7-ketocholesterol induces apoptosis in differentiated PC12 cells via reactive oxygen species-dependent activation of NF-κB and Akt pathways

Cholesterol oxidation products formed under the enhanced oxidative stress in the brain are suggested to induce neuronal cell death. However, it is still unknown whether oxysterol-induced apoptosis in neuronal cells is mediated by Akt and NF-κB pathways. We assessed the apoptotic effect of 7-ketocholesterol against differentiated PC12 cells in relation to activation of the reactive oxygen species-dependent nuclear factor (NF)-κB, which is mediated by the Akt pathway. 7-Ketocholesterol induced a decrease in cytosolic Bid and Bcl-2 levels, increase in cytosolic Bax levels, cytochrome c release, caspase-3 activation and upregulation of p53. 7-Ketocholesterol induced an increase in phosphorylated inhibitory κB-α, NF-κB p65 and NF-κB p50 levels, binding of NF-κB p65 to DNA, and activation of Akt. Treatment with Bay 11-7085 (an inhibitor of NF-κB activation) and oxidant scavengers, including N-acetylcysteine, prevented the 7-ketocholesterol-induced formation of reactive oxygen species, activation of NF-κB, Akt and apoptosis-related proteins, and cell death. Results from this study suggest that 7-ketocholesterol may exert an apoptotic effect against PC12 cells by inducing activation of the caspase-8-dependent pathway as well as activation of the mitochondria-mediated cell death pathway, leading to activation of caspases, via the reactive oxygen species-dependent activation of NF-κB, which is mediated by the Akt pathway.     

Tangeretin inhibits streptozotocin-induced cell apoptosis via regulating NF-κB pathway in INS-1 cells

Oxidative stress is considered to play an important role in inducing the pancreatic β-cells apoptosis and promoting the development of diabetes mellitus. Tangeretin is a plant-derived flavonoid that retains antidiabetic effects. However, the role of tangeretin in streptozotocin (STZ)-induced β-cell apoptosis remains unclear. In this study, we aimed to examine the effects of tangeretin on STZ-induced cell apoptosis and the underlying mechanisms implicated in vitro. Our results showed that tangeretin improved the cell viability in STZ-induced INS-1 cells. Tangeretin reduced the increase of apoptosis ratio and revered the altered expressions of Bax and Bcl-2 caused by STZ induction. Furthermore, the impairment of insulin secretion ability as well as a reduction in messenger RNA levels of insulin 1 and 2 was significantly attenuated by tangeretin in STZ-induced INS-1 cells. Moreover, tangeretin resulted in a significant decrease in reactive oxygen species content, accompanied by an evident increase in the activities of superoxide dismutase, catalase, and glutathione peroxidase. Mechanistic studies further revealed that tangeretin inhibited the NF-κB pathway in STZ-induced INS-1 cells. These data indicated that tangeretin improved the cell apoptosis induced by STZ in INS-1 cells, which might be partly due to its antioxidant potential. Furthermore, NF-κB was found to be involved in the protective effect of tangeretin. Collectively, the results indicated that tangeretin could be used as a therapeutic approach for diabetes mellitus treatment.     

Sensitization of endothelial cells to VEGI-induced apoptosis by inhibiting the NF-κB pathway

Vascular endothelial growth inhibitor (VEGI) is an endogenous inhibitor of endothelial cell growth and a promising candidate for cancer therapy. VEGI is able to inhibit tumor growth by specifically targeting the tumor neovasculature. Increasing the anti-angiogenic potential of this cytokine is of great interest for its therapeutic potential. NF-κB is known to have an integral role in TNF superfamily signaling, acting as a pro-survival factor. A role of VEGI-induced NF-κB activation in endothelial cells has yet to be described. Here we show that suppression of the NF-κB pathway can increase the apoptotic potential of VEGI. We used siRNA to deplete NF-κB or its activator IKK2 from adult bovine aortic endothelial cells. The siRNA treatments diminished VEGI-induced NF-κB activation, evidenced from a reduced extent of NF-κB nuclear translocation and diminished expression of NF-κB-target genes such as interleukins-6 and -1β. The siRNA-treated endothelial cells when exposed to VEGI exhibited a marked decrease in cell viability and a significant increase in apoptosis. These results confirm that VEGI utilizes NF-κB as a pro-survival role factor in endothelial cells. We then examined whether a combination of VEGI with NF-κB inhibitors would constitute a more potential therapeutic regiment. We found that in the presence of the NF-κB inhibitors curcumin or BMS-345541 there was a marked increase in the apoptotic potential of VEGI on endothelial cells. These findings indicate that a combination therapy using VEGI and NF-κB inhibitors could be a potent approach for cancer treatment.     

