Characterization of a novel curcumin analog P1 as potent inhibitor of the NF-κB signaling pathway with distinct mechanisms

Aim:

Curcumin has shown promising anticancer activity, which relies on its inhibition on NF-κB pathway. In this study, we characterized the pharmacological profile of a novel curcumin analog P1 and elucidate the related mechanisms.

Methods:

HEK293/NF-κB cells, stably transfected with an NF-κB-responsive luciferase reporter plasmid, were generated for high-throughput screen (HTS). Eight cancer cell lines, including PC3, COLO 205, HeLa cells etc. were tested. Cell viability was assessed using the sulforhodamine B (SRB) assays. Cell apoptosis was evaluated using FACS, immunocytochemistry, and Western blotting. H₂-DCFDA and MitoSOX Red were used to detect cellular and mitochondrial reactive oxygen species (ROS). The mitochondrial function was evaluated using mitochondrial oxygen consumption assay.

Results:

P1, a tropinone curcumin, was found in HTS targeting the NF-κB pathway. Its IC₅₀ value in inhibition of TNF-α-induced NF-κB activation was 0.8 μmol/L, whereas its IC₅₀ values in inhibiting the growth of A549 and HeLa cells were 1.24 and 0.69 μmol/L, respectively, which was 20- to 30-fold more potent than curcumin. The inhibition of P1 on the NF-κB pathway was further addressed in HeLa cells. The compound up to 10 μmol/L did not affect the binding of NF-κB to DNA, but markedly inhibited NF-κB nuclear translocation, IκB degradation and IκB kinase phosphorylation. The compound (1 and 3 μmol/L) concentration-dependently induced ROS generation, whereas curcumin up to 20 μmol/L had no effect. P1-induced ROS generation was mainly localized in mitochondria, and reversed by NAC. Moreover, the compound significantly enhanced TNF-α-induced apoptosis.

Conclusion:

P1 is a novel curcumin analog with potent anticancer activities, which exerts a distinct inhibition on the NF-κB pathway.     

Anti-inflammatory effects of a novel peptide designed to bind with NF-κB p50 subunit

AIM: To explore the anti-inflammatory effects of a novel peptide designed to bind with the NF-kappaB p50 subunit. METHODS: The affinity of the peptide binding with p50 was measured with a biosensor. Levels of tumor necrosis factor-alpha(TNF-alpha) and interleukin 6 (IL-6) from a human acute monocytic leukemia cell line (THP-1) treated with lipopolysaccharide (LPS) were measured using the ELISA method. In vivo anti-inflammatory effects of the peptide were tested with phorbol myristate acetate (PMA)-induced ear edema and zymosan A-induced peritonitis in mice. RESULTS: The peptide has the ability to interact with the NF-kappaB p50 subunit and can effectively inhibit TNF-alpha and IL-6 production in the THP-1 cell line, PMA-induced ear edema and zymosan A-induced peritonitis in mice. CONCLUSION: The peptide may have therapeutic potential for the treatment of local acute inflammation.     

Inhibitory action of salicylideneamino-2-thiophenol on NF-κB signaling cascade and cyclooxygenase-2 in HNE-treated endothelial cells

In the present study, the anti-inflammatory effect of salicylideneamino-2-thiophenol (SAL-2), a derivative of salicylate, on a potent oxidant 4-hydroxynonenal (HNE)-induced oxidative stress was investigated using rat prostate endothelial (YPEN-1) cells. We focused on anti-inflammatory activity of SAL-2 which was determined by its ability to suppress COX-2 and iNOS gene expression through suppression of NF-κB and redox regulation. We found that SAL-2 effectively inhibited HNE-induced reactive species generation, while upregulated GSH/GSSG ratio. Prostagrandin (PG) E2 production stimulated by arachidonic acid was suppressed by SAL-2. SAL-2 also downregulated COX-2 and iNOS expression induced by HNE, but salicylate did not. We found that SAL-2 inhibited HNE-mediated IKK phosphorylation, IκBα degradation and nuclear translocation of p65 which are linked to NF-κB activation. Furthermore, SAL-2 inhibited HNE-induced activation of mitogen-activated protein kinases. Collectively, SAL-2 inhibited COX-2 and iNOS gene expression through suppression of NF-κB leading to the inhibition of PGE₂ synthesis. Based on these data, we propose that with its combined effect on strong anti-oxidant and anti-inflammatory action, SAL-2 can be a potent anti-inflammatory agent for treatment of inflammatory-related diseases.     

