IKBKB siRNA-Encapsulated Poly (Lactic-co-Glycolic Acid) Nanoparticles Diminish Neuropathic Pain by Inhibiting Microglial Activation

Activation of nuclear factor-kappa B (NF-κB) in microglia plays a decisive role in the progress of neuropathic pain, and the inhibitor of kappa B (IκB) is a protein that blocks the activation of NF-κB and is degraded by the inhibitor of NF-κB kinase subunit beta (IKBKB). The role of IKBKB is to break down IκB, which blocks the activity of NF-kB. Therefore, it prevents the activity of NK-kB. This study investigated whether neuropathic pain can be reduced in spinal nerve ligation (SNL) rats by reducing the activity of microglia by delivering IKBKB small interfering RNA (siRNA)-encapsulated poly (lactic-co-glycolic acid) (PLGA) nanoparticles. PLGA nanoparticles, as a carrier for the delivery of IKBKB genes silencer, were used because they have shown potential to enhance microglial targeting. SNL rats were injected with IKBKB siRNA-encapsulated PLGA nanoparticles intrathecally for behavioral tests on pain response. IKBKB siRNA was delivered for suppressing the expression of IKBKB. In rats injected with IKBKB siRNA-encapsulated PLGA nanoparticles, allodynia caused by mechanical stimulation was reduced, and the secretion of pro-inflammatory mediators due to NF-κB was reduced. Delivering IKBKB siRNA through PLGA nanoparticles can effectively control the inflammatory response and is worth studying as a treatment for neuropathic pain.     

In Silico and In Vitro Screening of 50 Curcumin Compounds as EGFR and NF-κB Inhibitors

The improvement of cancer chemotherapy remains a major challenge, and thus new drugs are urgently required to develop new treatment regimes. Curcumin, a polyphenolic antioxidant derived from the rhizome of turmeric (Curcuma longa L.), has undergone extensive preclinical investigations and, thereby, displayed remarkable efficacy in vitro and in vivo against cancer and other disorders. However, pharmacological limitations of curcumin stimulated the synthesis of numerous novel curcumin analogs, which need to be evaluated for their therapeutic potential. In the present study, we calculated the binding affinities of 50 curcumin derivatives to known cancer-related target proteins of curcumin, i.e., epidermal growth factor receptor (EGFR) and nuclear factor κB (NF-κB) by using a molecular docking approach. The binding energies for EGFR were in a range of −12.12 (±0.21) to −7.34 (±0.07) kcal/mol and those for NF-κB ranged from −12.97 (±0.47) to −6.24 (±0.06) kcal/mol, indicating similar binding affinities of the curcumin compounds for both target proteins. The predicted receptor-ligand binding constants for EGFR and curcumin derivatives were in a range of 0.00013 (±0.00006) to 3.45 (±0.10) µM and for NF-κB in a range of 0.0004 (±0.0003) to 10.05 (±4.03) µM, indicating that the receptor-ligand binding was more stable for EGFR than for NF-κB. Twenty out of 50 curcumin compounds showed binding energies to NF-κB smaller than −10 kcal/mol, while curcumin as a lead compound revealed free binding energies of >−10 kcal/mol. Comparable data were obtained for EGFR: 15 out of 50 curcumin compounds were bound to EGFR with free binding energies of <−10 kcal/mol, while the binding affinity of curcumin itself was >−10 kcal/mol. This indicates that the derivatization of curcumin may indeed be a promising strategy to improve targe specificity and to obtain more effective anticancer drug candidates. The in silico results have been exemplarily validated using microscale thermophoresis. The bioactivity has been further investigated by using resazurin cell viability assay, lactate dehydrogenase assay, flow cytometric measurement of reactive oxygen species, and annexin V/propidium iodide assay. In conclusion, molecular docking represents a valuable approach to facilitate and speed up the identification of novel targeted curcumin-based drugs to treat cancer.     

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.     

