Parthenolide Inhibits the LPS‐induced Secretion of IL‐6 and TNF‐α and NF‐κB Nuclear Translocation in BV‐2 Microglia

Feverfew (Tanacetum parthenium [L.] Sch. Bip. [Asteraceae]) is a popular herbal treatment used to prevent and treat headache and migraine. Parthenolide (PTN), the sesquiterpene lactonic derivative that is the plant's major component, might be one of the ingredients that act on mediators of inflammation. In the present study, in cultured lipopolysaccharide (LPS)‐stimulated BV‐2 microglia pretreatment with PTN caused a dose‐dependent reduction of interleukin‐6 (IL‐6) secretion (29% by 200 nm, p < 0.001; 45% by 1 µm, p < 0.001; 98% by 5 µm, p < 0.001); at 5 µm, the highest concentration tested, it also reduced the secretion of TNF‐α (54%, p < 0.001). Western blotting analysis on separate cytoplasmic and nuclear extracts showed that PTN strongly reduced the translocation of nuclear factor (NF)‐κB to the cell nucleus. The reduction of microglial activation by inhibition of proinflammatory agents may help attenuate the onset and intensity of acute migraine attacks. These in vitro results provide an additional explanation for the efficacy of orally administered T. parthenium as an antimigraine agent. Copyright © 2012 John Wiley & Sons, Ltd.     

Quercetin reduces TNF‐α‐induced mesangial cell proliferation and inhibits PTX3 production: Involvement of NF‐κB signaling pathway

Lupus nephritis (LN) is an autoimmune disease caused by systemic lupus erythematosus. Excessive proliferation of mesangial cells is one of the most serious pathological manifestations of LN. In addition, the expression of PTX3 is elevated in the serum of patients with LN. Quercetin has good anti‐inflammatory effects and immunomodulatory activities. In this study, the result of MTT indicated that quercetin treatment alleviated the excessive proliferation of mesangial cells. ELISA and immunofluorescence experiments showed that quercetin treatment inhibited the expression of PTX3. Three doses of quercetin (20, 40, and 80 μM) were selected for the experiment. It is noteworthy that the efficacy of quercetin at 80 μM was significantly better than that of other dose groups. And the effect in inhibiting PTX3 expression was comparable with that of the PDTC (80 μM) positive control. Western blot and qRT‐PCR analysis revealed that quercetin treatment reduced the expression of nuclear factor‐κB p65 and IKKβ, increased the expression of IκBα, and inhibited the expression of PTX3. In conclusion, through inhibiting the activation of nuclear factor‐κB signaling pathway, quercetin treatment could reduce the expression of PTX3 and inhibit the excessive proliferation of mesangial cells, suggesting that quercetin is a potential therapeutic drug for LN.     

A Polyphenol‐rich Pomegranate Fruit Extract Suppresses NF‐κB and IL‐6 Expression by Blocking the Activation of IKKβ and NIK in Primary Human Chondrocytes

Pomegranate fruit extract (PE) rich in polyphenols has been shown to exert chondroprotective effects, but the mechanism is not established. Here, we used an in vitro model of inflammation in osteoarthritis (OA) to investigate the potential of PE to suppress interleukin 1 beta (IL‐1β)‐stimulated expression of inflammatory cytokine IL‐6, generation of reactive oxygen species (ROS) levels, and investigated the mechanism of NF‐κB inhibition by analyzing the activation of the kinases upstream of IκBα in primary human chondrocytes. Total and phosphorylated forms of kinases and expression of IL‐6 were determined at protein and mRNA levels by western immunoblotting and Taqman assay, respectively. Dihydrorhodamine 123 staining estimated ROS generation. Pomegranate fruit extract inhibited the mRNA and protein expression of IL‐6, generation of ROS, and inhibited the IL‐1β‐mediated phosphorylation of inhibitor of nuclear factor kappa‐B kinase subunit beta (IKKβ), expression of IKKβ mRNA, degradation of IκBα, and activation and nuclear translocation of NF‐κB/p65 in human chondrocytes. Importantly, phosphorylation of NF‐κB‐inducing kinase was blocked by PE in IL‐1β‐treated human OA chondrocytes. Taken together, these data suggest that PE exerts the chondroprotective effect(s) by suppressing the production of IL‐6 and ROS levels. Inhibition of NF‐κB activation by PE was blocked via modulation of activation of upstream kinases in human OA chondrocytes. Copyright © 2017 John Wiley & Sons, Ltd.     

