Curcumin inhibits NF-kappaB activation and reduces the severity of experimental steatohepatitis in mice

BACKGROUND/AIMS: While oxidative stress is a feature of non-alcoholic steatohepatitis, the causal link between oxidative stress and inflammatory recruitment has yet to be demonstrated. We analysed the role of NF-kappaB redox-sensitive signalling pathway of inflammatory recruitment in experimental steatohepatitis. METHODS: Mice were fed the methionine and choline deficient (MCD) or the control diet, with or without curcumin, an NF-kappaB inhibitor, for up to 4 weeks. Histopathology, lipoperoxides, NF-kappaB/DNA binding and expression of NF-kappaB-regulated genes were assessed. RESULTS: MCD-fed mice developed steatohepatitis accompanied by dramatic accumulation of hepatic lipoperoxides, activation of NF-kappaB and induction of pro-inflammatory ICAM-1, COX-2, MCP-1 and CINC mRNA. Curcumin significantly reduced MCD-induced inflammation but had no effect on steatosis or on the level of hepatic lipid peroxides. Curcumin prevented the MCD-induced activation of NF-kappaB and decreased downstream induction of ICAM-1, COX-2 and MCP-1. However, it failed to reduce activation of AP-1, MAPK pathways or CINC expression. CONCLUSIONS: Curcumin alleviates the severity of hepatic inflammation in experimental steatohepatitis induced by the MCD diet, an effect likely to be mediated via inhibition of NF-kB activation and dependent pro-inflammatory genes. The NF-kappaB pathway is one among several possible signalling pathways by which inflammation is recruited in experimental steatohepatitis.     

Vasoactive intestinal peptide induces cyclooxygenase-2 expression through nuclear factor-kappaB in human prostate cell lines Differential time-dependent responses in cancer progression

The effect of vasoactive intestinal peptide (VIP) on cyclooxygenase-2 (COX-2) expression was analyzed in human prostate non-neoplastic (RWPE-1) as well as cancer androgen-dependent (LNCaP) and independent (PC3) cells. The three cell lines expressed VIP mRNA and VIP peptide, as measured by RT-PCR and immunochemistry, which supports an autocrine/paracrine action of VIP in the prostate gland. VIP levels were progressively higher from non-neoplastic to androgen-dependent and independent cells. Real-time RT-PCR and Western-blotting showed that VIP stimulated both COX-2 mRNA and protein expression in a faster manner as prostate cancer stage progressed (i.e. RWPE1 < LNCaP < PC3 cells). Furthermore, VIP induced higher levels of COX-2 protein expression in cancer cells as compared with non-neoplastic cells. The anti-inflammatory agent curcumin blocked VIP-induced COX-2 expression in all cell lines studied supporting the involvement of nuclear factor-κB (NFκB) in such a response. In fact, VIP increased the translocation of the NFκB p50 subunit to the nucleus and the binding of the active form to its target gene promoter, as measured by Western-blotting and ELISA, respectively. VIP provoked faster responses according to the most aggressive status in cancer progression (androgen-independent situation). These results together with the existence of two NFκB sites in the COX-2 gene promoter together suggest that COX-2 may be a target for VIP in prostate cancer progression. On the other hand, VIP could be a proinflammatory cytokine acting through the NFκB/COX-2 system.     

Pharmacologic modulation of intracellular Na+ concentration with ranolazine impacts inflammatory response in humans and mice

Changes in Ca2⁺ influx during proinflammatory stimulation modulates cellular responses, including the subsequent activation of inflammation. Whereas the involvement of Ca²⁺ has been widely acknowledged, little is known about the role of Na⁺. Ranolazine, a piperazine derivative and established antianginal drug, is known to reduce intracellular Na⁺ as well as Ca2⁺ levels. In stable coronary artery disease patients (n = 51) we observed reduced levels of high-sensitive C-reactive protein (CRP) 3 mo after the start of ranolazine treatment (n = 25) as compared to the control group. Furthermore, we found that in 3,808 acute coronary syndrome patients of the MERLIN‐TIMI 36 trial, individuals treated with ranolazine (1,934 patients) showed reduced CRP values compared to placebo-treated patients. The antiinflammatory effects of sodium modulation were further confirmed in an atherosclerotic mouse model. LDL^−/− mice on a high-fat diet were treated with ranolazine, resulting in a reduced atherosclerotic plaque burden, increased plaque stability, and reduced activation of the immune system. Pharmacological Na⁺ inhibition by ranolazine led to reduced express of adhesion molecules and proinflammatory cytokines and reduced adhesion of leukocytes to activated endothelium both in vitro and in vivo. We demonstrate that functional Na⁺ shuttling is required for a full cellular response to inflammation and that inhibition of Na⁺ influx results in an attenuated inflammatory reaction. In conclusion, we demonstrate that inhibition of Na⁺–Ca²⁺ exchange during inflammation reduces the inflammatory response in human endothelial cells in vitro, in a mouse atherosclerotic disease model, and in human patients.

