Effects of emodin on the proliferation of the glomerular mesangial cell and correlative cytokines in rats

Objective：To investigate the effects of emodin（EMD） on cell proliferation and correlative cytokines secretion of glomerular mesangial in rats. Methods：The effects of EMD on cell proliferation and IL-6, TGF-β1 secretion of glomerular mesangial in rats were observed. Cell proliferation was measured by MTT method. IL-6 and TGF-β1 secretion was detected with ELISA. Results：EMD was able to inhibit the cell proliferation and down-regulate the IL-6 and TGF-β 1 secretion of glomerular mesangial, as compared to the model group in rats （P 〈 0.05）. Conclusion：EMD could significantly inhibit the cell proliferation, and reduce the creation of extracellular matrix（ECM）, this indicated that it could play an important role in alleviation and prevention of glomerular sclerosis. The mechanism may be that EMD can reduce the IL-6 and TGF-β1 secretion ofglomerular mesangial cell in rats.     

Interleukin-13 inhibits cytokines synthesis by blocking nuclear factor-κB and c-Jun N-terminal kinase in human mesangial cells

Objective： Monocytes/macrophages, proinflammatory cytokines and chemokines are important in the pathogenesis of glomerulonephritis. Interleukin （IL） -13 has been shown to exert potent anti-inflammatory properties. This study was designed to investigate the effect of IL-13 on the expression of proinflammatory cytokines, chemokines and profibrogenic cytokines and the involved molecular mechanism in cultured human mesangial cells （HMCs）. Methods： The expressions of proinflammatory cytokines, chemokines and profibrogenic cytokines were determined by ribonuclease protection assay （RPA）. Activity of nuclear factor-kappa B （NF-κB） and activa- tor protein-1 （AP-1） was examined by electrophoretic mobility shift assay （EMSA）. NF-κB subunit p65 nuclear transportation and c-Jun N-terminal kinase （JNK） activity were assayed by immunoblot. Results： Recombinant IL-13 inhibited tumor necrosis factor-α （TNF-u）, IL-1α, IL-1β, monocyte chemoattractant protein-1 （MCP-1）, IL-8, and transforming growth factor-β1 （TGF-β1） mRNA expressions in a dose-dependent manner. Lipopoly- sacchorides （LPS） dramatically increased NF-κB DNA binding activity of HMCs, which was inhibited by IL-13 in a dose-dependent manner. LPS-activated NF-κB contained p50 and p65 dimers, but not c-Rel subunit. IL-13 blocked LPS-induced NF-κB subunit p65. LPS stimulated JNK/AP-1 activation, which was inhibited by IL-13 in a dose-dependent manner. Conclusion： IL-13 inhibits proinflammatory cytokines, chemokines, and profibrogenic cytokines synthesis by blocking NF-κB and JNK/AP-1 activation. These observations point to the importance of IL-13 in the modulation of inflammatory processes in the renal glomerulus.     

Effect of amlodipine on apoptosis of human breast carcinoma MDA-MB-231 cells

Objective： To elucidate the effects of amlodipine on the proliferation and apoptosis of human breast carcinoma MDA-MB-231 cells. Methods： Light microscopy was used to determine the effects of amlodipine on cell morphology; Flow cytometry was used to quantitate cells undergoing apoptosis; the expression of a cell cycle-related protein, proliferating cell nuclear antigen （PCNA） and an antiapoptosis protein, Bcl-2 were assessed by immunocytochemistry. Results： Amlodipine concentration of 8.25umol/L （1/2 of ICs0） affected the morphology, decreased the expression of PCNA and Bcl-2 and induced apoptosis of human breast carcinoma MDA-MB-231 cells. Conclusion： The effect of amlodipine on the antiproliferation of human breast carcinoma MDA-MB-231 cells is related to inducement of apoptosis, and the decrease of the expression of Bcl-2 and PCNA may be the possible mechanism for proliferation inhibitory and inducement of apoptosis.     