Indigo Pulverata Levis (Chung-Dae,Persicaria tinctoria) Alleviates Atopic Dermatitis-like Inflammatory Responses In Vivo and In Vitro

Atopic dermatitis (AD) is a chronic inflammatory skin disease associated with a type 2 T helper cell (Th2) immune response. The Indigo Pulverata Levis extract (CHD) is used in traditional Southeast Asian medicine; however, its beneficial effects on AD remain uninvestigated. Therefore, we investigated the therapeutic effects of CHD in 2,4-dinitrochlorobenzene (DNCB)-induced BALB/c mice and tumor necrosis factor (TNF)-α- and interferon gamma (IFN)-γ-stimulated HaCaT cells. We evaluated immune cell infiltration, skin thickness, and the serum IgE and TNF-α levels in DNCB-induced AD mice. Moreover, we measured the expression levels of pro-inflammatory cytokines, mitogen-activated protein kinase (MAPK), and the nuclear factor-kappa B (NF-κB) in the mice dorsal skin. We also studied the effect of CHD on the translocation of NF-κB p65 and inflammatory chemokines in HaCaT cells. Our in vivo results revealed that CHD reduced the dermis and epidermis thicknesses and inhibited immune cell infiltration. Furthermore, it suppressed the proinflammatory cytokine expression and MAPK and NF-κB phosphorylations in the skin tissue and decreased serum IgE and TNF-α levels. In vitro results indicated that CHD downregulated inflammatory chemokines and blocked NF-κB p65 translocation. Thus, we deduced that CHD is a potential drug candidate for AD treatment.     

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.     

Phorbol 12-Myristate 13-Acetate Induced Toxicity Study and the Role of Tangeretin in Abrogating HIF-1α-NF-κB Crosstalk In Vitro and In Vivo

Phorbol 12-myristate 13-acetate (PMA) is a potent tumor promoter and highly inflammatory in nature. Here, we investigated the toxic effects of PMA on different model system. PMA (10 μg) caused chromosomal aberrations on the Allium cepa root tip and induced mitotic dysfunction. Similarly, PMA caused embryonic and larval deformities and a plummeted survivability rate on zebrafish embryo in a dose-dependent manner. Persistently, PMA treatment on immortalized human keratinocyte human keratinocyte (HaCaT) cells caused massive inflammatory rush at 4 h and a drop in cell survivability at 24 h. Concomitantly, we replicated a cutaneous inflammation similar to human psoriasis induced by PMA. Herein, we used tangeretin (TAN), as an antagonist to counteract the inflammatory response. Results from an in vivo experiment indicated that TAN (10 and 30 mg/kg) significantly inhibited PMA stimulated epidermal hyperplasia and intra-epidermal neutrophilic abscesses. In addition, its treatment effectively neutralized PMA induced elevated reactive oxygen species (ROS) generation on in vitro and in vivo systems, promoting antioxidant response. The association of hypoxia-inducible factor 1-alpha (HIF-1α)-nuclear factor kappa-light-chain-enhancer of activated b cells (NF-κB) crosstalk triggered by PMA enhanced PKCα-ERK1/2-NF-κB pathway; its activation was also significantly counteracted after TAN treatment. Conclusively, we demonstrated TAN inhibited the nuclear translocation of HIF-1α and NF-κB p65. Collectively, TAN treatment ameliorated PMA incited malignant inflammatory response by remodeling the cutaneous microenvironment.     

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.     

CK2 Down-Regulation Increases the Expression of Senescence-Associated Secretory Phenotype Factors through NF-κB Activation

