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.

Zerumin A attenuates the inflammatory responses in LPS-stimulated H9c2 cardiomyoblasts.

Zerumin A (ZA) is one of the potential components of Curcuma amada rhizomes, and it has been shown to possess a variety of pharmacological activities. This study deals with the beneficial activity of ZA in lipopolysaccharide (LPS)-stimulated inflammation in H9c2 cardiomyoblasts. Herein, H9c2 cells were preincubated with ZA for 1 h and stimulated with LPS for 24 h. The cells were analyzed for the expression of various pro-inflammatory mediators and signaling molecules. Results showed that the cell viability was significantly improved and reactive oxygen species production was alleviated remarkably with ZA pretreatment. We also found that ZA pretreatment significantly suppressed the upregulation of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2) protein levels, and nitric oxide (NO) release in LPS-stimulated cells. In addition, ZA significantly ameliorated LPS-elicited overexpression of pro-inflammatory chemokines and cytokines such as monocyte chemoattractant protein-1 (MCP-1), tumor necrosis factor α (TNF- α), interferon-γ (IFN-γ), and interleukin-1 (IL-1) in H9c2 cells, and it upregulated the synthesis of the anti-inflammatory cytokine interleukin-10 (IL-10). Moreover, pretreatment with ZA and the mitogen-activated protein kinases (MAPK) pathway inhibitors also reduced the phosphorylation of extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinases (JNK), and p38. ZA significantly inhibited IkB-a phosphorylation and nuclear factor (NF)-kB p65 subunit translocation into nuclei. Overall data demonstrated that ZA protects cardiomyocytes against LPS injury by inhibiting NF-kB p65 activation via the MAPK signaling pathway in vitro. These findings suggest that ZA may be a promising agent for a detailed study for the prevention or treatment of myocardial dysfunction in sepsis.

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.

Glucotoxicity results in apoptosis in H9c2 cells via alteration in redox homeostasis linked mitochondrial dynamics and polyol pathway and possible reversal with cinnamic acid.

Several mechanisms have been proposed for the heart dysfunction during hyperglycemia. The aim of the present in vitro study is to elucidate the role of alterations in redox homeostasis in the induction of apoptosis during hyperglycemia in H9c2 cells via dysfunction in mitochondria and polyol pathway and evaluation of the beneficial effect of cinnamic acid against the same. The H9c2 cells were incubated with 33 mM glucose for 48 h to simulate the diabetic condition. Cell injury was confirmed with a significant increase of atrial natriuretic peptide and lactate dehydrogenase release. Alterations in the innate antioxidant system, polyol pathway, mitochondrial integrity, dynamics and apoptosis were investigated. Hyperglycemic insult has significantly affected redox homeostasis via depletion of superoxide dismutase, glutathione and enhanced reactive oxygen species generation. It also caused dysregulation in mitochondrial dynamics (fusion, fission proteins), dissipation of mitochondrial transmembrane potential and increased sorbitol accumulation. Finally, apoptosis was observed with upregulation of Bax, activation of caspase-3 and downregulation of Bcl-2. Cinnamic acid cotreatment increased the innate antioxidant status, improved mitochondrial function and prevented apoptosis in H9c2 cardiomyoblasts. Moreover, this in vitro model is found to be ideal for the elucidation of mechanisms at the cellular and molecular level of any physiological, pharmacological and toxicological incidents in H9c2 cells.

Development of insulin resistance through sprouting of inflammatory markers during hypoxia in 3T3-L1 adipocytes and amelioration with curcumin.

The role of phytochemicals in general well-being has been recognized. Curcumin is an ideal example. Hypoxia in adipose tissue is a major cause of inflammation and insulin resistance in obesity. Herein we mainly explored inflammation, insulin resistance and angiogenesis in 3T3-L1 adipocytes and possible reversal with the curcumin during hypoxia. Hypoxia for 24h significantly increased (P ≤ 0.05) the secretion of monocyte chemotactic protein-1 (4.59 fold), leptin (2.96 fold) and reduced adiponectin (2.93 fold). mRNA level of resistin (6.8 fold) and toll-like receptor-4 (TLR-4) (8.8 fold) was upregulated. Increased serine phosphorylation of insulin receptor substrate 1 (IRS-1) (1.9 fold) and decreased expression of insulin receptor substrate 2 (IRS-2) (0.53 fold) in hypoxic group were observed. Hypoxia significantly increased (P ≤ 0.05) basal glucose uptake (3.3 fold), GLUT-1 expression and angiogenic factors but down regulated GLUT-4. Curcumin protected adipocytes from hypoxia induced inflammation and insulin resistance via reducing inflammatory adipokine, nuclear factor-κB (NF-κB)/c-jun N-terminal kinase (JNK) and serine phosphorylation of IRS-1 receptors and improving adiponectin secretion.

