Vulnerability to ROS-induced cell death in ageing articular cartilage: the role of antioxidant enzyme activity

OBJECTIVES: To test the hypothesis that age-related loss of chondrocytes in cartilage is associated with impaired reactive oxygen species (ROS) homeostasis resulting from reduced antioxidant defence. METHODS: Cell numbers: The total number of chondrocytes in the articular cartilage of the femoral head of young, mature and old rats was estimated using an unbiased stereological method. ROS quantification: Fluorescence intensity in chondrocytes was quantified using the oxygen free radical sensing probe dihydrorhodamine 123 (DHR 123), confocal laser scanning microscopy and densitometric image analysis. In order to delineate the reactive species, explants were pre-treated with N-acetylcysteine (NAC) or N(G)-nitro-l-arginine methyl ester (l-NAME) prior to ROS quantification. Induction of intracellular ROS: Explants were incubated in the redox-cycling drug menadione after which they underwent ROS quantification and cell-viability assay. Antioxidant enzyme activity: The activity of catalase, superoxide dismutase (SOD) and glutathione peroxidase (GPX) was measured. RESULTS: Chondrocyte numbers: A significant and progressive loss of chondrocytes was observed with ageing. Cellular ROS levels: A significant age-related increase in cellular ROS-induced fluorescence was demonstrated. NAC significantly reduced ROS levels in old chondrocytes only. Induction of intracellular ROS: Menadione increased cellular ROS levels dose-dependently in young and old chondrocytes, with a greater effect in the latter. Old chondrocytes were more vulnerable to menadione-induced cytotoxicity. Antioxidant enzymes: Catalase activity declined significantly in aged cartilage whilst SOD and GPX activities were unaltered. CONCLUSIONS: Substantial loss of chondrocytes occurs in rat articular cartilage which may result from increased vulnerability to elevated intracellular ROS levels, consequent upon a decline in antioxidant defence.     

Differential effects of tumor necrosis factor-alpha and interleukin-1beta on cell death in human articular chondrocytes

OBJECTIVE: The death of chondrocytes by apoptosis is characteristic of degenerative joint diseases, such as osteoarthritis (OA). Tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta) have been shown to play an important role in the development of OA. In this study we analyzed the effects of TNF-alpha and IL-1beta on cell death in normal human chondrocytes. METHODS: Normal human chondrocytes were isolated from knee cartilage obtained at autopsy from 30 adult cadaveric donors. The cells were stimulated with TNF-alpha (10 ng/ml) or IL-1beta (5 ng/ml) in the presence or absence of Ro 31-8220 (Ro: a structurally related analog of bisindolylmaleimide that inhibits mitogen-activated protein kinase phosphatase 1 [MKP-1]) (Ro; 10 microM), an MKP-1 inhibitor, which induces apoptosis in chondrocytes. Apoptosis was evaluated by flow cytometry (propidium iodide) and nuclear morphology was evaluated with 4',6'-dianidino-2-phenylindole dihydrochloride. The expressions of caspase-8, -7 and -3 and Bcl-2 were analyzed by Western blot and the activation of caspase-3 and -8 was measured by flow cytometry. Prostaglandin E2 (PGE2) was evaluated by enzyme-linked immunosorbent assay. RESULTS: At 24 h the percentage of apoptotic (hypodiploid) nuclei induced by TNF-alpha+Ro was higher than the level induced by Ro alone. The combination of IL-1beta (5 ng/ml) with Ro did not show a synergistic effect. A morphological analysis demonstrated that treatment with TNF-alpha+Ro resulted in a large number of cells with condensed nuclei and DNA fragmentation. Western blot studies indicated that IL-1beta+Ro did not induce the time-dependent activation of caspase-8, -7 and -3 as seen with TNF-alpha+Ro. As quantified by flow cytometry, TNF-alpha+Ro induced a higher level of caspase-3 and -8 activation than that seen with IL-1beta+Ro. Pre-incubation for 2h with caspase inhibitors for caspase-3, -7, -8 and pan-caspase significantly decreased the hypodiploid DNA peak induced by treatment with TNF-alpha+Ro at 24 h. Indomethacin increased the cell death induced by IL-1beta+Ro; however, apoptosis induced by TNF-alpha+Ro was not modified by indomethacin. CONCLUSIONS: These results confirm that TNF-alpha and IL-1beta regulate apoptosis differently in this human chondrocyte model and that the differing effects of these cytokines are PGE2-independent. Indomethacin potentiates the effect of IL-1 on cell death and this may explain the reported effect of indomethacin on the progression of joint destruction.     

