Phosphorylation of extracellular signal‐regulated kinase 1/2, p38 mitogen‐activated protein kinase and nuclear translocation of nuclear factor‐κB are involved in upregulation of matrix metalloproteinase‐9 by tumour necrosis factor‐α

Background: Upregulation of matrix metalloproteinase‐9 (MMP‐9) induced by tumour necrosis factor‐α (TNF‐α) is reportedly involved in a variety of non‐neoplastic and neoplastic diseases. In this study, we examined which signalling pathways are involved in TNF‐α‐induced MMP‐9 upregulation in cholangiocarcinoma (CC). Methods: We used two CC cell lines: HuCCT‐1 and CCKS‐1. Results: In an ex vivo study using HuCCT‐1 and CCKS‐1 cells, TNF‐α treatment induced MMP‐9 production and activation via interaction with TNF receptor‐1 (TNF‐R1) but not with TNF receptor‐2 (TNF‐R2), shown by zymography, and increased MMP‐9 promoter activity (luciferase assay). As for the signalling pathway, TNF‐α stimulation led to the phosphorylation of extracellular signal‐regulated kinase 1/2 (Erk1/2) and p38 mitogen‐activated protein kinase (p38MAPK) and translocation of nuclear factor κB (NF‐κB) (p65) into the nuclei. Inhibition studies using SB203580 (inhibitor of p38MAPK), U0126 (inhibitor of mitogen‐activated or extracellular signal‐regulated protein kinase 1/2) and MG132 (inhibitor of NF‐κB) showed that the phosphorylation of Erk1/2 and p38MAPK with activation of NF‐κB was closely related to MMP‐9 upregulation in both cell lines. Conclusion: These data suggest that TNF‐α/TNF‐R1 interaction leads to the phosphorylation of Erk1/2 and p38MAPK and nuclear translocation of NF‐κB, which is closely associated with the production and activation of MMP‐9 in cultured CC cells of HuCTT‐1 and CCKS‐1. Upregulation of MMP‐9 with NF‐κB activation may be involved in the tumour invasion of CC.     

Melatonin attenuates TNF‐α and IL‐1β expression in synovial fibroblasts and diminishes cartilage degradation: Implications for the treatment of rheumatoid arthritis

The hormone melatonin has many properties, including antioxidant, anti‐inflammatory, and immunomodulatory effects. Melatonin has been demonstrated to be beneficial in several inflammatory autoimmune diseases, but its effects in rheumatoid arthritis (RA) remain controversial. We sought to determine how melatonin regulates inflammation in RA. We found that melatonin dose‐dependently inhibits tumor necrosis factor‐α (TNF‐α) and interleukin (IL)‐1β expression through the PI3K/AKT, ERK, and NF‐κB signaling pathways. We also identified that melatonin inhibits TNF‐α and IL‐1β production by upregulating miR‐3150a‐3p expression. Synovial tissue specimens from RA patients and culture of human rheumatoid fibroblast‐like synoviocytes confirmed that the MT1 receptor is needed for the anti‐inflammatory activities of melatonin. Importantly, melatonin also significantly reduced paw swelling, cartilage degradation, and bone erosion in the collagen‐induced arthritis mouse model. Our results indicate that melatonin ameliorates RA by inhibiting TNF‐α and IL‐1β production through downregulation of the PI3K/AKT, ERK, NF‐κB signaling pathways, as well as miR‐3150a‐3p overexpression. The role of melatonin as an adjuvant treatment in patients with RA deserves further clinical studies.     

Transforming growth factor‐α attenuates hepatic fibrosis: possible involvement of matrix metalloproteinase‐1