Bilobalide abates inflammation, insulin resistance and secretion of angiogenic factors induced by hypoxia in 3T3-L1 adipocytes by controlling NF-κB and JNK activation.

Obesity leads to inflammation and insulin resistance in adipose tissue. Hypoxia, observed in obese adipose tissue is suggested as a major cause of inflammation and insulin resistance in obesity. However, the role of hypoxia in adipose tissue during obesity and insulin resistance was not well established. Here we mainly explored the crosstalk between hypoxia induced inflammation, and insulin resistance and also secretion of angiogenic factors in 3T3-L1 adipocytes and possible reversal with bilobalide. Hypoxia for 24h significantly (P≤0.05) increased the secretion of MCP-1 (4.59 fold), leptin (2.96 fold) and reduced adiponectin secretion (2.93 fold). In addition, the mRNA level of resistin (6.8 fold) and TLR4 receptors (8.8 fold) was upregulated in hypoxic adipocytes. The release of inflammatory cytokines and expression of TLR4 receptors led to activation of JNK and NF-κB signalling. We further investigated the effects of JNK and NF-κB activation on insulin signalling receptors. The present study showed increased (P≤0.05) serine 307 phosphorylation of IRS-1 (1.9 fold) and decreased expression of IRS-2 (0.53 fold) in hypoxic group showing hypoxia induced impairment in insulin signalling. Hypoxia significantly (P≤0.05) increased basal glucose uptake (3.3 fold) as well as GLUT-1 expression in adipocytes indicating GLUT-1 mediated glucose uptake. Hypoxia for 24h significantly increased (P≤0.05) the expression of angiogenic factors. Bilobalide protected adipocytes from hypoxia induced inflammation and insulin resistance mainly by reducing inflammatory adipokine secretion, improving adiponectin secretion, reducing NF-κB/JNK activation, and inhibiting serine phosphorylation of IRS-1 receptors of insulin signalling pathway.

Downregulation of inflammatory mediators and pro-inflammatory cytokines by alkaloids of Jeevaneeya rasayana in adjuvant-induced arthritis.

Jeevaneeya rasayana is an ayurvedic polyherbal formulation, with antirheumatic potential. The present study investigates the therapeutic efficacy of isolated total alkaloid fraction of Jeevaneeya Rasayana (AJR) in treating rheumatoid arthritis in a rat model of Adjuvant-induced arthritis (AIA). Paw swelling, inflammatory mediators such as cyclooxygenase-2 (COX-2), matrix metalloproteinase-9 (MMP-9), level of prostaglandin E2 (PGE2), expression of cytokines and serum nitric oxide (NO) level were analyzed in experimental rats after an experimental period of 21 days. Arthritic induction significantly increased paw edema, and up regulated the inflammatory mediators and cytokines. Administration of AJR significantly reversed the paw edema, reduced the level of PGE2, serum NO and decreased the COX-2 activity in the paw tissue. AJR treatment also downregulated mRNA expression of pro-inflammatory cytokines tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and MMP-9 in paw tissue. HPTLC analysis revealed the presence of 5 different alkaloid compounds in AJR. These findings suggest that the AJR have the therapeutic potential against adjuvant-induced arthritis.

Anti-inflammatory and antioxidative effects of mucilage of Trigonella foenum graecum (Fenugreek) on adjuvant induced arthritic rats.

