Translocation Of NF-κB P65 Subunit Research Articles

Ginkgolide C Alleviates Myocardial Ischemia/Reperfusion-Induced Inflammatory Injury via Inhibition of CD40-NF-κB Pathway.

Increasing evidence shows that inflammation plays a vital role in the occurrence and development of ischemia/reperfusion (I/R). Suppression of excessive inflammation can ameliorate impaired cardiac function, which shows therapeutic potential for clinical treatment of myocardial ischemia/reperfusion (MI/R) diseases. In this study, we investigated whether Ginkgolide C (GC), a potent anti-inflammatory flavone, extenuated MI/R injury through inhibition of inflammation. In vivo, rats with the occlusion of the left anterior descending (LAD) coronary artery were applied to mimic MI/R injury. In vitro, primary cultured neonatal ventricular myocytes exposed to hypoxia/reoxygenation (H/R) were applied to further discuss the anti-H/R injury property of GC. The results revealed that GC significantly improved the symptoms of MI/R injury, as evidenced by reducing infarct size, preventing myofibrillar degeneration and reversing the mitochondria dysfunction. Moreover, histological analysis and Myeloperoxidase (MPO) activity measurement showed that GC remarkably suppressed Polymorphonuclears (PMNs) infiltration and ameliorated the histopathological damage. Furthermore, GC pretreatment was shown to improve H/R-induced ventricular myocytes viability and enhance tolerance of inflammatory insult, as evidenced by suppressing expression of CD40, translocation of NF-κB p65 subunit, phosphorylation of IκB-α, as well as the activity of IKK-β. In addition, downstream inflammatory cytokines modulated by NF-κB signaling were effectively down-regulated both in vivo and in vitro, as determined by immunohistochemistry and ELISA. In conclusion, these results indicate that GC possesses a beneficial effect against MI/R injury via inflammation inhibition that may involve suppression of CD40-NF-κB signal pathway and downstream inflammatory cytokines expression, which may offer an alternative medication for MI/R diseases.

Ingredients from Litsea garrettii as Potential Preventive Agents against Oxidative Insult and Inflammatory Response.

Oxidative stress and inflammation undoubtedly contribute to the pathogenesis of many human diseases. The nuclear transcription factor erythroid 2-related factor (Nrf2) and the nuclear factor κB (NF-κB) play central roles in regulation of oxidative stress and inflammation and thus are targets for developing agents against oxidative stress- and inflammation-related diseases. Our previous study indicated that the EtOH extract of Litsea garrettii protected human bronchial epithelial cells against oxidative insult via the activation of Nrf2. In the present study, a systemic phytochemical investigation of L. garrettii led to the isolation of twenty-one chemical ingredients, which were further evaluated for their inhibitions on oxidative stress and inflammation using NAD(P)H:quinone reductase (QR) assay and nitric oxide (NO) production assay. Of these ingredients, 3-methoxy-5-pentyl-phenol (MPP, 5) was identified as an Nrf2 activator and an NF-κB inhibitor. Further studies demonstrated the following: (i) MPP upregulated the protein levels of Nrf2, NAD(P)H:quinone oxidoreductase 1 (NQO1), and glutamate-cysteine ligase regulatory subunit (GCLM); enhanced the nuclear translocation and stabilization of Nrf2; and inhibited arsenic [As(III)]-induced oxidative insult in normal human lung epithelial Beas-2B cells. And (ii) MPP suppressed the nuclear translocation of NF-κB p65 subunit; inhibited the lipopolysaccharide- (LPS-) stimulated increases of NF-κB p65 subunit, COX-2, iNOS, TNF-α, and IL-1β; and blocked the LPS-induced biodegrade of IκB-α in RAW 264.7 murine macrophages. Taken together, MPP displayed potential preventive effects against inflammation- and oxidative stress-related diseases.

Natural product celastrol suppressed macrophage M1 polarization against inflammation in diet-induced obese mice via regulating Nrf2/HO-1, MAP kinase and NF-κB pathways.