罗格列酮对大鼠肾缺血再灌注损伤的保护作用研究

目的研究罗格列酮对肾缺血再灌注损伤的保护作用及潜在机制。方法将60只大鼠随机分成假手术组、缺血再灌注损伤组、罗格列酮10 mg/kg组、罗格列酮20 mg/kg组、罗格列酮40 mg/kg组和罗格列酮80 mg/kg组,每组各10只。利用血管夹夹闭大鼠双侧肾蒂构建肾缺血再灌注损伤模型。ELISA检测大鼠血清肌酐(Cr)、尿素氮(BUN)、白介素-8(IL-8)、肿瘤坏死因子(TNF-α)和白介素-6(IL-6)表达量;Western blot检测过氧化物酶体增殖物激活受体γ(PPAR-γ)和p-PPAR-γ表达水平;RT-PCR检测肾脏IL-8、TNF-α和IL-6信使核酸序列(mRNA)水平;黄嘌呤氧化酶法检测过氧化氢酶(CAT)、谷胱甘肽过氧化物酶(GPX)和超氧化物歧化酶(SOD)活性;TBA法检测丙二醛(MDA)的含量;过碘酸雪夫氏(PAS)染色检测肾组织病理形态。结果与假手术组相比,缺血再灌注损伤组肾组织病理损伤和Cr、BUN、IL-8、TNF-α、IL-6、p-PPAR-γ以及MDA表达水平均明显增加,而CAT、GPX和SOD活性明显降低以及PPAR-γ表达差异无统计学意义;与缺血再灌注损伤组相比,罗格列酮预处理可明显减少肾组织病理损伤和Cr、BUN、IL-8、TNF-α、IL-6以及MDA表达水平,但可明显增加CAT、GPX和SOD活性以及p-PPAR-γ表达水平,但对PPAR-γ表达水平无影响。结论罗格列酮预处理对大鼠肾脏缺血再灌注损伤具有保护,其作用机制与激活PPAR-γ抑制氧化应激和炎症相关。     

An in-vitro screening assay for the detection of inhibitors of proinflammatory cytokine synthesis: a useful tool for the development of new antiarthritic and disease modifying drugs

OBJECTIVE: This work targets the development of a new tool to help develop new anticytokine drugs that prevent or reduce the progression of arthritic diseases. The specific aim of our study was to establish a fast and reliable in vitro screening assay of cytokine synthesis inhibitors (TNFalpha, IL-1beta) which shows better correlation with enzyme assays than previously reported in vitro assays. The test system should be able to detect p38-MAP kinase inhibitors. MATERIAL AND METHODS: Human peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll density gradient centrifugation from human EDTA-potassium whole blood. Cells were adjusted at 1 x 10(6) cells/ml. PBMCs were stimulated with lipopolysaccharide (LPS; E. coli serotype 026:B6: 1 microg/ml) in the presence of test compound (10(-5)-10(-8)M) for 4h at 37 degrees C in a 5% CO(2)-incubator. Induced TNFalpha and IL-1beta protein were measured by ELISA. RESULTS: The following are representative examples of inhibitors which effect cytokine synthesis. Corticoid Dexamethasone inhibits IL-1beta and TNFalpha synthesis at IC(50) of 38 nM and 25 nM, respectively. ERK1/ERK2 inhibitor U0126 effects cytokine synthesis at IC(50) of 0.34 microM for IL-1beta production and 0.26 microM for TNFalpha synthesis.p38-MAP kinase inhibitor SB 203580 inhibits IL-1beta- and TNF-alpha-synthesis (IC(50)sof 0.052 microM and 0.46 microM) in the same degree as p38-MAP kinase activity (IC(50): 0.34 microM). Same results could be shown for SB 210313, which had same efficacy on IL-1beta and TNFalpha biosynthesis (IC(50)'s: 1.88 microM and 1.01 microM) and on p38-MAP kinase (IC(50): 6.85 microM). Also for SB 202190 this correlation in inhibition of IL-1beta and TNFalpha synthesis (IC(50)'s: 0.055 microM and 1.01 microM) and p38-MAP kinase inhibition (IC(50): 0.088 microM) could be shown. CONCLUSION: This study shows the screening assay using PBMCs stimulated with LPS for IL-1beta and TNFalpha synthesis is a reliable test system for the quantification of the effectiveness of new drugs modulating IL-1beta and TNFalpha synthesis which is mainly mediated by p38-MAP Kinase. These assay allows fast detection of IL-1beta and TNFalpha synthesis inhibitors with different modes of action, including p38-MAP kinase inhibitors. The results obtained with our in-vitro screening assay show good correlation with results from enzyme assays.     

