N-(2-hydroxy phenyl) acetamide ameliorate inflammation and doxorubicin-induced nephrotoxicity in rats.

Doxorubicin (DOX) is an anthracyclin antibiotic used for the treatment of various cancers. Nephrotoxicity is among the serious side effects of DOX, therefore, DOX-induced nephrotoxic model has been widely used to study nephropathies. The objectives of this study is to investigate the possible anti-inflammatory and nephroprotective effects of salicylic acid derivative, N-(2-hydroxy phenyl) acetamide (NA-2), in a rat model of DOX-induced nephrotoxicity. The in vitro anti-inflammatory potential of NA-2 was manifested by whole blood oxidative burst and nitric oxide (NO) assays with no toxicity on normal human fibroblast (BJ) cells, human embryonic kidney (HEK-293) cells, and normal monkey kidney epithelial (Vero) cells. The in vivo study included five groups: Normal control, DOX (6 mg/kg DOX-i.v.via tail vein), NA-2 treated control-i.p., NA-2/DOX treated-i.p., and prednisolone/DOX treated. After 7 days of DOX administration, rats with urinary protein level of >50 mg/kg/day were selected. Treatment group rats received i.p. doses of NA-2 (10 mg/kg/day) for 3 weeks with weekly monitoring of urinary protein excretion and body weights. mRNA expression of interleukin (IL)-1ß, IL-6, tumor necrosis factor (TNF)-α, monocyte chemoattractant protein (MCP)-1, and kidney injury molecule (KIM)-1 was analyzed by quantitative polymerase chain reaction (qPCR). Protein expressions were analyzed by immunohistochemistry. NA-2 attenuated DOX-induced changes in serum and urine levels, and improved inflammatory profile of the renal tissue. Histopathological findings revealed protective effects of NA-2 showing lesser lesions. We conclude that NA-2 is able to protect against DOX-induced renal damage functionally, biochemically and histopathologically with corresponding improvement in the kidney inflammatory profile.

In-vitro inhibition of NLRP3 inflammasome by 3,6-dihydroxyflavone (3,6-DHF): a therapeutic strategy for the treatment of chronic inflammatory and autoimmune diseases.

Nucleotide-binding oligomerization domain (NOD)-like receptor family pyrin domain containing 3 (NLRP3) inflammasome complex has an important role in immune system and its abnormal activation is associated with the pathogenesis of various inflammatory and auto-immune diseases. The study reveals the anti-inflammatory effects of 3,6-dihydroxyflavone (3,6-DHF). Here, we aimed to determine the inhibitory effects of 3,6-DHF on NLRP3 inflammasome and its associated components, thereby determining the signaling pathways involved in the inhibition. Reactive oxygen species (ROS) and nitric oxide (NO) were quantified by chemiluminescence and Griess methods, respectively. Inflammatory cell model was induced in human leukemic monocytes (THP-1). mRNA levels were estimated through real-time RT-PCR, protein expressions were evaluated by protein slot blot and immunocytochemistry, MTT and alamar blue assays were employed for toxicity studies. The compound 3,6-DHF was found to be the potent inhibitor of NLRP3 inflammasome by targeting the molecules involve in its activation pathway. Anti-inflammatory effects were revealed by inhibition of ROS and NO, reduction in the transcription of caspase-1, ASC, IL-1ß and TLR-4 was observed along with the marked inhibition of NLRP3, IL-18, NF-κB and pNF-κB at translational level. 3,6-DHF was non-toxic on normal human fibroblast (BJ) and THP-1 cells and, could be a potential therapeutic agent in NLRP3 inflammasome driven diseases.

Design, Synthesis and Characterization of [G10a]-Temporin SHa Dendrimers as Dual Inhibitors of Cancer and Pathogenic Microbes.

