Mast Cells Differentiated in Synovial Fluid and Resident in Osteophytes Exalt the Inflammatory Pathology of Osteoarthritis.

INTRODUCTION: Osteophytes are a prominent feature of osteoarthritis (OA) joints and one of the clinical hallmarks of the disease progression. Research on osteophytes is fragmentary and modes of its contribution to OA pathology are obscure. AIM: To elucidate the role of osteophytes in OA pathology from a perspective of molecular and cellular events. METHODS: RNA-seq of fully grown osteophytes, collected from tibial plateau of six OA patients revealed patterns corresponding to active extracellular matrix re-modulation and prominent participation of mast cells. Presence of mast cells was further confirmed by immunohistochemistry, performed on the sections of the osteophytes using anti-tryptase alpha/beta-1 and anti-FC epsilon RI antibodies and the related key up-regulated genes were validated by qRT-PCR. To test the role of OA synovial fluid (SF) in mast cell maturation as proposed by the authors, hematopoietic stem cells (HSCs) and ThP1 cells were cultured in a media supplemented with 10% SF samples, obtained from various grades of OA patients and were monitored using specific cell surface markers by flow cytometry. Proteomics analysis of SF samples was performed to detect additional markers specific to mast cells and inflammation that drive the cell differentiation and maturation. RESULTS: Transcriptomics of osteophytes revealed a significant upregulation of mast cells specific genes such as chymase 1 (CMA1; 5-fold) carboxypeptidase A3 (CPA3; 4-fold), MS4A2/FCERI (FCERI; 4.2-fold) and interleukin 1 receptor-like 1 (IL1RL1; 2.5-fold) indicating their prominent involvement. (In IHC, anti-tryptase alpha/beta-1 and anti- FC epsilon RI-stained active mast cells were seen populated in cartilage, subchondral bone, and trabecular bone.) Based on these outcomes and previous learnings, the authors claim a possibility of mast cells invasion into osteophytes is mediated by SF and present in vitro cell differentiation assay results, wherein ThP1 and HSCs showed differentiation into HLA-DR+/CD206+ and FCERI+ phenotype, respectively, after exposing them to medium containing 10% SF for 9 days. Proteomics analysis of these SF samples showed an accumulation of mast cell-specific inflammatory proteins. CONCLUSIONS: RNA-seq analysis followed by IHC study on osteophyte samples showed a population of mast cells resident in them and may further accentuate inflammatory pathology of OA. Besides subchondral bone, the authors propose an alternative passage of mast cells invasion in osteophytes, wherein OA SF was found to be necessary and sufficient for maturation of mast cell precursor into effector cells.

New herbal composition (OA-F2) protects cartilage degeneration in a rat model of collagenase induced osteoarthritis.

BACKGROUND: Prevalence of osteoarthritis (OA) is on rise on the global scale. At present there are no satisfactory pharmacological agents for treating OA. Our previous study showed that Sida cordifolia L. and Zingiber officinale Rosc. had protective effect on cartilage. Here, we describe the effect of OA-F2, a herbal formulation prepared using combination of these two plants in alleviating OA associated symptoms in a rat model of collagenase-induced OA. METHODS: OA was induced by intra-articular injection of collagenase type II in wistar rats. Diclofenac (10 mg/kg) was used as a reference control. Rats (n = 6) were divided into 6 groups: Healthy control (HC), osteoarthritic control (OAC), diclofenac (DICLO), OA-F2L (135 mg/kg), OA-F2M (270 mg/kg) and OA-F2H (540 mg/kg). The effects of the 20 days treatment were monitored by parameters like knee diameter, paw volume, paw retraction; serum C-reactive protein (CRP), alkaline phosphatase (ALP) and glycosaminoglycan (GAG). Radiography and histopathology of knee joint were also studied. Additionally, gene expression was studied from isolated synovium tissue proving anti-osteoarthritic potential of OA-F2. RESULTS: Oral administration of OA-F2 has significantly prevented knee swelling compared to OAC; OA-F2 and DICLO, significantly reduced paw volume compared to OAC. Paw latency was remarkably increased by OA-F2 compared to OAC. OA-F2L (-0.670, p < 0.001), M (-0.110, p < 0.05) and H (0.073) has markedly reduced levels of CRP compared to DICLO. OA-F2L (p < 0.05), M (p < 0.001) and H (p < 0.05) significantly reduced ALP levels, compared to DICLO. GAG release in the serum was also significantly lowered in OA-F2 treated group compared to DICLO. Radiological and histopathological observations showed cartilage protection by OA-F2. OA-F2 has upregulated SOD and GPx. Upregulated CAT expression was observed in OA-F2M and H. Considerable down-regulation of expression of MMP-3 and MMP-9 was observed in all the groups. Up-regulation of TIMP-1 was observed in rats treated with OA-F2L, H and DICLO. CONCLUSION: OA-F2 has shown therapeutic effects in rat model of collagenase induced OA by demonstrating cartilage protection through controlling MMPs and improving anti-oxidant levels in arthritic synovium and is a potent candidate for further drug development and treatment for OA.