Hydroxy‐Safflower Yellow A inhibits the TNFR1‐Mediated Classical NF‐κB Pathway by Inducing Shedding of TNFR1

Hydroxy‐safflower yellow A (HSYA) is the major active component of safflower, a traditional Asia herbal medicine well known for its cardiovascular protective activities. The purpose of this study was to investigate the effect of HSYA on TNF‐α‐induced inflammatory responses in arterial endothelial cells (AECs) and to explore the mechanisms involved. The results showed that HSYA suppressed the up‐regulation of ICAM‐1 expression in TNF‐α‐stimulated AECs in a dose‐dependent manner. High concentration (120 μM) HSYA significantly inhibited the TNF‐α‐induced adhesion of RAW264.7 cells to AECs. HSYA blocked the TNFR1‐mediated phosphorylation and degradation of IκBα and also prevented the nuclear translocation of NF‐κB p65. Moreover, HSYA reduced the cell surface level of TNFR1 and increased the content of sTNFR1 in the culture media. TNF‐α processing inhibitor‐0 (TAPI‐0) prevented the HSYA inhibition of TNFR1‐induced IκBα degradation, implying the occurrence of TNFR1 shedding. Furthermore, HSYA induced phosphorylation of TNF‐α converting enzyme (TACE) at threonine 735, which is thought to be required for its activation. Conclusively, HSYA suppressed TNF‐α‐induced inflammatory responses in AECs, at least in part by inhibiting the TNFR1‐mediated classical NF‐κB pathway. TACE‐mediated TNFR1 shedding can be involved in this effect. Our study provides new evidence for the antiinflammatory and anti‐atherosclerotic effects of HSYA. Copyright © 2016 John Wiley & Sons, Ltd.     

Anti‐inflammatory Effects of Ginsenosides Rg5, Rz1, and Rk1: Inhibition of TNF‐α‐induced NF‐κB,COX‐2, and iNOS transcriptional expression

In the course of this experiment on the anti‐inflammatory effect of ginsenosides, protopanaxdiol ginsenosides have shown inhibition activities in inflammatory responses: NF‐κB, COX‐2, and iNOS were induced by TNF‐α. The responses of this experiment were evaluated by NF‐κB‐luciferase assay and RT‐PCR experiment of COX‐2 and iNOS genes. The NF‐κB expressions were inhibited by ginsenosides Rd, Rg₅, Rz₁, and Rk₁ in a dose‐dependent manner. The IC₅₀ values were 3.47, 0.61, 0.63, and 0.75 μM, respectively. Particularly, ginsenosides Rg₅, Rz₁, and Rk₁ as converted ginsenosides from primary protopanaxdiol ginsenosidess significantly inhibited COX‐2 and iNOS gene expression. These inhibition levels were similar to sulfasalazine as reference material. Copyright © 2014 John Wiley & Sons, Ltd.     

Praeruptorin a inhibits lipopolysaccharide‐induced inflammatory response in murine macrophages through inhibition of NF‐κB pathway activation

Praeruptorin A (PA) is a pyranocoumarin compound isolated from the dried root of Peucedanum praeruptorum Dunn (Umbelliferae). However, the antiinflammatory effect of PA has not been reported. The present study investigated the antiinflammatory effect of PA in lipopolysaccharide (LPS)‐stimulated RAW 264.7 macrophage cells. PA significantly inhibited the LPS‐induced production of nitric oxide (NO), interleukin‐1β (IL‐1β) and tumor necrosis factor‐α (TNF‐α). The mRNA and protein expressions of inducible nitric oxide synthase (iNOS), IL‐1β and TNF‐α were also suppressed by this compound. Further study showed that PA decreased the cytoplasmic loss of inhibitor κB‐α (IκB‐α) protein and inhibited the translocation of NF‐κB from cytoplasm to nucleus. Taken together, the results suggest that PA may exert antiinflammatory effects in vitro in LPS‐stimulated RAW 264.7 macrophages through inhibition of NF‐κB signal pathway activation. Copyright © 2010 John Wiley & Sons, Ltd.     

