A combination of proteasome inhibitors and antibiotics prevents lethality in a septic shock model

Our recent studies with lactacystin, a prototype proteasome inhibitor, have suggested that the proteasome is a key regulator of LPS-induced signaling pathways contributing to the inflammatory process. Moreover, lactacystin protects animals from LPS-induced shock. Therefore, we sought to identify other less toxic compounds that would block the chymotrypsin-like activity of the proteasome or LPS-induced nitric oxide (NO). After screening over 100 natural compounds (based on chemistry and inhibition of LPS-induced biological activities), we now report for the first time that quercetin, like lactacystin (the prototype proteasome inhibitor), and mevinolin are also inhibitors of the chymotrypsin-like activity of the cellular proteasome within living cells. In addition, this study also suggests that mevinolin and quercetin both have relatively potent anti-inflammatory effects on LPS-treated macrophages in vitro. Interestingly, both of these compounds behave like lactacystin in that they block LPS-induced NO to a greater extent than TNF-alpha. The results of our experiments clearly suggest that mevinolin, in combination with the antibiotic imipenem, can provide protection against polymicrobial septic lethality induced by cecal-ligation and puncture in mice. Collectively, these studies strongly support the conclusion that therapeutic targeting of cellular proteasomes, in conjunction with standard antimicrobial therapy, may be of considerable survival benefit in the treatment of septic shock.     

Basal protein phosphorylation is decreased and phosphatase activity increased by an antioxidant and a free radical trap in primary rat glia.

Reversible protein phosphorylation regulates a wide array of cellular functions. Cells respond to cytokines and various stressors via phosphorylation and thus activation of one or more of the mitogen-activated protein kinase (MAPK) pathways. Involvement of these signal transduction pathways has been implicated in numerous pathologies, including inflammation. Using a primary glia cell culture, we show here that the antioxidant N-acetylcysteine (NAC) and the nitrone-based free radical trap, alpha-phenyl-N-tert-butyl nitrone (PBN), reduce total basal protein phosphorylation in a concentration-dependent manner as assessed by phosphotyrosine analysis and by [gamma-32P]ATP transfer radioassay. In addition we show that NAC inhibits H2O2-induced phosphatase inactivation in glia cell lysate. The PBN- and NAC-induced reduction in protein phosphorylation is accompanied by an increase in phosphatase activity, suggesting that PBN and NAC reduce protein phosphorylation by globally augmenting oxidant-sensitive phosphatase activities. These results partly explain why certain antioxidants also possess anti-inflammatory actions.     

Biliverdin administration protects against endotoxin-induced acute lung injury in rats

Given the high morbidity and mortality rates associated with pulmonary inflammation in sepsis, there is a pressing need for new therapeutic modalities to prevent acute respiratory distress. The enzyme heme oxygenase-1 (HO-1) provides potent cytoprotection against lung injury; however, the mechanism by which it does so is unclear. HO-1 catabolizes heme into biliverdin (BV), which is rapidly converted to bilirubin by BV reductase. We tested the hypothesis that BV administration could substitute for the effects observed with HO-1. Using the well-described rat model of LPS-induced shock, we demonstrate that exposure to BV imparts a potent defense against lethal endotoxemia systemically, as well as in the lungs, and effectively abrogates the inflammatory response. BV administration before a lethal dose of LPS leads to a significant improvement in long-term survival: 87% vs. 20% in sham-treated controls. BV treatment suppressed LPS-induced increases in lung permeability and lung alveolitis and significantly reduced serum levels of the LPS-induced proinflammatory cytokine IL-6. Moreover, bilirubin administered just after LPS also abrogated lung inflammation. BV treatment also augmented expression of the anti-inflammatory cytokine IL-10. Similar effects on production were observed with BV treatment in vitro in mouse lung endothelial cells and RAW 264.7 macrophages treated with LPS. In conclusion, these data demonstrate that BV can modulate the inflammatory response and suppress pathophysiological changes in the lung and may therefore have therapeutic application in inflammatory disease states of the lung.     