Anti-fatigue effect of Amarkand on endurance exercise capacity in rats.

BACKGROUND: Amarkand tubers are routinely used by many Indian tribes as a specialized food for health and longevity but so far there is no scientific evidence for their activities. Taxonomically, Amarkand belong to genera Eulophia and Dioscorea. METHODS: In this communication, comparative antifatigue potential of Amarkand was analyzed using forced swimming model in rats and evaluated using biomarkers of physical fatigue. RESULTS: Methanol extracts of tubers of D. bulbifera, E. ochreata, E. leghapanensis and bulbils of D. bulbifera exhibited rich polyphenolic content. D. bulbifera bulbils and E. ochreata significantly prolonged the swimming endurance time. Creatine kinase and urea nitrogen were significantly reduced by treatment of D. bulbifera bulbils and E. ochreata as compared to negative control. D. bulbifera bulbils effectively increased creatine (p < 0.001), lactate dehydrogenase (p < 0.01) and hemoglobin (p < 0.001) compared to negative control. D. bulbifera bulbils and E. ochreata treatments significantly increased glycogen (p < 0.05, p < 0.01) and lowered malondialdehyde levels (p < 0.001) in muscles and in liver tissue compared to negative control. CONCLUSION: These results indicate that a treatment with D. bulbifera bulbils and tubers of E. ochreata facilitates aerobic glucose metabolism and endurance by improving various impairments associated with fatigue.

Proteomic analysis of protease resistant proteins in the diabetic rat kidney.

Glycation induced protein aggregation has been implicated in the development of diabetic complications and neurodegenerative diseases. These aggregates are known to be resistant to proteolytic digestion. Here we report the identification of protease resistant proteins from the streptozotocin induced diabetic rat kidney, which included enzymes in glucose metabolism and stress response proteins. These protease resistant proteins were characterized to be advanced glycation end products modified and ubiquitinated by immunological and mass spectrometry analysis. Further, diabetic rat kidney exhibited significantly impaired proteasomal activity. The functional analysis of identified physiologically important enzymes showed that their activity was reduced in diabetic condition. Loss of functional activity of these proteins was compensated by enhanced gene expression. Aggregation prone regions were predicted by in silico analysis and compared with advanced glycation end products modification sites. These findings suggested that the accumulation of protein aggregates is an inevitable consequence of impaired proteasomal activity and protease resistance due to advanced glycation end products modification.

Production of polyunsaturated fatty acids in recombinant Lipomyces starkeyi through submerged fermentation.

