Inhibition of glycolysis ameliorate arthritis in adjuvant arthritis rats by inhibiting synoviocyte activation through AMPK/NF-кB pathway

Objective

This study aimed to evaluate glycolysis inhibitor which can effectively ameliorate arthritis by inhibiting synoviocyte activation through AMPK/NF-кB pathway in AA rats.

Methods

Adjuvant arthritis (AA) rats were treated with 2-deoxyglucose (2-DG), glycolysis inhibitor. HE staining and radiological Examination were used for histopathology analysis and evaluation of joint destruction. HKII expression was quantified by immunostaining. Proliferation and migration of synoviocytes were assessed by synovicyte scores of joint, CCK8 and transwell assay. Inflammatory factors and levels of AMPK, p65 and IκBα were quantified by ELISA analysis and WB.

Results

We observed that HKII expression was positively correlated with synovial hyperplasia, inflammatory cell infiltration, and cartilage destruction, and glycolysis inhibitor reduces the joint swelling degree, alleviates bone destruction, inhibits the proliferation and migration of synoviocyte, and reduces secretory function of synoviocytes in AA rats. In addition, we investigated that glycolysis inhibitor may inhibit activation of the NF-κB signaling pathway by activating the AMPK pathway.

Conclusion

This study suggests the involvement of energy metabolism in the pathological inflammation process in RA joints. Glycolysis inhibitors might, therefore, provide an opportunity for therapeutic intervention.

     

The effect of treatment with recombinant γ-interferon on adjuvant-induced arthritis in rats

We have investigated the effects of recombinant rat -interferon (rIFN) on adjuvant-induced arthritis (AA). Lewis rats, inoculated in the left hind-paw with adjuvant (day 0), were given 10⁵ U/rat of rIFN daily (days 0 to 20), subcutaneously and intramuscularly on alternate days. rIFN suppressed the secondary phase of swelling of both hind-paw on and after day 18 without influencing the earlier phases, both primary and secondary, of swelling. rIFN also reduced the hind-paw bone lesions, the degree of splenomegaly, and the increase in erythrocyte sedimentation rate and plasma fibrinogen. These results indicate a new aspect of the regulatory role of IFN in chronic inflammation.     

Effect of Cornus officinalis glycoside on adjuvant-induced arthritis in rats

The aim of this study was to explore the effect of Cornus officinalis glucosides (COG) on adjuvant-induced arthritis in rats and its mechanism. Seventy-two rats were divided into six groups of normal, model, Dexasone (0.125 mg/kg), high-dose COG (240 mg/kg), mid-dose COG (120 mg/kg), and low-dose COG (60 mg/kg). Rat arthritis was induced by injection of Freund's complete adjuvant in the hind paws. All treatment started from the day the arthritis was induced. The edema degree of the adjuvant injection location was determined on days 1, 3, 5, 7, 9, 11, 13, 15, 17, 20, 23 and the opposite side was observed on days 11, 13, 15, 17, 20, 23 after the injection of adjuvant. All rats were sacrificed on day 24 after the injection of adjuvant for microscopic examination of the ankle, and for the study of the immunological molecular mechanism. The results showed that the COG significantly suppressed both the primary and secondary edema, improved pathological injuries of adjuvant arthritis (AA) rat ankles, significantly suppressed the proliferation of T lymphocytes and DTH reaction. It significantly suppressed IL-1, IL-6 and TNF-αproduction from peritoneal macrophages and PGE2 in plasma. In conclusion, the Cornus officinalis glucosides (COG) is able to prevent and cure the rat adjuvant-induced arthritis, and can suppress the production of pro-inflammatory cytokine IL-1, IL-6, TNF-αand PGE2.     