Senescent cells secrete pro-inflammatory factors, and a hallmark feature of senescence is senescence-associated secretory phenotype (SASP). The aim of this study is to investigate the protein kinase CK2 (CK2) effects on SASP factors expression in cellular senescence and organism aging. Here CK2 down-regulation induced the expression of SASP factors, including interleukin (IL)-1β, IL-6, and matrix metalloproteinase (MMP) 3, through the activation of nuclear factor-κB (NF-κB) signaling in MCF-7 and HCT116 cells. CK2 down-regulation-mediated SIRT1 inactivation promoted the degradation of inhibitors of NF-κB (IκB) by activating the AKT-IκB kinase (IKK) axis and increased the acetylation of lysine 310 on RelA/p65, an important site for the activity of NF-κB. kin-10 (the ortholog of CK2β) knockdown increased zmp-1, -2, and -3 (the orthologs of MMP) expression in nematodes, but AKT inhibitor triciribine and SIRT activator resveratrol significantly abrogated the increased expression of these genes. Finally, antisense inhibitors of miR-186, miR-216b, miR-337-3p, and miR-760 suppressed CK2α down-regulation, activation of the AKT-IKK-NF-κB axis, RelA/p65 acetylation, and expression of SASP genes in cells treated with lipopolysaccharide. Therefore, this study indicated that CK2 down-regulation induces the expression of SASP factors through NF-κB activation, which is mediated by both activation of the SIRT1-AKT-IKK axis and RelA/p65 acetylation, suggesting that the mixture of the four miRNA inhibitors can be used as anti-inflammatory agents.     

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.     

Protective Role of Andrographolide in Bleomycin-Induced Pulmonary Fibrosis in Mice

Idiopathic pulmonary fibrosis (IPF) is a chronic devastating disease with poor prognosis. Multiple pathological processes, including inflammation, epithelial mesenchymal transition (EMT), apoptosis, and oxidative stress, are involved in the pathogenesis of IPF. Recent findings suggested that nuclear factor-κB (NF-κB) is constitutively activated in IPF and acts as a central regulator in the pathogenesis of IPF. The aim of our study was to reveal the value of andrographolide on bleomycin-induced inflammation and fibrosis in mice. The indicated dosages of andrographolide were administered in mice with bleomycin-induced pulmonary fibrosis. On day 21, cell counts of total cells, macrophages, neutrophils and lymphocytes, alone with TNF-α in bronchoalveolar lavage fluid (BALF) were measured. HE staining and Masson’s trichrome (MT) staining were used to observe the histological alterations of lungs. The Ashcroft score and hydroxyproline content of lungs were also measured. TGF-β1 and α-SMA mRNA and protein were analyzed. Activation of NF-κB was determined by western blotting and electrophoretic mobility shift assay (EMSA). On day 21 after bleomycin stimulation, andrographolide dose-dependently inhibited the inflammatory cells and TNF-α in BALF. Meanwhile, our data demonstrated that the Ashcroft score and hydroxyproline content of the bleomycin-stimulated lung were reduced by andrographolide administration. Furthermore, andrographloide suppressed TGF-β1 and α-SMA mRNA and protein expression in bleomycin-induced pulmonary fibrosis. Meanwhile, andrographolide significantly dose-dependently inhibited the ratio of phospho-NF-κB p65/total NF-κB p65 and NF-κB p65 DNA binding activities. Our findings indicate that andrographolide compromised bleomycin-induced pulmonary inflammation and fibrosis possibly through inactivation of NF-κB. Andrographolide holds promise as a novel drug to treat the devastating disease of pulmonary fibrosis.     

Moracin C,A Phenolic Compound Isolated from Artocarpus heterophyllus,Suppresses Lipopolysaccharide-Activated Inflammatory Responses in Murine Raw264.7 Macrophages

Artocarpus heterophyllus, a popular tropical fruit commonly known as the jackfruit tree, is normally planted in subtropical or tropical areas. Since a variety of phytochemicals isolated from A. heterophyllus have been found to possess potently anti-inflammatory, antiviral and antimalarial activities, researchers have devoted much interest to its potential pharmaceutical value. However, the exact mechanism underlying its anti-inflammatory activity is not well characterized. In this study, seven natural products isolated from A. heterophyllus, including 25-Hydroxycycloart-23-en-3-one (HY), Artocarpin (AR), Dadahol A (DA), Morachalcone A (MA), Artoheterophyllin B (AB), Cycloheterophyllin (CY) and Moracin C (MC) were collected. Lipopolysaccharide (LPS)-stimulated inflammatory response in RAW264.7 macrophages were used in this study. Among these compounds, MC significantly inhibited LPS-activated reactive oxygen species (ROS) and nitric oxide (NO) release without marked cytotoxicity. Furthermore, MC effectively reduced LPS stimulated up-regulation of mRNA and protein expression of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), and serval pro-inflammatory cytokines (interleukin-1β (IL-1β), interleukin-6 (IL-6) and tumor necrosis factor α (TNF-α)). Mechanistic studies revealed that the anti-inflammatory effect of MC was associated with the activation of the mitogen activated protein kinases (MAPKs) (including p38, ERK and JNK) and nuclear factor-κB (NF-κB) pathways, especially reducing the nuclear translocation of NF-κB p65 subunit as revealed by nuclear separation experiment and confocal microscopy.     

