SC58125 通过抑制IκBα的降解调节TNF-α 诱导的人结肠癌HT29细胞的凋亡

目的： 了解SC58125与TNF-α是否具有协同诱导HT29细胞凋亡的作用，同时探讨其可能的分子机制。方法： 用MTT、Hoechst 33342染色及琼脂糖凝胶电泳，观察SC58125/TNF-α对HT29结肠癌细胞增殖与凋亡的作用；用EMSA及Western blotting检测转录因子NF-κB的结合活性、IκBα以及磷酸化IκBα的表达。结果： SC58125与TNF-α在抑制HT29细胞增殖及诱导其凋亡方面具有明显的协同作用；表现为细胞核浓缩、核碎裂及“DNA梯带”形成；凋亡过程中伴随caspase活性的激活。经TNF-α刺激后的HT29细胞，IκBα，磷酸化IκBα迅速降解，NF-κB的结合活性大大提高，而加入SC58125后，IκBα降解及NF-κB的结合活性明显受抑制。结论： SC58125与TNF-α在诱导HT29细胞凋亡方面具有明显的协同作用，这可能与激活caspase-3 及抑制IκBα降解有关。     

Inflammatory factor-specific sumoylation regulates NF-κB signalling in glomerular cells from diabetic rats

Objective

The activation of nuclear factor (NF)-κB by cytokines under hyperglycaemic conditions is a potential mechanism for complications in diabetes. We investigated whether small ubiquitin-like modifier 4 (SUMO4) regulates renal NF-κB signalling in diabetic rats.

Methods

Histological changes in kidney were analysed in diabetic GK rats. The expressions of tumour necrosis factor (TNF)-α, NF-κB (p65), IκBα and SUMO4 in renal tissues were examined by immunohistochemistry and Western blotting. Primary cultured glomerular endothelial cells from rats were stimulated by TNF-α or interleukin (IL)-2.

Results

The renal expression of TNF-α, NF-κB (p65), IκBα and SUMO4 was significantly higher in diabetic GK rats than in control rats. In control rats, no nuclear translocation was observed for IκBα or NF-κB (p65). However, in diabetic GK rats, translocation of NF-κB (p65) and IκBα into the nucleus was observed, and the expression of SUMO4 and IκBα was up-regulated in the glomerular endothelial cells. SUMO4 was localised in both the cytoplasm and nucleus, while IκBα was predominantly located in the nucleus after stimulation with TNF-α. In contrast, SUMO4 was localised in the nucleus, and increased cytoplasm SUMO4 localisation was found after stimulation with IL-2.

Conclusions

SUMO4 plays a role in regulating NF-κB signalling in glomerular cells. Cytokines have a unique effect in regulating the sumoylation of NF-κB.     

Calcium-sensing receptor in the T lymphocyte enhanced the apoptosis and cytokine secretion in sepsis

Calcium-sensing receptor (CaSR) is a member of the G protein-coupled receptor superfamily that existed in lymphocytes and promoted cytokine secretion. Lymphocytes are also involved in sepsis. However, the role of CaSR in lymphocytes in sepsis is unclear. In this study, we want to examine whether the CaSR in lymphocytes in sepsis is involved in the cytokine secretions and apoptosis and make clear the relationship between NF-κB and MAPK signal transduction pathways. We investigated the issues mentioned earlier using Western blotting, ELISA, and Flow Cytometry. The sepsis was remodeled by cecal ligation and puncture (CLP). We found that CaSR protein expression increased in the peripheral blood T lymphocytes in CLP rats. The calcimimetic R568 (NPS R568) promoted, whereas the calcilytic NPS 2143 attenuated, signaling pathways proteins P65 (subunit of NF-κB), ERK1/2, and JNK (one subgroup of MAPKs) phosphorylation. However, P-P38 and P-JAKs exhibit no significant changes. Furthermore, the production TNF-α and IL-4 was greater in CLP rats than in normal rats, and NPS R568 promoted secretion of these cytokines. Simultaneously, the apoptotic ratio of T cells in CLP increased, and NPS R 568 exacerbated the apoptosis degree. However, these effects could also be inhibited by U0126 or SP600125 (MAPKs pathway inhibitor) or Bay-11-7082 or (NF-κB pathway inhibitor). From these results, we can conclude that, in the sepsis, CaSR activation promoted T-cell apoptosis and the secretion of pro-inflammatory cytokine TNF-α and anti-inflammatory cytokines IL-4 probably through NF-κB and partial MAPK signal transduction pathways.     