Cardiovascular diseases(CVD) remain the number one cause of mortality in the Western world. According to the European Society of Cardiology, ischemic heart disease caused 45% of all deaths in females and 39% in males during the year 2019 (1). Similarly, the leading cause of death within the United States was heart disease (2). Patients with an underlying atherosclerotic process represent a substantial number among those individuals. The chance of recurrent vascular events was found to be of moderate to high risk of 59% in a large-scale model analysis (3, 4). The fact that atherosclerosis is not just a mere storage disease but rather a chronic inflammatory process has been known since the end of the last century (5), and numerous studies have shown that inflammatory markers like high-sensitive C-reactive protein (hsCRP) predict cardiovascular events (6). The CANTOS trial was able to show that antiinflammatory therapy targeting the interleukin‐1β (IL-1β) innate immunity pathway can lower the rate of recurrent cardiovascular events as compared to treatment with placebo in patients with a history of myocardial infarction and hsCRP ≥2mg/L. Importantly, these findings were independent of lipid levels and substantiate the importance of inflammation in the atherosclerotic process (7). However, the CANTOS trial also demonstrated the usability of hsCRP as an indirect marker as the significant reduction of vascular event rates was in proportion to the magnitude of hsCRP reduction achieved (8). The mode of action of canakinumab, the monoclonal antibody used in the CANTOS trial, is to prevent the downstream activation of IL‐1β targets and the nuclear factor “kappa-light-chain-enhancer” of activated B cells (nuclear factor κB [NFκB]) pathway. A similar antiinflammatory strategy in CVD was presented in the Colchicine Cardiovascular Outcomes Trial (COLCOT) in patients within 30 d after myocardial infarction and in the Low-Dose Colchicine 2 (LoDoCo2) Trial involving patients with chronic coronary disease which indicated a positive effect of the antiinflammatory drug colchicine on the development of ischemic cardiovascular events (9). In a following substudy hsCRP levels were found lower in patients treated with colchicine compared to placebo (10).The NFκB pathway plays a major role in the cellular inflammatory response and can be activated by numerous triggers, including IL‐1β (11). Intracellular electrolyte shifts, especially Ca²⁺ influx, and the Ca²⁺ concentration in itself are thought to be among those factors propagating the inflammatory signal. The association of intracellular Ca²⁺ levels with the activation of the NFκB pathway was shown in various cell lines (12–14). Intracellular calcium signaling might require several proteins, including stromal interaction molecule (STIM) and Orai (15). Interestingly, Orai signaling is also associated with Na⁺ influx (16). To our knowledge, there are no clear data on the influence of intracellular Na⁺ concentration on NFκB activation in vascular cells or on the effects of modulation of Na⁺ to control inflammation in vivo or in vitro.Ranolazine, a piperazine derivative and inhibitor of Na⁺ channel currents, is known to reduce intracellular Na⁺ and Ca²⁺ levels in cardiomyocytes (17). Even though ranolazine is indicated in patients with stable angina [class IIa recommendation for symptom relief in the American College of Cardiology/American Heart Association guidelines (18)], no information of ranolazine treatment regarding inflammation or atherosclerotic disease progression is available and a potential finding of inflammation‐modulating effects might be highly relevant in such patients. The purpose of this study was to investigate possible pleiotropic effects of an established antianginal drug in vivo and to evaluate a potential Na⁺- and Ca²⁺‐dependent inflammatory response via the NFκB pathway in endothelial cells in vitro.     

