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.     

Advanced glycosylation end products induce inducible nitric oxide synthase (iNOS) expression via a p38 MAPK-dependent pathway

BACKGROUND: Advanced glycosylation end products (AGEs) accumulation in tissue has been implicated in diabetic related complications, including diabetic nephropathy. Activation of peroxisome proliferator activated receptor-gamma (PPAR-gamma) ameliorates diabetic nephropathy. METHODS: In the present study, we investigated the effects of AGEs on inducible nitric oxide synthase (iNOS) expression and nitric oxide production, and the effects of rosiglitazone, an activator of PPAR-gamma, on AGE-induced iNOS expression and nitrite release in glomerular mesangial cells. RESULTS: AGEs caused a dose- and time-dependent increase of iNOS induction and nitrite accumulation in mesangial cells. A protein tyrosine kinase inhibitor (genistein), or a p38 mitogen-activated protein kinase (MAPK) inhibitor (SB203580) suppressed AGE-induced iNOS expression and nitrite release from mesangial cells. Addition of bovine serum albumin (BSA)-AGEs to mesangial cells increased p38 MAPK activities. Activation of PPAR-gamma by rosiglitazone inhibited AGE-induced iNOS expression, nitrite release, and p38 MAPK activation in mesangial cells. AGE-stimulated nitrite release was attenuated by pretreatment with anti-tumor necrosis factor-alpha (TNF-alpha) and anti-transforming growth factor-beta (TGF-beta) antibodies. AGE-induced iNOS expression was inhibited by treatment with a nuclear factor-kappaB (NF-kappaB) inhibitor, pyrrolidone dithiocarbamate. Addition of BSA-AGEs to mesangial cells stimulated p65 NF-kappaB translocation from the cytosol to the nucleus. CONCLUSION: These data suggest that cytokine release, NF-kappaB and p38 MAPK-dependent pathways may play a role in AGE-induced iNOS expression and subsequent nitric oxide production in mesangial cells. Rosiglitazone may prevent AGE-induced iNOS expression by interfering with p38 MAPK activity.     

p38 mitogen-activated protein kinase inhibition attenuates burn-induced liver injury in rats

This study was made to evaluate the effect of SB203580, a specific p38 MAP kinase inhibitor, on burn-induced hepatic injury as well as the activation of nuclear factor (NF)-kappaB in severely burned rats. Sprague-Dawley rats were divided into three groups: (1) sham group, rats underwent sham burn; (2) burn group, rats given third-degree burns over 30% total body surface area (TBSA) and treated with vehicle plus lactated Ringer solution for resuscitation 4 ml/(kg% TBSA); and (3) burn plus SB203580 group, rats given burn injury and fluid resuscitation plus SB203580 (10 mg/kg i.v., 15 min and 12 h after burn). Hepatocellular injury (measured by serum levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT)) and hepatocellular function (determined by the indocyanine green dye retention rate (ICG R15)) were assessed at 24 h post-burn. Liver histologic changes were also analyzed. Burn trauma resulted in increased serum aminotransferases concentrations, decreased ICG R15, elevated serum tumor necrosis factor (TNF)-alpha and interleukin (IL)-1beta levels and hepatic TNF-alpha and IL-1beta mRNA expressions, and worsen histologic condition. The level of Nuclear Factor (kappa) inhibitor (IkappaBalpha) in liver was decreased and DNA-binding activity of Nuclear Factor-kappaB (NF-kappaB) was increased after thermal injury. p38 MAP kinase was more significantly activated in liver harvested from burn rats than from shams. SB203580 inhibited the activation of p38 MAP kinase, reduced the levels of TNF-alpha and IL-1beta, and prevented burn-mediated liver injury. Both the IkappaBalpha level and NF-kappaB activity in the liver following burns was not affected by administration with SB203580. These findings suggest that (1) p38 MAP kinase activation is one important aspect of the signaling event that may mediate the release of TNF-alpha and IL-1beta and contributes to burn-induced liver injury and (2) p38 MAP kinase does not influence the activation of NF-kappaB directly in the liver of severely burned rats.     

