AKR1B1 drives hyperglycemia-induced metabolic reprogramming in MASLD-associated hepatocellular carcinoma.

Background & Aims: The mechanism behind the progressive pathological alteration in metabolic dysfunction-associated steatotic liver disease/steatohepatitis (MASLD/MASH)-associated hepatocellular carcinoma (HCC) is poorly understood. In the present study, we investigated the role of the polyol pathway enzyme AKR1B1 in metabolic switching associated with MASLD/MASH and in the progression of HCC. Methods: AKR1B1 expression was estimated in the tissue and plasma of patients with MASLD/MASH, HCC, and HCC with diabetes mellitus. The role of AKR1B1 in metabolic switching in vitro was assessed through media conditioning, lentiviral transfection, and pharmacological probes. A proteomic and metabolomic approach was applied for the in-depth investigation of metabolic pathways. Preclinically, mice were subjected to a high-fructose diet and diethylnitrosamine to investigate the role of AKR1B1 in the hyperglycemia-mediated metabolic switching characteristic of MASLD-HCC. Results: A significant increase in the expression of AKR1B1 was observed in tissue and plasma samples from patients with MASLD/MASH, HCC, and HCC with diabetes mellitus compared to normal samples. Mechanistically, in vitro assays revealed that AKR1B1 modulates the Warburg effect, mitochondrial dynamics, the tricarboxylic acid cycle, and lipogenesis to promote hyperglycemia-mediated MASLD and cancer progression. A pathological increase in the expression of AKR1B1 was observed in experimental MASLD-HCC, and expression was positively correlated with high blood glucose levels. High-fructose diet + diethylnitrosamine-treated animals also exhibited statistically significant elevation of metabolic markers and carcinogenesis markers. AKR1B1 inhibition with epalrestat or NARI-29 inhibited cellular metabolism in in vitro and in vivo models. Conclusions: Pathological AKR1B1 modulates hepatic metabolism to promote MASLD-associated hepatocarcinogenesis. Aldose reductase inhibition modulates the glycolytic pathway to prevent precancerous hepatocyte formation. Impact and implications: This research work highlights AKR1B1 as a druggable target in metabolic dysfunction-associated steatotic liver disease (MASLD) and hepatocellular carcinoma (HCC), which could provide the basis for the development of new chemotherapeutic agents. Moreover, our results indicate the potential of plasma AKR1B1 levels as a prognostic marker and diagnostic test for MASLD and associated HCC. Additionally, a major observation in this study was that AKR1B1 is associated with the promotion of the Warburg effect in HCC.

Integrative Placental Multi-Omics Analysis Reveals Perturbed Pathways and Potential Prognostic Biomarkers in Gestational Hypertension.

BACKGROUND: Gestational hypertension (GH) is a severe complication that occurs after 20 weeks of pregnancy; however, its molecular mechanisms are not yet fully understood. OBJECTIVE: Through this case-control discovery phase study, we aimed to find disease-specific candidate placental microRNAs (miRNAs) and metabolite markers for differentiating GH by integrating next-generation sequencing and metabolomics multi-omics analysis of placenta. Using small RNA sequencing and metabolomics of placental tissues of healthy pregnant (HP, n = 24) and GH subjects (n = 20), the transcriptome and metabolome were characterized in both groups. RESULTS: The study identified a total of 44 downregulated placental miRNAs which includes three novel, three mature and 38 precursor miRNAs. Six miRNAs including three mature (hsa-miR-181a-5p, hsa-miR-498-5p, and hsa-miR-26b-5p) and three novel (NC_000016.10_1061, NC_000005.10_475, and NC_000001.11_53) were considered for final target prediction and functional annotation. Integrative analysis of differentially expressed miRNAs and metabolites yielded five pathways such as purine, glutathione, glycerophospholipid, inositol phosphate and ß-alanine to be significantly perturbed in GH. We present fourteen genes (LPCAT1, LPCAT2, DGKH, PISD, GPAT2, PTEN, SACM1L, PGM2, AMPD3, AK7, AK3, CNDP1, IDH2, and ODC1) and eight metabolites (xanthosine, xanthine, spermine, glycine, CDP-Choline, glyceraldehyde 3-phosphate, ß-alanine, and histidine) with potential to distinguish GH and HP. CONCLUSION: The differential expression of miRNAs, their target genes, altered metabolites and metabolic pathways in GH patients were identified for the first time in our study. Further, the altered miRNAs and metabolites were integrated to build their inter-connectivity network. The findings obtained from our study may be used as a valuable source to further unravel the molecular pathways associated with GH and also for the evaluation of prognostic markers.

