Anti-Inflammatory Effects of Psoralen Derivatives on RAW264.7 Cells via Regulation of the NF-κB and MAPK Signaling Pathways

Using repositioning to find new indications for existing functional substances has become a global target of research. The objective of this study is to investigate the anti-inflammatory potential of psoralen derivatives (5-hydroxypsoralen, 5-methoxypsoralen, 8-hydroxypsoralen, and 8-methoxypsoralen) in macrophages cells. The results indicated that most psoralen derivatives exhibited significantly inhibited prostaglandin E₂ (PGE₂) production, particularly for 8-hydroxypsoralen (xanthotoxol) in lipopolysaccharide (LPS)-stimulated macrophage RAW 264.7 cells. In addition, xanthotoxol treatment decreased the PGE₂, IL-6, and IL-1β production caused by LPS stimulation in a concentration-dependent manner. Moreover, Western blot results showed that the protein levels of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), which activated with LPS treatment, were decreased by xanthotoxol treatment. Mechanistic studies revealed that xanthotoxol also suppressed LPS-stimulated phosphorylation of the inhibitor of κBα (IκBα), p38 mitogen-activated protein kinase (MAPK), and c-Jun N-terminal kinase (JNK) in RAW 264.7 cells. The Western blot assay results show that xanthotoxol suppresses LPS-induced p65 translocation from cytosol to the nucleus in RAW 264.7 cells. Moreover, we tested the potential application of xanthotoxol as a cosmetic material by performing human skin patch tests. In these tests, xanthotoxol did not induce any adverse reactions at a 100 μΜ concentration. These results demonstrate that xanthotoxol is a potential therapeutic agent for topical application that inhibits inflammation via the MAPK and NF-κB pathways.     

Prunetinoside Inhibits Lipopolysaccharide-Provoked Inflammatory Response via Suppressing NF-κB and Activating the JNK-Mediated Signaling Pathway in RAW264.7 Macrophage Cells

Inflammation is a multifaceted response of the immune system at the site of injury or infection caused by pathogens or stress via immune cells. Due to the adverse effects of chemical drugs, plant-based compounds are gaining interest in current research. Prunetinoside or prunetin-5-O-glucoside (PUG) is a plant-based active compound, which possesses anti-inflammatory effects on immune cells. In this study, we investigate the effect of PUG on mouse macrophage RAW264.7 cells with or without stimulation of lipopolysaccharide (LPS). Cytotoxicity results showed that PUG is non-cytotoxic to the cells and it reversed the cytotoxicity in LPS-stimulated cells. The levels of nitric oxide (NO) and interleukin-6 (IL-6) were determined using a NO detection kit and IL-6 ELISA kit, respectively, and showed a significant decrease in NO and IL-6 in PUG-treated cells. Western blot and qRT-PCR were performed for the expression of two important pro-inflammatory cytokines, COX2 and iNOS, and found that their expression was downregulated in a dose-dependent manner. Other pro-inflammatory cytokines, such as IL-1β, IL-6, and TNFα, had reduced mRNA expression after PUG treatment. Furthermore, a Western blot was performed to calculate the expression of NF-κB and MAPK pathway proteins. The results show that PUG administration dramatically reduced the phosphorylation of p-Iκbα, p-NF-κB 65, and p-JNK. Remarkably, after PUG treatment, p-P38 and p-ERK remain unchanged. Furthermore, docking studies revealed that PUG is covalently linked to NF-κB and suppresses inflammation. In conclusion, PUG exerted the anti-inflammatory mechanism by barring the NF-κB pathway and activating JNK. Thus, prunetinoside could be adopted as a therapeutic compound for inflammatory-related conditions.     