Distinct signaling pathways are involved in hypoxia- and IL-1-induced VEGF expression in human articular chondrocytes.

Recent data suggest that vascular endothelial growth factor (VEGF) is involved in the pathogenesis of osteoarthritis (OA). Cartilage is an avascular tissue, leading to a low cartilage O2 level. Thus, in a variety of pathologic or physiologic conditions, VEGF is partly regulated by hypoxic stress. The implications of hypoxia for VEGF expression by OA chondrocytes, however, are not known. We investigated the regulatory system of VEGF in OA chondrocytes under hypoxic conditions. Chondrocytes were obtained from articular cartilage of patients with OA. Cells were cultured and then incubated under hypoxic (95% N2, 5% CO2) or normoxic conditions, with or without interleukin (IL)-1 (10 ng/mL) stimulation. The mitogen activated protein kinase (MAPK) inhibitors were also used. VEGF levels in the culture supernatants were measured using an enzyme-linked immunosorbent assay. Western blot analysis was used to examine the expression of hypoxia inducible factor (HIF)-1alpha. Hypoxia significantly increased VEGF levels (p<0.05). Hypoxia-induced VEGF secretion was abolished by p38MAPK inhibitor, but not by JNK inhibitor. In contrast, IL-1-induced VEGF secretion was blocked by JNK inhibitor, and not by p38MAPK inhibitor. Both hypoxia and IL-1-induced HIF-1alpha were attenuated by p38 MAPK and JNK inhibitors. We demonstrate that hypoxia and IL-1 induce VEGF production in chondrocytes through distinct MAPK signaling pathways, indicating that VEGF is induced in a HIF-1-dependent or -independent manner in chondrocytes.     

Superoxide dismutase,catalase, glutathione peroxidase and NADPH oxidase in lead-induced hypertension

BACKGROUND: Earlier studies from this laboratory have revealed the presence of oxidative stress and its role in the pathogenesis of lead-induced hypertension (HTN). We have further shown evidence of increased hydroxyl radical (.OH) and superoxide production in lead-treated rats and cultured endothelial cells. This study was designed to determine whether oxidative stress in animals with lead-induced HTN is associated with dysregulation of the main antioxidant enzymes namely superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPX) or increased superoxide producing enzyme nicotinamide adenine dinucleotide (phosphate) oxidase [NAD(P)H]. METHODS: Male Sprague-Dawley rats were randomly assigned to lead-exposed and control groups. Animals in the lead-exposed group were provided with drinking water containing 100 ppm lead acetate for 12 weeks. The control group was provided with regular drinking water. At the conclusion of the experiment, immunodetectable Cu Zn SOD, Mn SOD, CAT, GPX and gp91 phox subunit of NAD(P)H oxidase were determined by Western analysis in the kidney, brain and left ventricle of control and lead-exposed rats. Subgroups of the study animals were treated with IV infusion (180 micromol/kg/h) of the superoxide trapping agent, tempol, and arterial pressure and urinary nitric oxide (NO) metabolite (NOx) excretion were determined. RESULTS: Lead exposure for 12 weeks resulted in a marked rise in systolic blood pressure, a significant reduction in urinary NOx excretion, a significant increase in kidney and brain Cu, Zn SOD, a significant increase in brain and insignificant increase in kidney and heart gp91 phox. In contrast, Mn SOD, CAT and GPX in the kidney, brain and left ventricle were unchanged. Incubation with lead acetate did not alter SOD activity in vitro. Infusion of tempol significantly lowered arterial pressure and raised urinary NOx excretion in the lead-exposed group (but had no effect in the control group) pointing to increased superoxide production in the lead-exposed animals. CONCLUSION: Animals with lead-induced hypertension exhibited oxidative stress which was associated with mild up-regulation of superoxide-generating enzyme, NAD(P)H oxidase, with no evidence of quantitative SOD, CAT or GPX deficiencies.     

