Uracil as a biomarker for spatial pyrimidine metabolism in the development of gingivobuccal oral squamous cell carcinoma.

No biomarker has yet been identified that allows accurate diagnosis and prognosis of oral cancers. In this study, we investigated the presence of key metabolites in oral cancer using proton nuclear magnetic resonance (NMR) spectroscopy to identify metabolic biomarkers of gingivobuccal oral squamous cell carcinoma (GB-OSCC). NMR spectroscopy revealed that uracil was expressed in 83.09% of tumor tissues and pyrimidine metabolism was active in GB-OSCC; these results correlated well with immunohistochemistry (IHC) and RNA sequencing data. Based on further gene and protein analyses, we proposed a pathway for the production of uracil in GB-OSCC tissues. Uridinetriphosphate (UTP) is hydrolyzed to uridine diphosphate (UDP) by CD39 in the tumor microenvironment (TME). We hypothesized that UDP enters the cell with the help of the UDP-specific P2Y6 receptor for further processing by ENTPD4/5 to produce uracil. As the ATP reserves diminish, the weakened immune cells in the TME utilize pyrimidine metabolism as fuel for antitumor activity, and the same mechanism is hijacked by the tumor cells to promote their survival. Correspondingly, the differential expression of ENTPD4 and ENTPD5 in immune and tumor cells, respectively, indicatedtheir involvement in disease progression. Furthermore, higher uracil levels were detected in patients with lymph node metastasis, indicating that metastatic potential is increased in the presence of uracil. The presence of uracil and/or expression patterns of intermediate molecules in purine and pyrimidine pathways, such asCD39, CD73, and P2Y6 receptors together with ENTPD4 and ENTPD5, hold promise as biomarker(s) for oral cancer diagnosis and prognosis.

Corrigendum to 'Wound healing efficacy of Jamun honey in diabetic mice model through reepithelialization, collagen deposition and angiogenesis' [J. Trad, Complementary Med 10 (6) (November 2020) 529-543].

[This corrects the article DOI: 10.1016/j.jtcme.2019.10.002.].

Impact of spatial metabolomics on immune-microenvironment in oral cancer prognosis: a clinical report.

MALDI imaging for metabolites and immunohistochemistry for 38 immune markers was used to characterize the spatial biology of 2 primary oral tumours, one from a patient with an early recurrence (Tumour R), and the other from a patient with no recurrence 2 years after treatment completion (Tumour NR). Tumour R had an increased purine nucleotide metabolism in different regions of tumour and adenosine-mediated suppression of immune cells compared to Tumour NR. The differentially expressed markers in the different spatial locations in tumour R were CD33, CD163, TGF-ß, COX2, PD-L1, CD8 and CD20. These results suggest that altered tumour metabolomics concomitant with a modified immune microenvironment could be a potential marker of recurrence.

Single-Cell Proteomics with Spatial Attributes: Tools and Techniques.

Now-a-days, the single-cell proteomics (SCP) concept is attracting interest, especially in clinical research, because it can identify the proteomic signature specific to diseased cells. This information is very essential when dealing with the progression of certain diseases, such as cancer, diabetes, Alzheimer's, etc. One of the major drawbacks of conventional destructive proteomics is that it gives an average idea about the protein expression profile in the disease condition. During the extraction of the protein from a biopsy or blood sample, proteins may come from both diseased cells and adjacent normal cells or any other cells from the disease environment. Again, SCP along with spatial attributes is utilized to learn about the heterogeneous function of a single protein. Before performing SCP, it is necessary to isolate single cells. This can be done by various techniques, including fluorescence-activated cell sorting (FACS), magnetic-activated cell sorting (MACS), laser capture microdissection (LCM), microfluidics, manual cell picking/micromanipulation, etc. Among the different approaches for proteomics, mass spectrometry-based proteomics tools are widely used for their high resolution as well as sensitivity. This Review mainly focuses on the mass spectrometry-based approaches for the study of single-cell proteomics.

Ameliorating effect of rutin against diclofenac-induced cardiac injury in rats with underlying function of FABP3, MYL3, and ANP.

