Total Hydroxyproline Research Articles

Quantification of extra-cardiac damage following acute myocardial infarction using cis- and trans-4-[18F]fluoro-L-proline PET imaging

The preclinical surgically-induced models of myocardial infarction (MI) have been extensively utilised for studying the wound healing response in the mammalian heart. It is well-established that these invasive procedures induce marked acute systemic inflammation and adverse haemodynamic derangements, which can result in extra-cardiac tissue injury and remodelling; however, the extent of this has not yet been robustly characterised. Here, using a rat permanent coronary artery ligation (PL) model of MI and the cis- and trans-4-[18F]fluoro-l-proline positron emission tomography (PET) probes, targeting active misfolded and triple-helical collagen biosynthesis, respectively, we conducted an exploratory time-course analysis of the associated extra-cardiac tissue remodelling post-injury. Our results showed an increased cis-4-[18F]fluoro-l-proline PET signal in the liver (P = 0.02; two sample t-test) and lung (P = 0.03; two sample t-test) at the acute day 7 timepoint in the MI group compared to the Sham group (n = 3-6), which was not apparent at the later timepoints (days 14, 28, and 84). Ex vivo quantification of the deposited collagen in extra-cardiac tissues using Sirius red histochemical staining and the total hydroxyproline assay validated the apparent time course trends observed with [18F]fluoro-l-proline PET. These findings suggest that the liver and lungs are susceptible to acute injury in the rat PL model of MI and align with numerous clinical reports of extracardiac injury in MI patients. Moreover, our results highlight the suitability of [18F]fluoro-l-proline PET as a systems-based imaging technique for investigating active collagen biosynthesis across multiple organs.

Interstitial electrical stimulation for middle-aged, and elderly adults with early stages of knee osteoarthritis

Introduction The adequate, pathogenetically substantiated pain management is essential for treatment of early stages of deforming osteoarthritis of the knee joint. There is a need to explore the effectiveness and mechanisms of modern methods of electrical therapy and their impact on the quality of life due to close cause-and-effect relationship between the pain, inflammatory and destructive components of osteoarthritis (OA) as one of the most common conditions.The objective was to evaluate the effect of interstitial electrical stimulation (IES) as a monotherapy in the course of conservative treatment of early stages of gonarthrosis based on clinical and biochemical findings.Materials and methods Radiographic findings, skin electrometry (objective parameters of pain intensity), limb weight-bearing capacity, biochemical parameters of connective tissue matrix degradation in blood serum and 24-hour urine samples, and treatment satisfaction were explored in 43 patients. Patients who received a course of IES were assigned to the treatment group (n = 22) and patients who received standard treatment without IES constituted the control group (n = 21).Results Electrometric analysis showed a higher effectiveness of pain relief in the treatment group compared to the controls with asymmetry coefficient measuring 3.2 ± 0.31 versus 1.9 ± 0.4 at p > 0.05. The weight‑bearing scored 3.59 ± 0.34 versus 3.26 ± 0.2 at p > 0.05. The treatment group showed shorter treatment length with 13.21 ± 1.2 days versus 18.3 ± 1.2 days in the control group at p > 0.05 and a higher degree of satisfaction with outcomes scored 96.2 ± 2.59 in the treatment group versus 86.2 ± 3.17 in the control group. A statistically significant difference (p > 0.05) was established in the levels of free and total hydroxyproline characterizing the different intensity of collagen breakdown in the groups at the end of treatment.Discussion The IES used as an analgesic and anti-inflammatory therapy was shown to be highly effective with changes in the hydroxyproline level in the media suggesting a chondroprotective effect. The analysis and comparison of objective parameters demonstrated high effectiveness of IES in the treatment of early stages of gonarthrosis in middle-aged and elderly adults.Conclusion The IES used for treatment of early stages of gonarthrosis helps pain reducing the intensity of collagen destruction and improving weight-bearing.