Eupatilin Suppresses OVA-Induced Asthma by Inhibiting NF-κB and MAPK and Activating Nrf2 Signaling Pathways in Mice

To investigate the effect of eupatilin in asthma treatment, we evaluated its therapeutic effect and related signal transduction in OVA-induced asthmatic mice and LPS-stimulated RAW264.7 cells. The BALF was tested for changes in lung inflammatory cells. Th2 cytokines in the BALF and OVA-IgE in the serum were measured by ELISA. H&E and PAS staining were used to evaluate histopathological changes in mouse lungs. The key proteins NF-κB, MAPK, and Nrf2 in lung tissues were quantitatively analyzed by Western blotting. Finally, we evaluated the effect of eupatilin on cytokines and related protein expression in LPS-stimulated RAW 264.7 cells in vitro. In OVA-induced asthmatic mice, eupatilin reduced the numbers of inflammatory cells, especially neutrophils and eosinophils. Eupatilin also decreased the levels of IL-5, IL-13 in the BALF and OVA-IgE in the serum. Furthermore, eupatilin inhibited the activation of NF-κB and MAPK pathways and increased the expression of Nrf2 in OVA-induced asthmatic mice. In vitro, eupatilin significantly reduced LPS-stimulated NO, IL-6, and ROS production. Additionally, the NF-κB, MAPK, and Nrf2 protein expression in LPS-stimulated RAW264.7 cells was consistent with that in OVA-induced asthmatic lung tissues. In summary, eupatilin attenuated OVA-induced asthma by regulating NF-κB, MAPK, and Nrf2 signaling pathways. These results suggest the utility of eupatilin as an anti-inflammatory drug for asthma treatment.     

Geranylgeraniol Inhibits Lipopolysaccharide-Induced Inflammation in Mouse-Derived MG6 Microglial Cells via NF-κB Signaling Modulation

Persistent inflammatory reactions in microglial cells are strongly associated with neurodegenerative pathogenesis. Additionally, geranylgeraniol (GGOH), a plant-derived isoprenoid, has been found to improve inflammatory conditions in several animal models. It has also been observed that its chemical structure is similar to that of the side chain of menaquinone-4, which is a vitamin K₂ sub-type that suppresses inflammation in mouse-derived microglial cells. In this study, we investigated whether GGOH has a similar anti-inflammatory effect in activated microglial cells. Particularly, mouse-derived MG6 cells pre-treated with GGOH were exposed to lipopolysaccharide (LPS). Thereafter, the mRNA levels of pro-inflammatory cytokines were determined via qRT-PCR, while protein expression levels, especially the expression of NF-κB signaling cascade-related proteins, were determined via Western blot analysis. The distribution of NF-κB p65 protein was also analyzed via fluorescence microscopy. Thus, it was observed that GGOH dose-dependently suppressed the LPS-induced increase in the mRNA levels of Il-1β, Tnf-α, Il-6, and Cox-2. Furthermore, GGOH inhibited the phosphorylation of TAK1, IKKα/β, and NF-κB p65 proteins as well as NF-κB nuclear translocation induced by LPS while maintaining IκBα expression. We showed that GGOH, similar to menaquinone-4, could alleviate LPS-induced microglial inflammation by targeting the NF-kB signaling pathway.     

α-Bisabolol Attenuates Doxorubicin Induced Renal Toxicity by Modulating NF-κB/MAPK Signaling and Caspase-Dependent Apoptosis in Rats

