Chromatographic separation by RPLC-ESI-MS of all hydroxyproline isomers for the characterization of collagens from different sources.

During collagen biosynthesis, proline is post-translationally converted to hydroxyproline by specific enzymes. This amino acid, unique to collagen, plays a crucial role in stabilizing the collagen triple helix structure and could serve as an important biomarker for collagen content and quality analysis. Hydroxyproline has four isomers, depending on whether proline is hydroxylated at position 4 or 3 and on whether the cis- or trans- conformation is formed. Moreover, as extensive hydrolysis of collagen is required for its amino acid analysis, epimerization may also occur, although to a lesser extent, giving a total of eight possible isomers. The aim of the present study was to develop a reversed-phase high-performance liquid chromatography-UV-mass spectrometry (RPLC-UV-MS) method for the separation and quantification of all eight hydroxyproline isomers. After the chiral derivatization of the hydroxyproline isomers with Nα-(2,4-dinitro-5-fluorophenyl)-L-valinamide (L-FDVA), to enable their UV detection, the derivatized diastereoisomers were separated by testing different C18 column technologies and morphologies and optimizing operative conditions such as the mobile phase composition (solvent, additives), elution mode, flow rate and temperature. Baseline resolution of all eight isomers was achieved on a HALO® ES-C18 reversed-phase column (150×1.5 mm, 2.7 µm, 160 Å) using isocratic elution and MS-compatible mobile phase. The optimized method was validated for the quantification of hydroxyproline isomers and then applied to different collagen hydrolysates to gain insight and a deeper understanding of hydroxyproline abundances in different species (human, chicken) and sources (native, recombinant).

Assessment of collagen content in fish skin - development of a flow analysis method for hydroxyproline determination.

This work describes the development of a flow injection method to determine hydroxyproline (HYP), one of collagen's most abundant amino acids. Collagen is a protein with several applications and high nutritional value. Evaluating the feasibility of using collagen from fish skin over its mammalian source is essential. The determination of HYP requires the pre-treatment and hydrolysis of the fish skin to break down collagen into its amino acids, and the HYP value quantified relates to the collagen content. The determination was based on the HYP oxidation with permanganate in an alkaline medium and the consequent decrease of colour intensity registered. Under optimal conditions, the developed method enables the determination of the HYP within the dynamic range of 23.8 to 500 mg L-1, with a limit of detection (LOD) of 2.6 mg L-1 and a limit of quantification (LOQ) of 23.8 mg L-1. Different samples were processed, and the digests were analysed by the proposed method and with the conventional procedure with good correlation (relative error < 7%). Moreover, the analyte quantification is performed faster, simpler, and more accurately, with less toxic solutions. The reproducibility of the developed method was also evaluated by calculating the relative standard deviation of the calibration curve slope (RSD < 1%).

Topical Cream Derived From Hylocereus polyrhizus (Red Dragon Fruit) Extract Accelerates Wound Healing Through Increased Hydroxyproline and Fibroblast Growth Factor 2 Levels: A Pilot Study.

The red dragon fruit (Hylocereus polyrhizus) extract (RDFE) is frequently used for a variety of therapeutic purposes (e.g., boosting the immune system, promoting a healthy gastrointestinal system, improving wound healing). We investigated the effects of a topical cream containing 7.5% RDFE on hydroxyproline and fibroblast growth factor 2 (FGF-2) levels and wound healing. On Day 0, we divided a total of 36 albino male Wistar rats into two equal groups. Using an 8-mm punch biopsy, we created a circular excision to fascial depth on the back of each rat. On Day 1, we treated the control group (n = 18) with 20 mg of base cream and the RDFE group (n = 18) with 20 mg of 7.5% RDFE cream. We measured hydroxyproline and FGF-2 levels in the wound tissue using an ELISA method on Days 3, 7, and 14. We found that on Day 3, hydroxyproline levels were significantly lower in the treatment group than in the control group (p = .031). We also found a significant correlation between FGF-2 levels in the treatment group and wound diameter (p = .02). On the basis of the results of this study, we concluded that using a topical cream containing 7.5% RDFE has the potential to accelerate wound healing by increasing levels of hydroxyproline and FGF-2 in the wound.

[Protective effect of intervention with cannabinoid type-2 receptor agonist JWH133 on pulmonary fibrosis in mice].

