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Hepatic encephalopathy (HE) is a debilitating neuropsychiatric complication associated with acute and chronic liver failure. It is characterized by diverse symptoms with variable severity that includes cognitive and motor deficits. The aim of the study is to assess metabolic alterations in the brain and liver using nuclear magnetic resonance (NMR) spectroscopy and subsequent multivariate analyses to characterize metabolic signatures associated with HE. HE was developed by bile duct ligation (BDL) that resulted in hepatic dysfunctions and cirrhosis as shown by liver function tests. Metabolic profiles from control and BDL rats indicated increased levels of lactate, branched-chain amino acids (BCAAs), glutamate, and choline in the liver, whereas levels of glucose, phenylalanine, and pyridoxine were decreased. In brain, the levels of lactate, acetate, succinate, citrate, and malate were increased, while glucose, creatine, isoleucine, leucine, and proline levels were decreased. Furthermore, neurotransmitters such as glutamate and GABA were increased, whereas choline and myo-inositol were decreased. The alterations in neurotransmitter levels resulted in cognitive and motor defects in BDL rats. A significant correlation was found among alterations in NAA/choline, choline/creatine, and NAA/creatine with behavioral deficits. Thus, the data suggests impairment in metabolic pathways such as the tricarboxylic acid (TCA) cycle, glycolysis, and ketogenesis in the liver and brain of animals with HE. The study highlights that metabolic signatures could be potential markers to monitor HE progression and to assess therapeutic interventions.

Gamma linolenic acid is a polyunsaturated fatty acid having selective anti-tumour properties with negligible systemic toxicity. In the present study, the anti-cancer potential of gamma linolenic acid and its effects on mitochondrial as well as hypoxia-associated marker was evaluated. The effect of gamma linolenic acid was scrutinised against ER + MCF-7 cells by using fluorescence microscopy, JC-1 staining, dot plot assay and cell cycle analysis. The in vitro results were also confirmed using carcinogen (n-methyl-n-nitrosourea) induced in vivo model. The early and late apoptotic signals in the conjugation with mitochondrial depolarisation were found once scrutinised through mitochondrial membrane potential and life death staining after gamma linolenic acid treatment. Gamma linolenic acid arrested the cell cycle in G0/G1 phase with the majority of cell populations in the early apoptotic stage. The translocation of phosphatidylserine was studied through annexin-V FITC dot plot assay. The markers of cellular proliferation (decreased alveolar bud count, histopathological architecture restoration and loss of tumour micro-vessels) were diminished after gamma linolenic acid treatment. Gamma linolenic acid ameliorates the biological effects of n-methyl-n-nitrosourea persuading the mitochondrial mediated death pathway and impeding the hypoxic microenvironment to make a halt in palmitic acid synthesis. SIGNIFICANCE: The present study elaborates the effect of gamma linolenic acid on mammary gland cancer by following mitochondrial-mediated death apoptosis pathway. Gamma linolenic acid also inhibits cell-wall synthesis by the curtailment of HIF-1α and FASN level in mammary gland cancer.

Peripheral nerves have the capacity for regeneration, but the rate of regeneration is so slow that many nerve injuries lead to incomplete recovery and permanent disability for patients. Macrophages play a critical role in the peripheral nerve response to injury, contributing to both Wallerian degeneration and nerve regeneration, and their function has recently been shown to be dependent on intracellular metabolism. To date, the impact of their intracellular metabolism on peripheral nerve regeneration has not been studied. We examined conditional transgenic mice with selective ablation in macrophages of solute carrier family 16, member 1 (Slc16a1), which encodes monocarboxylate transporter 1 (MCT1), and found that MCT1 contributed to macrophage metabolism, phenotype, and function, specifically in regard to phagocytosis and peripheral nerve regeneration. Adoptive cell transfer of wild-type macrophages ameliorated the impaired nerve regeneration in macrophage-selective MCT1-null mice. We also developed a mouse model that overexpressed MCT1 in macrophages and found that peripheral nerves in these mice regenerated more rapidly than in control mice. Our study provides further evidence that MCT1 has an important biological role in macrophages and that manipulations of macrophage metabolism can enhance recovery from peripheral nerve injuries, for which there are currently no approved medical therapies.

