Computational Deciphering of the Role of S100A8 and S100A9 Proteins and Their Changes in the Structure Assembly Influences Their Interaction with TLR4, RAGE, and CD36.

S100A8 and S100A9 belong to the calcium-binding, damage associated molecular pattern (DAMP) proteins shown to aggravate the pathogenesis of rheumatoid arthritis (RA) through their interaction with the TLR4, RAGE and CD36 receptors. S100A8 and S100A9 proteins tend to exist in monomeric, homo and heterodimeric forms, which have been implicated in the pathogenesis of RA, via interacting with Pattern Recognition receptors (PRRs). The study aims to assess the influence of changes in the structure and biological assembly of S100A8 and S100A9 proteins as well as their interaction with significant receptors in RA through computational methods and surface plasmon resonance (SPR) analysis. Molecular docking analysis revealed that the S100A9 homodimer and S100A8/A9 heterodimer showed higher binding affinity towards the target receptors. Most S100 proteins showed good binding affinity towards TLR4 compared to other receptors. Based on the 50 ns MD simulations, TLR4, RAGE, and CD36 formed stable complexes with the monomeric and dimeric forms of S100A8 and S100A9 proteins. However, SPR analysis showed that the S100A8/A9 heterodimers formed stable complexes and exhibited high binding affinity towards the receptors. SPR data also indicated that TLR4 and its interactions with S100A8/A9 proteins may play a primary role in the pathogenesis of RA, with additional contributions from CD36 and RAGE interactions. Subsequent in vitro and in vivo investigations are warranted to corroborate the involvement of S100A8/A9 and the expression of TLR4, RAGE, and CD36 in the pathophysiology of RA.

Methanolic extract of O.umbellata L. exhibits anti-osteoporotic effect via promoting osteoblast proliferation in MG-63 cells and inhibiting osteoclastogenesis in RANKL-stimulated RAW 264.7 cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Oldenlandia umbellata L., belonging to the Rubiaceae family, is an annual plant possessing anti-inflammatory and antipyretic, anti-nociceptive, anti-bacterial, anti-helminthic, antioxidant and hepatoprotective activities and used in traditional medicine to treat inflammation and respiratory diseases. AIM OF THE STUDY: The present study aims to evaluate the anti-osteoporotic effect of Methanolic extract of O.umbellata in MG-63 cells and RANKL-stimulated RAW 264.7 cells. MATERIALS AND METHODS: The methanolic extract from the aerial parts of O.umbellata was subjected to metabolite profiling. The anti-osteoporotic effect of MOU was assessed in MG-63 cells and RANKL-stimulated RAW 264.7 cells. In MG-63 cells, the proliferative effect of MOU was evaluated using MTT assay, ALP assay, Alizarin red staining, ELISA and western blot. Similarly, the anti-osteoclastogenic effect of MOU was assessed in RANKL-stimulated RAW 264.7 cells via MTT, TRAP staining and western blot. RESULTS: LC-MS metabolite profiling showed the presence of 59 phytoconstituents including scandoside, scandoside methyl ester, deacetylasperuloside, asperulosidic acid, and cedrelopsin in MOU. In MG-63 cells, MOU has increased the proliferation of osteoblast cells and ALP activity, thereby increasing bone mineralization. ELISA results showed increased levels of osteogenic markers such as osteocalcin and osteopontin in the culture media. Western blot analysis showed inhibition of GSK3ß protein expression and increased the expression levels of ß-catenin, Runx-2, col 1 and osterix, promoting osteoblast differentiation. In RANKL-stimulated RAW 264.7 cells, MOU did not elicit any significant cytotoxicity; instead, it suppressed the osteoclastogenesis reducing the osteoclast number. MOU has reduced TRAP activity in a dose-dependent manner. MOU inhibited the TRAF6, NFATc1, c-Jun, C-fos and cathepsin K expression, thereby inhibiting osteoclast formation. CONCLUSION: In conclusion, MOU promoted osteoblast differentiation via inhibiting GSK3ß and activating Wnt/ß catenin signalling and its transcription factors, including ß catenin, Runx2 and Osterix. Similarly, MOU inhibited osteoclast formation by inhibiting the expression of TRAF6, NFATc1, c-Jun, C-fos and cathepsin K in RANK-RANKL signalling. Finally, it can be emphasised that O.umbellata is a potential source of therapeutic leads for the treatment of osteoporosis.