Objective: JWH133, a cannabinoid type 2 receptor agonist, was tested for its ability to protect mice from bleomycin-induced pulmonary fibrosis. Methods: By using a random number generator, 24 C57BL/6J male mice were randomly divided into the control group, model group, JWH133 intervention group, and JWH133+a cannabinoid type-2 receptor antagonist (AM630) inhibitor group, with 6 mice in each group. A mouse pulmonary fibrosis model was established by tracheal instillation of bleomycin (5 mg/kg). Starting from the first day after modeling, the control group mice were intraperitoneally injected with 0.1 ml of 0.9% sodium chloride solution, and the model group mice were intraperitoneally injected with 0.1 ml of 0.9% sodium chloride solution. The JWH133 intervention group mice were intraperitoneally injected with 0.1 ml of JWH133 (2.5 mg/kg, dissolved in physiological saline), and the JWH133+AM630 antagonistic group mice were intraperitoneally injected with 0.1 ml of JWH133 (2.5 mg/kg) and AM630 (2.5 mg/kg). After 28 days, all mice were killed; the lung tissue was obtained, pathological changes were observed, and alveolar inflammation scores and Ashcroft scores were calculated. The content of type Ⅰ collagen in the lung tissue of the four groups of mice was measured using immunohistochemistry. The levels of interleukin 6 (IL-6) and tumor necrosis factor α (TNF-α) in the serum of the four groups of mice were measured using enzyme-linked immunosorbent assay (ELISA), and the content of hydroxyproline (HYP) in the lung tissue of the four groups of mice was measured. Western blotting was used to measure the protein expression levels of type Ⅲ collagen, α-smooth muscle actin (α-SMA), extracellular signal regulated kinase (ERK1/2), phosphorylated P-ERK1/2 (P-ERK1/2), and phosphorylated ribosome S6 kinase type 1 (P-p90RSK) in the lung tissue of mice in the four groups. Real-time quantitative polymerase chain reaction was used to measure the expression levels of collagen Ⅰ, collagen Ⅲ, and α-SMA mRNA in the lung tissue of the four groups of mice. Results: Compared with the control group, the pathological changes in the lung tissue of the model group mice worsened, with an increase in alveolar inflammation score (3.833±0.408 vs. 0.833±0.408, P<0.05), an increase in Ashcroft score (7.333±0.516 vs. 2.000±0.633, P<0.05), an increase in type Ⅰ collagen absorbance value (0.065±0.008 vs. 0.018±0.006, P<0.05), an increase in inflammatory cell infiltration, and an increase in hydroxyproline levels [(1.551±0.051) µg/mg vs. (0.974±0.060) µg/mg, P<0.05]. Compared with the model group, the JWH133 intervention group showed reduced pathological changes in lung tissue, decreased alveolar inflammation score (1.833±0.408, P<0.05), decreased Ashcroft score (4.167±0.753, P<0.05), decreased type Ⅰ collagen absorbance value (0.032±0.004, P<0.05), reduced inflammatory cell infiltration, and decreased hydroxyproline levels [(1.148±0.055) µg/mg, P<0.05]. Compared with the JWH133 intervention group, the JWH133+AM630 antagonistic group showed more severe pathological changes in the lung tissue of mice, increased alveolar inflammation score and Ashcroft score, increased type Ⅰ collagen absorbance value, increased inflammatory cell infiltration, and increased hydroxyproline levels. Compared with the control group, the expression of α-SMA, type Ⅲ collagen, P-ERK1/2, and P-p90RSK proteins in the lung tissue of the model group mice increased, while the expression of type Ⅰ collagen, type Ⅲ collagen, and α-SMA mRNA increased. Compared with the model group, the protein expression of α-SMA (relative expression 0.60±0.17 vs. 1.34±0.19, P<0.05), type Ⅲ collagen (relative expression 0.52±0.09 vs. 1.35±0.14, P<0.05), P-ERK1/2 (relative expression 0.32±0.11 vs. 1.14±0.14, P<0.05), and P-p90RSK (relative expression 0.43±0.14 vs. 1.15±0.07, P<0.05) decreased in the JWH133 intervention group. The type Ⅰ collagen mRNA (2.190±0.362 vs. 5.078±0.792, P<0.05), type Ⅲ collagen mRNA (1.750±0.290 vs. 4.935±0.456, P<0.05), and α-SMA mRNA (1.588±0.060 vs. 5.192±0.506, P<0.05) decreased. Compared with the JWH133 intervention group, the JWH133+AM630 antagonistic group increased the expression of α-SMA, type Ⅲ collagen, P-ERK1/2, and P-p90RSK protein in the lung tissue of mice, and increased the expression of type Ⅲ collagen and α-SMA mRNA. Conclusion: In mice with bleomycin-induced pulmonary fibrosis, the cannabinoid type-2 receptor agonist JWH133 inhibited inflammation and improved extracellular matrix deposition, which alleviated lung fibrosis. The underlying mechanism of action may be related to the activation of the ERK1/2-RSK1 signaling pathway.