Macrophage polarization is implicated in the inflammation in obesity. The aim of the present study was to examine the anti-inflammatory activities of botanical triterpene celastrol against diet-induced obesity. We treated diet-induced obese C57BL/6N male mice with celastrol (5, 7.5 mg/kg/d) for 3 weeks, and investigated macrophage M1/M2 polarization in adipose and hepatic tissues. Celastrol reduced fat accumulation and ameliorated glucose tolerance and insulin sensitivity. Celastrol down-regulated the mRNA levels of macrophage M1 biomarkers (e.g., IL-6, IL-1β, TNF-α, iNOS) in cell culture and in mice. The underlying mechanisms were investigated in murine macrophage RAW264.7 cells. Our results demonstrated that celastrol might control macrophage polarization through modulating the cross-talk between the following three mechanisms: 1) suppressing LPS-induced activation of MAP kinases (e.g., ERK1/2, p38, JNK) in a concentration dependent manner; 2) attenuating LPS-induced nuclear translocation of NF-κB p65 subunit in a time dependent manner; 3) activating Nrf2 and subsequently inducing HO-1 expression. HO-1 inhibitor SnPP diminished the inhibitory effects of celastrol on the activation of NF-κB pathway and the pro-inflammatory M1 macrophage polarization. Taken together, celastrol exhibited anti-obesity effects via suppressing pro-inflammatory M1 macrophage polarization. Thus, our results provide new evidence for the potential of celastrol in the treatment of obesity.

Omega-3 polyunsaturated fatty acids alleviate hepatic steatosis-induced inflammation through Sirt1-mediated nuclear translocation of NF-κB p65 subunit in hepatocytes of large yellow croaker (Larmichthys crocea)

Hepatic steatosis induced inflammation is becoming increasingly prevalent in farmed fish. This study was conducted to investigate the protective effects of omega-3 polyunsaturated fatty acids (ω-3 PUFAs) against hepatic steatosis-induced inflammation and its potential molecular mechanisms in hepatocyte of large yellow croaker (Larmichthys crocea). We found that the hepatic steatosis-induced inflammation was relieved by ω-3 PUFAs, meanwhile, the Sirt1 activity and transcript expression was increased by ω-3 PUFAs. The increased Sirt1 activity can decrease the hepatic steatosis-induced inflammation. The protective effects of ω-3 PUFAs against hepatic steatosis-induced inflammation was reversed by the treatment with Sirt1 inhibitor EX-527. The nuclear translocation of nuclear transcription factor kappa-B (NF-κB) p65 was significantly decreased after ω-3 PUFAs treatments compared to the palmitic acid stimulation group. The ω-3 PUFAs induced cytoplasm translocation of NF-κB p65 was reversed by EX-527. Together, ω-3 PUFAs alleviate hepatic steatosis-induced inflammation through Sirt1-mediated nuclear translocation of NF-κB p65 subunit in hepatocytes of large yellow croaker. The present study provides important insight into the mechanisms of the protective effects of ω-3 PUFAs, providing theory bases for alleviating the hepatic steatosis induced inflammation of farmed fish, thereby offering great benefits to the aquaculture industry and fish consumers.

Involvement of p38 MAPK and ATF-2 signaling pathway in anti-inflammatory effect of a novel compound bis[(5-methyl)2-furyl](4-nitrophenyl)methane on lipopolysaccharide-stimulated macrophages

Activated macrophages produce various pro-inflammatory mediators such as inducible nitric oxide synthase (iNOS)-derived nitric oxide (NO) and cyclooxygenase (COX)-2-derived prostaglandin E2 (PGE2) during inflammatory response. However, overproduction of NO and PGE2 appears to be involved in pathogenesis of various inflammatory diseases. Therefore, inhibition of NO and PGE2 production might be useful for the treatment of inflammatory-related diseases. In this study, the bis[(5-methyl)2-furyl](4-nitrophenyl)methane or BFNM was evaluated for the anti-inflammatory activity and mechanism of action in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophage. BFNM inhibited NO and PGE2 production in a concentration-dependent manner and down-regulated the expression of iNOS and COX-2 at mRNA and protein levels. BFNM suppressed nuclear translocation of NF-κB p65 subunit only very slightly, and failed to decrease NF-κB DNA binding activity. In contrast, the compound significantly reduced phosphorylation of p38 MAPK and ATF-2, a component of AP-1 known to be involved in the transcriptional regulation of iNOS and COX-2, in a dose-dependent manner in LPS-induced cells. Collectively, these results suggest that BFNM has an anti-inflammatory effect in RAW 264.7 macrophages, at least in part, by suppression of NO and PGE2 production. The inhibitory effect of BFNM is mediated mainly via the p38 MAPK/ATF-2 signaling pathway. Thus, BFNM would be a lead compound for the development of novel anti-inflammatory agents.