Underpinning Endogeneous Damp EDA-Fibronectin in the Activation of Molecular Targets of Rheumatoid Arthritis and Identifcation of its Effective Inhibitors by Computational Methods.

Rheumatoid arthritis (RA) is one of the most severe inflammatory diseases that cause swelling, stiffness and pain in the joints, which pose a significant threat worldwide. Damage-associated molecular patterns (DAMPs) are danger molecules of endogenous origin, released during cell injury or cell death, interacts with various Pattern recognition receptors (PRRs) and activates various inflammatory diseases. One of the DAMP molecules, so-called EDA-fibronectin (Fn) is also responsible for causing RA. EDA-Fn triggers RA through its interaction with TLR4. Apart from TLR4, it is divulged that certain other PRR's are also responsible for RA, but the identity and mechanism of those PRRs remain unknown until now. Hence, for the first time, we tried to reveal those PRR's interaction with EDA-Fn in RA through computational methods. Protein-protein interaction (PPI) was checked using ClusPro between EDA-Fn and certain Pattern recognition receptors (PRRs) to explore the binding affinities of the potential PRRs. Protein-protein docking unveiled that TLR5, TLR2 and RAGE has good interaction with EDA-Fn than the well-reported TLR4. Macromolecular simulation was performed for TLR5, TLR2 and RAGE complexes along with the control group TLR4 for 50 ns to further investigate the stability, leading to the identification of TLR2, TLR5 and RAGE as the stable complexes. Hence, TLR2, TLR5 and RAGE on interaction with EDA-Fn may lead to the progression of RA that may need additional validations through in vitro and in vivo animal models. Molecular docking was used to analyse the binding force of the top 33 active anti-arthritic compounds with the target protein EDA-Fn. Molecular docking study showed that withaferin A has a good binding activity with EDA-fibronectin target. Hence, it is emphasized that guggulsterone and berberine could modulate the EDA-Fn-mediated TLR5/TLR2/RAGE pathways, thereby it could inhibit the deteriorating effects of RA which needs further in vitro and in vivo experimental validations.

Phenolic acid bound arabinoxylans extracted from Little and Kodo millets modulate immune system mediators and pathways in RAW 264.7 cells.

The immunomodulating effect of Phenolic acid bound arabinoxylans (PCA-AXs) extracted from Little (PCA-AX-L) and Kodo (PCA-AX-K) millet seeds in RAW 264.7 cells were investigated. The PCA-AXs were extracted from millets and their chemical characterization were carried out by GC-MS, HPLC, and FT-IR. The immunomodulatory effect of PCA-AXs in RAW 264.7 cells were investigated by estimating ROS, NO, and cytokines TNF-α, IL-1ß, IL-6, and evaluation of molecular mechanism by q-PCR & western blotting techniques. The xylose: arabinose ratio of PCA-AX-L and PCA-AX-K were 1.48:1.0 and 2.26:1.0, respectively. The phenolic acids content was higher in PCA-AX-K than PCA-AX-L determined by HPLC. FT-IR analysis confirms the presence of α-glucosidic linkage with the degree of substitution of xylan backbone by arabinose residues. The evaluation of immunomodulating effect of PCA-AXs revealed that the PCA-AX-L-treated cells showed higher release of NO, ROS and cytokines than PCA-AX-K-treated cells. The mRNA expressions of TNF-α, iNOS and COX-2 were upregulated by PCA-AX-L and downregulated by PCA-AX-K in dose-dependent manner. Furthermore, in western blotting, the ERK and NF-κB were found to be activated by PCA-AX-L and inhibited by PCA-AX-K. Our findings suggest that the high branched arabinoxylans of PCA-AX-L could modulate the immune response in RAW 264.7 cells through activation of ERK and NF-κB signaling pathways and acts as an immunostimulant. The higher phenolic content in PCA-AX-K could modulate the immune response by downregulation of ERK and NF-κB signaling pathways and thus, it could act as an immunomodulator. PRACTICAL APPLICATIONS: Millets are the richest source of arabinoxylans in which they are known to be bound with phenolic acids (PCA-AX). Arabinoxylans derived from rice and wheat is known immunomodulators. This study was focused to evaluate the immunomodulatory property of PCA-AX derived from two different millets little and kodo. The study results clearly indicated the immune stimulatory action of PCA-AX-L and immunomodulatory action of PCA-AX-K. The explored mechanism indicated that the PCA-AXs modulate NF-κB & ERK pathways for their immunomodulatory action.