Development and biological evaluation of C(60) fulleropyrrolidine-thalidomide dyad as a new anti-inflammation agent

Research studies in the field of C(60) fullerene derivatives have significantly increased due to the broad range of biological activities that were found for these compounds. We designed and prepared a new C(60) fullerene hybrid bearing thalidomide as a potential double-action anti-inflammatory agent, capable of simultaneous inhibition of LPS-induced NO and TNF-alpha production. The C(60) fulleropyrrolidine-thalidomide dyad, CLT, was an effective agent to suppress the release of NO and TNF-alpha by the LPS-stimulated macrophages RAW 264.7. Ten micromolars of CLT effectively inhibited LPS-induced NO and TNF-alpha production by 47.3+/-4.2% and 70.2+/-4% with respected to the control, respectively. Furthermore, preliminary biochemical investigation revealed that CLT was a potent agent to suppress both LPS-induced intracellular ROS production and iNOS expression, and CLT also inhibited the phosphorylation of ERK which is an important protein kinase involved in the activation of TNF-alpha synthesis in LPS-activated macrophages. We believed that the studies herein would hold promise for future development of a new generation of potent anti-inflammatory agents.     

alpha-Phenyl-n-tert-butyl-nitrone reduces lipopolysaccharide-induced white matter injury in the neonatal rat brain

Lipopolysaccharide (LPS)-induced white matter injury in the neonatal rat brain is at least partially associated with oxidative stress. alpha-Phenyl-n-tert-butyl-nitrone (PBN) (100 mg/kg) significantly attenuated LPS (1 mg/kg)-induced brain injury, as indicated by the reduction in bilateral ventricular enlargement, apoptotic cell death of oligodendrocytes (OLs), and the loss of OL immunoreactivity in the neonatal rat brain. Protection of PBN was linked with the attenuated oxidative stress induced by LPS, as indicated by the decreased elevation of 8-isoprostane content and by the reduced number of 4-hydroxynonenal or malondialdehyde positive OLs following LPS exposure. Interestingly, while LPS exposure elevated, rather than depleted, levels of the reduced glutathione (GSH) and the GSH/GSSG (oxidized form) ratio, LPS exposure significantly suppressed glutathione peroxidase activity in the rat brain. PBN attenuated LPS-induced alterations in glutathione homeostasis in the rat brain. Additionally, the inflammatory responses were also reduced in the PBN-treated brain, as indicated by the decreased number of activated microglia following LPS exposure and by the consequently decreased elevation of interleukin1-beta and tumor necrosis factor-alpha contents in the rat brain. The overall results suggest that antioxidant PBN, more than a straightforward free radical scavenger, may also involve anti-inflammatory and anti-apoptotic properties in protection of the neonatal rat brain from LPS-induced injury.     

Scaffold compound L971 exhibits anti-inflammatory activities through inhibition of JAK/STAT and NFκB signalling pathways

JAK/STAT and NFκB signalling pathways play essential roles in regulating inflammatory responses, which are important pathogenic factors of various serious immune-related diseases, and function individually or synergistically. To find prodrugs that can treat inflammation, we performed a preliminary high-throughput screening of 18 840 small molecular compounds and identified scaffold compound L971 which significantly inhibited JAK/STAT and NFκB driven luciferase activities. L971 could inhibit the constitutive and stimuli-dependent activation of STAT1, STAT3 and IκBα and could significantly down-regulate the proinflammatory gene expression in mouse peritoneal macrophages stimulated by LPS. Gene expression profiles upon L971 treatment were determined using high-throughput RNA sequencing, and significant differentially up-regulated and down-regulated genes were identified by DESeq analysis. The bioinformatic studies confirmed the anti-inflammatory effects of L971. Finally, L971 anti-inflammatory character was further verified in LPS-induced sepsis shock mouse model in vivo. Taken together, these data indicated that L971 could down-regulate both JAK/STAT and NFκB signalling activities and has the potential to treat inflammatory diseases such as sepsis shock.     