Regulatory effect of IL-38 on NF-κB pathway in systemic lupus erythematosus

BackgroundIL-38 is a newly identified cytokine that exhibits immunosuppression effects. However, there are few studies focusing on the effects and mechanisms of IL-38 in the systemic lupus erythematosus (SLE).AimWe investigated the effects and mechanisms of IL-38 on NF-κB signaling pathway in SLE.MethodsLevels of IL-38, IL-36R, IL-1RAcP, IKKα/β, NF-κB, TNF-α and anti-dsDNA antibody levels in peripheral blood of SLE patients, and in peripheral blood and kidney tissues of MRL/lpr mice, were examined with real-time PCR, ELISA, Western blot and immunohistochemistry. Pathological changes of kidney were detected with PAS staining. Recombinant human IL-38 protein and IL-38 siRNA were used to intervene the PBMCs of SLE patients and MRL/lpr mice.ResultsThe mRNA and protein levels of IL-38 in peripheral blood of SLE patients decreased and were positively correlated. The mRNA and protein levels of IKKα/β, NF-κB, and TNF-α increased, especially in patients with active SLE. There was a negative correlation between IL-38 and the levels of IKKα/β, NF-κB and TNF-α in SLE patients. In vitro experiments showed that the levels of IKKα/β, NF-κB and TNF-α, and anti-dsDNA antibodies decreased in PBMCs of SLE patients after treatment with human recombinant IL-38 protein. These effects were reversed after IL-38 siRNA intervention. Consistent results were obtained on IL-38, IKKα/β, NF-κB, and TNF-α in MRL/lpr lupus mice after treatment with IL-38 protein or IL-38 shRNA. Additionally, kidney function (reflected by creatinine and blood urea nitrogen), anti-dsDNA antibody, complement C3, and urinary protein levels decreased after treatment with IL-38 protein but increased after IL-38 shRNA treatment. PAS staining showed IL-38 protein treatment induced mild hyperplasia of glomerular mesangial cells and a small amount of lymphocyte infiltration. However, these were aggravated after IL-38 shRNA treatment.ConclusionIL-38 may be involved in the occurrence and development of SLE by regulating the NF-κB signaling pathway. This study only discussed the relationship between IL-38 and NF-κB, and more biological functions of IL-38 need to be further studied.     

Damnacanthal inhibits the NF-κB/RIP-2/caspase-1 signal pathway by inhibiting p56lck tyrosine kinase

Damnacanthal is a major constituent of Morinda citrifolia L. (noni) and exhibits anti-cancer and anti-inflammatory activities. However, the effects of damnacanthal on allergic diseases have not been determined. In this study, we investigated the effect of damnacanthal on mast cell-mediated allergic inflammatory responses. Damnacanthal significantly and dose-dependently inhibited compound 48/80-induced systemic anaphylactic shock, histamine release and intracellular calcium levels. In particular, IgE-mediated passive cutaneous anaphylaxis was significantly inhibited by the oral administration of damnacanthal. In addition, we report for the first time that p56^lck tyrosine kinase was expressed in phorbol 12-myristate 13-acetate and calcium ionophore A23187 (PMACI)-stimulated mast cells. Furthermore, damnacanthal inhibited the up-regulation of p56^lck tyrosine kinase activity by PMACI and repressed PMACI-induced histidine decarboxylase expression and activity. Damnacanthal also inhibited PMACI-induced interleukin (IL)-1β, IL-6 and tumor necrosis factor-α expressions by suppressing nuclear factor-kappa B (NF-κB) activation and suppressed the activation of caspase-1 and the expression of receptor interacting protein-2. This study shows damnacanthal inhibits the NF-κB/receptor-interacting protein-2/caspase-1 signal pathway by inhibiting p56^lck tyrosine kinase and suggests that damnacanthal has potential for the treatment of mast cell-mediated allergic disorders.     