Interleukin-23 production in dendritic cells is negatively regulated by protein phosphatase 2A

IL-12 and IL-23 are produced by activated antigen-presenting cells but the two induce distinct immune responses by promoting Th1 and Th17 cell differentiation, respectively. IL-23 is a heterodimeric cytokine consisting of two subunits: p40 that is shared with IL-12 and p19 unique to IL-23. In this study, we showed that the production of IL-23 but not IL-12 was negatively regulated by protein phosphatase 2A (PP2A) in dendritic cells (DC). PP2A inhibits IL-23 production by suppressing the expression of the IL-23p19 gene. Treating DC with okadaic acid that inhibits the PP2A activity or knocking down the catalytic subunit of PP2A with siRNA enhanced IL-23 but not IL-12 production. Unlike PP2A, MAP kinase phosphatase-1 or CYLD did not show an effect on IL-23 production supporting the specificity of PP2A. PP2A-mediated inhibition requires a newly made protein that is likely responsible for bringing PP2A and IKKβ together upon LPS stimulation, which then results in the termination of IKK phosphorylation. Thus, our results uncovered an important role of the protein phosphatase in the regulation of IL-23 production and identified PP2A as a previously uncharacterized inhibitor of IL-23p19 expression in DC.     

Toll-like receptor-mediated signaling cascade as a regulator of the inflammation network during alcoholic liver disease

Chronic abuse of alcohol leads to various histological abnormalities in the liver. These are conditions collectively known as alcoholic liver disease(ALD). Currently, ALD is considered to be one of the major causes of death worldwide. An impaired intestinal barrier with related endotoxemia is among the various pathogenetic factors. This is mainly characterized by circulating levels of lipopolysaccharide(LPS), considered critical for the onset of intra-hepatic inflammation. This in turn promotes hepatocellular damage and fibrosis in ALD. Elevated levels of LPS exert their effects by binding to Toll-like receptors(TLRs) which are expressed by all liver-resident cells. The activation of TLR signaling triggers an overproduction and release of some cytokines, which promote an autocatalytic cascade of other proinflammatory signals. In this review, we provide an overview of the mechanisms that sustain LPS-mediated activation of TLR signaling, reporting current experimental and clinical evidence of its role during inflammation in ALD.     

Guggulsterone, a plant sterol, inhibits NF-kappaB activation and protects pancreatic beta cells from cytokine toxicity

Guggulsterone has been used to treat hyperlipidemia, arthritis, and obesity. Although its anti-inflammatory and anti-hyperlipidemic effects have been well documented, the effect of guggulsterone on pancreatic beta cells is unknown. Therefore, in this study, the effect of guggulsterone on IL-1beta- and IFN-gamma-induced beta-cell damage was investigated. Treatment of RINm5F (RIN) rat insulinoma cells with IL-1beta and IFN-gamma induced cell damage, and this damage was well correlated with nitric oxide (NO) and prostaglandin E2 (PGE2) production. However, guggulsterone completely prevented cytokines-mediated cytotoxicity, as well as NO and PGE2 production, and these effects were correlated with reduced levels of the inducible form of NO synthase (iNOS) and cyclooxygenase-2 (COX-2) mRNA and protein expressions. The molecular mechanism by which guggulsterone inhibits iNOS and COX-2 gene expressions appeared to involve the inhibition of NF-kappaB activation. The cytoprotective effects of guggulsterone were also mediated through the suppression of the JAK/STAT pathway. Cells treated with the cytokines downregulated the protein level of SOCS-3, however pretreatment with guggulsterone attenuated this decrease. Additionally, in a second set of experiments in which rat islets were used, the findings regarding the beta-cell protective effects of guggulsterone were essentially the same as those observed when RIN cells were used; guggulsterone prevented cytokines-induced NO and PGE2 production, iNOS and COX-2 expressions, JAK/STAT activation, NF-kappaB activation, downregulation of SOCS-3, and impairment of glucose-stimulated insulin secretion. Collectively, these results suggest that guggulsterone may be used to preserve functional beta-cell mass.     