Doxorubicin (DOX) is a well-known and effective antineoplastic agent of the anthracycline family. But, multiple organ toxicities compromise its invaluable therapeutic usage. Among many toxicity types, nephrotoxicity is one of the major concerns. In recent years many approaches, including bioactive agents of natural origin, have been explored to provide protective effects against chemotherapy-related complications. α-Bisabolol is a naturally occurring monocyclic sesquiterpene alcohol identified in the essential oils of various aromatic plants and possesses a wide range of pharmacological properties such as antioxidant, anti-inflammatory, analgesic, cardioprotective, antibiotic, anti-irritant, and anticancer activities. The present study aimed to evaluate the effects of α-Bisabolol on DOX-induced nephrotoxicity in Wistar male albino rats. Nephrotoxicity was induced in rats by injecting a single dose of DOX (12.5 mg/kg, i.p.), and the test compound, α-Bisabolol (25 mg/kg) was administered intraperitoneally along with DOX as a co-treatment daily for 5 days. DOX-injected rats showed reduction in body weight along with a concomitant fall in antioxidants and increased lipid peroxidation in the kidney. DOX-injection also increased levels/expressions of proinflammatory cytokines namely tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-1β (IL-1β) and inflammatory mediators like inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) and activated nuclear factor kappa-B (NF-κB)/mitogen-activated protein kinases (MAPK) signaling in the kidney tissues. DOX also triggered apoptotic cell death, evidenced by the increased expression of pro-apoptotic markers like BCL2-Associated X Protein (Bax), cleaved caspase-3, caspase- 9, and cytochrome-C) and a decrease in the expressions of anti-apoptotic markers namely B-cell lymphoma 2 (Bcl2) and B-cell lymphoma-extra large (Bcl-xL) in the kidney. These biochemical alterations were additionally supported by light microscopic findings, which revealed structural alterations in the kidney. However, treatment with α-Bisabolol prevented body weight loss, restored antioxidants, mitigated lipid peroxidation, and inhibited the rise in proinflammatory cytokines, as well as favorably modulated the expressions of NF-κB/MAPK signaling and apoptosis markers in DOX-induced nephrotoxicity. Based on the results observed, it can be concluded that α-Bisabolol has potential to attenuate DOX-induced nephrotoxicity by inhibiting oxidative stress and inflammation mediated activation of NF-κB/MAPK signaling alongwith intrinsic pathway of apoptosis in rats. The study findings are suggestive of protective potential of α-Bisabolol in DOX associated nephrotoxicity and this could be potentially useful in minimizing the adverse effects of DOX and may be a potential agent or adjuvant for renal protection.     

Isoleucilactucin Ameliorates Coal Fly Ash-Induced Inflammation through the NF-κB and MAPK Pathways in MH-S Cells

We investigated whether isoleucilactucin, an active constituent of Ixeridium dentatum, reduces inflammation caused by coal fly ash (CFA) in alveolar macrophages (MH-S). The anti-inflammatory effects of isoleucilactucin were assessed by measuring the concentration of nitric oxide (NO) and the expression of pro-inflammatory mediators in MH-S cells exposed to CFA-induced inflammation. We found that isoleucilactucin reduced CFA-induced NO generation dose-dependently in MH-S cells. Moreover, isoleucilactucin suppressed CFA-activated proinflammatory mediators, including cyclooxygenase-2 (COX2) and inducible NO synthase (iNOS), and the proinflammatory cytokines such as interleukin-(IL)-1β, IL-6, and tumor necrosis factor (TNF-α). The inhibiting properties of isoleucilactucin on the nuclear translocation of phosphorylated nuclear factor-kappa B (p-NF-κB) were observed. The effects of isoleucilactucin on the NF-κB and mitogen-activated protein kinase (MAPK) pathways were also measured in CFA-stimulated MH-S cells. These results indicate that isoleucilactucin suppressed CFA-stimulated inflammation in MH-S cells by inhibiting the NF-κB and MAPK pathways, which suggest it might exert anti-inflammatory properties in the lung.     

Selenium-Rich Yeast Peptide Fraction Ameliorates Imiquimod-Induced Psoriasis-like Dermatitis in Mice by Inhibiting Inflammation via MAPK and NF-κB Signaling Pathways