Hydrolysable tannin-rich fraction from Terminalia chebula Retz. fruits ameliorates collagen-induced arthritis in BALB/c mice.

Terminalia chebula Retz. (Fam: Combretaceae) (TC) is widely used in traditional system for the treatment of fever, asthma, urinary diseases, and rheumatism. The aim of the present study is to evaluate the efficacy of hydrolysable tannin-rich fraction (HTF) isolated from TC fruits in collagen-induced arthritic BALB/c mice. Type II collagen-induced arthritis was attained in BALB/c mice. HTF treatment at three gradual doses (100, 200, and 400 mg/kg) was started from 24th day on daily administration up to 4 weeks. Body weight, food intake, and increase in hind paw were monitored at weekly basis. At the end of the study, serum and joint tissues were estimated for proinflammatory cytokines and biochemical parameters and the hind limbs were removed for radiological and histopathological evaluations. Oral administration of HTF could significantly protect CIA in BALB/c mice by reducing paw volume, arthritic score, spleen index, serum and joint cytokine level, and biochemical markers. The anti-inflammatory efficacy of HTF was further demonstrated by morphological observation, histopathological , and radiological evaluations. HTF is a good therapeutic agent for the treatment of joint inflammatory condition, as its action is mediated through cytokine inhibition.

Chemical characterization and immunostimulatory activity of phenolic acid bound arabinoxylans derived from foxtail and barnyard millets.

The chemical characterization and evaluation of immunostimulating effect of phenolic acid bound arabinoxylan (PA-AXs) isolated from barnyard (PA-AX-B) and foxtail (PA-AX-F) millets were performed. The sugar composition analysis and bound phenolic acids' (caffeic acid, p-coumaric acid, and ferulic acid) content of PA-AXs were examined by gas chromatography and mass spectroscopy (GC-MS) and high performance liquid chromatography (HPLC), respectively. The immunostimulatory activity of PA-AXs was evaluated by studying the effect of PA-AXs on the release of nitric oxide (NO), ROS, and cytokine (TNF-α, IL-1ß, and IL-6) in RAW 264.7 murine macrophage cells. The GC-MS results revealed the xylose: arabinose ratio of PA-AX-F and PA-AX-B as 1.96:1.0 and 1.64:1.0, respectively. In HPLC analysis, PA-AX-B showed higher phenolic acid content than PA-AX-F. In RAW 264.7 cells, immunostimulatory activity was established by its increased release of NO, ROS, and cytokine (TNF-α, IL-1ß, and IL-6) in a dose-dependent manner. Both PA-AX-B and PA-AX-F exhibited significant immunostimulation in in vitro studies. PRACTICAL APPLICATIONS: Millets are known for the higher content of phenolic acid bound arabinoxylans (PA-AX). The composition of PA-AX varies with different types of millets. In general, rice bran and wheat arabinoxylans are well reported to have significant immunostimulatory and antitumor properties. The bound ferulic acid with arabinoxylan isolated from finger millet bran also possesses immunostimulatory property. As the millets grains, foxtail and barnyard are also rich in PA-AXs, the present study was focused to evaluate the immunostimulatory property of PA-AX derived from two different millets. The study results indicated the immune stimulatory action of millet PA-AX's and thus the purified PA-AX can be explored further to identify the mechanism of action with respect to its immune stimulation property.

