Protective effect of SKLB010 against d-galactosamine/lipopolysaccharide-induced acute liver failure via nuclear factor-κB signaling pathway in macrophages

Acute liver failure is characterized by the sudden loss of hepatic function and a high mortality. SKLB010, a derivative of thiazolidinediones, has been proved to be effective in protecting mice from acute liver failure caused by concanavalin A and carbon tetrachloride in our previous work. The purpose of the current study was to evaluate whether SKLB010 could prevent acute liver injury caused by d-galactosamine/lipopolysaccharide (LPS) in mice, and to investigate the underlying mechanisms. In the macrophage-mediated D-GalN/LPS model of acute liver injury, serum enzyme activity was suppressed and liver injury was attenuated by SKLB010. The serum levels of TNF-α and hepatic TNF-α mRNA expression were also markedly decreased after the treatment of SKLB010. In the liver of mice receiving injections of D-GalN/LPS, hepatocytes apoptosis and the infiltration of monocytes/macrophages were blocked by SKLB010. Furthermore, the survival rate of mice following D-GalN/LPS treatment was significantly improved by a single injection with SKLB010. In vivo, the luminescence intensity was suppressed by SKLB010 in NF-κB-luc mice after D-GalN/LPS treatment. In vitro, the production of tumor necrosis factor (TNF)-α and nitrite/nitrate in LPS-stimulated RAW264.7 macrophages was decreased by SKLB010 in a dose-dependent manner. Our further studies demonstrated that SKLB010 inhibited the phosphorylation of IκBα and p38MAPK, and the DNA binding activity of NF-κB in RAW264.7 cells. In conclusion, treatment with only a single injection of SKLB010 could significantly attenuate acute inflammation in mice induced by D-GalN/LPS, and these effects are likely associated with the inhibition of NF-κB activity.     

Murrayanine Attenuates Lipopolysaccharide‐induced Inflammation and Protects Mice from Sepsis‐associated Organ Failure

Murrayanine (MK) is the main compound isolated from Murraya koenigii, an aromatic plant belonging to the Rutaceae family, also known as curry leaf tree. Murrayanine was reported to possess potential antioxidant, antimycobacterial and antifungal effects. However, its effect in sepsis remains unclear. This study was designed to investigate the anti‐inflammatory effect of MK using both in vitro and in vivo assay. Results of this study indicated that MK decreased NO, TNF‐α and IL‐6 production in both lipopolysaccharide (LPS)‐stimulated RAW 264.7 cells and murine peritoneal macrophages. Moreover, iNOS and COX‐2 protein expression as well as their downstream product, PGE2, was also decreased effectively in RAW 264.7 cells. Furthermore, MK decreased the phosphorylation of IKB and repressed NF‐kB activity in LPS‐activated RAW 264.7 cells. Additionally, we evaluated MK efficacy in vivo using LPS‐induced sepsis, a systemic inflammation model in mice. Administration of MK inhibits pro‐inflammatory cytokines (TNF‐α and IL‐6) secretion; decreases AST, ALT, BUN and CRE level in mouse sera; mitigates lung, liver and kidney injuries; and also increases LPS‐challenged mice survival rate. Collectively, our results suggest that MK exerts potential as a new anti‐inflammatory and immunosuppressive drug in sepsis treatment.     

Cilostazol Inhibits Vascular Smooth Muscle Cell Proliferation and Reactive Oxygen Species Production through Activation of AMP-activated Protein Kinase Induced by Heme Oxygenase-1

Cilostazol is a selective inhibitor of phosphodiesterase 3 that increases intracellular cAMP levels and activates protein kinase A, thereby inhibiting vascular smooth muscle cell (VSMC) proliferation. We investigated whether AMP-activated protein kinase (AMPK) activation induced by heme oxygenase-1 (HO-1) is a mediator of the beneficial effects of cilostazol and whether cilostazol may prevent cell proliferation and reactive oxygen species (ROS) production by activating AMPK in VSMC. In the present study, we investigated VSMC with various concentrations of cilostazol. Treatment with cilostazol increased HO-1 expression and phosphorylation of AMPK in a dose- and time-dependent manner. Cilostazol also significantly decreased platelet-derived growth factor (PDGF)-induced VSMC proliferation and ROS production by activating AMPK induced by HO-1. Pharmacological and genetic inhibition of HO-1 and AMPK blocked the cilostazol-induced inhibition of cell proliferation and ROS production.These data suggest that cilostazol-induced HO-1 expression and AMPK activation might attenuate PDGF-induced VSMC proliferation and ROS production.     