Estrogen suppresses heptatic IκB expression during short-term alcohol exposure

Objective

To assess the effects of sex steroids on hepatic inflammatory pathways in short-term chronically ethanol-fed rats.

Methods

Ovariectomized female Wistar rats (8–12?weeks old, n?=?8 per treatment group) were implanted with osmotic pumps releasing 17β-estradiol (20?μg/24?h) or testosterone (25?μg/24?h) and fed liquid diets with or without ethanol (8?% w/v) for two?weeks. Hepatic expression of IκBα/β, TNF-α, and IL-6 mRNA was examined by real-time PCR. Liver (nuclear) NFκB, IκBα and β, IL-6, and IL-6Rα protein expression was examined by enzyme-linked immunosorbent assay (ELISA) or Western blot.

Results

Estrogen alone induced greater steatosis, NFκB translocation, TNF-α mRNA, as well as IL-6, and IL-6R protein. Alcohol consumption along with estrogen treatment further increased steatosis, NFκB translocation, TNF-α mRNA, and IL-6 protein. Conversely, neither estrogen nor ethanol consumption induced IκBα or IκBβ mRNA or protein expression, while testosterone robustly induced these inhibitory proteins regardless of treatment.

Conclusions

Estrogen exposure enhances alcohol-induced liver inflammation, and the anti-inflammatory effects of testosterone in the liver might be related to induction of IκB. Elevated inflammation in response to estrogen may overwhelm the regenerative influence of IL-6 in liver, leading to increased steatosis and greater liver damage.     

TNF-α对肾脏近端小管上皮细胞caspase-3的调控作用及其机制

目的:探讨肿瘤坏死因子-α(TNF-α)对大鼠原代肾小管上皮细胞半胱氨酸天冬氨酸蛋白酶-3(caspase-3)激活与表达的调控以及核因子-κB(NF-κB)在其中的作用。方法:体外分离与培养大鼠肾小管上皮细胞,以TNF-α(10μg/L)按不同时间给予刺激,Western blotting检测caspase-3及其活化裂解片段cleaved caspase-3,以及I-κBα和磷酸化I-κBα(pI-κBα)蛋白水平。分别以TNF-α(10μg/L)处理24h、NF-κB特异性抑制剂Bay11-7082(5μmol/L)处理25h、Bay11-7082(5μmol/L)预处理1h后加入TNF-α(10μg/L)刺激24h,检测caspase-3及cleaved caspase-3的变化。结果:TNF-α刺激6至24h,cleaved caspase-3与caspase-3的相对比值显著高于对照组;而caspase-3蛋白水平在TNF-α刺激后6h无明显增高,12h低于对照组,24h回升;Bay11-7082处理组和Bay11-7082+TNF-α处理组的caspase-3蛋白表达与活化水平显著低于TNF-α处理组和对照组。结论:TNF-α促进大鼠原代肾小管上皮细胞caspase-3的激活与表达,这一过程与NF-κB的激活有关。     

Aggregatibacter actinomycetemcomitans infection enhances apoptosis in vivo through a caspase-3-dependent mechanism in experimental periodontitis