Repeated oral dose toxicity study on hydrolysable tannin rich fraction isolated from fruit pericarps of Terminalia chebula Retz in Wistar albino rats.

Terminalia chebula fruits are one of the richest sources of hydrolysable tannins and it is well known medicinal agent in traditional systems of medicine for treatment of various chronic ailments. In the present study, hydrolysable tannin rich fraction (HTF) was isolated from 80% hydroalcoholic extract of Terminalia chebula fruit pericarps and it was studied for acute and repeated dose oral toxicity in Wistar albino rats. HTF did not show any toxic symptoms or mortality at single dose administration of 5000 mg/kg/p.o followed by observation for 14 days. On repeated dose 28 days oral toxicity study, administration of HTF at 1000 mg/kg showed marked reduction in body weight, food intake and water intake when compared with vehicle control. It was also observed that HTF could increase serum urea, glucose and AST level significantly when compared with vehicle control indicating mild disturbances in liver and kidney functions. On histopathological screening, HTF treatment showed a mild granulomatous inflammation in the liver and all other organs remained normal. It was concluded that following 28 days repeated dose oral administration, HTF caused mild disturbances in liver and kidney function which was indicated by reduced body weight, food and water intake, serum parameters and histological observations.

Anti-inflammatory effect of Naravelia zeylanica DC via suppression of inflammatory mediators in carrageenan-induced abdominal oedema in zebrafish model.

The traditional herbal medicines are receiving great importance in the health care sector, especially in Indian system of medicine, i.e, Ayurveda. The present study focused on the standardization of Naravelia zeylanica (L.) DC in terms of its active phytochemicals and to evaluate the anti-inflammatory activity of ethanol extract of N. zeylanica (ENZ). An analytical method was developed by high-performance liquid chromatography for simultaneous determination of ß-sitosterol, lupeol and oleanolic acid in ENZ. The cell viability of ENZ was investigated using MTT assay. IC50 value of ENZ on cell viability was found to be 653.01 µg/mL. To determine the anti-inflammatory activity of ENZ by in vitro method, LPS was added to the macrophage cells to induce activation and ENZ was further added to observe the recovery of inflamed cells. These cells when treated with ENZ, the percentage of viable cells were considerably increased to 74.68%. Loss of mitochondrial membrane potential on treatment with LPS and its recovery by ENZ was studied and found that the number of cells that were damaged on treatment with ENZ + LPS was comparatively lesser than treatment with LPS only. An in vivo anti-inflammatory study was carried out in carrageenan-induced abdominal oedema method in adult zebrafish which revealed the percentage inhibition of inflammation at graded dose levels of ENZ as 23.5% at 100 mg/kg, 62.4% at 200 mg/kg and 87.05% at 350 mg/kg when compared with standard of diclofenac which showed 85% inhibition at 100 mg/kg. The PCR amplification of DNA extracted from adult zebrafish showed that increased concentration of ENZ considerably downregulates the expression of TNF-α and iNOS, the mediators of inflammation.

In vivo antiarthritic activity of the ethanol extracts of stem bark and seeds of Calophyllum inophyllum in Freund's complete adjuvant induced arthritis.