Long chain polyunsaturated fatty acids (LCPUFA) are known to play an important role in human health and nutrition. Considering the limitation of LCPUFA sources, it is necessary to search new avenues for their production. Oleaginous yeasts are an attractive target for harvesting single cell oil, mainly because of the ease of cultivation with cheaper raw material. Lipomyces starkeyi is one such oleaginous yeast, which can accumulate oil to the extent of 60% of its biomass and where genetic transformation can be achieved. In our earlier work, Δ15 desaturase gene (AEP37840) from flax was transformed into L. starkeyi. In the present work, we report optimization of medium for the production of ω-3 enriched oil from this transformed yeast. A basic medium containing 20 g/l glucose as a carbon source and 10 g/l yeast extract as a nitrogen source was used during fermentation. At regular time intervals, glucose was fed to maintain high C:N ratio (65:10) during fermentation. Under the most favorable conditions, dry biomass and total lipid content were 18 and 7.29 g/l, respectively. Prior to genetic transformation, L. starkeyi contained 56.03 mg/l DHA along with 71.4 mg/l EPA and 42.2 mg/l ALA. Genetic engineering of this yeast resulted in a strain that produced 1080 mg/l DHA (17.4%) along with 74.28 mg/l EPA and 126.72 mg/l ALA possibly through modification of PUFA biosynthetic pathway. To the best of our knowledge, this is a first report of DHA enrichment and opens up avenues for LCPUFA production through L. starkeyi.

Phytochemicals in the treatment of inflammation-associated diseases: the journey from preclinical trials to clinical practice.

Advances in biomedical research have demonstrated that inflammation and its related diseases are the greatest threat to public health. Inflammatory action is the pathological response of the body towards the external stimuli such as infections, environmental factors, and autoimmune conditions to reduce tissue damage and improve patient comfort. However, when detrimental signal-transduction pathways are activated and inflammatory mediators are released over an extended period of time, the inflammatory process continues and a mild but persistent pro-inflammatory state may develop. Numerous degenerative disorders and chronic health issues including arthritis, diabetes, obesity, cancer, and cardiovascular diseases, among others, are associated with the emergence of a low-grade inflammatory state. Though, anti-inflammatory steroidal, as well as non-steroidal drugs, are extensively used against different inflammatory conditions, they show undesirable side effects upon long-term exposure, at times, leading to life-threatening consequences. Thus, drugs targeting chronic inflammation need to be developed to achieve better therapeutic management without or with a fewer side effects. Plants have been well known for their medicinal use for thousands of years due to their pharmacologically active phytochemicals belonging to diverse chemical classes with a number of these demonstrating potent anti-inflammatory activity. Some typical examples include colchicine (alkaloid), escin (triterpenoid saponin), capsaicin (methoxy phenol), bicyclol (lignan), borneol (monoterpene), and quercetin (flavonoid). These phytochemicals often act via regulating molecular mechanisms that synergize the anti-inflammatory pathways such as increased production of anti-inflammatory cytokines or interfere with the inflammatory pathways such as to reduce the production of pro-inflammatory cytokines and other modulators to improve the underlying pathological condition. This review describes the anti-inflammatory properties of a number of biologically active compounds derived from medicinal plants, and their mechanisms of pharmacological intervention to alleviate inflammation-associated diseases. The emphasis is given to information on anti-inflammatory phytochemicals that have been evaluated at the preclinical and clinical levels. Recent trends and gaps in the development of phytochemical-based anti-inflammatory drugs have also been included.

Synovial Fluid in Knee Osteoarthritis Extends Proinflammatory Niche for Macrophage Polarization.

Macrophage polarization is a steering factor of osteoarthritis (OA) progression. Synovial fluid (SF) obtained from OA patients with different Kellgren-Lawrence grades (KL grades) holds several proinflammatory factors and was hypothesized to induce macrophage differentiation and polarization by providing the needed microenvironment. U937 cells and peripheral-blood-mononuclear-cell-derived monocytes (PBMC-derived CD14+ cells) were induced with SFs of progressive KL grades for 48 h, and the status of the differentiated cells was evaluated by cell surface markers representing M1 and M2 macrophage phenotypes. Functional viability assessment of the differentiated cells was performed by cytokine estimation. The fraction of macrophages and their phenotypes were estimated by immunophenotyping of SF-isolated cells of different KL grades. A grade-wise proteome analysis of SFs was performed in search of the factors which are influential in macrophage differentiation and polarization. In the assay on U937 cells, induction with SF of KL grade III and IV showed a significant increase in M1 type (CD86+). The percentage of M2 phenotype (CD163+) was significantly higher after the induction with SF of KL grade II. A Significantly higher M1/M2 ratio was estimated in the cells induced with KL grade III and IV. The cell differentiation pattern in the assay on PBMC-derived CD14+ cells showed a grade-wise decline in both M1 (CD11C+, CD86+) and M2 phenotype (CD163+). Cytokine estimation specific to M1 (TNF-α, IL-6, IL-1ß, IFN-γ) and M2 (IL-4 and IL-10) macrophages corelated with the differentiation pattern in the U937 cell assay, while it did not reveal any significant changes in the PBMC-derived CD14+ cells assay. SF cells' immunophenotyping showed the highest percentage of CD14+ macrophages in KL grade II; CD86+ and CD163+ cells were minimal in all KL grades' SFs. The proteome analysis revealed significantly expressed MIF, CAPG/MCP, osteopontin, and RAS-related RAB proteins in KL grade III and IV samples, which are linked with macrophages' movement, polarization, and migration-behavior. In conclusion, this study demonstrated that SF in OA joints acts as a niche and facilitates M1 phenotype polarization by providing a proinflammatory microenvironment.

