Astragaloside IV ameliorates renal injury in streptozotocin-induced diabetic rats through inhibiting NF-κB-mediated inflammatory genes expression

Accumulating evidence suggests that inflammatory processes are involved in the development of diabetic nephropathy (DN). However, there are no effective interventions for inflammation in the diabetic kidneys. Here, we tested the hypothesis that Astragaloside IV(AS-IV), a novel saponin purified from Astragalus membranaceus (Fisch) Bge, ameliorates DN in streptozotocin (STZ)-induced diabetic rats through anti-inflammatory mechanisms. Diabetes was induced with STZ (65 mg/kg) by intraperitoneal injection in rats. Two weeks after STZ injection, rats were divided into three groups (n = 8/each group), namely, diabetic rats, diabetic rats treated with AS-IV at 5 and 10 mg kg^?1 d^?1, p.o., for 8 weeks. The normal rats were chosen as nondiabetic control group (n = 8). The rats were sacrificed 10 weeks after induction of diabetes. AS-IV ameliorated albuminuria, renal histopathology and podocyte foot process effacement in diabetic rats. Renal NF-κB activity, as wells as protein and mRNA expression were increased in diabetic kidneys, accompanied by an increase in mRNA expression and protein content of TNF-α, MCP-1 and ICAM-1 in kidney tissues. The α₁-chain type IV collagen mRNA was elevated in the kidneys of diabetic rats. All of these abnormalities were partially restored by AS-IV. AS-IV also decreased the serum levels of TNF-α, MCP-1 and ICAM-1 in diabetic rats. These findings suggest that AS-IV, a novel anti-inflammatory agent, attenuated DN in rats through inhibiting NF-κB mediated inflammatory genes expression.     

The spin trap 5,5-dimethyl-1-pyrroline N-oxide inhibits lipopolysaccharide-induced inflammatory response in RAW 264.7 cells

AimExposure of macrophages to lipopolysaccharide (LPS) induces oxidative and inflammatory stresses, which cause cell damage. Antioxidant and anti-inflammatory properties have been attributed to the nitrone spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO), commonly used in free radical analysis, but these aspects of DMPO have been little explored. In this study, we sought to establish the anti-inflammatory activity of DMPO, presumably by removing free radicals which otherwise help activate inflammatory response and damage cells.Main methodsRAW 264.7 macrophages were treated with LPS and/or DMPO for different time points, cell damage, production of inflammatory mediators, inducible nitric oxide synthase (iNOS) expression, NF-κB p65 activation, phosphorylation of MAPKs and Akt, and intracellular reactive oxygen species (ROS) were determined.Key findingsAfter cells were treated with LPS and/or DMPO for 24 h, DMPO reduced the LPS-induced inflammatory response as indicated by downregulated iNOS expression and production of inflammatory mediators. Accordingly, DMPO protected cells from LPS-induced cytotoxicity. In order to understand the mechanistic basis of these DMPO effects, the NF-κB p65 activation and the phosphorylation of MAPKs and Akt were examined. We found, by assaying cells treated with LPS and/or DMPO for 15–60 min, that DMPO inhibited the phosphorylation of MAPKs, Akt, and IκBα, and reduced the NF-κB p65 translocation. Furthermore, we demonstrated that DMPO inhibited LPS-induced ROS production.SignificanceDMPO showed the anti-inflammatory activity and attenuated LPS-induced cell damage, most likely by reducing ROS production and thus preventing the subsequent inflammatory activation and damage.     

