补肾活血泄浊汤对体外培养肾小球上皮细胞增殖影响

目的:探讨脂多糖(LPS)、硫酸鱼精蛋白、表皮生长因子(EGF)及肿瘤坏死因子-α(TNF-α)及补肾活血泄浊汤对体外培养大鼠肾小球上皮细胞生长的影响。方法:应用LPS、硫酸鱼精蛋白、EGF及TNF-α作为刺激因子,观察其对肾小球上皮细胞(GEC)增殖的影响;并采用血清药理学方法,提取动物的含药血清作用于上述受刺激的上皮细胞,观察补肾活血泄浊汤对GEC增殖的影响。结果:LPS、EGF+TNF-α和硫酸鱼精蛋白均可抑制GEC掺入[³H]-TdR,并呈一定的量效和时效关系;而补肾活血泄浊汤可使GEC掺入[³H]-TdR增加。结论:GEC是补肾活血泄浊汤发挥治疗作用的重要靶细胞,这可能是该方防治以蛋白尿为主要临床表现的肾小球疾病的机制之一。     

补肾活血泄浊汤治疗大鼠局灶性节段性肾小球硬化的研究

目的:观察中药补肾活血泄浊汤对大鼠局灶性节段性肾小球硬化的治疗作用及作用机制。方法:采用单侧肾切除术,两次尾静脉注射阿霉素法复制局灶性节段性肾小球硬化模型,分为补肾活血泄浊汤治疗组、模型组和正常组。实验的前6周每周测大鼠24h尿蛋白,后6周隔周测大鼠24h尿蛋白,第60d\,100d时测定血清白蛋白、胆固醇、肌酐、尿素氮含量,并进行光镜、电镜、原位杂交、免疫组化等观察。结果:治疗组各项指标与模型组相比均有显著性差异,形态学观察也显示治疗组损害轻于模型组。结论:补肾活血泄浊汤能够明显保护病鼠肾功能,明显减轻病鼠细胞外基质沉积,延缓病变肾小球硬化的发生。     

白细胞介素13抑制肾小球系膜细胞增殖及白细胞介素6表达

目的:探讨白细胞介素13(IL-13)对体外培养的大鼠系膜细胞的增殖及其产生白细胞介素6(IL-6)的影响。方法:用四甲基偶氮唑(MTT)法测定系膜细胞增殖,用逆转录聚合酶链反应(RT-PCR)及酶联免疫吸附法(ELISA)测定系膜细胞IL-6mRNA表达及其蛋白水平。结果:IL-13在1、10、100μg/L浓度范围呈剂量依赖性地抑制系膜细胞的增殖;5%FCSRPMI1640培养条件下系膜细胞IL-6mRNA表达及IL-6分泌水平较低,脂多糖(LPS)可刺激系膜细胞IL-6mRNA的表达及提高IL-6分泌水平,而IL-13可抑制LPS诱导的系膜细胞IL-6分泌及其mRNA表达。结论:IL-13抑制体外培养的系膜细胞增殖及LPS诱导的系膜细胞IL-6的产生,IL-13可能对于肾小球肾炎的系膜细胞炎症反应具有拮抗作用。     

活血化湿汤通过靶向TP53抑制M2巨噬细胞极化与非小细胞肺癌细胞的侵袭

目的：探讨活血化湿汤靶向TP53对M2巨噬细胞极化与非小细胞肺癌（NSCLC）细胞侵袭的作用。方法：网络药理学分析筛选出活血化湿汤治疗NSCLC的靶点TP53。qRT-PCR和Western blot检测TP53在NSCLC细胞中的表达水平。诱导THP-1细胞为M0巨噬细胞，使用活血化湿汤及TP53处理巨噬细胞，流式细胞术分析CD86、CD206表达，ELISA检测IL-1β、CCL18浓度。使用活血化湿汤、TP53过表达载体、巨噬细胞培养液处理NSCLC细胞，Transwell检测NSCLC细胞侵袭能力。结果：网络药理学分析发现活血化湿汤的靶点和NSCLC的发病风险基因靶点共交集17个基因，STRING分析显示TP53与其他基因的交互作用最强。与BEAS-2B细胞比较，NSCLC细胞中TP53表达增强，而活血化湿汤能抑制TP53表达。活血化湿汤可通过抑制TP53上调巨噬细胞中CD86阳性细胞百分比，下调CD206阳性细胞百分比，提高IL-1β浓度，降低CCL18浓度（均P<0.05）。此外，活血化湿汤能通过抑制TP53表达进而抑制NSCLC细胞的侵袭。结论：活血化湿汤通过抑制T...     

