FSGS疾病活动相关。尿沉渣足细胞nephrin mRNA表达可以把FSGS与MCD和MN区分开来,尿WT1蛋白可能是足细胞早期损伤指标。     

高糖通过蛋白激酶C改变肾小球系膜细胞的间隙连接与细胞表型

目的观察高糖环境下，蛋白激酶C（PKC）活性的变化对肾小球系膜细胞间隙连接与细胞表型的影响。方法将体外培养的大鼠肾小球系膜细胞分为低糖组、高糖组、高糖＋PKC抑制剂十字孢碱（SP）组，测定细胞间隙连接蛋白-43（connexin 43）、α-平滑肌肌动蛋白（α—SMA）的表达。结果①与低糖组相比，高糖组细胞PKC活性、mSMA mRNA表达增高，connexin 43 mRNA表达下降，差异有统计学意义（P〈0．05）；②与高糖组相比，高糖＋SP组细胞PKC活性、α—SMA mRNA表达下降，connexin 43 mRNA表达增高，差异有统计学意义（P〈0．05）。结论高糖通过PKC改变肾小球系膜细胞的间隙连接与细胞表型。     

脂多糖对人腹膜间皮细胞葡萄糖转运蛋白-1表达的影响

目的：研究脂多糖（lipopolysaccbaride，LPS）对人腹膜间皮细胞（HPMC）葡萄糖转运蛋白1（GLUT-1）表达的影响。方法：胰蛋白酶消化法进行FIPMC的原代培养及传代；免疫组化法（ABC）用于GLUT-1的鉴定和半定量分析：采用逆转录多聚酶链反应（RT—PCR）检测GLUT-1的mRNA表达；己糖激酶法用于葡萄糖浓度的测定，计算出培养液内葡萄糖的减少量作为细胞对糖的净利用。结果：（1）体外培养的HPMC细胞角蛋白用间接免疫荧光染色为绿色荧光．此为HPMC的特征；（2）LPS可以使正常糖浓度下（0、1％）FIPMC GLUT-1的蛋白和mRNA的表达增加（P〈0．05），呈时间和浓度依赖，并伴有细胞对葡萄糖净利用增加，其中作用24h后，浓度为100μg／ml的LPS使GLUT-1的蛋白和mRNA表达最大（P〈0．01），而100μg／ml组LPS作用24h后，GLUT-1的蛋白和mRNA的表达最大（P〈0．01），作用36h有所下降；（3）高糖条件下（4．25％），LPS可以使GLUT-1的蛋白和mRNA表达增加，与高糖组和正常对照相比有统计学差异（P〈0.05）。结论：LPS可以使HPMC GLUT-1表达和葡萄糖摄取增加，这种作用在高糖条件下更为显著。这为研究和防治腹膜炎时腹透失超滤提供了线索。     

脂多糖对离体大鼠胸主动脉和肺组织HO-1mRNA及蛋白表达的影响

目的 观察离体大鼠胸主动脉和肺组织脂多糖（Lipopolysaccharide,LPS)孵育后血红素氧合酶－1（HO－1）mRNA及蛋白表达时间依从性的变化。方法 24只Wistar大鼠颈椎脱臼处死，取其胸主动脉和肺组织，随机分成四组：对照组（n=6)，实验组包括LPS3、8、24组（均为n=6)，分别与LPS（1μg/ml）孵育3、8、24h。采用蛋白免疫杂交（Western blot）和半定量聚合酶链反应（RT－PCR）分别测定HO－1蛋白和mRNA表达，结果 与对照组相比，胸主动脉HO－1蛋白表达LPS3组即达最高值（P＜0．05），LPS8和LPS24组仍处于较高水平（P＜0．05）；肺组织HO－1蛋白表达LPS3组已开始升高（P＜0．05）,LPS8力LPS24组仍处于较高水平（P＜0．05）；肺组织HO－1蛋白表达LPS3组已开始升高（P＜0．05），LPS8组达最高水平（P＜0．01），LPS24组有下降趋势，但仍与对照组有差异（P＜0．05）。与对照组相比，胸主动脉HO－1mRNA表达LPS3组即达最高值（P＜0．05），LPS8组较LPS3组无明显变化（P＞0．05），而LPS24且则恢复至始水平；肺组织HO－1mRNA表达LPS3组开始升高（P＜0．05），LPS8组达到最大值（P＜0．01），LPS24组回到基础水平（P＞0．05）。结论 脂多糖可以明显促进大鼠胸主动脉和肺组织HO－1mRNA及蛋白表达，且两种组织表现出不同的时间依从性变化，可能与感染性休克体肺循环不同变化的病生理机制有关。     