Overexpression of microRNA-125b inhibits human acute myeloid leukemia cells invasion, proliferation and promotes cells apoptosis by targeting NF-κB signaling pathway

microRNA-125b has been reported to play an novel biological function in the progression and development of several kinds of leukemia. However, the detail role of miR-125b in acute myeloid leukemia (AML) is remains largely unknown. The present study aimed to investigate the biological role of miR-125b in AML and the potential molecular mechanism involved in this process. Our results showed that overexpression of miR-125b suppressed AML cells proliferation, invasion and promotes cells apoptosis in a dose-dependent manner, while the miR-NC did not show the same effect. In addition, miR-125b induced AML cells G2/M cell cycle arrest in vitro. Overexpression of miR-125b resulted in a significant decrease of the expression of p-IκB-α and inhibition of IκB-α degradation, and the nuclear translocation of NF-κB subunit p65 was abrogated by miR-125b simutaneously. To further verify that miR-125b targeted NF-κB signaling pathway, the NF-κB-regulated downstream genes that were associated with cell cycle arrest and apoptosis was also determined. The results showed that, miR-125b also affect NF-κB-regulated genes expression involved in cell cycle arrest and apoptosis. In conclusion, the present work certificates that miR-125b can significantly inhibit human AML cells invasion, proliferation and promotes cells apoptosis by targeting the NF-κB signaling pathway, and thus it can be viewed as an promising therapeutic target for AML.

C21-steroidal pregnane sapogenins and their derivatives as anti-inflammatory agents

During the screening of natural anti-inflammatory agent, we identified some C21-steroidal pregnane sapogenins or the derivatives to inhibit TLR2, TLR3, and TLR4-initiatedinflammatory responses respectively. Treatment with active compounds 10, 2j and 3p failed to impact tumor necrosis factor-α (TNF-α) induced nucleus translocation of NF-κB p65 subunit. However, these compounds regulated distinct canonical or non-canonical NF-κB family members. Ectopic expression of TNF receptor associated factor 6 (TRAF6) abrogated the inhibitory activity of the compounds on production of pro-inflammatory cytokines downstream of TLR4. These results suggested that compounds 10, 2j, and 3p suppressed TLR-initiated innate immunity through TRAF6 with differential regulation of NF-κB family proteins.

Immune-Stimulatory Effects of Althaea rosea Flower Extracts through the MAPK Signaling Pathway in RAW264.7 Cells.

Althaea rosea (Linn.) is a medicinal plant from China and Korea that has been traditionally used to control inflammation, to stop bedwetting and as a mouthwash in cases of bleeding gums. Its flowers are employed medicinally for their emollient, demulcent and diuretic properties, which make them useful in chest complaints. Furthermore, a flower extract decoction is used to improve blood circulation, for the treatment of constipation, dysmenorrhoea, haemorrhages, etc. However, the possible mechanisms of the immune-stimulatory effect remains to be elucidated. Therefore, we investigated the role of Althaea rosea flower (ARF) extracts in the immune-stimulatory effect of macrophages and the underlying mechanisms of action. ARF water extract (ARFW) could dose-dependently increase NO production and cytokines (IL-6 and TNF-α). We also found that ARFW significantly increased the expression of iNOS and COX-2 proteins in RAW264.7 cells. Consistent with these results, MAPK protein (JNK, ERK, p38) expression levels were induced after treatment with ARFW. Additionally, ARFW showed a marked increase in the phosphorylation level of IκBα and subsequent IκBα degradation allowing NF-κB nuclear translocation. These results suggest that the immune-stimulatory effect of A. rosea flower extracts is mediated through the translocation of NF-κB p65 subunit into the nucleus from the cytoplasm and subsequent activation of pro-inflammatory cytokines (IL-6 and TNF-α) and other mediators (iNOS and COX-2), which occurs mainly through MAPK signalling pathway. Thus, we suggest that ARFW could be considered as a potential therapeutic agent useful in the development of immune-stimulatory compounds.

Spermidine Protects against Oxidative Stress in Inflammation Models Using Macrophages and Zebrafish.