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。     

严重烧伤大鼠肝脏p38丝裂原活化蛋白激酶对肿瘤坏死因子α表达的调控及其在肝损伤中的作用

目的观察大鼠严重烧伤后肝脏p38丝裂原活化蛋白激酶(MAPK)对肿瘤坏死因子(TNF)α表达的调控及其在肝损伤中的作用。方法将健康成年雄性SD大鼠随机分为假伤组;烧伤 SB203580组:30%TBSAⅢ度烫伤(以下称烧伤)后15min和12h静脉注射p38MAPK的特异性抑制剂SB203580(10mg/kg);烧伤对照组:同前致伤后给予等量等渗盐水,每组8只。测定3组大鼠伤后24h血清天冬氨酸转氨酶(AST)和丙氨酸转氨酶(ALT)活性的变化,并分别采用实时逆转录聚合酶链反应(RT-PCR)法和蛋白印迹(Western blot)法检测肝脏TNF-αmRNA及p38MAPK、磷酸化p38MAPK的表达水平。结果烧伤对照组大鼠血清AST和ALT活性及肝脏TNF-αmRNA的表达水平均显著高于假伤组(P<0.05或0.01);烧伤 SB203580组此3项指标均显著低于烧伤对照组(P<0.05或0.01),但与假伤组比较差异无统计学意义(P>0.05).3组大鼠肝脏p38MAPK表达水平比较,差异无统计学意义(P>0.05);其磷酸化p38MAPK表达水平之比———假伤组∶烧伤对照组∶烧伤 SB203580组为1.00∶3.90∶1.10,烧伤 SB203580组与假伤组比较,差异无统计学意义(P>0.05),与烧伤对照组比较明显偏低(P<0.01).结论大鼠严重烧伤后,肝脏中活化的p38MAPK促进了TNF-αmRNA的表达,并参与了肝损伤的发生。     

p38丝裂原活化蛋白激酶信号转导通路和核因子-κB/抑制因子κB通路在烧伤后促炎性细胞因子产生中的相互作用

目的探讨烧伤后p38丝裂原活化蛋白激酶(MAPK)信号转导通路和核因子(NF)-κB/抑制因子(I)κB通路在调控细胞因子产生方面的地位及是否存在相互作用。方法人单核细胞株THP-1分为正常血清对照组、烧伤血清组、烧伤血清+SB203580组(p38 MAPK特异性抑制剂)和烧伤血清+PDTC(NF-κB特异性抑制剂)组,每组均实验8次。血清刺激24 h后,酶联免疫吸附法测定上清液中肿瘤坏死因子α(TNF-α)和白细胞介素1β(IL-1β)的含量,蛋白印迹(W estern b lot)法检测THP-1内p38 MAPK的活性和IκBα的表达,凝胶电泳迁移率变化分析法检测THP-1内NF-κB和激活蛋白(AP-1)活性的变化。结果和正常血清相比,烧伤血清刺激后24 h,THP-1上清液中TNF-α和IL-1β的含量均明显上升[分别为(7.30±0.84)ng/m l和(2.20±0.28)ng/m l,P<0.05;(2.88±0.38)ng/m l和(0.81±0.14)ng/m l,P<0.05],p38 MAPK活性升高(4728±582和1291±163,P<0.05),IκBα含量下降(1211±115和2658±318,P<0.05),THP-1中NF-κB活性和AP-1活性均较正常血清显著升高(1636±170和317±32,P<0.05;946±137和361±40,P<0.05),使用SB203580和PDTC均能抑制烧伤血清刺激后THP-1上清液中TNF-α和IL-1β的含量的上升。预先给予SB203580能抑制烧伤血清刺激后THP-1中p38 MAPK和AP-1活性升高,但对IκBα含量和NF-κB活性无显著影响;预先给予PDTC可以防止烧伤血清刺激后THP-1中IκBα含量的下降和NF-κB活性的升高,而对p38 MAPK和AP-1活性无影响。结论在烧伤后全身炎症反应的发生中,p38 MAPK信号转导通路和NF-κB/IκB通路是两个平行和独立的信号转导通路,彼此之间没有直接的联系,共同调节着烧伤后单核细胞TNF-α和IL-1β的产生。     

P38 mitogen-activated protein kinase inhibition attenuates pulmonary inflammatory response in a rat cardiopulmonary bypass model.