407 The OGT/O-GlcNAc Axis Regulates Fibrosis Resolution in Idiopathic Pulmonary Fibrosis

OBJECTIVES/GOALS: Idiopathic pulmonary fibrosis (IPF) is a chronic lung disease characterized by dysregulated collagen accumulation in the lung parenchyma. Our goal is to investigate the role of O-linked N-Acetylglucosamine (O-GlcNAc) transferase (OGT) in pulmonary fibrosis to ultimately discover novel therapies for fibrosis resolution. METHODS/STUDY POPULATION: Lung tissue from IPF and non-IPF donors was subjected to immunohistochemistry (IHC) to assess O-GlcNAc levels. Primary human lung fibroblasts were treated with OGT or O-GlcNAcase (OGA) inhibitors followed by transforming growth factor-beta 1 (TGF-β1) stimulation to assess O-GlcNAc regulation of fibroblast-to-myofibroblast transition (FMT) markers [alpha smooth muscle actin (α-SMA) and type 1 and type 3 collagen (COL1α1, COL3α1)] In Drosophila melanogaster, OGT knockdown (KD)/overexpression (OE) was conditionally induced to assess pericardin, a type IV collagen-like protein, regulation by immunofluorescence. Lastly, a mouse model of bleomycin-induced pulmonary fibrosis was examined following OGT KD and assessed for fibrosis resolution via histology, hydroxyproline assay, and western blotting. RESULTS/ANTICIPATED RESULTS: O-GlcNAc staining was increased in IPF lung tissue compared to non-IPF control lungs. In primary human lung fibroblasts, TGF-α1 administration resulted in increased FMT markers (α-SMA, COL1α1, and COL3α1), which were reduced or increased by OGT or OGA inhibition, respectively. Genetic manipulation in the Drosophila models showed decreased pericardin expression with OGT KD compared to the wild-type, whereas OGT OE increased pericardin compared to control. Additionally, OGT KD in bleomycin treated aged mice resulted in reduced collagen levels at the transcript and protein level and concurrent fibrosis resolution as assessed by Masson’s trichrome staining and total hydroxyproline analysis. Collectively, showing OGT/O-GlcNAc regulating collagen in fibrosis resolution. DISCUSSION/SIGNIFICANCE: These data suggest that the OGT/O-GlcNAc axis regulates collagen deposition in pulmonary fibrosis, and we show that O-GlcNAc is implicated in the pathogenesis of IPF. We identified OGT as a therapeutic target to overcome current drug limitations, opening new horizons for biomedical treatment.

A Comprehensive Investigation of Tenderization Methods: Evaluating the Efficacy of Enzymatic and Non-Enzymatic Methods in Improving the Texture of Squid Mantle — A Detailed Comparative Study

AbstractIn this study, enzymatic [papain (P), bromelain (B), and A. oryzae-derived fungal protease (FP); enzyme ratios: 0.012 (v/v), 38 °C, 50 min] and non-enzymatic [control (C; saline), mineral water (95%)–lemon juice (5%) (ML) (1/2:w/v), 38 °C, 50 min)] tenderizing methods on European squid mantle (Loligo vulgaris Lamarck, 1798) were investigated. Scanning electron microscope (SEM) imaging, water holding capacity (WHC), cooking loss (CL), total free amino acids (TFAA), total soluble protein (TSP), hydroxyproline (Hyp), and Fourier-transform infrared spectroscopy (FTIR) analyses were performed on uncooked samples. pH, water activity (aw), color, nutritional changes, amino acids (AA), total volatile basic-nitrogen (TVB-N), Warner-Bratzler shear (WBS), texture profile analysis (TPA), and sensory evaluations were performed on both uncooked and cooked samples. A significant decrease in protein content was observed in group P (12.86%) compared to untreated (U) squid (15.16%). During frying, group P absorbed more cooking oil (8.37%) than the other groups. A higher degree of hydrolysis was observed in the enzymatically tenderized groups than in non-enzymatic groups, and a shift in secondary protein structures in favor of random coils. Hydrolysis was confirmed by Hyp and AA analyses. The total AA content order was found as B < P < FP < C < ML < U. ML and FP groups have relatively higher total bitter and umami AA compositions, especially in uncooked samples. FP and ML groups had more favorable results across all analyses, especially in terms of the sensory results, nutritional values, and TPA results of cooked samples. It is concluded that both methods are very convenient regarding industrial usage.

A Novel Food Bore Protein Hydrogel with Silver Ions for Promoting Burn Wound Healing.

Hydrogels have emerged as a promising option for treating local scald wounds due to their unique physical and chemical properties. This study aims to evaluate the efficacy of ovalbumin/gelatin composite hydrogels in repairing deep II-degree scald wounds using a mouse dorsal skin model. Trauma tissues collected at various time points are analyzed for total protein content, hydroxyproline content, histological features, and expression of relevant markers. The results reveal that the hydrogel accelerates the healing process of scalded wounds, which is 17.27% higher than the control group. The hydrogel treatment also effectively prevents wound enlargement and redness of the edges caused by infection during the initial stage of scalding. The total protein and hydroxyproline content of the treated wounds are significantly elevated. Additionally, the hydrogel up-regulates the expression of VEGF (a crucial angiogenic factor) and down-regulates CD68 (a macrophage marker). In summary, this study provides valuable insights into the potential of multifunctional protein-based hydrogels in wound healing.