Spermidine is a naturally occurring polyamine compound that has recently emerged with anti-aging properties and suppresses inflammation and oxidation. However, its mechanisms of action on anti-inflammatory and antioxidant effects have not been fully elucidated. In this study, the potential of spermidine for reducing pro-inflammatory and oxidative effects in lipopolysaccharide (LPS)-stimulated macrophages and zebrafish was explored. Our data indicate that spermidine significantly inhibited the production of pro-inflammatory mediators such as nitric oxide (NO) and prostaglandin E2 (PGE2), and cytokines including tumor necrosis factor-α and interleukin-1β in RAW 264.7 macrophages without any significant cytotoxicity. The protective effects of spermidine accompanied by a marked suppression in their regulatory gene expression at the transcription levels. Spermidine also attenuated the nuclear translocation of NF-κB p65 subunit and reduced LPS-induced intracellular accumulation of reactive oxygen species (ROS) in RAW 264.7 macrophages. Moreover, spermidine prevented the LPS-induced NO production and ROS accumulation in zebrafish larvae and was found to be associated with a diminished recruitment of neutrophils and macrophages. Although more work is needed to fully understand the critical role of spermidine on the inhibition of inflammation-associated migration of immune cells, our findings clearly demonstrate that spermidine may be a potential therapeutic intervention for the treatment of inflammatory and oxidative disorders.

Sulforaphane enhances the anticancer activity of taxanes against triple negative breast cancer by killing cancer stem cells

Triple negative breast cancer (TNBC) typically exhibits rapid progression, high mortality and faster relapse rates relative to other breast cancer subtypes. In this report we examine the combination of taxanes (paclitaxel or docetaxel) with a breast cancer stem cell (CSC)-targeting agent sulforaphane for use against TNBC. We demonstrate that paclitaxel or docetaxel treatment induces IL-6 secretion and results in expansion of CSCs in TNBC cell lines. Conversely, sulforaphane is capable of preferentially eliminating CSCs, by inhibiting NF-κB p65 subunit translocation, downregulating p52 and consequent downstream transcriptional activity. Sulforaphane also reverses taxane-induced aldehyde dehydrogenase-positive (ALDH+) cell enrichment, and dramatically reduces the size and number of primary and secondary mammospheres formed. In vivo in an advanced treatment orthotopic mouse xenograft model together with extreme limiting dilution analysis (ELDA), the combination of docetaxel and sulforaphane exhibits a greater reduction in primary tumor volume and significantly reduces secondary tumor formation relative to either treatment alone. These results suggest that treatment of TNBCs with cytotoxic chemotherapy would be greatly benefited by the addition of sulforaphane to prevent expansion of and eliminate breast CSCs.

ZNF307 (Zinc Finger Protein 307) Acts as a Negative Regulator of Pressure Overload-Induced Cardiac Hypertrophy.

Pathological cardiac hypertrophy is a key risk factor for heart failure. We found that the protein expression levels of the ZNF307 (zinc finger protein 307) were significantly increased in heart samples from both human patients with dilated cardiomyopathy and mice subjected to aortic banding. Therefore, we aimed to elucidate the role of ZNF307 in the development of cardiac hypertrophy and to explore the signal transduction events that mediate the effect of ZNF307 on cardiac hypertrophy, using cardiac-specific ZNF307 transgenic (ZNF307-TG) mice and ZNF307 global knockout (ZNF307-KO) mice. The results showed that the deletion of ZNF307 potentiated aortic banding-induced pathological cardiac hypertrophy, fibrosis, and cardiac dysfunction; however, the aortic banding-induced cardiac hypertrophic phenotype was dramatically diminished by ZNF307 overexpression in mouse heart. Mechanistically, the antihypertrophic effects mediated by ZNF307 in response to pathological stimuli were associated with the direct inactivation of NF-κB (nuclear factor-κB) signaling and blockade of the nuclear translocation of NF-κB subunit p65. Furthermore, the overexpression of a degradation-resistant mutant of IκBα (IκBαS32A/S36A) reversed the exacerbation of cardiac hypertrophy, fibrosis, and dysfunction shown in aortic banding-treated ZNF307-KO mice. In conclusion, our findings demonstrate that ZNF307 ameliorates pressure overload-induced cardiac hypertrophy by inhibiting the activity of NF-κB-signaling pathway.

Vitamin D3 pretreatment protects against lipopolysaccharide-induced early embryo loss through its anti-inflammatory effects.

