Chlorogenic acid: A potent molecule that protects cardiomyocytes from TNF‐α–induced injury via inhibiting NF‐κB and JNK signals

The traditional Chinese herb Lonicerae Japonicae Flos has shown significant clinical benefits in the treatment of heart failure, but the mechanism remains unclear. As the main active ingredient found in the plasma after oral administration of Lonicerae Japonicae Flos, chlorogenic acid (CGA) has been reported to possess anti‐inflammatory, anti‐oxidant and anti‐apoptosis function. We firstly confirmed the cardioprotective effects of CGA in transverse aortic constriction (TAC)‐induced heart failure mouse model, through mitigating the TNF‐α–induced toxicity. We further used TNF‐α‐induced cardiac injury in human induced pluripotent stem cell‐derived cardiomyocytes (hiPSC‐CMs) to elucidate the underlying mechanisms. CGA pre‐treatment could reverse TNF‐α–induced cellular injuries, including improved cell viability, increased mitochondrial membrane potential and inhibited cardiomyocytes apoptosis. We then examined the NF‐κB/p65 and major mitogen‐activated protein kinases (MAPKs) signalling pathways involved in TNF‐α–induced apoptosis of hiPSC‐CMs. Importantly, CGA can directly inhibit NF‐κB signal by suppressing the phosphorylation of NF‐κB/p65. As for the MAPKs, CGA suppressed the activity of only c‐Jun N‐terminal kinase (JNK), but enhanced extracellular signal‐regulated kinase1/2 (ERK1/2) and had no effect on p38. In summary, our study revealed that CGA has profound cardioprotective effects through inhibiting the activation of NF‐κB and JNK pathway, providing a novel therapeutic alternative for prevention and treatment of heart failure.     

LIGHT/IFN‐γ triggers β cells apoptosis via NF‐κB/Bcl2‐dependent mitochondrial pathway

LIGHT recruits and activates naive T cells in the islets at the onset of diabetes. IFN‐γ secreted by activated T lymphocytes is involved in beta cell apoptosis. However, whether LIGHT sensitizes IFNγ‐induced beta cells destruction remains unclear. In this study, we used the murine beta cell line MIN6 and primary islet cells as models for investigating the underlying cellular mechanisms involved in LIGHT/IFNγ – induced pancreatic beta cell destruction. LIGHT and IFN‐γ synergistically reduced MIN6 and primary islet cells viability; decreased cell viability was due to apoptosis, as demonstrated by a significant increase in Annexin V⁺ cell percentage, detected by flow cytometry. In addition to marked increases in cytochrome c release and NF‐κB activation, the combination of LIGHT and IFN‐γ caused an obvious decrease in expression of the anti‐apoptotic proteins Bcl‐2 and Bcl‐xL, but an increase in expression of the pro‐apoptotic proteins Bak and Bax in MIN6 cells. Accordingly, LIGHT deficiency led to a decrease in NF‐κB activation and Bak expression, and peri‐insulitis in non‐obese diabetes mice. Inhibition of NF‐κB activation with the specific NF‐κB inhibitor, PDTC (pyrrolidine dithiocarbamate), reversed Bcl‐xL down‐regulation and Bax up‐regulation, and led to a significant increase in LIGHT‐ and IFN‐γ‐treated cell viability. Moreover, cleaved caspase‐9, ‐3, and PARP (poly (ADP‐ribose) polymerase) were observed after LIGHT and IFN‐γ treatment. Pretreatment with caspase inhibitors remarkably attenuated LIGHT‐ and IFNγ‐induced cell apoptosis. Taken together, our results indicate that LIGHT signalling pathway combined with IFN‐γ induces beta cells apoptosis via an NF‐κB/Bcl2‐dependent mitochondrial pathway.     