EOTAXIN AND EOTAXIN-2 EXPRESSION IN HUMAN BRONCHIAL EPITHELIAL CELL

Objective To study the role of eotaxin and eotaxin-2 expression by Th2 cytokine and analyze their relationship in normal human bronchial epithelial cell line-BEAS-2B cell. Methods Levels of eotaxin mRNA and protein expression in the bronchial epithelial cell line BEAS-2B cell were determined with RT-PCR and ELISA. We also used RT-PCR to evaluate eotaxin-2 expression under the regulation of Th2 cytokine IL-4 and IL-13 as well as proinflammatory agent-TNFα. Results Eotaxin mRNA expression was the highest at the time point of 12h under the stimulation of TNF-α. While Th2 cytokine IL-4 and IL-13 had the amplification effect on the expression. Eotaxin protein was also elevated with the combination stimulation of proinflammatory agent TNF-α and IL-4 in dose and time dependent manner（ P 〈 0. 01 ）. These results were also seen when the cells were stimulated by TNF-α and 1L- 13. Eotaxin-2 mRNA expression was the highest at the time point of 8h. The expression evaluated by semi-quantitative RT-PCR also elevated under the co-stimulation of TNF-α and IL-4 or TNF-α and IL-13 and it should significantly correlate with Eotaxin （ P 〈 0. 05 ）. Conclusion This study demonstrated that Th2 cytokine like IL-4 and IL-13 enhances eotaxin and eotaxin-2 expression when co-stimulated with proinflammatory agent TNF-α. These results showed that Th2 cytokines existence is the strong evidence for bronchial epithelial cells taking part in the allergic inflammation especially in eosinophils recruitment.     

Kawasaki Disease on PDGF Expression and VSMC Proliferation

The effect of serum of patients with Kawasaki disease (KD) on expression of platelet-derived growth factor (PDGF) B chain protein in vascular endothelial cells (VEC) was studied by immunocytochemical method. Meanwhile,the effects of the endothelial cell conditioned media (ECM) on expression of PDGF receptor mRNA in vascular smooth muscle cells (VSMC) and on cell cycle of VSMC were investigated by the methods of nucleic acid hybridization and flow cytometry (FCM). The results showed that the serum of patients with KD induced the expression of PDGF-B chain protein significantly. ECM significantly promoted the expression of PDGF receptor mRNA and induced the proliferation of VSMC. These data suggest that the activation of PDGF-PDGF receptor may play a role in the pathogenesis of coronary complication of KD.     

Inhibitory effects of polymyxin B on NF-κB activation and expression of procollagen I, III in pre-eclamptic umbilical artery smooth muscle cells

Background It has been identified that in patients with pre-eclampsia, several factors were released into the serum, which can regulate the proliferation of vascular smooth muscle cells and stimulate the synthesis of extracellular matrix （ECM）. However, the signal transduction pathway of the growth factors and cytokines synthesized by endothelial cells leading to the proliferation and ECM synthesis of human umbilical arterial smooth muscle cell （HUASMC） is unknown. The aim of this study was to research the effects of protein kinase C （PKC） inhibitor, polymyxin B （PMB）, on the proliferation, nuclear factor-kappa B （NF-KB） activation, and expression of procollagen Ⅰ and Ⅲ mRNA in HUASMC cultured with pre-eclamptic umbilical sera. Methods Normal HUASMCs were treated with pre-eclamptic umbilical serum （pre-eclamptic group）, preeclamptic umbilical serum plus PMB （PMB group）, or normal umbilical serum （normal group）. The expression of Ⅰ-kB, NF-kB was detected by Western blotting after the HUASMC was incubated for 2 hours. The proliferation of HUASMC was evaluated by MTT, the cell cycle was analyzed by flow cytomerty, and the expression of procollagen Ⅰ,Ⅲ mRNA was measured by RT-PCR assay after HUASMC was incubated for 48 hours. ANOVA was used for statistical analysis. Results Umbilical sera in pre-eclampsia could stimulate the proliferation, the DNA synthesis, the transition of G0＋G1 phase or G2/M phase to S and phase, the activation of NF-kB, and the expression of procollagen Ⅰ mRNA of HUASMC as compared with normal umbilical sera （P〈0.01）. PMB could inhibit the proliferation, the DNA synthesis, the transition of G0＋G1 or G2/M phase phase to S phase, the activation of NF-KB, and the expressions of procollagen Ⅰ mRNA of HUASMC stimulated by umbilical sera in pre-eclampsia （P〈0.01）. Conclusion PKC-NF-KB signal transduction pathway may play a key role in SMC phenotype modulation, which is more important in the pathogenesis of placental blood vessel in pre-eclampsia.     