Obovatol attenuates LPS-induced memory impairments in mice via inhibition of NF-κB signaling pathway

Neuroinflammation and accumulation of β-amyloid are critical pathogenic mechanisms of Alzheimer’s disease (AD). In the previous study, we have shown that systemic lipopolysaccharide (LPS) caused neuroinflammation with concomitant increase in β-amyloid and memory impairments in mice. In an attempt to investigate anti-neuroinflammatory properties of obovatol isolated from Magnolia obovata, we administered obovatol (0.2, 0.5 and 1.0 mg/kg/day, p.o.) to animals for 21 days before injection of LPS (0.25 mg/kg, i.p.). We found that obovatol dose-dependently attenuates LPS-induced memory deficit in the Morris water maze and passive avoidance tasks. Consistent with the results of memory tasks, the compound prevented LPS-induced increases in Aβ_1-42 formation, β- and γ-secretases activities and levels of amyloid precursor protein, neuronal β-secretase 1 (BACE1), and C99 (a product of BACE1) in the cortex and hippocampus. The LPS-mediated neuroinflammation as determined by Western blots and immunostainings was significantly ameliorated by the compound. Furthermore, LPS-induced nuclear factor (NF)-κB DNA binding activity was drastically abolished by obovatol as shown by the electrophoretic mobility shift assay. The anti-neuroinflammation and anti-amyloidogenesis by obovatol were replicated in in vitro studies. These results show that obovatol mitigates LPS-induced amyloidogenesis and memory impairment via inhibiting NF-κB signal pathway, suggesting that the compound might be plausible therapeutic intervention for neuroinflammation-related diseases such as AD.     

Inhibition of LPS-induced apoptosis in differentiated-PC12 cells by new triazine derivatives through NF-κB-mediated suppression of COX-2

Anti-inflammatory therapy approaches have been in the focus of attention in the treatment of neurodegenerative diseases, such as Alzheimer's disease (AD). In this study, we examined the role of new 1,2,4-triazine derivatives against cytotoxicity exerted by lipopolysaccharide (LPS) in differentiated rat pheochromocytoma (PC12) cell line. Our results indicated that LPS-induced cell death can be inhibited in the presence of some of these compounds, as measured by MTT test, acridine orange/ethidium bromide staining and caspase-3 expression assay. We further showed that these compounds exert their protective effects through the inhibition of LPS-induced generation of nitric oxide and reactive oxygen species. Triazine derivatives inhibited LPS-induced nuclear translocation of nuclear factor- κB, a known regulator of a host of genes involved in specific stress and inflammatory responses. Pretreatment of PC12 cells with triazine derivatives also suppressed LPS-induced cyclooxygenase-2 expression while up-regulated heat shock protein-70 (Hsp-70). Moreover, the treatment of brain diseases is limited by the insufficiency in delivering therapeutic drugs into brain relating to highly limited transport of compounds through blood-brain barrier (BBB). Using a reliable model based on the artificial neural network, we indicated that these compounds are capable of penetrating BBB and may be useful agents for preventing neuroinflammatory diseases like AD.     