p38丝裂原活化蛋白激酶在应激性溃疡发病中的作用

目的探讨p38丝裂原活化蛋白激酶(MAPK)在应激性溃疡发生中的作用。方法80只SD大鼠分成正常对照组(10只)、浸水-束缚(WIR)应激模型组(0.5、1、2、4和6h时间点各10只)和p38特异性抑制剂CNI1493处理组(低剂量和高剂量组各10只)。CNI1493处理组于应激前2h尾静脉注射1或10mg/kg的CNI1493。胃黏膜病变程度采用溃疡指数计分评价,磷酸化和总p38MAPK表达采用蛋白印迹法(Westernblot)检测,肿瘤坏死因子α(TNFα)和白细胞介素1β(IL1β)基因表达采用Northernblot方法检测。结果WIR应激后大鼠胃黏膜p38MAPK发生持续活化,其最大活化发生于应激后1h,为正常水平的(6.8±3.2)倍(P<0.01)。使用CNI1493阻断p38MAPK活化后,胃黏膜损伤程度显著减轻,同时促炎性细胞因子TNFα和IL1β基因表达也明显下调。结论p38MAPK活化在实验性应激性溃疡的发生中发挥了重要作用。     

Osteoblasts from the sclerotic subchondral bone downregulate aggrecan but upregulate metalloproteinases expression by chondrocytes. This effect is mimicked by interleukin-6, -1beta and oncostatin M pre-treated non-sclerotic osteoblasts

OBJECTIVE: To determine the effects of osteoarthritic (OA) subchondral osteoblasts on the metabolism of human OA chondrocytes in alginate beads. METHODS: Human chondrocytes were isolated from OA cartilage and cultured in alginate beads for 4 days in the absence or in the presence of osteoblasts isolated from non-sclerotic (N) or sclerotic (SC) zones of human OA subchondral bone in monolayer (co-culture system). Before co-culture, osteoblasts were incubated for 72 h with or without 1.7ng/ml interleukin (IL)-1beta, 100 ng/ml IL-6 with its soluble receptor (50 ng/ml) or 10 ng/ml oncostatin M (OSM). Aggrecan (AGG) and matrix metalloproteases (MMP)-3 and -13 mRNA levels in chondrocytes were quantified by real-time polymerase chain reaction. AGG production was assayed by a specific enzyme amplified sensitivity immunoassay. RESULTS: SC, but not N, osteoblasts induced a significant inhibition of AGG production and AGG gene expression by human OA chondrocytes in alginate beads, and significantly increased MMP-3 and MMP-13 gene expression by chondrocytes. When they were pre-incubated with IL-1beta, IL-6 or OSM, N osteoblasts inhibited AGG synthesis and increased MMP-3 and -13 gene expression by chondrocytes in alginate beads in a same order of magnitude as SC osteoblasts. CONCLUSIONS: These results demonstrate that SC OA subchondral osteoblasts could contribute to cartilage degradation by stimulating chondrocytes to produce more MMP and also by inhibiting AGG synthesis.     

Reactive oxygen species and superoxide dismutases: role in joint diseases

Reactive oxygen species (ROS) are produced in many normal and abnormal processes in humans, including atheroma, asthma, joint diseases, aging, and cancer. The superoxide anion O(2)(-) is the main ROS. Increased ROS production leads to tissue damage associated with inflammation. Superoxide dismutases (SODs) convert superoxide to hydrogen peroxide, which is then removed by glutathione peroxidase or catalase. Thus, SODs prevent the formation of highly aggressive ROS, such as peroxynitrite or the hydroxyl radical. Experimental models involving SOD knockout or overexpression are beginning to shed light on the pathophysiological role of SOD in humans. Although the antiinflammatory effects of exogenous native SOD (orgotein) are modest, synthetic SOD mimetics hold considerable promise for modulating the inflammatory response. In this review, we discuss new knowledge about the role of the superoxide anion and its derivates as mediators of inflammation and the role of SODs and SOD mimetics as antioxidant treatments in joint diseases such as rheumatoid arthritis, osteoarthritis, and crystal-induced arthropathies.     

Modulation of interleukin-6 by beta2-adrenoceptor in endotoxin-stimulated renal macrophage cells.