Unlocking The Mysteries of DNA Adducts with Artificial Intelligence.

Cellular genome is considered a dynamic blueprint of a cell since it encodes genetic information that gets temporally altered due to various endogenous and exogenous insults. Largely, the extent of genomic dynamicity is controlled by the trade-off between DNA repair processes and the genotoxic potential of the causative agent (genotoxins or potential carcinogens). A subset of genotoxins form DNA adducts by covalently binding to the cellular DNA, triggering structural or functional changes that lead to significant alterations in cellular processes via genetic (e. g., mutations) or non-genetic (e. g., epigenome) routes. Identification, quantification, and characterization of DNA adducts are indispensable for their comprehensive understanding and could expedite the ongoing efforts in predicting carcinogenicity and their mode of action. In this review, we elaborate on using Artificial Intelligence (AI)-based modeling in adducts biology and present multiple computational strategies to gain advancements in decoding DNA adducts. The proposed AI-based strategies encompass predictive modeling for adduct formation via metabolic activation, novel adducts' identification, prediction of biochemical routes for adduct formation, adducts' half-life predictions within biological ecosystems, and, establishing methods to predict the link between adducts chemistry and its location within the genomic DNA. In summary, we discuss some futuristic AI-based approaches in DNA adduct biology.

From co-delivery to synergistic anti-inflammatory effect: Studies on chitosan-stabilized Janus emulsions having chloroquine phosphate and flavopiridol in Complete Freund's Adjuvant induced arthritis rat model.

For the first time, the co-delivery of chloroquine phosphate and flavopiridol by intra-articular route was achieved to provide local joint targeting in Complete Freund's Adjuvant-induced arthritis rat model. The presence of paired-bean structure onto the dispersed oil droplets of o/w nanosized emulsions allows efficient entrapment of two drugs (85.86-96.22 %). The dual drug-loaded emulsions displayed a differential in vitro drug release behavior, near normal cell viability in MTT assay, better cell uptake (internalization) and better reducing effect of mean immunofluorescence intensity of inflammatory proteins such as NF-κB and iNOS at in vitro RAW264.7 macrophage cell line. The radiographical study, ELISA test, RT-PCR study and H & E staining also indicated a reduction in joint tissue swelling, IL-6 and TNF-α levels diminution, fold change diminution in the mRNA expressions for NF-κB, IL-1ß, IL-6 and PGE2 and maintenance of near normal histology at bone cartilage interface respectively. The results of metabolomic pathway analysis performed by LC-MS/MS method using the rat blood (plasma) collected from disease control and dual drug-loaded emulsions treatment groups revealed a new follow-up study to understand not only the disease progression but also the formulation therapeutic efficacy assessment.

Preclinical pharmacokinetic and in vitro metabolic stability study of lysosomotropic autophagy inhibitor, IITZ-01 in mice by using UPLC-MS/MS.

The present study evaluates the pharmacokinetics and metabolic stability of a novel lysosomotropic autophagy inhibitor, IITZ-01 using an ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS-MS). It is required as this lead molecule awaits pre-clinical studies for development because of significant therapeutic outcomes in triple-negative breast cancer and renal cancer. A bioanalytical method for the quantitative determination of IITZ-01 in the plasma of mice was developed using the UPLC-MS/MS technique. The UPLC-MS/MS method was validated according to US-FDA bioanalytical guidance and successfully applied to study the pharmacokinetics and metabolic stability. Separation of IITZ- 01 and ZSTK474 (IS) from endogenous components with high selectivity and sensitivity (0.5 ng/mL) was achieved using Waters Acquity BEH C-18 column (50 mm × 2.1 mm, 1.7 µm). A gradient mobile phase consisting of 0.1 % formic acid in water and 0.1 % formic acid in acetonitrile was applied at a flow rate of 0.2 mL/min. Electrospray ionization was employed in positive ion mode for detection, while quantification utilized the multiple reaction monitoring (MRM) mode. This involved using [M+H]+fragment ions at m/z 483.19 â†’ 235.09 for IITZ-01 and m/z 418 â†’ 138 for the internal standard (IS). The method was validated over the calibration range of 0.5-800 ng/mL. The LLOQ of IITZ-01 was 0.5 ng/mL in mice plasma. The method demonstrated good in terms of intra- and inter-day precision and accuracy. The matrix effect was found to be negligible, and the stability data were within acceptable limits. The validated technique supports suitability, reliability, reproducibility, and sensitivity for the pre-clinical investigation of IITZ-01 pharmacokinetics in mice and metabolic stability in human liver microsomes.