Tanshinone ⅡA Inhibits Dendritic Cell-mediated Adaptive Immunity: Potential Role in Anti-atherosclerotic Activity

Objective：Antigen-presenting cells such as monocytes and dendritic cells（DCs） stimulate T-ceil proliferation and activation during adaptive immunity.This cellular interaction plays a role in the growth of atherosclerotic plaques.Tanshinone ⅡA（TSN） had been shown to decrease the growth of atherosclerotic lesions.We therefore investigated the ability of TSN to inhibit human monocyte-derived DCs and their T-cellstimulatory capacity.Methods：DCs derived from human monocytes cultured with recombinant human interieukin（IL）-4 and recombinant human granulocyte-macrophage colony-stimulating factor were co-cultured with TSN and lipopolysaccharide for 48 h.Phosphate-buffered saline was used as a negative control.Activation markers and the capacity of DCs for endocytosis were measured by flow cytometry,and proinflammatory cytokines were measured by enzyme-linked immunosorbent assays.DCs were co-cultured with lymphocytes to measure T-cell proliferation and IL-2 secretion by mixed lymphocyte reactions.Results：TSN dose-dependently attenuated DC expression of costimulatory molecules（CD86）,and decreased expression of major histocompatibility complex class I（human loukocyte antigen-DR） and adhesion molecules（CD54）.Moreover,TSN reduced secretion of the proinflammatory cytokines IL-12 and IL-1 by human DCs,and restored the capacity for endocytosis.Finally,TSN-preincubated DCs showed a reduced capacity to stimulate T-cell proliferation and cytokine secretion.Conclusions：TSN inhibits DC maturation and decreases the expression of proinflammatory cytokines,while impairing their capacity to stimulate T-cell proliferation and cytokine secretion.These effects may contribute to the influence of TSN on the progression of atherosclerotic lesions.     

Study on Effects of Extracts from Salvia Miltiorrhiza and Curcuma Longa in Inhibiting Phosphorylated Extracellular Signal Regulated Kinase Expression in Rat''''s Hepatic Stellate Cells

Objective: To study the effect of salvianolic acid B (SAB) and curcumin, the extracts of Salvia Miltiorrhiza and Curcuma Longa, on the proliferation and activation of hepatic stellate cell (HSC), and the extracellular signal regulated kinase (ERK) expression in it. Methods: Rat's HSC-T6 were cultured and treated by SAB or curcumin. The inhibitory effect on cell proliferation was determined by 3-(4, 5-dimthyl-2-2thiazoly)-2,5-diphenyl-2H-tetrazolium bromide (MTT) colorimetry, and the expression levels of a smooth actin (α-SMA), collagen typeⅠ, and ERK were determined by Western blot. Results: SAB and curcumin inhibited the proliferation and activation of rat's HSC-T6 in dose-dependent fashion and significantly reduced the expression level ofα-SMA (P<0.01). Curcumin significantly reduced the expression of collagen typeⅠ(P<0.05). Both SAB and curcumin showed insignificant effect on the ERK expression level, but they could significantly reduce the level of phosphorylated-ERK expression, showing significant difference as compared with that in the control group (P<0.01 and P<0.05 respectively). Conclusion: SAB and curcumin could significantly inhibit the proliferation, activation of HSC, and the production of typeⅠcollagen in HSC, the mechanism may be associated with their inhibition on ERK phosphorylation.     