Background Diabetes is among the most prevalent diseases worldwide, of all the affected individuals a significant proportion of the population remains undiagnosed due to lack of specific symptoms early in this disorder and inadequate diagnostics. Diabetes and its associated sequela, i.e., comorbidity are associated with microvascular and macrovascular complications. As diabetes is characterized by an altered metabolism of key metabolites and regulatory pathways. Metabolic phenotyping can provide us with a better understanding of the unique set of regulatory perturbations that predispose to diabetes and its associated complication/comorbidities. Methodology The present study utilizes the analytical platform NMR spectroscopy coupled with Random Forest statistical analysis to identify the discriminatory metabolites in diabetes (DB = 38) vs. diabetes-related complication (DC = 35) along with the healthy control (HC = 50) subjects. A combined and pairwise analysis was performed to identify the discriminatory metabolites responsible for class separation. The perturbed metabolites were further rigorously validated using t-test, AUROC analysis to examine the statistical significance of the identified metabolites. Results The DB and DC patients were well discriminated from HC. However, 15 metabolites were found to be significantly perturbed in DC patients compared to DB, the identified panel of metabolites are TCA cycle (succinate, citrate), methylamine metabolism (trimethylamine, methylamine, betaine), -intermediates; energy metabolites (glucose, lactate, pyruvate); and amino acids (valine, arginine, glutamate, methionine, proline, and threonine). Conclusion The 1H NMR metabolomics may prove a promising technique to differentiate and predict diabetes and its complication on their onset or progression by determining the altered levels of the metabolites in serum.

In this study, we isolated two compounds from the leaves of Salacia oblonga (SA1, mangiferin and SA2, naringenin), and their structures were confirmed by infrared spectroscopy, nuclear magnetic resonance (NMR) spectroscopy, and mass spectrometry. SA1 and SA2 were orally administered to streptozotocin-induced diabetic rats at 50 and 100 mg/kg daily for 15 days. Blood glucose level, serum lipid profile, oxidative stress parameters, histopathology, docking, molecular parameters, and NMR-based metabolic perturbation studies were performed to investigate the pharmacological activities of SA1 and SA2. Results suggested that both compounds reduced blood glucose level, restored body weight, and normalized lipid concentrations in the serum and oxidative stress biomarkers in the liver and pancreas. In addition, the docking study on several diabetes-associated targets revealed that both compounds had a strong binding affinity towards peroxisome proliferator-activated receptor gamma (PPARγ) and glucose transporter type 4 (GLUT4). Further real-time reverse transcription polymerase chain reaction and western blot analyses were performed to confirm the gene and protein expression levels of PPARγ and GLUT4 in the pancreatic tissues. Data obtained from the molecular studies showed that both compounds exhibited antidiabetic effects through dual activation of PPARγ/GLUT4 signaling pathways. Finally, the NMR-based metabolic studies showed that both compounds normalized the diabetogenic metabolites in the serum. Altogether, we concluded that SA1 and SA2 might be potential antidiabetic lead compounds for future drug development.