CONTEXT: Calophyllum inophyllum Linn. (Clusiaceae) (CI) is traditionally used to treat pain, inflammation, eye disorders and rheumatism. OBJECTIVE: The present study evaluates the antiarthritic activity of the ethanol extract of the stem bark (ESBCI) and seeds (ESCI) of Calophyllum inophyllum in Freund's adjuvant induced arthritic Wistar albino rat model. MATERIALS AND METHODS: ESBCI and ESCI were screened for in vitro anti-inflammatory activity by proteinase inhibition and membrane stabilization assays. Acute oral toxicity studies were conducted according to OECD-425 guidelines. Antiarthritic activity of ESBCI and ESCI at the dose of 250 mg/kg/p.o. was evaluated by Freund's adjuvant induced arthritic rat model. RESULTS: ESBCI and ESCI have shown maximum inhibition at 250 µg/mL in proteinase inhibition and haemolysis assays. The LD50 of ESBCI and ESCI was found to be greater than 5000 and 2000 mg/kg/p.o., respectively. In Freund's adjuvant induced arthritic rat model ESBCI, ESCI and Diclofenac treatment have shown 28.57, 36.36, and 43.51% as maximum reduction in rat paw oedema volume respectively when compared with the arthritic control rats. ESBCI and ESCI treatment at the dose level of 250 mg/kg/p.o. normalized the altered haematological and biochemical parameters of arthritic control rats. Histological and radiological evaluation confirmed the antiarthritic effect of ESBCI and ESCI. DISCUSSION: ESBCI and ESCI were found to show significant antiarthritic activity evidenced with clinical, biochemical, histological and radiological evaluations. CONCLUSION: The present study indicates the antiarthritic activity of ESBCI and ESCI, however its mechanism of action has to be studied in the future.

Antibacterial synergy between rosmarinic acid and antibiotics against methicillin-resistant Staphylococcus aureus.

AIM/BACKGROUND: Medicinal plants have ability to resist microorganisms by synthesizing secondary metabolites such as phenols. Rosmarinic acid (RA) is a phenylpropanoid widely distributed in plants and well known as therapeutic and cosmetic agent. Methicillin-resistant Staphylococcus aureus (MRSA) which is resistant to all kinds of ß-lactams, threatens even most potent antibiotics. To improve the efficiency of antibiotics against multi-drug resistant bacteria and to reduce the antibiotic dose, the antibacterial activity and the synergistic effect of RA with standard antibiotics against S. aureus and MRSA was investigated. MATERIALS AND METHODS: Antibacterial activity of RA against S. aureus and a clinical isolate of MRSA was evaluated by agar well diffusion method. Minimum inhibitory concentration (MIC) of RA was determined by broth dilution method. Synergism of RA with various antibiotics against S. aureus and MRSA was studied by broth checkerboard method and time-kill kinetic assay. Effect of RA on microbial surface components recognizing adhesive matrix molecules (MSCRAMM's) of S. aureus and MRSA was studied using sodium dodecyl sulfate - polyacrylamide gel electrophoresis. RESULTS: MIC of RA was found to be 0.8 and 10 mg/ml against S. aureus and MRSA, respectively. RA was synergistic with vancomycin, ofloxacin, and amoxicillin against S. aureus and only with vancomycin against MRSA. The time-kill analysis revealed that synergistic combinations were a more effective than individual antibiotics. MSCRAMM's protein expression of S. aureus and MRSA was markedly suppressed by RA + vancomycin combination rather than RA alone. CONCLUSION: The synergistic effects of RA with antibiotics were observed against S. aureus and MRSA. RA showed inhibitory effect on the surface proteins MSCRAMM's. Even though RA was shown to exhibit a synergistic effect with antibiotics, the MIC was found to be higher. Thus, further studies on increasing the efficacy of RA can develop it as an adjuvant for antibiotics.

Scope of Hydrolysable Tannins as Possible Antimicrobial Agent.

Hydrolysable tannins (HTs) are secondary metabolites from plants, which are roughly classified into gallotannins and ellagitannins having gallic acid and ellagic acid residues respectively attached to the hydroxyl group of glucose by ester linkage. The presence of hexahydroxydiphenoyl and nonahydroxyterphenoyl moieties is considered to render antimicrobial property to HTs. HTs also show considerable synergy with antibiotics. Nevertheless, they have low pharmacokinetic property. The present review presents the scope of HTs as future antimicrobial agent. Copyright © 2016 John Wiley & Sons, Ltd.

Decolorization and biodegradation of remazol reactive dyes by Clostridium species.