BACKGROUND: Activation of the cAMP signaling pathway by means of beta2-adrenoceptor agonists has been shown to up-regulate interleukin-6 (IL-6) gene expression and to stimulate IL-6 production in macrophage cells. However, whether beta2-adrenoceptor activation can also modify the rate of IL-6 production in macrophage cells activated by the bacterial endotoxins has not yet been determined. Using renal resident macrophage cells treated with endotoxin, lipopolysaccharide (LPS), and beta2-adrenoceptor agonist, terbutaline, we investigated the role of cAMP pathway, tumor necrosis factor (TNF)-alpha and mitogen-activated protein kinase (MAPK) pathway (p42/p44) in regulating IL-6 production. METHODS: IL-6 protein, mRNA, and promoter activity were measured in these cells exposed to LPS (1 microg/ml) and/or terbutaline (10(-9) to 10(-6) M). Furthermore, the time course effects of terbutaline on cAMP, MAPK (p42/p44), and TNF-alpha release were evaluated in the cells. RESULTS: Terbutaline at high concentrations (10(-6) M) significantly up-regulated IL-6 by approximately 25% (P<0.05), whereas at a lower concentration (10(-8) M), it down-regulated IL-6 production by 42% (P<0.05). Terbutaline (10(-8) and 10(-6) M) caused a concentration- and time-dependent stimulation of cAMP (P<0.05) and TNF production (P<0.05) and a time-dependent decrease in MAPK activity (P<0.05). Following the addition of a cAMP inhibitor, IL-6 promoter activity was correlated with TNF-alpha levels and MAPK activity. CONCLUSIONS: A biphasic effect of beta2-adrenoceptor agonist on IL-6 production in renal resident macrophage cells became apparent when LPS was exposed to the cells. The terbutaline-induced down-regulation of IL-6 gene production was mediated by an inhibitory effect of terbutaline on TNF-alpha, which was exerted through the MAPK and cAMP pathways, whereas the up-regulation appeared to be due to a direct action of intracellular cAMP.     

Differential regulation of chondrocyte metabolism by oncostatin M and interleukin-6

OBJECTIVE: To determine the effects of interleukin (IL)-6 and oncostatin M (OSM) added separately or in combination with IL-1beta on human osteoarthritic (OA) chondrocytes in alginate beads. DESIGN: Human chondrocytes were isolated from OA cartilage and cultured in alginate beads for 12 days, in the absence or in the presence of increasing amounts of IL-6 (20-500ng/ml) with its soluble receptor or OSM (0.1-10ng/ml) and with or without IL-1beta (1.7ng/ml). Aggrecan (AGG), transforming growth factor-beta1 (TGF-beta1), stromelysin-1 [matrix metalloprotease (MMP)-3], tissue inhibitor of metalloproteinases-1 (TIMP-1), macrophage inflammatory protein-1 beta (MIP-1beta), IL-6 and IL-8 productions were assayed by specific enzyme amplified sensitivity immunoassays. Prostaglandin (PG)E(2) was measured by a specific radioimmunoassay and nitrite (NO(2)(-)) by a spectrophotometric method based upon the Griess reaction. RESULTS: OSM, but not IL-6, decreased basal AGG and TGF-beta1 synthesis. Although IL-6 stimulated basal TIMP-1 production, it did not significantly modify MMP-3/TIMP-1 ratio. In contrast, 10ng/ml OSM highly increased TIMP-1 production, and decreased by half the ratio MMP-3/TIMP-1. IL-1beta highly stimulated *NO, IL-8, IL-6, MIP-1beta and PGE(2) synthesis but decreased AGG and TGF-beta1 production. Neither IL-6 nor OSM modulated IL-1beta-inhibitory effect on AGG production. IL-6, but not OSM, reversed IL-1beta-induced TGF-beta1 inhibition. At 1-10ng/ml, OSM significantly decreased IL-1beta-stimulated IL-8, MIP-1beta, PGE(2) and *NO production but amplified IL-1beta stimulating effect on IL-6 production. IL-6 had no effect on these parameters. CONCLUSIONS: OSM and IL-6, two glycoprotein 130 binding cytokines, show different activity profiles on OA chondrocytes, indicating that these cytokines could play different roles in the OA disease process.     