Effect of β-carotene on the development of adjuvant-induced arthritis and production of interleukin-1 in rats

Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 116, N ^o 12, pp. 611–613, December 1993     

Suppression of puerarin on polymethylmethacrylate-induced lesion of peri-implant by inhibiting NF-κB activation in vitro and in vivo

Puerarin (PR), a natural isoflavone isolated from Chinese traditional plant pueraria lobata, has attracted considerable attention due to its important biological and pharmacological activities. However, its effects on lesion of peri-implant and related mechanism of action are still not clear, which require further investigation. In this study, we evaluated the effects of PR on polymethylmethacrylate (PMMA)-induced lesion of peri-implant in vitro and in vivo, and explored its possible mechanism of action. Our results indicated that PR could inhibit PMMA-induced osteoclastogenesis in RAW264.7 cells with a dose-dependent manner in vitro and effectively down-regulate mRNA and protein expressions of matrix metalloprotein 9 (MMP-9), tumor necrosis factor (TNF)-α, interleukin (IL)-6, and receptor activator of nuclear factor (NF)-κB (RANK), primarily via the suppression of NF-κB signaling. Furthermore, we found that PMMA induction could directly cause the phosphorylation of IκB and significantly promote the nuclear translocation of p65 in RAW264.7 cells. In other words, PR was able to dose-dependently attenuate the PMMA-induced nuclear translocation of p65 in RAW264.7 cells. In vivo, PR was observed to attenuate PMMA-induced osteoclastogenesis, osteolysis, mRNA expressions of receptor activator of nuclear factor (NF)-κB ligand (RANKL) and RANK, as well as protein levels of MMP-9, TNF-α, IL-6, and p65 in a murine calvarial osteolysis model. These findings suggested that PR might be a potential therapeutic drug to lesion of peri-implant, and provided new insights for understanding its possible mechanism.     

Triphala herbal extract suppresses inflammatory responses in LPS-stimulated RAW 264.7 macrophages and adjuvant-induced arthritic rats via inhibition of NF-κB pathway

This study sought to explore the mechanism of anti-inflammatory effect of triphala in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages and in adjuvant-induced arthritic rats. In stimulated RAW 264.7 cells, triphala (100–300 μg/ml) significantly suppressed production of inflammatory mediators (e.g. TNFα, IL-1β, IL-6, MCP-1, VEGF, NO, PGE₂), intracellular free radicals and release of lysosomal enzymes (e.g. acid phosphatase, β-galactosidase, N-acetyl glucosamindase and cathepsin D) in a dose-related manner. With triphala, mRNA levels of genes for pro-inflammatory TNFα, IL-1β, IL-6 and MCP-1, inflammatory iNOS and COX-2 enzymes and NF-κBp65 were down-regulated in the stimulated cells; in contrast, there was up-regulation of heme oxygenase-1 (HO-1) expression. Western blot analyses revealed that triphala suppressed the protein expression of NF-κB p65 and p-NF-κB p65 in the stimulated cells, which subsequently reduced over-expression of TNFα, IL-17, iNOS and COX-2 in a manner similar to that observed with BAY 11-7082, an IκB kinase inhibitor. Immunofluorescence analysis revealed inhibition of p-NF-κB p65 nuclear translocation and COX-2 protein expression caused by triphala. Consistent with these findings, the animal studies presented confirmed that triphala exhibited anti-inflammatory effects in a rat adjuvant-induced arthritis model by reducing of inflammatory mediator (e.g. IL-17, COX-2 and RANKL) expression via inhibition of NF-κB activation. Taken together, the results here demonstrated that triphala has potential anti-inflammatory applications that could be used for the treatment of inflammatory disorders, including rheumatoid arthritis.     

miR-595 suppresses cell proliferation and metastasis in hepatocellular carcinoma by inhibiting NF-κB signalling pathway