The purpose of this study was to test the hypothesis that diabetes aggravates periodontal destruction induced by Aggregatibacter actinomycetemcomitans infection. Thirty-eight diabetic and 33 normal rats were inoculated with A. actinomycetemcomitans and euthanized at baseline and at 4, 5, and 6 weeks after inoculation. Bone loss and the infiltration of polymorphonuclear leukocytes (PMNs) in gingival epithelium were measured in hematoxylin-eosin-stained sections. The induction of tumor necrosis factor alpha (TNF-α) was evaluated by immunohistochemistry and of apoptotic cells by a TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling) assay. After A. actinomycetemcomitans infection, the bone loss in diabetic rats was 1.7-fold and the PMN infiltration 1.6-fold higher than in normoglycemic rats (P < 0.05). The induction of TNF-α was 1.5-fold higher and of apoptotic cells was up to 3-fold higher in diabetic versus normoglycemic rats (P < 0.05). Treatment with a caspase-3 inhibitor significantly blocked noninflammatory cell apoptosis induced by A. actinomycetemcomitans infection in gingival epithelium and connective tissue (P < 0.05). These results provide new insight into how diabetes aggravates A. actinomycetemcomitans-induced periodontal destruction in rats by significantly increasing the inflammatory response, leading to increased bone loss and enhancing apoptosis of gingival epithelial and connective tissue cells through a caspase-3-dependent mechanism. Antibiotics had a more pronounced effect on many of these parameters in diabetic than in normoglycemic rats, suggesting a deficiency in the capacity of diabetic animals to resist infection.     

Xaf1调节TNFR信号转导而诱导细胞凋亡的机制研究

目的： 利用基因开关调节的Xaf1-Saos诱导细胞株， 检测Xaf1对TNFR信号转导通路的影响，探索Xaf1与TNF-α协同诱导细胞凋亡的机制。方法： 以免疫印迹法和RT-PCR检测Xaf1对TNFR1 表达的影响，细胞周期 DNA 含量流式细胞术检测NF-κB对Xaf1诱导细胞凋亡的影响，gel mobility shift assay 检测NF-κB的DNA结合活性， luciferase 活性检测法及RT-PCR检测NF-κB的转录活性，激酶分析法检测SAPK/JNK 激酶的活性。结果： Xaf1不影响TNFR1蛋白及mRNA水平的表达，细胞内诱导活性的NF-κB可抑制Xaf1诱导的细胞凋亡， Xaf1的表达抑制TNF-α所介导的NF-κB的DNA结合活性和转录活性，也抑制了SAPK/JNK 激酶的活性。结论： Xaf1 对TNFR信号转导的抑制是Xaf1协同TNF-α诱导细胞凋亡的机制之一。     

Effect of aspirin on the expression of hepatocyte NF-κB and serum TNF-α in streptozotocin-induced type 2 diabetic rats

Aspirin is a kind of anti-inflammatory drug and may be used to reverse hyperglycemia, hyperinsulinemia, and dyslipidemia by improving insulin resistance. We hypothesized that aspirin improves insulin resistance in type 2 diabetes by inhibiting hepatic nuclear factor kappa-β (NF-κB) activation and serum tumor necrosis factor-α (TNF-α). Adult male Wistar rats were randomly divided into four groups: control, untreated diabetic, diabetic treated with metformin (100 mg/kg/day), and diabetic treated with aspirin (120 mg/kg/day). Diabetes was induced by high-fat feeding and a low dose of streptozotocin (30 mg/kg). After treatment, plasma glucose, insulin, lipids, free fatty acids (FFAs) concentrations and serum TNF-α were determined. The expression of NF-κB in hepatocytes was analyzed by immunohistochemistry and western blot. The results showed administration of aspirin caused no significant lowering in fasting glucose level but significant reduction of hepatic NF-κB expression and serum TNF-α level with improved insulin resistance compared to the diabetic group. The relevant analysis showed positive correlation between the expression of homeostasis model assessment-insulin resistance (HOMA-IR) and NF-κB (r = 0.799, P < 0.01); HOMA-IR and serum TNF-α (r = 0.790, P < 0.01). It is concluded that aspirin improves insulin resistance by inhibiting hepatic NF-κB activation and TNF-α level in streptozotocin-induced type 2 diabetic rats.     

