Objective To investigate effect of tumor necrosis factor-or （TNF-α） on the Src-suppressed C kinase substrate （SSeCKS） in C6 glioma cells. Methods Cultured C6 glioma cells were randomly divided into two groups. In...Objective To investigate effect of tumor necrosis factor-or （TNF-α） on the Src-suppressed C kinase substrate （SSeCKS） in C6 glioma cells. Methods Cultured C6 glioma cells were randomly divided into two groups. In time-dependent group, cells were cultured with TNF-α （2 ng/mL） for 0 h, 1 h, 3 h, 6 h, 12 or 24 h, respectively; in dose-dependent group, cells were cultured with TNF-α （0 ng/mL, 0.02 ng/mL, 0.2 ng/mL, or 2 ng/mL） for 6 h. The expression of SSeCKS was detected by Realtime PCR and Western blot analysis, and immunocytochemistry was used to investigate SSeCKS＇s subcellular localization. Results TNF-α induced rapid phosphorylations of protein kinase C （PKC） substrates in C6 glioma cells, and upregulated SSeCKS expression in a time and concentration dependent manner. Immunocytochemistry suggested that SSeCKS was localized in the cyroplasm and the leading end of podosomal extensions in control groups, while TNF-α induced translocation of SSeCKS perinuclear. This effect could be partly reversed by PKC inhibitor Ro-31-8220. Conclusion TNF-α activates PKC and upregulates SSeCKS expression in C6 glioma cells. These effects are associated with PKC activity, suggesting that SSeCKS plays a role in response to glia activation in PKC mediated pathway.展开更多
AIM: To investigate the effects of FR167653 on the development of dextran sulfate sodium (DSS)-induced colitis in mice. METHODS: BALB/c mice were fed rodent chow containing 3.5% (wt/wt) DSS. The recipient mice underwe...AIM: To investigate the effects of FR167653 on the development of dextran sulfate sodium (DSS)-induced colitis in mice. METHODS: BALB/c mice were fed rodent chow containing 3.5% (wt/wt) DSS. The recipient mice underwent intra-peritoneal injection of vehicles or FR167653 (30 mg/kg per day). The mice were sacrificed on day 14, and the degree of colitis was assessed. Immunohistochemical analyses for CD4+ T cell and F4/80+ macrophage infiltration were also performed. Mucosal cytokine expression was analyzed by RT-PCR. RESULTS: The body weight loss was more apparent in the FR167653-treated DSS mice than in the vehicle- treated DSS mice. The colon length was shorter in the FR167653-treated DSS mice than in the vehicle-treated DSS mice. Disease activity index and histological colitis score were significantly higher in FR167653- than in vehicle-treated DSS animals. Microscopically, mucosal edema, cellular infiltration (CD4 T cells and F4/80 macrophages), and the disruption of the epithelium were much more severe in FR167653-treated mice than in controls. Mucosal mRNA expression for interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) were found to be markedly reduced in FR167653-treated DSS mice. CONCLUSION: Treatment with FR167653 aggravated DSS colitis in mice. This effect was accompanied by a reduction of mucosal IL-1β and TNF-α expression, suggesting a role of p38 mitogen-activated protein kinase (MAPK)-mediated proinflammatory cytokine induction in host defense mechanisms.展开更多
Mixed lineage kinase 3 （MLK3） is a mitogen-activated protein kinase kinase kinase that is activated by tumor necrosis factor-α （TNF-α） and specifically activates c-Jun N-terminal kinase （JNK） on TNF-a stimulat...Mixed lineage kinase 3 （MLK3） is a mitogen-activated protein kinase kinase kinase that is activated by tumor necrosis factor-α （TNF-α） and specifically activates c-Jun N-terminal kinase （JNK） on TNF-a stimulation. The mecha- nism by which TNF-α activates MLK3 is still not known. TNF receptor-associated factors （TRAFs） are adapter molecules that are recruited to cytoplasmic end of TNF receptor and mediate the downstream signaling, including activation of JNK. Here, we report that MLK3 associates with TRAF2, TRAF5 and TRAF6; however only TRAF2 can significantly induce the kinase activity of MLK3. The interaction domain of TRAF2 maps to the TRAF domain and for MLK3 to its C-terminal half （amino acids 511-847）. Endogenous TRAF2 and MLK3 associate with each other in response to TNF-α treatment in a time-dependent manner. The association between MLK3 and TRAF2 mediates MLK3 activation and competition with the TRAF2 deletion mutant that binds to MLK3 attenuates MLK3 kinase activity in a dose-dependent manner, on TNF-α treatment. Furthermore the downstream target of MLK3, JNK was activated by TNF-α in a TRAF2-dependent manner. Hence, our data show that the direct interaction between TRAF2 and MLK3 is required for TNF-α-induced activation of MLK3 and its downstream target, JNK.展开更多