As the technologies for peptide synthesis and development continue to mature, antimicrobial peptides (AMPs) are being widely studied as significant contributors in medicinal chemistry research. Furthermore, the advancement in the synthesis of dendrimers' design makes dendrimers wonderful nanostructures with distinguishing properties. This study foregrounds a temporin SHa analog, [G10a]-SHa, and its dendrimers as globular macromolecules possessing anticancer and antibacterial activities. These architectures of temporin SHa, named as [G10a]-SHa, its dendrimeric analogs [G10a]2-SHa and [G10a]3-SHa, and [G10a]2-SHa conjugated with a polymer molecule, i.e., Jeff-[G10a]2-SHa, were synthesized, purified on RP-HPLC and UPLC and fully characterized by mass, NMR spectroscopic techniques, circular dichroism, ultraviolet, infrared, dynamic light scattering, and atomic force microscopic studies. In pH- and temperature-dependent studies, all of the peptide dendrimers were found to be stable in the temperature range up to 40-60 °C and pH values in the range of 6-12. Biological-activity studies showed these peptide dendrimers possessed improved antibacterial activity against different strains of both Gram-positive and Gram-negative strains. Together, these dendrimers also possessed potent selective antiproliferative activity against human cancer cells originating from different organs (breast, lung, prostate, pancreas, and liver). The high hemolytic activity of [G10a]2-SHa and [G10a]3-SHa dendrimers, however, limits their use for topical treatment, such as in the case of skin infection. On the contrary, the antibacterial and anticancer activities of Jeff-[G10a]2-SHa, associated with its low hemolytic action, make it potentially suitable for systemic treatment.

Serum Stable and Low Hemolytic Temporin-SHa Peptide Analogs Disrupt Cell Membrane of Methicillin-Resistant Staphylococcus aureus (MRSA).

Anti-microbial peptides (AMPs) have attracted major attention due to their potential bio-activities against some multidrug resistant pathogens. The present study evaluated the mechanism of actions of highly potent AMP temporin-SHa analogs, i.e., [G4a]-SHa, [G7a]-SHa, and [G10a]-SHa, against methicillin-resistant Staphylococcus aureus (MRSA) NCTC (13277) with minimum inhibitory concentrations (MICs) of 14.35, 7.16, and 3.58 µM, respectively. These analogs exhibited significant anti-MRSA activity at physiological salt concentration, 30% fetal bovine serum, and 30% human serum. [G4a]-SHa and [G7a]-SHa were non-hemolytic and non-cytotoxic to normal mouse fibroblast 3T3 cell and human Caco-2 cell line. Atomic force microscopy revealed that these analogs have profound effect on the morphological changes in MRSA surface with significant leakage of cell cytoplasmic content. Propidium iodide uptake kinetic assay and (bis-(1,3-dibutylbarbituric acid) trimethine oxonol) DiBAC4(3) membrane depolarization assay demonstrated that these analogs display a membrane disrupting property, characterized by elevation of plasma membrane permeability and rapid transmembrane potential depolarization. [G10a]-SHa showed a significant anti-biofilm activity against biofilm forming S. aureus (ATCC 6538). Acute in vivo toxicity studies revealed that [G10a]-SHa possesses some toxic effect at 100-mg/kg dose. While [G4a]-SHa at 100 mg/kg, i.p. has no toxic effect even after 48 h, [G7a]-SHa also did not show any toxic effect at the dose of 100 mg/kg, i.p. during 24-h observation of animals. In conclusion, [G4a]-SHa, [G7a]-SHa, and [G10a]-SHa show improved activity against MRSA and stability compared to SHa peptide. Although highly potent, [G10a]-SHa, due to its hemolytic activity, might be more suitable for topical application, whereas [G4a]-SHa and [G7a]-SHa have potential to be used for systemic application.

Antimicrobial and biofilm inhibiting potential of an amide derivative [N-(2', 4'-dinitrophenyl)-3ß-hydroxyurs-12-en-28-carbonamide] of ursolic acid by modulating membrane potential and quorum sensing against colistin resistant Acinetobacter baumannii.