The Role of Nuclear Factor Kappa B (NF-κB) in Development and Treatment of COVID-19: Review

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes Coronavirus Disease 19 (COVID-19), a disease that has affected more than 500 million people worldwide since the end of 2019. Due to its high complications and death rates, there is still a need to find the best therapy for SARS-CoV-2 infection. The dysregulation of the inflammatory response in COVID-19 plays a very important role in disease progression. It has been observed that abnormal activity of Nuclear Factor kappa B (NF-κB) is directly associated with, inter alia, increased synthesis of proinflammatory factors. Therefore, this review paper focuses on the functions of NF-κB in the development of SARS-CoV-2 infection and potential application of NF-κB inhibitors in COVID-19 immunotherapy. A comprehensive literature search was performed using the MEDLINE/PubMed database. In the current review, it is highlighted that NF-κB plays important functions in the modulation of an adaptive inflammatory response, including inducing the expression of proinflammatory genes. Increased activation of NF-κB in SARS-CoV-2 infection was observed. The association between NF-κB activation and the expression of SARS-CoV-2 structural and non-structural proteins were also reported. It was observed that modulation of NF-κB using, e.g., traditional Chinese medicine or glucocorticosteroids resulted in decreased synthesis of proinflammatory factors caused by SARS-CoV-2 infection. This review summarizes the role of NF-κB in COVID-19 and describes its potential immunotherapeutic target in treatment of SARS-CoV-2 infection. However, indisputably more studies involving patients with a severe course of COVID-19 are sorely needed.     

Possible Mechanisms of Di(2-ethylhexyl) Phthalate-Induced MMP-2 and MMP-9 Expression in A7r5 Rat Vascular Smooth Muscle Cells

Proliferation and migration of vascular smooth muscle cells (VSMC) are important in the development and/or progression of many cardiovascular diseases, including atherosclerosis. Evidence shows that matrix metalloproteinase (MMP)-2 and MMP-9 are related to the pathogenesis of atherosclerosis. The expressions of MMP-2 and MMP-9 in atherosclerosis are regulated via various pathways, such as p38 mitogen activated protein kinase (MAPK), extracellular signal regulated kinase 1 and 2 (ERK1/2), Akt, and nuclear factor kappa (NF-κB). Di(2-ethylhexyl) phthalate (DEHP) has been shown to induce atherosclerosis by increasing tumor necrosis factor (TNF)-α, interleukin (IL)-6, and intercellular adhesion molecule (ICAM) productions. However, whether DEHP poses any effects on MMP-2 or MMP-9 expression in VSMC has not yet been answered. In our studies, rat aorta VSMC was treated with DEHP (between 2 and 17.5 ppm) and p38 MAPK, ERK1/2, Akt, NF-κB, and MMP-2 and MMP-9 proteins and activities were measured. Results showed that the presence of DEHP can induce higher MMP-2 and MMP-9 expression than the controls. Similar results on MMP-regulating proteins, i.e., p38 MAPK, ERK1/2, Akt, and NF-κB, were also observed. In summary, our current results have showed that DEHP can be a potent inducer of atherosclerosis by increasing MMP-2 and MMP-9 expression at least through the regulations of p38 MAPK, ERK1/2, Akt, and NF-κB.