// Subhadeep Roy 1 , Atul Kumar Rawat 2 , Shreesh Raj Sammi 3 , Uma Devi 4 , Manjari Singh 1 , Swetlana Gautam 1 , Rajnish Kumar Yadav 1 , Jitendra Kumar Rawat 1 , Lakhveer Singh 1 , Mohd. Nazam Ansari 5 , Abdulaziz S. Saeedan 5 , Rakesh Pandey 3 , Dinesh Kumar 2 and Gaurav Kaithwas 1 1 Department of Pharmaceutical Sciences, Babasaheb Bhimrao Ambedkar University, Lucknow (UP), India 2 Central for Biomedical Research, Sanjay Gandhi Post Graduate Institute of Medical Sciences Campus, Lucknow (UP), India 3 Department of Microbial Technology and Nematology, CSIR-Central Institute of Medicinal and Aromatic Plants, Lucknow (UP), India 4 Department of Pharmaceutical Sciences, Faculty of Health and Medical Sciences, Sam Higginbottom Institute of Agricultural Sciences and Technology, Allahabad (UP), India 5 Department of Pharmacology, College of Pharmacy, Prince Sattam Bin Abdulaziz University, Al-Kharj, KSA Correspondence to: Gaurav Kaithwas, email: gauravpharm@hotmail.com Keywords: alpha linolenic acid, apoptosis, polyunsaturated fatty acid, hypoxia, fatty acid synthase Received: February 06, 2017 Accepted: June 12, 2017 Published: July 25, 2017 ABSTRACT Alpha linolenic acid is an essential polyunsaturated fatty acid and is reported to have the anti-cancer potential with no defined hypothesis or mechanism/s. Henceforth present study was in-quested to validate the effect of alpha linolenic acid on mitochondrial apoptosis, hypoxic microenvironment and de novo fatty acid synthesis using in-vitro and in-vivo studies. The IC 50 value of alpha linolenic acid was recorded to be 17.55μM against ER+MCF-7 cells. Treatment with alpha linolenic acid was evident for the presence of early and late apoptotic signals along with mitochondrial depolarization, when studied through acridine orange/ethidium bromide and JC-1 staining. Alpha linolenic acid arrested the cell cycle in G2/M phase. Subsequently, the in-vivo efficacy was examined against 7, 12-dimethylbenz anthracene induced carcinogenesis. Treatment with alpha linolenic acid demarcated significant effect upon the cellular proliferation as evidenced through decreased in alveolar bud count, restoration of the histopathological architecture and loss of tumor micro vessels. Alpha linolenic acid restored the metabolic changes to normal when scrutinized through 1 H NMR studies. The immunoblotting and qRT-PCR studies revealed participation of mitochondrial mediated death apoptosis pathway and curtailment of hypoxic microenvironment after treatment with alpha linolenic acid. With all above, it was concluded that alpha linolenic acid mediates mitochondrial apoptosis, curtails hypoxic microenvironment along with inhibition of de novo fatty acid synthesis to impart anticancer effects.

Collagen containing wound-care dressings are extensively used. However, the mechanism of action of these dressings remain unclear. Earlier studies utilizing a modified collagen gel (MCG) dressing demonstrated improved vascularization of ischemic wounds and better healing outcomes. Wound macrophages are pivotal in facilitating wound angiogenesis and timely healing. The current study was designed to investigate the effect of MCG on wound macrophage phenotype and function. MCG augmented recruitment of macrophage at the wound-site, attenuated pro-inflammatory and promoted anti-inflammatory macrophage polarization. Additionally, MCG increased anti-inflammatory IL-10, IL-4 and pro-angiogenic VEGF production, indicating a direct role of MCG in resolving wound inflammation and improving angiogenesis. At the wound-site, impairment in clearance of apoptotic cell bioburden enables chronic inflammation. Engulfment of apoptotic cells by macrophages (efferocytosis) resolves inflammation via a miR-21-PDCD4-IL-10 pathway. MCG-treated wound macrophages exhibited a significantly bolstered efferocytosis index. Such favorable outcome significantly induced miR-21 expression. MCG-mediated IL-10 production was dampened under conditions of miR-21 knockdown pointing towards miR-21 as a causative factor. Pharmacological inhibition of JNK attenuated IL-10 production by MCG, implicating miR-21-JNK pathway in MCG-mediated IL-10 production by macrophages. This work provides direct evidence demonstrating that a collagen-based wound-care dressing may influence wound macrophage function and therefore modify wound inflammation outcomes.