A semi-synthetic derivative of artemisinin,artesunate inhibits prostaglandin E2 production in LPS/IFNγ-activated BV2 microglia

Artesunate is a semi-synthetic derivative of artemisinin used to treat malaria, and has been shown to possess anti-inflammatory activity. In this study, we have investigated the effect of artesunate on PGE₂ production/COX-2 protein expression in LPS + IFNγ-activated BV2 microglia. To further understand the mechanism of action of this compound, we investigated its interference with NF-κB and p38 MAPK signalling pathways. PGE₂ production was determined using EIA, while protein expressions of inflammatory targets like COX-2, mPGES-1, IκB, p38 and MAPKAPK2 were evaluated using western blot. An NF-κB-bearing luciferase reporter gene assay was used to test the effect of artesunate on NF-κB-mediated pro-inflammatory gene expression in HEK293 cells stimulated with TNFα (1 ng/ml). Artesunate (2 and 4 μM), significantly (p <0.01) suppressed PGE₂ production in LPS + IFNγ-activated BV2 microglia. This effect was found to be mediated via reduction in COX-2 and mPGES-1 proteins. Artesunate also produced significant inhibition of TNFα and IL-6 production in activated BV2 microglia. Further investigations showed that artesunate (0.5–4 μM) significantly (p <0.001) reduced NF-κB-driven luciferase expression, and inhibited IκB phosphorylation and degradation, through inhibition of IKK. Artesunate inhibited phosphorylation of p38 MAPK and its substrate MAPKAPK2 following stimulation of microglia with LPS + IFNγ. Taken together, we have shown that artesunate prevents neuroinflammation in BV2 microglia by interfering with NF-κB and p38 MAPK signalling.     

Expression of IL-32 modulates NF-κB and p38 MAP kinase pathways in human esophageal cancer

BackgroundEsophageal cancer is the seventh leading cause of cancer death in males in USA, and there is a strong link has been demonstrated between inflammation and esophageal cancer, interleukin (IL)-32 is a recently described pro-inflammatory cytokine characterized by the induction of nuclear factor NF-κB activation, the p38MAPK also plays an important role in key cellular processes related to inflammation and cancer. We investigated whether the IL-32 expression may be involved in esophageal carcinogenesis through modulates the activity of NF-κB and p-p38 MAPK.MethodMalignant esophageal tissue and blood samples were obtained from 65 operated untreated patients, normal samples was obtained from 35 patients operated for other reasons as control. IL-32 expression visualized by immunohistochemistry, Real time RT–PCR for IL-32 mRNA expression, NF-κB phosphorylation and phosphorylated p38mapk were analyzed by immunoblotting, ELISA for further detection IL-32 and cytokines (TNF-α, IL-1β, IL-6 and IL-8) concentration in the patient’s sera.ResultsIL-32 expression was increased in immunohistochemical staining for malignant esophageal tissue and it’s correlated with the relative expression level of IL-32 mRNA P = 0.007, the P-NF-κB level elevated in tumor tissue compared with control and no difference in the total NF-κB level P = 0.003 while the IL-32 up-regulated the P-pNF-κB in the esophageal tumor P = 0.005. There is increase in p-p38MAPK activation underlying IL-32 expression in tumor P = 0.004, but no change in total p38 MAPK in malignant esophagus. The plasma level of IL-32 expression was increased in malignant esophageal patients P = 0.01, with increased in the levels of the cytokines TNF-α, IL-6, and IL-1β P<0.05.ConclusionsUnderstanding the pathway of IL-32 expression to stimulate the secretion cytokines via the activation of NF-κB and up-regulation of p-p38MAPK may or may not prove to be a therapeutic target, or a biomarker, and future studies will finally answer this hypothesis generated.     

1,8-cineole (eucalyptol) ameliorates cerulein-induced acute pancreatitis via modulation of cytokines,oxidative stress and NF-κB activity in mice