氯化镧对内毒素/脂多糖诱导巨噬细胞株一氧化氮合酶表达的抑制作用

目的了解氯化镧（LaCl3）对内毒素／脂多糖（LPS）刺激的巨噬细胞诱导型一氧化氮合酶（iNOS）表达的影响，并探讨其机制。方法将小鼠巨噬细胞株RAW264．7分为空白对照组、LaCl、组、LPS组和LaCl3＋LPS组。前3组细胞分别用常规培养液、含2．50μmol／L LaCl3的培养液、含1mg／L LPS的培养液培养24h，LaCl3＋LPS组用含2．5μmol／LLaCl，的培养液培养24h后，换为含1mg／L LPS的培养液培养24h。采用免疫细胞化学染色法检测iNOS在各组细胞中的表达强度；蛋白质印迹法检测iNOS的蛋白表达水平；反转录一PCR测定iNOS的mRNA表达水平；硝酸还原酶法测定各组细胞培养上清液中一氧化氮（NO）含量。结果免疫细胞化学染色结果显示，iNOS主要分布于各组细胞的胞质中，空白对照组和LaCl3组荧光强度极弱；LPS组荧光强度最强，阳性细胞百分率为44．4％，明显高于LaCl3＋LPS组（11．8％，P〈0．05）。LPS组iNOS蛋白及其mRNA表达量和细胞培养上清液中NO含量均高于其余各组（P〈0．05）。结论LaCl3可在mRNA水平和蛋白水平抑制LPS诱导的iNOS过度表达，减少NO生成，提示LaCl3能拮抗LPS诱导的iNOS过度活化。     

异丙酚或氯胺酮对内毒素性急性肺损伤大鼠肺小动脉BMP-2表达的影响

目的评价异丙酚或氯胺酮对内毒素（LPS）性急性肺损伤大鼠肺小动脉骨形态构建蛋白-2（BMP-2）表达的影响。方法雌性Wistar大鼠60只，体重220—260g，6—7周龄。随机分为6组，每组10只。对照组（C组）：1．5h内经股静脉输注生理盐水5ml；LPS组（L组）：1h内输注生理盐水3ml，然后30min内输注内毒素1mg／kg（溶于2ml生理盐水中）；异丙酚20mg／kg＋LPS组（P1组）、异丙酚50mg／kg＋LPS组（P2组）、氯胺酮20mg／kg＋LPS组（K1组）、氯胺酮50mg／kg＋LPS组（K组）1h内经股静脉分别输注不同剂量的药物，然后30min内输注LPS1mg／kg（溶于2ml生理盐水中）。输注完毕后72b断头处死大鼠。RT-PCR法测定肺小动脉BMP-2mRNA、BaxmRNA表达。免疫组织化学法、Western blot测定肺小动脉BMP-2、Bax、Bcl-2表达。显微成像分析系统测量肺小动脉中膜厚度。结果各组肺小动脉BMP-2、Bax、Bcl-2均有表达。与C组比较，其余组BMP-2、Bax蛋白水平和mRNA表达降低，Bcl-2水平升高（P〈0．05或0．01）。与L组比较，异丙酚或氯胺酮使BMP-2、Bax蛋白水平及mRNA表达升高，Bcl-2水平降低（P〈0．05）。各组肺小动脉中膜厚度较C组增加（P〈0．05）；异丙酚或氯胺酮减轻了肺小动脉中膜的增厚（P〈0．05）。结论异丙酚或氯胺酮可以抑制大鼠LPS致急性肺损伤肺血管的重建，其机制可能与BMP-2、Bax基因表达上调，Bcl-2基因表达下调有关。     