Diclofenac is a widely prescribed anti-inflammatory drug having cardiovascular complications as one of the main liabilities that restrict its therapeutic use. We aimed to investigate for any role of rutin against diclofenac-induced cardiac injury with underlying mechanisms as there is no such precedent to date. The effect of rutin (10 and 20 mg/kg) was evaluated upon concomitant oral administration for fifteen days with diclofenac (10 mg/kg). Rutin significantly attenuated diclofenac-induced alterations in the serum cardiac markers (LDH, CK-MB, and SGOT), serum cytokine levels (TNF-α and IL-6), and oxidative stress markers (MDA and GSH) in the cardiac tissue. Histopathological examination and Scanning Electron Microscopy (SEM) findings displayed a marked effect of rutin to prevent diclofenac-mediated cardiac injury. Altered protein expression of myocardial injury markers (cTnT, FABP3, and ANP) and apoptotic markers (Bcl-2 and Caspase-3) in the cardiac tissue upon diclofenac treatment was considerably shielded by rutin treatment. MYL3 was unaffected due to diclofenac or rutin treatment. Rutin also significantly improved diclofenac-induced gastrointestinal and hepatic alterations based on the observed ameliorative effects in key mediators, oxidative stress markers, histopathology examination, and SEM findings. Overall results suggest that rutin can protect the diclofenac-induced cardiac injury by lowering oxidative stress, inhibiting inflammation, and reducing apoptosis. Further research work directs toward the development of phytotherapeutics for cardioprotection.

Glabridin attenuates paracetamol-induced liver injury in mice via CYP2E1-mediated inhibition of oxidative stress.

CYP2E1 plays a crucial role in the bio-activation of toxic substances leading to liver damage. In this context, CYP2E1 converts paracetamol (PCM) to N-acetyl-p-benzoquinone imine (NAPQI), which is prone to cause hepatotoxicity. Hence, we aimed to explore the protective effect of glabridin on widely used PCM-induced liver injury model in the present study and, after that, correlated with the role of CYP2E1 toward its efficacy. Glabridin was isolated from Glycyrrhiza glabra and characterized before the investigation in an in-vivo mice model of PCM-induced liver injury. Glabridin after oral treatment at 5-20 mg/kg showed a considerable improvement in serum biochemical parameters (ALT and AST) and oxidative stress markers (MDA, GSH, SOD, and catalase) in comparison to only PCM-treatment. Histopathological examination of the liver depicted that glabridin exhibited substantial protection from PCM-induced liver injury compared to the disease control group. Significant down-regulation of CYP2E1 protein and its mRNA expression levels were observed in the glabridin-treated groups compared to PCM-induced respective elevation of CYP2E1. Moreover, activation of NF-κB was significantly inhibited by glabridin. Therefore, glabridin has the potential to protect PCM-induced liver injury through CYP2E1 inhibition-mediated normalization of oxidative stress. Further research is warranted to establish glabridin as a phytotherapeutics for liver protection for which no effective and safe oral drug is available to date.

Glabridin ameliorates methotrexate-induced liver injury via attenuation of oxidative stress, inflammation, and apoptosis.

Despite unprecedented advances in modern medicine, no safe and effective drug is available to date for oral administration to combat drug-induced liver injury, which is a vital concern nowadays. The present study deals with the hepatoprotective effect of pure glabridin, a key phytoconstituent from Glycyrrhiza glabra with mechanistic investigations using an in-vivo methotrexate-induced liver injury model as there is no such precedent. The study was performed in the Swiss mice model where a single dose of methotrexate (40 mg/kg) was given on the 7th day through an intraperitoneal route to induce hepatotoxicity, and glabridin as a test compound was administered orally for eleven consecutive days at 10 to 40 mg/kg. Glabridin markedly improved serum biochemical parameters (SGPT, SGOT), proinflammatory cytokine (TNF-α) level, oxidative stress markers (MDA, GSH, SOD, CAT) as compared to methotrexate alone. Alterations in methotrexate-induced liver architecture were considerably prevented by glabridin treatment as suggested by liver histopathological examination and SEM investigation. Glabridin substantially prevented methotrexate-induced down-regulation of Nrf2, & activation of NF-κB, and caused up-regulation of BAX at different dose levels. Overall, glabridin is found to protect methotrexate-induced hepatotoxicity by improving important factors for oxidative stress, inflammation, and apoptosis.