Vasoactive hormone adrenomedullin and its binding protein: anti-inflammatory effects by up-regulating peroxisome proliferator-activated receptor-gamma

Sepsis is a critical inflammatory condition from which numerous patients die due to multiple organ failure and septic shock. The vasoactive hormone adrenomedullin (AM) and its binding protein (AMBP-1) are beneficial in sepsis by abrogating the progression to irreversible shock and decreasing proinflammatory cytokine release. To investigate the anti-inflammatory mechanism, we studied to determine the effect of the AM/AMBP-1 complex on peroxisome proliferator-activated receptor-gamma (PPAR-gamma) expression and activation by using RAW264.7 cells and a rat endotoxemia model. LPS treatment significantly decreased PPAR-gamma expression in vivo and in vitro and was associated with increased TNF-alpha production. Treatment with AM/AMBP-1 for 4 h completely restored PPAR-gamma levels in both models, resulting in TNF-alpha suppression. In a knockdown model using small interfering RNA in RAW264.7 macrophages, AM/AMBP-1 failed to suppress TNF-alpha production in the absence of PPAR-gamma. LPS caused the suppression of intracellular cyclic AMP (cAMP), which was prevented by simultaneous AM/AMBP-1 treatment. Although incubation with dibutyryl cAMP significantly decreased LPS-induced TauNuF-alpha release, it did not alter PPAR-gamma expression. Through inhibition studies using genistein and PD98059 we found that the Pyk-2 tyrosine kinase-ERK1/2 pathway is in part responsible for the AM/AMBP-1-mediated induction of PPAR-gamma and the anti-inflammatory effect. We conclude that AM/AMBP-1 is protective in sepsis due to its vasoactive properties and direct anti-inflammatory effects mediated through both the cAMP-dependent pathway and Pyk-2-ERK1/2-dependent induction of PPAR-gamma.     

Anti-inflammatory effects of novel curcumin analogs in experimental acute lung injury

Background

Acute lung injury (ALI) and its most severe form acute respiratory distress syndrome (ARDS) have been the leading cause of morbidity and mortality in intensive care units (ICU). Currently, there is no effective pharmacological treatment for acute lung injury. Curcumin, extracted from turmeric, exhibits broad anti-inflammatory properties through down-regulating inflammatory cytokines. However, the instability of curcumin limits its clinical application.

Methods

A series of new curcumin analogs were synthesized and screened for their inhibitory effects on the production of TNF-α and IL-6 in mouse peritoneal macrophages by ELISA. The evaluation of stability and mechanism of active compounds was determined using UV-assay and Western Blot, respectively. In vivo, SD rats were pretreatment with c26 for seven days and then intratracheally injected with LPS to induce ALI. Pulmonary edema, protein concentration in BALF, injury of lung tissue, inflammatory cytokines in serum and BALF, inflammatory cell infiltration, inflammatory cytokines mRNA expression, and MAPKs phosphorylation were analyzed. We also measured the inflammatory gene expression in human pulmonary epithelial cells.

Results

In the study, we synthesized 30 curcumin analogs. The bioscreeening assay showed that most compounds inhibited LPS-induced production of TNF-α and IL-6. The active compounds, a17, a18, c9 and c26, exhibited their anti-inflammatory activity in a dose-dependent manner and exhibited greater stability than curcumin in vitro. Furthermore, the active compound c26 dose-dependently inhibited ERK phosphorylation. In vivo, LPS significantly increased protein concentration and number of inflammatory cells in BALF, pulmonary edema, pathological changes of lung tissue, inflammatory cytokines in serum and BALF, macrophage infiltration, inflammatory gene expression, and MAPKs phosphorylation . However, pretreatment with c26 attenuated the LPS induced increase through ERK pathway in vivo. Meanwhile, compound c26 reduced the LPS-induced inflammatory gene expression in human pulmonary epithelial cells.

Conclusions

These results suggest that the novel curcumin analog c26 has remarkable protective effects on LPS-induced ALI in rat. These effects may be related to its ability to suppress production of inflammatory cytokines through ERK pathway. Compound c26, with improved chemical stability and bioactivity, may have the potential to be further developed into an anti-inflammatory candidate for the prevention and treatment of ALI.