Background: The effect of transforming growth factor (TGF)‐α on fibrosis varies between cell types and the role of TGF‐α in hepatic fibrosis has not been fully elucidated. Methods: We examined the effect of TGF‐α on hepatic fibrosis using TGF‐α‐expressing transgenic mice fed a methionine‐ and choline‐deficient (MCD) diet and human hepatic stellate cells (HSCs) line LX‐2, rat and human primary HSCs. Results: Although the expression levels of the tissue inhibitor of metalloproteinases‐1 and α1(I) collagen mRNA were unchanged, feeding the TGF‐α transgenic mice the MCD diet resulted in greater expression of the murine functional analogue of matrix metalloproteinase‐1 (MMP‐1), MMP‐13 mRNA and protein and attenuated hepatic fibrosis compared with wild‐type mice. TGF‐α overexpression did not affect the extent of the steatosis, oxidative stress and hepatic inflammation in the MCD diet‐fed mice. The effect of TGF‐α on the fibrogenic and anti‐fibrogenic gene expressions varied between cell types in vitro. TGF‐α increased MMP‐1 mRNA expressions that were completely blocked by gefitinib in LX‐2 cells. The extracellular signal‐regulated kinase (ERK) 1/2, c‐Jun N‐terminal kinase and p38 pathways were involved in MMP‐1 mRNA expression in LX‐2 cells. Although TGF‐α increased the phosphorylation of p38, the p38 inhibitor activated the RAS‐ERK pathway and increased TGF‐α‐induced MMP‐1 mRNA expression, which suggested that there may be a crosstalk between the RAS‐ERK and the p38 pathways in LX‐2 cells. Conclusions: The TGF‐α may attenuate hepatic fibrosis in part because of upregulation of the expression of MMP‐1. The balance between fibrogenic and anti‐fibrogenic gene expression and between the activity of the RAS‐ERK and the p38 pathways may be crucial for the fibrotic process.     

Acute liver injury upregulates microRNA‐491–5p in mice,and its overexpression sensitizes Hep G2 cells for tumour necrosis factor‐α‐induced apoptosis

Background: MicroRNAs (miRNAs) have emerged as novel genetic regulators of cell functions such as proliferation, apoptosis and cancer. Aims: The aim of this study was to evaluate the role of a specific miRNA in modulating hepatic cell functions. Methods: C57Bl/6 mice were administered anti‐fas receptor antibodies to induce liver cell apoptosis. miRNAs were purified from the liver tissue and evaluated using an miRNA microarray. The role of miRNA‐491_5p, which was overexpressed in the model, in modulating hepatic cell functions was evaluated. miRNA‐491_5p was overexpressed in Hep G2 cells, followed by the addition of tumour necrosis factor (TNF)‐α, and induction of apoptosis as well as genes involved in apoptosis pathways were evaluated. The effect of miRNA‐491_5p target genes on apoptosis was also analysed by inhibiting their expression by siRNA‐induced gene silencing. Results: Upregulation of miRNA‐491_5p was found in a high‐dose anti‐fas receptor antibody group. Overexpression of microRNA‐491_5p sensitized Hep G2 cells for TNF‐α‐induced apoptosis, and also caused an inhibition of α‐fetoprotein, (AFP), heat shock protein‐90 (hsp‐90) and nuclear factor‐κB (NF‐κB). Overexpression of miRNA‐491_5p or inhibition of AFP and hsp‐90 resulted in an increased apoptosis in TNF‐α‐treated Hep G2 cells. Conclusions: One of the miRNAs that is associated with the acute liver injury mouse model, miRNA‐491_5p, sensitizes Hep G2 cells for TNF‐α‐induced apoptosis, at least in part, by inhibiting AFP, hsp‐90 and NF‐κB.     

Increased apoptosis in HepG2.2.15 cells with hepatitis B virus expression by synergistic induction of interferon‐γ and tumour necrosis factor‐α

Background: Interferon‐γ (IFN‐γ) and tumour necrosis factor‐α (TNF‐α) were thought to be important immune mediators in host defence against hepatitis B virus (HBV) infection. Aims: To examine the synergistic effect of IFN‐γ and TNF‐α on HBV‐expressing HepG2.2.15 cells and its potential mechanisms. Methods: Cell viability was quantitatively measured by 3‐[4,5‐dimethylthiazol‐2‐yl]‐2,5‐diphenyl tetrazolium bromide assay. Cell morphology was captured using light microscopy. The typical DNA ladder test was performed using agarose gel electrophoresis. HBsAg and HBeAg titre changes were quantified by the enzyme‐linked immunosorbent assay method. Gene expression was analysed using cDNA macroarrays. Results: Interferon‐γ (1000 U/ml) alone or combined with TNF‐α (5 ng/ml) treatment resulted in apoptosis in HepG2.2.15 cells, but no significant apoptosis in the parent non‐virus expressing HepG2 cells. IFN‐γ‐ and TNF‐α‐mediated apoptosis was reduced by lamivudine treatment in HepG2.2.15 cells. IFN‐γ combined with TNF‐α reduced the titre of hepatitis B surface antigen and hepatitis B e antigen in the HepG2.2.15 cell line. For apoptosis‐related gene changes, IFN regulatory factor 1 (IRF‐1) (12.2‐fold), c‐myc (V00568 4.7‐fold, L00058 2.4‐fold) and caspase 7 (2.3‐fold) genes were upregulated in the combination treatment group. Conclusion: Interferon‐γ and TNF‐α play a role in the cell death of HBV‐expressing HepG2.2.15 cells. Expression of HBV leads to IFN‐γ‐ and TNF‐α‐mediated apoptosis in the cells. Increased IRF‐1, c‐myc and caspase 7 gene expression may be responsible for the synergistic induction of apoptosis by IFN‐γ and TNF‐α.     