Salvia miltiorrhiza compounds protect the liver from acute injury by regulation of p38 and NFκB signaling in Kupffer cells

Context: Salvia miltiorrhiza Bunge is a traditional Asian medicine used to treat cerebral and cardiac ischemia. However, the effects of the active compounds of S. miltiorrhiza on liver damage are unclear.

Objective: In this study, we tested the effects on acute liver injury of crude S. miltiorrhiza extracts from roots as well as neotanshinone B, dehydromiltirone, tanshinol A, tanshinone I, dihydrotanshinono I, neotanshinone A, cryptanshinono, tanshinone II A, and salvianolie acid B from purified S. miltiorrhiza extracts.

Materials and methods: Various compounds or ethanol extract of S. miltiorrhiza (50, 100, and 200?mg/kg, p.o.) were administered to rats for five consecutive days. After acute carbon tetrachloride (CCl₄)-induced liver injury by treatment of rats with a single dose of CCl₄ (0.75?mL/kg, p.o), rat liver function was tested by measuring serum biochemical parameters. Serum cytokine concentrations were assessed by enzyme-linked immunosorbent assay (ELISA). Expression of p38 and NFκB was evaluated by western blot.

Results: All S. miltiorrhiza components showed their effects on liver function from the dose from 50 to 200?mg/kg. At the dose of 200?mg/kg, they reduced serum levels of alkaline phosphatase (ALP) by 34–77%, alanine aminotransferase (ALT) by 30–57%, aspartate aminotransferase (AST) by 43–72%, creatine total bilirubin (BIL-T) by 33–81%, albumin (ALB) by 37–67%, indicating that S. miltiorrhiza extracts protected liver from CCl₄-induced damage. Moreover, S. miltiorrhiza extracts at 200?mg/kg reduced the increase in the proinflammatory cytokines tumor necrosis factor-α (TNF-α) by 25–82%, interleukin-1 (IL-1) by 42–74% and interleukin-6 (IL-6) by 67–83%, indicating an effect on alleviating liver inflammation. Furthermore, in vitro, S. miltiorrhiza extracts inhibited p38 and NFκB signaling in Kupffer cells. This effect could be a main mechanism by which S. miltiorrhiza protects against acute liver toxicity.

Discussion and conclusion: Active compounds of S. miltiorrhiza protected the liver from CCl₄-induced injury. Protection might have been due to inhibition of p38 and NFκB signaling in Kupffer cells, which subsequently reduced inflammation in the liver.     

Suppression of NF-κB pathway by crocetin contributes to attenuation of lipopolysaccharide-induced acute lung injury in mice

Crocetin, a carotenoid compound, has been shown to reduce expression of inflammation and inhibit the production of reactive oxygen species. In the present study, the effect of crocetin on acute lung injury induced by lipopolysaccharide (LPS) was investigated in vivo. In the mouse model, pretreatment with crocetin at dosages of 50 and 100 mg/kg reduced the LPS-induced lung oedema and histological changes, increased LPS-impaired superoxide dismutase (SOD) activity, and decreased lung myeloperoxidase (MPO) activity. Furthermore, treatment with crocetin significantly attenuated LPS-induced mRNA and the protein expressions of interleukin-6 (IL-6), macrophage chemoattractant protein-1 (MCP-1), and tumour necrosis factor-α (TNF-α) in lung tissue. In addition, crocetin at different dosages reduced phospho-IκB expression and NF-κB activity in LPS-induced lung tissue alteration. These results indicate that crocetin can provide protection against LPS-induced acute lung injury in mice.     

Possible underlying influence of p38MAPK and NF-κB in the diminished anti-anxiety effect of diazepam in stressed mice

The present study was designed to explore the possible nitriergic influence and role of p38MAPK and NF-κB in the diminished anti-anxiety effect of diazepam in stressed mice, using the elevated plus maze and light/dark box to assess anxiety. Immobilization stress for 6 h enhanced an anxiety-like behavior and increased plasma nitrite levels in mice. Diazepam (2 mg/kg, i.p.) produced an anti-anxiety effect in unstressed mice, but could not produce any change in anxiety levels of stressed mice. SB-203580 (2 mg/kg, i.p.), a specific inhibitor of p38MAPK, per se produced a significant antianxiety-like activity in stressed mice. Administration of a combination of SB-203580 (2 mg/kg, i.p.) and diazepam (2 mg/kg) in stressed mice produced a significantly higher antianxiety-like activity than that produced by SB-203580 alone. Pyrrolidine dithiocarbamate (PDTC), an inhibitor of the activation of NF-κB, per se produced a significant antianxiety-like activity in stressed mice. Combination of PDTC and diazepam also served to produce a higher significant antianxiety-like activity in stressed mice than that produced by PDTC alone. Diazepam could not produce any change in plasma nitrite levels in both unstressed and stressed mice. Both SB-203580 (2 mg/kg, i.p.) and PDTC (100 mg/kg, i.p.) significantly decreased plasma nitrite levels in stressed mice. The observations indicate that the diminished anti-anxiety effect of diazepam in stressed mice may involve strong nitriergic influence and may further be p38MAPK- and NF-κB-dependent.     