MicroRNAs (miRNAs) have been proven to be critical regulators of cancer development. To date, many of them are still in urgent need of characterisation, and role of miR-595 in hepatocellular carcinoma (HCC) remains unknown. To better understand the mechanism of miR-595 in HCC development, a series of experiments were carried out to explore the effects of miR-595 on malignant behaviour in HCC. First, we found that miR-595 was downregulated in HCC tissues and cells and tightly associated with poor overall survival in HCC patients. Then, we further demonstrated that miR-595 inhibited cell proliferation, migration and invasion in vitro. Additionally, animal experimental results demonstrated that miR-595 inhibited HCC carcinogenesis in vivo. Moreover, we demonstrated that upregulation of miR-595 expression inhibited the NF-κB signalling pathway in HCC cells. To further uncover the molecular mechanism of miR-595 action on the NF-κB signalling pathway, we identified ABCB1 as a direct target of miR-595 through bioinformatics prediction and supported our results with luciferase assays. Finally, we showed that miR-595 inhibited the NF-κB pathway by suppressing ABCB1 expression in HCC cells. Taken together, our findings uncover a pivotal role for the miR-595/ABCB1/NF-κB axis in HCC development, and this novel axis may be a suitable target for diagnostic or therapeutic interventions in HCC.     

Malvidin attenuates pain and inflammation in rats with osteoarthritis by suppressing NF-κB signaling pathway

Objective

Malvidin is one of the most widespread anthocyanidins which exhibits significant antioxidant and anti-inflammatory activity. The aim of this paper is to investigate the effects of Malvidin on osteoarthritis (OA).

Materials and methods

We created an animal model of OA using Wistar rats administered by monosodium iodoacetate (MIA). Effects of Malvidin on hyperalgesia were evaluated by paw pressure tests and compression threshold test. Articular chondrocytes were isolated from the OA rats to detect the apoptotic chondrocytes using senescence-associated β-galactosidase (SA-β-gal) staining kit. The expression levels of pro-inflammatory cytokines and matrix metalloproteinase (MMPs) were assessed by western blot and qPCR. Luciferase assay was used to determine the impact of Malvidin on nuclear factor-kappa B (NF-κB) pathway.

Results

Malvidin treatment exhibited significant pain-relieving effects in OA rats and decreased the expression level of apoptotic marker SA-β-gal in chondrocytes. We found that the upregulated expressions of interleukin (IL)-1β, IL-6, tumor necrosis factor-α (TNF-α), and MMPs induced by MIA in cartilage tissues were significantly reversed by Malvidin. Furthermore, Malvidin inhibited NF-κB pathway via an NF-κB inhibitor (IκBα)-independent manner through suppressing p65 nuclear transportation in vitro.

Conclusions

Our findings suggest that Malvidin significantly attenuates the OA-induced pain and inflammation by inhibiting NF-κB signaling pathway and suppressing pro-inflammatory cytokine expression and chondrocyte apoptosis.

     

Suppression of NF-κB p65 nuclear translocation and tumor necrosis factor-α by Pongamia pinnata seed extract in adjuvant-induced arthritis

Abstract

Pongamia pinnata is a plant known for its therapeutic usage in Indian traditional medicine. Despite the controversy regarding toxic flavonoid and erucic acid content, the seed of this plant is consumed in tribal medicine and its oil is used in Ayurveda to treat psoriasis and arthritis. This study explored the potential anti-arthritic effects of a P. pinnata seed (hexane) extract (PSE) at non-lethal doses in an adjuvant-induced arthritic rat model; possible mechanisms of any observed effects were also explored. After establishing the lethal doses arising from oral exposure to the extract, the material was administered per os daily at two doses (0.3?g/kg/day; 0.5?g/kg/day) to arthritic rats. Other rats received indomethacin or vehicle (control). Treatments were performed for a total of 14 days. One day after the final exposure, the rats were euthanized to permit harvest of various cells, blood, and tissues for analyses. Paw diameter and tissue myeloperoxidase activity in the paws were evaluated as indices for edema and neutrophil infiltration into the tissue. The severity of arthritis in the experimental rats was assessed via measures of urinary hydroxyproline (HP) and glucosamine, and of serum pro-inflammatory TNFα and anti-inflammatory IL-10. The extent of NF-κB p65 nuclear translocation in peritoneal macrophages harvested from naïve rats and then treated in vitro was also assessed. The results indicated that exposure to PSE significantly decreased paw diameter, tissue myeloperoxidase level, and levels of urinary HP and glucosamine, as well as of serum TNFα and IL-10 in adjuvant-injected (arthritic) rats. In vitro PSE treatment also resulted in a marked inhibition of NF-κB p65 nuclear translocation in primary cultures of peritoneal macrophages. Thus, PSE appears to be able to prevent experimental arthritis, in part, by helping to maintain the balance between pro- and anti-inflammatory cytokines and by inhibiting NF-κB activation.     