A hydrogen bonding based SERS method for direct label-free L-hydroxyproline detection.

The detection of non-protein nitrogen adulterants is a major challenge in dairy testing. As a marker molecule of animal hydrolyzed protein, the presence of non-edible L-hydroxyproline (L-Hyp) molecules can be used to identify low-quality milk containing components of animal hydrolyzed protein. However, it is still difficult to detect L-Hyp directly in milk. The Ag@COF-COOH substrate in this paper can be used to realize label-free L-Hyp sensitive detection based on the hydrogen bond transition mechanism. To explore the mechanism, the binding sites of hydrogen bond interaction have been verified experimentally and computationally, and the charge transfer process was also explained in terms of HOMO/LOMO energy level. In conclusion, the quantitative models for L-Hyp in an aqueous environment and in milk were developed. The limit of detection (LOD) of L-Hyp in an aqueous environment could reach 8.18 ng/mL, with R2 of 0.982. The linear range of quantitative detection in milk was 0.5-1000 µg/mL and the LOD was as low as 0.13 µg/mL. In this work, a hydrogen bond interaction based Surface-enhanced Raman spectroscopy (SERS) method for the label-free detection of L-Hyp was proposed, which complemented the application of SERS technology in the detection of dairy products.

Evaluation of Wound Healing Potential of Root Bark Extract of Berberis aristata and Molecular Docking Analysis of Berberis Phytoconstituents.

INTRODUCTION: The root bark of Berberis aristata has been utilized by indigenous peoples for wound treatment for centuries. The mature root barks are crushed into a paste and applied to the wound's surface. OBJECTIVE: The focus of this research is to analyse the wound healing activities of an ethanolic extract of Berberis aristata, as well as to use molecular docking to establish the likely mechanism of the potent phytochemical. There is no scientific evidence to support the usage of root bark extract of Berberis aristata. METHODS: The Herbal ointment, which comprises (1%, 2%, and 4% w/w) ethanolic extract of root bark, was developed to test the wound healing ability of incision and excision wounds, and the molecular mechanism was established using Auto-Dock software. RESULTS: Epithelization stage, wound index, % wound contraction area, hydroxyproline content, DNA estimate, and histopathological assessments were performed on the incision wound model. Tensile strength was assessed in an excision wound model. TLC was used to identify the samples after successive extractions with different solvents based on polarity. CONCLUSION: Berberine and tetrahydropalmatine were major active phytoconstituent found in root barks of Berberis aristata as secondary metabolites. Animals treated with 4% w/w formulation demonstrated considerable wound contraction, epithelization time, and wound index in the excision model. In contrast, to control and standardize the concentrations of hydroxyproline, total amino acids, and DNA in recovering tissue were higher. At 4% w/w extract formulation, the parameters studied indicated a substantial result. Berberine and tetrahydropalmatine, active metabolites which are present in the ethanolic extract of Berberis aristata, were found to be responsible for wound healing. Based on ligand interactions, the findings verified Berberis aristata ethnomedicinal claim in a wound healing capacity.

Flavonoid constituents and protective efficacy of Citrus reticulate (Blanco) leaves ethanolic extract on thioacetamide-induced liver injury rats.