Ethnopharmacological relevance Ficus racemosa (FR) has been used for thousands of years in Ayurvedic system of medicine in India and is closely associated with prevention, treatment and cure of various human ailments like obesity and diabetes. It is popularly known as gular. A vast and wide range of chemical compounds like polyphenols, friedelane-type triterpenes, norfriedelane type triterpene, eudesmane-type sesquiterpene including various glycosides had been isolated from this plant. However, no detail studies related to isolation of flavonoids has been reported previously with their antidiabetic, hypolipidemic and toxicological consequences. Aim of the study The present study was undertaken to evaluate antidiabetic, hypolipidemic and toxicological assessments of flavonoids isolated from Ficus racemosa (FR) stem bark. Materials and methods We isolated four flavonoids from stem bark of FR and structures were confirmed by Infrared spectroscopy (IR), Nuclear Magnetic Resonance (NMR) (both 1D and 2D), mass spectroscopy (MS). Later, these flavonoids were administered to streptozotocin (STZ) rats once in a day for a period of seven days at 100 mg/kg dose. We measured blood glucose level and body weight changes at different days (1st, 3rd, 5th and 7th days). Serum lipid profiles were also estimated to investigate the hypolipidemic potential of flavonoids in the similar experiment. Various oxidative stress parameters in pancreas and liver and hepatic biomarker enzymes in plasma were also determined to investigate the toxicity potential of isolated flavonoids. Finally, we performed docking studies to find out the mechanism of action. Results Our results collectively suggested that four flavonoids reduced blood glucose level and restored body weight, signifying antidiabetic action. There were reduction of other lipid profile parameters and increase of high density lipoprotein (HDL) during administration of flavonoids, also signifying hypolipidemic action. Various oxidative stress biomarkers and hepatic enzymes levels were also normalized with respect to diabetic control at the same time. Docking studies revealed that isolated flavonoids showed their antidiabetic potential via binding to PPARγ and GLUT1 receptors. Conclusion The isolated four flavonoids demonstrated good antidiabetic, hypolipidemic and antioxidant properties in STZ diabetic rats which supported the use of FR stem bark as useful supplementary drug for future antidiabetic therapy.

Introduction: Erythromycin (ERY) is known to induce hepatic toxicity which mimics other liver diseases. Thus, ERY is often used to produce experimental models of drug-induced liver-toxicity. The serum metabolic profiles can be used to evaluate the liver-toxicity and to further improve the understanding of underlying mechanism. Objective: To establish the serum metabolic patterns of Erythromycin induced hepatotoxicity in albino wistar rats using 1H NMR based serum metabolomics. Experimental: Fourteen male rats were randomly divided into two groups (n = 7 in each group): control and ERY treated. After 28 days of intervention, the metabolic profiles of sera obtained from ERY and control groups were analyzed using high-resolution 1D 1H CPMG and diffusion-edited nuclear magnetic resonance (NMR) spectra. The histopathological and SEM examinations were employed to evaluate the liver toxicity in ERY treated group. Results: The serum metabolic profiles of control and ERY treated rats were compared using multivariate statistical analysis and the metabolic patterns specific to ERY-induced liver toxicity were established. The toxic response of ERY was characterized with: (a) increased serum levels of Glucose, glutamine, dimethylamine, malonate, choline, phosphocholine and phospholipids and (b) decreased levels of isoleucine, leucine, valine, alanine, glutamate, citrate, glycerol, lactate, threonine, circulating lipoproteins, N-acetyl glycoproteins, and poly-unsaturated lipids. These metabolic alterations were found to be associated with (a) decreased TCA cycle activity and enhanced fatty acid oxidation, (b) dysfunction of lipid and amino acid metabolism and (c) oxidative stress. Conclusion and Recommendations: Erythromycin is often used to produce experimental models of liver toxicity; therefore, the established NMR-based metabolic patterns will form the basis for future studies aiming to evaluate the efficacy of anti-hepatotoxic agents or the hepatotoxicity of new drug-formulations.

The present study was undertaken to investigate the antiproliferative action of isolated M1 (6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid) from Mucuna pruriens seeds using human hepatic carcinoma cell line (Huh-7 cells). Initially, docking studies was performed to find out the binding affinities of M1 to caspase-3 and 8 enzymes. Later, cytotoxic action of M1 was measured by cell growth inhibition (MTT), followed by caspase-3 and 8 enzymes assay colorimetrically. Our results collectively suggested that M1 had strong binding affinity to caspase-8 in molecular modelling. M1 possessed antiproliferative activity on Huh-7 cells (EC50 = 13.97 μM) and also inhibited the action of caspase-8 enzyme, signified process of apoptosis. M1 was active against Huh-7 cells that may be useful for future hepatic cancer treatment.