OBJECTIVE: Cardiopulmonary bypass (CPB) produces an inflammatory response associated with pulmonary dysfunction. P38 mitogen-activated protein kinase (P38MAPK) have been shown to mediate pulmonary inflammatory response after CPB, we examined the effect of SB203580, a specific p38 MAPK inhibitor, on CPB-induced pulmonary inflammatory response. METHODS: Sprague-Dawley rats (n=54) were randomized into three groups (each n=18): (1) S group, rats underwent sham CPB; (2) CPB group, rats underwent CPB; (3) SB group, rats underwent CPB plus pretreatment with SB203580 (10 mg/kg, i.v., 30 min before CPB). The lung samples were collected after 10 min, 60 min, and 150 min lung reperfusion (each n=6) in CPB group and SB group, and after 70 min, 120 min, and 210 min observation in S group as the control. RESULTS: The level of lung phospho-IkappaBalpha, nuclear factor (NF)-kappaB activity and activating protein (AP)-1 activity in CPB group was increased than S group. CPB resulted in increased pulmonary tissue tumor necrosis factor (TNF)-alpha and interleukin (IL)-1beta expression and production, increased pulmonary inflammatory response. The in vivo administration of SB203580 prevented up-regulation of lung-phosphorylated p38 MAP kinase, decreased pulmonary tissue level of proinflammatory cytokines expression and production, and reduced lung inflammation. CONCLUSIONS: These findings suggested that (1) p38 MAP kinase activation is one of the important aspects of the signaling event that mediate the release of TNF-alpha and IL-1beta and contributes to CPB-induced pulmonary inflammatory response, (2) SB203580 selectively inhibiting p38 MAP kinase activation efficaciously reduces pulmonary inflammatory response after CPB, and (3) p38 MAP kinase influence the activation of NF-kappaB in the lung during and after CPB.     

Interleukin-17--induced interleukin-8 release in human airway smooth muscle cells: role for mitogen-activated kinases and nuclear factor-kappaB.

BACKGROUND: It has recently become clear that interleukin (IL)-8 plays a role in chronic neutrophilic inflammatory disorders, such as chronic rejection after lung transplantation. We have shown that IL-17--stimulated human airway smooth muscle cells (HASMC) are able to produce IL-8. The aim of this study was to determine whether p38 mitogen-activated protein kinase (MAPK), c-Jun amino-terminal kinase (JNK), p42/p44 extracellular signal-related kinase (ERK) and nuclear factor-kappaB (NF-kappaB) are involved in IL-17--induced IL-8 production in HASMC in vitro. METHODS: We used human airway smooth muscle cells in culture. Western blotting was done to obtain data regarding activation of MAPK. Furthermore, we used specific inhibitors of MAPK to investigate their involvement in IL-17--induced IL-8 release, which was measured by enzyme-linked immunosorbent assay (ELISA). RESULTS: Western blotting clearly demonstrated that p38 MAPK, JNK and p42/p44 ERK were activated by IL-17 in HASMC. Using SB203580, a specific inhibitor of p38 MAPK, we detected a concentration-dependent inhibition of IL-17--induced IL-8 production with a maximal decrease of 63 +/- 5% (n=8, p<0.01). Curcumin, a specific inhibitor of JNK, also concentration-dependently reduced IL-17--induced IL-8 production, with a maximal decrease of 82+/-4% (n=8, p<0.01). U0126, a specific inhibitor of p42/p44 ERK, induced a maximal decrease of 84+/-5% (n=8, p<0.001). Pyrrolydine dithiocarbamate (PDTC), an inhibitor of NF-kappaB, caused a 70+/-5% (n=8, p<0.01) decrease in IL-17--induced IL-8 production. CONCLUSIONS: We found that IL-17 induces activation of p38MAPK, JNK and p42/p44ERK in HASMC. We also found that p38MAPK, JNK, p42/p44 ERK and NF-kappaB play an important role in IL-17--induced IL-8 production in HASMC in vitro. This may open up new opportunities for further treatment of this disease.     