Label-Free Method Development for Hydroxyproline PTM Mapping in Human Plasma Proteome.

Post-translational modifications (PTMs) impart structural heterogeneities that can alter plasma proteins' functions in various pathophysiological processes. However, the identification and mapping of PTMs in untargeted plasma proteomics is still a challenge due to the presence of diverse components in blood. Here, we report a label-free method for identifying and mapping hydroxylated proteins using tandem mass spectrometry (MS/MS) in the human plasma sample. Our untargeted proteomics approach led us to identify 676 de novo sequenced peptides in human plasma that correspond to 201 proteins, out of which 11 plasma proteins were found to be hydroxylated. Among these hydroxylated proteins, Immunoglobulin A1 (IgA1) heavy chain was found to be modified at residue 285 (Pro285 to Hyp285), which was further validated by MS/MS study. Molecular dynamics (MD) simulation analysis demonstrated that this proline hydroxylation in IgA1 caused both local and global structural changes. Overall, this study provides a comprehensive understanding of the protein profile containing Hyp PTMs in human plasma and shows the future perspective of identifying and discriminating Hyp PTM in the normal and the diseased proteomes.

Flavonoids as potential phytotherapeutics to combat cytokine storm in SARS-CoV-2.

Emergence of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection, COVID-19, has become the global panic since December 2019, which urges the global healthcare professionals to identify novel therapeutics to counteract this pandemic. So far, there is no approved treatment available to control this public health issue; however, a few antiviral agents and repurposed drugs support the patients under medical supervision by compromising their adverse effects, especially in emergency conditions. Only a few vaccines have been approved to date. In this context, several plant natural products-based research studies are evidenced to play a crucial role in immunomodulation that can prevent the chances of infection as well as combat the cytokine release storm (CRS) generated during COVID-19 infection. In this present review, we have focused on flavonoids, especially epicatechin, epigallocatechin gallate, hesperidin, naringenin, quercetin, rutin, luteolin, baicalin, diosmin, ge nistein, biochanin A, and silymarin, which can counteract the virus-mediated elevated levels of inflammatory cytokines leading to multiple organ failure. In addition, a comprehensive discussion on available in silico, in vitro, and in vivo findings with critical analysis has also been evaluated, which might pave the way for further development of phytotherapeutics to identify the potential lead candidatetoward effective and safe management of the SARS-CoV-2 disease.

Wound healing efficacy of Jamun honey in diabetic mice model through reepithelialization, collagen deposition and angiogenesis.

Diabetic patients are frequently afflicted with impaired wound healing where linear progression of molecular and cellular events compromised. Despite of meaningful progress in diabetic treatment, management of diabetic chronic wounds is still challenging. Jamun (Syzygium cumini) honey may be a promising candidate for diabetic wound healing and need to explore in detail. So present study was designed to evaluate the efficacy of Jamun honey (JH) for diabetic wound healing in in vitro wound (primary fibroblasts) model and in in vivo of diabetic mice (Streptozotocin induced) model. The fibroblast cell model was studied for migratory behaviour and myofibrolasts infiltration under honey interventions via scratch/migration assay, immuno-cytochemistry and western blot. We applied FDA approved Manuka honey (MH) as positive control and JH as test honey to evaluate wound re-epithelialization, sub-epithelial connective tissue modification and angiogenesis via histo-pathological and immuno-histochemical analysis. JH (0.1% v/v) dilution has notably improved wound closure, migration with concomitant α-SMA expressions in vitro. Topical application of JH in diabetic mice model showed significant (*p ≤ 0.05) wound closure, reepithelialization, collagen deposition (I/III) and balanced the myofibroblasts formation. It also modulated vital angiogenic markers (viz HIF-1α, VEGF, VEGF R-II) significantly (*p ≤ 0.05). All these observations depicted that JH promotes sequential stages of wound healing in diabetic mice model. The results of the present study established Jamun honey as good as Manuka honey considering wound closure, re-epithelialization, collagen deposition and pro-angiogenic potential.

Density of CD3+ and CD8+ cells in gingivo-buccal oral squamous cell carcinoma is associated with lymph node metastases and survival.