Expression of the chemokines MCP-1/CCL2 and RANTES/CCL5 is differentially regulated by infiltrating inflammatory cells

BACKGROUND: Chemokines are involved in the regulation of the cellular renal infiltrate in glomerulonephritis; however, it is unclear to which degree resident glomerular cells or infiltrating leukocytes contribute to the formation of chemokines in glomerular inflammatory lesions. We therefore examined whether monocytes/macrophages play a role in the expression of the C-C chemokines MCP-1/CCL2 and RANTES/CCL5 in renal tissue in a lipopolysaccharide (LPS)-induced model of inflammation, where previously we have shown increased glomerular RANTES expression and glomerular infiltration of ED-1-positive cells. METHODS: Inflammatory lesions were induced by an intraperitoneal injection of LPS. The infiltration of monocytes into the glomerulus was reduced by two experimental approaches. First, rats were depleted of monocytes by the use of specific monocyte-antisera or by cytotoxic drugs. Second, the infiltration of monocytes into the kidney was reduced by using intercellular adhesion molecule-1 (ICAM-1) knockout mice. RESULTS: Both experimental approaches demonstrated a significant reduction in the number of infiltrating monocytes/macrophages after lipopolysaccharide injection. This reduction in the infiltration of inflammatory cells was associated with significantly reduced RANTES/CCL5 mRNA expression. However, MCP-1/CCL2 mRNA expression was not inhibited after the LPS injection by monocyte/macrophage depletion. Also, the increase in nuclear factor-kappaB (NF-kappaB) binding activity after the LPS injection was not reduced in pretreated animals. The experiments therefore demonstrate that infiltrating monocytes/macrophages contribute to increased RANTES/CCL5 mRNA expression in inflammatory renal lesions, whereas MCP-1/CCL2 mRNA expression and NF-kappaB activation were not reduced by monocyte/macrophage depletion. CONCLUSION: MCP-1/CCL2 released from renal tissue upon stimulation plays a major role in the regulation of monocyte/macrophage infiltration, which contributes significantly to increased renal RANTES/CCL5 expression. This cross-talk between resident renal cells and monocytes/macrophages is therefore likely to boost the number of infiltrating inflammatory cells.     

Effect of unilateral ureteral occlusion on fibrin deposition in the kidney and renal blood flow during intravascular coagulation in rat

The effect of unilateral ureteral occlusion on fibrin deposition in the kidney and the interrelation of the fibrin deposition and the renal blood flow was studied in rat. Intravascular coagulation in the kidney was induced by infusion of thrombin and inhibition of fibrinolysis with tranexamic acid. The effects unilateral occlusion of the ureter for 1 and 24 h on fibrin deposition and renal blood flow were studied. Fibrin in the kidneys was quantitated by intravenous injection of 125I-labelled fibrinogen 24 h before the experiment. The renal blood flow was measured before and after infusion of thrombin by injection of 85Sr- and 141Ce-labelled microspheres into the left ventricle. After ureteral occlusion for 1 h the deposition of fibrin in the kidneys was unaffected. After 24 h substantially less fibrin deposition was found in the occluded than in the unoccluded kidney (0.3 +/- 0.2 and 5.7 +/- 1.6 mg, respectively; p less than 0.05). Before thrombin infusion the blood flow to the occluded kidney was less than that in the unoccluded kidney (2.1 +/- 0.8 and 3.7 +/- 1.2 ml/min, 100 g body weight, respectively; p less than 0.05). The blood flow after infusion of thrombin was equally reduced in both kidneys. The results contradict the hypothesis that vasoconstriction increases the amount of fibrin in the kidneys in thrombin-induced intravascular coagulation.     

