MicroRNA-532-3p Regulates Pro-Inflammatory Human THP-1 Macrophages by Targeting ASK1/p38 MAPK Pathway.

Inflammation is a complex biological process which alters the normal physiological function of the immune system resulting in an abnormal microenvironment that leads to several clinical complications. The process of inflammation is mediated through various intracellular signaling factors inside the cells. Apoptosis signal-regulating kinase 1 (ASK1) is an inflammation-derived kinase that controls the activation of other family of kinases such as p38 mitogen-activated protein kinases (p38 MAPKs), which mediates various the inflammatory processes. In this study, we cultured THP-1 macrophage cells to undergo inflammatory proliferation with LPS (1 µg/ml) and TNFα (10 ng/ml) stimulation. Initial in silico analysis was utilized to predict novel microRNAs (miRNAs) that target ASK1 signaling and its expression levels in LPS and TNFα stimulated THP-1 cells were estimated. Among the miRNAs, miR-532-3p showcased the highest binding affinity towards ASK1 kinase. We witnessed that transient transfection of miR-532-3p diminished the levels of ASK1 and downstream phosphorylation/translocation of p38 MAPK. Furthermore, direct targeting of ASK1 resulted in regulation of uncontrolled release of cytokines (TNFα, IL-6, and IL-23) and chemokines (GM-CSF and MIP-2α). Overall, we suggest that miR-532-3p attenuates the pro-inflammatory nature of macrophages by targeting ASK1/p38 MAPK signaling pathway and can be used as a molecular intervention for treating inflammatory diseases.

miR-506-3p alleviates uncontrolled osteoclastogenesis via repression of RANKL/NFATc1 signaling pathway.

Bone erosion is the major cause of deformities in autoimmune disease conditions such as osteoporosis and rheumatoid arthritis. Aberrant receptor activator of nuclear factor kappa B ligand (RANKL) secretion in bone disorders have been implicated to promote uncontrolled osteoclast differentiation through the regulation of nuclear factor of activated T cells 1 (NFATc1) transcription factor. This phenomenon is governed by several molecular factors including microRNAs, which are under-expressed during disease progression. This report focuses on elucidating the molecular mechanism of miR-506-3p towards the RANKL/NFATc1 pathway. miR-506-3p showed high binding affinity towards NFATc1 (ΔG = -22.4 kcal/mol). Bone marrow-derived macrophages (BMMs) isolated from rats stimulated with RANKL (100 ng/ml) showed active expression of NFATc1 which differentiated into mature osteoclasts. Moreover, NFATc1 activation resulted in downstream secretion of various bone resorptive enzymes (cathepsin K, carbonic anhydrase II, tartarate acid phosphatase, and matrix metalloproteinase 9) which lead to active bone resorption. However, transfection of miR-506-3p resulted in selective repression of NFATc1 inside the cells. This further resulted in the diminished release of bone resorptive enzymes that were essential for the degradation of the bone. Overall, we predict that miR-506-3p can be used as a molecular intervention for RANKL/NFATc1 mediated osteoclastogenesis.

MiR-145-5p mitigates dysregulated Wnt1/ß-catenin signaling pathway in rheumatoid arthritis.

Fibroblast-like synoviocytes (FLS) lining the arthritic synovial joint region have been implicated to be a key player in bone remodeling. The uncontrolled proliferation of this cell subtype is strictly regulated by various molecular elements including microRNAs (miRNAs). The Wnt1/ß-catenin signaling pathway plays a crucial role in the survival of FLS cells. This study explores the underlying mechanism of miR-145-5p towards the Wnt1/ß-catenin pathway. MiR-145-5p depicted a strong binding affinity towards frizzled class receptor 4 (FZD4) 3' UTR, a key receptor complex essential for recognizing circulating Wnt1 molecules. Adjuvant induced arthritic fibroblast-like synoviocytes (AA-FLS) isolated from rats stimulated with Wnt1 (10 ng/ml) elicited active Wnt1/ß-catenin signaling. Transfection of miR-145-5p mimic (50 pmol) to AA-FLS stimulated with Wnt1 elicited reduced expression levels of various factors of Wnt1/ß-catenin signaling including low-density lipoprotein receptor-related protein 5 (LRP5), dishevelled segment polarity protein 1 (Dvl1) and ß-catenin transcription factor. Moreover, pro-inflammatory cytokines (TNFα, IL-1ß, IL-6 and IL-23) were regulated compared to the diseased groups. Furthermore, miR-145-5p counterbalanced the levels of receptor activator of nuclear factor kappa B ligand (RANKL) and osteoprotegerin (OPG) at the cellular level, essential for bone remodeling. Hence, we suggest that miR-145-5p regulates the survival/proliferation of FLS cells in RA disease condition through attenuation of Wnt1/ß-catenin signaling.

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-1ß, IL-6, MCP-1, VEGF, NO, PGE2), intracellular free radicals and release of lysosomal enzymes (e.g. acid phosphatase, ß-galactosidase, N-acetyl glucosamindase and cathepsin D) in a dose-related manner. With triphala, mRNA levels of genes for pro-inflammatory TNFα, IL-1ß, 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.

Morin, a Bioflavonoid Suppresses Monosodium Urate Crystal-Induced Inflammatory Immune Response in RAW 264.7 Macrophages through the Inhibition of Inflammatory Mediators, Intracellular ROS Levels and NF-κB Activation.