AimsAcute pancreatitis (AP) is an inflammatory condition wherein pro-inflammatory mediators, oxidative stress, and NF-κB signaling play a key role. Currently, no specific therapy exists and treatment is mainly supportive and targeted to prevent local pancreatic injury and systemic inflammatory complications. This study was aimed to examine whether 1,8-cineole, a plant monoterpene with antioxidant and anti-inflammatory properties could ameliorate cerulein-induced acute pancreatitis.Main methodsAP was induced in Swiss mice by six one hourly injections of cerulein (50 μg/kg, i.p.). 1,8-cineole (100, 200 and 400 mg/kg, p.o.) was administered 1 h prior to first cerulein injection, keeping vehicle and thalidomide treated groups as controls. Blood samples were taken 6-h later to determine serum levels of amylase and lipase, and cytokines. The pancreas was removed for morphological examination, myeloperoxidase (MPO) and malondialdehyde (MDA) assays, reduced glutathione (GSH) levels, and for nuclear factor (NF)-κB immunostaining.Key findings1,8-cineole effectively reduced the cerulein-induced histological damage, pancreatic edema and NF-κB expression, levels of MPO activity and MDA, and replenished the GSH depletion. Cerulein increased serum levels of amylase and lipase, and pro-inflammatory cytokines TNF-α, IL-1β, and IL-6 were also decreased by 1,8-cineole pretreatment, similar to thalidomide, a TNF-α inhibitor. The anti-inflammatory IL-10 cytokine level was, however, enhanced by 1,8-cineole.SignificanceThese findings indicate that 1,8-cineole can attenuate cerulein-induced AP via an anti-inflammatory mechanism and by combating oxidative stress. Further studies are needed to clearly elucidate its benefits in patients on acute pancreatitis.     

S-allyl cysteine inhibits TNFα-induced skeletal muscle wasting through suppressing proteolysis and expression of inflammatory molecules

Background

Elevated levels of inflammatory molecules are key players in muscle wasting/atrophy leading to human morbidity. TNFα is a well-known pro-inflammatory cytokine implicated in the pathogenesis of muscle wasting under diverse clinical settings. S-allyl cysteine (SAC), an active component of garlic (Allium sativum), has established anti-oxidant and anti-inflammatory effects in various cell types. However, the impact of SAC on skeletal muscle pathology remains unexplored. Owing to the known anti-inflammatory properties of SAC, we investigated whether pre-treatment with SAC has a protective role in TNFα-induced atrophy in cultured myotubes.

Downregulation of nuclear factor-kappa B (NF-κB) pathway by silibinin in human monocytes challenged with Paracoccidioides brasiliensis

AimsSilibinin is the major active component of silymarin, a polyphenolic plant flavonoid that has anti-inflammatory effects. The modulatory effect of silibinin on monocyte function against Paracoccidioides brasiliensis (Pb18) has not yet been demonstrated. The present study investigated whether the effect of silibinin on nuclear factor-kappa B (NF-κB) pathways may affect the production of tumor necrosis factor-alpha (TNF-α), interleukin-10 (IL-10), transforming growth factor beta (TGF-β1), prostaglandin E₂ (PGE₂), nitric oxide (NO) and fungicidal activity of human monocytes challenged in vitro with Pb18.Main methodsPeripheral blood monocytes from healthy individuals were treated with silibinin and challenged with Pb18 for 18 h. TNF-α, IL-10, TGF-β1 and PGE₂ expression were determined by immunoenzymatic assay (ELISA) and NO release was determined by the accumulation of nitrite in culture supernatants. Fungicidal activity of monocytes was analyzed after treatment with interferon-gamma plus silibinin and challenge with Pb18. NF-κB activation in cultured monocytes was evaluated by flow cytometry and ELISA.Key findingsSilibinin partially inhibited p65NF-κB activation as the number of cells expressing this factor was reduced and the concentration of nuclear p65NF-κB was low, compared to untreated controls. The addition of silibinin also resulted in suppression of TNF-α, IL-10, TGF-β1, PGE₂ and NO production but did not affect the fungicidal activity of monocytes against Pb18.SignificanceSilibinin exerts anti-inflammatory and anti-fibrotic effects on CD14± human monocytes challenged by Pb18 by partial inhibition of p65NF-κB activation.     

Design and synthesis of pregnenolone/2-cyanoacryloyl conjugates with dual NF-κB inhibitory and anti-proliferative activities

Twenty-five novel pregnenolone/2-cyanoacryloyl conjugates (6–30) were designed and prepared, with the aim of developing novel anticancer drugs with dual NF-κB inhibitory and anti-proliferative activities. Compounds 22 and 27–30 showed inhibition against TNF-α-induced NF-κB activation in luciferase assay, which was confirmed by Western blotting. Among them, compound 30 showed potent NF-κB inhibitory activity (IC₅₀ = 2.5 μM) and anti-proliferative against MCF-7, A549, H157, and HL-60 cell lines (IC₅₀ = 6.5–36.2 μM). The present study indicated that pregnenolone/2-cyanoacryloyl conjugate I can server as a novel scaffold for developing NF-κB inhibitors and anti-proliferative agents in cancer chemotherapy.     