The aim of the present study is to evaluate the effect of gamma linolenic acid (GLA) on mitochondrial mediated death apoptosis, hypoxic microenvironment and cholinergic anti-inflammatory pathway against 7, 12-dimethylbenz (a) anthracene (DMBA) induced mammary gland carcinoma. The effects of GLA were evaluated morphologically and biochemically against DMBA induced mammary gland carcinoma. The metabolic study was done for evaluation of biomarkers using 1H NMR. The present study was also verified through immunoblotting and qRT-PCR studies for the evaluation of various pathways. GLA treatment has a delineate implementation upon morphology of the tissues when evaluated through carmine staining, hematoxyline and eosin staining and scanning electron microscopy. GLA also demarked a commendatory proclamation of the fifteen key serum metabolites analogous with amino acid metabolism and fatty acid metabolism when recognized through1H NMR studies. The immunoblotting and qRT-PCR studies accomplished that GLA mediated mitochondrial death apoptosis, curtail hypoxic microenvironment along with hindrance of de novo fatty acid synthesis and also mediate the cholinergic anti-inflammatory pathway to proclaim its anticancer effects.

Background Takayasu arteritis (TA) is a chronic large vessel vasculitis of unknown etiopathogenesis. The serological and radiological parameters currently used to assess the disease activity are not highly specific and there is a pertinent need for a biomarker discovery. In our previous study [1], NMR based serum metabolomics had revealed distinctive metabolic signatures in patients with TA compared to age/sex matched healthy controls. In this study we sought to investigate whether these distinctive metabolites correlate with disease activity. Objectives To identify the discriminatory serum metabolic profiles and their correlation with disease activity. Methods Patients with TA fulfilling ACR criteria were assessed for disease activity by ITAS 2010, with a score of 4 or more, considered as active. The serum metabolic profiles of active and inactive TA patients were obtained at 800 MHZ NMR spectrometer and were compared using multivariate orthogonal partial least-squares discriminant analysis (OPLS-DA) to identify metabolites that changed in response to disease activity [based on PLS-DA VIP (variable importance on projection) score >2.0 and permutation test, p-value Results 88 patients were categorized into active (34) and inactive (54) groups. Median age in active and inactive groups was 25 years and 27 years respectively. Female to male ratio was 3.4:1 in the active group and 5:1 in the inactive group. Majority had class V disease. Mean duration of illness was 4.0±3.5 years in active TA and 6.5±5.5 years in inactive TA group. An exquisite separation in OPLS-DA score plot showed metabolic differences between active and inactive TA patients (Fig. 1A). The key metabolite entities identified with highest discriminatory potential (VIP score >2) were glucose, glutamine, glycine, N-acetyl glycoprotein (NAG), choline, and low/very-low density lipoproteins (LDL/VLDL). Of them glucose, glycine, and NAGs were elevated in the sera of active TA patients, whereas glutamine, choline and LDL levels were decreased in these patients. Receiver operating characteristic (ROC) curve analysis revealed NAG has the highest potential to discriminate active from inactive TA patients (area under the ROC curve was 0.75 (p-value Conclusions The study revealed discriminatory metabolites between active and inactive TA patients and evaluated the possibility of NAG as a clinical biomarker for activity judgment in this disease. However, more work needs to be done to validate the results in a large cohort of patients in a longitudinal manner. References Guleria A, Misra DP, Rawat A, Dubey D, Khetrapal CL, Bacon PA, Misra R, and Kumar D, NMR based serum metabolomics discriminates Takayasu Arteritis from Healthy Individuals: A proof of principle study” Journal of Proteome Research (2015), 14 (8), 3372–3381. Disclosure of Interest None declared