Visfatin regulates Pg LPS-induced proinflammatory/prodegradative effects in healthy and inflammatory periodontal cells partially via NF-κB pathway

Periodontitis is a widespread chronic infectious-inflammatory disease associated with multiple systemic diseases. Visfatin is an adipokine-enzyme that can be locally produced by human periodontal ligament cells (hPDLCs) and human gingival fibroblasts (hGFs). It can upregulate proinflammatory cytokines and matrix metalloproteinases (MMPs) in various types of cells. However, the effects of visfatin on healthy and inflammatory human periodontal cells as well as the underlying molecular mechanisms remain unclear. This study firstly demonstrated visfatin expression was highly elevated in inflamed human gingiva and Pg LPS-treated hPDLCs. Moreover, recombinant visfatin significantly upregulated the expression of proinflammatory cytokines (TNF-α, IL-1β and IL-6) and prodegradative factors (EMPPRIN, MMP1, MMP3 and MMP13) in hPDLCs. Next, we found the levels of proinflammatory and prodegradative cytokines were significantly increased in visfatin-overexpressing hPDLCs, and decreased in visfatin-silencing inflammatory hGFs (iGFs) when treated with Pg LPS. In the absence of Pg LPS, visfatin silencing failed to affect the expression of these factors in iGFs, and overexpression of visfatin upregulated MMPs but no other factors in hPDLCs. Furthermore, marked NF-κB pathway activation with increased phosphorylation of p65 was observed in visfatin-overexpressing hPDLCs. BAY11-7082, a specific inhibitor of NF-κB, partially reversed the upregulation proinflammatory and prodegradative factors induced by visfatin overexpression. Taken together, this study showed that visfatin critically regulates Pg LPS-induced proinflammatory/prodegradative effects in healthy and inflammatory periodontal cells partially via NF-κB pathway. The findings suggest that visfatin is closely involved in the development of periodontitis, and may serve as a promising novel biomarker and therapeutic target for periodontitis management.     

miR-488 mediates negative regulation of the AKT/NF-κB pathway by targeting Rac1 in LPS-induced inflammation

Endometritis is an inflammatory change in the structure of the endometrium due to various causes and is a common cause of infertility. Studies have confirmed that microRNAs (miRNAs) play a key regulatory role in various inflammatory diseases. However, the miRNA-mediated mechanism of endometrial inflammation induced by lipopolysaccharides (LPS) remains unclear. In this study, real-time quantitative polymerase chain reaction, Western blot analysis, immunofluorescence and Rac family small GTPase 1 (Rac1) interference were used to reveal the overexpression of miR-488 in the LPS-induced bovine uterus, and the effect of protein kinase B κ-light chain enhancement of the nuclear factor-activated B cells (AKT/NF-κB) pathway in intimal epithelial cells. The results showed that the expression of inflammatory cytokines such as interleukin-1β (IL-1β), IL-6, and tumor necrosis factor-α in the experimental group was significantly lower than that in the control group when miR-488 was overexpressed. Similar results were observed in the expression levels of p-AKT, p-IKK, and p-p65 proteins. In addition, the dual-luciferase reporter system confirmed that miRNA-488 may directly target the 3′-untranslated region of Rac1. In turn, the expression of Rac1 was inhibited. Moreover, the nuclear translocation of NF-κB was inhibited, and meanwhile, the accumulation of reactive oxygen species (ROS) in the cells was reduced. Thus, we provide basic data for the negative regulation of miR-488 in LPS-induced inflammation by inhibiting ROS production and the AKT/NF-kB pathway in intimal epithelial cells.     