Investigating the impact of inbuilt cold atmospheric pressure plasma on molecular assemblies of tryptophan enantiomers: in vitro fabrication of self-assembled supramolecular structures.

The advancements in understanding the phenomenon of plasma interactions with matter, coupled with the development of CAPP devices, have resulted in an interdisciplinary research topic of significant importance. This has led to the integration of various fields of science, including plasma physics, chemistry, biomedical sciences, and engineering. The reactive oxygen species and reactive nitrogen species generated from cold atmospheric plasma on interaction with biomolecules like proteins and peptides form various supramolecular structures. CAPP treatment of amino acids, which are the fundamental building blocks of proteins, holds potential in creating self-assembled supramolecular architectures. In this work, we demonstrate the process of self-assembly of aromatic amino acid tryptophan (Trp) enantiomers (l-tryptophan and d-tryptophan) into ordered supramolecular assemblies induced by the reactive species generated by a cold atmospheric pressure helium plasma jet. These enantiomers of tryptophan form organized structures as evidenced by FE-SEM. To assess the impact of CAPP treatment on the observed assemblies, we employed various analytical techniques such as zeta potential, dynamic light scattering and FTIR spectroscopy. Also, photoluminescence and time-resolved lifetime measurements revealed the transfiguration of individual Trp enantiomers. The LC-ESI-QTOF-MS analysis demonstrated that CAPP irradiation led to the incorporation of oxygenated ions into the pure Trp molecule. These studies of the self-assembly of Trp due to ROS and RNS interactions will help us to understand the assembly environment. This knowledge may be utilized to artificially design and synthesize highly ordered functional supramolecular structures using CAPP.

A pharmacokinetic study to correlate the hypoglycemic effect of phlorizin in rats: Identification of metabolites as inhibitors of sodium/glucose cotransporters.

Phlorizin (PRZ) is a natural product that belongs to a class of dihydrochalcones. The unique pharmacological property of PRZ is to block glucose absorption or reabsorption through specific and competitive inhibitors of the sodium/glucose cotransporters (SGLTs) in the intestine (SGLT1) and kidney (SGLT2). This results in glycosuria by inhibiting renal reabsorption of glucose and can be used as an adjuvant treatment for type 2 diabetes. The pharmacokinetic profile, metabolites of the PRZ, and efficacy of metabolites towards SGLTs are unknown. Therefore, the present study on the characterization of hitherto unknown in vivo metabolites of PRZ and pharmacokinetic profiling using liquid chromatography-electrospray ionization tandem mass spectrometry (LC/ESI/MS/MS) and accurate mass measurements is undertaken. Plasma, urine, and feces samples were collected after oral administration of PRZ to Sprague-Dawley rats to identify in vivo metabolites. Furthermore, in silico efficacy of the identified metabolites was evaluated by docking study. PRZ at an intraperitoneal dose of 400 mg/kg showed maximum concentration in the blood to 439.32 ± 8.84 ng/mL at 1 h, while phloretin showed 14.38 ± 0.33 ng/mL at 6 h. The pharmacokinetic profile of PRZ showed that the maximum concentration lies between 1 and 2 h after dosing. Decreased blood glucose levels and maximum excretion of glucose in the urine were observed when the PRZ and metabolites were observed in plasma. The identification and characterization of PRZ metabolites by LC/ESI/MS/MS further revealed that the phase I metabolites of PRZ are hydroxy (mono-, di-, and tri-) and reduction. Phase II metabolites are O-methylated, O-acetylated, O-sulfated, and glucuronide metabolites of PRZ. Further docking study revealed that the metabolites diglucuronide metabolite of mono-hydroxylated PRZ and mono-glucuronidation of PRZ could be considered novel inhibitors of SGLT1 and SGLT2, respectively, which show better binding affinities than their parent compound PRZ and the known inhibitors.