Electroacupuncture modulated the inflammatory reaction in MCAO rats via inhibiting the TLR4/NF-κB signaling pathway in microglia

In this study, we aim to investigate the effects of electroacupuncture on the TLR4/NF-κB signaling pathway in microglia. Male Wistar rat of SPF grade (weighing 200±20 g) were randomly divided into (i): sham control group, which was subjected to sham operation (ii) vehicle group, which underwent the occlusion of middle cerebral artery; (iii-v): acupuncture groups, which were subjected to the occlusion of middle cerebral artery and treated with acupuncture on the Neiguan acupoint (P6), Quchi acupoint (LI11), and Diji acupoint (SP8), respectively. HE staining was performed to detect the necrotic rate of neurons. Mediators of inflammation were measured using ELISA. Immunofluorescence was performed to measure the expression of TLR4, HMGB1, TRAF6, IKKβ and NF-κB p65 in microglia. Severe decrease was noticed in the neurological score, necrotic rates of neuron, expression of IL-1β, IL-6, TLR4, HMGB1, TRAF6, IKKβ and NF-κB p65 in microglia. Compared with the vehicle group, significant decrease was revealed in the neurological score, necrotic rate, IL-1β, TLR4, TRAF6, IKKβ and NF-κB p65 in Neiguan group and Quchi group, respectively. In addition, remarkable decrease was observed in the expression of TNF-α and IL-6 in Quchi group. Compared with the Diji group, the necrotic rate of neurons in hippocampus region was significantly decreased in the Quchi group (P < 0.05). In Neiguan group, the expression of TLR4 and IKKβ was significantly attenuated (P < 0.05). The expression of TRAF6 was remarkably decreased in the Neiguan group and Quchi group, respectively. Electroacupuncture on Neiguan and Quchi could improve the neurological injury, attenuate the inflammation, and inhibit the activity of TLR4/NF-κB signaling pathway in microglia.     

Anti-inflammatory effect of methyl dehydrojasmonate (J2) is mediated by the NF-κB pathway

Inflammation as a major defense mechanism against pathogens is modulated by diverse microbial products. A variety of plant and microbial products interacting with Toll-like receptors initiate a wide spectrum of responses from phagocytosis to cytokine production, which modulates inflammation. Jasmonates are fatty acid-derived cyclopentanones produced by plants and lower eukaryotes that play an important role in the defense against insects. In this study, we are set up to define the molecular targets of J2 action. While the lipopolysaccharide (LPS) stimulation of macrophage cell line RAW264.7 induced TNF-α, IL-6, iNOS, and COX-2 that were associated with an increase in miR-155 and miR-146a, the J2 suppressed the induction of these inflammatory cytokines and enzymes as well as miR-155 in a dose-dependent manner. To assess the associations of miR-155 with inflammatory markers, we overexpressed miR-155 and found attenuation of COX-2 suppression with J2 treatment. Furthermore, J2 inhibited NF-κB, p65, and IκB but had no or only minimal effects on the mitogen-activated protein kinase pathway. In conclusion, the present study demonstrates that J2 suppresses LPS stimulation of RAW264.7 cells by targeting NF-κB pathways.     