Rectal gel application of Withania somnifera root extract expounds anti-inflammatory and muco-restorative activity in TNBS-induced inflammatory bowel disease.

BACKGROUND: Inflammatory Bowel Disease (IBD) is marked with chronic inflammation of intestinal epithelium driven by oxidative stress. Traditional treatments with plant extracts gained renewed interest due to their ability to ameliorate the multi factorial conditions like inflammation. We investigated the beneficial effects of Withania somnifera in Trinitro Benzyl Sulfonic Acid (TNBS) induced experimental IBD through a rectally applicable formulation. METHODS: The study included (i) preparation of gel formulation from aqueous Withania somnifera root extract (WSRE), (ii) biochemical assays to determine its performance potential, (iii) testing of formulation efficacy in TNBS-induced IBD rat model, and (iv) histo-patholgical studies to assess its healing and muco-regenerative effect in IBD-induced rats. For this purpose, concentration dependant antioxidant activity of the extracts were evaluated using biochemical assays like (a) inhibition of lipid peroxidation, (b) NO scavenging, (c) H2O2 scavenging, and (d) ferric reducing power assay. RESULTS: The extract, at 500 µg/ml, the highest concentration tested, showed 95.6% inhibition of lipid peroxidation, 14.8% NO scavenging, 81.79% H2O2 scavenging and a reducing capacity of 0.80. The results were comparable with standard antioxidants, ascorbic acid and curcumin. WSRE treatment positively scored on histopathological parameters like necrosis, edema, neutrophil infiltration. The post treatment intestinal features showed restoration at par with the healthy intestine. In view of these results, gel formulation containing an aqueous extract of W. somnifera, prepared for rectal application was tested for its anti-inflammatory activity in TNBS-induced rat models for IBD. Commercially available anti-inflammatory drug Mesalamine was used as the standard in this assay. CONCLUSIONS: Dose of the rectal gel applied at 1000 mg of WSRE per kg rat weight showed significant muco-restorative efficacy in the IBD-induced rats, validated by histo-pathological studies.

Functional and bioinformatic characterisation of sequence variants of Fad3 gene from flax.

BACKGROUND: Desaturases are enzymes that drive the multi-step fatty acid biosynthetic pathway. As evident from directed mutagenesis, single base changes in their polypeptide can potentially alter their structure and may result in altered substrate specificity, regioselectivity and even loss of function. The authors have previously isolated several sequence variants of Δ15 desaturase from flax while attempting to clone that gene. The aim of the present study was to analyse these gene variants for their functionality and to predict the tertiary structure of the protein in order to correlate the functional differences with the protein structure. RESULTS: The variants differed in the rate at which they could convert linoleic acid to α-linolenic acid. The highest conversion rate was 7.03%, while the lowest was 2.39%. The overall shape of the predicted 3D model of the protein is a compact cylinder containing α-helices and ß-sheets. The Ramchandran plot of this model revealed that 98.5% of the residues are located in allowed region, which denotes a stable structure. CONCLUSION: Although the structures of the variants are apparently similar, subtle changes account for variation in their activity. Besides, these substitutions may alter their cross-talk with other proteins and thus differentially influence their specificity, localisation and stability, which in turn may explain the diversity in their function.

Pathophysiological landscape of osteoarthritis.