Regulation of adhesion molecules expression in TNF‐α‐stimulated brain microvascular endothelial cells by tanshinone IIA: involvement of NF‐κB and ROS generation

Pro‐inflammatory cytokine‐mediated expression of cell surface adhesion molecules plays a key role in endothelial cell injury, leading to vascular inflammation and the development of many cerebrovascular diseases. Thus, antiinflammatory agents targeting these adhesion molecules may represent potential drugs for the prevention and treatment of cerebrovascular diseases. The present study explored the effects of tanshinone IIA (Tan IIA), an active ingredient present in the Salvia miltiorrhiza root, on the expression of cellular adhesion molecules in TNF‐α‐stimulated brain microvascular endothelial cells (BMVECs). Treatment with Tan IIA was found to suppress the expression of vascular cell adhesion molecule‐1 (VCAM‐1) and intercellular adhesion molecule‐1 (ICAM‐1), resulting in inhibition of TNF‐α‐induced adhesion of neutrophils to BMVECs in a dose‐dependent manner. In addition, Tan IIA significantly inhibited TNF‐α‐induced production of reactive oxygen species (ROS), which was accompanied by decreased malondialdehyde (MDA) levels. Treatment with Tan IIA also inhibited nuclear factor‐kappa B (NF‐κB) activation. Together, these results suggest that Tan IIA regulates TNF‐α‐induced expression of VCAM‐1 and ICAM‐1 through inhibition of NF‐κB activation and ROS generation in BMVECs. Copyright © 2010 John Wiley & Sons, Ltd.     

Inhibition of TNF‐α‐Induced MUC5AC Mucin Gene Expression and Production by Wogonin Through the Inactivation of NF‐κB Signaling in Airway Epithelial Cells

In this study, we investigated whether wogonin significantly affects MUC5AC mucin gene expression and production in human airway epithelial cells. Confluent NCI‐H292 cells were pretreated with wogonin for 30 min and then stimulated with tumor necrosis factor‐α (TNF‐α) for 24 h or the indicated periods. The MUC5AC mucin gene expression and mucin protein production were measured by RT‐PCR and ELISA, respectively. We found that incubation of NCI‐H292 cells with wogonin significantly inhibited mucin production and down‐regulated MUC5AC gene expression induced by TNF‐α in a dose‐dependent fashion. To elucidate the action mechanism of wogonin, effect of wogonin on TNF‐α‐induced NF‐κB signaling pathway was investigated by western blot analysis. Wogonin inhibited NF‐κB activation induced by TNF‐α. Inhibition of IKK by wogonin led to the suppression of IκB phosphorylation and degradation, p65 nuclear translocation and NF‐κB‐regulated gene expression. This, in turn, led to the down‐regulation of MUC5AC protein production in NCI‐H292 cells. Wogonin also inhibited the gene products involved in cell survival (Bcl‐2) and proliferation (cyclooxygenase‐2). These results suggest that wogonin inhibits the NF‐κB signaling pathway, which may explain its role in the inhibition of MUC5AC mucin gene expression and production. Copyright © 2013 John Wiley & Sons, Ltd.     