Radioprotective effect of geraniin via the inhibition of apoptosis triggered by γ-radiation-induced oxidative stress

The radioprotective effect of geraniin, a tannin compound isolated from Nymphaea tetragona Georgi var. (Nymphaeaceae), against γ-radiation-induced damage was investigated in Chinese hamster lung fibroblast (V79-4) cells. Geraniin recovered cell viability detected by MTT test and colony formation assay, which was compromised by γ-radiation, and reduced the γ-radiation-induced apoptosis by the inhibition of loss of the mitochondrial membrane potential. Geraniin protected cellular components (lipid membrane, cellular protein, and DNA) damaged by γ-radiation, which was detected by lipid peroxidation, protein carbonyl formation, and comet assay. Geraniin significantly reduced the level of intracellular reactive oxygen species generated by γ-radiation, which was detected using spectrofluorometer, flow cytometer, and confocal microscope after 2′,7′-dichlorodihydrofluorescein diacetate staining. Geraniin normalized the superoxide dismutase and catalase activities, which were decreased by γ-radiation. These results suggest that geraniin protects cells against radiation-induced oxidative stress via enhancing of antioxidant enzyme activities and attenuating of cellular damage.     

Platycodin D,a triterpenoid saponin from Platycodon grandiflorum,suppresses the growth and invasion of human oral squamous cell carcinoma cells via the NF‐κB pathway

This work was undertaken to explore the effects of platycodin D, a triterpenoid saponin from Platycodon grandiflorum, on the growth and invasiveness of human oral squamous cell carcinoma (OSCC). Platycodin D caused a significant, concentration‐dependent inhibition of cell viability and induced significant apoptosis in OSCC cells. Moreover, platycodin D significantly inhibited OSCC cell invasion. At the molecular level, platycodin D increased the amounts of IκBα protein and reduced the expression of phosphorylated NF‐κB p65, MMP‐2, and MMP‐9. Ectopic expression of constitutively active NF‐κB p65 prevented platycodin D‐mediated induction of apoptosis and suppression of invasion in OSCC cells. In vivo studies confirmed that platycodin D retarded the growth of subcutaneous SCC‐4 xenograft tumors and reduced phosphorylation of NF‐κB p65. Altogether, platycodin D shows inhibitory activity on OSCC growth and invasion through inactivation of the NF‐κB pathway and might provide therapeutic benefits in the treatment of OSCC.     

Attenuating Smac mimetic compound 3‐induced NF‐κB activation by luteolin leads to synergistic cytotoxicity in cancer cells

Smac mimetics are potential anticancer therapeutics selectively killing cancer cells through autocrine tumor necrosis factor (TNF)‐mediated apoptosis pathway. Our recent study reveal that the Smac mimetic compound 3 (SMC3)‐activated NF‐κB protects cancer cells against apoptosis, thus blunting SMC3's anticancer activity. Based on our previous observations that the nutrient flavonoid luteolin potently blocks TNF‐induced NF‐κB activation in cancer cells, we investigated if the combination of SMC3 and luteolin would achieve a synergistic anticancer activity. The results show that luteolin had no effect on autocrine TNF but it effectively blocked SMC3‐induced nuclear factor kappa B (NF‐κB) activation and expression of anti‐apoptotic NF‐κB targets. When SMC3 and luteolin were combined in treating cancer cells derived from lung and liver tumors, the activation of TNF‐dependent apoptosis was markedly sensitized and a synergistic cytotoxic effect was achieved. In addition, the SMC3 and luteolin co‐treatment had marginal effect on immortalized normal human bronchial epithelial cells. The results suggest that combination of SMC3 and luteolin is an effective approach for improving the anticancer value of SMC3, which has implications in cancer prevention and therapy. J. Cell. Biochem. 108: 1125–1131, 2009. © 2009 Wiley‐Liss, Inc.     