Ruscogenin exerts beneficial effects on monocrotaline-induced pulmonary hypertension by inhibiting NF-κB expression

This study aims to examine the effect of ruscogenin on pulmonary arterial hypertension (PAH) and to determine the mechanism underlying this effect. We isolated pulmonary vascular smooth muscle cells (PVSMCs) from the pulmonary artery of the rats; the PVSMCs were cultured in vitro and then were treated with platelet-derived growth factor (PDGF), PDGF + ruscogenin, or PDGF + ruscogenin + parthenolide. We randomized Sprague-Dawley rats into five groups as follows: control group, PAH group, low-dose group, medium-dose group, and high-dose group; the rats in the low-, medium-, and high-dose groups received the vehicle and ruscogenin 0.1, 0.4, and 0.7 mg/kg, respectively, from day 1 to day 21 after injection of monocrotaline (MCT). We measured the mean pulmonary arterial pressure (mPAP), right ventricular systolic pressure (RVSP), and medial wall thickness of the pulmonary artery (PAWT). We examined the levels of the nuclear factor kappa B (NF-κB) protein by using immunohistochemistry and western blot analysis, and the mRNA levels of NF-κB in PVSMCs were evaluated using real-time polymerase chain reaction (PCR). The mPAP, RVSP, and PAWT and the protein and mRNA levels of NF-κB were significantly higher in the PAH model group than in the control group (P < 0.05). Ruscogenin induced a significant dose-dependent decrease in the mPAP, RVSP, and PAWT and in the NF-κB expression in the PAH group (P < 0.05), which suggests that ruscogenin will also exert dose-dependent effects on MCT-induced PAH through the inhibition of NF-κB.     

Dioscin protects against diabetic nephropathy by inhibiting renal inflammation through TLR4/NF-κB pathway in mice

Diabetic nephropathy (DN) is a chronic kidney disease caused by the long-term loss of renal function, which occurs in 20% - 40% of all diabetes and is also the primary cause of end-stage renal diseases. DN is related with other lethal diseases, particularly cardiovascular diseases, leading to an increased risk of death. Therefore, an effective treatment for DN is required. Here we tested the protective effect of dioscin in a mouse model of streptozocin (STZ)-induced DN. First, STZ was intraperitoneally injected into C57BL/6 J mice and TLR4^-/- mice respectively, on a daily basis for 5 days to induce diabetes. Dioscin was then orally administered into diabetic mice daily for 8 weeks. Our results show that STZ injection effectively induced diabetes in mice as indicated by the increased blood glucose levels in C57BL/6 J mice, whereas it did not cause diabetes in TLR4^-/- mice. Dioscin significantly ameliorated STZ-induced renal damage via reducing inflammatory responses in diabetic mice and antagonizing the activation of TLR4/NF-κB pathway and the production of inflammatory cytokines. In conclusion, our study highlights the potential of dioscin as a novel approach to treat DN in diabetic patients.     

Normoxic induction of cerebral HIF-1α by acetazolamide in rats: Role of acidosis

Acetazolamide has been recognized as an effective treatment for acute mountain sickness. The efficacy of acetazolamide is related to metabolic acidosis, which promotes chemoreceptors to respond to hypoxic stimuli at altitude. In this study, adult male Sprague–Dawley rats were treated with acetazolamide (100 mg/kg or 50 mg/kg, I.P.) for 3 days. Primary cultured cortical neurons and PC12 cell lines were exposed to acidosis-permissive (pH 6.5) or standard (pH 7.2) media for 20 h. HIF-1α and its target genes were assayed by Western blot, real-time PCR, HIF-1 DNA-binding assay and chloramphenicol acetyltransferase reporter gene assay. HIF-1α protein level and HIF-1 DNA-binding activities were increased in cerebral cortices of rats treated with acetazolamide. Moreover, the mRNA levels of erythropoietin, vascular endothelial growth factor, and glucose transporter-1 also increased. The HIF-1α protein level and activity of HIF-driven chloramphenicol acetyltransferase reporters of cortical neurons and PC12 cells treated with acidosis media were significantly enhanced. We conclude that the normoxic induction of HIF-1α and HIF-1 mediated genes by acetazolamide may mediate the effect of acetazolamide in the reduction of symptoms of acute mountain sickness.