Isoforskolin and forskolin attenuate lipopolysaccharide‐induced inflammation through TLR4/MyD88/NF‐κB cascades in human mononuclear leukocytes

The principal active component of isoforskolin (ISOF) is from the plant Coleus forskohlii, native to China, which has attracted much attention for its biological effects. We hypothesize that ISOF and forskolin (FSK) pretreatment attenuates inflammation induced by lipopolysaccharide (LPS) related to toll‐like receptor 4 (TLR4), myeloid differentiation factor 88 (MyD88), and nuclear factor kappa B (NF‐κB) signaling. Mononuclear leukocytes (MLs) from healthy donors' blood samples were separated by using density gradient centrifugation. Protein levels of TLR4, MyD88, and NF‐κB were detected using western blot and inflammatory cytokines interleukin (IL) 1β, IL‐2, IL‐6, IL‐21, IL‐23, tumor necrosis factor (TNF) α, and TNF‐β were tested by enzyme‐linked immunosorbent assay and Quantibody array in MLs. Our results showed that LPS augmented the protein levels of TLR4, MyD88, and NF‐κB in MLs and the production of IL‐1β, IL‐2, IL‐6, IL‐21, IL‐23, TNF‐α, and TNF‐β in supernatants of MLs. Despite treatment with ISOF and FSK prior to LPS, the protein levels of TLR4, MyD88, NF‐κB, IL‐1β, IL‐2, IL‐6, IL‐21, IL‐23, TNF‐α, and TNF‐β in MLs were apparently decreased. roflumilast (RF) and dexamethasone (DM) had a similar effect on MLs with ISOF and FSK. Our results, for the first time, have shown that ISOF and FSK attenuate inflammation in MLs induced by LPS through down‐regulating protein levels of IL‐1β and TNF‐α, in which TLR4/MyD88/NF‐κB signal pathway could be involved.     

Astragaloside IV inhibits TGF‐β1‐induced epithelial‐mesenchymal transition through inhibition of the PI3K/Akt/NF‐κB pathway in gastric cancer cells

Astragaloside IV (AS‐IV) has been reported to possess anti‐metastasis activity in cancer cells. However, it is unknown whether AS‐IV could inhibit epithelial‐mesenchymal transition (EMT), a cellular de‐differentiation program that promotes metastasis, in cancer cells. The aim of this study was to study the effect and mechanism of AS‐IV on EMT in gastric cancer (GC) cells. The results showed that AS‐IV significantly inhibited cell viability, invasion, and migration of GC cells. The E‐cadherin to N‐cadherin switch and expression of Vimentin and metastasis‐related genes were induced by transforming growth factor β1 (TGF‐β1), whereas AS‐IV reversed the induction. In addition, AS‐IV inhibited TGF‐β1‐induced activation of PI3K/Akt/NF‐κB. Inhibition of the PI3K/Akt/NF‐κB pathway reversed TGF‐β1‐induced EMT. In conclusion, AS‐IV inhibited TGF‐β1‐induced EMT through inhibition of the PI3K/Akt/NF‐κB pathway in GC cells. AS‐IV might be an effective candidate for the treatment for GC.     

Calycosin attenuates MPTP‐induced Parkinson's disease by suppressing the activation of TLR/NF‐κB and MAPK pathways

Parkinson is the second common neurodegenerative disease. The characteristics of Parkinson's disease (PD) are the dopamin neurons loss caused by neuroinflammation responses. C alycosin, an isoflavone phytoestrogen isolated from Astragalus membranaceus, has multiple pharmacological activities, such as anti‐inflammation, anti‐tumor, and neuroprotective effects. However, it is unknown whether calycosin can mitigate PD symptoms. This study aims to explore whether calycosin can alleviate PD symptoms and the underlying mechanisms. PD was induced in mice by 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP) injection, and calycosin was given intracerebroventricularly to these mice. A cell model of nerve inflammation was established by BV2 microglia cells injected with lipopolysaccharide (LPS). The motor states were evaluated by stepping, whisker, and cylinder experiments. The states of dopaminergic neurons and microglia were detected by immunostainning of tyrosine hydroxylase and cluster of differentiation molecule 11b (CD11b). The expression levels of inflammatory factors were detected by qPCR. Toll‐like receptor (TLR)/nuclear factor kappa B (NF‐κB) and mitogen‐activated protein kinase (MAPK) pathways were investigated by western blot. We found that calycosin treatment mitigated the behavioral dysfunctions and inflammatory responses in MPTP‐induced PD mice. The TLR/NF‐κB and MAPK pathways in MPTP‐induced PD mice were inhibited by calycosin treatment, which was coincident with experiments in LPS‐induced BV2 cells. Above all, calycosin mitigates PD symptoms through TLR/NF‐κB and MAPK pathways in mice and cell lines.     