A sharp rise in osteoarthritis (OA) incidence is expected as over 25% of world population ages in the coming decade. Although OA is considered a degenerative disease, mounting evidence suggests a strong connection with chronic metabolic conditions and low-grade inflammation. OA pathology is increasingly understood as a complex interplay of multiple pathological events including oxidative stress, synovitis and immune responses revealing its intricate nature. Cellular, biochemical and molecular aspects of these pathological events along with major outcomes of the relevant research studies in this area are discussed in the present review. With reference to their published and unpublished work, the authors strongly propose synovitis as a central OA pathology and the key OA pathological events are described in connection with it. Recent research outcomes also have succeeded to establish a linkage between metabolic syndrome and OA, which has been precisely included in the present review. Impact of aging process cannot be neglected in OA. Cell senescence is an important mechanism of aging through which it facilitates development of OA like other degenerative disorders, also discussed within a frame of OA. Conclusively, the reviewers urge low-grade inflammation linked to aging and derailed immune function as a pathological platform for OA development and progression. Thus, interventions targeted to prevent inflammaging hold a promising potential in effective OA management and efforts should be invested in this direction.

Synovium-Synovial Fluid Axis in Osteoarthritis Pathology: A Key Regulator of the Cartilage Degradation Process.

Failure of conventional anti-inflammatory therapies in osteoarthritis (OA) underlines the insufficient knowledge about inflammatory mechanisms, patterns and their relationship with cartilage degradation. Considering non-linear nature of cartilage loss in OA, a better understanding of inflammatory milieu and MMP status at different stages of OA is required to design early-stage therapies or personalized disease management. For this, an investigation based on a synovium-synovial fluid (SF) axis was planned to study OA associated changes in synovium and SF along the progressive grades of OA. Gene expressions in synovial-biopsies from different grades OA patients (N = 26) revealed a peak of IL-1ß, IL-15, PGE2 and NGF in early OA (Kellgren-Lawrence (KL) grade-I and II); the highest MMP levels were found in advanced stages (KL grade-III and IV). MMPs (MMP-1, 13, 2 and 9) abundance and FALGPA activity estimated in forty SFs of progressive grades showed the maximum protein levels and activity in KL grade-II and III. In an SF challenge test, SW982 and THP1 cells were treated with progressive grade SFs to study the dynamics of MMPs modulation in inflammatory microenvironment; the test yielded a result pattern, which matched with FALGPA and the protein-levels estimation. Inflammatory mediators in SFs served as steering factor for MMP up-regulation. A correlation-matrix of IL-1ß and MMPs revealed expressional negative correlation.

Gender dependent differences in lipid metabolism in individuals with type 2 diabetes mellitus.

AIM: The present study investigates gender dependent effects of insulin resistance on lipid profile and adipocytokines in individuals with diabetes receiving oral antidiabetic drugs (OADs). The aim was also to reveal the changes in the expression of genes involved in lipid metabolism and inflammation. METHODS: Lipid profile, adipocytokine levels and homeostatic model assessment of insulin resistance (HOMA-IR) was assessed in 100 patients with diabetes (M = 43, F = 57) matched for age and gender with healthy individuals (M = 45, F = 55). The expression pattern of genes was analyzed by quantitative real time PCR. RESULTS: Males consuming metformin with other drugs exhibited a positive association between HOMA-IR and cholesterol, triglyceride and very low density lipoprotein (VLDL). Females consuming only metformin and metformin with other drugs, showed a positive association of HOMA-IR with cholesterol and a negative association with adiponectin. In males and females with diabetes, a comparable expression of peroxisome proliferator activated receptor γ (PPARγ) while higher expression of sterol regulatory element binding protein 1 (SREBP1) was observed. Expression of fatty acid synthase (FAS), long chain acyl CoA Synthetases (ACSL), malonyl-CoA-acyl carrier protein transacylase (MCAT) and nuclear factor kappa ß (NFkß) was higher in men with diabetes than healthy males. Expression of tumor necrosis factor α (TNF-α) was higher in males and females with diabetes than respective healthy genders. CONCLUSION: Insulin resistance adversely affects lipid profile, adipocytokines in males with type 2 diabetes. Expression of genes involved in lipid metabolism and inflammation is found to be undesirably and differentially altered in both the genders.