Synthesis and anti-inflammatory evaluations of β-lapachone derivatives

β-Lapachone (β-LAPA), a natural product from the lapacho tree in South America, is a potential chemotherapeutic agent that exhibit a wide variety of pharmacological effects such as anti-virus, anti-parasitic, anti-cancer, and anti-inflammatory activities. In order to discover novel anti-inflammatory agents, we have synthesized a series of β-LAPA derivatives for evaluation. Among them, 4-(4-methoxyphenoxy)naphthalene-1,2-dione (6b) was found to be able to inhibit NO and TNF-α released in LPS-induced Raw 264.7 cells. Inhibition of iNOS and COX-2 was also observed in compound 6b treated cells. Mechanism studies indicated that 6b exhibited anti-inflammatory properties by suppressing the release of pro-inflammatory factors through down-regulating NF-κB activation. In addition, it suppressed NF-κB translocation by inhibiting the phosphorylation of p38 kinase. Our results also indicate that the inhibitory effect of 6b on LPS-stimulated inflammatory mediator production in Raw 264.7 cell is associated with the suppression of the NF-κB and MAPK signaling pathways. A low cytotoxicity (IC₅₀ = 31.70 μM) and the potent anti-inflammatory activity exhibited by compound 6b make this compound a potential lead for developing new anti-inflammatory agents. Further structural optimization of compound 6b is on-going.     

The modulation of hepatic adenosine triphosphate and inflammation by eicosapentaenoic acid during severe fibrotic progression in the SHRSP5/Dmcr rat model

AimsEicosapentaenoic acid (EPA) can ameliorate certain liver lesions involved in non-alcoholic steatohepatitis (NASH). A previous study has found that stroke-prone spontaneously hypertensive 5/Dmcr (SHRSP5/Dmcr) rats fed a high fat-cholesterol (HFC) diet developed fibrotic steatohepatitis with histological similarities to NASH. This study evaluated the potential effects and mechanisms of action of EPA supplementation using this rodent model.Main methodsMale rats were randomly assigned to groups that were fed with either the stroke-prone (SP) diet or HFC diet with or without EPA for 2, 8 and 14 weeks, respectively. The liver histopathology, biochemical features, mRNA and protein levels, and nuclear factor-κB (NF-κB) DNA binding activity were determined.Key findingsThe SP diet-fed rats presented normal livers. Conversely, the HFC diet-fed rats developed microvesicular/macrovesicular steatosis, inflammation, ballooning degeneration and severe fibrosis. At 2 weeks, the administration of EPA inhibited hepatic inflammatory recruitment by blocking the phosphorylation of inhibitor of κB-α (IκBα), which antagonizes the NF-κB activation pathway. The dietary supplementation of EPA for 8 weeks ameliorated hepatic triglyceride accumulation and macrovesicular steatosis by inhibiting the HFC diet-induced decrease in the protein levels of enzymes involved in fatty acid β-oxidation including carnitine palmitoyltransferase 1, very long chain acyl-CoA dehydrogenase and peroxisomal bifunctional protein. Although the administration of EPA elicited no histologically detectable effects on severe fibrosis at 14 weeks, it restored an HFC diet-induced decline in hepatic adenosine triphosphate (ATP) levels and suppressed ballooning degeneration, suggesting that EPA may inhibit HFC diet-induced ATP loss and cell death.SignificanceInitial amelioration of the inflammation and steatosis in the rats after EPA supplementation indicates a possibility to treat steatohepatitis. Additionally, this study provides new insights into the roles of EPA in hepatic ATP depletion and subsequent hepatocellular injury during severe fibrosis.     