Electronic supplementary material

The online version of this article (doi:10.1186/s12931-015-0199-1) contains supplementary material, which is available to authorized users.     

Lysophosphatidic acid inhibits bacterial endotoxin-induced pro-inflammatory response: potential anti-inflammatory signaling pathways

Previous studies have demonstrated that heterotrimeric guanine nucleotide-binding regulatory (Gi) protein-deficient mice exhibit augmented inflammatory responses to lipopolysaccharide (LPS). These findings suggest that Gi protein agonists will suppress LPS-induced inflammatory gene expression. Lysophosphatidic acid (LPA) activates G protein-coupled receptors leading to Gi protein activation. We hypothesized that LPA will inhibit LPS-induced inflammatory responses through activation of Gi-coupled anti-inflammatory signaling pathways. We examined the anti-inflammatory effect of LPA on LPS responses both in vivo and in vitro in CD-1 mice. The mice were injected intravenously with LPA (10 mg/kg) followed by intraperitoneal injection of LPS (75 mg/kg for survival and 25 mg/kg for other studies). LPA significantly increased the mice survival to endotoxemia (P < 0.05). LPA injection reduced LPS-induced plasma TNF-alpha production (69 +/- 6%, P < 0.05) and myeloperoxidase (MPO) activity in lung (33 +/- 9%, P < 0.05) as compared to vehicle injection. LPS-induced plasma IL-6 was unchanged by LPA. In vitro studies with peritoneal macrophages paralleled results from in vivo studies. LPA (1 and 10 microM) significantly inhibited LPS-induced TNFalpha production (61 +/- 9% and 72 +/- 9%, respectively, P < 0.05) but not IL-6. We further demonstrated that the anti-inflammatory effect of LPA was reversed by ERK 1/2 and phosphatase inhibitors, suggesting that ERK 1/2 pathway and serine/threonine phosphatases are involved. Inhibition of phosphatidylinositol 3 (PI3) kinase signaling pathways also partially reversed the LPA anti-inflammatory response. However, LPA did not alter NFkappaB and peroxisome proliferator-activated receptor gamma (PPARgamma) activation. Inhibitors of PPARgamma did not alter LPA-induced inhibition of LPS signaling. These studies demonstrate that LPA has significant anti-inflammatory activities involving activation of ERK 1/2, serine/threonine phosphatases, and PI3 kinase signaling pathways.     

Cardamonin inhibits LPS-induced inflammatory responses and prevents acute lung injury by targeting myeloid differentiation factor 2

BackgroundAcute lung injury (ALI) is a systemic inflammatory process, which has no pharmacological therapy in clinic. Accumulating evidence has demonstrated that natural compounds from herbs have potent anti-inflammatory efficacy in several disease models, which could be the potential candidates for the treatment of ALI.Hypothesis/PurposeAnti-inflammatory screening from natural product bank may provide new anti-inflammatory compounds for therapeutic target discovery and ALI treatment.Methods165 natural compounds were screened for their anti-inflammatory activity in LPS-stimulated macrophages. PCR array, SPR and ELISA were used to determine the potential target of the most active compound, Cardamonin (CAR). The pharmacological effect of CAR was further evaluated in both LPS-stimulated macrophages and ALI mice model.ResultsOut of the screened 165 compounds, CAR significantly inhibited LPS-induced inflammatory cytokine secretion in macrophages. We further showed that CAR significantly inhibited NF-κB and JNK signaling activation, and thereby inflammatory cytokine production via directly interacting with MD2 in vitro. In vivo, our data show that CAR treatment inhibited LPS-induced lung damage, systemic inflammatory cytokine production, and reduced macrophage infiltration in the lungs, accompanied with reduced TLR4/MD2 complex in lung tissues, Treatment with CAR also dose-dependently increased survival in the septic mice induced by DH5α bacterial infection.ConclusionWe demonstrate that a natural product, CAR, attenuates LPS-induced lung injury and sepsis by inhibiting inflammation via interacting with MD2, leading to the inactivation of the TLR4/MD2-MyD88-MAPK/NF-κB pathway.     