Leukotriene B(4) inhibits neutrophil apoptosis via NADPH oxidase activity: redox control of NF-κB pathway and mitochondrial stability

Leukotriene B(4), an arachidonic acid-derived lipid mediator, is a known proinflammatory agent that has a direct effect upon neutrophil physiology, inducing reactive oxygen species generation by the NADPH oxidase complex and impairing neutrophil spontaneous apoptosis, which in turn may corroborate to the onset of chronic inflammation. Despite those facts, a direct link between inhibition of neutrophil spontaneous apoptosis and NADPH oxidase activation by leukotriene B(4) has not been addressed so far. In this study, we aim to elucidate the putative role of NADPH oxidase-derived reactive oxygen species in leukotriene B(4)-induced anti-apoptotic effect. Our results indicate that NADPH oxidase-derived reactive oxygen species are critical to leukotriene B(4) pro-survival effect on neutrophils. This effect also relies on redox modulation of nuclear factor kappaB signaling pathway. We have also observed that LTB(4)-induced Bad degradation and mitochondrial stability require NADPH oxidase activity. All together, our results strongly suggest that LTB(4)-induced anti-apoptotic effect in neutrophils occurs in a reactive oxygen species-dependent manner. We do believe that a better knowledge of the molecular mechanisms underlying neutrophil spontaneous apoptosis may contribute to the development of more successful strategies to control chronic inflammatory conditions such as rheumatoid arthritis.     

Mechanism of NF-κB signaling pathway and autophagy in the regulation of osteoblast differentiation

Objective: The objective of the present work was to investigate a possible mechanism of NF-κB signaling pathway and autophagy in the regulation of osteoblast differentiation, and provide experimental basis for the study of tooth eruption disorder.

Methods: Mouse osteoblast-like (MC3T3-E1) cells were inoculated with a cell density of 70%. According to the grouping experimental design, Western blot and monodansylcadaverine (MDC) detection were conducted after dosing for 24?h. The cells were divided into the following five groups: blank control group; 6.25?µg/mL SN50 group; 12.5?µg/mL SN50 group; 25?µg/mL SN50 group and 50?µg/mL SN50 group.

Results: Western blot analysis revealed that the expression of LC3 protein was present in the blank control group; 6.25?µg/mL SN50 group; 12.5?µg/mL SN50 group and 50?µg/mL SN50 group, with no significant differences among these groups. However, the expression of LC3 protein was significantly lower in the 25?µg/mL SN50 group. MDC detection showed that, in the blank control group; 6.25?µg/mL SN50 group; 12.5?µg/mL SN50 group and 50?µg/mL SN50 group, there was obvious green fluorescence in the cytoplasm of the osteoblasts. However, in the 25?µg/mL SN50 group, it was found that there were significantly fewer green fluorescent particles.

Conclusion: The osteoblast itself had a strong function of autophagy. The appropriate concentration of SN50 in blocking the NF-κB pathway of the osteoblast was associated with the obvious inhibition of autophagy. However, the relationship between NF-κB signaling pathway and autophagy in the process of tooth eruption requires further study.     

Construction of NF-κB-targeting RNAi adenovirus vector and the effect of NF-κB pathway on proliferation and apoptosis of vascular endothelial cells

To construct a recombinant adenovirus vector expressing a RNAi for the Nuclear Factor kappa B (NF-κB)/p65 gene and use it to explore the role of the NF-κB pathway on the regulation of proliferation and apoptosis of vascular endothelial cells. A recombinant adenovirus containing a RNAi cassette targeting the p65 gene was constructed, and its silencing effect on p65 was detected by Western blot analysis in ECV304 cells. Expression of the p65 protein in ECV304 cells was efficiently down-regulated by the RNAi adenovirus for more than 6 days. ECV304 cells proliferation and apoptosis were measured using the MTT assay and flow cytometry, respectively. Blocking the NF-κB pathway with the RNAi adenovirus substantially decreased the proliferation of ECV304 cells, but only slightly affected cell apoptosis. We used a NF-κB/p65-targeting RNAi adenovirus to demonstrate the role of the NF-κB pathway in the regulation of ECV304 cell proliferation. This adenovirus may serve as an important tool to study the NF-κB pathway.     