Ambient particulate matter (PM2.5) exposure contributes to neurodegeneration through the microbiome-gut-brain axis: Therapeutic role of melatonin.

Exposure to ambient particulate matter (PM2.5) has been shown to disturb the gut microbiome homeostasis and cause initiation of neuroinflammation and neurodegeneration via gut-brain bi-directional axis. Polyaromatic hydrocarbons (PAHs), which are carcinogenic and mutagenic, are important organic constituents of PM2.5 that could be involved in the microbiome-gut-brain axis-mediated neurodegeneration. Melatonin (ML) has been shown to modulate the microbiome and curb inflammation in the gut and brain. However, no studies have been reported for its effect on PM2.5-induced neuroinflammation. In the current study, it was observed that treatment with ML at 100 µM significantly inhibits microglial activation (HMC-3 cells) and colonic inflammation (CCD-841 cells) by the conditioned media from PM2.5 exposed BEAS2B cells. Further, melatonin treatment at a dose of 50 mg/kg to C57BL/6 mice exposed to PM2.5 (at a dose of 60 µg/animal) for 90 days significantly alleviated the neuroinflammation and neurodegeneration caused by PAHs in PM2.5 by modulating olfactory-brain and microbiome-gut-brain axis.

Analysis of NSAIDs in Rat Plasma Using 3D-Printed Sorbents by LC-MS/MS: An Approach to Pre-Clinical Pharmacokinetic Studies.

Analytical sample preparation techniques are essential for assessing chemicals in various biological matrices. The development of extraction techniques is a modern trend in the bioanalytical sciences. We fabricated customized filaments using hot-melt extrusion techniques followed by fused filament fabrication-mediated 3D printing technology to rapidly prototype sorbents that extract non-steroidal anti-inflammatory drugs from rat plasma for determining pharmacokinetic profiles. The filament was prototyped as a 3D-printed sorbent for extracting small molecules using AffinisolTM, polyvinyl alcohol, and triethyl citrate. The optimized extraction procedure and parameters influencing the sorbent extraction were systematically investigated by the validated LC-MS/MS method. Furthermore, a bioanalytical method was successfully implemented after oral administration to determine the pharmacokinetic profiles of indomethacin and acetaminophen in rat plasma. The Cmax was found to be 0.33 ± 0.04 µg/mL and 27.27 ± 9.9 µg/mL for indomethacin and acetaminophen, respectively, at the maximum time (Tmax) (h) of 0.5-1 h. The mean area under the curve (AUC0-t) for indomethacin was 0.93 ± 0.17 µg h/mL, and for acetaminophen was 32.33± 10.8 µg h/mL. Owing to their newly customizable size and shape, 3D-printed sorbents have opened new opportunities for extracting small molecules from biological matrices in preclinical studies.

Urinary Metabolomics Identified Metabolic Perturbations Associated with Gutka, a Smokeless Form of Tobacco.

Gutka, a form of smokeless tobacco, is widely used in the Indian subcontinent and in other regions of South Asia. Smokeless tobacco exposure is most likely to increase the incidence of oral cancer in the Indian population, and metabolic changes are a hallmark of cancer. The development of biomarkers for early detection and better prevention measures for smokeless tobacco users at risk of oral cancer can be aided by studying urinary metabolomics and offering insight into altered metabolic profiles. This study aimed to investigate urine metabolic alterations among smokeless tobacco users using targeted LC-ESI-MS/MS metabolomics approaches to better understand the effects of smokeless tobacco on human metabolism. Smokeless tobacco users' specific urinary metabolomics signatures were extracted using univariate, multivariate analysis and machine learning methods. Statistical analysis identified 30 urine metabolites significantly associated with metabolomic alterations in humans who chew smokeless tobacco. Receiver operator characteristic (ROC) curve analysis evidenced the 5 most discriminatory metabolites from each approach that could differentiate between smokeless tobacco users and controls with higher sensitivity and specificity. An analysis of multiple-metabolite machine learning models and single-metabolite ROC curves revealed discriminatory metabolites capable of distinguishing smokeless tobacco users from nonusers more effectively with higher sensitivity and specificity. Furthermore, metabolic pathway analysis depicted several dysregulated pathways in smokeless tobacco users, including arginine biosynthesis, beta-alanine metabolism, TCA cycle, etc. This study devised a novel strategy to identify exposure biomarkers among smokeless tobacco users by combining metabolomics and machine learning algorithms.