白细胞介素1β通过p38信号通路上调系膜细胞表达白细胞介素6

目的：探讨p38信号通路(p38MAPK)在白细胞介素1(IL-1)β上调肾小球系膜细胞表达白细胞介素6(IL-6)中的作用。方法：应用Western Blotting检测p38MAPK在IL-1β诱导的肾小球系膜细胞炎症反应中的活化程度，应用逆转录-聚合酶链反应(RT-PCR)和ELJSA法检测IL-1β诱导的系膜细胞促炎症介质IL-6的表达水平并观察p38MAPK特异性抑制剂SB203580对其mRNA的转录和蛋白质生成的影响。结果：IL-1β以时间和剂量依赖方式刺激系膜细胞引起p38MAPK的活化，并明显上调系膜细胞IL-6表达。SB203580以剂量依赖方式从基因转录和翻译水平显著抑制IL-1β诱导的IL-6表达。结论：p38MAPK在IL-1β诱导的肾小球系膜细胞炎症反应产生炎症介质IL-6中起重要作用。     

补肾活血方联合非布司他对高尿酸性肾病大鼠炎症损伤的影响及可能机制

目的 探讨补肾活血方联合非布司他对高尿酸性肾病大鼠炎症反应和肾损伤的改善作用及可能机制。方法 雄性Sprague-Dawley大鼠随机分为对照组(Control)、模型组(Model)、补肾活血汤组(BSHXDS)、非布司他组(Febuxostat)、补肾活血汤联合非布司他组(BSHXDS+Febuxostat),每组12只。生化分析检测各组大鼠血清尿酸和肌酐水平;HE染色检测各组大鼠肾脏组织损伤;ELISA检测各组大鼠血清IL-6和IL-1β水平;Western blot检测肾组织TLR4、MyD88、NF-kappa B p65蛋白的表达。结果 补肾活血汤、非布司他和补肾活血方联合非布司他均能降低高尿酸性肾病大鼠肾组织炎症性损伤、血清尿酸、肌酐、炎症因子水平,同时下调大鼠肾组织TLR4、MyD88、NF-kappa B的表达水平;与单独使用补肾活血汤或非布司他相比,补肾活血汤联合非布司他组的作用更为明显。结论 补肾活血方联合非布司他可通过调控TLR4/MyD88/NF-kappa B通路抑制高尿酸性肾病大鼠炎症反应,改善肾功能。     

蕨麻多糖通过促进环状RNA AKT3(circ-AKT3)表达减轻高糖诱导的小鼠肾小球系膜细胞损伤

目的 探讨蕨麻多糖对高糖诱导的肾小球系膜细胞损伤的影响及环状RNA-AKT3(circ-AKT3)在其中的作用。方法采用高糖诱导SV40 MES13小鼠肾小球系膜细胞建立细胞损伤模型,加入(0.1、 0.3、 0.6)mg/mL蕨麻多糖处理细胞;分别采用pcDNA和pcDNA-circ-AKT3质粒、小干涉RNA阴性对照(si-NC)、环状RNA AKT3小分子RNA(si-circ-AKT3)转染高糖诱导的肾小球系膜细胞后,加入蕨麻多糖处理细胞。根据试剂盒检测超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)、丙二醛(MDA)的水平;ELISA检测白细胞介素6(IL-6)、 IL-18、单核细胞趋化蛋白1(MCP-1)的水平;流式细胞术检测细胞凋亡率;实时定量PCR检测circ-AKT3的表达量。结果 高糖诱导的肾小球系膜细胞中SOD、 GSH-Px的水平降低,MDA、 IL-6、 IL-18、 MCP-1的水平升高,凋亡率升高,circ-AKT3的表达水平降低;蕨麻多糖处理高糖诱导的肾小球系膜细胞后,SOD、 GSH-Px的水平升高,MDA、 IL-6、 IL-18、 M...     