Context: Oxidative stress leads to deleterious processes in the liver that resulted in liver diseases.Objective: To evaluate antioxidant activity and hepatoprotective potential of ethanolic leaves extract of Citrus reticulate against hepatic dysfunction induced by thioacetamide (TAA).Materials and Methods: Flavonoid constituents were isolated from the ethanol extract by chromatographic techniques and identified by the spectroscopic analyses. Antioxidant activity was determined using DPPH assay. Hepatotoxicity was induced in rats via intraperitoneal injection of TAA and the ethanol extract was orally administrated at a dose of 100 mg/kg/day for four weeks. Serum biomarkers, hepatic antioxidant enzymes, tumour necrosis factor-alpha (TNF-α), hepatic hydroxyproline levels, and histopathology were examined.Results: Ten known flavonoids were identified, among of them, 6,3`-dimethoxyluteolin and 8,3`-dimethoxyluteolin possessed the highest antioxidant activity. The substantially elevated serum enzymatic levels of ALT, ALP, and bilirubin were found to be restored towards normalisation significantly by the plant extract. Furthermore, the markers including MDA, GSH, SOD, NO, and protein carbonyl which were close to oxidative damage, were restored. Meanwhile, the extract treatment decreased TNF-α level and also was able to reverse the induced fibrosis by significantly reducing the hydroxyproline content. Moreover, histopathological studies further substantiate the protective effect of the extract.Conclusion: C. reticulate leaves extract is a rich source of phytochemicals with in vitro and in vivo protective effects.

Interactions of two phosphate ester monomers with hydroxyapatite and collagen fibers and their contributions to dentine bond performance.

OBJECTIVES: To evaluate the interactions of two phosphate ester monomers [10-methacryloyloxydecyl dihydrogen phosphate (10-MDP) and dipentaerythritol penta-acrylate phosphate (PENTA)] with hydroxyapatite and collagen and understand their influence on dentine bonding. METHODS: Fourier transform infrared spectroscopy, X-ray photoelectron spectroscopy, X-ray diffraction, nuclear magnetic resonance, ultraviolet-visible, and molecular docking were applied for separately evaluating the interactions of two monomers with hydroxyapatite and collagen. Hydrophilicity tests and morphological observation were employed to characterize pretreated dentine. Microtensile bond strength (µTBS) and nanoleakage were investigated to evaluate the bonding performance. Hydroxyproline assay, in situ zymography, and matrix metalloproteinase-9 (MMP-9) activity assay were used to confirm the MMP inhibition. RESULTS: Chemoanalytic characterization confirmed the interactions of 10-MDP and PENTA with hydroxyapatite and collagen. The interactions of PENTA were weaker than 10-MDP. PENTA possessed better dentine tubule sealing after etching than 10-MDP. Dentine treated with PENTA was more hydrophilic than 10-MDP. 10-MDP and PENTA treating significantly increased the initial µTBS than the control group without primer conditioning. µTBS decreased significantly during aging, and the decrease was more severe in the PENTA group than 10-MDP. The 10-MDP and PENTA groups exhibited relatively less fluorescence than the control. The relative inhibition percentages of MMP-9 decreased in the order of 10-MDP-Ca salt, 10-MDP and PENTA. The 10-MDP, PENTA, and 10-MDP-Ca salt groups showed significantly lower hydroxyproline contents than the control. CONCLUSIONS: Although PENTA adsorbed on hydroxyapatite, it did not form a stable calcium salt. The interactions of 10-MDP with hydroxyapatite and collagen are different than those of PENTA. CLINICAL SIGNIFICANCE: The sealing of dentinal tubules by PENTA and the inhibition of MMP by 10-MDP and its calcium salts contribute to improving the dentine bonding durability.

Dentin collagen denaturation status assessed by collagen hybridizing peptide and its effect on bio-stabilization of proanthocyanidins.

OBJECTIVE: To assess dentin collagen denaturation from phosphoric acid and enzyme treatments using collagen hybridizing peptide (CHP) and to investigate the effect of collagen denaturation on bio-stabilization promoted by proanthocyanidins (PA). METHODS: Human molars were sectioned into 7-µm-thick dentin films, demineralized, and assigned to six groups: control with/without PA modification, H3PO4-treated collagen with/without PA modification, enzyme-treated collagen with/without PA modification. PA modification involved immersing collagen films in 0.65% PA for 30 s. H3PO4 and enzyme treatments were used to experimentally induce collagen denaturation, which was quantitated by fluorescence intensity (FI) from the fluorescently-conjugated-CHP (F-CHP) staining (n = 4). FTIR was used to characterize collagen structures. All groups were subject to collagenase digestion to test the bio-stabilization effect of PA on denatured collagen using weight loss analysis and hydroxyproline assay (n = 6). Data were analyzed using two-factor ANOVA and Games-Howell post hoc tests (α = 0.05). RESULTS: FTIR showed collagen secondary structural changes after denaturation treatments and confirmed the incorporation and cross-linking of PA in control and treated collagen. F-CHP staining indicated high-degree, medium-degree, and low-degree collagen denaturation from H3PO4-treatment (FI = 83.22), enzyme-treatment (FI = 36.54), and control (FI = 6.01) respectively. PA modification significantly reduced the weight loss and hydroxyproline release of all groups after digestion (p < 0.0001), with the results correlated with FI values at r = 0.96-0.98. SIGNIFICANCE: A molecular method CHP is introduced as a sensitive technique to quantitate dentin collagen denaturation for the first time. PA modification is shown to effectively stabilize denatured collagen against collagenase digestion, with the stabilization effect negatively associated with the collagen denaturation degree.