Cloning of a novel omega-6 desaturase from flax (Linum usitatissimum L.) and its functional analysis in Saccharomyces cerevisiae.

The Delta12 desaturase represents a diverse gene family in plants and is responsible for conversion of oleic acid (18:1) to linoleic acid (18:2). Several members of this family are known from plants like Arabidopsis and Soybean. Using primers from conserved C- and N-terminal regions, we have cloned a novel Delta12 desaturase gene amplified from flax genomic DNA, denoted as LuFAD2-2. This intron-less gene is 1,149-base pair long encoding 382 amino acids-putative membrane-bound Delta12 desaturase protein. Sequence comparisons show that the novel sequence has 85% similarity with previously reported flax Delta12 desaturase at amino acid level and shows typical features of membrane-bound desaturase such as three conserved histidine boxes along with four membrane-spanning regions that are universally present among plant desaturases. The signature amino acid sequence 'YNNKL' was also found to be present at the N terminus of the protein, which is necessary and sufficient for ER localization of enzyme. Neighbor-Joining tree generated from the sequence alignment grouped LuFAD2-2 among the other FAD2 sequences from Ricinus, Hevea, Jatropha, and Vernicia. When LuFAD2-2 and LuFAD2 were expressed in Saccharomyces cerevisiae, they could convert the oleic acid to linoleic acid, with an average conversion rate of 5.25 and 8.85%, respectively. However, exogenously supplied linoleic acid was feebly converted to linolenic acid suggesting that LuFAD2-2 encodes a functional FAD2 enzyme and has substrate specificity similar to LuFAD2.

High Energy Intake Induced Overexpression of Transcription Factors and Its Regulatory Genes Involved in Acceleration of Hepatic Lipogenesis: A Rat Model for Type 2 Diabetes.

Type 2 diabetes mellitus (T2DM) is a metabolic disorder characterized by impaired insulin action and its secretion. The objectives of the present study were to establish an economical and efficient animal model, mimicking pathophysiology of human T2DM to understand probable molecular mechanisms in context with lipid metabolism. In the present study, male Wistar rats were randomly divided into three groups. Animals were fed with high fat diet (HFD) except healthy control (HC) for 12 weeks. After eight weeks, intra peritoneal glucose tolerance test was performed. After confirmation of glucose intolerance, diabetic control (DC) group was injected with streptozotocin (STZ) (35 mg/kg b.w., i.p.). HFD fed rats showed increase (p ≤ 0.001) in glucose tolerance and HOMA-IR as compared to HC. Diabetes rats showed abnormal (p ≤ 0.001) lipid profile as compared to HC. The hepatocyte expression of transcription factors SREBP-1c and NFκß, and their target genes were found to be upregulated, while PPAR-γ, CPT1A and FABP expressions were downregulated as compared to the HC. A number of animal models have been raised for studying T2DM, but the study has been restricted to only the biochemical level. The model is validated at biochemical, molecular and histopathological levels, which can be used for screening new therapeutics for the effective management of T2DM.

Reduced synovial inflammation and inhibition of matrix metalloproteinases explicates anti-osteoarthritis activity of polyherbal formulations.