Increasing evidence demonstrates that inflammatory cytokines are involved in LPS-induced adverse pregnant outcomes including early embryo loss. Vitamin D3 (VitD3) has anti-inflammatory activity. We aimed to investigate the effects of vitamin D3 (VitD3) on LPS-induced early embryo loss in mice. All pregnant mice except controls were intraperitoneally (ip) injected with LPS on GD7. In VitD3 alone and LPS+VitD3 groups, pregnant mice were pretreated with VitD3 by gavage daily from GD5 to GD7. LPS caused 62.5% pregnant mice with early embryo loss. Interestingly, the rate of abortion dropped to 14.3% when pregnant mice were pretreated with VitD3. Additional experiment showed that VitD3 significantly attenuated LPS-evoked elevation on TNF-α, IFN-γ, MIP-2, and nitrate plus nitrite in maternal serum. In addition, VitD3 alleviated LPS-induced COX-2 expression in the decidua and attenuated the elevation of PGF2α in maternal serum. Although VitD3 had no effect on IL-10 in maternal serum, it induced further elevation of serum IL-10 level in LPS-treated mice. Further analysis showed that VitD3 activated VDR signaling, simultaneously inhibited LPS-induced nuclear translocation of NF-κB p65 subunits in the decidua. VitD3 protects mice from LPS-induced early embryo loss at least partially through its anti-inflammatory effects.

Inhibitory effect ofPetalonia binghamiaeon neuroinflammation in LPS-stimulated microglial cells

Purpose: Neuroinflammation is mediated by activation of microglia implicated in the pathogenesis of neurodegenerative disorders such as Alzheimers disease and Parkinsons disease. Inhibition of neuroinflammation may be an effective solution to treat these brain disorders. Petalonia binghamiae is known as a traditional food, based on multiple biological activities such as anti-oxidant and anti-obesity. In present study, the anti-neuroinflammatory potential of Petalonia binghamiae was investigated in LPS-stimulated BV2 microglial cells. Methods: Cell viability was measured by MTT assay. Production of nitric oxide (NO) was examined using Griess reagent. Expression of inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2) was detected by Western blot analysis. Activation of nuclear factor κB (NF-κB) signaling was examined by nuclear translocation of NF-κB p65 subunit and phosphorylation of IκB. Results: Extract of Petalonia binghamiae significantly inhibited LPS-stimulated NO production and iNOS/COX-2 protein expression in a dose-dependent manner without cytotoxicity. Pretreatment with Petalonia binghamiae suppressed LPS-induced NF-κB p65 nuclear translocation and phosphorylation of IκB. Co-treatment with Petalonia binghamiae and pyrrolidine duthiocarbamate (PDTC), an NF-κB inhibitor, reduced LPS-stimulated NO release compared to that in PB-treated or PDTC-treated cells. Conclusion: The present results indicate that extract of Petalonia binghamiae exerts anti-neuroinflammation activities, partly through inhibition of NF-κB signaling. These findings suggest that Petalonia binghamiae might have therapeutic potential in relation to neuroinflammation and neurodegenerative diseases.

Kukoamine A Prevents Radiation-Induced Neuroinflammation and Preserves Hippocampal Neurogenesis in Rats by Inhibiting Activation of NF-κB and AP-1.

Impaired hippocampal neurogenesis and neuroinflammation are involved in the pathogenesis of radiation-induced brain injury. Kukoamine A (KuA) was demonstrated to have neuroprotective effects through inhibiting oxidative stress and apoptosis after whole-brain irradiation (WBI) in rats. The aim of this study was to investigate whether administration of KuA would prevent radiation-induced neuroinflammation and the detrimental effect on hippocampal neurogenesis. For this study, male Wistar rats received either sham irradiation or WBI (30Gy single dose of X-rays) followed by the immediate injection of either KuA or vehicle intravenously. The dose of KuA was 5, 10, and 20mg/kg, respectively. The levels of pro-inflammatory cytokines were assayed by ELISA kits. The newborn neurons were detected by 5-bromo-2-deoxyuridine (BrdU)/neuronal nuclei (NeuN) double immunofluorescence. Microglial activation was measured by Iba-1 immunofluorescence. The expression of Cox-2 and the activation of nuclear factor κB (NF-κB), activating protein 1(AP-1), and PPARδ were evaluated by western blot. WBI led to a significant increase in the level of TNF-α, IL-1β, and Cox-2, and it was alleviated by KuA administration. KuA attenuated microglial activation in rat hippocampus after WBI. Neurogenesis impairment induced by WBI was ameliorated by KuA. Additionally, KuA alleviated the increased translocation of NF-κB p65 subunit and phosphorylation of c-jun induced by WBI and elevated the expression of PPARδ. These data indicate that KuA could ameliorate the neuroinflammatory response and protect neurogenesis after WBI, partially through regulating the activation of NF-κB, AP-1, and PPARδ.