Nuclear factor‐kappa B as a promising target for selenium chemoprevention in rat hepatocarcinogenesis

Background and Aims: Selenium's molecular mechanism for cancer chemoprevention remains unknown. We aimed to study the gene expression of nuclear factor‐κB (NF‐κB), tumor growth factor‐α (TGF‐α) and cyclin D1 and the effects of sodium selenite using preventive and therapeutic approaches in chemically‐induced hepatocarcinogenesis in rats. Methods: Rats were divided randomly into six groups: negative control, positive control (diethyl nitrosamine [DEN] + 2‐acetylaminofluorene [2‐AAF]), preventive group, preventive control (respective control for preventive group), therapeutic group and therapeutic control (respective control for therapeutic group). The relative gene expression of NF‐κB, TGF‐α and cyclin D1 in liver tissues were measured using real‐time polymerase chain reaction. Results: The findings showed that the gene expression of NF‐κB in the preventive group and its respective control was significantly lower (P < 0.05) when compared with both the negative and positive controls. However, the expression of NF‐κB in the positive controls and therapeutic group was significantly higher (P < 0.05) when compared with the negative controls. The expression of TGF‐α and cyclin D1 was insignificant in all groups. Conclusion: The inhibition of the NF‐κB pathway in the initiation phase of hepatocarcinogenesis could be a promising target for selenium chemoprevention. However, further studies are required.     

Effects of gut barrier dysfunction and NF‐κB activation on aggravating mechanism of severe acute pancreatitis

OBJECTIVE: To study the effects of gut‐derived endotoxin translocation and NF‐κB activation on the aggravating mechanism of severe acute pancreatitis (SAP) and of treatment with pyrrolidine dithiocarbamate (PDTC) on rats with SAP. METHODS: SD rats were randomly divided into sham operation group (SO), SAP group, SAP + lipopolysaccharide(LPS) group, pyrrolidine dithiocarbamate (PDTC) treatment group and LPS group. Biochemical parameters and cytokines were examined in the serum. Multiple organs pathological slices were examined. Expression of NF‐κB mRNA in the liver tissue was detected by RT‐PCR. Activation of NF‐κB by the method of streptomycin avidin‐peroxidase (SP) and expression of NF‐κB p65 protein and its binding activity were analyzed by Western blot and electrophoretic mobidity shift assay (EMSA). RESULTS: Compared with sham operation group, the concentration of TNF‐α, alanine aminotransferase (ALT), and diamine oxidase (DAO) in serum significantly increased in SAP + LPS group (P < 0.05). Pathological changes were markedly observed in tissues and the expression of NF‐κB mRNA in the liver significantly increased (P < 0.05) also, the activation of NF‐κB and binding activity of NF‐κB p65 protein in the liver markedly increased (P < 0.01) in SAP + LPS group. Treatment with PDTC markedly reduced concentration of ALT, DAO and TNF‐α, and the expression of NF‐κB, and the pathologic scores, as well as significantly decreased the expression of NF‐κB p65 protein. CONCLUSION: The activation and overexpression of NF‐κB may participate in the aggravating mechanism of SAP. Treatment with PDTC has a protective effect on multiple organs damage in SAP.     

Steatosis correlates with hepatic expression of death receptors and activation of nuclear factor‐κB in chronic hepatitis C