Acinetobacter baumannii is Gram-negative, an opportunistic pathogen responsible for life-threatening ventilator-associated pneumonia. World Health Organization (WHO) enlisted it as a priority pathogen for which therapeutic options need speculations. Biofilm further benefits this pathogen and aids 100-1000 folds more resistant against antimicrobials and the host immune system. In this study, ursolic acid (1) and its amide derivatives (2-4) explored for their antimicrobial and antibiofilm potential against colistin-resistant A. baumannii (CRAB) reference and clinical strains. Viability, crystal violet, microscopic, and gene expression assays further detailed the active compounds' antimicrobial and biofilm inhibition potential. Compound 4 [N-(2',4'-dinitrophenyl)-3ß-hydroxyurs-12-en-28-carbonamide)], a synthetic amide derivate of ursolic acid significantly inhibits bacterial growth with MIC in the range of 78-156 µg/mL against CRAB isolates. This compound failed to completely kill the CRAB isolates even at 500 µg/mL concentration, suggesting the compound's anti-virulence and bacteriostatic nature. Short and prolonged exposure of 4 inhibited or delayed the bacterial growth at sub MIC, MIC, and 2× MIC, as evident in time-kill and post-antibacterial assay. It significantly inhibited and eradicated >70% of biofilm formation at MIC and sub MIC levels compared to colistin required in high concentrations. Microscopic analysis showed disintegrated biofilm after treatment with the 4 further strengthened its antibiofilm potential. Atomic force microscopy (AFM) hinted the membrane disrupting effect of 4 at MIC's. Further it was confirmed by DiBAC4 using fluorescence-activating cells sorting (FACS), suggesting a depolarized membrane at MIC. Gene expression analysis also supported our data as it showed reduced expression of biofilm-forming (bap) and quorum sensing (abaR) genes after treatment with sub MIC of 4. The results suggest that 4 significantly inhibit bacterial growth and biofilm mode of colistin-resistant A. baumannii. Thus, further studies are required to decipher the complete mechanism of action to develop 4 as a new pharmacophore against A. baumannii.

Novel Mannich base 3FB3FA8H induces apoptosis by upregulating P53 pathway in neuroblastoma cells.

P53 plays an important role in maintaining genetic stability and development of resistance against tumors. Dysregulation of P53 gene is one of the key factors contributing to the etiology of neuroblastoma which causes cells to evade apoptosis. Activating P53 pathway can be a therapeutic alternative to the currently available medicinal strategies. Mannich bases have been known to possess various biological activities including the anticancer activity. In this study, we have targeted the P53 pathway by novel Mannich base (3FB3FA8H) which can be a future prospect to cure neuroblastoma. 3FB3FA8H has shown modulation of P53 pathway leading to apoptosis of neuroblastoma cells. Mitochondrial membrane permeability is also increased by 3FB3FA8H which may be a consequence of P53 pathway modulation. 3FB3FA8H increases the mRNA levels of P53 leading to activation of BAX. Inclining BAX/BCL2 ratio towards apoptotic BAX leads to cleavage of caspase 3, ultimately, causing apoptosis. Series of experiments provide the evidence that Mannich base 3FB3FA8H leads to P53-mediated apoptosis. Inducing apoptosis by this mechanism could be of central importance in reducing tumor burden which can be a good prospect for neuroblastoma patients.

Antiproliferative and apoptotic effects of proteins from black seeds (Nigella sativa) on human breast MCF-7 cancer cell line.

BACKGROUND: Nigella sativa (NS), a member of family Ranunculaceae is commonly known as black seed or kalonji. It has been well studied for its therapeutic role in various diseases, particularly cancer. Literature is full of bioactive compounds from NS seed. However, fewer studies have been reported on the pharmacological activity of proteins. The current study was designed to evaluate the anticancer property of NS seed proteins on the MCF-7 cell line. METHODS: NS seed extract was prepared in phosphate-buffered saline (PBS), and proteins were precipitated using 80% ammonium sulfate. The crude seed proteins were partially purified using gel filtration chromatography, and peaks were resolved by SDS-PAGE. MTT assay was used to screen the crude proteins and peaks for their cytotoxic effects on MCF-7 cell line. Active Peaks (P1 and P4) were further studied for their role in modulating the expression of genes associated with apoptosis by real-time reverse transcription PCR. For protein identification, proteins were digested, separated, and analyzed with LC-MS/MS. Data analysis was performed using online Mascot, ExPASy ProtParam, and UniProt Knowledgebase (UniProtKB) gene ontology (GO) bioinformatics tools. RESULTS: Gel filtration chromatography separated seed proteins into seven peaks, and SDS-PAGE profile revealed the presence of multiple protein bands. Among all test samples, P1 and P4 depicted potent dose-dependent inhibitory effect on MCF-7 cells exhibiting IC50 values of 14.25 ± 0.84 and 8.05 ± 0.22 µg/ml, respectively. Gene expression analysis demonstrated apoptosis as a possible cell killing mechanism. A total of 11 and 24 proteins were identified in P1 and P4, respectively. The majority of the proteins identified are located in the cytosol, associate with biological metabolic processes, and their molecular functions are binding and catalysis. Hydropathicity values were mostly in the hydrophilic range. CONCLUSION: Our findings suggest NS seed proteins as a potential therapeutic agent for cancer. To our knowledge, it is the first study to report the anticancer property of NS seed proteins.