Time course of antioxidant enzyme activities in liver transplant recipients

Reactive oxygen species (ROS) play a central role in ischemia-reperfusion injury after organ transplantation. They are degraded by endogenous radical scavengers such as antioxidant enzymes. The purpose of this study was to evaluate the temporal variations of antioxidant enzyme activities in liver transplant recipients. The study was performed in 13 liver transplant patients (11 men and 2 women). Blood samples were obtained pre- and postsurgical intervention: before transplant (T(0)), and 1, 6, 12, 24, 48, and 72 hours, as well as 5 and 7 days thereafter. We determined total and specific superoxide dismutase (SOD) activity, catalase (CAT), glutathione peroxidase (GPX), and glutathione reductase (GR) activities as well as malondialdehyde (MDA) and low-density lipoproteins (LDL). The results showed increased SOD and mainly GPX activities after liver transplantation, which correlated with MDA levels. Total SOD activity was mainly represented by Mn-SOD (75%) and Cu,Zn-SOD (25%), whereas Fe-SOD was not detected. In conclusion, the enhanced antioxidant enzyme activities reported in this study indicated a control of oxidative stress generated in liver transplantation. In this sense, although MDA levels showed an enormeous increase at 1 hour after transplantation, the lipid peroxidation was compensated for by GPX activity.     

Effects of dexamethasone on proteoglycan content and gene expression of IL-1beta-stimulated osteoarthrotic chondrocytes in vitro

We studied the effects of dexamethasone on proteoglycan (PG) concentration and gene expression in human osteoarthrotic chondrocytes stimulated with IL-1beta. Cartilage samples were taken from 7 patients with osteoarthrosis of the knee and chondrocytes cultivated in alginate beads. Dexamethasone was added in three concentrations (10(-5), 10(-6), 10(-7) M) to IL-1beta (100 pg/nL)-stimulated chondrocytes. PG concentration was estimated by a dimethylmethylene blue assay. To assess cell proliferation, DNA content was measured fluorometrically. Quantitative Lightcycler-PCR was used to estimate the mRNA levels of stromelysin-1 (MMP-3) and aggrecan (AGG). The proliferation rate was unchanged in all treatment groups. IL-1beta increased MMP-3 expression by 44% and inhibited AGG expression by 16%, but PG-concentration was reduced by 7%. The addition of dexamethasone to IL-1beta-stimulated chondrocytes further reduced the PG concentration by 19% at 10(-5) M and by 17% at 10(-7) M. The MMP-3 expression was inhibited between 27-53% and the AGG expression between 30-46% by dexamethasone. In osteoarthrotic chondrocytes, dexamethasone in an appropriate dose range reduced the expression of MMP-3 and AGG at the same time. The resulting decrease in PG concentration should be considered when using intraarticular corticosteroids to treat an osteoarthrotic joint.     

IL-17, IL-1beta and TNF-alpha stimulate VEGF production by dedifferentiated chondrocytes

OBJECTIVE: To verify the involvement of proinflammatory cytokines IL-17, IL-1beta and tumor necrosis factor alpha (TNF-alpha) in cartilage vascularization by stimulating the production of vascular endothelial growth factor (VEGF) by chondrocytes isolated from patients with osteoarthritis (OA), in comparison with patients with rheumatoid arthritis (RA) and patients with femoral or humeral neck fracture (FP). DESIGN: Chondrocytes isolated from patients with OA were maintained in monolayer culture for several passages. Chondrocyte dedifferentiation was monitored by the synthesis of cathepsin B by these cells. Chondrocytes freshly isolated at each subculture (subcultures 1-3) were stimulated with IL-17, IL-1beta or TNF-alpha. Supernatants were collected, immunoassayed for the production of VEGF and cathepsin B and assayed as the source of VEGF on the VEGF sensible ECV304 cell line. The cells were used to quantify intracellular cathepsin B enzymatic activity. RESULTS: In differentiated conditions IL-1beta and TNF-alpha, but not IL-17, can inhibit the spontaneous secretion of VEGF by human OA, RA and FP chondrocytes, and IL-17 can restore the decrease in VEGF secretion caused by TNF-alpha. IL-17, together with IL-1beta and TNF-alpha, can enhance VEGF secretion to various extents by dedifferentiated OA chondrocytes. This change in effect with respect to primary culture was observable for all cytokines at the beginning of dedifferentiation, when the production of VEGF by chondrocytes had dramatically fallen and the cathepsin B synthesis had increased. The amount of VEGF induced by cytokines on dedifferentiated chondrocytes never reached the amount of VEGF produced by differentiated chondrocytes. VEGF produced by chondrocytes stimulated the ECV304 cell line proliferation. CONCLUSIONS: These results indicate that dedifferentiated OA chondrocytes secrete VEGF after stimulation with proinflammatory cytokines. This event may be responsible for neovascularization found in OA cartilage.     