Celastrol-induced apoptosis in human HaCaT keratinocytes involves the inhibition of NF-κB activity

Psoriasis is a chronic inflammatory skin disease affecting 1-3% of the world's population. Traditional Chinese medicines have been extensively used for treating psoriasis with promising clinical results. Celastrol, a triterpenoid isolated from a Chinese herb Celastrus orbiculatus caulis, has been known to have diverse pharmacological effects such as anti-inflammatory, anti-cancer and antioxidant activities. The present study aimed at evaluating the anti-proliferative action of celastrol on cultured HaCaT cells and elucidating the mechanisms of action involved. Celastrol was shown to inhibit HaCaT cells growth with an IC?? value of 1.1 μM as measured by MTT assay. The ability of celastrol to induce apoptosis was studied by flow cytometric and western blot analyses. Celastrol was found to be capable of inducing apoptosis in HaCaT cells as characterized by phosphatidyl-serine (PS) externalization, depolarization of mitochondrial membrane potential and activation of caspase-3. The apoptosis induced by celastrol could be suppressed by Z-IETD-FMK and Z-LEHD-FMK, the respective caspase-8 and caspase-9 inhibitor. In addition, western blot analysis revealed a significant augmentation in the protein expression of Bax and attenuation in Bcl-2, suggesting that the celastrol-induced apoptosis acts through both death receptor and mitochondrial pathways. Moreover, western blot analysis on the expression of Rel/NF-κB demonstrated that the celastrol-mediated apoptosis on HaCaT cells was associated with the inhibition of the NF-κB pathway. Taken together, the present project has for the first time identified celastrol as a naturally occurring compound with potent apoptogenic action on cultured human keratinocytes, rendering it a promising candidate for further development into an anti-psoriatic agent.     

IL-38 alleviates the inflammatory response and the degeneration of nucleus pulposus cells via inhibition of the NF-κB signaling pathway in vitro

Previous studies have suggested that the inflammatory response contributes to the onset of intervertebral disc degeneration (IVDD). Interleukin (IL)-38, a newly discovered cytokine of the IL-1 family, has been demonstrated to play an anti-inflammatory role in autoimmune diseases, such as Crohn’s disease, rheumatoid arthritis and psoriasis. However, whether IL-38 participates in the pathogenesis of IVDD remains unknown. In this study, human disc tissues from IVDD patients and rat disc tissues from an IVDD model were collected to measure the expression of IL-38 in the IVDD groups and the control groups by western blot and immunohistochemical staining. To further determine the role of IL-38 in IVDD, human nucleus pulposus cells (HNPCs) were stimulated with TNF-α to generate an in vitro model of inflammation to mimic the local inflammatory environment of the lumbar disc. The inflammatory response and HNPC degeneration markers were measured after stimulation with TNF-α and IL-38. IL-38 was upregulated in both the human and rat degenerated disc tissues compared with the control tissues. In vitro, IL-38 significantly decreased the TNF-α-induced expression of IL-1β, IL-6, COX-2, MMP-13 and ADAMTS-5 in the HNPCs, and IL-38 also alleviated the TNF-α-induced reductions in type II collagen and aggrecan. Moreover, IL-38 inhibited the activation of the NF-κB signaling pathway in the HNPC-based model of inflammation by reducing the expression level of the NF-κB P-P65 protein. In conclusion, IL-38 could alleviate the inflammatory response and HNPC degeneration in vitro via the inhibition of the NF-κB signaling pathway. These results suggest that IL-38 may be a new strategy for the treatment of IVDD.     