Studies on spray dried topical ophthalmic emulsions containing cyclosporin A (0.05% w/w): systematic optimization, in vitro preclinical toxicity and in vivo assessments.

Cyclosporin A (CsA, 0.05% w/w)-loaded positively charged emulsions were prepared based on castor oil, chitosan, poloxamer 188, glycerin and double-distilled water. To augment the shelf/storage-stability of original emulsions, the solid-dry powder for reconstitution was made by spray drying technique. The screening (Taguchi OA) and optimization (face-centered central composite) designs produced the optimized conditions for spray drying: 40 Nm3/h aspirator flow rate, 15 ml/min feed rate, 115 °C inlet temperature, 10% mannitol and 1.25% trehalose. The % drug entrapment efficiency values of original and reconstituted emulsions ranged from 73.20 ± 0.13 to 71.55 ± 1.25%. At 20 min post-dissolution, two times higher CsA release was seen from reconstituted emulsions than the original emulsions (85.78 ± 1.14 vs. 42.25 ± 1.84%) in simulated tear fluid. Using MTT assay, the reconstituted emulsions with or without CsA produced 94.512 ± 2.12 to 99.941 ± 1.89% cell viability values in HCE-2 cells. No appreciable change in capillary integrity was visualized in HET CAM following reconstituted emulsions treatment. At equivalent 15 µg drug, the in vitro protein denaturation assay showed augmented inhibition value (~ 85%) for tested CsA emulsions compared to diclofenac reference (68.30 ± 2.05) indicating enhanced anti-inflammatory activity. The CsA concentrations in multiple ocular matrices of rabbit eyes determined by the UPLC-MS/MS method attained the therapeutic drug level of 50-300 ng/ml even at 90 min post-topical instillation of both emulsions. Overall, the CsA emulsion eyedrops can be supplied as a spray dried storable intermediate product for reconstitution.

Environmental phenol exposure associates with urine metabolome alteration in young Northeast Indian females.

Urinary biomonitoring delivers the most accurate environmental phenols exposure assessment. However, environmental phenol exposure-related biomarkers are required to improve risk assessment to understand the internal processes perturbed, which may link exposure to specific health outcomes. This study aimed to investigate the association between environmental phenols exposure and the metabolome of young adult females from India. Urinary metabolomics was performed using liquid chromatography-mass spectrometry. Environmental phenols-related metabolic biomarkers were investigated by comparing the low and high exposure of environmental phenols. Seven potential biomarkers, namely histidine, cysteine-s-sulfate, 12-KETE, malonic acid, p-hydroxybenzoic acid, PE (36:2), and PS (36:0), were identified, revealing that environmental phenol exposure altered the metabolic pathways such as histidine metabolism, beta-Alanine metabolism, glycerophospholipid metabolism, and other pathways. This study also conceived an innovative strategy for the early prediction of diseases by combining urinary metabolomics with machine learning (ML) algorithms. The differential metabolites predictive accuracy by ML models was >80%. This is the first mass spectrometry-based metabolomics study on young adult females from India with environmental phenols exposure. The study is valuable in demonstrating multiple urine metabolic changes linked to environmental phenol exposure and a better understanding of the mechanisms behind environmental phenol-induced effects in young female adults.

Extrusion 3D printing of minicaplets for evaluating in vitro & in vivo praziquantel delivery capability.

This study aimed to explore extrusion three dimensional (3D) printing technology to develop praziquantel (PZQ)-loaded minicaplets and evaluate their in vitro and in vivo delivery capabilities. PZQ-loaded minicaplets were 3D printed using a fused deposition modelling (FDM) principle-based extrusion 3D printer and were further characterized by different in vitro physicochemical and sophisticated analytical techniques. In addition, the % PZQ entrapment and in vitro PZQ release performance were evaluated using chromatographic techniques. It was in vitro observed that PZQ was fully released in the gastric pH medium within the period of gastric emptying, that is, 120 min, from the PZQ-loaded 3D printed minicaplets. Furthermore, in vivo pharmacokinetic (PK) profiles of PZQ-loaded 3D printed minicaplets were systematically evaluated using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The PK profile of the PZQ-loaded 3D printed minicaplets was established using different parameters such as Cmax, Tmax, AUC0-t, AUC0-∞, and oral relative bioavailability (RBA). The Cmax value of pristine PZQ was found at 64.79 ± 13.99 ng/ml, while PZQ-loaded 3D printed minicaplets showed a Cmax of 263.16 ± 47.85 ng/ml. Finally, the PZQ-loaded 3D printed minicaplets showed 9.0-fold improved oral RBA compared with that of pristine PZQ (1.0-fold). Together, these observations potentiate the desired in vitro and improved in vivo delivery capabilities of PZQ from the PZQ-loaded 3D printed minicaplets.