Bioactive eicosanoids: Role of prostaglandin F2α and F2-isoprostanes in inflammation and oxidative stress related pathology

Oxidative stress and inflammation are supposed to be the key players of several acute and chronic diseases, and also for progressive aging process. Eicosanoids, especially prostaglandin F_2α (PGF_2α) and F₂-isoprostanes are endogenous compounds that are involved both in physiology and the above mentioned pathologies. These compounds are biosynthesized mainly from esterified arachidonic acid through both enzymatic and non-enzymatic free radical-catalysed reactions in vivo, respectively. They have shown to possess potent biological activities in addition to their application as biomarkers of oxidative stress and inflammation. Recent advancement of methodologies has made it possible to quantify these compounds more reliably and apply them in various in vivo studies successfully. Today, experimental and clinical studies have revealed that both PGF_2α and F₂-isoprostanes are involved in severe acute or chronic inflammatory conditions such as rheumatic diseases, asthma, risk factors of atherosclerosis, diabetes, ischemia-reperfusion, septic shock and many others. These evidences promote that assessment of bioactive PGF_2α and F₂-isoprostanes simultaneously in body fluids offers unique non-invasive analytical opportunity to study the function of these eicosanoids in physiology, oxidative stress-related and inflammatory diseases, and also in the determination of potency of various radical scavengers, anti-inflammatory compounds, drugs, antioxidants and diet.     

Differential regulation of interleukin-12 and interleukin-10 by heat shock response in murine peritoneal macrophages

Heat shock response is a conserved stress response and has been shown to have anti-inflammatory effects. We investigated the effect of heat shock response on LPS-induced production of IL-12 and IL-10, which are two important cytokines playing contradictory roles in regulation of immune response, by murine peritoneal macrophages. The data showed that induction of heat shock response strongly suppressed LPS-induced production of IL-12 while augmented that of IL-10, suggesting the pleiotropic effects of heat shock response on immune regulatory gene expression. Also, the novel observation on up-regulation of IL-10 by heat shock response adds to the mechanism by which heat shock response exerts its anti-inflammatory effects.     

MCPIP1 negatively regulates toll-like receptor 4 signaling and protects mice from LPS-induced septic shock

Septic shock is one of leading causes of morbidity and mortality in hospital patients. However, genetic factors predisposing to septic shock are not fully understood. Our previous work showed that MCP-induced protein 1 (MCPIP1) was induced by lipopolysaccharides (LPSs), which then negatively regulates LPS-induced inflammatory signaling in vitro. Here we report that although MCPIP1 was induced by various toll-like receptor (TLR) ligands in macrophages, MCPIP1-deficient mice are extremely susceptible to TLR4 ligand (LPS)-induced septic shock and death, but not to the TLR2, 3, 5 and 9 ligands-induced septic shock. Consistently, LPS induced tumor necrosis factor α (TNFα) production in MCPIP1-deficient mice was 20-fold greater than that in their wild-type littermates. Further analysis revealed that MCPIP1-deficient mice developed severe acute lung injury after LPS injection and JNK signaling was highly activated in MCPIP1-deficient lungs after LPS stimulation. Finally, macrophage-specific MCPIP1 transgenic mice were partially protected from LPS-induced septic shock, suggesting that inflammatory cytokines from sources other than macrophages may significantly contribute to the pathogenesis of LPS-induced septic shock. Taken together, these results suggest that MCPIP1 selectively suppresses TLR4 signaling pathway and protects mice from LPS-induced septic shock.     