The Effect of Variation in Dietary Cation-Anion Difference on Calcium Status, Blood Metabolites and Rumen Activity during the Transition Period of Holstein Dairy Cows

A negative dietary cation-anion difference (DCAD) induces a compensated metabolic acidosis, stimulating calcium (Ca) absorption and mobilization before calving, thereby decreasing clinical and subclinical hypocalcemia postpartum. The study was designed to determine the effects of varying pre- and postpartum DCAD diets on serum total calcium, ionized calcium, blood and ruminal fluid metabolites, and milk production in prepartum and postpartum Holstein cows. Fifty-four multiparous dry Holstein cows n= 54, were enrolled in a completely randomized block experimental design at 29 days prior to expected parturition through 86 days in milk. A 3 x 2 factorial arrangement of treatments was utilized. Three DCAD levels were fed precalving (0, -120 and -200 mEq/kg DM), n=18 cows per treatment and two DCAD levels were fed post calving (+200 and +400 mEq/kg DM), n=27 cows per treatment. Prepartum urine pH was lower for cows fed -200 DCAD compared with those fed -120 or 0 DCAD. Postpartum urine pH was higher for cows fed +400 mEq/kg compared to cows fed +200 mEq/kg DCAD. Prepartum serum total calcium, ionized calcium, and hydroxyproline was highest for cows fed -200 DCAD compared to those fed -120 and 0 DCAD. Parathyroid hormone was highest for cows fed 0 DCAD compared to those fed -120 and -200 DCAD. Prepartum dry matter intake (DMI) was lower for -200 and -120 DCAD compared with 0 DCAD. Postpartum DMI was not different among treatments. Pre- and postpartum DCAD treatments did not affect total milk yield or milk fat, percentage of milk protein not affected by different pre-and postpartum DCAD levels. Prepartum anionic diets lowered urine pH and parathyroid hormone and raised serum hydroxyproline, resulting in improved Ca availability after parturition. Postpartum blood metabolites were unaffected in cows given positive DCAD (+200 and +400 mEq/kg DM). Calves born to cows fed low DCAD had no change in calf bieth weight. Colostrum amount and IgG concentrations were unaffected by treatments. No effects of pre-or postpartum DCAD treatments were observed for milk yield and fat-corrected milk. Feeding prepartum an acidogenic diet improved postpartum Ca status in multiparous Holstein cows.

Topical Application of Siberian Pine Essential Oil Formulations Enhance Diabetic Wound Healing.

This study aimed to develop novel topical formulations based on a natural component (0.5% of Siberian pine essential oil) and to assess its wound-healing capacity through macroscopic, histopathological, and biochemical examination. The phytochemical profile of Pinus sibirica essential oil (PSEO) and rheological analysis and safety potential of formulations were determined. The wound-healing effect was evaluated on an excision wound model in diabetic Wistar albino rats randomly divided into the following groups topically treated with (1) untreated, (2) 1% silver sulfadiazine, (3) ointment base, (4) gel base, (5) PSEO ointment, and (6) PSEO gel. Formulations containing PSEO were stable and safe for skin application. Three weeks of treatment with both PSEO formulations (ointment and gel) led to a significant reduction in wound size (98.14% and 96.28%, respectively) and a remarkably higher level of total hydroxyproline content (9.69 µg/mg and 7.26 µg/mg dry tissue, respectively) relative to the control group (65.97%; 1.81 µg/mg dry tissue). These findings were in correlation with histopathological results. Topically applied PSEO formulations were associated with a significant reduction in most of the measured pro-oxidants and enhanced activity of the antioxidant defense system enzymes (p < 0.05). Our findings showed that gel and ointment with PSEO demonstrated significant wound-repairing capabilities in the excision wound model.

Manilkara zapota L. extract topical ointment application to skin wounds in rats speeds up the healing process.