Silencing of ATP2B1‐AS1 contributes to protection against myocardial infarction in mouse via blocking NFKBIA‐mediated NF‐κB signalling pathway

Myocardial infarction (MI) is an acute coronary syndrome that refers to tissue infarction of the myocardium. This study aimed to investigate the effect of long intergenic non‐protein‐coding RNA (lincRNA) ATPase plasma membrane Ca²⁺ transporting 1 antisense RNA 1 (ATP2B1‐AS1) against MI by targeting nuclear factor‐kappa‐B inhibitor alpha (NFKBIA) and mediating the nuclear factor‐kappa‐B (NF‐κB) signalling pathway. An MI mouse model was established and idenepsied by cardiac function evaluation. It was determined that ATP2B1‐AS1 was highly expressed, while NFKBIA was poorly expressed and NF‐κB signalling pathway was activated in MI mice. Cardiomyocytes were extracted from mice and introduced with a series of mouse ATP2B1‐AS1 vector, NFKBIA vector, siRNA‐mouse ATP2B1‐AS1 and siRNA‐NFKBIA. The expression of NF‐κBp50, NF‐κBp65 and IKKβ was determined to idenepsy whether ATP2B1‐AS1 and NFKBIA affect the NF‐κB signalling pathway, the results of which suggested that ATP2B1‐AS1 down‐regulated the expression of NFKBIA and activated the NF‐κB signalling pathway in MI mice. Based on the data from assessment of cell viability, cell cycle, apoptosis and levels of inflammatory cytokines, either silencing of mouse ATP2B1‐AS1 or overexpression of NFKBIA was suggested to result in reduced cardiomyocyte apoptosis and expression of inflammatory cytokines, as well as enhanced cardiomyocyte viability. Our study provided evidence that mouse ATP2B1‐AS1 silencing may have the potency to protect against MI in mice through inhibiting cardiomyocyte apoptosis and inflammation, highlighting a great promise as a novel therapeutic target for MI.     

Inhibition of DNM1L and mitochondrial fission attenuates inflammatory response in fibroblast‐like synoviocytes of rheumatoid arthritis

Mitochondrial fission and fusion are important for mitochondrial function, and dynamin 1‐like protein (DNM1L) is a key regulator of mitochondrial fission. We investigated the effect of mitochondrial fission on mitochondrial function and inflammation in fibroblast‐like synoviocytes (FLSs) during rheumatoid arthritis (RA). DNM1L expression was determined in synovial tissues (STs) from RA and non‐RA patients. FLSs were isolated from STs and treated with a DNM1L inhibitor (mdivi‐1, mitochondrial division inhibitor 1) or transfected with DNM1L‐specific siRNA. Mitochondrial morphology, DNM1L expression, cell viability, mitochondrial membrane potential, reactive oxygen species (ROS), apoptosis, inflammatory cytokine expression and autophagy were examined. The impact of mdivi‐1 treatment on development and severity of collagen‐induced arthritis (CIA) was determined in mice. Up‐regulated DNM1L expression was associated with reduced mitochondrial length in STs from patients with RA and increased RA severity. Inhibition of DNM1L in FLSs triggered mitochondrial depolarization, mitochondrial elongation, decreased cell viability, production of ROS, IL‐8 and COX‐2, and increased apoptosis. DNM1L deficiency inhibited IL‐1β–mediated AKT/IKK activation, NF‐κBp65 nuclear translocation and LC3B‐related autophagy, but enhanced NFKBIA expression. Treatment of CIA mice with mdivi‐1 decreased disease severity by modulating inflammatory cytokine and ROS production. Our major results are that up‐regulated DNM1L and mitochondrial fission promoted survival, LC3B‐related autophagy and ROS production in FLSs, factors that lead to inflammation by regulating AKT/IKK/NFKBIA/NF‐κB signalling. Thus, inhibition of DNM1L may be a new strategy for treatment of RA.     