NF-κB介导脂氧素A4拮抗TNF-α所致的系膜细胞白介素的合成

为研究脂氧素A4(LXA4)拮抗肿瘤坏死因子α(TNF-α)对肾小球系膜细胞的白介素(IL)-1β(IL-1β)、IL-6合成的作用。对体外培养大鼠肾小球系膜细胞,用不同浓度的LXA4预刺激,再加入TNF-α共同孵育;或单用TNF-α刺激肾小球系膜细胞。在孵育后用ELISA法检测培养上清中的IL-1β、IL-6蛋白表达量;用RT-PCR法检测IL-1β、IL-6的mRNA表达量。应用凝胶电泳迁移率试验(EMSA)测定核因子-κB(NF-κB)的DNA结合活性。结果发现,LXA4呈剂量依赖性地抑制TNF-α诱导的肾小球系膜细胞IL-1β和IL-6蛋白的合成与mRNA表达,抑制NF-κB的DNA结合活性。说明LXA4通过下调NF-κB的DNA结合活性,拮抗TNF-α对肾小球系膜细胞的IL-1β、IL-6合成的促进作用。     

IL-13对LPS诱导人系膜细胞分泌IL-12的影响

目的: 探讨白细胞介素13（IL-13）对肾小球系膜细胞分泌白细胞介素12（IL-12）的影响作用。方法: 用酶联免疫吸附（ELISA）法和逆转录聚合酶链反应（RT-PCR）法检测系膜细胞IL-12蛋白和L-12p40 mRNA表达。结果: LPS诱导系膜细胞的IL-12p40 mRNA表达及其蛋白分泌（P<0.01）。IL-13在1-100 μg/L浓度范围内呈剂量依赖性抑制LPS诱导的系膜细胞IL-12分泌及其mRNA表达（P<0.05或P<0.01）。结论: IL-13可能通过抑制LPS诱导系膜细胞分泌IL-12，而调整了体内Th1/Th2细胞因子平衡。     

NF-kB介导脂氧素A4拮抗TNF-α所致的系膜细胞白介素的合成

为研究脂氧素A4(LXA4)拈抗肿瘤坏步E因子α(TNF-α)对肾小球系膜细胞的白介素(IL）-β(IL-1β)、IL-6合成的作用。对体外培养大鼠肾小球系膜细胞，用不同浓度的LXA4预刺激，再加入TNF-α共同孵育；或单用TNF-α刺激肾小球系膜细胞。在孵育后用ELISA法检测培养上清中的IL-1β,IL-6蛋白表达量；用RT-PCR法检测IL-1β、IL-6的mRNA表达量。应用凝胶电泳迁移率试验（EMSA)测定核因子-kB(NF-KB)的DNA结合活性。结果发现，LXA4呈剂量依赖性地抑制TNF-α诱导的肾小球系膜细胞IL-1β和IL-6蛋白的合成与mRNA表达，抑制NF-kB的DNA结合活性。说明LXA4通过下调NF-kB的DNA结合活性，拮抗TNF-α对肾小球系膜细胞的IL-1β、IL-6合成的促进作用。     

白细胞介素-13对肾小球系膜细胞炎症反应的拮抗作用

目的:探讨白细胞介素-13(IL-13)对肾小球系膜细胞(MC)炎症反应的调节作用。方法:ELISA法测定体外培养MC上清中TNF-α浓度。流式细胞术检测MC膜表面ICAM-1表达。逆转录聚合酶链式反应(RT-PCR)评估MCTNF-αmRNA及ICAM-1mRNA表达。结果:未受刺激的MC无TNF-αmRNA及其蛋白表达。经脂多糖(LPS)(10mg/L)刺激后,MC可高表达TNF-αmRNA及其蛋白。IL-13浓度为1μg/L、10μg/L时显著抑制LPS诱导MC表达TNF-αmRNA及其蛋白。IL-13(0.1μg/L)无抑制作用,IL-13(100μg/L)时完全抑制LPS诱导MC分泌TNF-α。无任何刺激时,MC低表达ICAM-1。TNF-α(100μg/L)诱导MC高表达ICAM-1mRNA及其蛋白。IL-13(10μg/L)和TNF-α(100μg/L)共作用MC,各时点均显示IL-13对TNF-α诱导MC表达ICAM-1mRNA及蛋白有抑制作用。结论:IL-13既抑制LPS刺激MC分泌TNF-α,也抑制TNF-α诱导MC表达膜糖蛋白ICAM-1。提示IL-13能从多个环节抑制MC的炎症反应。     