Angiotensin II type-1 receptor antagonist attenuates LPS-induced acute lung injury

Angiotensin II is able to trigger inflammatory responses through an angiotensin II type 1 (AT1) receptor. The role of AT1 receptor in acute lung injury (ALI) is poorly understood. Mice were randomly divided into three groups (n = 40 each groups): NS group; LPS group (2 mg/kg LPS intratracheally); and LPS + ZD 7155 group, 10 mg/kg ZD 7155 (an AT1 receptor antagonist) intraperitoneally 30 min prior to LPS exposure. Samples from the lung were isolated and assayed for histopathology analyses or proinflammatory gene expressions, angiotensin II receptors expressions and nuclear factors activities. LPS exposure resulted in severe ALI, elevated levels of TNF-α and IL-1β mRNA expressions, and increased activities of NF-κB and activated protein (AP)-1. Upregulation of AT1 receptor and down-regulation of AT2 receptor were also observed after LPS challenge. Pretreatment with ZD 7155 significantly inhibited the increase of AT1 receptor expression and upregulated AT2 receptor expression. ZD 7155 also reduced the mRNA expression of TNF-α and IL-1β, inhibited the activation of NF-κB and AP-1, and improved lung histopathology. These findings suggest that antagonism of AT1 receptor inhibits the activation of NF-κB and AP-1 in the lung, which may mediate the release of TNF-α and IL-1β and contribute to LPS-induced ALI.     

Promising role of ferulic acid,atorvastatin and their combination in ameliorating high fat diet-induced stress in mice

AimsThe present study evaluated a comparative and combined hepatoprotective effect of atorvastatin (AS) and ferulic acid (F) against high fat diet (HFD) induced oxidative stress in terms of hyperlipidemia, anti-oxidative status, lipid peroxidation and inflammation.Main methodsMale Swiss albino mice were given a diet containing high fat (H) (23.9% wt/wt), supplemented with AS (10 mg/kg) or F (100 mg/kg) and both (10 and 100 mg/kg) for 8 weeks. The control mice (C) were fed with normal diet.Key findingsThe H mice exhibited increased body weight; hyperlipidemia; serum level of tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6); hepatic lipid profile; lipid accumulation; reactive oxygen species (ROS) of hepatocytes, lipid peroxidation and liver antioxidant capacity was decreased. Immunofluorescent and Western blot assay revealed activation of nuclear factor kappa B (NF-κB) signaling pathway. The addition of F or AS and both in the diet significantly counteracted HFD induced body weight gain; hyperlipidemia; TNF-α, IL-6; hepatic lipid profile; fatty infiltration; NF-κB signaling pathway; ROS; lipid peroxidation and moreover elevated levels of hepatic antioxidant enzymes activity were observed.SignificanceSimultaneous treatment with AS, F and their combination protected against HFD induced weight gain and oxidative stress. The protection may be attributed to the hypolipidemic and free radical scavenging activity of AS or F and their combination. This study illustrates that AS and F have relatively similar hypolipidemic, antioxidative, anti-inflammatory actions and the AS + F combination along with HFD has shown outstanding effects as compared to other treated groups.     

Lipoic acid effects on established atherosclerosis

AimsAlpha-lipoic acid (LA) is a commonly used dietary supplement that exerts anti-oxidant and anti-inflammatory effects in vivo and in vitro. We investigated the mechanisms by which LA may confer protection in models of established atherosclerosis.Main methodsWatanabe heritable hyperlipidemic (WHHL) rabbits were fed with high cholesterol chow for 6 weeks and then randomized to receive either high cholesterol diet alone or combined with LA (20 mg/kg/day) for 12 weeks. Vascular function was analyzed by myography. The effects of LA on T cell migration to chemokine gradients was assessed by Boyden chamber. NF-κB activation was determined by measuring translocation and electrophoresis migration shift assay (EMSA).Key findingsLA decreased body weight by 15 ± 5% without alterations in lipid parameters. Magnetic Resonance Imaging (MRI) analysis demonstrated that LA reduced atherosclerotic plaques in the abdominal aorta, with morphological analysis revealing reduced lipid and inflammatory cell content. Consistent with its effect on atherosclerosis, LA improved vascular reactivity (decreased constriction to angiotensin II and increased relaxation to acetylcholine and insulin), inhibited NF-κB activation, and decreased oxidative stress and expression of key adhesion molecules in the vasculature. LA reduced T cell content in atherosclerotic plaque in conjunction with decreasing ICAM and CD62L (l-selectin) expression. These effects were confirmed by demonstration of a direct effect of LA in reducing T cell migration in response to CCL5 and SDF-1 and decreasing T cell adhesion to the endothelium by intra-vital microscopy.SignificanceThe present findings offer a mechanistic insight into the therapeutic effects of LA on atherosclerosis.     