Therapeutic effects of KANK2 in myocardial infarction rats might be associated with the NF-κB p65 inhibition

ObjectiveKN motif and ankyrin repeat domains 2 (KANK2) may inhibit the activation of (NF-kappaB) p65, which plays a role in myocardial injury. Thus, our study aims to discover the effect of KANK2 on myocardial infarction (MI) induced by ligating the left anterior descending coronary artery (LAD) through regulating NF-κB p65 in vivo.MethodsMI rats underwent LAD ligation were administered with intramyocardial injections of KANK2/Control activation plasmids. Six weeks after MI, pressure-volume (P/V) loops was used to investigate the cardiac function of rats, then the following detections were performed, including TTC staining, HE staining, immunofluorescence, Masson’ s trichrome staining, ELISA assay, TUNEL staining, immunohistochemistry, qRT-PCR and Western blotting.ResultsMI rats decreased in maximum pressure (p_max), ejection fraction (EF%), peak rate of pressure rise (dpdt_max) and decline (-dpdt_max) with increased end diastolic pressure (EDP), which was partially reversed by KANK2 overexpression. Besides, KANK2 CRISPR activation plasmids reduced infarct size with less collagen fiber proliferation and neutrophil infiltration in infarct tissues, as well as suppressed pro-inflammatory factors expressions in MI rats. Moreover, injection of KANK2 activation plasmid decreased collagen deposition, aggravated cardiomyocyte apoptosis, enhanced the capillary density, and increased the expressions of VEGF and bFGF in the infarct and peri-infarct regions of MI rats. KANK2 lowered myocardial NF-κB p65 expression in MI rats.ConclusionKANK2 may play its therapeutic role in MI through improving cardiac function, decreasing myocardial collagen deposition, reducing cardiomyocyte apoptosis, and increasing angiogenesis, which might be associated with the reduction of NF-κB p65 expression.     

Ascorbic acid suppresses endotoxemia and NF-κB signaling cascade in alcoholic liver fibrosis in guinea pigs: A mechanistic approach

Alcohol consumption increases the small intestinal bacterial overgrowth (SIBO) and intestinal permeability of endotoxin. The endotoxin mediated inflammatory signaling plays a major role in alcoholic liver fibrosis. We evaluated the effect of ascorbic acid (AA), silymarin and alcohol abstention on the alcohol induced endotoxemia and NF-κB activation cascade pathway in guinea pigs (Cavia porcellus). Guinea pigs were administered ethanol at a daily dose of 4 g/kg b.wt for 90 days. After 90 days, ethanol administration was stopped. The ethanol treated animals were divided into abstention, silymarin (250 mg/kg b.wt) and AA (250 mg/kg b.wt) supplemented groups and maintained for 30 days. The SIBO, intestinal permeability and endotoxin were significantly increased in the ethanol group. The mRNA expressions of intestinal proteins claudin, occludin and zona occludens-1 were significantly decreased in ethanol group. The mRNA levels of inflammatory receptors, activity of IKKβ and the protein expressions of phospho-IκBα, NF-κB, TNF-α, TGF-β₁ and IL-6 were also altered in ethanol group. The expressions of fibrosis markers α-SMA, α₁ (I) collagen and sirius red staining in the liver revealed the induction of fibrosis. But the supplementation of AA could induce greater reduction of ethanol induced SIBO, intestinal barrier defects, NF-κB activation and liver fibrosis than silymarin. The possible mechanism may be the inhibitory effect of AA on SIBO, intestinal barrier defect and IKKβ, which decreased the activation of NF-κB and synthesis of cytokines. This might have led to suppression of HSCs activation and liver fibrosis.     

Anti-inflammatory effects of aromatic-turmerone through blocking of NF-κB, JNK, and p38 MAPK signaling pathways in amyloid β-stimulated microglia

Amyloid β (Aβ) induces the production of neuroinflammatory molecules, which may contribute to the pathogenesis of numerous neurodegenerative diseases. Therefore, suppression of neuroinflammatory molecules could be developed as a therapeutic method. Aromatic (ar)-turmerone, turmeric oil isolated from Curcuma longa, has long been used in Southeast Asia as both a remedy and a food. In this study, we investigated the anti-inflammatory effects of ar-turmerone in BV2 microglial cells. Aβ-stimulated microglial cells were tested for the expression and activation of MMP-9, iNOS, and COX-2, the production of proinflammatory cytokines, chemokines, and ROS, as well as the underlying signaling pathways. Ar-turmerone significantly suppressed Aβ-induced expression and activation of MMP-9, iNOS, and COX-2, but not MMP-2. Ar-turmerone also reduced TNF-α, IL-1β, IL-6, and MCP-1 production in Aβ-stimulated microglial cells. Further, ar-turmerone markedly inhibited the production of ROS. Impaired translocation and activation of NF-κB were observed in Aβ-stimulated microglial cells exposed to ar-turmerone. Furthermore, ar-turmerone inhibited the phosphorylation and degradation of IκB-α as well as the phosphorylation of JNK and p38 MAPK. These results suggest that ar-turmerone impaired the Aβ-induced inflammatory response of microglial cells by inhibiting the NF-κB, JNK, and p38 MAPK signaling pathways. Lastly, ar-turmerone protected hippocampal HT-22 cells from indirect neuronal toxicity induced by activated microglial cells. These novel findings provide new insights into the development of ar-turmerone as a therapeutic agent for the treatment of neurodegenerative disorders.     