Downregulation of p38 MAPK involved in inhibition of LDL-induced proliferation of mesangial cells and matrix by curcumin

Curcumin, as a main pharmacological component in the traditional Chinese medicine-- tttrmeric, has shown anti-inflammatory, anti-oxidation, anti-tumor and anti-fibrotic effects. This study aimed to investigate the possible underlying signaling pathway which was involved in the inhibition of LDL-induced proliferation of mesangial cells and matrix by curcumin. Rat mesangial cells in vitro were incubated with low-density lipoprotein （LDL） and different concentrations of curcumin （0, 6.25, 12.5, 25.0 9mol/L） or p38 MAPK inhibitor, SB203580 （10 μmol/L）. Under LDL incubation, mesangial cells proliferated, the expression of MMP-2 mRNA and protein was decreased, the expression of COX-2 mRNA and protein was increased, reactive oxygen species （ROS） generation was increased and p38 MAPK was activated significantly （P〈0.05）. When LDL-induced cells were treated with curcumin in the concentration of 12.5 or 25.0 μmol/L, LDL-induced proliferation ofmesangial cells was suppressed, the expression of MMP-2 mRNA and protein increased, the expression of COX-2 mRNA and protein downregulated, the production of ROS inhibited and p38 MAPK inactivated （P〈0.05）. In conclusion, curcumin can inhibit the LDL-induced proliferation of mesangial cells and up-regulate the expression of MMP-2, which may be related with the inhibitory effect of curcumin on COX-2 expression, ROS pro- duction and p38 MAPK.     

洛伐他汀对肾小球系膜细胞增殖及细胞周期时相的影响

目的探讨洛伐他汀对人肾小球系膜细胞（HMC）增殖和细胞周期时相的影响及机制。方法用３H-TdR掺入量法测定HMC增殖水平,以流式细胞术测定HMC的细胞周期。结果洛伐他汀剂量依赖性抑制HMC增殖,从细胞周期水平,洛伐他汀可使HMC由G１期向S期的进展受阻,这些抑制作用可被外源甲羟戊酸和法呢醇恢复。结论洛伐他汀是HMC增殖的抑制剂,本研究为重新认识和评价他汀类药物在治疗肾脏疾病中的作用提供了一定的理论及实验依据。     

NF-κB/IκB信号通路介导血管紧张素Ⅱ诱导的系膜细胞促炎性细胞因子表达

目的：探讨核因子—κB(NF—κB)／IκB信号通路在血管紧张素Ⅱ(AngⅡ)诱导的肾小球系膜细胞促炎性细胞因子表达中的作用。方法：应用核酸酶保护法检测系膜细胞肿瘤坏死因子α(TNF—α)、IL-1α和IL-1β mRNA表达；应用凝胶电泳迁移率和Western blot检测肾小球系膜细胞中NF—κB活化、p65亚基核转位以及IκBα和IκBβ的降解。结果：正常培养状态下，系膜细胞可组成型表达TNF—α和IL—1β，而不表达IL-1αmRNA，AngⅡ刺激后促炎性细胞因子表达显著上调，NF—κB特异性抑制剂2-硫代氨基甲酸吡咯烷显著抑制AngⅡ诱导的促炎性细胞因子基因表达；AngⅡ诱导系膜细胞NF—κB活化，p65核转位及胞质内IκBα和IκBα的降解。结论：AngⅡ诱导肾小球系膜细胞中促炎性细胞因子表达可能通过NF—κB／IκB信号转导通路来实现。     

护肾固精方下调核因子-κB活化对系膜细胞肿瘤坏死因子-α表达的影响

目的 :研究护肾固精方 (Hushengujingfang ,HSGJF)对脂多糖 (LSP)刺激大鼠肾小球系膜细胞(mesangialcell,MC)表达炎性细胞因子的影响及其作用机制。方法 :分别以酶联免疫吸附法 (ELISA)检测MCIL 1β、TNF α蛋白水平 ;电泳迁移率变动分析 (EMSA)检测MC核因子 κB(nuclearfactor κB ,NF κB)的活性 ;反转录多聚酶链反应技术 (RT PCR)检测MCTNF αmRNA表达。结果 :HSGJF能抑制LSP刺激肾小球MC分泌IL 1β、TNF α蛋白质增加的同时 ,亦能抑制LSP刺激肾小球MC中NF κB活化 ,在HSGJF作用下炎性细胞因子表达均呈不同程度减弱。结论 :HSGJF通过抑制MCNF κB的活性 ,下调炎性细胞因子的表达。此结果可作为进一步认识和评价该方对肾脏疾病防治作用的实验依据。     