Anti-citrullinated Protein Antibodies Activated ERK1/2 and JNK Mitogen-activated Protein Kinases via Binding to Surface-expressed Citrullinated GRP78 on Mononuclear Cells

In a previous study, we found that anti-citrullinated protein antibodies (ACPAs) enhance nuclear factor (NF)-κB activity and tumor necrosis factor (TNF)-α production by normal human peripheral blood mononuclear cells (PBMCs) and U937 cells via binding to surface-expressed citrullinated glucose-regulated protein 78 (cit-GRP78). However, the downstream signaling pathways remain unclear after binding. In the present study, we firstly measured the effects of different kinase inhibitors on ACPA-mediated TNF-α production from normal PBMCs and monocytes. Then, the native and phosphorylated mitogen-activated protein kinases (MAPKs) were detected in ACPA-activated U937 cells by Western blotting. We also explored the role of the phosphoinositide 3-kinase (PI3K)-Akt pathway in activating IκB kinase alpha (IKK-α) in ACPA-stimulated U937 cells. Finally, we measured the amount of cit-GRP78 from PBMC membrane extracts in RA patients and controls. We found that MAPK and Akt inhibitors, but not PI3K inhibitor, remarkably suppressed ACPA-mediated TNF-α production. Interestingly, ACPAs selectively activated extracellular signal-regulated kinase 1/2 (ERK1/2) and c-jun N-terminal kinase (JNK), but not p38 MAPK, in U937 cells. This activation was suppressed by cit-GRP78, but not GRP78. The JNK activation further enhanced the phosphorylation of Akt and IKK-α. The expression of cit-GRP78 on cell membrane was higher in RA than normal PBMCs. Taken together; these results suggest that through binding to surface, over-expressed cit-GRP78 on RA PBMCs, ACPAs selectively activate ERK1/2 and JNK signaling pathways to enhance IKK-α phosphorylation, which leads to the activation of NF-κB and the production of TNF-α .     

Chikusetsusaponin V attenuates lipopolysaccharide-induced liver injury in mice

Chikusetsusaponin V (CsV), a saponin from Panax japonicus, has been reported to inhibit inflammatory responses in lipopolysaccharide (LPS)-induced macrophage cells. However, whether CsV could alleviate LPS-induced liver injury in vivo and the potential mechanisms involved remain unclear. In the present study, we investigated the anti-inflammatory effects of CsV on LPS-induced acute liver injury in mice and further explored the potential mechanisms involved. Our results showed that CsV significantly attenuated elevation of alanine transaminase (ALT) and aspartate aminotransferase (AST) levels and improved liver histopathological changes in LPS-induced mice. In addition, CsV decreased serum tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) levels and inhibited mRNA expressions of inducible nitric oxide synthase (iNOS), TNF-α and IL-1β in LPS challenged mice. Furthermore, CsV inhibited nuclear factor kappa B (NF-κB) activation by downregulating phosphorylated NF-κB, IκB-α, ERK, c-Jun N-terminal kinase (JNK) and p38 levels in the liver tissue, which ultimately decreased nucleus NF-κB protein level. In conclusion, our data suggested that CsV could be a promising drug for preventing LPS challenged liver injury since it attenuated LPS-induced inflammatory responses, partly via inhibiting NF-κB and MAPK signaling pathways.     

Ginsenosides compound K and Rh2 inhibit tumor necrosis factor-α-induced activation of the NF-κB and JNK pathways in human astroglial cells

Ginsenosides, the main component of Panax ginseng, have been known for the anti-inflammatory and anti-proliferative activities. In this study, we investigated the molecular mechanisms responsible for the anti-inflammatory effects of ginsenosides on activated astroglial cells. Among 13 different ginsenosides, intestinal bacterial metabolites Rh₂ and compound K (C-K) showed a significant inhibitory effect on tumor necrosis factor-α (TNF-α)-induced expression of intercellular adhesion molecule-1 in human astroglial cells. Pretreatment with C-K or Rh₂ suppressed TNF-α-induced phosphorylation of IκBα kinase and the subsequent phosphorylation and degradation of IκBα. Additionally, the same treatment inhibited TNF-α-induced phosphorylation of MKK4 and the subsequent activation of the JNK-AP-1 pathway. The inhibitory effect of ginsenosides on TNF-α-induced activation of the NF-κB and JNK pathways was not observed in human monocytic U937 cells. These results collectively indicate that ginsenoside metabolites C-K and Rh₂ exert anti-inflammatory effects by the inhibition of both NF-κB and JNK pathways in a cell-specific manner.     