Poor circulation, unresolved inflammation, neuropathy, and infection make wound care difficult. Manilkara zapota (M. zapota) antibacterial and antioxidant properties may help speed up the healing process. The present investigation aimed to evaluate the wound healing activity of M. zapota bark ethanolic extract (MZE) by employing in-vitro migration scratch assay and in-vivo animal models. Wistar albino rats were used for the in-vivo wound healing models. No treatment was given to Group I; Group II received povidone-iodine (5% W/W); Group III received MZE (5% W/W); and Group IV received MZE (10% W/W). Linear incision models and excision wound models were used to induce injury. The ointments were applied immediately to the wounds after causing the injury. The percentage of wound contraction, the length of the epithelization period, and the wound's tensile strength were all calculated. The scratch assay assessed the test drug's potential for wound healing in-vitro. H2O2 and DPPH scavenging assays were used to measure antioxidant activity. A p < 0.05 was used to define statistical significance. On days 4, 8, 12, 16, and 20, the wound contraction potential of animals treated with MZE ointment was significantly higher (p < 0.001) than that of the control group. On day 20, the proportion of wound contraction in MZE-treated animals was 99.88%, compared to 83.86% in untreated animals. The test group had a significantly (p < 0.01) faster time to full epithelization than the control group. In the incision model, the control group had considerably lower mechanical strength (p < 0.001) than animals treated with MZE. In addition, MZE caused a significant increase (p < 0.001) in total protein and hydroxyproline levels. In the scratch experiment, test drug-treated cells showed a higher rate of cell migration than untreated cells. Furthermore, animals treated with MZE showed increased levels of epithelial tissue, collagen proliferation, and keratinization. To summarize, the current study found that M. zapota improved wound healing activity both in vitro and in vivo, as evidenced by the study results. M. zapota extract has significant wound-healing potential and could be a viable source of wound-healing nutraceuticals.

Urinary collagen peptides: Source of markers for bone metabolic processes in kidney transplant recipients.

Kidney transplant recipients (KTRs) are at an increased risk of fractures. Total urinary hydroxyproline excretion served as marker for bone resorption (BR) but was replaced by β-CrossLaps (CTX), a C-terminal collagen α-1(I) chain (COL1A1) telopeptide. We investigated the low-molecular-weight urinary proteome for peptides associated with changes in bone metabolism after kidney transplantation. Clinical and laboratory data including serum levels of CTX in 96 KTR from two nephrology centers were correlated with signal intensities of urinary peptides identified by capillary electrophoresis mass spectrometry. Eighty-two urinary peptides were significantly correlated with serum CTX levels. COL1A1 was the predominant peptide source. Oral bisphosphonates were administered for decreased bone density in an independent group of 11 KTR and their effect was evaluated on the aforementioned peptides. Study of the peptides cleavage sites revealed a signature of Cathepsin K and MMP9. Seventeen of these peptides were significantly associated with bisphosphonate treatment, all showing a marked reduction in their excretion levels compared to baseline. This study provides strong evidence for the presence of collagen peptides in the urine of KTR that are associated with BR and that are sensitive to bisphosphonate treatment. Their assessment might become a valuable tool to monitor bone status in KTR.

Total sesquiterpenoids from Eupatorium lindleyanum DC. attenuate bleomycin-induced lung fibrosis by suppressing myofibroblast transition

Eupatorium lindleyanum DC. has been used as a functional food in China for a long time. However, the antifibrotic activity of total sesquiterpenoids from Eupatorium lindleyanum DC. (TS-EL) is still unknown. In this study, we discovered that TS-EL reduced the increase in α-smooth muscle actin (α-SMA), type I collagen and fibronectin content, the formation of cell filaments and collagen gel contraction in transforming growth factor-β1-stimulated human lung fibroblasts. Intriguingly, TS-EL did not change the phosphorylation of Smad2/3 and Erk1/2. TS-EL decreased the levels of serum response factor (SRF), a critical transcription factor of α-SMA, and SRF knockdown alleviated the transition of lung myofibroblasts. Furthermore, TS-EL significantly attenuated bleomycin (BLM)-induced lung pathology and collagen deposition and reduced the levels of two profibrotic markers, total lung hydroxyproline and α-SMA. TS-EL also decreased the levels of SRF protein expression in BLM-induced mice. These results suggested that TS-EL attenuates pulmonary fibrosis by inhibiting myofibroblast transition via the downregulation of SRF.

Removal of Proteoglycans in Animal Hides by Glycosidase to Promote Opening-Up of Collagen Fiber Bundles