Quercetin modulates iron homeostasis and iNOS expression of splenic macrophages in a rat model of iron deficiency anemia.

Iron deficiency anemia is one of the most common micronutrient deficient conditions around the globe with various consequences, including the weakened immune system. Quercetin is widely distributed bioflavonoid; it has been debated for its dual roles in iron regulation. Quercetin-iron interaction in the body is a complex mechanism which has not been completely understood. The present study aimed to investigate the effect of quercetin on iron supplementation in iron deficiency anemia and on iNOS expression in splenic macrophages. The rat model of iron deficiency anemia was induced by feeding low iron diet to weanling rats for 20 days. The animals were then administered with ferrous sulfate, quercetin, and their combination for 30 days. Blood parameters, histopathological analysis, iron storage, CD68, iNOS and SLC40 expression in rat spleen were investigated. Our results showed that quercetin regulated iron absorption, despite SLC40 down-expression, indicating possible alternate route of iron transport, and that quercetin modulated iNOS production in splenic macrophages.

Synthesis of breast cancer targeting conjugate of temporin-SHa analog and its effect on pro- and anti-apoptotic protein expression in MCF-7 cells.

The frog natural product temporin-SHa (FLSGIVGMLGKLFamide) is a potent antimicrobial peptide, as is the analog [S3K]SHa. By solid-phase synthesis, we prepared temporin-SHa and several temporin-SHa analogs with one or more D-alanine residues incorporated. The natural product and the analog [G10a]SHa were found to be cytotoxic in mammalian cell lines and induce cell death. To achieve selectivity, we conjugated the analog [G10a]SHa with a breast cancer targeting peptide (BCTP). The resulting peptide temporin [G10a]SHa-BCTP conjugate was selectively active against the MCF-7 breast cancer cell line with no cytotoxicity in NIH-3T3 fibroblasts. Unlike the natural product or [G10a]SHa, the conjugated peptide induced apoptosis, downregulating the expression of Bcl-2 and survivin and upregulating Bax and caspase-3.

Verapamil potentiates anti-glioblastoma efficacy of temozolomide by modulating apoptotic signaling.

Glioblastoma Multiforme (GBM) is the most malignant and invasive tumor of the CNS. Although temozolomide (TMZ) has improved the survival, long-lasting responses have not been reported. Therefore, there is a need to develop improved treatments, one of which might be newly identified drugs which can be used in combination therapy with low doses of standard drugs. Verapamil (VP) a known antihypertensive drug has been shown to enhance the activity of bis-chloroethylnitrosourea (BCNU), a drug used to treat GBM. Since, TMZ has replaced BCNU as the standard GBM chemotherapy; therefore, we aimed to study in vitro interaction of VP and TMZ against GBM. Anti-proliferative and apoptotic activities were studied using MTT, TUNEL assay and DAPI staining. Synergy was assessed using combination index method. Apoptotic markers were evaluated by RT-PCR, and immunocytochemistry. Both VP and TMZ significantly inhibited the growth of U87 cells in dose dependent manner. The combine effect of TMZ with VP was synergistic with a CDI value of <1. Combination of TMZ and VP increased the ratio of Bax to Bcl-2 expression and thus shifted the equilibrium of cells towards apoptosis. Our findings suggest that the synergistic growth inhibition that was observed in combination treatment group may in part relate to increase in apoptosis. The combine administration of VP and TMZ may be therapeutically exploited for the management of GBM.