Objective:To explore the effect of herb-partitioned moxibustion(HPM)on tight junctions(TJs)of intestinal epithelial cells in Crohn disease(CD)mediated by tumor necrosis factor-α(TNF-α)-nuclear factor kappa B(NF-κB)...Objective:To explore the effect of herb-partitioned moxibustion(HPM)on tight junctions(TJs)of intestinal epithelial cells in Crohn disease(CD)mediated by tumor necrosis factor-α(TNF-α)-nuclear factor kappa B(NF-κB)-myosin-light-chain kinase(MLCK)pathway.Methods:Forty-eight male Sprague-Dawley rats were randomly divided into a normal control(NC)group,a model control(MC)group,an HPM group and a mesalazine(MESA)group,with 12 rats in each group.Trinitrobenzene sulfonic acid(TNBS)was administered to establish CD models.When the model was confirmed a success,the HPM group rats were treated with HPM at Tianshu(ST 25)and Qihai(CV 6),while the MESA group rats were given MESA solution by lavage.When the intervention finished,the colonic epithelial tissues were separated,purified and cultured in each group to establish the intestinal epithelial barrier model in vitro,and TNF-αwas added(100 ng/mL)in the culture medium and maintained for 24 h to establish an increased epithelial permeability model.Transepithelial electrical resistance(TEER)was used to examine the permeability of the barrier;Western blot was used to observe the expressions of the proteins related to TJs of intestinal epithelial cells mediated by TNF-α-NF-κB-MLCK pathway;immunofluorescence staining was used to observe the expressions and distributions of tight junction proteins in the intestinal epithelium.Results:After TNF-αinduction,compared with the MC+TNF-αgroup,the TEER value increased significantly in the HPM+TNF-αand MESA+TNF-αgroups(both P<0.001);the expressions of nuclear factor kappa B(NF-κB)p65,MLCK,myosin light chain(MLC),tumor necrosis factor receptor-associated factor 6(TRAF6)and receptor interaction protein-1(RIP1)decreased significantly(P<0.01 or P<0.05),and the expression of zinc finger protein A20(A20)increased significantly(P<0.01);the expressions of occludin,claudin-1,zonula occludens protein 1(ZO-1)and F-actin also increased significantly(all P<0.01).Compared with the MESA+TNF-αgroup,the expressions of MLC,occludin,claudin-1,ZO-1 and F-actin increased significantly in the HPM+TNF-αgroup(P<0.01 or P<0.05).Conclusion:HPM can protect or repair the damage of intestinal epithelial barrier in CD rats,which may be achieved through modulating the abnormal TJs in intestinal epithelium mediated by TNF-α-NF-κB-MLCK pathway.展开更多
基金supported by the National Natural Science Foundation of China(No.30300099)the Natural Science Foundation of Jiangsu Province,China(No.BK2003035)the Natural Science Research Program in College and University of Jiangsu Province(No.03KJB180109).
文摘Objective To investigate effect of tumor necrosis factor-or （TNF-α） on the Src-suppressed C kinase substrate （SSeCKS） in C6 glioma cells. Methods Cultured C6 glioma cells were randomly divided into two groups. In time-dependent group, cells were cultured with TNF-α （2 ng/mL） for 0 h, 1 h, 3 h, 6 h, 12 or 24 h, respectively; in dose-dependent group, cells were cultured with TNF-α （0 ng/mL, 0.02 ng/mL, 0.2 ng/mL, or 2 ng/mL） for 6 h. The expression of SSeCKS was detected by Realtime PCR and Western blot analysis, and immunocytochemistry was used to investigate SSeCKS＇s subcellular localization. Results TNF-α induced rapid phosphorylations of protein kinase C （PKC） substrates in C6 glioma cells, and upregulated SSeCKS expression in a time and concentration dependent manner. Immunocytochemistry suggested that SSeCKS was localized in the cyroplasm and the leading end of podosomal extensions in control groups, while TNF-α induced translocation of SSeCKS perinuclear. This effect could be partly reversed by PKC inhibitor Ro-31-8220. Conclusion TNF-α activates PKC and upregulates SSeCKS expression in C6 glioma cells. These effects are associated with PKC activity, suggesting that SSeCKS plays a role in response to glia activation in PKC mediated pathway.