Protective Effect of Spin Trap Agent,N - tert -butyl-α-phenylnitrone on Hyperoxia-induced Oxidative Stress and Its Potential As a Nitric Oxide Donor

We have previously suggested that the spin trap agent, N - tert -butyl- &#102 -phenylnitrone (PBN) can function not only as an antioxidant but also as a nitric oxide (NO) donor. To characterize the pharmacological activities of PBN against oxidative damage, we examined the effect of PBN on NO generation under hyperoxic conditions. The formation of NO in mice exposed to 95% oxygen was determined using a NOx analyzer and electron spin resonance (ESR). Levels of NOx, an oxidative product of NO, increased in the blood of mice under these conditions. However, the increase was returned to a normal level by the NOS (nitric oxide synthase) inhibitor, L-NMMA, indicating that the NO was formed via a biosynthetic pathway. In addition, ESR spectra of the liver and brain of control and experimental mice that were measured using Fe(DETC) 2 as an NO trap reagent showed strong ESR signals from NO complexes in the livers of mice exposed to 95% oxygen. When examining the effect of PBN in mice, PBN reduced the NOx formation in the blood under the same hyperoxic conditions. In addition, the ESR intensity of the NO complex was weaker in the PBN-treated mice than in the non-treated mice, showing that PBN possess anti-inflammatory properties. However, under a normal atmosphere, NOx and ESR analyses showed that NO levels increased in PBN-treated mice but not in control mice. These findings suggested that PBN functions as an NO donor under specific physiological conditions. PBN appears to protect against hyperoxia-induced NO toxicity by anti-inflammatory action rather than by serving as an NO donor.     

Antioxidants, oxidative stress, and degenerative neurological disorders

Recently, clinical trials of several neurodegenerative diseases have increasingly targeted the evaluation of the effectiveness of various antioxidants. The results so far are encouraging but variable and thus confusing. Rationale for the possible clinical effectiveness of antioxidants in several degenerative conditions has arisen out of the many years of basic science generally showing that reactive oxygen species (ROS) and oxidative damage are important factors in the processes involved. Aging is one of the most significant risk factors for degenerative neurological disorders. Basic science efforts in our laboratory have centered on exploring the role of ROS and oxidative stress in neurodegenerative processes. The present review brings together some of the basic concepts we have learned by following this approach for the last 20 years and specifically the results we have obtained by following up on our serendipitous findings that a nitrone-based free radical trap, alpha-phenyl-tert-butylnitrone (PBN), has neuroprotective activity in several experimental neurodegenerative models. The mechanistic basis of the neuroprotective activity of PBN does not appear to rely on its general free radical trapping or antioxidant activity per se, but its activity in mediating the suppression of genes induced by pro-inflammatory cytokines and other mediators associated with enhanced neuroinflammatory processes. Neuroinflammatory processes, induced in part by pro-inflammatory cytokines, yield enhanced ROS and reactive nitric oxide species (RNS) as well as other unknown components that have neurotoxic properties. Neurotoxic amounts of RNS are formed by the activity of inducible nitric oxide synthase (iNOS). The demonstration of enhanced 3-nitro-tyrosine formation in affected regions of the Alzheimer's brain, in comparison to age-matched controls, reinforces the importance of neuroinflammatory processes. iNOS induction involves activation by phosphorylation of the MAP kinase p38 and can be induced in cultured astrocytes by IL-1beta or H2O2. The action of PBN and N-acetyl cysteine to suppress the activation of p38 was demonstrated in cultured astrocytes. The demonstration of activated p38 in neurons surrounding amyloid plaques in affected regions of the Alzheimer's brain attest to enhanced signal transduction processes in this neurodegenerative condition. The major themes of ROS and RNS formation associated with neuroinflammation processes and the suppression of these processes by antioxidants and PBN continue to yield promising leads for new therapies. Outcomes of clinical trials on antioxidants will become less confusing as more knowledge is amassed on the basic processes involved.     

