The metabolic fingerprint of chronic hepatitis C progression: Metabolome shifts and cutting-edge diagnostic options.

Hepatitis C virus infection causes chronic diseases such as cirrhosis and hepatocellular carcinoma. Metabolomics research has been shown to be linked to pathophysiologic pathways in liver illnesses. The aim of this study was to investigate the serum metabolic profile of patients with chronic hepatitis C (CHC) infection and to identify underlying mechanisms as well as potential biomarkers associated with the disease. Nuclear magnetic resonance (NMR) was used to evaluate the sera of 83 patients with CHC virus and 52 healthy control volunteers (NMR). Then, multivariate statistical analysis was used to find distinguishing metabolites between the two groups. Sixteen out of 40 metabolites including include 3-HB, betaine, carnitine, creatinine, fucose, glutamine, glycerol, isopropanol, lysine, mannose, methanol, methionine, ornithine, proline, serine, and valine-were shown to be significantly different between the CHC and normal control (NC) groups (variable importance in projection >1 and p < 0.05). All the metabolic perturbations in this disease are associated with pathways of Glycine, serine, and threonine metabolism, glycerolipid metabolism, arginine and proline metabolism, aminoacyl-tRNA biosynthesis, cysteine and methionine metabolism, alanine, aspartate, and glutamate metabolism. Multivariate statistical analysis constructed using these expressed metabolites showed CHC patients can be discriminated from NCs with high sensitivity (90%) and specificity (99%). The metabolomics approach may expand the diagnostic armamentarium for patients with CHC while contributing to a comprehensive understanding of disease mechanisms.

Adoptive cell transfer of macrophages following peripheral nerve injury in mice.

Macrophages are key innate immune cells involved in multiple biological processes, including peripheral nerve regeneration. Here, we describe a protocol for the adoptive cell transfer of bone-marrow-derived macrophages (BMDMs) following sciatic nerve crush injury (SNCI). This procedure involves isolating BMDMs from a donor mouse, potentially manipulating them ex vivo, and reintroducing them into an animal following SNCI. Preclinical studies show that BMDMs can infiltrate injured nerves and impact functional recovery, potentially providing a novel therapy for nerve injuries. For complete details on the use and execution of this protocol, please refer to Jha et al.1.

Macrophage monocarboxylate transporter 1 promotes peripheral nerve regeneration after injury in mice.

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.

Mitochondrial apoptosis and curtailment of hypoxia-inducible factor-1α/fatty acid synthase: A dual edge perspective of gamma linolenic acid in ER+ mammary gland cancer.

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.

A Modified Collagen Dressing Induces Transition of Inflammatory to Reparative Phenotype of Wound Macrophages.

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.

DuCLOX-2/5 Inhibition Attenuates Inflammatory Response and Induces Mitochondrial Apoptosis for Mammary Gland Chemoprevention.

The present study is a pursuit to define implications of dual cyclooxygenase-2 (COX-2) and 5-lipoxygenase (5-LOX) (DuCLOX-2/5) inhibition on various aspects of cancer augmentation and chemoprevention. The monotherapy and combination therapy of zaltoprofen (COX-2 inhibitor) and zileuton (5-LOX inhibitor) were validated for their effect against methyl nitrosourea (MNU) induced mammary gland carcinoma in albino wistar rats. The combination therapy demarcated significant effect upon the cellular proliferation as evidenced through decreased in alveolar bud count and restoration of the histopathological architecture when compared to toxic control. DuCLOX-2/5 inhibition also upregulated levels of caspase-3 and caspase-8, and restored oxidative stress markers (GSH, TBARs, protein carbonyl, SOD and catalase). The immunoblotting and qRT-PCR studies revealed the participation of the mitochondrial mediated death apoptosis pathway along with favorable regulation of COX-2, 5-LOX. Aforementioned combination restored the metabolic changes to normal when scrutinized through 1H NMR studies. Henceforth, the DuCLOX-2/5 inhibition was recorded to import significant anticancer effects in comparison to either of the individual treatments.

GLA supplementation regulates PHD2 mediated hypoxia and mitochondrial apoptosis in DMBA induced mammary gland carcinoma.

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.

Alpha-linolenic acid stabilizes HIF-1 α and downregulates FASN to promote mitochondrial apoptosis for mammary gland chemoprevention.

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 IC50 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 1H 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.

6,7-dimethoxy-1,2,3,4-tetrahydro-isoquinoline-3-carboxylic acid attenuates heptatocellular carcinoma in rats with NMR-based metabolic perturbations.

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.

p-TSA-promoted syntheses of 5H-benzo[h] thiazolo[2,3-b]quinazoline and indeno[1,2-d] thiazolo[3,2-a]pyrimidine analogs: molecular modeling and in vitro antitumor activity against hepatocellular carcinoma.