核因子—kB调控白细胞介素1β刺激人肾小球系膜细胞表达细胞间粘 …

目的 研究因子－ｋＢ（ＮＦ－ｋＢ）对白细胞介素１β（ＩＬ－１β）引起的体外培养的人肾小球系膜细胞表达细胞间粘附分子（ＩＣＡＭ－１）的基因调控机理。方法 采用凝胶迁移率法观测ＮＦ－ｋＢ的活化。以Ｎｏｒｔｈｅｒｎ杂交方法检测细胞间粘附分子的基因表达,用细胞ＥＬＩＳＡ法检测ＩＣＡＭ－１蛋白水平的表达。结果 ｒｈＩＬ－１β１０ｎｇ／ｍｌ刺激肾小球系膜细胞１,２,４小时,均引起ＮＦ－ｋＢ活化,且１小时为最强     

Nuclear factor- κB in signal conduction of protein kinase C in T lymphocytes from an asthmatic guinea pig model

Objective To explore the role of nuclear factor- κB (NF- κB) in the signal conduction of protein kinase C (PKC) regulated proliferation, apoptosis and expression of Th2 cytokines - interleukin- 4 (IL- 4) and interleukin- 5 (IL- 5) of T lymphocytes in the bronchial alveolus lavage fluid (BALF).Methods T lymphocytes were isolated and purified from BALF of asthmatic guinea pigs in normal and asthmatic groups, and were stimulated with PKC agitator phorbol 12- myristate 13- acetate (PMA) and NF- κB inhibitor pyrrolidine dithiocarbamate (PDTC), respectively.The expressions of NF- κB, IL- 4 and IL- 5 mRNA and protein, the proliferation and apoptosis of T lymphocytes were observed by immunohistochemistry, in situ hybridization, ELISA, MTT and TUNEL, respectively. Results The activation of NF- κB, proliferation response, and expression of IL- 4 and IL- 5 mRNA and protein in T lymphocytes stimulated by PMA were significantly higher than those of their blank control (P&lt;0.01), while those indexes of T lymphocytes stimulated by PMA and PDTC simultaneously were significantly lower than those stimulated by PMA alone (P&lt;0.01).The apoptotic index of T lymphocytes stimulated with PMA were significantly lower than that of their blank control (P&lt;0.01), and the apoptotic index of asthmatic guinea pig T lymphocytes stimulated with PMA and PDTC simultaneously were significantly higher than that stimulated by PMA alone (P&lt;0.01).The significant positive correlations were found between the activation of NF- κB and the proliferation (r=0.64, P&lt;0.001), and the expression of IL- 4 and IL- 5 mRNA and protein of T lymphocytes, respectively (r=0.55-0.68, P&lt;0.001).There was also significant negative correlation between the activation of NF- κB and apoptosis of T lymphocytes (r=0.62, P&lt;0.001). Conclusions NF- κB may participate in the signal conduction of PKC regulated proliferation, apoptosis and expression of IL- 4 and IL- 5 of T lymphocytes in asthma.The activation of NF- κB in PKC signal conduction pathway of T lymphocytes may play an important role in the pathogenesis of asthma.     