PPARdelta modulates lipopolysaccharide-induced TNFalpha inflammation signaling in cultured cardiomyocytes

Peroxisome proliferator activated receptors (PPARs: PPARalpha, gamma and delta) regulate fatty acid metabolism, glucose homeostasis, cell proliferation, differentiation and inflammation. Tumor necrosis factor alpha (TNFalpha) is one of the important pathological factors in inflammatory responses during the pathological progression of myocardial ischemic/reperfusion and hypertrophy. Accumulating evidence shows that synthetic ligands of PPARalpha and PPARgamma suppress myocardial inflammatory responses, such as the production of TNFalpha, thus exerting beneficial effects in animals who had undergone ischemia/reperfusion injury or cardiac hypertrophy. However, it remains obscured if PPARdelta and its ligands exert any effect on the production of TNFalpha, thus influencing cardiac inflammatory responses. In this study, we investigated the effects of PPARdelta and its synthetic ligand GW0742 on TNFalpha production in cultured cardiomyocytes. Our studies indicate that a PPARdelta-selective ligand inhibited lipopolysaccharide (LPS)-induced TNFalpha production from cardiomyocytes. Adenoviral-mediated overexpression of PPARdelta substantially inhibited TNFalpha expression in cultured cardiomyocytes compared to controls, whereas overexpression of a PPARdelta mutant with ablated ligand binding domain did not show similar effect. Conversely, absence of PPARdelta in cardiomyocytes further exaggerated LPS-induced TNFalpha production. Moreover, activation of PPARdelta abrogated LPS-induced degradation of IkappaBs, thus suppressing LPS-induced nuclear factor kappaB (NF-kappaB) activities. Therefore, PPARdelta is an important determinant of TNFalpha expression via the NF-kappaB signaling pathway, thus serving as therapeutic targets to attenuate inflammation that are involved in cardiac pathological progression.     

HuR regulates the expression of stress-sensitive genes and mediates inflammatory response in human umbilical vein endothelial cells

An important aspect of vascular biology is the identification of regulators of stress-sensitive genes that play critical roles in mediating inflammatory response. Here, we show that expression of HuR in human umbilical vein endothelial cells is regulated by shear stress and statin treatment; HuR, in turn, regulates other stress-sensitive genes such as Kruppel-like factor 2 (Klf2), endothelial nitric oxide synthase (eNOS), and bone morphogenic protein 4 (BMP-4). We found that siRNA knockdown of HuR-inhibited inflammatory responses in endothelial cells, including ICAM-1 and VCAM-1 up-regulation, NFκB phosphorylation, and adhesion of monocytes. Tissue staining of the mouse aorta revealed increased HuR expression in the lesser curvature region of the arch that is exposed to disturbed flow, consistent with our in vitro data. Taken together, these results suggest that HuR plays a critical role in inducing inflammatory response of endothelial cells under mechanical and biochemical stresses.     

Protective effect of bicyclol on lipopolysaccharide-induced acute lung injury in mice

Bicyclol is synthesized based on schisandrin, which is one of the main active components of Chinese herb Fructus Schisandrae. The purpose of this study is to investigate whether bicyclol has a beneficial effect on lipopolysaccharide (LPS)-induced acute lung injury (ALI) in mice. Bicyclol was given to mice by gavage for three times. ALI was induced by vena caudalis injection of LPS. The last dose of bicyclol was administrated 1 h before LPS given. Mice in each group were sacrificed at different time point after LPS administration. As revealed by survival study, pretreatment with high doses of bicyclol reduced the mortality of mice from ALI. Bicyclol pretreatment significantly improved LPS-induced lung pathological changes, inhibited myeloperoxidase (MPO) activity, and reduced lung/body and lung wet/dry weight ratios. Bicyclol also inhibited the release of TNF-α, IL-1β and HMGB1, whereas simultaneously increased the expression of IL-10. Furthermore, the phosphorylation level of NF-κB p65 was markedly decreased by bicyclol. Taken together, our study showed that bicyclol improves survival rate and attenuates LPS-induced ALI. The protective mechanism may be due to the inhibition of NF-κB activation and regulation of cytokine secretion.     