Our previous studies had reported that morin, a bioflavanoid exhibited potent anti-inflammatory effect against adjuvant-induced arthritic rats. In this current study, we investigated the anti-inflammatory mechanism of morin against monosodium urate crystal (MSU)-induced inflammation in RAW 264.7 macrophage cells, an in vitro model for acute gouty arthritis. For comparison purpose, colchicine was used as a reference drug. We have observed that morin (100-300 µM) treatment significantly suppressed the levels of inflammatory cytokines (TNF-α, IL-1ß, IL-6, MCP-1 and VEGF), inflammatory mediators (NO and PEG2), and lysosomal enzymes (acid phosphatase, ß-galactosidase, N-acetyl glucosamindase and cathepsin D) in MSU-crystals stimulated macrophage cells. The mRNA expression of pro-inflammatory cytokines (TNF-α, IL-1ß, IL-6, and MCP-1), inflammatory enzymes (iNOS and COX-2), and NF-κBp65 was found downregulated in MSU crystal stimulated macrophage cells by morin treatment, however, the mRNA expression of hypoxanthine phospho ribosyl transferse (HPRT) was found to be increased. The flow cytometry analysis revealed that morin treatment decreased intracellular reactive oxygen species levels in MSU crystal stimulated macrophage cells. The western blot analysis clearly showed that morin mainly exerts its anti-inflammatory effects by inhibiting the MSU crystal-induced COX-2 and TNF-α protein expression through the inactivation of NF-κB signaling pathway in RAW 264.7 macrophage cells similar to that of BAY 11-7082 (IκB kinase inhibitor). Our results collectively suggest that morin can be a potential therapeutic agent for inflammatory disorders like acute gouty arthritis.

Triphala exhibits anti-arthritic effect by ameliorating bone and cartilage degradation in adjuvant-induced arthritic rats.

The present study was aimed to investigate the anti-arthritic effect of triphala and its underlying mechanism on adjuvant-induced rat model. For comparison purpose, non-steroidal anti-inflammatory drug indomethacin was used. Arthritis was induced by intradermal injection of complete Freund's adjuvant (0.1 ml) into the right hind paw of the Wistar albino rats. Triphala (100 mg/kg body weight [bwt]) was administered intraperitoneally (from 11th to 20th day) after the arthritis induction. Arthritis induction increased the levels of reactive oxygen species (LPO and NO), elastase, and mRNA expression of pro-inflammatory cytokines (TNF-α, IL-ß, IL-17, IL-6 and MCP-1), inflammatory marker enzymes (iNOS and COX-2), receptor activator of nuclear factor kappa-B ligand (RANKL), and transcription factors (NF-kB p65 and AP-1) in the paw tissues of rats. The levels of bone collagen were found to decrease with increased urinary constituents (hydroxyproline and total glycosaminoglycans) in arthritic rats. In addition, the immunohistochemistry analysis revealed increased expression of NF-kBp65 and COX-2 in the paw tissues of arthritic rats. However, administration of triphala significantly inhibited the biochemical and molecular alterations in adjuvant-induced arthritic rats compared to indomethacin (3 mg/kg bwt) as evidenced by the radiological and histopathological analysis. In conclusion, our results suggest that triphala administration ameliorate bone and cartilage degradation during rheumatoid arthritis.

The anti-inflammatory effect of triphala in arthritic-induced rats.

CONTEXT: Triphala, an Indian Ayurvedic herbal formulation which contains Terminalia chebula Retz. (Combretaceae), Terminalia bellerica (Gaertn.) Roxb. (Combretaceae) and Emblica officinalis L. (Phyllanthaceae), is used for treating bowel-related complications, inflammatory disorders, and gastritis. OBJECTIVE: To determine the anti-arthritic effect of triphala in arthritis-induced rats. For comparison purpose, the non-steroidal anti-inflammatory drug indomethacin was used. MATERIALS AND METHODS: Arthritis was induced in Wistar albino rats by intradermal injection of complete Freund's adjuvant (0.1 ml) into the foot pad of right hind paw. Triphala (100 mg/kg b wt, i.p.) was administered from day 11 to 18 after the administration of complete Freund's adjuvant. The activities/levels of lysosomal enzymes, glycoproteins, antioxidant status, and lipid peroxidation were determined in the paw tissues of arthritic rats. In addition, the inflammatory mediators were also measured in both the serum and the paw tissue of arthritic rats. RESULTS: The levels/activities of lipid peroxidation (∼41.5%), glycoproteins (hexose ∼43.3%, hexosamine ∼36.5%, and sialic acid ∼33.7%), lysosomal enzymes (acid phosphatase ∼52.4%, ß-galactosidase ∼22.9%, N-acetyl ß-glucosaminidase ∼22.1%, and cathepsin-D ∼27.7%) were found to be decreased and the antioxidant status (SOD ∼75.6%, CAT ∼62.7%, GPx ∼55.8%, GST ∼82.1%, and GSH ∼72.7%) was increased in the paw tissues of triphala-treated arthritic rats. In addition, the inflammatory mediator levels in serum (TNF-α ∼75.5%, IL-1ß âˆ¼99%, VEGF ∼75.2%, MCP-1 ∼76.4%, and PGE2 ∼69.9%) and in paw tissues (TNF-α ∼71.6%, IL-1ß âˆ¼75.5%, VEGF ∼55.1%, MCP-1 ∼69.1%, and PGE2 ∼66.8%) were found to be suppressed. CONCLUSION: Triphala has a promising anti-inflammatory effect in the inflamed paw of arthritis-induced rats.