Pyrazinamide (PYZ)-an essential component of primary drug regimen used for the treatment and management of multidrug resistant or latent tuberculosis-is well known for its hepatoxicity. However, the mechanism of PYZ-induced hepatotoxicity is still unknown to researchers. Studies have shown that the drug is metabolized in the liver to pyrazinoic acid (PA) and 5-hydroxy pyrazinoic acid (5-OHPA) which individually may cause different degrees of hepatotoxicity. To evaluate this hypothesis, PYZ, PA, and 5-OHPA were administered to albino Wistar rats orally (respectively, at 250, 125, and 125 mg kg-1 for 28 days). Compared to normal rats, PYZ and its metabolic products decreased the weights of dosed rats and induced liver injury and a status of oxidative stress as assessed by combined histopathological and biochemical analysis. Compared to normal controls, the biochemical and morphological changes were more aberrant in PA- and 5-OHPA-dosed rats with respect to those dosed with PYZ. Finally, the serum metabolic profiles of rats dosed with PYZ, PA, and 5-OHPA were measured and compared with those of normal control rats. With respect to normal control rats, the rats dosed with PYZ and 5-OHPA showed most aberrant metabolic perturbations in their sera as compared to those dosed with PA. Altogether, the study suggests that PYZ-induced hepatotoxicity might be associated with its metabolized products, where 5-OHPA contributes to a higher degree in its overall toxicity than PA.

Aim: 6,7-dimethoxy-1,2,3,4-tetrahydro-isoquinoline-3-carboxylic acid (M1) was synthesized and evaluated for in-vivo antiproliferative action in diethylnitrosamine-induced hepatocarcinogenic rats. Materials & methods: The antiproliferative effect of M1 was assessed by various biochemical parameters, histopathology of liver and HPLC analysis. Proton nuclear magnetic resonance-based serum metabolic study was implemented on rat sera to explore the effects of M1 on hepatocellular carcinoma-induced metabolic alterations. Results: M1 showed protective action on liver and restored the arrangement of liver tissues in normal proportion. HPLC analysis displayed a good plasma drug concentration after its oral administration. Score plots of partial least squares discriminate analysis models exhibited that M1 therapy ameliorated hepatocellular carcinoma-induced metabolic alterations which signified its antiproliferative potential. Conclusion: M1 manifested notable antiproliferative profile, and warrants further investigation for future anticancer therapy., Lay abstract: Our previous study unveiled that an isoquinoline alkaloid (M1) isolated from Mucuna pruriens seeds had potent antiproliferative action on human hepatoma cells in vitro. In the present investigation we synthesized M1 and studied its in vivo antiproliferative effects in liver carcinogenic rats. To observe the effects of M1, various studies were conducted which include biochemical parameters of plasma and serum, histopathology of liver and HPLC analysis of plasma. In addition, 1H-NMR-based serum metabolic profiling was carried out to identify the effects of M1 on hepatocellular carcinoma-induced metabolic alterations. Our findings showed that compound M1 exemplifies remarkable efficacy against hepatocellular carcinoma.

Management of patient with Lupus Nephritis (LN) continues to remain a challenge for the treating physicians because of considerable morbidity and even mortality. The search of biomarkers in serum and urine is a focus of researchers to unravel new targets for therapy. In the present study, the utility of NMR-based serum metabolomics has been evaluated for the first time in discriminating LN patients from non-nephritis lupus patients (SLE) and further to get new insights into the underlying disease processes for better clinical management. Metabolic profiling of sera obtained from 22 SLE patients, 40 LN patients and 30 healthy controls (HC) were performed using high resolution 1D 1H-CPMG and diffusion edited NMR spectra to identify the potential molecular biomarkers. Using multivariate analysis, we could distinguish SLE and LN patients from HC and LN from SLE patients. Compared to SLE patients, the LN patients had increased serum levels of lipid metabolites (including LDL/VLDL lipoproteins), creatinine and decreased levels of acetate. Our results revealed that metabolic markers especially lipids and acetate derived from NMR spectroscopy has high sensitivity and specificity to distinguish LN among SLE patients and has the potential to be a useful adjunctive tool in diagnosis and clinical management of LN.