The effect of lysophosphatidic acid on Toll-like receptor 4 expression and the nuclear factor-κB signaling pathway in THP-1 cells.

Toll-like receptors (TLRs) are major receptors that mediate the innate immune and inflammatory responses, of which TLR4 has been found most closely related to human atherosclerosis. After ligands are polymerized and activated by TLR, the mitogen-activated protein kinase and nuclear factor-κB (NF-κB) pathways are activated, leading to promotion of NF-κB-regulated transcription of inflammatory factors, thus playing a role in the physiological and pathological processes in atherosclerosis. Oxidized lipoproteins or their components, oxidized lipids, have been confirmed as endogenous TLR receptors. Lysophosphatidic acid (LPA) is an active component of low-density lipoprotein that induces vascular endothelial lesions. However, the mechanism of the TLR4/NF-κB signaling system involved in LPA-induced atherosclerosis has not been fully elucidated. In this study, we investigated the effects of LPA on TLR4 expression, nuclear translocation of NF-κB p65 subunit, and changes in the cytokine tumor necrosis factor α (TNF-α) in human THP-1 cells. LPA upregulated expression of the TLR4 mRNA and protein in THP-1 cells in a dose- and time-dependent manner, induced NF-κB p65 activation synchronously in THP-1 cells, and increased TNF-α secretion. After TLR4 was blocked using TLR4 monoclonal antibody, NF-κB p65 expression and TNF-α secretion were inhibited significantly. These data suggest that LPA can significantly upregulate TLR4 expression and promote NF-κB activation and proinflammatory cytokine secretion in THP-1 cells; it is possible that the TLR4/NF-κB signaling pathway mediates the atherogenic effect of LPA.

Quercetin attenuates atherosclerotic inflammation and adhesion molecule expression by modulating TLR-NF-κB signaling pathway

Adhesion molecules expressed by activated endothelial cells play key role in regulating leukocyte trafficking to sites of inflammation. The present study attempted to explore whether the polyphenolic flavonoid quercetin influence leukocyte endothelial attraction and the involvement of TLR-NF-κB signaling pathway in the expression of adhesion molecules involved in the early development of atherosclerosis. Quercetin at 25μM concentration significantly reduced the HUVEC expression of VCAM-1 and ICAM-1 evidently enhanced by oxLDL. In addition, quercetin significantly downregulated the mRNA expression of MCP-1 and alleviated the nuclear translocation of NF-κB p65 subunit in oxLDL induced HUVECs. Western blot and PCR analyses revealed that quercetin significantly attenuated the expression of both protein and mRNA expression of TLR2 and TLR4. Quercetin supplementation significantly decreased the inflammatory mediators like COX, 5-LOX, MPO, NOS, CRP and the mRNA expression of the cytokine; IL-6 in hypercholesterolemic diet (HCD) fed atherosclerotic rats. The results demonstrate that quercetin is effective to regulate the atherosclerotic inflammatory process by inhibiting oxLDL induced endothelial leukocyte adhesion by attenuating the TLR-NF-κB signaling pathway in endothelial cells and decrease the inflammatory process induced by HCD in rats. Therefore, quercetin acts as anti-inflammatory and anti-atherogenic agent, which may have implications for strategies attenuating endothelial dysfunction-related atherosclerosis.

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.