The tumor immune microenvironment is emerging as a critical player in predicting cancer prognosis and response to therapies. However, the prognostic value of tumor-infiltrating immune cells in Gingivo-Buccal Oral Squamous Cell Carcinoma (GBOSCC) and their association with tumor size or lymph node metastases status require further elucidation. To study the relationship of tumor-infiltrating immune cells with tumor size (T stage) and lymph node metastases (N stages), we analyzed the density of tumor-infiltrating immune cells in archived, whole tumor resections from 94 patients. We characterized these sections by immune-histochemistry using 12 markers and enumerated tumor-infiltrating immune cells at the invasive margins (IM) and centers of tumors (CT). We observed that a higher density of CD3+ cells in the IM and CT was associated with smaller tumor size (T1-T2 stage). Fewer CD3+ cells was associated with larger tumor size (T3-T4 stage). High infiltration of CD3+and CD8+ cells in IM and CT as well as high CD4+ cell infiltrates in the IM was significantly associated with the absence of lymph node metastases. High infiltrates of CD3+ and CD8+ cells in CT was associated with significantly improved survival. Our results illustrate that the densities and spatial distribution of CD3+ and CD8+ cell infiltrates in primary GBOSCC tumors is predictive of disease progression and survival. Based on our findings, we recommend incorporating immune cell quantification in the TNM classification and routine histopathology reporting of GBOSCC. Immune cell quantification in CT and IM may help predict the efficacy of future therapies.

Hypoxic Transformation of Immune Cell Metabolism Within the Microenvironment of Oral Cancers.

Oral squamous cell carcinoma (OSCC) includes tumors of the lips, tongue, gingivobuccal complex, and floor of the mouth. Prognosis for OSCC is highly heterogeneous, with overall 5-year survival of ~50%, but median survival of just 8-10 months for patients with locoregional recurrence or metastatic disease. A key feature of OSCC is microenvironmental oxygen depletion due to rapid growth of constituent tumor cells, which triggers hypoxia-associated signaling events and metabolic adaptations that influence subsequent tumor progression. Better understanding of leukocyte responses to tissue hypoxia and onco-metabolite expression under low-oxygen conditions will therefore be essential to develop more effective methods of diagnosing and treating patients with OSCC. This review assesses recent literature on metabolic reprogramming, redox homeostasis, and associated signaling pathways that mediate crosstalk of OSCC with immune cells in the hypoxic tumor microenvironment. The likely functional consequences of this metabolic interface between oxygen-starved OSCC and infiltrating leukocytes are also discussed. The hypoxic microenvironment of OSCC modifies redox signaling and alters the metabolic profile of tumor-infiltrating immune cells. Improved understanding of heterotypic interactions between host leukocytes, tumor cells, and hypoxia-induced onco-metabolites will inform the development of novel theranostic strategies for OSCC.

Elucidation of Differential Nano-Textural Attributes for Normal Oral Mucosa and Pre-Cancer.

Computational analysis on altered micro-nano-textural attributes of the oral mucosa may provide precise diagnostic information about oral potentially malignant disorders (OPMDs) instead of an existing handful of qualitative reports. This study evaluated micro-nano-textural features of oral epithelium from scanning electron microscopic (SEM) images and the sub-epithelial connective tissue from light microscopic (LM) and atomic force microscopic (AFM) images for normal and OPMD (namely oral sub-mucous fibrosis, i.e., OSF). Objective textural descriptors, namely discrete wavelet transform, gray-level co-occurrence matrix (GLCM), and local binary pattern (LBP), were extracted and fed to standard classifiers. Best classification accuracy of 87.28 and 93.21%; sensitivity of 93 and 96%; specificity of 80 and 91% were achieved, respectively, for SEM and AFM. In the study groups, SEM analysis showed a significant (p < 0.01) variation for all the considered textural descriptors, while for AFM, a remarkable alteration (p < 0.01) was only found in GLCM and LBP. Interestingly, sub-epithelial collagen nanoscale and microscale textural information from AFM and LM images, respectively, were complementary, namely microlevel contrast was more in normal (0.251) than OSF (0.193), while nanolevel contrast was more in OSF (0.283) than normal (0.204). This work, thus, illustrated differential micro-nano-textural attributes for oral epithelium and sub-epithelium to distinguish OPMD precisely and may be contributory in early cancer diagnostics.

