In vitro and in vivo anti-inflammatory and anti-arthritic effect of Tinospora cordifolia via modulation of JAK/STAT pathway.

BACKGROUND: Rheumatoid arthritis (RA) is a chronic inflammatory disorder causing cartilage and joint degeneration. In spite of the availability of several robust drugs like biologics, most of the patients are unresponsive, and reports of severe adverse effects following long-term use are also there. Subsequently the use of natural plant-based products in RA therapy is broadening over the years. Tinospora cordifolia is a widely used medicinal plant in Ayurveda against various inflammatory disorders including RA. However, there is very limited knowledge regarding the actual molecular events responsible for its therapeutic effect, and this has limited its acceptance among the professionals. PURPOSE: To explore the anti-inflammatory and anti-arthritic effect of hydro-alcoholic extract from Tinospora cordifolia. METHODS: The rich polyphenol nature of the extract was elucidated using HPLC. LPS-stimulated murine macrophage cell line RAW 264.7 was used for in vitro studies, and collagen-induced arthritis (CIA) model was used for in vivo studies. RESULTS: The polyphenols in TCE were identified using HPLC. TCE effectively downregulated the level of pro-inflammatory mediators (IL-6, TNF-α, PGE2, and NO) in LPS-stimulated RAW 264.7 cells. Subsequently the upregulated expression of COX-2 and iNOS following LPS stimulation were also downregulated by TCE. Furthermore, TCE targeted the upstream kinases of the JAK/STAT pathway, a crucial inflammatory pathway. The expression of VEGF, a key angiogenic factor as well as an inflammatory mediator was also decreased following pre-treatment with TCE. The anti-arthritic effect of TCE (150 mg/kg) was evaluated in the CIA model as well. From the results of histopathology, oral administration of TCE was found to be effective in reducing the clinical symptoms of arthritis including paw edema, erythema, and hyperplasia. In vivo results validated the in vitro results and there was a significant reduction in serum level of pro-inflammatory cytokines and mediators (IL-6, TNF-α, IL-17, NO, and PGE2). The phosphorylation of STAT3 and the expression of VEGF were also downregulated following TCE treatment. CONCLUSION: Our study provided a detailed insight into the molecular events associated with anti-inflammatory and anti-arthritic effect of Tinospora cordifolia.

Downregulation of TLR4/MyD88/p38MAPK and JAK/STAT pathway in RAW 264.7 cells by Alpinia galanga reveals its beneficial effects in inflammation.

ETHNOPHARMACOLOGICAL RELEVANCE: Alpinia galanga, commonly known as greater galangal or raasna, is widely used in Ayurveda against various inflammatory disorders. It is also known as Kulinjan, Aratha, Rasna or Sugandhamula. Some of the Ayurvedic preparations using the rhizome of Alpinia galanga are Rasnadi kashayam, Rasna panchakam, Rasnapthakam, and Rasnarendadi. The aromatic rhizome is the source of the drug greater galangal and it is also used as a spice in South and South East Asia. However, the molecular mechanism of action of A galanga against inflammation remains poorly understood. AIM OF THE STUDY: To elucidate the anti-inflammatory effect of hydroalcoholic extract of Alpinia galanga rhizome. STUDY DESIGN/METHOD: The mechanism of the anti-inflammatory effect of hydroalcoholic extract of Alpinia galanga (AGE) was investigated by enzyme-linked immunosorbent assay (ELISA), Western blot, and immunofluorescence in LPS stimulated murine macrophage cell line (RAW 264.7). HPLC analysis was done to elucidate the rich polyphenolic nature of AGE. RESULTS: The study showed that pre-treatment with AGE downregulated the release of pro-inflammatory mediators (IL-6, TNF-α, NO, and ROS) and stimulated the release of anti-inflammatory mediator IL-10 in LPS stimulated RAW 264.7 cells. The vital enzymes of inflammation (iNOS, COX-2, and MMP-9) were also downregulated by pre-treatment with AGE. AGE targeted the upstream elements of the inflammatory cascade by blocking LPS induced activation of TLR4 and JAK/STAT pathway. The phosphorylation of downstream kinases was significantly affected. The inhibition of nuclear translocation of NFκB further confirmed the specific inhibition of the TLR4 pathway. Particularly AGE inhibited the phosphorylation of JNK, p38, IκBα, and STAT. HPLC analysis of the AGE showed the polyphenol-rich nature of the extract. CONCLUSIONS: The results from this study provide firm evidence that AGE exerts its anti-inflammatory effect via modulation of TLR4 and JAK/STAT pathway.