Inhibition of TNF‐α‐mediated endothelial cell–monocyte cell adhesion and adhesion molecules expression by the resveratrol derivative,trans‐3,5,4′‐trimethoxystilbene

Resveratrol (RSV) has been shown to have anti‐inflammatory activity and to have a protective role against atherosclerosis. Here it is shown, for the first time, that its derivative trans‐3,5,4′‐trimethoxystilbene (TMS) may be a more potent anti‐inflammatory agent than resveratrol. A comparative analysis of the inhibitory activities of related stilbenes, resveratrol, TMS and polydatin (PD), on monocyte adhesion to TNF‐α‐activated endothelial cells showed TMS to be the most effective, with PD being the least effective. RSV and its analogues inhibited, albeit differentially, the protein and mRNA expression levels of inducible cell adhesion molecules, ICAM‐1 and VCAM‐1, in cultured endothelial cells. The mechanism of the inhibitory effects of these stilbenes on endothelial cell–monocyte cell adhesion can be attributed mainly to inhibition of NF‐κB pathway activation. The results demonstrate that all three investigated stilbene compounds, especially TMS, exhibit a potent inhibitory effect on inflammation‐induced cell–cell adhesion, expression of adhesion molecules and activation of the NF‐κB pathway. Copyright © 2010 John Wiley & Sons, Ltd.     

Celastrol attenuates renal injury in diabetic rats via MAPK/NF‐κB pathway

The purpose of this study was to investigate the renal protective effect of celastrol on diabetic rats. Furthermore, the mechanism of its action was discussed whether it was related to MAPK/NF‐κB signaling pathway. There were a total of 36 rats. Six rats were randomly chosen as the control group. The remaining 30 rats were given 1% streptozotocin intraperitoneal injection (50 mg/kg) and were randomly divided into five groups: the model control group, the low‐dose celastrol group, the high‐dose celastrol group, the Tripterygium wilfordii polyglycosides group, and the MAPK/NF‐κB inhibitor group. After 4 weeks of continuous administration, 24‐hr urine volume, urinary protein, blood urea nitrogen, and serum creatinine content were observed, and hematoxylin–eosin (HE) staining of the kidney and liver were evaluated. p38MAPK was designated by immunohistochemical method, and NF‐κB p65 in renal tissue was detected by western blotting. Our results showed that celastrol could not only reduce contents of creatinine and urea nitrogen in blood but also reduce excretion of urinary protein in diabetic rats, improve renal pathological injury, and down‐regulate the expression of p38MAPK and NF‐κB p65. In conclusion, celastrol could protect kidney of diabetic rats by regulating the signal pathway of MAPK/NF‐κB, inhibiting inflammation and delaying renal injury.     

Eugenol inhibits non‐small cell lung cancer by repressing expression of NF‐κB‐regulated TRIM59