Suppression of P2X7/NF-κB pathways by Schisandrin B contributes to attenuation of lipopolysaccharide-induced inflammatory responses in acute lung injury

The aim of the present study was to assess the effects and mechanisms of Schisandrin B (SchB) on lipopolysaccharide (LPS)-induced acute lung injury (ALI). ALI was induced in mice by intratracheal instillation of LPS (1 mg/kg), and SchB (25, 50, and 75 mg/kg) was injected 1 h before LPS challenge by gavage. After 12 h, bronchoalveolar lavage fluid (BALF) samples and lung tissues were collected. Histological studies demonstrated that SchB attenuated LPS-induced interstitial edema, hemorrhage, and infiltration of neutrophils in the lung tissue. SchB pretreatment at doses of 25, 50, and 75 mg/kg was shown to reduce LPS-induced lung wet-to-dry weight ratio and lung myeloperoxidase activity. In addition, pretreatment with SchB lowered the number of inflammatory cells and pro-inflammatory cytokines including tumor necrosis factor-α, interleukin-1β, and interleukin-6 in BALF. The mRNA and protein expression levels of nuclear factor kappa B (NF-κB) signaling-related molecules activated by P2X7 were investigated to determine the molecular mechanism of SchB. The findings presented here suggest that the protective mechanism of SchB may be attributed partly to the decreased production of pro-inflammatory cytokines through the inhibition of P2X7/NF-κB activation.     

Lobolide,a diterpene,blockades the NF-κB pathway and p38 and ERK MAPK activity in macrophages in vitro

Aim:

Recent studies have shown that constitutive activation of the nuclear factor κB (NF-κB) plays a key role in chronic inflammation and cancers. The aim of this study was to characterize lobolide, a cembrane diterpene, as a drug candidate targeting the NF-κB signaling pathway.

Methods:

A HEK 293/NF-κB-Luc stable cell line was constructed to evaluate the effect of lobolide on NF-κB activation. THP-1 human monocytes and peripheral blood mononuclear cells (PBMCs) from healthy volunteers were tested. Lipopolysaccharide (LPS)-induced TNFα and IL-1β production and activation of the TAK1-IKK-NF-κB pathway were studied using ELISA and Western blot analysis.

Results:

In HEK 293/NF-κB-Luc stable cells, lobolide (0.19–50 μmol/L) inhibited NF-κB activation in a concentration-dependent manner with an IC₅₀ value of 4.2±0.3 μmol/L. Treatment with lobolide (2.5–10 μmol/L) significantly suppressed LPS-induced production of TNFα and IL-1β in both THP-1 cells and PBMCs. In THP-1 cells, the suppression was partially caused by blockade of the translocation of NF-κB from the cytoplasm to the nucleus via affecting the TAK1-IKK-NF-κB pathway and p38 and ERK MAPK activity.

Conclusion:

Lobolide is a potential inhibitor of the NF-κB pathway, which blocks the translocation of NF-κB from the cytoplasm to the nucleus. Lobolide inhibits LPS-stimulated TNFα and IL-1β release, suggesting that the compound might be an anti-inflammatory compound.     

Anti-inflammatory effect of a novel formulation of coconut inflorescence sap against ox-LDL induced inflammatory responses in human peripheral blood mononuclear cells by modulating TLR-NF-κB signaling pathway

Oxidized low density lipoprotein (ox LDL) induced inflammatory response was reported to play an important role in the pathogenesis of atherosclerosis. The purpose of this study was to explore the anti-inflammatory effect of a novel formulation of coconut inflorescence sap (CSP); COCOZEN? against ox-LDL induced inflammatory responses in human peripheral blood mononuclear cells (hPBMCs). The hPBMCs were isolated from healthy human volunteers and cultured in collagen coated plates at 37?°C. The cells were grouped as Group I (Control), Group II (ox-LDL treated) and Group III (ox-LDL?+?CSP treated). Further analysis of inflammatory markers, reactive oxygen species, mRNA and protein expression levels indicated increased expressions of TLR-4, TNF-α, IL-6 and VCAM-1 in ox-LDL treated group along with the nuclear translocation of NF-κB. Other inflammatory markers such as LOX, PGE2, NO, total COX and lipid peroxidation level were also found to be significantly (p?via TLR-NF-κB signaling pathway.