Gastrokine 1 regulates NF‐κB signaling pathway and cytokine expression in gastric cancers

Gastrokine 1 (GKN1) plays an important role in the gastric mucosal defense mechanism and also acts as a functional gastric tumor suppressor. In this study, we examined the effect of GKN1 on the expression of inflammatory mediators, including NF‐κB, COX‐2, and cytokines in GKN1‐transfected AGS cells and shGKN1‐transfected HFE‐145 cells. Lymphocyte migration and cell viability were also analyzed after treatment with GKN1 and inflammatory cytokines in AGS cells by transwell chemotaxis and an MTT assay, respectively. In GKN1‐transfected AGS cells, we observed inactivation and reduced expression of NF‐κB and COX‐2, whereas shGKN1‐transfected HFE‐145 cells showed activation and increased expression of NF‐κB and COX‐2. GKN1 expression induced production of inflammatory cytokines including IL‐8 and ‐17A, but decreased expression of IL‐6 and ‐10. We also found IL‐17A expression in 9 (13.6%) out of 166 gastric cancer tissues and its expression was closely associated with GKN1 expression. GKN1 also acted as a chemoattractant for the migration of Jurkat T cells and peripheral B lymphocytes in the transwell assay. In addition, GKN1 significantly reduced cell viability in both AGS and HFE‐145 cells. These data suggest that the GKN1 gene may inhibit progression of gastric epithelial cells to cancer cells by regulating NF‐κB signaling pathway and cytokine expression. J. Cell. Biochem. 114: 1800–1809, 2013. © 2013 Wiley Periodicals, Inc.     

N‐methyl‐N‐nitrosourea induces retinal degeneration in the rat via the inhibition of NF‐κB activation

Retinitis pigmentosa (RP) is a group of inherited neurodegenerative diseases characterized by the loss of photoreceptor cells through apoptosis. N‐methyl‐N‐nitrosourea (MNU) is an alkylating toxicant that induces photoreceptor cell death resembling hereditary RP. This study aimed to investigate the role of nuclear factor κB (NF‐κB) in MNU‐induced photoreceptor degeneration. Adult rats received a single intraperitoneal injection of MNU (60 mg/kg bodyweight). Hematoxylin and eosin staining demonstrated progressive outer nuclear layer (ONL) loss after MNU treatment. Transmission electron microscopy revealed nuclear pyknosis, chromatin margination in the photoreceptors, increased secondary lysosomes, and lobulated retinal‐pigmented epithelial cells in MNU‐treated rats. Numerous photoreceptor cells in the ONL showed positive TUNEL staining and apoptosis rate peaked at 24 hours. Enhanced depth imaging spectral‐domain optical coherence tomography showed ONL thinning and decreased choroid thickness. Electroretinograms showed decreased A wave amplitude that predominated in scotopic conditions. Western blot analysis showed that nuclear IκBα level increased, whereas nuclear NF‐κB p65 decreased significantly in the retinas of MNU‐treated rats. These findings indicate that MNU leads to selective photoreceptor degradation, and this is associated with the inhibition of NF‐κB activation.     

Central role of PAFR signalling in ExoU‐induced NF‐κB activation

ExoU is an important virulence factor in acute Pseudomonas aeruginosa infections. Here, we unveiled the mechanisms of ExoU‐driven NF‐κB activation by using human airway cells and mice infected with P. aeruginosa strains. Several approaches showed that PAFR was crucially implicated in the activation of the canonical NF‐κB pathway. Confocal microscopy of lungs from infected mice revealed that PAFR‐dependent NF‐κB activation occurred mainly in respiratory epithelial cells, and reduced p65 nuclear translocation was detected in mice PAFR?/? or treated with the PAFR antagonist WEB 2086. Several evidences showed that ExoU‐induced NF‐κB activation regulated PAFR expression. First, ExoU increased p65 occupation of PAFR promoter, as assessed by ChIP. Second, luciferase assays in cultures transfected with different plasmid constructs revealed that ExoU promoted p65 binding to the three κB sites in PAFR promoter. Third, treatment of cell cultures with the NF‐κB inhibitor Bay 11–7082, or transfection with IκBα negative‐dominant, significantly decreased PAFR mRNA. Finally, reduction in PAFR expression was observed in mice treated with Bay 11–7082 or WEB 2086 prior to infection. Together, our data demonstrate that ExoU activates NF‐κB by PAFR signalling, which in turns enhances PAFR expression, highlighting an important mechanism of amplification of response to this P. aeruginosa toxin.     