Inhibition of p38 Pathway Suppresses Human Islet Production of Pro-Inflammatory Cytokines and Improves Islet Graft Function

Nonspecific inflammation is associated with primary graft nonfunction (PNF). Inflammatory islet damage is mediated at least partially by pro-inflammatory cytokines, such as interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) produced by resident islet macrophages. The p38 pathway is known to be involved in cytokine production in the cells of the monocyte-macrophage lineage. Therefore, inhibition of the p38 pathway may prevent pro-inflammatory cytokine production by resident islet macrophages and possibly reduce the incidence of PNF. Our present study has demonstrated that inhibition of the p38 pathway by a chemical p38 inhibitor, SB203580, suppresses IL-1beta and TNF-alpha production in human islets exposed to lipopolysaccharide (LPS) and/or inflammatory cytokines. Although IL-1beta is predominantly produced by resident macrophages, ductal cells and islet vascular endothelial cells were found to be another cellular source of IL-1beta in isolated human islets. SB203580 also inhibited the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in the treated islets. Furthermore, human islets treated with SB203580 for 1 h prior to transplantation showed significantly improved graft function. These results suggest that inhibition of the p38 pathway may become a new therapeutic strategy to improve graft survival in clinical islet transplantation.     

Requirement for p38 and p44/p42 mitogen-activated protein kinases in RAGE-mediated nuclear factor-kappaB transcriptional activation and cytokine secretion

Advanced glycation end product (AGE) activation of the signal-transducing receptor for AGE (RAGE) has been linked to a proinflammatory phenotypic change within cells. However, the precise intracellular signaling pathways involved have not been elucidated. We demonstrate here that human serum albumin modified with N(epsilon)-(carboxymethyl)lysine (CML), a major AGE adduct that progressively accumulates with aging, diabetes, and renal failure, induced nuclear factor (NF)-kappaB-driven reporter gene expression in human monocytic THP-1 cells. The NF-kappaB response was blocked with a synthetic peptide corresponding to the putative ligand-binding domain of RAGE, with anti-RAGE antiserum, and by coexpression of truncated receptors lacking the intracellular domain. Signal transduction from RAGE to NF-kappaB involved the generation of reactive oxygen species, since reporter gene expression was blocked with the antioxidant N-acetyl-L-cysteine. CML-modified albumin produced rapid transient activation of tyrosine phosphorylation, extracellular signal-regulated kinase 1 and 2, and p38 mitogen-activated protein kinase (MAPK), but not c-Jun NH(2)-terminal kinase. RAGE-mediated NF-kappaB activation was suppressed by the selective p38 MAPK inhibitor SB203580 and by coexpression of a kinase-dead p38 dominant-negative mutant. Activation of NF-kappaB by CML-modified albumin increased secretion of proinflammatory cytokines (tumor necrosis factor-alpha, interleukin-1beta, and monocyte chemoattractant protein-1) severalfold, and inhibition of p38 MAPK blocked these increases. These results indicate that p38 MAPK activation mediates RAGE-induced NF-kappaB-dependent secretion of proinflammatory cytokines and suggest that accelerated inflammation may be a consequence of cellular activation induced by this receptor.     

Tumor necrosis factor-alpha induces intestinal insulin resistance and stimulates the overproduction of intestinal apolipoprotein B48-containing lipoproteins

There is growing evidence suggesting intestinal insulin resistance and overproduction of apolipoprotein (apo) B48-containing chylomicrons in insulin-resistant states. In the current study, we investigated the potential role of the inflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) in the development of insulin resistance and aberrant lipoprotein metabolism in the small intestine in a Syrian golden hamster model. TNF-alpha infusion decreased whole-body insulin sensitivity, based on in vivo euglycemic clamp studies in chow-fed hamsters. Analysis of intestinal tissue in TNF-alpha-treated hamsters indicated impaired phosphorylation of insulin receptor-beta, insulin receptor substrate-1, Akt, and Shc and increased phosphorylation of p38, extracellular signal-related kinase-1/2, and Jun NH(2)-terminal kinase. TNF-alpha infusion also increased intestinal production of total apoB48, triglyceride-rich lipoprotein apoB48, and serum triglyceride levels in both fasting and postprandial (fat load) states. The effects of TNF-alpha on plasma apoB48 levels could be blocked by the p38 inhibitor SB203580. Ex vivo experiments using freshly isolated enterocytes also showed TNF-alpha-induced p38 phosphorylation and intestinal apoB48 overproduction, effects that could be blocked by SB203580. Interestingly, TNF-alpha increased the mRNA and protein mass of intestinal microsomal triglyceride transfer protein without altering apoB mRNA levels. Enterocytes were found to have detectable levels of both TNF-alpha receptor types (p55 and p75), and antibodies against either of the two TNF-alpha receptors partially blocked the stimulatory effect of TNF-alpha on apoB48 production and p38 phosphorylation. In summary, these data suggest that intestinal insulin resistance can be induced in hamsters by TNF-alpha infusion, and it is accompanied by intestinal overproduction of apoB48-containing lipoproteins. TNF-alpha-induced stimulation of intestinal lipoprotein production appears to be mediated via TNF-alpha receptors and the p38 mitogen-activated protein kinase pathway.     