Psoriasis, a chronic and immune-mediated inflammatory disease, adversely affects patients’ lives. We previously prepared selenium-rich yeast peptide fraction (SeP) from selenium-rich yeast protein hydrolysate and found that SeP could effectively alleviate ultraviolet radiation-induced skin damage in mice and inhibited H₂O₂-induced cytotoxicity in cultured human epidermal keratinocyte (HaCaT) cells. This study aimed to investigate whether SeP had a protective effect on imiquimod (IMQ)-induced psoriasis-like dermatitis in mice and the underlying mechanisms. Results showed that SeP significantly ameliorated the severity of skin lesion in IMQ-induced psoriasis-like mouse model. Moreover, SeP treatment significantly attenuated the expression of key inflammatory cytokines, including interleukin (IL)-23, IL-17A, and IL-17F, in the dorsal skin of mice. Mechanistically, SeP application not only inhibited the activation of JNK and p38 MAPK, but also the translocation of NF-κB into the nucleus in the dorsal skin. Furthermore, SeP treatment inhibited the levels of inflammatory cytokines and the activation of MAPK and NF-κB signaling induced by lipopolysaccharide in HaCaT cells and macrophage cell line RAW264.7. Overall, our findings showed that SeP alleviated psoriasis-like skin inflammation by inhibiting MAPK and NF-κB signaling pathways, which suggested that SeP would have a potential therapeutic effect against psoriasis.     

Nrf2-Activating Bioactive Peptides Exert Anti-Inflammatory Activity through Inhibition of the NF-κB Pathway

Redox status and inflammation are related to the pathogenesis of the majority of diseases. Therefore, understanding the role of specific food-derived molecules in the regulation of their specific pathways is a relevant issue. Our previous studies indicated that K-8-K and S-10-S, milk and soy-derived bioactive peptides, respectively, exert antioxidant effects through activation of the Keap1/Nrf2 pathway. A crosstalk between Nrf2 and NF-κB, mediated by the action of heme oxygenase (HO-1), is well known. On this basis, we studied if these peptides, in addition to their antioxidant activity, could exert anti-inflammatory effects in human cells. First, we observed an increase of HO-1 expression in Caco-2 cells treated with K-8-K and S-10-S, following the activation of the Keap1/Nrf2 pathway. Moreover, when cells are treated with the two peptides and stimulated by TNF-α, the levels of NF-κB in the nucleus decreased in comparison with TNF-α alone. In the same conditions, we observed the downregulation of the gene expression of proinflammatory cytokines (IL1B, IL6, and TNF), while the anti-inflammatory cytokine gene, IL1RN, was upregulated in Caco-2 cells processed as reported above. Then, when the cells were pretreated with the two peptides and stimulated with LPS, a different proinflammatory factor, (TNF-α) was estimated to have a lower secretion in the supernatant of cells. In conclusion, these observations confirmed that Nrf2-activating bioactive peptides, K-8-K and S-10-S, exerted anti-inflammatory effects by inhibiting the NF-κB pathway.     

Naegleria fowleri Cathepsin B Induces a Pro-Inflammatory Immune Response in BV-2 Microglial Cells via NF-κB and AP-1 Dependent-MAPK Signaling Pathway

Naegleria fowleri is a ubiquitous protozoa parasite that can cause primary amoebic meningoencephalitis (PAM), a fatal brain infection in humans. Cathepsin Bs of N. fowleri (NfCBs) are multifamily enzymes. Although their pathogenic mechanism in PAM is not clearly understood yet, NfCBs have been proposed as pathogenic factors involved in the pathogenicity of amoeba. In this study, the immune response of BV-2 microglial cells induced by NfCB was analyzed. Recombinant NfCB (rNfCB) evoked enhanced expressions of TLR-2, TLR-4, and MyD88 in BV-2 microglial cells. This enzyme also induced an elevated production of several pro-inflammatory cytokines such as TNF-α, IL-1α, IL-1β, and IL-6 and iNOS in cells. The inhibition of mitogen-activated protein kinases (MAPKs), including JNK, p38, and ERK, effectively reduced the production of these pro-inflammatory cytokines. The rNfCB-induced production of pro-inflammatory cytokines in BV-2 microglial cells was suppressed by inhibiting NF-kB and AP-1. Phosphorylation and nuclear translocation of p65 in cells were also enhanced by rNfCB. These results suggest that NfCB can induce a pro-inflammatory immune response in BV-2 microglial cells via the NF-κB- and AP-1-dependent MAPK signaling pathways. Such a NfCB-induced pro-inflammatory immune response in BV-2 microglial cells might contribute to the pathogenesis of PAM caused by amoeba, by exacerbating deleterious immune responses and tissue damages in N. fowleri-infected foci of the brain.     