Wenshen Xiaozheng Tang induces apoptosis and inhibits migration of ectopic endometriotic stromal cells

Ethnopharmacological relevanceWenshen Xiaozheng Tang (WXT), a traditional Chinese medicine prescription, exerted a good therapeutic effect on endometriosis. However, the underlying mechanism is unclear. In the present study, we sought to evaluate the effect of WXT on the proliferation and migration of ectopic endometriotic stromal cells and explore the potential molecular mechanism. Materials and methodsPrimary stromal cells derived from ectopic endometriotic lesions of patients with endometriosis were isolated and cultured. The inhibition effect of WXT on cell proliferation was determined by MTT. Apoptosis of ectopic endometriotic cells treated with WXT was analyzed with Annexin V-FITC/7-AAD staining. The activation of caspases was detected by western blot analysis. The influence of WXT on migration of ectopic endometriotic cells was measured by scratch wound healing assay and Transwell assay. The DNA binding activity of NF-κB and the expression of nuclear p65 protein were determined by electrophoretic mobility shift assay and western blot analysis, respectively. The impact of WXT on the expression of NF-κB regulated gene products involved in apoptosis and migration was determined by western blot analysis. ResultsWXT inhibited the proliferation of ectopic endometriotic cells in a time- and dose-dependent manner. In addition, WXT treatment resulted in significant induction of apoptosis through the activation of caspases and inhibition of migration in ectopic endometriotic cells. WXT notably suppressed constitutive NF-κB-DNA-binding activity as well as TNF-α induced nuclear translocation of NF-κB p65 subunit in ectopic endometriotic cells. Moreover, WXT diminished the expression of NF-κB regulated gene products involved in apoptosis and migration, including c-IAP1, c-IAP2, XIAP, survivin, Mcl-1, COX-2 and MMP-9. ConclusionsOur results indicate that WXT induces apoptosis and inhibits migration of ectopic endometriotic stromal cells.

Naringin lauroyl ester inhibits lipopolysaccharide-induced activation of nuclear factor κB signaling in macrophages.

Naringin (Nar) has antioxidant and anti-inflammatory properties. It was recently reported that enzymatic modification of Nar enhanced its functions. Here, we acylated Nar with fatty acids of different sizes (C2-C18) using immobilized lipase from Rhizomucor miehei and investigated the anti-inflammatory effects of these molecules. Treatment of murine macrophage RAW264.7 cells with Nar alkyl esters inhibited lipopolysaccharide (LPS)-induced nitric oxide (NO) production, with Nar lauroyl ester (Nar-C12) showing the strongest effect. Furthermore, Nar-C12 suppressed the LPS-induced expression of inducible NO synthase by blocking the phosphorylation of inhibitor of nuclear factor (NF)-κB-α as well as the nuclear translocation of NF-κB subunit p65 in macrophage cells. Analysis of Nar-C12 uptake in macrophage cells revealed that Nar-C12 ester bond was partially degraded in the cell membrane and free Nar was translocated to the cytosol. These results indicate that Nar released from Nar-C12 exerts anti-inflammatory effects by suppressing NF-κB signaling pathway.

Pretreatment with Astragaloside IV protects human umbilical vein endothelial cells from hydrogen peroxide induced oxidative stress and cell dysfunction via inhibiting eNOS uncoupling and NADPH oxidase - ROS - NF-κB pathway.

Endothelial cell injury caused by reactive oxygen species (ROS) plays a critical role in the pathogenesis of cardiovascular disorders. Astragaloside IV (AsIV) possesses potent antioxidant properties against oxidative stress through undefined mechanism(s). We sought to investigate whether AsIV protects human umbilical vein endothelial cells (HUVECs) from hydrogen peroxide (H2O2) induced oxidative stress focusing on eNOS uncoupling and the NADPH oxidase - ROS - NF-κB pathway. Compared with HUVECs incubated with H2O2 alone, pretreatment with AsIV significantly increased the viability of HUVECs, which was accompanied with apparent increase in nitric oxide (NO) production and decrease in intracellular superoxide anion production. Furthermore, pretreatment with AsIV increased endothelial nitric oxide synthase (eNOS) dimer/monomer ratio and its critical cofactor tetrahydrobiopterin (BH4) content, decreased Nox4 protein expression (the most abundant Nox isoform in HUVECs), inhibited translocation of NF-κB p65 subunit into nuclear fraction while enhanced the protein expression of IκB-α (the inhibitor of NF-κB p65), reduced the levels of IL-1β, IL-6, and TNF-α in HUVECs medium, and decreased iNOS protein expression. These results suggest that AsIV may protect HUVECs from H2O2-induced oxidative stress via inhibiting NADPH oxidase - ROS - NF-κB pathway and eNOS uncoupling.