The degree of proteoglycan removal from animal hides is closely related to the opening-up of collagen fibers and the properties of leather, but conventional leather-making processes are not very effective at removing proteoglycans. By rationally selecting and utilizing glycosidases during the leather-making process, proteoglycans can be efficiently removed without causing collagen damage due to glycosidases specifically hydrolyzing glycosidic bonds. In spite of this, glycosidases are highly specific and come in a wide variety of types. To solve the blindness of optimizing glycosidases and its application in the soaking process, in this study, seven typical commercial glycosidases capable of hydrolyzing β-glycosidic bonds were selected according to the types of major glycosidic bonds in animal hide proteoglycans. The basic properties of the selected glycosidases and its effectiveness in removing proteoglycans from the hide during the soaking process were investigated. Then, the effects of the dosage of the two optimal glycosidases on the dissolution of polysaccharide, total soluble protein and hydroxyproline, as well as the properties of leather during the soaking process were further studied. The results showed that although the optimal pH of the selected glycosidases was between pH 5 - 7, they maintained more than 50% of their activity under weak alkaline conditions and could be applied to the soaking process. The removal of proteoglycan by different glycosidases is closely related to their specificity for glycosidic bonds, and the penetrability of the enzyme proteins, namely the molecular weight and isoelectric point of the enzymes. Among them, β-Mannase-AKT and Xylanase-AKT are more effective in removing proteoglycans. When the dosage of β-Mannase-AKT and Xylanase-AKT reached 47 U/mL, almost all the glycosaminoglycan was removed from the hide, the opening-up of collagen fibers and the softness and physical properties of the crust leather were significantly improved without damage to the structural proteins.

The vulnerability of the human taenia coli to alterations in total collagen within the colon of the elderly

The structure of the colonic wall relies on collagen, distributed within the submucosa and the muscularis externa. A recent analysis of total collagen in human ascending colon (AC) suggests that the muscularis externa is more susceptible to age-related increases in collagen among the elderly. However, it is not clear if this change also occurs in the descending colon (DC) or if the circular and longitudinal muscle layers are similarly affected in either region of colon. The aim of this study is to determine the total collagen content in the DC and its distribution between the circular muscle (CM) and taenia coli (TC) of the AC and DC of adults and compare the same with tissue from the elderly. Masson’s trichrome and Picrosirius red were used to assess total collagen content in the AC and DC; aged 22 – 91 years. Macroscopically normal AC from 22 patients (adults: 22–60 years; 6 male, 6 female; elderly: 70 – 91 years; 6 male, 4 female) and DC from 23 patients (adults: 23–63 years; 6 male, 7 female; elderly: 66 – 88 years; 6 male, 4 female) were obtained following surgery for non-obstructed bowel cancer. The total hydroxyproline content in DC samples was also evaluated. In the DC, tinctorial staining demonstrated an increased occurrence of total collagen fibres in the submucosa of the elderly (159.8 ± 9.6 in elderly vs. 126.9 ± 6.1 in the adults; p 0.05) and in the muscularis externa (respectively 37.4 ± 4.1 vs. 18.8 ± 2.4; p 0.01). In the adult AC and DC, there were no statistically significant differences in the amount of collagen within the CM and TC. In the elderly, the total collagen fibres within the TC was greater in the AC (mean grey intensity: 63.4 ± 3.9% in the elderly vs. 36.6 ± 1.6% in adults; p 0.05) and DC (mean grey intensity: respectively, 59.82 ± 2.4 vs. 40.2 ± 0.9%; p 0.05). In both AC and DC of the elderly samples, several thickened collagen fibrils were microscopically identified within the TC infiltrating to the myenteric plexus. In the TC of the elderly AC, the total collagen fibres were increased by approximately 4% compared to that of the DC. The total collagen concentration in the elderly DC assessed by hydroxyproline assay was increased by approximately 15% compared to the adult. Sex related differences were not found when data combined. We concluded that the total collagen content in the muscularis externa particularly of the TC of human colon increases with age. The subtle change in collagen distribution with age between AC and DC may differentially affect the tensile strength of the colon.

Characterization, antimicrobial and cytotoxic activity of polymer blends based on chitosan and fish collagen

This study aims to produce, characterize, and assess the antimicrobial activity and cytotoxicity of polymer blends based on chitosan (CT) and fish collagen (COL) produced by different precipitation methods. Polymer blends were obtained in alkaline (NaOH), saline (NaCl), and alkaline/saline (NaOH/NaCl) solutions with different CT:COL concentration ratios (20:80, 50:50, and 80:20). The polymer blends were characterized by various physicochemical methods and subsequently evaluated in terms of their in vitro antimicrobial and cytotoxicity activity. In this study, the degree of chitosan deacetylation was 82%. The total hydroxyproline and collagen content in the fish matrix was 47.56 mg. g−1 and 394.75 mg. g−1, respectively. The highest yield was 44% and was obtained for a CT:COL (80:20) blend prepared by precipitation in NaOH. High concentrations of hydroxyproline and collagen in the blends were observed when NaOH precipitation was used. Microbiological analysis revealed that the strains used in this work were sensitive to the biomaterial; this sensitivity was dose-dependent and increased with increasing chitosan concentration in the products. The biocompatibility test showed that the blends did not reduce the viability of fibroblast cells after 48 h of culture. An analysis of the microbiological activity of the all-polymer blends showed a decrease in the values of minimal inhibitory concentration (MIC) and minimal bactericidal concentrations (MBC) for S. aureus and P. aeruginosa. The blends showed biocompatibility with NIH-3T3 murine fibroblast cells and demonstrated their potential for use in biomedical applications such as wound healing, implants, and scaffolds.