Identification of α-enolase as a prognostic and diagnostic precancer biomarker in oral submucous fibrosis.

AIMS: Diagnostic ambiguities regarding the malignant potentiality of oral submucous fibrosis (OSF), an oral precancerous condition having dysplastic and non-dysplastic isoforms are the major failure for early intervention of oral squamous cell carcinoma (OSCC) patients. Our goal is to identify proteomic signatures from biopsies that can be used as precancer diagnostic marker for patient suffering from OSF. METHODS: The high throughput techniques adopting de novo peptide sequencing (1D SDS-PAGE coupled nanoLC MALDI tandem mass spectrometry (MS/MS)-based peptide mass fingerprint), immunohistochemistry (IHC), Western blot (WB) and real-time PCR (RT-PCR) analysis are considered for such biomarker identification and multilevel validations. RESULTS: Alpha-enolase is identified as an overexpressed protein in biopsies of oral submucous fibrosis with dysplasia (OSFWD) compared with oral submucous fibrosis without dysplasia (OSFWT) and normal oral mucosa (NOM). Total proteome analysis of an overexpressed protein band around 47 kDa of OSFWD identifies 334 peptides corresponding to 61 human proteins. Among them α-enolase is identified as a prime protein with highest number of peptides (44 out of 334 peptides) and sequence coverage (66.4%). Furthermore, RT-PCR, WB and IHC analysis also show mRNA and tissue level upregulation of α-enolase in OSFWD validating α-enolase as precancer marker. CONCLUSIONS: This study for the first time identifies and validates α-enolase as a novel biomarker for early diagnosis of malignant potentiality of OSF. Hence, the identified protein marker, α-enolase can help in early therapeutic intervention of OSF patients leading to the reduction of patient's pain, treatment cost and enhancement of patient's quality of life.

Spectropathology-corroborated multimodal quantitative imaging biomarkers for neuroretinal degeneration in diabetic retinopathy.

INTRODUCTION: Image-based early detection for diabetic retinopathy (DR) needs value addition due to lack of well-defined disease-specific quantitative imaging biomarkers (QIBs) for neuroretinal degeneration and spectropathological information at the systemic level. Retinal neurodegeneration is an early event in the pathogenesis of DR. Therefore, development of an integrated assessment method for detecting neuroretinal degeneration using spectropathology and QIBs is necessary for the early diagnosis of DR. METHODS: The present work explored the efficacy of intensity and textural features extracted from optical coherence tomography (OCT) images after selecting a specific subset of features for the precise classification of retinal layers using variants of support vector machine (SVM). Fourier transform infrared (FTIR) spectroscopy and nuclear magnetic resonance (NMR) spectroscopy were also performed to confirm the spectropathological attributes of serum for further value addition to the OCT, fundoscopy, and fluorescein angiography (FA) findings. The serum metabolomic findings were also incorporated for characterizing retinal layer thickness alterations and vascular asymmetries. RESULTS: Results suggested that OCT features could differentiate the retinal lesions indicating retinal neurodegeneration with high sensitivity and specificity. OCT, fundoscopy, and FA provided geometrical as well as optical features. NMR revealed elevated levels of ribitol, glycerophosphocholine, and uridine diphosphate N-acetyl glucosamine, while the FTIR of serum samples confirmed the higher expressions of lipids and ß-sheet-containing proteins responsible for neoangiogenesis, vascular fragility, vascular asymmetry, and subsequent neuroretinal degeneration in DR. CONCLUSION: Our data indicated that disease-specific spectropathological alterations could be the major phenomena behind the vascular attenuations observed through fundoscopy and FA, as well as the variations in the intensity and textural features observed in OCT images. Finally, we propose a model that uses spectropathology corroborated with specific QIBs for detecting neuroretinal degeneration in early diagnosis of DR.

Honey Extracted Polyphenolics Reduce Experimental Hypoxia in Human Keratinocytes Culture.