Effects of florfenicol on early cytokine responses and survival in murine endotoxemia

Some antibacterials have been reported to regulate the host immune and inflammatory responses both in vitro and in vivo. Florfenicol is an antibiotics used in treatment of infection. We investigated the effects of florfenicol on cytokine production by lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages in vitro, and the results showed that florfenicol reduced tumor necrosis factor (TNF) and interleukin-6 (IL-6) production but had little effect on interleukin-1beta (IL-1beta) and interleukin IL-10 (IL-10) secretion. This inspired us to further study the effects of florfenicol in vivo. Florfenicol significantly attenuated TNF and IL-6 production in serum from mice challenged with LPS, and in consistent with the results in vitro. In murine model of endotoxemia, mice were prophylactically or therapeutically treated with florfenicol prior to or after LPS challenge. The results showed that florfenicol significantly increased mouse survival. Further studies revealed that florfenicol prevented the LPS-induced nuclear factor-kappaB (NF-kappaB) translocation from cytoplasm into nuclear in RAW 264.7 macrophages. These observations indicate that florfenicol modulates early cytokine responses by blocking NF-kappaB pathway, and thus, increases mouse survival. This effect of the drug may be of potential usefulness in treatment of bacterial shock.     

Asperlin from the marine-derived fungus Aspergillus sp. SF-5044 exerts anti-inflammatory effects through heme oxygenase-1 expression in murine macrophages

Asperlin is a fungal metabolite isolated from Aspergillus sp. SF-5044. In the present study, we isolated asperlin from the marine-derived fungus Aspergillus sp. SF-5044 and demonstrated that it inhibited inducible nitric oxide synthase (iNOS) expression, reduced iNOS-derived NO, suppressed cyclooxygenase (COX)-2 expression, and reduced COX-derived prostaglandin (PG) E? production in lipopolysaccharide (LPS)-stimulated RAW264.7 and murine peritoneal macrophages. Similarly, asperlin reduced the production of tumor necrosis factor (TNF)-α and interleukin (IL)-1β. In addition, asperlin inhibited the phosphorylation and degradation of IκB-α, as well as the nuclear translocation of p65 caused by the stimulation of LPS in RAW264.7 macrophages. Furthermore, asperlin induced heme oxygenase (HO)-1 expression through nuclear translocation of nuclear factor E2-related factor 2 and increased HO activity in RAW264.7 macrophages. The effects of asperlin on the LPS-induced expression of iNOS and COX-2 and production of NO, PGE?, TNF-α, and IL-1β were partially reversed by a HO-1 inhibitor, tin protoporphyrin. These findings suggest that asperlin-induced HO-1 expression plays a role in the anti-inflammatory effects of asperlin in macrophages.     

Berberine protects liver from ethanol-induced oxidative stress and steatosis in mice

Alcohol consumption is customary in many cultures and it is a common human behavior worldwide. Binge ethanol and chronic alcohol consumption, two usual drinking patterns of human beings, produce a state of oxidative stress in liver and disturb the liver function. However, a safe and effective therapy for alcoholic liver disease in humans is still elusive. This study identified the natural product berberine as a potential agent for treating or preventing ethanol-induced liver injury. We demonstrated that berberine attenuated oxidative stress resulted from binge drinking in liver by reducing hepatic lipid peroxidation, glutathione exhaust and mitochondrial oxidative damage. Furthermore, berberine also prevented the oxidative stress and macrosteatosis in response to chronic ethanol exposure in mice. Either the total cytochrome P450 2E1 or the mitochondria-located cytochrome P450 2E1, which is implicated in ethanol-mediated oxidative stress, was suppressed by berberine. On the other hand, berberine significantly blunted the lipid accumulation in liver due to chronic alcohol consumption, at least partially, through restoring peroxisome proliferator-activated receptor α/peroxisome proliferator-activated receptor-gamma Co-activator-1α and hepatocyte nuclear factor 4α/microsomal triglyceride transfer protein pathways. These findings suggested that berberine could serve as a potential agent for preventing or treating human alcoholic liver disease.     