Inhibition of mesangial cell proliferation by antisense oligodeoxynucleotide targeting preproendothelin-1 mRNA in vitro

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洛伐他汀联合环孢素A对人外周血单个核细胞增殖、细胞因子表达及NK细胞毒作用的影响

目的 探讨洛伐他汀联合环孢素A（CsA)对体外培养的人淋巴细胞增殖、对某些细胞因子表达及NK细胞活性的影响。方法 Ficoll-Hypague密度梯度离心法提取人外周血单个核细胞（PBMC）;乳酸脱氢酶（LDH）释放试验测定细胞活性;^3H-标记甲基胸腺嘧啶（^3H-TdR)掺入法观察药物对细胞增殖及细胞毒作用的影响;ELISA-斑点法（ELISA-SPOT）检测细胞分泌的IL-2;放免法（RIA）测定细胞上清IFN-γ的浓度;半定量反转录-聚合酶链反应（RT-PCR）显示IL-2及IFN-γmRNA的表达。结果 洛伐他汀及CsA均可剂量依赖性地抑制体外培养的人淋巴细胞的增殖,IL-2和IFN-γ表达及NK细胞活性。两药联合使用具有协同作用。结论 他汀类药物可与CsA协同使用减缓移植排斥反应发生发展,延长移植物寿命。     

洛伐他汀对TNF-α诱导人脐静脉内皮细胞MCP-1和IL-10表达的影响

目的：观察洛伐他汀(LVT)对肿瘤坏死因子α(TNF-α)刺激人脐静脉内皮细胞(HUVECs)后单核细胞趋化蛋白-1 (MCP-1)和白细胞介素-10 (IL-10)表达的影响,探讨其抗动脉粥样硬化(AS)的可能机制。方法：体外常规培养HUVECs,分为对照组,用含10% FBS的RPMI 1640培养液培养;TNF-α处理组,用含10% FBS的RPMI 1640培养液+TNF-α (20 μg/L) 处理;应用活性氧检测试剂盒检测活性氧含量;LVT干预组,用含10% FBS的 RPMI 1640 培养液+TNF-α (20 μg/L)+LVT (10 μmol/L) 处理。实时定量RT-PCR方法检测各组细胞MCP-1和IL-10 mRNA的表达;Western blotting方法检测各组HUVECs内MCP-1、IL-10和NF-κB蛋白表达。结果：与对照组相比较,TNF-α处理组ROS含量明显增高 (P<0.05);与TNF-α处理组比较,LVT干预组ROS含量明显降低(P<0.05);与对照组比较,TNF-α处理组MCP-1和NF-κB mRNA及蛋白表达水平明显增高(P<0.05),而IL-10 mRNA和蛋白的表达水平明显降低(P<0.05);与TNF-α处理组比较,LVT干预组细胞MCP-1和NF-κB mRNA及蛋白表达水平明显降低(P<0.05),而IL-10 mRNA和蛋白表达水平明显增高(P<0.05)。结论：LVT抑制HUVECs中TNF-α诱导的MCP-1 mRNA和蛋白上调,增强IL-10蛋白表达。LVT可能通过NF-κB途径对抗炎症反应从而防止AS的形成。
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氧化低密度脂蛋白对人肾小球系膜细胞A型清道夫受体表达的调节作用

目的研究氧化低密度脂蛋白(Ox-LDL)对人肾小球系膜细胞(HMC)A型清道夫受体(SR-A)表达的调节作用,探讨在慢性肾脏疾病中Ox-LDL加重肾病进展的作用机制.方法脂质体转染含SR-A cDNA的表达质粒,建立稳定高表达SR-A的人肾小球系膜细胞株(HMCL),油红"O"染色检测HMCL对Ox-LDL的摄取;RT-PCR法检测Ox-LDL和LDL对HMC SR-A mRNA表达的作用.结果稳定高水平表达SR-A的HMCL对Ox-LDL的摄取明显强于未转染细胞;Ox-LDL上调HMC SR-A mRNA的表达,作用于24 h达到高峰;Ox-LDL(10～100μg/ml)作用24 h对HMC SR-A mRNA的上调作用呈剂量依赖性;天然LDL对SR-A的表达无明显影响.结论在HMC,SR-A是Ox-LDL进入细胞内的主要受体之一,Ox-LDL具有上调HMC SR-A mRNA表达的作用,推测在慢性肾脏疾病进展中,局部沉积的LDL经氧化修饰后可能通过刺激SR-A的表达进入细胞内增强其对HMC的毒性作用,进而加重肾小球硬化的进展.