The aim of this study was to investigate anti-inflammatory and antioxidant effects of mucilage from fenugreek in adjuvant induced arthritis in rats. Arthritis was induced by intradermal injection of complete Freund's adjuvant into the right hind paw produce inflammation of the joint. The activities of inflammatory enzymes like cyclooxygenase, lipoxygenase and myeloperoxidase, and levels of nitrite and C-reactive protein were observed. Also oxidative stress was measured by analyzing the activity of catalase, superoxide dismutase, glutathione peroxidase and the levels of glutathione and vitamin C and lipid peroxidation. The blood parameters like ESR, total WBC, RBC and hemoglobin content was checked. Fenugreek mucilage exhibited maximum percentage of edema inhibition at a dose of 75 mg/kg on 21st day of adjuvant arthritis. The effect was higher than that of standard drug indomethacin. The activities of cyclooxygenase-2 and myeloperoxidase and concentration of thiobarbituric acid reactive substance (TBARS) were decreased and the activities of antioxidant enzymes, vitamins C and reduced glutathione level were increased on treatment with fenugreek mucilage. The increment in ESR and total WBC, reduction in RBC count and hemoglobin and aberrant changes to the C-reactive protein (CRP) levels observed in the arthritic animals were also found to be significantly restored in fenugreek mucilage treated rats. Histopathology of paw tissue showed decreased edema formation and cellular infiltration on supplementation with fenugreek mucilage. Thus the results demonstrated the potential beneficiary effect of fenugreek mucilage on adjuvant induced arthritis in rats.

Inhibitory effect of Ruta graveolens L. on oxidative damage, inflammation and aortic pathology in hypercholesteromic rats.

The purpose of the study was to investigate the efficacy of methanolic extract of Ruta graveolens L. in reducing oxidative damage, inflammation and aortic pathology in hypercholesteremic rats. For the study rats were divided into three groups - control group, hypercholesteremic group and treatment group (20 mg MER/kg/d orally) - and were fed for 90 days. Serum total cholesterol, LDL-C, total WBC count, CRP level, TBARS, atherogenic index, activities of COX, 15 LOX in monocyte and serum myeloperoxidase were increased in cholesterol fed rats. Activities of antioxidant enzymes and the concentration reduced glutathione in liver and heart tissue and serum HDL-C were decreased in cholesterol fed rats. The results showed that level of total cholesterol, LDL-C, atherogenic index was decreased and HDL-C was increased in MER treated rats. Activities of antioxidant enzymes were found to be increased and the activity of MPO, COX and 15 LOX were decreased on supplementation with MER. Concentration of TBARS and total WBC count were decreased and GSH was increased on supplementation with MER. Histopathology of aorta of cholesterol fed rat showed marked alterations whereas the aorta of MER administrated rat showed no significant changes. These results suggested that MER reduces oxidative stress, inflammation and aortic pathology in hypercholesteremic rats. Thus the plant may therefore be useful for therapeutic treatment of clinical conditions associated atherosclerosis.

Efficacy of Bacopa monniera (L.) Wettst in alleviating lysosomal instability in adjuvant-induced arthritis in rats.

The present study was aimed to assess the effect of Bacopa monniera extract against lysosomal instability during adjuvant-induced arthritis in rats. B. monniera extract was administered orally at a dose of 100 mg/kg body weight for 30 days to the experimental rats after the induction of adjuvant arthritis. The ability of B. monniera extract to stabilize lysosomal enzyme activities in the cartilage of control and experimental rats was done by monitoring the activities of pathophysiological enzymes such as ß-glucuronidase, ß-glucosaminidase, cathepsin D, hyaluronidase, collagenase and the level of protein bound carbohydrates and glycosaminoglycans (GAGs) in arthritic rats. B. monniera extract supplementation significantly inhibited lysosomal instability in different tissues studies and improved the level of glycoproteins in synovial effusate and GAG in the cartilage. To evaluate whether anti-inflammatory property of B. monniera extract was due to lysosomal stability and GAG protection, purified chloroform fraction of B. monniera (CF) was assayed for its role to modulate interleukin-6 (IL-6) expression and prostaglandin E2 (PGE2) production. Administration of CF (50 mg/kg) to arthritic rats significantly downregulated the expression of IL-6 in blood mononuclear cells and PGE2 levels in cartilage tissue. The possible mechanism of action of the B. monniera extract may be through its stabilizing action on lysosomal membranes and hence the decrease in spread of inflammation.

Anti-inflammatory and antioxidant effects of Jeevaneeya Rasayana: an ayurvedic polyherbal formulation on acute and chronic models of inflammation.