p38丝裂原活化蛋白激酶在烧伤大鼠急性肺损伤中的作用机制

目的　研究 p38丝裂原活化蛋白激酶 (MAPK)信号转导通路在严重烧伤大鼠肺部促炎细胞因子肿瘤坏死因子α(TNF α)、白细胞介素 1β(IL 1β)产生及肺血管内皮细胞损伤中的作用和相关机制。　方法　健康成年雄性SD大鼠 4 8只 ,随机分为假烫 (A)组、烫伤对照 (B)组和烫伤 p38MAPK抑制剂SB2 0 35 80(C)组 ,每组各 16只。观察烫伤 (以下称烧伤 ) 2 4h后大鼠血清和支气管肺泡灌洗液 (BALF)中TNF α和IL 1β含量、血浆和肺脏微血管vonWillebrand因子 (vWF)含量、肺脏激活蛋白 1(AP 1)活性等指标的改变。　 结果　B组大鼠烧伤后 2 4h血清和BALF中TNF α和IL 1β含量明显增高 ,血浆vWF含量为 (194 .2± 2 8.3) % ,显著高于A组的 (93.2± 14 .3) % (P <0.0 1);肺脏微血管vWF含量的积分值为 1.1± 0 .3,显著低于A组的 3.3± 0 .4 (P <0.0 1);其肺脏AP 1活性上升。C组血清和BALF中TNF α和IL 1β含量、血浆及肺脏微血管vWF含量、肺脏AP 1的活性较B组变化幅度明显偏小。　结论　p38MAPK活化后 ,通过活化转录因子AP 1,介导了严重烧伤后肺脏促炎性细胞因子TNF α和IL 1β的产生和肺血管内皮细胞的损伤     

Role of p38 mitogen-activated protein kinase in Kupffer cell secretion of the proinflammatory cytokines after burn trauma

This study was designed to investigate the role of p38 mitogen-activated protein (MAP) kinase on Kupffer cells (KCs) secretion of proinflammatory cytokines such as tumor necrosis factor (TNF)-alpha and interleukin (IL)-1beta and hepatic injury following burn trauma. Sprague-Dawley rats were randomized into four groups: (1) sham burn rats given vehicle, (2) sham burn rats given the p38 MAP kinase inhibitor SB203580 (10mg/kg i.v., 15min and 12h after sham burn), (3) rats given a 30% total body surface area (TBSA) full-thickness burn and fluid resuscitation plus vehicle, and (4) burn rats given injury and fluid resuscitation plus SB203580. Rats from each group were killed at 24h post-burn to examine plasma aspartate transaminase (AST) and alanine transaminase (ALT) and KCs were isolated. The KCs secretion of TNF-alpha and IL-1beta and p38 MAP kinase activity (by Western blot analysis) were also examined. These studies showed by more significant activation of p38 MAP kinase in KCs harvested from burn rats than from shams. Burn trauma resulted in hepatic dysfunction and promoted KCs secretion of TNF-alpha and IL-1beta. SB203580 inhibited p38 MAP kinase activity, reduced KCs secretion of proinflammatory cytokines, and alleviated burn-mediated hepatic dysfunction. These data suggest p38 MAP kinase activation is one important aspect of the signaling event that may mediate the KCs secretion of proinflammatory cytokines TNF-alpha and IL-1beta following burn trauma.