Rutin and rutin-conjugated gold nanoparticles ameliorate collagen-induced arthritis in rats through inhibition of NF-κB and iNOS activation.

Numerous studies have suggested that nuclear factor-κB (NF-κB) and inducible nitric oxide synthase (iNOS) are important mediators of inflammatory response in human and animal models of arthritis. Besides, oxidative stress markers, nitric oxide (NO) and peroxide (PO) are also major contributors in the pathogenesis of rheumatoid arthritis (RA). Over expression of these inflammatory mediators leads to the extracellular matrix degradation, and excessive cartilage and bone resorption, ultimately leading to the irreversible damage to joints. The aim of the present study was to investigate the anti-arthritic mechanism of bioflavonoids, rutin and rutin-conjugated gold nanoparticles (R-AuNPs) by determining their role in the modulation of NF-κB and iNOS expression in collagen-induced arthritis (CIA) model of rats. Arthritis was induced by the subcutaneous administration of bovine type II collagen. Treatment was started with rutin, indomethacin + rutin (I + R) and R-AuNPs on the day of CIA induction. The severity of arthritis was determined by measuring the arthritic score on alternate days until mean arthritic score of 4 was observed. The NO and PO levels were also analyzed in serum samples. NF-κB and iNOS expression levels were determined in spleen tissue samples by real time RT-PCR and immunohistochemistry. Marked reduction in the arthritic score as well as in the NO and PO levels was observed in the treated groups. A significant downregulation in the NF-κB and iNOS expression levels was also observed in the treatment groups compared to the arthritic control group. Collectively, the findings suggest potential clinical role of rutin and R-AuNPs in the treatment of rheumatoid arthritis.

A novel, semi-synthetic diterpenoid 16(R and S)-phenylamino-cleroda-3,13(14), Z-dien-15,16 olide (PGEA-AN) inhibits the growth and cell survival of human neuroblastoma cell line SH-SY5Y by modulating P53 pathway.

Neuroblastoma being the most common extracranial pediatric solid tumor accounts for 15% of overall cancer-related childhood mortalities. Resistance to chemotherapeutic drugs is one of the limiting factors for positive prognosis for neuroblastoma. Therefore, there is always a need for developing new therapeutic moieties which can become a future prospect of neuroblastoma therapy. Terpenoids being the largest natural compounds have demonstrated many biological activities including anticancer activity. Keeping in mind the role of terpenoids in biological system, we aimed to identify novel semi-synthetic terpenoid derived from cleroda diterpene, 16-oxo-cleroda-3,13(14)E-diene-15-oic acid (1) as a potential anticancer moiety against neuroblastoma. We choose γ-amino γ-lactone (PGEA-AN, 2) of 1 to study further because it exhibited the most potent cytotoxic activity in preliminary screening. In comparison to cisplatin, PGEA-AN significantly decreased the nuclear area factor which suggest the potential apoptosis as cause of cell death. PGEA-AN demonstrated a significant increase in the percent of late apoptosis and necrotic cell death at 48-h treatment with IC50 dose. PGEA-AN significantly increased expression of P53 and BAX with no or little effect on BCL2 shifting BAX/BCL2 towards BAX promoting apoptosis. Increment in mitochondrial permeability supports P53 pathway involvement. Despite similarity in actions with cisplatin, PGEA-AN has found to have no effect on renal system. Based on these observations, we suggest that PGEA-AN modulates P53 system which further leads to the death of the neuroblastoma cells with no effect on renal system in vivo owing it to be a future prospect for development of anticancer moiety against neuroblastoma.

N-(2-Hydroxyphenyl)acetamide: a Novel Suppressor of RANK/RANKL Pathway in Collagen-Induced Arthritis Model in Rats.