p38丝裂原活化蛋白激酶信号转导通路在严重烧伤大鼠枯否细胞肿瘤坏死因子α和白细胞介素1β产生中的作用

目的 探讨p38丝裂原活化蛋白激酶(MAPK)信号转导通路在严重烧伤大鼠枯否细胞(KCs)促炎性细胞因子肿瘤坏死因子α(TNF-α)和白细胞介素1β(IL-1β)产生中的作用。方法 健康成年的雄性SD大鼠32只,随机分为：假烫组;假烫 SB203580组;烧伤对照组;烧伤 SB203580组,每组8只。假烫或烧伤24h后分离出肝脏KCs,培养18h后加入50ng／ml的LPS进行刺激,18h后取上清液,用酶联免疫吸附法(ELISA)测定TNF-α和IL-1β的含量,并收集KCs,实时逆转录聚合酶链反应检测KCs内TNF-α和IL-1β mRNA表达的改变,蛋白印迹(Western blot)法检测KCs中p38MAPK和JNK活性的变化。结果 烧伤大鼠分离出的KCs培养上清液中TNF-α和IL-1β含量、KCs中TNF-α和IL-1β mRNA的表达均较假烫组的明显增强,同时KCs中p38 MAPK活性和JNK活性升高,SB203580能显著抑制大鼠KCs上清液中TNF-α和IL-1β含量、KCs中TNF-α和IL-1β mRNA的表达和p38MAPK活性的升高,对JNK活性无影响。结论p38MAPK信号转导通路介导了严重烧伤大鼠KCs促炎性细胞因子TNF-α和IL-1β的产生。     

微小RNA-27b-3p与基质金属蛋白酶13在人软骨细胞的对应关系

目的探讨微小RNA-27b-3p(miR-27b-3p)与基质金属蛋白酶-13(MMP-13)在人软骨细胞表达及其靶向对应关系。 方法运用蛋白质印迹法(WB)与实时定量PCR技术(qRT-PCR)明确miR-27b-3p与MMP13在正常和骨关节炎(OA)人软骨细胞的表达。利用不同浓度的白介素(IL)1β干预原代人软骨细胞24 h,或利用不同时间点的IL-1β(10 ng/ml)干预原代人软骨细胞。利用原位杂交、转染及双荧光素酶报告技术确定miR-27b-3p与MMP13的靶向对应关系;结合运用核转录因子-κB(NF-κB)和丝裂原活化蛋白激酶(MAPK)信号通路抑制剂评估其作用机制。两组资料比较采用独立样本t检验,多组资料比较采用单因素方差分析,LSD法多重比较检验。 结果WB、qRT-PCR和原位杂交检测结果显示,与正常软骨相比,OA软骨中miR-27b-3p表达降低(t＝5.07,P<0.01),MMP13表达升高(t＝－6.31,P<0.01)。IL-1β干扰后的结果显示miR-27b-3p表达降低(F＝129.54,P<0.05),MMP-13表达升高(F＝394.50,P<0.05)。通过TargetScan数据库和荧光素酶报告基因检测结果分析,野生型-MMP13组荧光素酶活性降低(F＝55.27,P<0.001),突变型-MMP-13荧光素酶活性变化没有统计学意义(P＝0.654)。利用特异性MAPK信号抑制剂和NF-kB抑制剂干预IL-1β诱导软骨细胞模型结果提示,与对照组相比,抑制剂组的MMP13表达水平降低(F＝28.43,P<0.001),miR-27b-3p表达水平增高(F＝35.04,P<0.001)。 结论miR-27b-3p在OA软骨细胞呈现低表达,并负向调控MMP13的表达,其作用机制可能是通过NF-κB和MAPK信号通路,这结果提示这miR-27b-3p可能作为OA诊断与治疗的潜在靶点。     

Role of MAP kinase pathways in mediating IL-6 production in human primary mesangial and proximal tubular cells.