TNF-α and TGF-β1 regulate Syndecan-4 expression in nucleus pulposus cells: role of the mitogen-activated protein kinase and NF-κB pathways

Syndecan-4 is emerging as an important player in cell interaction with the extracellular environment and has been shown to be involved in the progression of intervertebral disc degeneration. However, the mechanism of syndecan-4 regulation by TNF-α and the role of TGF-β1 in regulating syndecan-4 expression remain poorly understood in nucleus pulposus (NP) cells. The aim of this study was to investigate these mechanisms. We exposed NP cells to TNF-α and the gene, protein expression, and promoter activity levels of syndecan-4 were measured by qPCR, western blotting, and the luciferase reporter assay, respectively. The activation of the MAPK and NF-κB pathways was detected using western blot analysis. Syndecan-4 expression in rat NP cells was increased by TNF-α, but this was neither time nor dose dependent in response to TNF-α. ERK1/2, JNK, and NF-κB pathways were activated following TNF-α treatment. Treatment with ERK1/2 and NF-κB inhibitors decreased the up-regulation of syndecan-4 by TNF-α. However, JNK inhibition showed no effect on syndecan-4 expression induced by TNF-α. TNF-α mediated up-regulation of syndecan-4 was antagonized by TGF-β1. This study provided evidence for the differential regulation by MAPK and NF-κB pathways in the over-expression of syndecan-4 promoted by TNF-α in NP cells. Our results demonstrate that TGF-β1 exerts anabolic effects on intervertebral discs by inhibiting the expression of syndecan-4.     

Synthetic liver X receptor agonist T0901317 attenuates high glucose-induced oxidative stress,mitochondrial damage and apoptosis in cardiomyocytes

The aim of the present study was to investigate the protective effects of T0901317 (T0), a potent agonist of liver X receptors (LXRs), on high glucose-induced oxidative stress and apoptosis in H9c2 cardiac cells. Exposure of H9c2 cells to high glucose alone, not only caused a significant increase in apoptosis and reactive oxygen species (ROS) generation, but also led to a decrease in mitochondrial membrane potential (ΔΨm), release of cytochrome c, decrease in Bcl-2, increase in Bax expression and the activation of caspase-3, caspase-9, poly (ADP-ribose) polymerase (PARP) and nuclear factor (NF)-κB. However, pretreatment with T0 effectively decreased apoptosis, reduced the levels of ROS, abrogated ΔΨm, inhibited cytochrome c release and NF-κB activation, increased Bcl-2 and decreased Bax expression. In conclusion, our data suggest that T0 exerts protective effects against high glucose-induced apoptosis in H9C2 cardiac muscle cells via inhibition of ROS production, mitochondrial death and NF-κB activation.     

Thymol Inhibits LPS-Stimulated Inflammatory Response via Down-Regulation of NF-κB and MAPK Signaling Pathways in Mouse Mammary Epithelial Cells

Thymol is a natural monoterpene phenol primarily found in thyme, oregano, and tangerine peel. It has been shown to possess anti-inflammatory property both in vivo and in vitro. In the present paper, we studied the anti-inflammatory effect of thymol in lipopolysaccharide (LPS)-stimulated mouse mammary epithelial cells (mMECs). The mMECs were stimulated with LPS in the presence or absence of thymol (10, 20, 40 μg/mL). The concentrations of tumor necrosis factor α (TNF-α), interleukin (IL)-6, and IL-1β in the supernatants of culture were determined using enzyme-linked immunosorbent assay. Cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), extracellular signal-regulated protein kinase (ERK), c-Jun N-terminal kinase (JNK), nuclear factor-κB (NF-κB), and inhibitor protein of NF-κB (IκBα) were measured using western blot. The results showed that thymol markedly inhibited the production of TNF-α and IL-6 in LPS-stimulated mMECs. The expression of iNOS and COX-2 was also suppressed by thymol in a dose-dependent manner. Furthermore, thymol blocked the phosphorylation of IκBα, NF-κB p65, ERK, JNK, and p38 mitogen-activated protein kinases (MAPKs) in LPS-stimulated mMECs. These results indicate that thymol exerted anti-inflammatory property in LPS-stimulated mMECs by interfering the activation of NF-κB and MAPK signaling pathways. Thereby, thymol may be a potential therapeutic agent against mastitis.     