Integrated metabolomics and machine learning approach to predict hypertensive disorders of pregnancy.

BACKGROUND: Hypertensive disorders of pregnancy account for 3% to 10% of maternal-fetal morbidity and mortality worldwide. This condition has been considered one of the leading causes of maternal deaths in developing countries, such as India. OBJECTIVE: This study aimed to discover hypertensive disorders of pregnancy-specific candidate urine metabolites as markers for hypertensive disorders of pregnancy by applying integrated metabolomics and machine learning approaches. STUDY DESIGN: The targeted urinary metabolomics study was conducted in 70 healthy pregnant controls and 133 pregnant patients having hypertension as cases. Hypertensive disorders of pregnancy-specific metabolites for disease prediction were further extracted using univariate and multivariate statistical analyses. For machine learning analysis, 80% of the data were used for training (79 for hypertensive disorders of pregnancy and 42 for healthy pregnancy) and validation (27 for hypertensive disorders of pregnancy and 14 for healthy pregnancy), and 20% of the data were used for test sets (27 for hypertensive disorders of pregnancy and 14 for healthy pregnancy). RESULTS: The statistical analysis using an unpaired t test revealed 44 differential metabolites. Pathway analysis showed mainly that purine and thiamine metabolism were altered in the group with hypertensive disorders of pregnancy compared with the healthy pregnancy group. The area under the receiver operating characteristic curves of the 5 most predominant metabolites were 0.98 (adenosine), 0.92 (adenosine monophosphate), 0.89 (deoxyadenosine), 0.81 (thiamine), and 0.81 (thiamine monophosphate). The best prediction accuracies were obtained using 2 machine learning models (95% for the gradient boost model and 98% for the decision tree) among the 5 used models. The machine learning models showed higher predictive performance for 3 metabolites (ie, thiamine monophosphate, adenosine monophosphate, and thiamine) among 5 metabolites. The combined accuracies of adenosine from all models were 98.6 in the training set and 95.6 in the test set. Moreover, the predictive performance of adenosine was higher than other metabolites. The relative feature importance of adenosine was also observed in the decision tree and the gradient boost model. CONCLUSION: Among other metabolites, adenosine and thiamine metabolites were found to differentiate participants with hypertensive disorders of pregnancy from participants with healthy pregnancies; hence, these metabolites can serve as a promising noninvasive marker for the detection of hypertensive disorders of pregnancy.

Alternanthera brasiliana L. extract alleviates carbon tetrachloride-induced liver injury and fibrotic changes in mice: Role of matrix metalloproteinases and TGF-ß/Smad axis.