IL-13抑制人肾小球系膜细胞IL-12的表达

为了探讨白细胞介素 13(IL 13)对体外培养的人肾小球系膜细胞产生白细胞介素 12 (IL 12 )的影响。我们用脂多糖(LPS 10 μg/ml) ,不同浓度的IL 13对系膜细胞培养 ,分别采用ELISA法和半定量RT PCR法检测细胞上清液的IL 12和系膜细胞IL 12p4 0mRNA表达。结果提示 5 %NCSRPMI 16 4 0基础培养条件下的系膜细胞未检测到IL 12蛋白分泌及其mRNA表达。在LPS刺激下系膜细胞的IL 12p4 0mRNA表达加强 ,并分泌出大量的IL 12。IL 13在 1～ 10 0ng/ml浓度范围内对LPS诱导的系膜细胞IL 12分泌及IL 12p4 0mRNA表达的抑制作用呈剂量依赖趋势。本研究认为IL 13可能通过抑制IL 12的产生 ,而调整了体内Th1/Th2细胞因子平衡 ,作为抗炎性细胞因子在肾小球肾炎发病机制中发挥一定作用     

Anti-dsDNA antibody up-regulates interleukin 6, but not cyclooxygenase, gene expression in glomerular mesangial cells: a marker of immune-mediated renal damage?

OBJECTIVE AND DESIGN: To determine whether anti-double stranded DNA antibody (anti-dsDNA) can affect the synthesis of eicosanoids and cytokines in rat glomerular mesangial cells (RMC). MATERIALS OR SUBJECTS: Glomerular mesangial cells were isolated and subcultured from Sprague-Dawley rats. Monoclonal anti-dsDNA (12B3 clone) was derived from autoimmune MRL-lpr/lpr mouse by hybridoma technology. METHODS: The mRNA expression of cyclo-oxygenase type 1 (COX-1), type 2 (COX-2), Th1 (IL-2 and IFN-gamma)/Th2 (IL-4 and IL-10) and proinflammatory (IL-6 and TNF-alpha) and anti-inflammatory (TGF-beta) cytokines of RMC +/- anti-dsDNA was detected by RT-PCR. The PGE2 production by RMC +/- anti-dsDNA was measured by ELISA. The statistical significance was assessed by non-parametric Wilcoxon signed rank test. RESULTS: We found RMC spontaneously expressed COX-1, but not COX-2. The incubation of RMC with anti-dsDNA (50 ng/ml) did not affect COX expression and PGE2 production by RMC. RMC also spontaneously expressed IL-6, TNF-alpha and TGF-beta mRNA. However, only IL-6 was up-regulated by anti-dsDNA. CONCLUSIONS: Increased IL-6 expression in RMC may become a marker of anti-dsDNA-mediated immune damage of mesangial cells.     

NF-κB活化在IL-1β诱导的小鼠肾小球系膜细胞表达IL-6中的作用

目的:研究NF-κB在rhIL-1β刺激引起的体外培养的鼠肾小球系膜细胞表达IL-6中的作用。方法:NF-κΒ活性检测采用电泳迁移率改变法(EMSA),IL-6mRNA表达采用逆转录/聚合酶链反应(RT/PR)检测,培养上清IL-6蛋白含量采用ELISA检测。结果:rhIL-1β刺激肾小球系膜细胞时,在上调IL-6蛋白和基因表达的同时亦激活NF-κB,而且这种上调作用可被NF-κB特异性抑制剂PDTC所阻抑。结论:IL-1β诱导鼠肾小球系膜细胞表达IL-6是通过NF-κB调控,NF-κB可能参与肾小球肾炎的免疫炎症反应。     