BACKGROUND: Both interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha) are pleiotropic cytokines that have been implicated in the development of glomerular and tubular injury in various forms of immune-mediated renal disease, including glomerulonephritis. Although TNF-alpha has been shown to stimulate IL-6 production in renal cells in culture, the signaling mechanisms that regulate IL-6 production are not fully understood. The aim of this study was to examine the role of the p38 and extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK) pathways in regulating TNF-alpha-mediated IL-6 production from both primary human mesangial cells (HMCs) and human proximal tubular (HPT) cells. METHODS: Primary mesangial and proximal tubular cells were prepared from nephrectomized human kidney tissue. Cells were treated for 24 hours with TNF-alpha in the presence and absence of the specific p38 and ERK1,2 MAPK inhibitors SB203580 and PD98059, respectively, either alone or in combination. IL-6 levels in the cell culture media were measured by enzyme-linked immunosorbent assay. MAPK activation was demonstrated by immunoblot for the active kinase (tyrosine/threonine phosphorylated) in whole cell extracts using phospho-specific antibodies. p38 MAPK activity in HPT cells was measured using an in vitro immunokinase assay using ATF2 as the substrate. RESULTS: TNF-alpha (0.1 to 100 ng/ml) stimulated a dose-dependent increase in IL-6 production in both renal cell types. The activation of the p38 and the ERK1,2 MAPKs occurred following TNF-alpha stimulation. The role of these activations in IL-6 production was confirmed by the ability of both inhibitors SB203580 (1 to 30 microM) and PD98059 (0.01 to 10 microM) to inhibit basal and TNF-alpha-stimulated IL-6 production in both cell types. The addition of both inhibitors in combination caused greater decreases in IL-6 production compared with either inhibitor alone. Pretreatment with SB203580 (10 microM) had no effect on basal or TNF-alpha-stimulated phosphorylation of p38 MAPK but completely abolished TNF-alpha-stimulated p38 MAPK activity. PD98059 decreased both basal and TNF-alpha-stimulated phosphorylation of ERK1,2. CONCLUSIONS: This study provides evidence that both the p38 and ERK MAPK pathways are important for the regulation of the production of IL-6 from the proximal tubular and glomerular mesangial regions of the nephron. In response to TNF-alpha, the activation of both pathways leads to IL-6 production. These findings could aid in an understanding of the cellular mechanisms that regulate IL-6 production and could provide insights into possible pharmacological strategies in inflammatory renal disease.     

RelA is required for IL-1beta stimulation of Matrix Metalloproteinase-1 expression in chondrocytes

OBJECTIVE: Interleukin-1beta (IL-1beta) stimulates collagenase-1 (Matrix Metalloproteinase-1 (MMP-1)) expression in articular chondrocytes, leading to cleavage of type II collagen and irreversible cartilage degradation. The nuclear factor-kappa B (NF-kappaB) pathway is potently activated in IL-1beta-stimulated cells and has been implicated as an intermediate in MMP-1 gene expression. However, the roles of individual NF-kappaB family members during IL-1beta-induced MMP-1 gene expression have not been defined. RESULTS: To address the relationship between the NF-kappaB pathway and MMP-1 gene activation in chondrocytes, primary cultured human articular chondrocyte cultures (HAC) and SW-1353 cells were stimulated with IL-1beta over a 24-h time course and MMP-1, NF-kappaB1, NF-kappaB2 and RelA gene expression was assayed. IL-1beta-induced MMP-1 expression was comparable in HAC and SW-1353 cells both temporally and quantitatively. MMP-1 gene expression was mirrored by increases in NF-kappaB gene expression, and inhibition of NF-kappaB nuclear translocation with dominant-negative IkappaBalpha reduced IL-1beta-dependent MMP-1 gene expression. IL-1beta activated the NF-kappaB pathway in chondrocytes, both through phosphorylation and transient degradation of IkappaBalpha, as well as through sustained phosphorylation of RelA. Small inhibitory RNAs (siRNA) specific for RelA resulted in significant reduction of MMP-1 mRNA, whereas siRNA for NF-kappaB1 and NF-kappaB2 augmented IL-1beta-induced MMP-1 expression. CONCLUSIONS: Our data demonstrate that IL-1beta activation of the NF-kappaB pathway is required for IL-1beta induction of MMP-1 in chondrocytes and that RelA can work independently of NF-kappaB1 or NF-kappaB2 to activate this gene expression program.