The involvement of β-actin in the signaling of transmembrane TNF-α-mediated cytotoxicity

Actin cytoskeleton has been shown to play a regulating role in several signaling pathways, and disruption of actin filament has been reported to increase sTNF-α-induced cell death. However, whether actin is involved in tmTNF-α-mediated cytotoxicity remains unclear. Here, we demonstrated that pretreatment of HL-60 with CytD or LatA to depolymerize actin significantly suppressed tmTNF-α-mediated apoptosis. Interestingly, tmTNF-α increased the actin immunoprecipitated by anti-TNFR2 but not anti-TNFR1 antibody, and disruption of the actin filament totally blocked this effect. In addition, TNFR1 knockdown by siRNA did not affect tmTNF-α-mediated cytotoxicity and the inhibitory effect of CytD, suggesting that the involvement of actin in the tmTNF-α-induced apoptosis is linked to the TNFR2 pathway. Our results revealed further that tmTNF-α signaled the inhibition of IκB degradation and NF-κB activity by recruiting RIP1 to and uncoupling TRAF2 from the TNFR2 complex. Nevertheless, CytD totally reversed the tmTNF-α signaling and activated NF-κB by recruiting TRAF2 to and dissociating RIP1 from the TNFR2 complex. Furthermore, tmTNF-α led to activation of caspase-8 by dissociation of cFLIP from TNFR2 and inhibition of the cFLIP expression. Activated caspase-8 cleft RIP1 to suppress NF-κB activity and also mediated tmTNF-α-induced apoptosis. However, CytD blocked the tmTNF-α-induced uncoupling of cFLIP from TNFR2 and prevented caspase-8 activation and the resulting cleavage of RIP1, converting the signaling for tmTNF-α-mediated apoptosis into one for activating NF-κB to survive. These results suggest that the actin cytoskeleton functions in transmitting signals via TNFR2 to mediate tmTNF-α-induced apoptosis.     

Differential roles of JNK in ConA/GalN and ConA-induced liver injury in mice

Tumor necrosis factor-α-mediated liver injury can be induced by several different means; however, the signaling events and mechanisms of cell death are likely different. We investigated the mechanism of both apoptotic and necrotic hepatocyte cell death as well as the role of c-Jun NH₂-terminal kinase (JNK) in the ConA and ConA/D-galactosamine (GalN) models of murine liver injury. ConA alone induced primarily necrotic cell death with no caspase activation, whereas ConA/GalN induced apoptosis in addition to necrotic cell death. The bi-modal death pattern in the ConA/GalN model was confirmed by the use of transgenic mice expressing a dominant-negative form of Fas-associated death domain in which the mice were resistant to apoptotic but not necrotic cell death. JNK1 and, more significantly, JNK2 participated in the induction of hepatocyte apoptosis in response to ConA/GalN. Deletion of JNK led to the stabilization of FLIP_L, reduced caspase-8 activation, decreased Bid cleavage, and inhibition of the mitochondrial apoptosis pathway. In contrast, JNK did not participate in necrotic death induced by ConA either alone or in combination with GalN. As such, JNK-deficient mice remained susceptible to necrotic liver injury in both model systems. Thus, ConA and ConA/GalN mouse models induce liver injury with different mechanisms of cell death, and JNK contributes to apoptotic but not necrotic cell death. These findings further elucidate the specific pathways involved in tumor necrosis factor-α-mediated liver injury.     

紫草素对非酒精性脂肪性肝炎的影响及分子机制研究

目的 探究紫草素对非酒精性脂肪性肝炎(nonalcoholic steatohepatitis,NASH)是否具有改善作用及其作用机制.方法 给予C57BL/6小鼠胆碱和蛋氨酸缺乏(methionine choline deficient,MCD)饮食诱导NASH模型,检测小鼠体质量、血清转氨酶、血脂以及肝脏中脂质含量...