ROR1 is essential for proper innervation of auditory hair cells and hearing in humans and mice

Hair cells of the inner ear, the mechanosensory receptors, convert sound waves into neural signals that are passed to the brain via the auditory nerve. Little is known about the molecular mechanisms that govern the development of hair cell–neuronal connections. We ascertained a family with autosomal recessive deafness associated with a common cavity inner ear malformation and auditory neuropathy. Via whole-exome sequencing, we identified a variant (c.2207G>C, p.R736T) in ROR1 (receptor tyrosine kinase-like orphan receptor 1), cosegregating with deafness in the family and absent in ethnicity-matched controls. ROR1 is a tyrosine kinase-like receptor localized at the plasma membrane. At the cellular level, the mutation prevents the protein from reaching the cellular membrane. In the presence of WNT5A, a known ROR1 ligand, the mutated ROR1 fails to activate NF-κB. Ror1 is expressed in the inner ear during development at embryonic and postnatal stages. We demonstrate that Ror1 mutant mice are severely deaf, with preserved otoacoustic emissions. Anatomically, mutant mice display malformed cochleae. Axons of spiral ganglion neurons show fasciculation defects. Type I neurons show impaired synapses with inner hair cells, and type II neurons display aberrant projections through the cochlear sensory epithelium. We conclude that Ror1 is crucial for spiral ganglion neurons to innervate auditory hair cells. Impairment of ROR1 function largely affects development of the inner ear and hearing in humans and mice.Sensorineural hearing loss (SNHL) is diagnosed in approximately 1 per 500 newborns (1). A genetic etiology is present in more than half of the cases. Inner ear anomalies (IEAs), demonstrated with computerized tomography or magnetic resonance imaging, are associated with SNHL in about one-third of individuals (2). Although IEAs can be diagnosed in patients with other clinical manifestations, such as those seen in Waardenburg [Mendelian Inheritance in Man (MIM) 193500], Pendred (MIM 274600), or BOR (MIM 113650) syndromes, the majority of cases fall into the category of nonsyndromic deafness. Despite recent progress in identifying genes that determine many forms of hearing loss (hereditaryhearingloss.org/), the genetic basis of IEAs in humans remains largely unknown.The inner ear is a complex organ that is built from a simple structure, referred to as the otocyst, through a series of morphogenetic events. Roughly, it consists of a dorsal vestibular and a ventral auditory component (3). Studies in model organisms have identified a number of genes that play roles in proper development of the inner ear. Mouse models have been particularly relevant because the anatomy and physiology of the murine auditory system are similar to those of humans. Mutations in human orthologs of many of these genes have been reported to cause deafness in humans as well (4).Next-generation sequencing technologies have allowed rapid identification of novel human deafness genes. Approximately 85% of disease-causing mutations in Mendelian disorders have been found in the protein-coding regions, despite the fact that this portion accounts for less than 2% of the entire human genome (5). Accordingly, whole exome sequencing (WES) has been frequently used because it allows for a targeted enrichment and resequencing of nearly all exons of protein-coding genes.In this study, via WES, we detected a mutation in ROR1 (receptor tyrosine kinase-like orphan receptor 1; MIM 602336), encoding receptor tyrosine kinase-like orphan receptor 1, that associates with an IEA and nonsyndromic deafness in a family. Further characterization of Ror1 mutant mice revealed that Ror1 deficiency results in defective hair cell innervation and abnormal cochlear development.     

Damaged spermatogenic cells induce inflammatory gene expression in mouse Sertoli cells through the activation of Toll-like receptors 2 and 4

Testicular inflammation, including noninfectious inflammatory responses in the testis, may impair male fertility. Mechanisms underlying the initiation of noninfectious testicular inflammation are poorly understood. In the current study, we demonstrate that damaged spermatogenic cell products (DSCPs) induce expression of various inflammatory mediators, including TNF-α, IL-1β, IL-6, and macrophage chemotactic protein 1 (MCP-1), in Sertoli cells. Notably, the DSCP-induced inflammatory gene expression was significantly reduced by knockout Toll-like receptor (TLR)2 or TLR4, and abolished by double knockout TLR2 and TLR4 (TLR2^−/−TLR4^−/−). MCP-1 secreted by Sertoli cells after stimulation with DSCPs promotes macrophage migration. We also provide evidence that busulfan-induced spermatogenic cell damages in vivo upregulate TNF-α and MCP-1 expression in Sertoli cells, and facilitate macrophage infiltration into the testis in wild-type mice. These phenomena were not observed in TLR2^−/−TLR4^−/− mice. Data indicate that DSCPs induce inflammatory gene expression in Sertoli cells via the activation of TLR2 and TLR4, which may initiate noninfectious inflammatory responses in the testis. The results provide novel insights into the mechanisms underlying damaged spermatogenic cell-induced testicular inflammation.     