In view of the recognized anti‐tumor properties of eugenol against non‐small cell lung cancer (NSCLC) in cell culture, here we further set out to investigate the potential therapeutic effect of eugenol in vivo and elucidate the underlying molecular mechanism. The relative expression levels of TRIM59 and p65 in NSCLC were quantified by real‐time polymerase chain reaction. Xenograft tumor model was established with TRIM59‐deficient H1975 cells, and tumor progression was monitored. Kaplan–Meier's analysis was performed to measure overall survival. Protein levels of TRIM59 and p65 in xenograft tumor were determined by western blot. Direct binding of p65 on the TRIM59 promoter was analyzed by chromatin immunoprecipitation assay, and the regulatory effect was interrogated with luciferase reporter assay. Both TRIM59 and p65 were up‐regulated in NSCLC. Eugenol treatment significantly inhibited xenograft tumor progression and prolonged the overall survival of tumor‐bearing mice. Mechanistically, eugenol suppressed p65 expression, which subsequently decreased TRIM59 expression. TRIM59 deficiency fully recapitulated the anti‐tumoral phenotype elicited by eugenol. Ectopic expression of TRIM59 completely abolished the tumor suppressive effect of eugenol, which underlined the predominant role of TRIM59 in mediating the signaling downstream of eugenol treatment. Eugenol inhibited NSCLC via repression NF‐κB‐TRIM59 pathway.     

Formula Optimization of the Jiashitang Scar Removal Ointment and Antiinflammatory Compounds Screening by NF‐κB Bioactivity‐guided Dual‐luciferase Reporter Assay System

Inflammation plays a role in scar formation; therefore, decreasing inflammation benefits scar removal. Jiashitang scar removal ointment (JST) is a commercially available traditional Chinese medicinal formulation. It is composed of extracts from Carthamus tinctorius L. (Car), Rheum officinale Baill. (Rhe), Salvia miltiorrhiza Beg. (Sal), and Panax notoginseng (Burk.) F. H. Chen (Pan), which are all herbs with potent antiinflammatory activities. Our aims are to optimize the formula of JST and to elucidate its antiinflammatory active components. Response surface methodology was applied to optimize proportions of the four herb extracts. The antiinflammatory effects were evaluated using in vitro and in vivo models. To screen for active components in this formula, a bioactivity‐based ultra‐performance liquid chromatography/quadrupole time‐of‐flight mass spectrometry analysis was performed. After optimization, the antiinflammatory effects of the new formula were significantly superior to the original one. Screening identified 13 active ingredients: a series of saffiomin, emodin, salvianolic acid, tanshinone, and triterpenoid saponin derivatives. These active ingredients were predicted to exert nuclear factor‐κB inhibiting effects through MAPK, PI3K/AKT, and NIK‐IKK pathways. In conclusion, the original formula was successfully optimized with more potent antiinflammatory activity. These methods can be applied to researches of other formulas. Copyright © 2014 John Wiley & Sons, Ltd.     

Nerolidol Protects Against LPS‐induced Acute Kidney Injury via Inhibiting TLR4/NF‐κB Signaling

Acute kidney injury (AKI) is a critical care syndrome, resulting in acute reduction of renal function and up to 22% mortality of hospitalized patients. Nerolidol is a major component in several essential oils that possesses various pharmacological properties. The present study aimed to investigate the potential effect of nerolidol on lipopolysaccharide (LPS)‐induced AKI. Nerolidol dose‐dependently reduced the pathological injuries of kidney induced by LPS in rats. Nerolidol significantly decreased the levels of blood urea nitrogen and creatinine in LPS‐treated rats in a dose‐dependent manner. In addition, nerolidol inhibited LPS‐induced decrease of cell viability in NRK‐52E rat proximal tubular cells, which effect was concentration dependent. Nerolidol notably inhibited the increase of TNFα and IL‐1β in LPS‐treated rats and the mRNA expression of TNFα and IL‐1β in LPS‐treated NRK‐52E cells. Nerolidol suppressed the increase of toll‐like receptor 4 (TLR4) expression, phosphorylation and nuclear translocation of p65 NF‐κB in kidneys of LPS‐treated rats and LPS‐treated NRK‐52E cells. Overexpression of TLR4 and p65 NF‐κB significantly suppressed nerolidol‐induced inhibition of TNFα and IL‐1β expression and increase of cell viability in LPS‐treated cells. In summary, we found that nerolidol played a critical anti‐inflammatory effects through inhibition of TLR4/NF‐κB signaling and protected against LPS‐induced AKI. Copyright © 2017 John Wiley & Sons, Ltd.