RANKL and RANK are potential contributors of inflammatory cascade in human and animal model of arthritis. The current study aims to investigate the effect of N-(2-hydroxyphenyl)acetamide (NA-2) on regulation of RANKL pathway in collagen-induced arthritis (CIA) model in rats. CIA was induced using bovine type II collagen in female Wistar rats. The clinical parameters, level of pro-inflammatory and oxidative stress markers were measured to determine the progression of the disease. The mRNA level of RANKL and RANK and downstream mediators of inflammation i.e. c-fos, c-jun, NF-κB and Akt were analysed in spleen tissue using real-time PCR. Immunohistochemical analysis of iNOS, pAkt and c-Fos was also done in spleen tissue. Treatment with NA-2 and indomethacin showed increase in body weight and significant reduction in paw volume and arthritic score (p < 0.0001). Marked reduction in the level of oxidative stress markers, NO, PO and GSH (p < 0.0001), and pro-inflammatory markers, IL-1ß (p < 0.0001) and TNF-α (p < 0.01), was also observed. Likewise, NA-2 and indomethacin treatment also significantly suppressed the mRNA expression of RANKL, RANK, c-fos, c-jun, NF-κB (p < 0.0001) and Akt (p < 0.01) and protein expression of iNOS, pAkt and c-Fos (p < 0.0001) compared to the arthritic control group. Our findings suggest that NA-2 is an antiarthritic agent acting in a pleiotropic manner in CIA rats by not only reducing the clinical signs of arthritis, inflammatory cytokines and free radical production but also attenuating the RANK/RANKL signaling pathway.

Glioblastoma Multiforme: A Review of its Epidemiology and Pathogenesis through Clinical Presentation and Treatment

Glioblastoma multiforme (GBM) is one of the most malignant types of central nervous system tumors. Despite advances in treatment modalities it remains largely incurable. The objective of our review is to provide a holistic picture of GBM epidemiology, etiology, pathogenesis, clinical findings and treatment. A literature search was conducted for GBM at PubMed and Google Scholar, with relevant key words like glioblastoma multiforme, pathogenesis, signs and symptoms, treatment etc., and papers published until 2015 were reviewed. It was found that radiation and certain genetic syndromes are the only risk factors identified to date for GBM. Depending on the tumor site patients may present to the clinic with varying symptoms. To confirm the presence and the extent of tumor, various invasive and non-invasive imaging techniques require employment. The literature survey revealed the pathogenesis to involve aberrations of multiple signaling pathways through multiple genetic mutations and altered gene expression. Although several treatment options are available, including surgery, along with adjuvant chemo- and radio-therapy, the disease has a poor prognosis and patients generally succumb within 14 months of diagnosis.

Comparative studies on the effects of clinically used anticonvulsants on the oxidative stress biomarkers in pentylenetetrazole-induced kindling model of epileptogenesis in mice.

BACKGROUND: Oxidative stress plays a key role in the pathogenesis of epilepsy and contributes in underlying epileptogenesis process. Anticonvulsant drugs targeting the oxidative stress domain of epileptogenesis may provide better control of seizure. The present study was carried out to investigate the effect of clinically used anti-epileptic drugs (AEDs) on the course of pentylenetetrazole (PTZ)-induced kindling and oxidative stress markers in mice. METHODS: Six mechanistically heterogeneous anticonvulsants: phenobarbital, phenytoin, levetiracetam, pregabalin, topiramate, and felbamate were selected and their redox profiles were determined. Diazepam was used as a drug control for comparison. Kindling was induced by repeated injections of a sub-convulsive dose of PTZ (50 mg/kg, s.c.) on alternate days until seizure score 5 was evoked in the control kindled group. Anticonvulsants were administered daily. Following PTZ kindling, oxidative stress biomarkers were assessed in homogenized whole brain samples and estimated for the levels of nitric oxide, peroxide, malondialdehyde, protein carbonyl, reduced glutathione, and activities of nitric oxide synthase and superoxide dismutase. RESULTS: Biochemical analysis revealed a significant increase in the levels of reactive oxygen species with a parallel decrease in endogenous anti-oxidants in PTZ-kindled control animals. Daily treatment with levetiracetam and felbamate significantly decreased the PTZ-induced seizure score as well as the levels of nitric oxide (p<0.001), nitric oxide synthase activity (p<0.05), peroxide levels (p<0.05), and malondialdehyde (p<0.05). Levetiracetam and felbamate significantly decreased lipid and protein peroxidation whereas topiramate was found to reduce lipid peroxidation only. CONCLUSIONS: An AED that produces anticonvulsant effect by the diversified mechanism of action such as levetiracetam, felbamate, and topiramate exhibited superior anti-oxidative stress activity in addition to their anticonvulsant activity.