Co‐expression of NF‐κB‐p65 and phosphorylated NF‐κB‐p105 is associated with poor prognosis in surgically resectable non‐small cell lung cancer

Nuclear factor‐kappa B (NF‐κB) as a prognostic marker remains unclear in non‐small cell lung cancer (NSCLC). Here, we studied NF‐κB‐p65 (p65) expression and phosphorylated NF‐κB‐p105 (p‐p105) expression in NSCLC and correlated the finding with overall survival (OS) and clinicopathological features. A total of 186 archival samples from patients with surgically resectable NSCLC were probed with p65 and p‐p105 (Ser 932). The p65‐positive expression and p‐p105‐positive expression were defined as distinct nuclear p65 and cytoplasmic p‐p105 labelling in at least 1% of tumour cells, respectively. The positive staining of p65 alone, p‐p105 alone and co‐expression of p65 and p‐p105 were observed in 61 (32.8%), 90 (48.4%) and 35 (18.8%) patients, respectively. Co‐expression of p65 and p‐p105 but not of either p65 or p‐p105 alone was associated with a poor prognosis. Patients with co‐expression of p65 and p‐p105 had a shorter OS than others, median OS 26.5 months versus 64.1 months, HR 1.85 (95% CI: 1.18–2.91), P = 0.007. There was no statistically significant association between clinicopathological characteristics and either p65 or p‐p105 alone or co‐expression of p65 and p‐p105. This indicates that co‐expression of p65 and p‐p105 was a poor prognostic factor, and pathologic studies of NF‐κB expression could include multiple pathway components in NSCLC.     

Brain‐derived neurotrophic factor protects cementoblasts from serum starvation‐induced cell death

Our previous studies have shown that brain‐derived neurotrophic factor (BDNF) enhances bone/cementum‐related protein gene expression through the TrkB‐c‐Raf‐ERK1/2‐Elk‐1 signaling pathway in cementoblasts, which play a critical role in the establishment of a functional periodontal ligament. To clarify how BDNF regulates survival in cementoblasts, we examined its effects on cell death induced by serum starvation in immortalized human cementoblast‐like (HCEM) cells. BDNF inhibited the death of HCEM cells. Small‐interfering RNA (siRNA) for TRKB, a high affinity receptor for BDNF, and for Bcl‐2, countered the BDNF‐induced decrease in dead cell number. In addition, LY294002, a PI3‐kinase inhibitor; SH‐6, an Akt inhibitor; and PDTC, a nuclear factor kappa B (NF‐κB) inhibitor, but not PD98059, an ERK1/2 inhibitor, abolished the protective effect of BDNF against cell death. BDNF enhanced phosphorylated Akt levels, NF‐κB activity in the nucleus, Bcl‐2 mRNA levels, and mitochondrial membrane potential. The blocking of BDNF's actions by treatment with siRNA in all cases for TRKB and Bcl‐2, LY294002, SH‐6, and PDTC suppressed the enhancement. These findings provide the first evidence that a TrkB‐PI3‐kinase‐Akt‐NF‐κB‐Bcl‐2 signaling pathway triggered by BDNF and the subsequent protective effect of BDNF on mitochondrial membrane potential are required to rescue HCEM cells from serum starvation‐induced cell death. Furthermore, the survival and increased expression of bone/cementum‐related proteins induced by BDNF in HCEM cells occur through different signaling pathways. J. Cell. Physiol. 221: 696–706, 2009. © 2009 Wiley‐Liss, Inc.