IInvestigation of possible oxidative damage caused by formaldehyde exposure in the rat's heart and aorta tissue / Investigación de posible daño oxidativo causado por la exposición al formaldehído en el corazón de la rata y tejido de la aorta

SUMMARY: Formaldehyde (FA) is a toxic substance used frequently in the field of medicine as well as in many industrial areas. Especially people working in the field of anatomy, histology, and pathology are in high risk group because of the use of the FA. Studies showing the effects of FA on the cardiovascular system are few in number. The purpose of the present study was to investigate the effects of FA exposure, which we believe can cause oxidative stress, on the heart and aorta with various biochemical analyses. A total of 24 Wistar Albino rats were used in our study. We divided the rats into 3 groups as the Control Group (CG), the group exposed to low-dose FA (avg. 1 ppm) (DDG) Group, and the group exposed to high-dose FA (avg. 10 ppm) (YDG). At the end of the subchronic FA exposure, the blood samples, heart and aorta tissues of the rats were taken and subjected to biochemical analyses. As a result of the analyses, statistically significant differences were detected between CG (2.96?0.85 ng/mg), and HDG (2.08?0.77 ng/mg) in aortic tissues in TXNIP analysis (p<0.05). In heart tissues, significant differences were detected between CG (0.73?0.27 ng/mg) and LDG (1.13?0.22 ng/mg) (p<0.05). Statistically significant differences were also detected between CG (1.98?0.31 mM/ml) and YDG (2.43?0.31 mM/ml) in serum MDA analyses (p<0.05). It was shown that subchronic application of FA to LDG rats through inhalation had no effects on apoptosis markers in heart tissues. More studies are required to show FA toxicity and the mechanism of action of pathology on the cardiovascular system. We believe that our study will contribute to clarifying the roles of mild and subchronic exposure of FA in heart and aortic tissues in terms of oxidative stress risk.

RESUMEN: El formaldehído es una sustancia tóxica que se utiliza con frecuencia en el campo de la medicina, así como en muchas áreas industriales. Especialmente las personas que trabajan en el area de la anatomía, y patología se encuentran en el grupo de alto riesgo debido al uso de esta sustancia. Pocos son los estudios que muestran los efectos del formaldehído en el sistema cardiovascular. El propósito del presente estudio fue investigar a través de análisis bioquímicos, los efectos de la exposición a formaldehído, que podría causar estrés oxidativo, en el corazón y la aorta. Se utilizaron un total de 24 ratas Albinas Wistar. Dividimos a las ratas en 3 grupos: grupo control (GC), grupo expuesto a dosis bajas de AG (promedio 1 ppm) (DDG) y grupo expuesto a dosis altas de AG (promedio 10 ppm) (YDG). Al término de la exposición a FA subcrónica, se tomaron muestras de sangre, tejido cardíaco y aorta de las ratas y se sometieron a análisis bioquímicos. Como resultado de los análisis, se detec- taron diferencias estadísticamente significativas entre GC (2,96 ? 0,85 ng / mg) y HDG (2,08 ? 0,77 ng / mg) en los tejidos aórticos en el análisis TXNIP (p <0,05). En los tejidos cardíacos se detectaron diferencias significativas entre GC (0,73 ? 0,27 ng / mg) y LDG (1,13 ? 0,22 ng / mg) (p <0,05). También se detectaron diferencias estadísticamente significativas entre CG (1,98 ? 0,31 mM / ml) y YDG (2,43 ? 0,31 mM / ml) en los análisis de MDA en suero (p <0,05). Se demostró que la aplicación subcrónica de formaldehído a ratas LDG a través de la inhalación no tuvo efectos sobre los marcadores de apoptosis en los tejidos del corazón. Se requieren más estudios para demostrar la toxicidad de los AG y el mecanismo de acción de la patología en el sistema cardiovascular. Creemos que nuestro estudio contribuirá a aclarar las funciones de la exposición leve y subcrónica de formaldehído en los tejidos cardíacos y aórticos en términos de riesgo al estrés oxidativo.