Natural killer cells trigger osteoclastogenesis and bone destruction in arthritis

Osteoclasts are bone-eroding cells that develop from monocytic precursor cells in the presence of receptor activator of NF-κB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF). Osteoclasts are essential for physiological bone remodeling, but localized excessive osteoclast activity is responsible for the periarticular bone destruction that characteristically occurs in patients with rheumatoid arthritis (RA). The origin of osteoclasts at sites of bone erosion in RA is unknown. Natural killer (NK) cells, as well as monocytes, are abundant in the inflamed joints of patients with RA. We show here that such NK cells express both RANKL and M-CSF and are frequently associated with CD14⁺ monocytes in the RA synovium. Moreover, when synovial NK cells are cocultured with monocytes in vitro, they trigger their differentiation into osteoclasts, a process dependent on RANKL and M-CSF. As in RA, NK cells in the joints of mice with collagen-induced arthritis (CIA) express RANKL. Depletion of NK cells from mice before the induction of CIA reduces the severity of subsequent arthritis and almost completely prevents bone erosion. These results suggest that NK cells may play an important role in the destruction of bone associated with inflammatory arthritis.     

Highly potent,synthetically accessible prostratin analogs induce latent HIV expression in vitro and ex vivo

Highly active antiretroviral therapy (HAART) decreases plasma viremia below the limits of detection in the majority of HIV-infected individuals, thus serving to slow disease progression. However, HAART targets only actively replicating virus and is unable to eliminate latently infected, resting CD4⁺ T cells. Such infected cells are potentially capable of reinitiating virus replication upon cessation of HAART, thus leading to viral rebound. Agents that would eliminate these reservoirs, when used in combination with HAART, could thus provide a strategy for the eradication of HIV. Prostratin is a preclinical candidate that induces HIV expression from latently infected CD4⁺ T cells, potentially leading to their elimination through a virus-induced cytopathic effect or host anti-HIV immunity. Here, we report the synthesis of a series of designed prostratin analogs and report in vitro and ex vivo studies of their activity relevant to induction of HIV expression. Members of this series are up to 100-fold more potent than the preclinical lead (prostratin) in binding to cell-free PKC, and in inducing HIV expression in a latently infected cell line and prostratin-like modulation of cell surface receptor expression in primary cells from HIV-negative donors. Significantly, selected members were also tested for HIV induction in resting CD4⁺ T cells isolated from infected individuals receiving HAART and were found to exhibit potent induction activity. These more potent agents and by extension related tunable analogs now accessible through the studies described herein should facilitate research and preclinical advancement of this strategy for HIV/AIDS eradication.     

Inhibitory effect of glycyrrhizin on NF-κB binding activity in CC14- plus ethanol-induced liver cirrhosis in rats

ABSTRACT— Aims/Background: Potenlini is an injectable compound whose active component is glycyrrhizin, which is extracted from licorice. Previous studies showed that it could reduce liver injury, improve alanine aminotransferase (ALT) levels and act as an antifibrotic agent. However, the mechanism of its action remains unclear. The aim of this study was to determine the molecular mechanism of its action by investigating the effects of potenlini on nuclear factor-κB(NF-κB) binding activity in an animal model of liver cirrhosis. Methods: Rats were randomly allocated into a normal control group, a model control group, and a potenlini group. Rats in the latter two groups were treated with CCl₄ and ethanol solution in order to induce chronic liver injury. Rats in the potenlini group were given potenlini treatment at the same time. Results: Serum ALT levels were significantly reduced in rats treated with potenlini compared with levels in rats of the model control group, which had dramatically increased ALT levels. Histologically, liver steatosis and fibrosis were severe in the rats of the model group, but were significantly improved in rats of the potenlini group. NF-κB binding activity was markedly increased in the liver specimens taken from the rats of the model control group in comparison with the binding of normal livers, but the binding levels were nearly normal in the livers of the potenlini group. Conclusions: The results suggest that potenlini can inhibit the NF-κB binding activity in CCl₄ and ethanol-induced chronic liver injury, and that effect may be a possible mechanism by which potenlini protects the liver from hepatotoxin-induced liver injury and cirrhosis.