纤维蛋白对肾小球内皮细胞表达纤溶酶原激活物及纤溶酶原激活物抑制物的作用

目的：观察体外培养人肾小球内皮细胞（GEC）表面原位形成的纤维蛋白对GEC表达纤溶酶原激活物及纤溶酶原激活物抑制物（PA／PAI）的影响。方法：应用逆转录聚合酶链反应（RT－PCR）,酶谱分析法与反向酶谱法分别在基因转录水平与蛋白质活性水平上检测纤维蛋白对GEC表达tPA,uPA gn PAI-1r 作用,纤维蛋白平板法检测纤维蛋白对GEC PA／PAI系统的综合效应,结果：纤维蛋白能够明显促进tPA,uPA与PAI－1的mRNA表达上调,无血清RPMI 1640培养下的GEC几乎检测不到PAI知性,但可检测到PAI－1的活性。纤维蛋白能够浓度依赖性刺激GEC tPA与uPA活性增加以及PAI01的活性增加,呈浓度依赖性与时间依赖性,相同剂量的纤维蛋白原与纤维蛋白的作用相似,放线菌酮与放线菌素D均可抑制纤维蛋白上调GEC表达tPA,uPA与PAI的作用,纤维蛋白平板法显示,纤维蛋白对GEC PA／PAI系统的综合效应是以升高PA活性为主,其活性能够被抑肽酶完全阻断。结论：肾脏局部毛细血[管内沉积的纤维蛋白可能通过对GEC PA／PAI系统的调节发挥其病理作用。     

MONOCYTE CHEMOATTRACTANT PROTEIN-1 EXPRESSION CORRELATES WITH MONOCYTE INFILTRATION IN THE POST-ISCHEMIC KIDNEY

Chemokines play a prominent role in the acute inflammatory response in several models of kidney disease. We reported that monocyte chemotactic peptide-1 (MCP-1) mRNA is increased by ischemia-reperfusion injury. In this report, we examined the effects of ischemia-reperfusion injury on the kinetics and location of MCP-1 protein expression, the excretion of MCP-1 protein in the urine and on the infiltration of mononuclear cells in the kidney. Pair-fed Sprague-Dawley rats underwent bilateral renal ischemia (50 min) or sham ischemia and placed in metabolic cages for daily urine collections. Kidneys were harvested at d. 1, 3, 7, and 10 after ischemia-reperfusion (I-R) or sham-ischemia (S-I). Kidney MCP-1 mRNA levels were increased on d. 1 and 3 post-ischemia. Kidney MCP-1 protein levels were increased in the I-R group on d. 1 and 3. MCP-1 expression occurred predominantly in the distal tubule segments by immunohistology. There was an increase in monocytes/macrophages infiltration in the I-R group, compared to the S-I or controls by d. 1. Urinary MCP-1 excretion increased 3-fold in the I-R group, and remained elevated above the S-I group and baseline levels, on d. 3 through d. 8. Kidney MCP-1 mRNA levels, protein levels and urinary MCP-1 excretion rates are increased by ischemia-reperfusion injury. The areas of increase in MCP-1 chemoattractant expression correlates with an increase in monocyte infiltration in the kidney. Although its pathophysiologic role remains to be determined, MCP-1 may participate in, and be a biomarker for, the mononuclear inflammatory processes that occur after ischemia-induced acute renal failure.     