Anti-TNF-α and anti-arthritic effect of patuletin: A rare flavonoid from Tagetes patula.

Rheumatoid arthritis (RA) poses a serious health problem as a chronic autoimmune joint disease with significant mortality and morbidity. Proinflammatory cytokines TNF-α and IL-1ß, reactive oxygen species (ROS), and activated CD4(+) T-cells play key roles in the progression of arthritis. The aim of the study is to evaluate the in vitro and in vivo immunomodulatory and anti-arthritic effect of flavonoid patuletin, isolated from Tagetes patula. ELISA was applied for quantification of TNF-α and IL-1ß. Intracellular and extracellular ROS production from phagocytes was measured by the chemiluminescence technique. Proliferation of T-cells was observed using a liquid scintillation counter. Cytotoxicity was assessed by a MTT assay. The serological and histological analysis studies were performed using a rodent model of adjuvant-induced arthritis (AIA). Expression of p38 and NF-κB after treatment of compound was observed by western blotting. Patuletin showed potent inhibitory effects on TNF-α in vitro as well as inhibited the production of both cytokines in vivo. It also showed potent suppression of proliferation of T-cells and significantly inhibited the extracellular and intracellular ROS production. Patuletin revealed significant anti-inflammatory and anti-arthritic activities in the rodent model of adjuvant-induced arthritis (AIA). Histologically, it causes mild bone destruction compared to the arthritic control group, thus representing its anti-arthritic potential. Based on these studies, patuletin could be considered as a potential immunosuppressive and anti-arthritic lead candidate.

N-(2-hydroxy phenyl) acetamide produces profound inhibition of c-Fos protein and mRNA expression in the brain of adjuvant-induced arthritic rats.

Chronic pain and cognitive decline are characteristic symptoms of rheumatoid arthritis. One of the immediate early gene c-fos is overexpressed during peripheral and central noxious conditions and can be used as a marker for neuronal activity/excitability. In the adjuvant-induced arthritis Sprague-Dawley rat model, we examined the dynamics of c-Fos protein and mRNA expression in the amygdala, cortex, hippocampus, and thalamus and evaluated the effects of N-(2-hydroxy phenyl) acetamide (NA-2), a derivative of salicylic acid. The paw volume was assessed as an indicator of peripheral edema and the hyperalgesia associated with arthritis was monitored by gait analysis. The region of interests of the brain from arthritic and non-arthritic animals were used to isolate the RNA and were then reverse transcribed into cDNA. The PCR products were electrophoresed on 1% agarose gel and the gels were visualized in gel-doc system. The frozen brain sections were stained for c-Fos using immunohistochemistry. Negative control experiments were performed without the primary and secondary antibodies to rule out the nonspecific tissue binding of antibodies. We report a significant increase in the c-Fos expression in the arthritic control animals. In comparison to the control group, the treatment of NA-2 treatment was found to block the development of the arthritis-induced c-Fos protein and mRNA expression and peripheral edema. It also significantly reduces the gait deficits which were otherwise observed in the arthritic control group. Both the immunohistochemistry and PCR analysis revealed NA-2 to be more potent in comparison to member of non-steroidal anti-inflammatory drug.

N-(2-hydroxyphenyl)acetamide (NA-2) and Temozolomide synergistically induce apoptosis in human glioblastoma cell line U87.