Overexpression of tissue factor pathway inhibitor in CHO-K1 cells results in increased activation of NF-κB and apoptosis mediated by a caspase-3 independent pathway

There is now circumstantial evidence that tissue factor pathway inhibitor (TFPI) is not only a major anticoagulant, but also has proapoptotic properties. The current study was designed to address the role of TFPI on signalling pathways and apoptosis. The non-TFPI expressing cell line CHO-K1 was stably transfected with pcDNA3.1/V5-His-TOPO-TFPI and control cells were established by transfecting the CHO-K1 cells with pcDNA3.1/V5-His-TOPO. Sodium butyrate (NaBut) has been shown to induce the expression of recombinant proteins. Here we have used NaBut to increase the expression of TFPI as assessed by qRT-PCR and ELISA. Compared to the control cells, TFPI induced apoptosis in a concentration dependent manner as measured by a cell death detection assay. Independent of caspase-3 activation an increased cleavage of PARP was detected in the TFPI expressing cells. This was accompanied by downregulation of Bcl-XL, elevated levels of Bax, and increased translocation of the apoptosis initiating factor. Increased DNA binding activity of NF-κB was revealed by electrophoretic mobility shift assay when the TFPI level was elevated by NaBut together with an increased translocation of the NF-κB subunit p65. The results indicate that TFPI affected the apoptotic activity through a process independent of caspase-3, and was also able to increase the activation of the NF- κB pathway.     

A critical role of redox state in determining HL-60 cell granulocytic differentiation and apoptosis via involvement of PKC and NF-κB

The modifications of intracellular redox balance leads to important cellular changes in many cell types. Here, a causal relationship among redox state, granulocytic differentiation induced by all-trans retinoic acid (RA) or dibutyryl cAMP (dbcAMP) and apoptosis have been studied in the human acute promyelocytic leukaemia HL-60 cells. The modulation of intracellular reactive oxygen species levels by d, l-buthionine-(S, R) sulfoximide (BSO), and n-acetyl-l-cysteine (NAC) caused inducer- and time-dependent or stage-specific effects on HL-60 cell growth inhibition, differentiation and subsequent apoptosis. The presence of BSO during the commitment stage suppressed RA—but not dbcAMP-mediated differentiation, while NAC inhibited both. BSO alone and in combination with RA or dbcAMP-induced apoptosis, which was prevented by NAC in dbcAMP—but not in RA-treated cells. Using protein kinase C inhibitor, calphostin C, cross-talk effects between the intracellular redox state and PKC signalling was identified by demonstrating inducer-dependent changes in cell differentiation or apoptosis, which were associated with the changes in DNA-NF-κB binding activity. These observations suggest a critical role of redox state in determining HL-60 cell behaviour and provide new insights into the complex effects of redox perturbations on the intracellular signalling network via the involvement of PKC and NF-κB.     

Chlorogenic acid inhibits proliferation in human hepatoma cells by suppressing noncanonical NF-κB signaling pathway and triggering mitochondrial apoptosis

Chlorogenic acid (CGA), a phenylpropanoid derived from Eucommia ulmoides Oliver, has been shown to exhibit potent cytotoxic and anti-proliferative activities against several human cancers. However, the effects of CGA on hepatocellular carcinoma (HCC) and the underlying mechanisms have not been intensively studied. In this study, the CGA treatment effects on the viability of human hepatoma cells were investigated by MTT assay. Our data showed that CGA could dose-dependently inhibit the activity of human hepatoma cells Hep-G2 and Huh-7, but did not affect the activity and growth of normal human hepatocyte QSG-7701. The genes and pathways influenced by CGA treatment were explored by RNA sequencing and bioinformatics analysis, which identified 323 differentially expressed genes (DEGs) involved in multiple pharmacological signaling pathways such as MAPK, NF-κB, apoptosis and TGF-β signaling pathways. Further analyses by real-time quantitative PCR, Western blot and flow cytometry revealed that CGA effectually suppressed the noncanonical NF-κB signaling pathway, meanwhile it activated the mitochondrial apoptosis of HCC by upregulation of the BH3-only protein Bcl-2 binding component 3 (BBC3). Our findings demonstrated the potential of CGA in suppressing human hepatoma cells and provided a new insight into the anti-cancer mechanism of CGA.