Monocyte chemoattractant protein-1 expression correlates with monocyte infiltration in the post-ischemic kidney

Chemokines play a prominent role in the acute inflammatory response in several models of kidney disease. We reported that monocyte chemotactic peptide-1 (MCP-1) mRNA is increased by ischemia-reperfusion injury. In this report, we examined the effects of ischemia-reperfusion injury on the kinetics and location of MCP-1 protein expression, the excretion of MCP- 1 protein in the urine and on the infiltration of mononuclear cells in the kidney. Pair-fed Sprague-Dawley rats underwent bilateral renal ischemia (50 min) or sham ischemia and placed in metabolic cages for daily urine collections. Kidneys were harvested at d. 1, 3, 7, and 10 after ischemia-reperfusion (I-R) or sham-ischemia (S-I). Kidney MCP-1 mRNA levels were increased on d. I and 3 post-ischemia. Kidney MCP-1 protein levels were increased in the I-R group on d. 1 and 3. MCP-1 expression occurred predominantly in the distal tubule segments by immunohistology. There was an increase in monocytes/macrophages infiltration in the I-R group, compared to the S-I or controls by d. 1. Urinary MCP-1 excretion increased 3-fold in the I-R group, and remained elevated above the S-I group and baseline levels, on d. 3 through d. 8. Kidney MCP-1 mRNA levels, protein levels and urinary MCP-1 excretion rates are increased by ischemia-reperfusion injury. The areas of increase in MCP-1 chemoattractant expression correlates with an increase in monocyte infiltration in the kidney. Although its pathophysiologic role remains to be determined, MCP-1 may participate in, and be a biomarker for, the mononuclear inflammatory processes that occur after ischemia-induced acute renal failure.     

姜黄素对内毒素所致肾脏炎症的抑制作用

目的：肾脏的急慢性炎症与肾脏疾病的进展密切相关,本研究拟探讨姜黄素对内毒素（LPS）所致肾脏炎症的抑制作用。方法：SPF级昆明种小鼠40只,鼠龄6～8周,体重20～25g。小鼠分为3组：（1）正常对照组：腹腔注射生理盐水;（2）模型组（LPS组）：腹腔注射LPS（1mg/kg和5mg/kg）;（3）治疗组（LPS＋姜黄素）：动物先腹腔注射姜黄素（1mg/kg或5mg/kg）3d,然后腹腔注射LPS1mg/kg或5mg/kg。于处理后6h取材,切取部分肾组织用10%甲醛固定,HE染色,进行病理学观察。部分肾组织用于MCP-1mRNA检测。将肾小管上皮细胞（HK-2细胞）培养于KSF培养液中,设正常对照组给予正常培养液,LPS刺激组（1ng/ml,100ng/ml和10μg/ml）,LPS刺激＋姜黄素（5μmol/L和50μmol/L）处理组,分别培养4h和24h后收集细胞,应用Real-timePCR检测各组细胞MCP-1的表达。ELISA检测细胞培养上清液中MCP-1的表达。EMSA检测肾小管上皮细胞NF-κB的活性。结果：腹腔注射LPS（1mg/kg和5mg/kg）虽未引起光镜下肾脏组织的病理改变,但可显著增加小鼠肾脏MCP-1 mRNA的表达,分别增加20倍和26倍,而姜黄素处理可降低LPS所诱导的肾脏MCP-1 mRNA的表达,尤以LPS（1mg/kg）＋姜黄素（1mg/kg）为明显（下降至基础值的2.5倍）。应用不同浓度的LPS刺激HK-2细胞4h,HK-2细胞MCP-1 mRNA表达量显著增加,且呈剂量依赖的效应（分别增加1.60、2.15和14.7倍）。而以100ng/ml的LPS刺激HK-2细胞4h,观察姜黄素的干预作用,可见不同浓度的姜黄素（5μmol/L和50μmol/L）可显著抑制LPS所诱导的HK-2细胞MCP-1 mRNA的表达,且以50μmol/L姜黄素干预组为明显。同时ELSA检测细胞上清液中MCP-1的表达结果相似。EMSA结果显示NF-κB的DNA结合活性随LPS的浓度增加而增加,而应用姜黄素预处理后,由LPS所诱导的增高的NF-κB的DNA结合活性随之下降。结论：早期给予姜黄素治疗,能明显改善LPS诱导的小鼠肾脏趋化因子MCP-1的表达,从而发挥其肾脏损伤的保护功能;而体外研究表明姜黄素可抑制LPS所诱导的肾小管上皮细胞MCP-1表达,其作用可能与抑制细胞NF-κB的活性有关。     