GPI-80 Augments NF-κB Activation in Tumor Cells

Recent studies have discovered a relationship between glycosylphosphatidylinositol (GPI)-anchored protein 80 (GPI-80)/VNN2 (80 kDa GPI-anchored protein) and malignant tumors. GPI-80 is known to regulate neutrophil adhesion; however, the action of GPI-80 on tumors is still obscure. In this study, although the expression of GPI-80 mRNA was detectable in several tumor cell lines, the levels of GPI-80 protein were significantly lower than that in neutrophils. To clarify the function of GPI-80 in tumor cells, GPI-80-expressing cells and GPI-80/VNN2 gene-deleted cells were established using PC3 prostate cancer cells. In GPI-80-expressing cells, GPI-80 was mainly detected in vesicles. Furthermore, soluble GPI-80 in the conditioned medium was associated with the exosome marker CD63 and was also detected in the plasma obtained from prostate cancer patients. Unexpectedly, cell adhesion and migration of GPI-80-expressing PC3 cells were not modulated by anti-GPI-80 antibody treatment. However, similar to the GPI-80 family molecule, VNN1, the pantetheinase activity and oxidative state were augmented in GPI-80-expressing cells. GPI-80-expressing cells facilitated non-adhesive proliferation, slow cell proliferation, NF-κB activation and IL-1β production. These phenomena are known to be induced by physiological elevation of the oxidative state. Thus, these observations indicated that GPI-80 affects various tumor responses related to oxidation.     

The Links between ALS and NF-κB

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease wherein motor neuron degeneration leads to muscle weakness, progressive paralysis, and death within 3–5 years of diagnosis. Currently, the cause of ALS is unknown but, as with several neurodegenerative diseases, the potential role of neuroinflammation has become an increasingly popular hypothesis in ALS research. Indeed, upregulation of neuroinflammatory factors have been observed in both ALS patients and animal models. One such factor is the inflammatory inducer NF-κB. Besides its connection to inflammation, NF-κB activity can be linked to several genes associated to familial forms of ALS, and many of the environmental risk factors of the disease stimulate NF-κB activation. Collectively, this has led many to hypothesize that NF-κB proteins may play a role in ALS pathogenesis. In this review, we discuss the genetic and environmental connections between NF-κB and ALS, as well as how this pathway may affect different CNS cell types, and finally how this may lead to motor neuron degeneration.     

OSMI-1 Enhances TRAIL-Induced Apoptosis through ER Stress and NF-κB Signaling in Colon Cancer Cells

Levels of O-GlcNAc transferase (OGT) and hyper-O-GlcNAcylation expression levels are associated with cancer pathogenesis. This study aimed to find conditions that maximize the therapeutic effect of cancer and minimize tissue damage by combining an OGT inhibitor (OSMI-1) and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). We found that OSMI-1 treatment in HCT116 human colon cancer cells has a potent synergistic effect on TRAIL-induced apoptosis signaling. Interestingly, OSMI-1 significantly increased TRAIL-mediated apoptosis by increasing the expression of the cell surface receptor DR5. ROS-induced endoplasmic reticulum (ER) stress by OSMI-1 not only upregulated CHOP-DR5 signaling but also activated Jun-N-terminal kinase (JNK), resulting in a decrease in Bcl2 and the release of cytochrome c from mitochondria. TRAIL induced the activation of NF-κB and played a role in resistance as an antiapoptotic factor. During this process, O-GlcNAcylation of IκB kinase (IKK) and IκBα degradation occurred, followed by translocation of p65 into the nucleus. However, combination treatment with OSMI-1 counteracted the effect of TRAIL-mediated NF-κB signaling, resulting in a more synergistic effect on apoptosis. Therefore, the combined treatment of OSMI-1 and TRAIL synergistically increased TRAIL-induced apoptosis through caspase-8 activation. Conclusively, OSMI-1 potentially sensitizes TRAIL-induced cell death in HCT116 cells through the blockade of NF-κB signaling and activation of apoptosis through ER stress response.     