Current and novel therapeutic targets in the treatment of rheumatoid arthritis.

Rheumatoid arthritis (RA), a multifactorial disease characterized by synovitis, cartilage destruction, bone erosion, and periarticular decalcification, finally results in impairment of joint function. Both genetic and environmental factors are risk factors in the development of RA. Unwanted side effects accompany most of the current treatment strategies, and around 20-40% of patients with RA do not clinically benefit from these treatments. The unmet need for new treatment options for RA has prompted research in the development of novel agents acting through physiologically and pharmacologically relevant targets. Here we discuss in detail three critical pathways, Janus kinase/signal transducer and activator of transcription (JAK/STAT), Th17, and hypoxia-inducible factor (HIF), and their roles as unique therapeutic targets in the field of RA. Some of the less developed but potential targets like nucleotide-binding and oligomerization domain-like receptor containing protein 3 (NLRP3) inflammasome and histone deacetylase 1 (HDAC1) are also discussed.

Plasma anti-α-galactoside antibody mediates lipoprotein(a) binding to macrophages.

Lipoprotein (a) [Lp(a)] is the dominant lipid in atherosclerotic plaques though it is much less numerous than LDL or HDL in circulation. Molecular mechanism of selective uptake of Lp(a) into macrophages is unclear. Lp(a) was reported to form circulating immune complexes with the IgG-dominated plasma anti-α-galactoside antibody (anti-Gal) using the serine- and threonine-rich peptide sequences ( STPS) on its apo(a) subunit as surrogate ligand but left the other binding site of antibody free. We examined if these monovalent immune complexes could bind to smaller STPS-containing molecules on macrophage surface. Using placental membrane O-glycosylated proteins (PMOP) isolated by lectin affinity chromatography as model it was shown that human cell surface glycoproteins were small enough to occupy both binding sites of anti-Gal since they increased the fluorescence of FITC label at Fc part of anti-Gal and inhibited binding of anti-Gal and Griffonia simplicifolia lectin of similar specificity to immobilized ligands. Pre-incubation with anti-Gal facilitated Lp(a) attachment to macrophages unless anti-Gal-specific sugar was present. Anti-Gal-mediated attachment of apo(a) to macrophages increased with the number of apo(a) subunits. Further, anti-Gal-mediated binding of the same sample of apo(a) increased with the specific activity of anti-Gal sample. Finally binding of anti-Gal and anti-Gal-apo(a) complex to PMOP and macrophages respectively was mostly inhibited by LDL suggesting STPS as major anti-Gal epitopes on the cell surface. Results indicated that circulating Lp(a)-anti-Gal immune complexes anchor on macrophages using STPS-bearing cell surface glycoproteins as ligands and offer a pathway for Lp(a) sequestration into macrophages.

Antigen-Induced Activation of Antibody Measured by Fluorescence Enhancement of FITC Label at Fc.

Three anti-carbohydrate antibodies of defined specificity isolated from plasma were used to demonstrate that macromolecular antigen binding caused considerable enhancement of fluorescence of FITC-labeled antibody. Mono and disaccharide antigens which could compete with the large antigens in antibody binding could not however produce any increase in fluorescence. Fluorescence enhancement in a given antibody sample increased with the size of the occupying macromolecular antigen. Conversely in antibody samples of same ligand specificity isolated from plasma of different individuals, fluorescence enhancement produced by the same antigen correlated with specific activity of the antibody sample. Removal of Fc part of antibody, confirmed by electrophoresis and Fc-specific antibody binding, caused abolition of most of the antigen-driven fluorescence increase. Since antigen binding sites of antibodies were protected during FITC labeling, the above results suggest that conformational shift in Fc produced by occupation of binding sites by large antigens resulted in the enhancement of fluorescence of FITC tags on Fc. Data provides a tool for detection and measurement of specific ligands using fluorolabeled whole antibodies.