CQMUH-011, a novel adamantane sulfonamide compound,inhibits lipopolysaccharide- and D-galactosamine-induced fulminant hepatic failure in mice

CQMUH-011, a novel adamantane sulfonamide compound, was shown to suppress macrophage activation and proliferation in our previous study. However, it is unknown whether CQMUH-011 has anti-inflammatory and hepatoprotective properties. In this study, we investigated the potential effects and mechanisms of CQMUH-011 on lipopolysaccharide (LPS)-induced RAW264.7 cell activation in vitro and LPS- and D-galactosamine (D-GalN)-induced fulminant hepatic failure (FHF) in vivo. The results showed that in RAW264.7 cells challenged by LPS, CQMUH-011 inhibited cell proliferation and induced cell cycle arrest and apoptosis. Furthermore, CQMUH-011 reduced tumor necrosis factor (TNF)-α and interleukin (IL)-1β production and down-regulated the overexpression of toll-like receptor 4 (TLR4) and nuclear factor (NF)-κB induced by LPS in RAW264.7 cells. In vivo, CQMUH-011 reduced serum levels of aspartic aminotransferase and alanine transaminase and improved the mortality and hepatic pathological damage induced by LPS/D-GalN in mice. Moreover, CQMUH-011 significantly inhibited the serum levels of proinflammatory mediators, including TNF-α, IL-6, IL-1β, nitric oxide (NO), and prostaglandin E2 (PGE₂), and down-regulated the protein expression of TLR4, p38 mitogen-activated protein kinases, NF-κB, NF-κB inhibitor α (IκBα), IκB kinase β (IKKβ), cyclooxygenase-2 (COX-2) and inducible NO synthases (iNOS) induced by LPS/D-GalN in mice. In conclusion, these results demonstrated that CQMUH-011 has a notable anti-inflammatory effect and protects mice from LPS/D-GalN-induced FHF and that the molecular mechanisms might be related to the inhibition of the TLR4/NF-κB signaling pathway activation, the subsequent decrease in proinflammatory mediator production, and the inhibition of macrophage activation.     

Lycorine inhibits lipopolysaccharide-induced iNOS and COX-2 up-regulation in RAW264.7 cells through suppressing P38 and STATs activation and increases the survival rate of mice after LPS challenge

As a natural alkaloid extracted from Amaryllidaceae, lycorine shows various biological effects on tumor cells. Here we show that lycorine dose-dependently inhibited the LPS-induced up-regulation of nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) protein level in RAW264.7 cells. Besides, it also inhibited NO, PGE(2), TNF-α and IL-6 release from LPS-treated RAW264.7 cells. RT-PCR experiments showed that lycorine suppressed LPS-induced iNOS but not COX-2 gene expression. Moreover, lycorine decreased LPS-induced mortality in mice. Mechanistically, LPS-induced activation of P38 and STATs pathways was suppressed significantly by lycorine. In addition, lycorine did not interfere with the phosphorylation of ERK1/2, JNK1/2 and NF-κB pathways. In conclusion, lycorine inhibits LPS-induced production of pro-inflammatory mediators and increases the survival rate of mice after LPS challenge, suggesting that lycorine could play an anti-inflammatory role in response to LPS.     