Topical Application of Premna integrifolia Linn on Skin Wound Injury in Rats Accelerates the Wound Healing Process: Evidence from In Vitro and In Vivo Experimental Models.

Background When the skin and tissues within the body are injured, the healing process begins. Medicinal herbs have been used to cure wounds since time immemorial. The antimicrobial and antioxidant activity possessed by P. integrifolia may accelerate wound healing. Objectives To assess the wound healing activity of Premna integrifolia extract (PIE) by employing in-vivo experimental animal models and an in-vitro migration scratch assay. Furthermore, to assess its cytotoxicity using the MTT assay. Methods Wistar albino rats were used for the in vivo wound healing models. The animals were divided into four groups at random: Group I was untreated. Group II was vehicle control (ointment base). Group III was PIE ointment (5% W/W). Group IV was standard (povidone-iodine ointment) (5% W/W). The ointments were applied directly to the wounds as described above until they healed completely. The wound contraction percentage and tensile strength were calculated. The MTT test was used to determine the viability of the test extract against the fibroblast cells. The scratch assay was used in vitro to determine the wound healing potential of the test drug. P ≤ 0.05 values were considered statistically significant. Results Premna integrifolia extract did not possess any noticeable cytotoxicity to the cell line and showed an IC50 of 185.98 μg/ml. The wound contraction potential of PIE ointment-treated animals was considerably greater (P ≤ 0.001) on days 4, 8, 12, 16, and 20 when compared to the control group. The percentage of wound contraction on day 20 was 99.92% in PIE-treated animals compared to 83.23% in untreated animals. Compared to the untreated group, the duration of full epithelization was significantly (P ≤ 0.01) shorter in the test group. When compared to the incision control group, the animals treated with PIE ointment had significantly higher (P ≤ 0.001) tensile strength. In addition, animals given the test drug had a significant (P ≤ 0.001) increase in total protein and hydroxyproline. In the in vitro scratch assay, test drug-treated cells demonstrated greater cell migration. Histology images confirmed that the test drug-treated group had epithelial tissue proliferation and keratinization. Conclusion The current study found that Premna integrifolia improved wound healing activity both in vitro and in vivo. These findings indicate that Premna integrifolia extract has wound-healing potential and could be a viable source of nutraceuticals with wound-healing properties.

Modulation of H4K16Ac levels reduces pro-fibrotic gene expression and mitigates lung fibrosis in aged mice.

Histone H4 lysine16 acetylation (H4K16Ac) modulates chromatin structure by serving as a switch from a repressive to a transcriptionally active state. This euchromatin mark is associated with active transcription. In this study, we investigated the effects of H4K16Ac on the expression of pro-fibrotic genes in lung fibroblasts from patients with idiopathic pulmonary fibrosis (IPF) and in an aging murine model of lung fibrosis.Methods: The lung tissues and fibroblasts from human IPF/non-IPF donors and from aged mice with/without bleomycin induced lung fibrosis were used in this study. The H4K16Ac levels were examined by immunohistochemistry or western blots. RNA silencing of H4K16Ac acetyltransferase Mof was used to reduce H4K16Ac levels in IPF fibroblasts. The effects of reduced H4K16Ac on pro-fibrotic gene expression were examined by western blots and real-time PCR. The association of H4K16Ac with these genes' promoter region were evaluated by ChIP assays. The gene expression profile in siRNA Mof transfected IPF cells were determined by RNA-Seq. The impact of H4K16Ac levels on lung fibrosis was evaluated in an aging murine model.Results: Aged mice with bleomycin induced lung fibrosis showed increased H4K16Ac levels. Human lung fibroblasts with siRNA Mof silencing demonstrated reduced H4K16Ac, and significantly down-regulated profibrotic genes, such as α-smooth muscle actin (α-SMA), collagen I, Nox4, and survivin. ChIP assays confirmed the associations of these pro-fibrotic genes' promoter region with H4K16Ac, while in siRNA Mof transfected cells the promoter/H4K16Ac associations were depleted. RNA-seq data demonstrated that Mof knockdown altered gene expression and cellular pathways, including cell damage and repair. In the aging mice model of persistent lung fibrosis, 18-month old mice given intra-nasal siRNA Mof from week 3 to 6 following bleomycin injury showed improved lung architecture, decreased total hydroxyproline content and lower levels of H4K16Ac.Conclusions: These results indicate a critical epigenetic regulatory role for histone H4K16Ac in the pathogenesis of pulmonary fibrosis, which will aid in the development of novel therapeutic strategies for age-related diseases such as IPF.