Genistein attenuates D-galactose-induced oxidative damage through decreased reactive oxygen species and NF-κB binding activity in neuronal PC12 cells

AimsWe investigated the mechanism of D-galactose (DG)-induced oxidative damage and the neuroprotective action of genistein in PC12 cells.Main methodsPC12 cells were treated with 40 mM DG dissolved in medium containing 85% RPMI1640, 10% HBS and 5% FBS with or without genistein. We measured the protein expression of β-amyloid (Aβ), advanced glycation end products (AGEs), IκB-α and manganese-superoxide dismutase (MnSOD) by western blotting, intracellular reactive oxygen species (ROS) by 2, 7-dichlorofluorescin-diacetate, and the binding activity of nuclear factor kappa B (NF-κB) by electrophortic mobility shift assay.Key findingsDG (40 mM) completely retarded cell growth after incubation for 72 h, and this effect was not due to osmotic changes, as 40 mM mannitol had no effect. Mechanistically, we found that DG increased intracellular ROS starting at 4 h and increased Aβ and AGEs at 24 h. DG treatment for 24 h also increased the binding activity of NF-κB but strongly decreased the expression of IκB-α protein. Furthermore, DG treatment for 48 h increased MnSOD protein expression. All these effects of DG were effectively inhibited by genistein (0.5–10 μM).SignificanceThe present study indicates that the protection of genistein against DG-induced oxidative stress in PC12 cells, and the effect is likely mediated by decreased intracellular ROS and binding activity of NF-κB.     

Ohioensin F suppresses TNF-α-induced adhesion molecule expression by inactivation of the MAPK, Akt and NF-κB pathways in vascular smooth muscle cells

AimsThe expression of cell adhesion molecules on vascular smooth muscle cells is central to leukocyte recruitment and progression of atherosclerotic disease. Ohioensin F, a chemical compound of the Antarctic moss Polyerichastrum alpinum, exhibited inhibitory activity against protein tyrosine phosphatase 1B and antioxidant activity. However, published scientific information regarding other biological activities and pharmacological function of ohioensin F is scarce. In the present study, we aimed to examine the in vitro effects of ohioensin F on the ability to suppress TNF-α-induced adhesion molecule expression in vascular smooth muscle cells (VSMCs).Main methodsThe inhibitory effect of ohioensin F on TNF-α-induced upregulation in expression of adhesion molecules was investigated by enzyme-linked immunosorbent assay, cell adhesion assay, RT-PCR, western blot analysis, immunofluorescence, and transfection and reporter assay, respectively.Key findingsPretreatment of VSMCs with ohioensin F at nontoxic concentrations of 0.1–10 μg/ml dose-dependently inhibited TNF-α-induced expression of vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1). In addition, ohioensin F suppressed adhesion of THP-1 monocytes to TNF-α-stimulated VSMCs. Ohioensin F reduced TNF-α-induced production of intracellular reactive oxygen species (ROS) and phosphorylation of p38, ERK, JNK and Akt. Finally, ohioensin F inhibited TNF-α-induced CAM mRNA expression and NK-κB translocation.SignificanceThese results suggest a new mechanism of ohioensin F's anti-inflammatory action, owing to the negative regulation of TNF-α-induced adhesion molecule expression, monocyte adhesion and ROS production in vascular smooth muscle cells. Our finding also supports ohioensin F as a potential pharmacological, anti-inflammatory molecule that has a protective effect on the atherosclerotic lesion.