ETHNOPHARMACOLOGICAL RELEVANCE: Alternanthera brasiliana L. is a flowering plant belonging to the family Amaranthaceae and is popularly known as "penicillin". It is used in folk medicine to treat infections, coughs, wound healing, and inflammatory diseases. AIM OF THE STUDY: We investigated the effect of Alternanthera brasiliana L. leaves hydroalcoholic extract (AB) against oxidative stress, inflammation, and fibrotic changes in an experimental model of carbon tetrachloride (CCl4)-induced liver injury and fibrosis in mice. MATERIALS AND METHODS: Thirty-six male Balb/C mice were randomized into five groups: normal control, AB control, CCl4 control, CCl4 + AB-200 mg/kg, and CCl4 + AB-400 mg/kg. In mice, liver injury was induced by intraperitoneal injection of CCl4 (20% in corn oil, 5 ml/kg body weight) thrice a week for six consecutive weeks. AB extract at two doses (200 mg/kg and 400 mg/kg body weight) was administered orally for six consecutive weeks. Liver injury-related serum markers (ALT, AST, ALP), antioxidants (GSH, GST, SOD, and vitamin C), pro-inflammatory cytokines including tumor necrosis factor-α (TNF-α), interleukin (IL)-1ß, and IL-18, ultrasonographic and histological alterations, proteins of matrix metalloproteinases (MMPs) and tissue inhibitors of matrix metalloproteinase-1 (TIMP-1), nuclear factor-κB (p65) (NF-κB), nod-like receptor protein 3 (NLRP3), and TGF-ß/Smad signaling were accessed. LC-Q-TOF-MS/MS analysis of AB was performed. RESULTS: AB treatment significantly decreased the CCl4-induced rise in serum ALT, AST, and ALP activities and improved the histological alterations. Compared with the CCl4-treated group, treatment with AB significantly restored the hepatic antioxidants and reduced the pro-inflammatory cytokines in the liver. The antioxidant activity of AB may be attributed to its terpenoid constituents, which was confirmed by LC-Q-TOF-MS/MS analysis. The CCl4-induced rise in expression of MMP-2 and MMP-9 and decrease in TIMP-1 were markedly restored in the AB-treated groups. Further findings revealed a significant reduction in the protein levels of phospho-NF-κB (p65), NLRP3, TGF-ß, pSmad2/3, collagen I, and α-smooth muscle actin (α-SMA) in the AB treatment groups. CONCLUSIONS: The hepatoprotective effect of AB may be attributed to the high content of terpenoid compounds and alleviates liver injury and associated fibrotic changes through modulating MMPs, NF-κB (p65), and the TGF-ß/Smad axis.

Dual Delivery of Cyclosporin A and Etodolac Using Polymeric Nanocapsules in a Rabbit Eye Model: Ocular Biodistribution and Pharmacokinetic Study.

Purpose: Commercially available eye drops are loaded only with a single drug. By using the polymeric nanocapsules, dual delivery of 0.05% w/w cyclosporin A (CsA) and 0.2% w/w etodolac (Edc) was achieved. An ultraperformance liquid chromatography/tandem mass spectrometry (UPLC-MS/MS) method was developed for determining simultaneously the biodistribution and pharmacokinetic profile of CsA and Edc in ocular tissues. Methods: After one single drop instillation of nanocapsules into healthy right eyes of rabbits, the eyeballs were enucleated at 5, 15, 30, 60, and 90 min time periods to separate the 5 different ocular tissues. A liquid/liquid extraction method was used for ocular sample extraction using darunavir as internal standard. Using 3 diverse conditions such as bench-top, autosampler, and freeze-thaw, the stability of the analytes at 3 quality control samples in ocular tissues was also checked. Results: Intra- and interday precisions for both CsA and Edc in multiple ocular tissues were <10.32%, and the accuracy was <11.98%. The % bias and % RSD values for CsA and Edc were found within the acceptable limit of ±15%. The highest Cmax values were attained in cornea for both the drugs at 60 min postinstillation time point. Despite molecular size and structural differences, both CsA and Edc after liberation from nanocapsule drops can permeate into the tissues of the anterior as well as posterior segments of the eye. Conclusion: The biodistribution and pharmacokinetic data might help and strengthen our understanding of synergetic anti-inflammatory activity of CsA and Edc from nanocapsules after its ocular topical application for managing keratoconjunctivitis sicca.

Phyto-metabolomics of phlogacanthus thyrsiformis by using LC-ESI-QTOF-MS/MS and GC/QTOF-MS: Evaluation of antioxidant and enzyme inhibition potential of extracts.

Phlogacanthus thyrsiformis (P. thyrsiformis) is a non-conventional edible plant that has been used as a vegetable and as a traditional medicine to treat various diseases. This non-conventional edible plant is widespread in India, Yunnan-Chinese provinces, and Vietnam. Despite this potential claim, the phytochemical investigation of plants remains incomplete. In the present study, the chemical profile analysis of P. thyrsiformis leaves extracts was performed using LC-ESI-QTOF-MS/MS and GC/QTOF-MS and assessed for its antioxidants and enzyme inhibitory potential. A cursory examination of the phytometabolites of different solvents extracts revealed chloroform extract has different metabolites, including phenols, terpenes, glycosides and alkaloids. 113 and 138 metabolites, including primary and secondary metabolites, were identified using LC-ESI-QTOF-MS/MS and GC/QTOF-MS, respectively. Total phenolic content was observed highest in chloroform extract (60.59 ± 1.25 mg GAE/g). Maximum antioxidant activity was found to be in chloroform extract, which was identified by DPPH (0.88 ± 0.29 mg/mL), ABTS (0.54 ± 0.1 mg/mL) and FRAP assay (26.46 ± 1.65 mg AA/g extract). Ethyl acetate extract have the highest α-amylase (0.09 ± 0.11 mg/mL) and α-glucosidase (0.088 ± 0.13 mg/mL) enzyme inhibition potential. For all the detected molecules, docking and molecular dynamics studies were performed. To the best of our knowledge, this is the first report that discusses LC-ESI-QTOF-MS/MS and GC/QTOF-MS-based metabolites profiling of P. thyrsiformis that can help to treat various diseases.

