Inhibition of Nuclear Transcription Factor-κB by Specific IκB Kinase Peptide Inhibitor

Pharmaceutical Research -     

Tetrachlorobenzoquinone exhibits neurotoxicity by inducing inflammatory responses through ROS-mediated IKK/IκB/NF-κB signaling

Tetrachlorobenzoquinone (TCBQ) is a joint metabolite of persistent organic pollutants (POPs), hexachlorobenzene (HCB) and pentachlorophenol (PCP). Previous studies have been reported that TCBQ contributes to acute hepatic damage due to its pro-oxidative nature. In the current study, TCBQ showed the highest capacity on the cytotoxicity, ROS formation and inflammatory cytokines release among four compounds, i.e., HCB, PCP, tetrachlorohydroquinone (TCHQ, reduced form of TCBQ) and TCBQ, in PC 12 cells. Further mechanistic study illustrated TCBQ activates nuclear factor-kappa B (NF-κB) signaling. The activation of NF-κB was identified by measuring the protein expressions of inhibitor of nuclear factor kappa-B kinase (IKK) α/β, p-IKKα/β, an inhibitor of NF-κB (IκB) α, p-IκBα, NF-κB (p65) and p-p65. The translocation of NF-κB was assessed by Western blotting of p65 in nuclear/cytosolic fractions, electrophoretic mobility shift assay (EMSA) and luciferase reporter gene assay. In addition, TCBQ significantly induced protein and mRNA expressions of inflammatory cytokines and mediators, such as interleukin-1 beta (IL-1β), IL-6, tumor necrosis factor-alpha (TNF-α), inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2) and the production of nitric oxide (NO) and prostaglandin E2 (PGE2). Pyrrolidine dithiocarbamate (PDTC), a specific NF-κB inhibitor inhibited these effects efficiently, further suggested TCBQ-induced inflammatory responses involve NF-κB signaling. Moreover, antioxidants, i.e., N-acetyl-l-cysteine (NAC), Vitamin E and curcumin, ameliorated TCBQ-induced ROS generation as well as the activation of NF-κB, which implied that ROS serve as the upstream molecule of NF-κB signaling. In summary, TCBQ exhibits a neurotoxic effect by inducing oxidative stress-mediated inflammatory responses via the activation of IKK/IκB/NF-κB pathway in PC12 cells.     

LXA4 protects against hypoxic-ischemic damage in neonatal rats by reducing the inflammatory response via the IκB/NF-κB pathway

Hypoxia and the resultant decreases in cerebral blood flow in the perinatal period can lead to neonatal hypoxic-ischemic (HI) brain injury, which can, in turn, cause severe disability or even death. However, the efficacy of current treatment strategies remains limited. Several studies have demonstrated that lipoxin A4 (LXA4), as one of the earliest types of endogenous lipid mediators, can inhibit the accumulation of neutrophils, arrest inflammation, and promote the resolution of inflammation. However, research on LXA4 in the nervous system has rarely been carried out. In the present study, we sought to investigate the protective effect of LXA4 on HI brain damage in neonatal rats, as well as the underlying mechanisms. Through experiments conducted using an HI animal model, we found that the LXA4 intervention promoted the recovery of neuronal function and tissue structure following brain injury while maintaining the integrity of the blood–brain barrier in addition to reducing cerebral edema, infarct volume, and inflammatory responses. Our results suggest that LXA4 interfered with neuronal oxygen-glucose deprivation insults, reduced the expression of inflammatory factors, inhibited apoptosis, and promoted neuronal survival in vitro. Finally, the LXA4 intervention attenuated HI-induced activation of inhibitor kappa B (IκB) and degradation of nuclear factor-κB (NF-κB). In conclusion, our data suggest that LXA4 exerts a neuroprotective effect against neonatal HI brain damage through the IκB/NF-κB pathway. Our findings will help inform future studies regarding the effects of LXA4 on neuroinflammation, blood–brain barrier integrity, and neuronal apoptosis.     