OBJECTIVES: Current osteoarthritis (OA) research experiences an incline toward Ayurveda to attain a complete cure without notable adverse effects. Ayurveda uses natural products, which are known to perform the multi-faceted role, a much demanding approach for OA management. However, lack of scientific evidence is a major drawback hindering their wider use. The present work investigated the anti-arthritic potential of Ashwagandharishta, Balarishta, Dashmoolarishta, and Triphala-extract to establish molecular-evidence for their clinical use. MATERIALS AND METHODS: Rabbit synoviocytes were induced using interleukin-1 beta (IL-1 ß) and lipopolysaccharide (LPS) separately and were further treated with study formulations to test anti-inflammatory and anti-oxidant potential, using nitric oxide (NO) and malondialdehyde (MDA) assays. Collagenase inhibition activity was estimated with N-(3-[2-Furyl] acryloyl)-Leu-Gly-Pro-Ala (FALGPA)-substrate and gelatinase spot assays. Data were analyzed with GraphPad Prism using one-way ANOVA followed by Bonferroni's multiple comparison. RESULTS: The study formulations were effective against synovitis, oxidative-stress, and inhibiting collagenase. They caused NO reduction in selected concentrations. DA showed the maximum NO decline of 0.02 ± 0 and 0.97 ± 0.62 µM/ml with IL-1 ß and LPS induction at 5 and 20 µg/ml concentrations, respectively. Estimated by FALGPA assay, increasing collagenase inhibition was observed as the function of concentration. All formulations showed a significant MDA decline, in dose-dependent manner. CONCLUSION: We assessed the anti-OA efficacy of conventionally prescribed Ayurvedic drugs using relevant biochemical assays. The studied formulations revealed potential to restrain synovitis, cartilage degeneration and to reduce oxidative stress, and the signature OA features. With established molecular authenticity, Ayurvedic drugs can offer a safer and affordable therapeutic option for OA.

Chondroprotective potential of root extracts of Withania somnifera in osteoarthritis.

This is the first report describing two novel chondroprotective activities of aqueous extracts of Withania somnifera root powder.First,these extracts had a statistically significant,short-term chondroprotective effect on damaged human osteoarthritic cartilage matrix in 50% of the patients tested. Second,these extracts caused a significant and reproducible inhibition of the gelatinase activity of collagenase type 2 enzyme in vitro.

Hyaluronidase and collagenase inhibitory activities of the herbal formulation Triphala guggulu.

Myrrh (guggulu) oleoresin from the Commiphora mukul tree is an important component of antiarthritic drugs in Ayurvedic medicine. Clinical data suggest that elevated levels of hyaluronidase and collagenase type 2 enzymes contribute significantly to cartilage degradation. Triphala guggulu (TG) is a guggulu-based formulation used for the treatment of arthritis. We assessed the chondroprotective potential of TG by examining its effects on the activities of pure hyaluronidase and collagenase type 2 enzymes. Triphala shodith guggulu (TSG), an intermediate in the production of TG, was also examined. A spectrophotometric method was used to assay Hyaluronidase activity, and to detect potential Hyaluronidase inhibitors. Aqueous and hydro-alcoholic extracts of TSG showed weak but dose-dependent inhibition of hyaluronidase activity. In contrast, the TG formulation was 50 times more potent than the TSG extract with respect to hyaluronidase inhibitory activity. A validated X-ray film-based assay was used to measure the gelatinase activity of pure collagenase type 2. Hydro-alcoholic extracts of the TG formulation were 4 times more potent than TSG with respect to collagenase inhibitory activity. Components of Triphala were also evaluated for their inhibitory activities on hyaluronidase and collagenase. This is the first report to show that the T2 component of Triphala (T.chebula) is a highly potent hyaluronidase and collagenase inhibitor. Thus, the TG formulation inhibits two major enzymes that can degrade cartilage matrix. Our study provides the first in vitro preclinical evidence of the chondroprotective properties of TG.

Gender dependent effects of fasting blood glucose levels and disease duration on biochemical markers in type 2 diabetics: A pilot study.

AIM: The impact of fasting blood glucose levels (FBG) and disease duration on type 2 diabetes in Indian population is still unclear. The present study examines gender-dependent effects of FBG and disease duration on lipid profile, adipocytokines and related biochemical parameters in diabetic individuals. METHODS: Type 2 diabetic individuals (n=100) were classified depending on FBG: patients with normal FBG (Glucose<126mg/dl) and patients with high FBG (Glucose≥126mg/dl); and disease duration: ≥0-≤3yr, >3-≤7yr, >7yr. RESULTS: Males with high FBG had significantly higher serum glucose, triglycerides, very low density lipoprotein (VLDL), serum glutamic oxaloacetic transaminase (SGOT), serum glutamic pyruvic transaminase (SGPT) and waist hip ratio (WHR) than males with normal FBG. Females with high FBG had significant increase in serum glucose, adiponectin and creatinine while decrease in leptin levels than females with normal FBG. Males with high FBG had higher WHR, superoxide dismutase, SGOT, SGPT and lower adiponectin, leptin than females with high FBG. Significant positive association was observed between glucose and cholesterol, triglyceride, VLDL and urea in males with high FBG. With chronic diabetes for >7yr, males had increased systolic blood pressure, glucose, LDL, urea and low catalase activity as compared to other disease duration groups. However, females had higher adiponectin, creatinine and lower body mass index and cholesterol. CONCLUSIONS: High FBG in males adversely affects lipid profile, adipocytokines and liver function. Some of these effects exacerbate as disease progresses. Higher adiponectin may have desirable effects on metabolic markers in females.