Pifithrin-α, a pharmacological inhibitor of p53, downregulates lipopolysaccharide-induced nitric oxide production via impairment of the MyD88-independent pathway

The effect of pifithrin (PFT)-α, a pharmacological inhibitor of p53, on lipopolysaccharide (LPS)-induced nitric oxide (NO) production in RAW 264.7 macrophage-like cells was examined. PFT-α inhibited the production of NO but not tumor necrosis factor (TNF)-α in response to LPS. PFT-α inhibited LPS-induced NO production via reduced expression of an inducible NO synthase (iNOS). Moreover, PFT-α inhibited LPS-induced iNOS expression in p53-silenced cells. PFT-α inhibited the production of interferon (IFN)-β, characteristic of the MyD88-independent pathway of LPS signaling, whereas it did not affect the activation of nuclear factor (NF)-κB and mitogen-activated protein kinases in the MyD88-dependent pathway. PFT-α inhibited poly I:C-induced NO production whereas it did not inhibit IFN-β-induced NO production. Further, PFT-α reduced the expression of IFN regulatory factor 3 that leads to the IFN-β production in the MyD88-independent pathway. The most upstream event impaired by PFT-α was the reduced expression of TNF receptor-associated factor (TRAF) 3 in the MyD88-independent pathway. PFT-α also reduced the in vivo expression of iNOS in the livers of mice injected with LPS. Taken together, PFT-α was suggested to inhibit LPS-induced NO production via impairment of the MyD88-independent pathway and attenuated LPS-mediated inflammatory response.     

Triptolide suppresses lipopolysaccharide-induced activity of toll-like receptor 4 in mouse macrophage cell line RAW 264.7

Toll-like receptor 4 (TLR4) has attracted a great deal of attention in ischemia–reperfusion injury in recent years. Triptolide has potent anti-inflammatory and immunosuppressive effects; however, the mechanism has not been fully delineated. The objective of this study was to evaluate the effects of triptolide on TLR4 expression in the mouse macrophage cell line RAW 264.7. In the first part of this study, the mouse macrophage cell line RAW 264.7 was treated with different concentrations of lipopolysaccharide (LPS) for 6?h to find the concentration of LPS to be used in the second part of this study. In the second part of the study, mouse macrophage cell line RAW 264.7 was pre-treated with triptolide at different concentrations for 1?h, and then exposed to LPS. The expression of the TLR4 and HSP70 mRNA and protein, as well as the levels of tumour necrosis factor (TNF)-α and interleukin (IL)-6, were assessed. In the first part of this study, the expression level of TLR4 mRNA was increased significantly in the macrophage cell line treated with LPS, and reached a plateau at 100?ng/ml. In the second part of this study, the mouse macrophage cell line RAW 264.7 pre-treated with different concentrations of triptolide, showed a dose-dependent inhibition of the levels of TLR4 mRNA and protein, TNF-α and IL-6. Triptolide can suppress LPS-induced TLR4 expression at both the mRNA and protein levels in the mouse macrophage cell line RAW 264.7.     

Inhibition of lipopolysaccharide-induced nitric oxide production by flavonoids in RAW264.7 macrophages involves heme oxygenase-1

The role of heme oxygenase-1 (HO-1) played in the inhibitory mechanism of flavonoids in lipopolysaccharide (LPS)-induced responses remained unresolved. In the present study, flavonoids, including 3-OH flavone, baicalein, kaempferol, and quercetin, induced HO-1 gene expression at the protein and mRNA levels in the presence or absence of LPS in RAW264.7 macrophages. This effect was associated with suppression of LPS-induced nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) protein expression. Hemin induced HO-1 protein expression and this was associated with the suppression of LPS-induced NO production and iNOS protein expression in a dose-dependent manner. In addition, an increase in bilirubin production was found in flavonoid- and hemin-treated cells. Hemin, at the doses of 10, 20, and 50 microM, dose-dependently stimulated the flavonoid (50 microM)-induced HO-1 protein expression, and enhanced their inhibitory effects on LPS-induced NO production and iNOS protein expression. Pretreatment of the HO-1 inhibitor, tin protoporphyrin (10 microM), attenuated the inhibitory activities of the indicated flavonoids on LPS-induced NO production. Morphologic analysis showed that 3-OH flavone, baicalein, kaempferol, quercetin, hemin, and tin protoporphyrin did not cause any change in cell viability in the presence or absence of LPS. In contrast, only 3-OH flavone showed a significant inhibition of cell growth using the MTT assay. Transfection of an HO-1 vector in macrophages (HO-1/RAW264.7) resulted in a 3-fold increase in HO-1 protein compared with that the parental RAW264.7 cells. NO production mediated by LPS in HO-1 over-expressed RAW264.7 cells (HO-1/RAW264.7) was significant less than that in parental RAW264.7 cells. 3-OH Flavone, baicalein, kaempferol, and quercetin showed a more significant inhibition on LPS-induced NO production in HO-1/RAW264.7 cells than in parental RAW264.7 cells. These results provide evidence on the role of HO-1 in the inhibition of LPS-induced NO production by flavonoids. A combination of HO-1 inducers (i.e. hemin) and flavonoids might be an effective strategy for the suppression of LPS-induced NO production.     