文摘AIM: To investigate the effects of FR167653 on the development of dextran sulfate sodium (DSS)-induced colitis in mice. METHODS: BALB/c mice were fed rodent chow containing 3.5% (wt/wt) DSS. The recipient mice underwent intra-peritoneal injection of vehicles or FR167653 (30 mg/kg per day). The mice were sacrificed on day 14, and the degree of colitis was assessed. Immunohistochemical analyses for CD4+ T cell and F4/80+ macrophage infiltration were also performed. Mucosal cytokine expression was analyzed by RT-PCR. RESULTS: The body weight loss was more apparent in the FR167653-treated DSS mice than in the vehicle- treated DSS mice. The colon length was shorter in the FR167653-treated DSS mice than in the vehicle-treated DSS mice. Disease activity index and histological colitis score were significantly higher in FR167653- than in vehicle-treated DSS animals. Microscopically, mucosal edema, cellular infiltration (CD4 T cells and F4/80 macrophages), and the disruption of the epithelium were much more severe in FR167653-treated mice than in controls. Mucosal mRNA expression for interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) were found to be markedly reduced in FR167653-treated DSS mice. CONCLUSION: Treatment with FR167653 aggravated DSS colitis in mice. This effect was accompanied by a reduction of mucosal IL-1β and TNF-α expression, suggesting a role of p38 mitogen-activated protein kinase (MAPK)-mediated proinflammatory cytokine induction in host defense mechanisms.
文摘Mixed lineage kinase 3 （MLK3） is a mitogen-activated protein kinase kinase kinase that is activated by tumor necrosis factor-α （TNF-α） and specifically activates c-Jun N-terminal kinase （JNK） on TNF-a stimulation. The mecha- nism by which TNF-α activates MLK3 is still not known. TNF receptor-associated factors （TRAFs） are adapter molecules that are recruited to cytoplasmic end of TNF receptor and mediate the downstream signaling, including activation of JNK. Here, we report that MLK3 associates with TRAF2, TRAF5 and TRAF6; however only TRAF2 can significantly induce the kinase activity of MLK3. The interaction domain of TRAF2 maps to the TRAF domain and for MLK3 to its C-terminal half （amino acids 511-847）. Endogenous TRAF2 and MLK3 associate with each other in response to TNF-α treatment in a time-dependent manner. The association between MLK3 and TRAF2 mediates MLK3 activation and competition with the TRAF2 deletion mutant that binds to MLK3 attenuates MLK3 kinase activity in a dose-dependent manner, on TNF-α treatment. Furthermore the downstream target of MLK3, JNK was activated by TNF-α in a TRAF2-dependent manner. Hence, our data show that the direct interaction between TRAF2 and MLK3 is required for TNF-α-induced activation of MLK3 and its downstream target, JNK.
基金国家自然科学基金项目,No.81674069 and No.81473757973 Program,国家重点基础研究发展计划项目,No.2015CB554500。
文摘Objective:To explore the effect of herb-partitioned moxibustion(HPM)on tight junctions(TJs)of intestinal epithelial cells in Crohn disease(CD)mediated by tumor necrosis factor-α(TNF-α)-nuclear factor kappa B(NF-κB)-myosin-light-chain kinase(MLCK)pathway.Methods:Forty-eight male Sprague-Dawley rats were randomly divided into a normal control(NC)group,a model control(MC)group,an HPM group and a mesalazine(MESA)group,with 12 rats in each group.Trinitrobenzene sulfonic acid(TNBS)was administered to establish CD models.When the model was confirmed a success,the HPM group rats were treated with HPM at Tianshu(ST 25)and Qihai(CV 6),while the MESA group rats were given MESA solution by lavage.When the intervention finished,the colonic epithelial tissues were separated,purified and cultured in each group to establish the intestinal epithelial barrier model in vitro,and TNF-αwas added(100 ng/mL)in the culture medium and maintained for 24 h to establish an increased epithelial permeability model.Transepithelial electrical resistance(TEER)was used to examine the permeability of the barrier;Western blot was used to observe the expressions of the proteins related to TJs of intestinal epithelial cells mediated by TNF-α-NF-κB-MLCK pathway;immunofluorescence staining was used to observe the expressions and distributions of tight junction proteins in the intestinal epithelium.Results:After TNF-αinduction,compared with the MC+TNF-αgroup,the TEER value increased significantly in the HPM+TNF-αand MESA+TNF-αgroups(both P<0.001);the expressions of nuclear factor kappa B(NF-κB)p65,MLCK,myosin light chain(MLC),tumor necrosis factor receptor-associated factor 6(TRAF6)and receptor interaction protein-1(RIP1)decreased significantly(P<0.01 or P<0.05),and the expression of zinc finger protein A20(A20)increased significantly(P<0.01);the expressions of occludin,claudin-1,zonula occludens protein 1(ZO-1)and F-actin also increased significantly(all P<0.01).Compared with the MESA+TNF-αgroup,the expressions of MLC,occludin,claudin-1,ZO-1 and F-actin increased significantly in the HPM+TNF-αgroup(P<0.01 or P<0.05).Conclusion:HPM can protect or repair the damage of intestinal epithelial barrier in CD rats,which may be achieved through modulating the abnormal TJs in intestinal epithelium mediated by TNF-α-NF-κB-MLCK pathway.