BACKGROUND: Despite the modern therapies available for treating glioblastoma multiforme (GBM), it is still a deadly disease. The development of new therapeutic strategies for the management of gliomas is therefore crucial. The present study is designed to analyze the therapeutic potentials of synthetic compound N-(2-hydroxyphenyl)acetamide (NA-2) in the treatment of GBM as a single agent or in combination with Temozolomide (TMZ) on glioblastoma cells. METHODS: MTT and TUNEL assays were used to detect the growth inhibitory effect and apoptotic activity of NA-2 alone and in combination with TMZ. Synergy was assessed using combination Index method. The expression of apoptosis related markers Bax, Bcl-2 and caspase-3 were assessed by RT-PCR, whereas, the active caspase-3 protein expression was determined using imunocytochemistry. RESULTS: Both NA-2 and TMZ inhibited the growth of U87 in a dose dependent manner. The combine administration of NA-2 (0.33 mM) and temozolomide (0.1 mM) significantly enhanced the cell growth inhibition and apoptosis. Furthermore RT-PCR and imunocytochemistry data revealed that cooperative apoptosis induction was associated with increased ratio of Bax to Bcl-2 and active Caspase-3 expression. CONCLUSION: Our findings support that NA-2 possesses strong apoptotic activity and the combined administration of NA-2 and TMZ may be therapeutically exploited for the management of GBM.

The anti-arthritic and immune-modulatory effects of NHAG: a novel glucosamine analogue in adjuvant-induced arthritis.

Rheumatoid arthritis (RA) is potentially devastating condition which lacks good treatment options. Pro-inflammatory cytokines interleukin-1beta (IL-1 ß ), tumor necrosis factor-alpha (TNF- α ), and oxidative stress markers such as nitric oxide (NO) and peroxide (PO) are mediators of RA pathogenesis. In the present study N-[2,4,5-trihydroxy-6-(hydroxymethyl) tetrahydro-2H-pyran-3-yl]acrylamide (NHAG), analogue of glucosamine, was evaluated in adjuvant-induced arthritic model of rats. The disease progression was monitored by analysing arthritis scoring, loss of body weight, paw oedema, and histological changes in joints. RA associated hyperalgesia was evaluated by gait analysis. The serum or plasma levels of NO, PO, glutathione (GSH) superoxide dismutase (SOD) IL-1 ß and TNF- α were analyzed to monitor the state of disease severity. The arthritic control animals exhibited significant increase in arthritic score (P < 0.003) and paw oedema (P < 0.001) with parallel loss in body weight (P < 0.04). The NHAG-treated arthritic animals exhibited refinement in the gait changes associated with arthritis. NHAG also significantly decreased the NO (P < 0.02) and PO (P < 0.03) with concurrent increased in GSH (P < 0.04) and SOD (P < 0.007). Both IL-1 ß (P < 0.001) and TNF- α (P < 0.001), were significantly decreased in NHAG-treated group. Thus NHAG might have a therapeutic potential for arthritis by exerting antioxidative and immunomodulatory effects.

Protective efficacy of N-(2-hydroxyphenyl) acetamide against adjuvant-induced arthritis in rats.

Rheumatoid arthritis is a chronic inflammatory joint disease characterized by synovial proliferation and tissue destruction. Proinflammatory cytokines like interleukin-1beta (IL-1ß) and tumor necrosis factor-alpha (TNF-α) play a key role in the disease process and elevate energy expenditure, which further increases the joint pain and stiffness. To explore the effects of N-(2-hydroxyphenyl) acetamide (NA-2) on the development of arthritis, clinical signs, histopathology of knee joints, nociception analysis, and the serum levels of IL-1ß and TNF-α were monitored. Arthritis was induced by intradermal administration of heat-killed adjuvant Mycobacterium tuberculosis H37Ra in rats. NA-2 and indomethacin treatments were started in their respective group on the same day when adjuvant was administered. Experiments were terminated when arthritic score of 4 was observed in arthritic control group. NA-2 (5 mg/kg) treatment significantly ameliorated the disease severity. Reduction in body weight and increase in paw oedema were significantly reversed in arthritic animal receiving NA-2. The nociceptive sensation was also inhibited in the NA-2 treated arthritic rats. Remission was associated with improved histology and significant decreased expression of serum proinflammatory cytokines (P < 0.05 for IL-1ß and TNF-α). Based on our observations, it can be suggested that NA-2 possesses promising anti-arthritic property, and it can be used as a therapeutic agent for arthritis.