Background: Steatosis is recognized as a predictor of the severity as well as the progression of fibrosis in chronic hepatitis C. The mechanisms that cause increased hepatocellular injury associated with steatosis remain largely unknown. Methods: We studied the correlation of hepatic expression of death receptors: Fas and tumour necrosis factor‐α receptor 1 (TNF‐R1), and downstream caspase (caspase‐3) with hepatic steatosis by immunohistochemical study in chronic hepatitis C and determined the role of nuclear factor‐κB (NF‐κB). Results: Ninety patients (49 males and 41 females, mean age of 50.5 ± 10.4 years, genotype 1 or 2) with chronic hepatitis C virus infection were recruited. The factors associated with steatosis grade were body mass index (P=0.004) and fibrosis stage (P=0.034). Moderate/severe steatosis was an independent variable associated with advanced fibrosis stage by stepwise logistic regression analysis. The expression of immunoreactivity for Fas, TNF‐R1 and active caspases‐3 in liver tissues was significantly correlated with the steatosis grade (P<0.001, P<0.001 and P<0.001 respectively). The extent of active caspases‐3 correlated significantly with the expression of Fas (r=0.659, P<0.001) and TNF‐R1 (r=0.617, P<0.001). NF‐κB p65 expression correlated significantly with the extent of Fas (r=0.405, P<0.001), TNF‐R1 (r=0.448, P=0.002) and active caspase‐3 (r=0.313, P=0.003), and correlated with steatosis grade (P<0.001) but not with inflammatory and fibrosis scores. Conclusion: Our observations suggest a mechanism whereby steatosis contributes to the progression of liver injury in chronic hepatitis C through upregulation of death receptors and activation of NF‐κB.     

Melatonin ameliorates Aβ42‐induced alteration of βAPP‐processing secretases via the melatonin receptor through the Pin1/GSK3β/NF‐κB pathway in SH‐SY5Y cells

Melatonin is involved in the physiological regulation of the β‐amyloid precursor protein (βAPP)‐cleaving secretases which are responsible for generation of the neurotoxic amyloid beta (Aβ) peptide, one of the hallmarks of Alzheimer's disease (AD) pathology. In this study, we aimed to determine the underlying mechanisms of this regulation under pathological conditions. We establish that melatonin prevents Aβ₄₂‐induced downregulation of a disintegrin and metalloproteinase domain‐containing protein 10 (ADAM10) as well as upregulation of β‐site APP‐cleaving enzyme 1 (BACE1) and presenilin 1 (PS1) in SH‐SY5Y cell cultures. We also demonstrate that the intrinsic mechanisms of the observed effects occurred via regulation of nuclear factor kappa‐light‐chain‐enhancer of activated B cells (NF‐κB) and glycogen synthase kinase (GSK)‐3β as melatonin reversed Aβ₄₂‐induced upregulation and nuclear translocation of NF‐κBp65 as well as activation of GSK3β via its receptor activation. Furthermore, specific blocking of the NF‐κB and GSK3β pathways partially abrogated the Aβ₄₂‐induced reduction in the BACE1 and PS1 levels. In addition, GSK3β blockage affected α‐secretase cleavage and modulated nuclear translocation of NF‐κB. Importantly, our study for the first time shows that peptidyl‐prolyl cis‐trans isomerase NIMA‐interacting 1 (Pin1) is a crucial target of melatonin. The compromised levels and/or genetic variation of Pin1 are associated with age‐dependent tau and Aβ pathologies and neuronal degeneration. Interestingly, melatonin alleviated the Aβ₄₂‐induced reduction of nuclear Pin1 levels and preserved the functional integrity of this isomerase. Our findings illustrate that melatonin attenuates Aβ₄₂‐induced alterations of βAPP‐cleaving secretases possibly via the Pin1/GSK3β/NF‐κB pathway.     

Tumour necrosis factor‐α promoter region polymorphisms affect the course of spontaneous HBsAg clearance

Background: This study aimed to investigate the roles of tumour necrosis factor‐α (TNF‐α) gene polymorphisms in the spontaneous clearance of HBsAg after a hepatitis B virus (HBV) infection. Methods: Polymorphisms in the TNF‐α (?1031 T to C, ?863 C to A, ?857 C to T, ?308 G to A and ?238 G to A transition) gene were evaluated in 274 chronic HBV‐infected patients and 194 patients with resolved HBV infection. The peripheral blood mononuclear cells (PBMC) isolated from 77 (28%) of the 274 chronic HBV‐infected patients with negative HBeAg and positive antibody to HBeAg were stimulated with HBcAg. Data on TNF‐α genotypes and phenotypes in subjects with/without the A allele at the TNF‐α?863 promoter single nucleotide polymorphism (rs1800630) were compared. Results: The A allele in the ?863 promoter region of the TNF‐α gene was present in 154 (56.2%) chronic HBV‐infected patients and 87 (44.8%) patients who recovered from HBV infection (odds ratio 1.58; P<0.01). The TNF‐α?863 A allele genotype predicted lower TNF‐α production by PBMC after in vitro HBcAg stimulation (P<0.02). Conclusions: The A allele at the ?863 locus of the promoter region of the TNF‐α gene predicts lower HBcAg‐inducible TNF‐α secretion. It is also associated with chronicity of HBV infection.