NF-κB活化在IL-1β诱导的小鼠肾小球系膜细胞表达IL-6中的作用

目的：研究NF-κB在rhIL-1β刺激引起的体外培养的鼠肾小球系膜细胞表达IL-6中的作用。方法： NF-κΒ活性检测采用电泳迁移率改变法(EMSA),IL-6 mRNA表达采用逆转录/聚合酶链反应(RT/PR)检测,培养上清IL-6蛋白含量采用ELISA检测。结果：rhIL-1β刺激肾小球系膜细胞时,在上调IL-6蛋白和基因表达的同时亦激活NF-κB,而且这种上调作用可被NF-κB特异性抑制剂PDTC所阻抑。结论： IL-1β诱导鼠肾小球系膜细胞表达IL-6是通过NF-κB调控,NF-κB可能参与肾小球肾炎的免疫炎症反应。     

Interleukin-12 is synthesized by mesangial cells and stimulates platelet-activating factor synthesis, cytoskeletal reorganization, and cell shape change

Preliminary studies indicate the involvement of interleukin (IL)-12 in experimental renal pathology. In the present study, we evaluated whether cultured glomerular mesangial cells are able to produce IL-12 and whether IL-12 may regulate some of their functions, including the cytoskeletal reorganization, the change in cell shape, and the production of platelet-activating factor (PAF). The results obtained indicate that pro-inflammatory stimuli, such as tumor necrosis factor-alpha and bacterial polysaccharides, induce the expression of IL-12 mRNA and the synthesis of the protein by cultured mesangial cells. Moreover, cultured mesangial cells were shown to bind IL-12 and to express the human low-affinity IL-12 beta1-chain receptor. When challenged with IL-12, mesangial cells produced PAF in a dose- and time-dependent manner and superoxide anions. No production of tumor necrosis factor-alpha and IL-8 was observed. Moreover, we demonstrate that IL-12 induced a delayed and sustained shape change of mesangial cells that reached its maximum between 90 and 120 minutes of incubation. The changes in cell shape occurred concomitantly with cytoskeletal rearrangements and may be consistent with cell contraction. As IL-12-dependent shape change of mesangial cells was concomitant with the synthesis of PAF, which is known to promote mesangial cell contraction, we investigated the role of PAF using two chemically different PAF receptor antagonists. Both antagonists inhibited almost completely the cell shape change induced by IL-12, whereas they were ineffective on angiotensin-II-induced cell shape change. In conclusion, our results suggest that mesangial cells can either produce IL-12 or be stimulated by this cytokine to synthesize PAF and to undergo shape changes compatible with cell contraction.     

Constitutive expression of endothelin gene in cultured human mesangial cells and its modulation by transforming growth factor-beta, thrombin, and a thromboxane A2 analogue.

The present study was designed to assess whether human glomerular mesangial cells in culture express preproendothelin gene and whether endothelin gene expression in the mesangium is regulated by factors potentially released by inflammatory cells and platelets infiltrating the glomerular tuft during the course of various types of glomerulonephritis. For this purpose mesangial cells were incubated for 6 hours in the presence of absence of interleukin 1 beta (IL-1 beta), transforming growth factor-beta (TBF-beta), the thromboxane A2 analogue U-46619, and thrombin. Resting mesangial cells expressed a 2.3-kilobase mRNA on blot hybridization analysis with a human cDNA preproendothelin probe, indicating that this type of cells, in addition to glomerular endothelial cells, constitutively expresses endothelin gene. IL-1 beta did not change endothelin mRNA levels in respect to unstimulated mesangial cells. At variance, TGF-beta, U-46619, and thrombin had a marked effect on endothelin mRNA, stimulating a 3- to 8-fold increase over basal levels. Quantification of actin mRNA and analysis of the autoradiographic signals provided validation of the difference in the endothelin mRNA levels. Expression of preproendothelin mRNA in either resting or stimulated mesangial cells was associated with synthesis and release of the corresponding peptide in the cell supernatant as determined by a specific radioimmunoassay for endothelin. Endothelin production from IL-1 beta stimulated mesangial cells was not different from that of unstimulated cells, whereas a significant (p less than 0.01) increase in endothelin production was observed after cell stimulation with TGF-beta, U-46619, and thrombin. The demonstration that mesangial cells constitutively express mRNA for preproendothelin and release endothelin into culture medium, together with the finding that endothelin gene expression and production in mesangial cells are regulated by molecules potentially released at glomerular level during an inflammatory reaction may suggest that endothelin participates in the complex process of glomerular disease progression.