Human Caspase 12 Enhances NF-κB Activity through Activation of IKK in Nasopharyngeal Carcinoma Cells

Human nasopharyngeal carcinoma (NPC) is a highly invasive cancer associated with proinflammation. Caspase-12 (Casp12), an inflammatory caspase, is implicated in the regulation of NF-κB-mediated cellular invasion via the modulation of the IκBα protein in NPC cells. However, the effect mechanisms of Casp12 need to be elucidated. NPC cells were transfected with the full length of human Casp12 cDNA (pC12) and the effect of human Casp12 (hCasp12) on the NF-κB activity was investigated. We found ectopic expression of hCasp12 increased the NF-κB activity accompanied by an increased p-IκBα expression and a decreased IκBα expression. Treatment of BMS, a specific IKK inhibitor, and pC12-transfected cells markedly decreased the NF-κB activity and ameliorated the expression level of IκBα reduced by hCasp12. Co-immunoprecipitation assays validated the physical interaction of hCasp12 with IKKα/β, but not with NEMO. Furthermore, the NF-κB activity of ΔCasp12-Q (a mutated catalytic of hCasp12) transfected cells was concentration-dependently induced, but lower than that of hCasp12-transfected cells. Importantly, the hCasp12-mediated NF-kB activity was enhanced by TNFα stimulation. That indicated a role of the catalytic motif of hCasp12 in the regulation of the NF-κB activity. This study indicated hCasp12 activated the NF-κB pathway through the activation of IKK in human NPC cells.     

PARP Inhibitor Inhibits the Vasculogenic Mimicry through a NF-κB-PTX3 Axis Signaling in Breast Cancer Cells

Poly (ADP-ribose) polymerase inhibitors (PARPi) are targeted therapies that inhibit PARP proteins which are involved in a variety of cell functions. PARPi may act as modulators of angiogenesis; however, the relationship between PARPi and the vasculogenic mimicry (VM) in breast cancer remains unclear. To determine whether PARPi regulate the vascular channel formation, we assessed whether the treatment with olaparib, talazoparib and veliparib inhibits the vascular channel formation by breast cancer cell lines. Here, we found that PARPi act as potent inhibitors of the VM formation in triple negative breast cancer cells, independently of the BRCA status. Mechanistically, we find that PARPi trigger and inhibit the NF-κB signaling, leading to the inhibition of the VM. We further show that PARPi decrease the expression of the angiogenic factor PTX3. Moreover, PTX3 rescued the PARPi-inhibited VM inhibition. In conclusion, our results indicate that PARPi, by targeting the VM, may provide a new therapeutic approach for triple negative breast cancer.     

Proteasome Inhibitors Interrupt the Activation of Non-Canonical NF-κB Signaling Pathway and Induce Cell Apoptosis in Cytarabine-Resistant HL60 Cells

Over half of older patients with acute myeloid leukemia (AML) do not respond to cytotoxic chemotherapy, and most responders relapse because of drug resistance. Cytarabine is the main drug used for the treatment of AML. Intensive treatment with high-dose cytarabine can increase the overall survival rate and reduce the relapse rate, but it also increases the likelihood of drug-related side effects. To optimize cytarabine treatment, understanding the mechanism underlying cytarabine resistance in leukemia is necessary. In this study, the gene expression profiles of parental HL60 cells and cytarabine-resistant HL60 (R-HL60) cells were compared through gene expression arrays. Then, the differential gene expression between parental HL60 and R-HL60 cells was measured using KEGG software. The expression of numerous genes associated with the nuclear factor κB (NF-κB) signaling pathway changed during the development of cytarabine resistance. Proteasome inhibitors inhibited the activity of non-canonical NF-κB signaling pathway and induced the apoptosis of R-HL60 cells. The study results support the application and possible mechanism of proteasome inhibitors in patients with relapsed or refractory leukemia.