蟛蜞菊内酯对脂多糖诱导RAW264.7细胞环氧化酶2、NO及TNF-α的影响

目的:研究蟛蜞菊内酯对脂多糖(lipopo-lysaccharide,LPS)诱导RAW264.7巨噬细胞环氧化酶2(COX-2)、NO及TNF-α的作用。方法:ELISA方法检测0.2、2、20μmol/L不同浓度蟛蜞菊内酯对终浓度为10μg/mL LPS诱导RAW264.7细胞产生TNF-α、NO及前列腺素E2(PGE2)的影响,Western blot方法检测蟛蜞菊内酯对LPS诱导COX-2酶蛋白表达的影响。结果:LPS能够明显诱导小鼠RAW264.7细胞产生的COX-2酶蛋白,蟛蜞菊内酯低中高3个浓度均能抑制LPS诱导产生的COX-2酶蛋白表达。PGE2可以被LPS诱导增加,与空白组比有显著差异。蟛蜞菊内酯低中高3个浓度均能抑制LPS诱导产生的PGE2、NO和TNF-α,呈现剂量依赖性。结论:蟛蜞菊内酯抗炎的作用机制可能为抑制COX-2的蛋白表达,进而抑制PGE2的生成,也可能与抑制NO和TNF-α生成有关。     

人参总皂苷在巨噬细胞RAW264.7中降低脂多糖诱导的炎症和氧化应激

目的:通过脂多糖诱导巨噬细胞RAW264.7建立炎症和氧化应激模型,探讨人参总皂苷的抗炎和抗氧化效果以及对脂多糖诱导的巨噬细胞RAW264.7自噬水平的影响。方法:培养RAW264.7细胞,给予脂多糖刺激并加入不同浓度人参总皂苷,使用硝酸还原酶法检测细胞内一氧化氮水平;ELISA法检测小鼠肿瘤坏死因子α(tumor necrosis factor-α,TNF-α)的分泌;DCFH-DA荧光探针法检测人参总皂苷处理后活性氧的变化;超氧化物阴离子荧光探针(dihydroethidium, DHE)检测细胞内超氧化物阴离子水平;吖啶橙染色法检测自噬体的形成;Western Blot法检测COX-2、NF-кB、Nrf2、HO-1、GCLC、GCLM、Beclin1、ATG7和LC3的蛋白表达。结果:与脂多糖模型组相比,随着TGS的浓度增加,细胞一氧化氮水平、TNF-α的分泌降低,细胞内活性氧和超氧阴离子的含量降低,巨噬细胞RAW264.7酸性自噬体的数量增加;Nrf2、HO-1、GCLC、GCLM、Beclin1、ATG7及LC3的蛋白表达提高,COX-2、NF-кB的蛋白表达降低。结论:在...     

Differential effect of phosphodiesterase IV inhibitor RP73401 on various inflammatory and immune responses relevant to rheumatoid arthritis

Phosphodiesterase (PDE) IV inhibitors have been reported to possess potent anti-inflammatory activities through enhancement of cAMP. In this study, the immunopharmacological effect of PDE IV inhibitor (RP73401) was further carefully evaluated. RP73401 strongly blocked the production of tumor necrosis factor (TNF)-α from lipopolysaccharide (LPS)-stimulated murine macrophages (RAW264.7) and human peripheral blood mononuclear cells (PBMC) and LPS-primed mice. RP73401 did not relieve joint inflammation in adjuvant-arthritis (RA) model, whereas the compound attenuated arachidonic acid-induced inflammation. RP73401 displayed weak or no modulatory effects on the activation of macrophage and lymphocytes (assessed by proliferation, nitric oxide (NO) release and cell–cell adhesion, TNF-α production upon phorbol 12-myristate 13-acetate (PMA) treatment), and fluorescein-isothiocynate (FITC)-induced ear oedema. Collectively, these data suggest that PDE IV inhibitor RP73401 may differentially modulate various immune responses and these may explain its inability to inhibit adjuvant-induced joint inflammation or FITC-induced ear oedema.     