The present study was aimed to establish the efficacy of Jeevaneeya Rasayana (JR), an ayurvedic polyherbal formulation, in adjuvant-induced arthritic (AIA) rat model with reference to mediators of inflammation. The methanolic (MJR), ethanolic (EJR), and water extracts (WJR) of JR were prepared and their anti-inflammatory activity in carrageenan-induced acute model was evaluated. MJR at a dose of 25 mg/kg showed significantly higher anti-inflammatory effect than EJR, WJR, and standard drug diclofenac. MJR also significantly decreased the paw edema in AIA rats. Activities of cyclooxygenase, 5-lipoxygenase, and myeloperoxidase were decreased significantly on treatment with MJR. Supplementation with MJR increases the activities of antioxidant enzymes and the level of glutathione content. The increment in the concentration of C-reactive protein, thiobarbituric acid reactive substance, and ceruloplasmin observed in arthritic rats were found to be significantly restored in MJR treated rats. Thus, the results demonstrated the potential beneficiary effect of methanolic extract of Jeevaneeya Rasayana on acute and chronic models of inflammation.

Protective effects of isolated polyphenolic and alkaloid fractions of Ruta graveolens L. on acute and chronic models of inflammation.

Ruta graveolens L. (Rutaceae) are traditionally used for the treatment of rheumatism, arthritis and other inflammatory conditions in the traditional medicine of India, were evaluated for their protective effect in acute and chronic models of inflammation. Carrageenan induced rat paw edema and adjuvant induced arthritis were employed as the experimental models of acute and chronic inflammation respectively. Isolated polyphenolic and alkaloid fraction (AFR) from Ruta graveolens and evaluated its anti inflammatory activity in carrageenan induced acute model. AFR with a dose 10 mg/kg showed higher anti inflammatory effect than polyphenols and standard drug diclofenec. AFR significantly decreased the paw edema in arthritic rats. TBARS, COX-2, 5-LOX and MPO level were decreased and the levels of antioxidant enzymes and GSH level were increased on treatment with AFR. The increment in CRP level and ceruloplasmin level observed in arthritic animals were also found to be significantly restored in AFR treated rats. The results demonstrated the potential beneficiary effect of isolated polyphenolic and alkaloid fraction of Ruta graveolens L. on acute and chronic models of inflammation in rats.

Inhibitory effects of Cynodon dactylon L. on inflammation and oxidative stress in adjuvant treated rats.

Cynodon dactylon is one of the 10 auspicious herbs that constitute the group Dasapushpam in Ayurveda. Traditionally Cynodon dactylon L. is used against many chronic inflammatory diseases in India. The present study was carried out to evaluate the protective effect of Cynodon dactylon against rats with adjuvant- induced arthritis. Arthritis was induced by intradermal injection of complete Freund's adjuvant into the right hind paw produce inflammation of the joint. A significant increase in the levels of inflammatory mediators, myeloperoxidase, nitrite, C-reactive protein, ceruloplasmin was observed. This was associated with oxidative stress with a marked reduction in the activity of catalase, superoxide dismutase, glutathione peroxidase and the levels of glutathione, vitamins C and E and an increase in the lipid peroxidation as indicated by the higher levels of thiobarbituric acid reactive substances. Cynodon dactylon (20mg/kg/b.wt) was orally administered to arthritic rats after adjuvant injection produced a significant attenuation in the inflammatory response, oxidative stress and ameliorated the arthritic changes to near normal conditions. Hence, the results of this study clearly indicate that Cynodon dactylon extract has a promising protective role against arthritis.

Methanolic extract of Ruta graveolens L. inhibits inflammation and oxidative stress in adjuvant induced model of arthritis in rats.

Ruta graveolens L. (Rutaceae) are traditionally used for the treatment of rheumatism, arthritis and other inflammatory conditions in the traditional medicine of India. The purpose of this study was to investigate anti-inflammatory and anti-oxidant effects of methanolic extract of Ruta graveolens L. in adjuvant induced arthritis in rats. Methanolic extract of Ruta graveolens (MER) exhibited maximum percentage of oedema inhibition at a dose of 20 mg/kg on 21st day of adjuvant arthritis. The effect was higher than that of standard drug indomethacin. The activities of cycloxygenase-2 and myeloperoxidase and concentration of thiobarbituric acid reactive substance (TBARS) were decreased and the activities of antioxidant enzymes, vitamins C & E and reduced glutathione level were increased on treatment with MER. The increment in ESR and total WBC, reduction in RBC count and haemoglobin and aberrant changes to the C-reactive protein (CRP) and ceruloplasmin levels observed in the arthritic animals were also found to be significantly restored in MER treated rats. Histopathology of paw tissue showed decreased oedema formation and cellular infiltration on supplementation with MER. Thus the results demonstrated the potential beneficiary effect of methanolic extract of Ruta graveolens on adjuvant induced arthritis in rats.