Inhibition of LPS-induced apoptosis in differentiated-PC12 cells by new triazine derivatives through NF-κB-mediated suppression of COX-2

Anti-inflammatory therapy approaches have been in the focus of attention in the treatment of neurodegenerative diseases, such as Alzheimer's disease (AD). In this study, we examined the role of new 1,2,4-triazine derivatives against cytotoxicity exerted by lipopolysaccharide (LPS) in differentiated rat pheochromocytoma (PC12) cell line. Our results indicated that LPS-induced cell death can be inhibited in the presence of some of these compounds, as measured by MTT test, acridine orange/ethidium bromide staining and caspase-3 expression assay. We further showed that these compounds exert their protective effects through the inhibition of LPS-induced generation of nitric oxide and reactive oxygen species. Triazine derivatives inhibited LPS-induced nuclear translocation of nuclear factor- κB, a known regulator of a host of genes involved in specific stress and inflammatory responses. Pretreatment of PC12 cells with triazine derivatives also suppressed LPS-induced cyclooxygenase-2 expression while up-regulated heat shock protein-70 (Hsp-70). Moreover, the treatment of brain diseases is limited by the insufficiency in delivering therapeutic drugs into brain relating to highly limited transport of compounds through blood-brain barrier (BBB). Using a reliable model based on the artificial neural network, we indicated that these compounds are capable of penetrating BBB and may be useful agents for preventing neuroinflammatory diseases like AD.     

Ginsenoside Rg1, a novel glucocorticoid receptor agonist of plant origin, maintains glucocorticoid efficacy with reduced side effects

Glucocorticoids (GCs) are widely used to treat inflammatory diseases. However, they cause debilitating side effects, which limit the use of these compounds. In the past decade, many researchers have attempted to find so-called dissociated GCs that have separate distinct transactivation and transrepression activities. Anti-inflammation of GCs is a result of glucocorticoid receptor (GR)-mediated transactivation and transrepression in some tissues, similar to their side effects; therefore, the goal to discover a compound that has anti-inflammatory properties, but lacks the negative side effects seen with GCs, has yet to be achieved. In the present study, we introduce a plant-derived compound, ginsenoside Rg1, which possesses GC and estrogen-like activities. In this study, we show that Rg1 downmodulates LPS-induced proinflammatory cytokine release and inhibits NF-κB nuclear translocation and DNA binding activity. The negative effects on NF-κB activation are due to a decrease in IκB phosphorylation and protein stabilization. Furthermore, the inhibitory effect of Rg1 on NF-κB is GR-dependent, as small interfering RNA knockdown of GR abrogated this function. Rg1 also displayed profound inhibitory effects on LPS-induced MAPK activation. Importantly, Rg1 did not impair proliferation or differentiation of mouse osteoblasts. Finally, we show that Rg1 can effectively inhibit acute and chronic inflammation in vivo, but it does not cause hyperglycemia or osteoporosis as seen with dexamethasone. These results suggest that ginsenoside Rg1 may serve as a novel anti-inflammatory agent and may exhibit a potential profile for therapeutic intervention in inflammatory diseases.     

Myricetin relieves LPS-induced mastitis by inhibiting inflammatory response and repairing the blood–milk barrier

Mastitis, an inflammation of mammary gland, is a serious disease that affects the health of dairy cows around the world. Myricetin, a flavonoid from Bayberry, has been reported to suppress various inflammatory response. The aim of this study was to evaluate the effect of myricetin on lipopolysaccharide (LPS)-induced in vivo and in vitro mastitis model and clarify the underlying mechanism. In vivo experiments, myricetin attenuated the severity of inflammatory lesion and neutrophil infiltration. Moreover, myricetin pretreatment induced a significant decrease in the activity of myeloperoxidase (MPO) and the production of TNF-α, IL-6, and IL-1β triggered by LPS. Myricetin pretreatment could also increase the integrity of the blood–milk barrier and upregulate the tight junction proteins in LPS-induced mice mastitis. In vitro, myricetin inhibited LPS-induced inflammatory response in mice mammary epithelial cells (mMECs). In the further mechanism studies, we found that the anti-inflammatory effect of myricetin was mediated by inhibiting LPS-induced phosphorylation of AKT, IKK-α, IκB-α, and P65 in vivo and in vitro. Collectively, these data suggested that myricetin effectively ameliorated the inflammatory response by inhibiting the AKT/IKK/NF-κB signaling pathway and repairing the integrity of blood–milk barrier in LPS-induced mice mastitis.