Conditioned medium from a human adipose-derived stem cell line ameliorates inflammation and fibrosis in a lung experimental model of idiopathic pulmonary fibrosis

Idiopathic pulmonary fibrosis is a chronic, progressive parenchymal lung disease that results in fibrogenesis and the conditioned medium from adipose-derived mesenchymal stem cells (CM-ADSCs) has been shown to be efficacious in pulmonary fibrosis animal models. The aim of the present study is to evaluate the effect of CM-ADSCs on lung inflammation and fibrosis in a Bleomycin (BLM)-induced pulmonary fibrosis model. CM-ADSCs safety and toxicity were evaluated in Sprague Dawley rats and no adverse effects were observed. Six-week-old female C57BL/6J mice were employed in the BLM-induced pulmonary fibrosis model and were divided into four groups: Group 1 (Sham): animals were kept without BLM and treatment, Group 2 (Control): BLM with vehicle DMEM, Group 3: 10 μg/kg CM-ADSCs and Group 4: 100 μg/kg CM-ADSCs. Body weight, fibrosis and inflammation histological analyses, mRNA and protein pro-inflammatory cytokine, and total hydroxyproline content calculation were performed in all groups upon sacrifice. The 100 μg/kg CM-ADSCs showed a significant increase in mean body weight compared to Controls. CM-ADSCs doses resulted in the amelioration of fibrosis, as seen by Masson's Trichrome-staining, Ashcroft scoring, and Sirius red-staining. Compared to Controls, inflammation was also significantly reduced in CM-ADSCs-treated mice, with reduced F4/80 macrophage antigen staining, TNF-α mRNA and IL-6 and IL-10 protein levels. Total hydroxyproline content was found significantly reduced in both groups of CM-ADSCs-treated mice. Overall, our study shows that the CM-ADSCs is safe and efficient against pulmonary fibrosis, as it significantly reduced inflammation and fibrosis, with the larger dose of 100 μg/kg CM-ADSCs being the most efficient one.

Gastrophysical and chemical characterization of structural changes in cooked squid mantle.

Squid (Loligo forbesii and Loligo vulgaris) mantles were cooked by sous vide cooking using different temperatures (46°C, 55°C, 77°C) and times (30 s, 2min, 15min, 1 h, 5h, 24h), including samples of raw tissue. Macroscopic textural properties were characterized by texture analysis (TA) conducted with Meullenet-Owens razor shear blade and compared to analysis results from differential scanning calorimetry. The collagen content of raw tissues of squid was quantified as amount of total hydroxyproline using ultra-high-performance liquid chromatography. Structural changes were monitored by Raman spectroscopy and small-angle X-ray scattering and visualized by second harmonic generation microscopy. Collagen in the squid tissue was found to be highest in arms (4.3% of total protein), then fins (3.0%), and lowest in the mantle (1.5%), the content of the mantle being very low compared to that of other species of squid. Collagen was found to be the major protein responsible for cooking loss, whereas both collagen and actin were found to be key to mechanical textural changes. A significant decreased amount of cooking loss was obtained using a lower cooking temperature of 55°C compared to 77°C, without yielding significant textural changes in most TA parameters, except for TA hardness which was significantly less reduced. An optimized sous vide cooking time and temperature around 55-77°C and 0.5-5h deserves further investigation, preferably coupled to sensory consumer evaluation. PRACTICAL APPLICATION: The study provides knowledge about structural changes during sous vide cooking of squid mantle. The results may be translated into gastronomic use, promoting the use of an underutilized resource of delicious and nutritious protein (Loligo vulgaris and Loligo forbesii).

Intracellular hydroxyproline imprinting following resolution of bleomycin-induced pulmonary fibrosis.