Hypoxic assault affects fundamental cellular processes and generates oxidative stress on healthy cells/molecules. Honey extracted polyphenolics (HEP) as a natural antioxidant reduced hypoxic cytotoxicity in this study. Different honey samples were physicochemically characterized to identify preferred (jamun) honey [pH 3.55 ± 0.04, conductivity (µs/cm) = 6.66 ± 0.14, water content % (w/w) = 14.70 ± 0.35, total solid content % (w/w) = 85.30 ± 0.35, phenol content (mg GAE/100 g) = 403.55 ± 0.35, flavonoid content (mg QE/100 g) = 276.76 ± 4.10, radical scavenging activity (% 500 µL) = 147.75 ± 3.13, catalase activity (absorbance at 620 nm) = 0.226 ± 0.01]. HEP was tested in different doses on hypoxic and normoxic cells (HaCaT) using viability and antioxidant assays. Cardinal molecular expressions such as cadherin-catenin-cytoskeleton complex (namely, E-cadherin, ß-catenin, and F-actin), hypoxia marker (Hif 1 α), proliferation marker (Ki67), and epithelial master regulator (p63) were studied by immuno-cytochemisty (ICC) and qRT-PCR. The 0.063 mg/mL HEP demonstrated better vitality and functionality of HaCaT cells as per viability assay (*, P < 0.01) even under hypoxia. ICC and qRT-PCR observations indicated restoration of cellular survival and homeostasis under 0.063 mg/mL HEP after hypoxic assault. Furthermore, major spectral changes for nucleic acid and membrane phospholipid reorganizations by Fourier transform infrared spectroscopy illustrated a positive impact of 0.063 mg/mL HEP on hypoxic cells considering proliferation and cellular integrity. It was concluded that a specific dose of jamun HEP reduces hypoxic cytotoxicity.

Computer-aided molecular pathology interpretation in exploring prospective markers for oral submucous fibrosis progression.

BACKGROUND: Evaluation of molecular pathology markers using a computer-aided quantitative assessment framework would help to assess the altered states of cellular proliferation, hypoxia, and neoangiogenesis in oral submucous fibrosis and could improve diagnostic interpretation in gauging its malignant potentiality. METHODS: Immunohistochemical (IHC) expression of c-Myc, hypoxia-inducible factor-1-alpha (HIF-1α), vascular endothelial growth factor (VEGF), VEGFRII, and CD105 were evaluated in 58 biopsies of oral submucous fibrosis using computer-aided quantification. After digital stain separation of original chromogenic IHC images, quantification of the diaminobenzidine (DAB) reaction pattern was performed based on intensity and extent of cytoplasmic, nuclear, and stromal expression. RESULTS: Assessment of molecular expression proposed that c-Myc and HIF-1α may be used as strong screening markers, VEGF for risk-stratification and VEGFRII and CD105 for prognosis of precancer into oral cancer. CONCLUSION: Our analysis indicated that the proposed method can help in establishing IHC as an effective quantitative immunoassay for molecular pathology and alleviate diagnostic ambiguities in the clinical decision process.

Connecting cyto-nano-architectural attributes and epithelial molecular expression in oral submucous fibrosis progression to cancer.

OBJECTIVE: Problems in pre-cancer diagnosis complicate cancer theragnosis as well as life expectancy. There is uncertainty regarding malignant transformation of oral submucous fibrosis (OSF), an oral pre-cancer with dysplastic (OSFWD) and non-dysplastic (OSFWT) subtypes. Understanding the structural, molecular and physical aspects of epithelial homeostasis may be useful. MATERIALS AND METHODS: Histopathological grading of biopsy sections was performed using H&E staining. Alterations in epithelial surface architecture in different groups was evaluated using scanning electron microscopy (SEM). The expression of crucial epithelial genes (p63, CK-5/6, CK-10, E-cadherin and ß-catenin) was studied by immunohistochemistry, Western blot and RT-PCR analysis. RESULTS: SEM observations revealed that the surface epithelial ridge pattern became thick and dense, and pit pattern gradually decreased in OSFWD and oral squamous cell carcinoma (OSCC). p63, ΔNp63 and CK-5/6 were up-regulated in OSFWD and OSCC but down-regulated in OSFWT. CK-10 was down-regulated in OSFWD compared to OSFWT. Cytoplasmic expression of E-cadherin and ß-catenin was elevated in dysplastic and cancerous conditions. Moreover, statistical correlation between SEM features (ridges and pits) and molecular attributes demonstrated a significant positive relationship between the ridge-to-pit ratio and p63 population density (r=0.85) and the ridge-to-pit ratio and CK-5/6 intensity (r=0.63). CONCLUSIONS: Molecular changes related to epithelial progressive maturation and cellular proliferation are correlated with concomitant alteration of epithelial surface architecture which helps to predict the malignant potentiality of OSF.