NF-κB的研究进展

核因子κB(NF-κB)是一种广泛存在于真核细胞内的基因多向性转录因子,属于Rel家族,于1986年首次在小鼠B淋巴细胞的核抽提物中发现,因能与免疫球蛋白κ链基因的     

Regulation of cytochrome P450 4F11 by nuclear transcription factor-κB

Although the mechanisms that regulate CYP4F genes have been and are currently being studied in a number of laboratories, the specific mechanisms for the regulation of these genes are not yet fully understood. This study shows that nuclear factor κB of the light-chain-enhancer in activated B cells (NF-κB) can inhibit CYP4F11 expression in human liver carcinoma cell line (HepG2) as summarized below. Tumor necrosis factor-α (TNF-α), a proinflammatory cytokine, has been shown to activate NF-κB signaling while also activating the c-Jun NH(2)-terminal kinase (JNK) signaling pathway. Other studies have reported that JNK signaling can up-regulate CYP4F11 expression. The results of this study demonstrate that in the presence of TNF-α and the specific NF-κB translocation inhibitor N-[3,5-bis(trifluoromethyl)phenyl]-5-chloro-2-hydroxybenzamide (IMD-0354), there is a greater increase in CYP4F11 expression than that elicited by TNF-α alone, indicating that NF-κB plays an inhibitory role. Moreover, NF-κB stimulation by overexpression of mitogen-activated protein kinase kinase kinase inhibited CYP4F11 promoter expression. CYP4F11 promoter inhibition can also be rescued in the presence of TNF-α when p65, a NF-κB protein, is knocked down. Thus, NF-κB signaling pathways negatively regulate the CYP4F11 gene.     

Naringin attenuates acute lung injury in LPS-treated mice by inhibiting NF-κB pathway

Naringin has been reported as an effective anti-inflammatory compound. We previously showed that naringin had antitussive effect on experimentally induced cough in guinea pigs. However, the effects and mechanism of naringin on lipopolysaccharide (LPS)-induced acute lung injury (ALI) in mice are not fully understood. In this study, our aim was to evaluate the anti-inflammatory activities of naringin on LPS-induced ALI in mice and clarify its underlying mechanisms of action. We found that in vivo pretreatment with naringin markedly decreased the lung wet weight to dry weight ratio, and led to significant attenuation of LPS-induced evident lung histopathological changes. Meanwhile, naringin significantly reduced bronchoalveolar lavage fluid (BALF) total cell and neutrophil (PMN) counts after LPS challenge. Furthermore, naringin inhibited myeloperoxidase (MPO: a marker enzyme of neutrophil granule) and inducible nitric oxide synthase (iNOS) activities in lung tissue and alleviated LPS-induced tumor neurosis factor-α (TNF-α) secretion in BALF in a dose-dependent manner. Additionally, Western blotting showed that naringin efficiently blunt NF-κB activation by inhibiting the degradation of I?B-α and the translocation of p65. Taken together, these results suggest that naringin shows anti-inflammatory effects through inhibiting lung edema, MPO and iNOS activities, TNF-α secretion and pulmonary neutrophil infiltration by blockade of NF-κB in LPS-induced ALI.     

Tetramethylpyrazine protected photoreceptor cells of rats by modulating nuclear translocation of NF-κB

Aim: To evaluate the effect of tetramethylpyrazine (TMP) injection on retinal damage induced by N-methyl-N-nitrosourea (MNU) in rats and on nuclear factorkappa B (NF-κB) family members. Methods: Female Sprague-Dawley (SD) rats were randomly divided into groups: (i), control group; (ii), model group; and (iii), TMP-injection groups, in which the rats were subdivided into 40 mg/kg, 80 mg/kg and 160 mg/kg groups. Drugs were injected ip into 47-day-old SD rats once a day. At 50 days of age, all rats in the model group and drug groups also received a single ip injection of 60 mg/kg MNU. Rats in group 1 received ip injection of physiological saline. All rats were killed at different times after MNU or physiological saline treatment. The apoptotic index of photoreceptor ceils was calculated by TUNEL labeling; retinal damage was evaluated based on retinal thickness and the expression of NF-nB family members was detected by Western blot. Results: TMP injections, in a dose-dependent manner, suppressed photoreceptor cell apoptosis and decreased its loss in the peripheral retina. As compared with the MNU-treated group, TMP injection at a dose of 160 mg/kg also timedependently upregulated the NF-κB/p65 protein level in the nucleus and downregulated the IκBα protein level in the cytoplasm. However, no protective effect of TMP injection on MNU-induced central retinal damage was found. Conclusion: TMP injection partially protects against MNU-induced retinal damage by upregulating the nuclear translocation of p65 to inhibit photoreceptor cells apoptosis.     