BackgroundIdiopathic pulmonary fibrosis (IPF) is a fatal lung disease with few treatment options. The poor success in developing anti-IPF strategies has impelled researchers to reconsider the importance of the choice of animal model and assessment methodologies. Currently, it is still not settled whether the bleomycin-induced lung fibrosis mouse model finally returns to resolution.MethodsThis study aimed to follow the dynamic fibrotic features of bleomycin-treated mouse lungs over extended durations through a combination of the latest technologies (micro-computed tomography imaging and histological detection of degraded collagens) and traditional methods. In addition, we also applied immunohistochemistry to explore the distribution of all hydroxyproline-containing molecules.ResultsAs determined by classical biochemical methods, total lung hydroxyproline contents reached a peak at 4 weeks after bleomycin injury and maintained a steady high level thereafter until the end of the experiments (16 weeks). This result seemed to partially contradict with the changes of other fibrosis evaluation parameters, which indicated a gradual degradation of collagens and a recovery of lung aeration after the fibrosis peak. This inconsistency was well reconciled by our data from immunostaining against hydroxyproline and fluorescent peptide staining against degraded collagen, together showing large amounts of hydroxyproline-rich degraded collagen fragments detained and enriched within the intracellular regions at 10 or 16 weeks rather than at 4 weeks after bleomycin treatment.ConclusionsOur present data not only offer respiratory researchers a new perspective towards the resolution nature of mouse lung fibrosis, but also remind them to be cautious when using the hydroxyproline content assay to evaluate the severity of fibrosis.

Formulation and Evaluation of Helichrysum italicum Essential Oil-Based Topical Formulations for Wound Healing in Diabetic Rats.

As proper wound management is crucial to reducing morbidity and improving quality of life, this study evaluated for the first time the wound healing potential of H. italicum essential oil (HIEO) prepared in the form of ointment and gel in streptozotocin-induced diabetic wound models in rats. After creating full-thickness cutaneous wounds, forty-eight diabetic rats were divided into six groups: (1) negative control; (2) positive control; (3) ointment base; (4) gel base; (5) 0.5% HIEO ointment (6) 0.5% HIEO gel. Wound healing potential was determined by the percentage of wound contraction, hydroxyproline content, redox status, and histological observation. A significant decrease in the wound size was observed in animals treated with HIEO formulations compared with other groups. The HIEO groups also showed a higher level of total hydroxyproline content, and more pronounced restitution of adnexal structures with only the underlying muscle defect indicating the incision site. Hence, our results legitimate the traditional data of the pro-healing effect of HIEO because HIEO in both formulations such as gel and ointment exhibited the significant wound repairing effect in the incision wound model.

Evaluation of in vivo Wound Healing Activity of Ursolic Acid Rich Chloroform Extract of Hedyotis herbacea Linn Ointment

Aim: This study evaluates the effect of in vivo wound healing potential of Ursolic acid rich chloroform extract of Hedyotis herbacea Linn ointment using incision and excision wound model.&#x0D; Study Design: Wound healing potentials of Hedyotis herbacea were analysed by Incision and excision wound model.&#x0D; Place of Study: Nandha College of Pharmacy, Erode, Tamilnadu.&#x0D; Methodology: Hedyotis herbacea was subjected to extraction (cold maceration), with solvents of increasing polarity. All the extracts were estimated for the presence of phytoconstituents by HPTLC. As the study has been focused on the phytoconstituent based biological activity, the Ursolic acid rich chloroform extract was chosen for the study of wound healing activity. The Chloroform extract of Hedyotis herbacea was incorporated into ointment base, to prepare the ointment. The ointments prepared with Chloroform extract (2.5% and 5%) of Hedyotis herbacea (CEHH) were subjected for evaluation of excision and incision wound model. Wistar albino rats were divided into four groups each consisting of six animals; group I (left untreated) considered as untreated control, group II treated with 5% w/w povidone-iodine ointment served as standard, group III treated with CEHH 2.5% w/w ointment, and group IV treated with CEHH 5% w/w ointment were considered as test groups. All the treatments were given once daily. Wound healing effect was assessed by the rate of wound contraction, level of total protein, Hydroxy proline, Hexosamine and Hexuronic acid and histopathology studies in an excision wound model. Tensile strength was also measured in both excision and incision wound.&#x0D; Results: Wound healing activity of CEHH 5% w/w ointment treated group was greater than CEHH 2.5% w/w and untreated groups in both excision and incision wound model. The high rate of wound contraction (*P&lt; 0.001), high tensile strength (*P&lt; 0.001), and elevated total protein, Hydroxyproline, Hexosamine, and Hexuronic acid content were observed in animals treated with CEHH ointments when compared to the untreated control group of animals. Histopathological studies of the CEHH ointments treated groups also revealed the effectiveness in wound healing.&#x0D; Conclusion: These results justified the claimed traditional use of the Hedyotis herbacea as wound healing plant.