Perillyl alcohol attenuates chronic restraint stress aggravated dextran sulfate sodium-induced ulcerative colitis by modulating TLR4/NF-κB and JAK2/STAT3 signaling pathways.

BACKGROUND: Ulcerative colitis (UC) is the most prevalent chronic inflammatory immune bowel disease. The modernization of lifestyle accompanied by the stress to cope with the competition has resulted in a new range of complications where stress became a critical contributing factor for many diseases, including UC. Hence there is an urgent need to develop a dual role in curtailing both systemic and neuroinflammation. Perillyl alcohol (POH) is a natural essential oil found in lavender, peppermint, cherries etc and has been widely studied for its strong anti-inflammatory, antioxidant and anti-stress properties. HYPOTHESIS/PURPOSE: POH regulates the various inflammatory signaling cascades involved in chronic inflammation by inhibiting farnesyltransferase  enzyme. Several studies reported that POH could inhibit the phosphorylation of  NF-κB, STAT3 and promote the endogenous antioxidant enzymes like Nrf2 via farnesyltransferase enzyme inhibition.  Also, the effects of POH against UC is not known yet. Thus, this study aims to explore the anti-ulcerative properties of POH on stress aggravated ulcerative colitis in C57BL/6 mice. METHODS: Ulcerative colitis was induced by duel exposure of chronic restraint stress (day 1 to day 28) and 2.5% dextran sulphate sodium (day8 to day14) in mice. POH treatment 100 and 200 mg/kg was administred from day14 ti day28 following oral route of administration. Disease activity index, colonoscopy, western blot analysis and histological analysis, neurotransmitter analysis and Gene expression studies were perofomerd to asses the anti-colitis effects of POH. RESULTS: The treatment reversed the oxidative stress and inflammatory response by inhibiting TLR4/NF-kB pathway, and IL-6/JAK2/STAT3 pathway in both isolated mice colons and brains. The inhibition of these pathways resulted in a decrease in pro-inflammatory cytokines like IL-6, IL-1ß and TNF-α. The treatment improved the physiological and histological changes with decreased ulcerations as examined by colonic endoscopy and Haematoxylin and Eosin staining. The treatment also improved the behavior response as it increased mobility time which was reduced by chronic restrained stress. This was due to increased satiety neurotransmitters like dopamine and serotonin and decreased cortisol in mice brains. CONCLUSION: These results infer that POH has significant anti-colitis activity on chronic restraint stress aggravated DSS-induced UC in mice.

Implications of endocrine-disrupting chemicals on polycystic ovarian syndrome: A comprehensive review.

Polycystic ovarian syndrome (PCOS) is a complex multifactorial disorder of unknown pathogenesis in which genetic and environmental factors contribute synergistically to its phenotypic expressions. Endocrine-disrupting chemicals (EDCs), a group of widespread pollutants freely available in the environment and consumer products, can interfere with normal endocrine signals. Extensive evidence has shown that EDCs, environmental contributors to PCOS, can frequently induce ovarian and metabolic abnormalities at low doses. The current research on environmental EDCs suggests that there may be link between EDC exposure and PCOS, which calls for more human bio-monitoring of EDCs using highly sophisticated analytical techniques for the identification and quantification and to discover the underlying pathophysiology of the disease. This review briefly elaborated on the general etiology of PCOS and listed various epidemiological and experimental data from human and animal studies correlating EDCs and PCOS. This review also provides insights into various analytical tools and sample preparation techniques for biomonitoring studies for PCOS risk assessment. Furthermore, we highlight the role of metabolomics in disease-specific biomarker discovery and its use in clinical practice. It also suggests the way forward to integrate biomonitoring studies and metabolomics to underpin the role of EDCs in PCOS pathophysiology.