In our efforts to address the rising incidence of hepatocellular carcinoma (HCC), we have made a commitment to the synthesis of novel molecules to combat Hep-G2 cells. A facile and highly efficient one-pot, multicomponent reaction has been successfully devised utilizing a p-toluenesulfonic acid (p-TSA)-catalyzed domino Knoevenagel/Michael/intramolecular cyclization approach for the synthesis of novel 5H-benzo[h]thiazolo[2,3-b]quinazoline and indeno[1,2-d] thiazolo[3,2-a]pyrimidine analogs bearing a bridgehead nitrogen atom. This domino protocol constructed one new ring by the concomitant formation of multiple bonds (C-C, C-N, and C=N) involving multiple steps without the use of any metal catalysts in one-pot, with all reactants effi-ciently exploited. All the newly synthesized compounds were authenticated by means of Fourier transform infrared spectroscopy, liquid chromatography-mass spectrometry, proton nuclear magnetic resonance spectroscopy, and carbon-13 nuclear magnetic resonance spectroscopy, together with elemental analysis, and their antitumor activity was evaluated in vitro on a Hep-G2 human cancer cell line by sulforhodamine B assay. Computational molecular modeling studies were carried out on cancer-related targets, including interleukin-2, interleukin-6, Caspase-3, and Caspase-8. Two compounds (4A and 6A) showed growth inhibitory activity comparable to the positive control Adriamycin, with growth inhibition of 50% <10 µg/mL. The results of the comprehensive structure-activity relationship study confirmed the assumption that two or more electronegative groups on the phenyl ring attached to the thiazolo[2,3-b]quinazoline system showed the optimum effect. The in silico simulations suggested crucial hydrogen bond and π-π stacking interactions, with a good ADMET (absorption, distribution, metabolism, excretion, and toxicity) profile and molecular dynamics, in order to explore the molecular targets of HCC which were in complete agreement with the in vitro findings. Considering their significant anticancer activity, 4A and 6A are potential drug candidates for the management of HCC.

Ameliorative effects of pyrazinoic acid against oxidative and metabolic stress manifested in rats with dimethylhydrazine induced colonic carcinoma.

Pyrazinoic acid (PA) is structurally similar to nicotinic acid which acts on G-protein-coupled receptor (GPR109A). GPR109A expresses in colonic and intestinal epithelial sites, and involves in DNA methylation and cellular apoptosis. Therefore, it may be assumed that PA has similar action like nicotinic acid and may be effective against colorectal carcinoma (CRC). CRC was produced via subcutaneous injection of dimethylhydrazine (DMH) at 40 mg/kg body weight once in a week for 4 weeks. After that, PA was administered orally at 2 doses of 10 and 25 mg/kg daily for 15 d to observe the antiproliferative effect. Various physiologic, oxidative stress, molecular parameters, histopathology, RT-PCR and NMR based metabolomics were performed to evaluate the antiproliferative potential of PA. Our results collectively suggested that PA reduced body weight, tumor volume and incidence no. to normal. It restored various oxidative stress parameters and normalized IL-2, IL-6, and COX-2 as compared with carcinogen control. In molecular level, overexpressed IL-6 and COX-2 genes became normal after PA administration. Again, normal tissue architecture was prominent after PA administration. Score plots of PLS-DA models exhibited that PA treated groups were significantly different from CRC group. We found that CRC rat sera have increased levels of acetate, glutamine, o-acetyl-glycoprotein, succinate, citrulline, choline, o-acetyl choline, tryptophan, glycerol, creatinine, lactate, citrate and decreased levels of 3-hydroxy butyrate, dimethyl amine, glucose, maltose, myoinositol. Further the PA therapy has ameliorated the CRC-induced metabolic alterations, signifying its antiproliferative properties. In conclusion, our study provided the evidence that PA demonstrated good antiproliferative effect on DMH induced CRC and thus demonstrated the potential of PA as a useful drug for future anticancer therapy.

Isolated flavonoids from Ficus racemosa stem bark possess antidiabetic, hypolipidemic and protective effects in albino Wistar rats.

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 100mg/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.

Antiproliferative effect of isolated isoquinoline alkaloid from Mucuna pruriens seeds in hepatic carcinoma cells.

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.

NMR investigations of structural and dynamics features of natively unstructured drug peptide - salmon calcitonin: implication to rational design of potent sCT analogs.

Backbone dynamics and conformational properties of drug peptide salmon calcitonin have been studied in aqueous solution using nuclear magnetic resonance (NMR). Although salmon calcitonin (sCT) is largely unfolded in solution (as has been reported in several circular dichroism studies), the secondary H(α) chemical shifts and three bond H(N) -H(α) coupling constants indicated that most of the residues of the peptide are populating the α-helical region of the Ramachandran (ϕ, ψ) map. Further, the peptide in solution has been found to exhibit multiple conformational states exchanging slowly on the NMR timescale (10(2) -10(3) s(-1) ), inferred by the multiple chemical shift assignments in the region Leu4-Leu12 and around Pro23 (for residues Gln20-Tyr22 and Arg24). Possibly, these slowly exchanging multiple conformational states might inhibit symmetric self-association of the peptide and, in part, may account for its reduced aggregation propensity compared with human calcitonin (which lacks this property). The (15) N NMR-relaxation data revealed (i) the presence of slow (microsecond-to-millisecond) timescale dynamics in the N-terminal region (Cys1-Ser5) and core residues His17 and Asn26 and (ii) the presence of high frequency (nanosecond-to-picosecond) motions in the C-terminal arm. Put together, the various results suggested that (i) the flexible C-terminal of sCT (from Thr25-Thr31) is involved in identification of specific target receptors, (ii) whereas the N-terminal of sCT (from Cys1-Gln20) in solution - exhibiting significant amount of conformational plasticity and strong bias towards biologically active α-helical structure - facilitates favorable conformational adaptations while interacting with the intermembrane domains of these target receptors. Thus, we believe that the structural and dynamics features of sCT presented here will be useful guiding attributes for the rational design of biologically active sCT analogs.