Effect of Glucocorticoid Pretreatment on Oxidative Liver Injury and Survival in Jaundiced Rats with Endotoxin Cholangitis

Introduction Biliary tract infection is associated with high mortality. This study investigated the effect of glucocorticoid pretreatment on lipopolysaccharide (LPS)-induced cholangitis. Methods Rats undergoing either sham operation or ligation of the extrahepatic bile duct (BDL) for 2 weeks were randomly assigned to receive intravenous injections of dexamethasone (DX) or normal saline (NS) prior to infusing LPS into the biliary tract. The plasma levels of tumor necrosis factor-α (TNFα), chemokines monocyte chemoattractant protein-1 (MCP-1) and macrophage inflammatory protein-2 (MIP-2) as well as liver mRNA expression of MCP-1 and MIP-2 were determined. Infiltration of monocytes, Kupffer cells, and neutrophils in rat liver were studied with immunohistochemistry. Oxidative liver injury was measured by the malondialdehyde (MDA) content. Results Dexamethasone pretreatment resulted in significantly decreased plasma levels of TNFα at 1 hour, MCP-1 and MIP-2 at 2 and 3 hours, and decreased liver MCP-1 mRNA expression at 3 hours following LPS infusion in BDL-DX rats than in BDL-NS rats. The number of inflammatory cells in the liver was significantly different between sham- and BDL-treated rats but was not affected by DX pretreatment. Pretreatment with DX resulted in significantly decreased liver MDA contents in the BDL-DX group than that in the BDL-NS group. Jaundiced rats pretreated with 5 mg DX prior to infusion of 1 g of LPS were 6.8 times more likely to survive than those that were not pretreated. Conclusions Pretreatment of jaundiced, LPS-treated rats with a supraphysiological dose of dexamethasone may rescue their lives by suppression of chemokine expression and alleviation of oxidative liver injury.     

表面活性蛋白D高表达对脂多糖诱导人肾小管上皮细胞单核细胞趋化蛋白1表达的影响

目的 研究表面活性蛋白D(SP-D)高表达对脂多糖诱导人肾小管上皮细胞（HK-2）单核细胞趋化蛋白1（MCP-1）表达的影响及其机制。 方法 间接免疫荧光、Western印迹及RT-PCR检测SP-D蛋白及mRNA在HK-2细胞内的表达。脂多糖在不同浓度（0、0.1、1、2、5、10 mg/L）作用8 h及5 mg/L脂多糖作用HK-2细胞不同时间（0、2、4、8、16、24 h）,采用Western印迹和实时定量PCR检测SP-D表达,ELISA和实时定量PCR检测MCP-1表达。脂质体转染法将pEE14-hSP-D质粒转染HK-2细胞,筛选稳定转染细胞株,Western印迹检测转染后SP-D蛋白表达。采用5 mg/L脂多糖刺激pEE14-hSP-D稳定转染的HK-2细胞8 h,ELISA和实时定量PCR检测HK-2细胞MCP-1表达。 结果 HK-2细胞表达SP-D。脂多糖可诱导MCP-1 mRNA及蛋白表达显著增高（P < 0.05）;并可诱导SP-D mRNA及蛋白表达显著降低（P < 0.05）。转染pEE14-hSP-D质粒使HK-2细胞高表达SP-D,并可下调脂多糖诱导的MCP-1表达（P < 0.01）。 结论 SP-D可通过下调脂多糖诱导的HK-2细胞MCP-1的表达,从而在肾脏炎性反应中起重要作用。