Salvianolic acid A suppress lipopolysaccharide-induced NF-κB signaling pathway by targeting IKKβ

Salvianolic acid A, an active compound present in Salvia miltiorrhiza, is a phenolic carboxylic acid derivative, ((2R)-3-(3,4-Dihydroxyphenyl)-2-[(E)-3-[2-[(E)-2-(3,4-dihydroxyphenyl) ethenyl]-3,4-dihydroxyphenyl] prop-2-enoyl]oxypropanoic acid). The present study was performed to investigate the underlying mechanisms of anti-inflammatory effects with salvianolic acid A, specially focused on nuclear factor κB (NF-κB) signaling pathway by targeting the IκB kinase β (IKKβ). The effect of salvianolic acid A for IKKβ activity was analyzed using an immobilized metal affinity for phosphochemicals (IMAP)-based time-resolved fluorescence resonance energy transfer (TR-FRET) assay. The underlying mechanisms of salvianolic acid A were examined using lipopolysaccharide (LPS)-stimulated RAW264.7 cells. IKKβ studies based on IMAP-TR-FRET showed that salvianolic acid A possesses a potent IKKβ inhibitory activity with Ki value of 3.63 μM in an ATP-noncompetitive manner. Pretreatment with salvianolic acid A (10, 30 μM) decreased LPS-induced expression of iNOS and COX-2, thereby inhibiting production of nitric oxide and prostaglandin E₂, respectively. In addition, salvianolic acid A (10, 30 μM) also attenuated the LPS-induced IκBα phosphorylation and degradation, and NF-κB translocation. These results suggest that salvianolic acid A modulates NF-κB-dependent inflammatory pathways through IKKβ inhibition and these anti-inflammatory effects will aid in understanding the pharmacology and mode of action of salvianolic acid A.     

Induction of Fas receptor and Fas ligand by nodularin is mediated by NF-κB in HepG2 cells

Nodularin is a natural toxin with multiple features, including inhibitor of protein phosphatases 1 and 2A as well as tumor initiator and promoter. One unique feature of nodularin is that this chemical is a hepatotoxin. It can accumulate into the liver after contact and lead to severe damage to hepatocyte, such as apoptosis. Fas receptor (Fas) and Fas ligand (FasL) system is a critical signaling network triggering apoptosis. In current study, we investigated whether nodularin can induce Fas and FasL expression in HepG2 cell, a well used in vitro model for the study of human hepatocytes. Our data showed nodularin induced Fas and FasL expression, at both mRNA and protein level, in a time- and dose-dependent manner. We also found nodularin induced apoptosis at the concentration and incubation time that Fas and FasL were significantly induced. Neutralizing antibody to FasL reduced nodularin-induced apoptosis. Further studies demonstrated that nodularin promoted nuclear translocation and activation of p65 subunit of NF-κB. By applying siRNA targeting p65, which knocked down p65 in HepG2 cells, we successfully impaired the activation of NF-κB by nodularin. In these p65 knockdown cells, we observed that Fas and FasL expression and apoptosis induced by nodularin were significantly reduced. These findings suggest the induction of Fas and FasL expression and thus cell apoptosis in HepG2 cells by nodularin is mediated through NF-κB pathway.     

Icariin potentiates the antitumor activity of gemcitabine in gallbladder cancer by suppressing NF-κB

Aim:

Gemcitabine has been increasingly prescribed for the treatment of gallbladder cancer. However, the response rate is low. The aim of this study is to determine whether icariin, a flavonoid isolated from Epimedi herba, could potentiate the antitumor activity of gemcitabine in gallbladder cancer.

Methods:

Human gallbladder carcinoma cell lines GBC-SD and SGC-996 were tested. Cell proliferation and apoptosis were analyzed using MTT assay and flow cytometry, respectively. The expression of apoptosis- and proliferation-related molecules was detected with Western blotting. Caspase-3 activity was analyzed using colorimetric assay, and NF-κB activity was measured with ELISA. A gallbladder cancer xenograft model was established in female BALB/c (nu/nu) mice. The mice were intraperitoneally administered gemcitabine (125 mg/kg) in combination with icariin (40 mg/kg) for 2 weeks.

Results:

Icariin (40–160 μg/mL) dose-dependently suppressed cell proliferation and induced apoptosis in both GBC-SD and SGC-996 cells, with SGC-996 cells being less sensitive to the drug. Icariin (40 μg/mL) significantly enhanced the antitumor activity of gemcitabine (0.5 μmol/L) in both GBC-SD and SGC-996 cells. The mice bearing gallbladder cancer xenograft treated with gemcitabine in combination with icariin exhibited significantly smaller tumor size than the mice treated with either drug alone. In GBC-SD cells, icariin significantly inhibited both the constitutive and gemcitabine-induced NF-κB activity, enhanced caspase-3 activity, induced G₀-G₁ phase arrest, and suppressed the expression of Bcl-2, Bcl-xL and surviving proteins.

Conclusion:

Icariin, by suppressing NF-κB activity, exerts antitumor activity, and potentiates the antitumor activity of gemcitabine in gallbladder cancer. Combined administration of gemcitabine and icariin may offer a better therapeutic option for the patients with gallbladder cancer.