Chronic maternal calcium and 25-hydroxyvitamin D deficiency in Wistar rats programs abnormal hepatic gene expression leading to hepatic steatosis in female offspring.

Importance of calcium and vitamin D deficiency is well established in adult dyslipidemia. We hypothesized that maternal calcium and vitamin D deficiency could alter offspring's lipid metabolism. Our objective was to investigate the effect of maternal dietary calcium and vitamin D deficiency on lipid metabolism and liver function of the F1 generation offspring. intergenerational calcium-deficient (CaD) and vitamin D-deficient (VDD) models were developed by mating normal male rats with deficient females and continuing maternal-deficient diets through pregnancy and lactation. Offspring were fed on control diet post-weaning and studied till 30 weeks. Lipid profile, serum glutamate pyruvate transaminase (SGPT), calcium and vitamin D levels were analyzed. Liver fat deposition, omega-3 fatty acids level and mRNA expression levels of peroxisome proliferator-activated receptor-alpha (PPAR-α), sterol regulatory element-binding protein 1c (SREBP-1c), interleukin 6 (IL-6), superoxide dismutase 1 (SOD-1) and uncoupling protein 2 (UCP2) were determined. Low serum vitamin D levels with an increase in SGPT and TG levels in CaD and VDD female offspring were observed. Severe liver steatosis with down-regulation of PPAR-α and UCP2 and up-regulation of SREBP-1c, IL-6 and SOD-1 was observed in the female offspring born to deficient dams. CaD and VDD male offspring showed mild steatosis and down-regulation of UCP2 and SOD-1. We conclude that maternal calcium and vitamin D deficiency programs abnormal lipid metabolism and hepatic gene expression in the F1 generation female offspring leading to hepatic steatosis, despite feeding them on control diet post-weaning.

Glycosaminoglycan measured from synovial fluid serves as a useful indicator for progression of Osteoarthritis and complements Kellgren-Lawrence Score.

BACKGROUND: Plain radiography is the first choice for diagnosis and monitoring of knee-osteoarthritis (OA) while, Kellgren-Lawrence score (KL) is most widely used to grade OA severity. However, incompetency for reproducibility of joint space measurement in longitudinal assessment and non-linearity of KL-score system, limits radiography-based early diagnosis of the disease. Glycosaminoglycan (GAG) is direct cartilage-degradation product, which can be measured biochemically. We strived to correlate KL-score and GAG from OA patients to compliment KL-system. METHODS: We obtained 34 synovial-fluid (SF) samples from 28 OA patients (few bilateral) with different disease severity using arthrocetesis. All patients were categorised using radiographic KL-score-system. SFs were further analysed for GAG estimation using 1,2-dimethylmethylene blue (DMMB) assay. RESULTS: A substantial increase in GAG was noted in KL-grade-II and III, comparing grade-I patients, indicating amplified cartilage-degradation. KL-grade-IV patients revealed further rise in GAG reflecting more cartilage-loss. Another category of grade-IV patients with lower GAG were also detected, indicating close to total cartilage-loss. CONCLUSIONS: Accurate diagnosis of cartilage-loss remains a challenge with OA due to limitations of KL-system; thus no target intervention is available to arrest active cartilage-loss. We propose, GAG-estimation in OA patients, characterizes accurate biochemical depiction of cartilage degeneration. GENERAL SIGNIFICANCE: Radiology often fails to reveal an accurate cartilage loss, associated with OA. GAG levels from the SFs of OA patients' serve as a useful marker, which parallels cartilage degeneration and strengthen radiographic grading system, ultimately.