Decolorisation and biodegradation efficacy of potential strains isolated from dyeing effluent collected from Tirupur region, Tamil Nadu, India were studied in remazol reactive dyes. Two potential strains Clostridium butyricum (EI05) and Clostridium acetobutylicum (EI25) identified by biochemical tests in our previous study were studied for their decolorising efficiency on various remazol reactive dyes (Remazol Blue RGB, Remazol Blue RR, Remazol Navy RGB and Remazol Orange RR). The synthetic dyes showed complete decolorization after 24-72 h by two potential strains EI05 and EI25. Clostridium acetobutylicum (EI25) was found to be the most potential strain isolated. The spectral analysis was performed by UV-Visible spectroscopy and FT-IR spectroscopy to study biodegradation. The peak disappearance in UV spectrum, peak shifts and disappearance in FTIR spectrum of treated samples indicated biodegradation. Thus Clostridium species could able to decolorize the remazol reactive dyes.

Analytical method development and validation of simultaneous estimation of rabeprazole, pantoprazole, and itopride by reverse-phase high-performance liquid chromatography.

A simple, selective, rapid, and precise reverse-phase high-performance liquid chromatography (RP-HPLC) method for the simultaneous estimation of rabeprazole (RP), pantoprazole (PP), and itopride (IP) has been developed. The compounds were well separated on a Phenomenex C18 (Luna) column (250 mm × 4.6 mm, dp = 5 µm) with C18 guard column (4 mm × 3 mm × 5 µm) with a mobile phase consisting of buffer containing 10 mM potassium dihydrogen orthophosphate (adjusted to pH 6.8): acetonitrile (70:30 v/v) at a flow rate of 1.0 mL/min and ultraviolet detection at 288 nm. The retention time of RP, PP, and IP were 5.35, 7.92, and 11.16 minutes, respectively. Validation of the proposed method was carried out according to International Conference on Harmonisation (ICH) guidelines. Linearity range was obtained for RP, PP, and IP over the concentration range of 2.5-25, 1-30, and 3-35 µg/mL and the r2 values were 0.994, 0.978, and 0.991, respectively. The calculated limit of detection (LOD) values were 1, 0.3, and 1 µg/mL and limit of quantitation (LOQ) values were 2.5, 1, and 3 µg/mL for RP, PP, and IP correspondingly. Thus, the current study showed that the developed reverse-phase liquid chromatography method is sensitive and selective for the estimation of RP, PP, and IP in combined dosage form.

Evaluation of antiarthritic activity of Strychnos potatorum Linn seeds in Freund's adjuvant induced arthritic rat model.

BACKGROUND: Strychnos potatorum Linn (Loganiaceae) is a moderate sized tree found in southern and central parts of India, Sri Lanka and Burma. In traditional system of medicine, Strychnos potatorum Linn seeds were used for various ailments including inflammation, diabetes etc. To investigate the folkloric use of the seeds the present study was carried out on Freund's adjuvant induced arthritic rats. METHODS: The present study states the effect of the aqueous extract (SPE) and the whole seed powder (SPP) of Strychnos potatorum Linn seeds on the Freund's complete adjuvant (FCA) induced arthritic rat paw edema, body weight changes and alterations in haematological and biochemical parameters in both developing and developed phases of arthritis. Histopathology of proximal interphalangeal joints and radiology of hind legs were studied. RESULTS: In FCA induced arthritic rats, there was significant increase in rat paw volume and decrease in body weight increment, whereas SPP and SPE treated groups, showed significant reduction in paw volume and normal gain in body weight. The altered haematological parameters (Hb, RBC, WBC and ESR) and biochemical parameters (blood urea, serum creatinine, total proteins and acute phase proteins) in the arthritic rats were significantly brought back to near normal by the SPP and SPE treatment at the dose of 200 mg/kg/p.o in both developing and developed phases of arthritis. Further the histopathological and radiological studies revealed the antiarthritic activity of SPP and SPE by indicating fewer abnormalities in these groups when compared to the arthritic control group. CONCLUSION: In conclusion, both SPP and SPE at the specified dose level of 200 mg/kg, p.o. showed reduction in rat paw edema volume and it could significantly normalize the haematological and biochemical abnormalities in adjuvant induced arthritic rats in both developing and developed phases of FCA induced arthritis. Further the histopathological and radiological studies confirmed the antiarthritic activity of SPP and SPE.