Honey dilution impact on in vitro wound healing: Normoxic and hypoxic condition.

Honey is known as a popular healing agent against tropical infections and wounds. However, the effects of honey dilutions on keratinocyte (HaCaT) wound healing under hypoxic condition is still not explored. In this study, we examined whether honey dilution have wound healing potential under hypoxic stress. The antioxidant potential and healing efficacy of honey dilution on in vitro wound of human epidermal keratinocyte (HaCaT cells) under hypoxia (3% O2 ), and normoxia is explored by nitro blue tetrazolium assay. The cell survival % quantified by MTT assay to select four honey dilutions like 10, 1, 0.1, and 0.01 v/v% and the changes in cellular function was observed microscopically. Further, the cell proliferation, migration, cell-cell adhesion, and relevant gene expression were studied by flow cytometry, migration/scratch assay, immunocytochemistry, and reverse transcription-polymerase chain reaction, respectively. The expression pattern of cardinal molecular features viz. E-cadherin, cytoskeletal protein F-actin, p63, and hypoxia marker Hif 1α were examined. Honey dilution in 0.1% v/v combat wound healing limitations in vitro under normoxia and hypoxia (3%). Its wound healing potential was quantified by immunocytochemistry and real-time PCR for the associated molecular features that were responsible for cell proliferation and migration. Our data showed that honey dilution can be effective in hypoxic wound healing. Additionally, it reduced superoxide generation and supplied favorable bioambience for cell proliferation, migration, and differentiation during hypoxic wound healing. These findings may reveal the importance of honey as an alternative and cost effective therapeutic natural product for wound healing in hypoxic condition.

Modulating prime molecular expressions and in vitro wound healing rate in keratinocyte (HaCaT) population under characteristic honey dilutions.

ETHNOPHARMACOLOGY RELEVANCE: In traditional medicines honey is known for healing efficacy and vividly used as "Anupan" in Ayurvedic medicines appreciating roles in dilutions. Validating efficacy of physico-chemically characterized honey in dilutions, studies on in vitro wound healing and attainment of cellular confluence epithelial cells including expressions of cardinal genes is crucial. To evaluate effects of characterized honey in varied dilutions on cellular viability, in vitro wound healing and modulation of prime epithelial gene expressions. MATERIALS AND METHODS: Six Indian honey-samples from different sources were physico-chemically characterized and optimal one was explored in dilutions (v/v%) through in vitro studies on human epithelial (HaCaT) cells for viability, wound healing and expressions of genes p63, E-cadherin, ß-catenin, GnT-III and GnT-V. RESULTS: Studied honey samples (i.e. A-F) depicted range of pH (2-4), water (12.48-23.95), electrical conductivity (2.57-14.34), carbohydrate (68.73-98.65), protein (.316-5.36) and antioxidant potential. Though sample A and F showed physico-chemical proximity, but overall bio-impact of the earlier was better, thus studied in 8-.1% (v/v) dilution range. Four dilutions (.01, .04, .1, .25 v/v%) augmented cellular viability but in vitro wound healing was fastest (p<.05) under .1%. Such efficacy was further documented for p63 up-regulation by immunocytochemistry and mRNA studies. The E-cadherin and ß-catenin mRNA-expressions were also up-regulated and their proteins were predominantly cytoplasmic. E-cadherin up-regulation was corroborative with down-regulation and up-regulation of GnT-III and GnT-V respectively. CONCLUSION: Present study illustrated efficacy of particular honey dilution (.1%) with characteristic free radical scavenging activity in facilitating cell proliferation and attainment of confluence towards faster wound healing and modulation of cardinal epithelial genes (viz. p63, E-cadherin, ß-catenin, Gnt-III and V).