Eudesmin inhibits tumor necrosis factor-α production and T cell proliferation

Possible antiinflammatory effects of eudesmin were examined by assessing the effects on tumor necrosis factor (TNF)-alpha production and lymphocyte proliferation as well as cytotoxicity against murine and human macrophages. The compound significantly inhibited TNF-alpha production by lipopolysaccharide (LPS)-stimulated murine macrophage RAW264.7 without displaying cytotoxicity suggesting that eudesmin may inhibit TNF-alpha production without any interference of normal cell function. It also significantly attenuated T cell proliferation stimulated by concanavalin A (Con A) in a dose-dependent manner.     

Involvement of cytokines in the hepatic metallothionein expression by alpha-hederin

Alpha-hederin, a triterpenoid saponin, has been reported to induce hepatic metallothionein (MT). However, the mechanism underlying its effects is unknown. This study investigated the effects of alpha-hederin on the regulation of MT expression in an in vitro model using the murine hepatoma cell line, Hepa-1c1c7, and the murine macrophage cell line, RAW 264.7. Alpha-hederin that was added directly to Hepa-1c1c7 cells had no effect on MT induction. However, MT and its mRNA levels increased markedly when the Hepa-1c1c7 cells were cultured with the alpha-hederin-treated, conditioned medium from the RAW 264.7 cells. Co-treating the RAW 264.7 cells with alpha-hederin and pentoxifylline, a TNF-alpha synthesis inhibitor, resulted in decreased effects of alpha-hederin on MT induction. In the alpha-hederin-exposed RAW 264.7 cell cultures, production and mRNA levels of TNF-alpha and IL-6 were increased. Accordingly, it was found that the MT induction activity was inhibited when antibodies to TNF-alpha and/or IL-6 were added to the alpha-hederin-treated, conditioned medium from the RAW 264.7 cells. These results suggest that the upregulation of MT expression by alpha-hederin is mediated by TNF-alpha and IL-6.     

Treatment of intermittent claudication with pentoxifylline and cilostazol.

The pathophysiology of intermittent claudication (IC) and the role of pentoxifylline and cilostazol for treating IC are discussed. IC, a result of inadequate blood flow to the musculature, is the primary symptom of occlusive peripheral vascular disease (PVD). Patients with IC often have a decreased quality of life because of mobility limitations. PVD is a sign of generalized atherosclerosis and increases the risk of cardiac morbidity and mortality. Smoking, hypertension, diabetes mellitus, and increasing age may hasten the progression of PVD. Strategies for treating IC are aimed at improving symptoms and reducing the progression of atherosclerosis and include risk-factor modification, exercise, and antiplatelet therapy. Cilostazol and pentoxifylline are the only two drugs with FDA-approved labeling for use in treating IC. Both drugs have been shown to increase pain-free walking time and total distance walked, although there is some conflicting evidence for pentoxifylline. Cilostazol and pentoxi-fylline are fairly well tolerated; the most common adverse effects involve the gastrointestinal tract and central nervous system. Inhibitors of cytochrome P-450 isoenzymes 3A4 and 2C19 should be used cautiously in patients taking cilostazol, and this drug is contraindicated in patients with congestive heart failure. Cilostazol is more costly than pentoxifylline. Initiation of therapy with either pentoxifylline or cilostazol may be reasonable if risk-factor modifications, lifestyle changes, and antiplatelet therapy are not effective. The mainstays of therapy for IC are risk-factor modification, exercise, and antiplatelet therapy. If these prove inadequate, treatment with pentoxifylline or cilostazol may be reasonable.