Effect of Adipose-Derived Stem Cells on Colonic Anastomosis in Rats Immunosuppressed With Everolimus: An Experimental Study.

OBJECTIVES: Immunosuppressed patients sometimes require colorectal surgery. We investigated whether adipose tissue-derived stem cells contributed to anastomosis healing in rats immunosuppressed with the mTOR inhibitor everolimus. MATERIALS AND METHODS: Sixty male Sprague-Dawley rats were randomly divided into 4 groups of 14 each, with all groups undergoing descending colon anastomosis; the 4 remaining rats were used for stem cell retrieval. Group 1 (control) underwent surgery only, group 2 received stem cell injection, group 3 received everolimus only, and group 4 received everolimus plus stem cell injection. After treatment, each group was randomly divided into 2 equal subgroups according to the day of euthanasia (posttreatment day 4 or day 7). We measured anastomosis bursting pressure and tissue hydroxyproline level and performed histopathological evaluation. RESULTS: At both posttreatment days 4 and 7, median weight loss in group 3 was higher than in group 1, group 3 had higher severity of intraabdominal adhesion than group 4, and group 2 had mean hydroxyproline level higher than the other groups. At posttreatment day 4, mean bursting pressure was significantly different in group 1 versus groups 2 and 4 (P = .002) and group 2 versus groups 3 and 4 (P < .001). No significant differences were shown in pathological analysis except for vascular proliferation on day 7 (P = .003). CONCLUSIONS: Injection of adipose tissue-derived stem cells in the anastomosis site prevented anastomosis leakage by contributing to healing. Injection of adipose tissue-derived stem cells in the anastomosis region, especially in the early period after solid-organ transplant in recipients and after gastrointestinal surgery in immunosuppressed patients, may help reduce mortality and morbidity.

Dating the last Middle Palaeolithic of the Crimean Peninsula: New hydroxyproline AMS dates from the site of Kabazi II.

Radiocarbon dating of bone and charcoal from sites dating to the Middle and Upper Paleolithic is challenging due to low residual levels of radiocarbon. This means that small amounts of contaminating carbon can wield a great influence over accuracy unless they are fully removed. The site of Kabazi II in the Crimea is important because radiocarbon dates previously obtained from bones in archaeological horizons that date to the Western Crimean Mousterian (WCM) are surprisingly young. We redated the same samples using a single compound dating method that focuses on extracting and dating the amino acid hydroxyproline. We show that single amino acid dates produce significantly older determinations than those that use bulk collagen pretreatment procedures. Our results suggest that instead of dating to 35,000-40,000 cal BP, the bones actually date to >50,000 cal BP. This implies that the WCM at this site is much older than previously thought. In light of these current findings, we considered the dates of other key Crimean sites and concluded that in the absence of reliable pretreatment methods, it would be wise to consider many of them minimum ages. We conclude that there is little robust evidence to suggest Neanderthals were present in the Crimea after 40,000 cal BP.

The study of Yangyinyiqi mixture anti bleomycin-induced pulmonary fibrosis on rats by intervening matrix metalloproteinase-9 and tissue inhibitors of matrix metallopreoteinases-1 / El estudio de la mezcla Yangyinyiqi en fibrosis pulmonar inducida por anti-bleomicina en ratas por intervención en la matriz metaloproteinasa-9 e inhibidores de tejido de matriz metaloproteinasa-1

To investigate effectsof Yangyinyiqi Mixture on pulmonary fibrosis caused by bleomycin. SD ratswere divided randomly into: model group(distilled water,1 mL·0.1 kg-1), dexamethasone acetate group (dexamethasone acetate, the dosage was reduced gradually), low-dose group (Yangyinyiqi Mixture, 11 g·kg-1), moderate-dose group (Yangyinyiqi Mixture, 22 g·kg-1), high-dose group (Yangyinyiqi Mixture, 44 g·kg-1) and control group (distilled water, 1 mL·0.1 kg-1). Yangyinyiqi Mixture and dexamethasone acetate were intragastrically administrated. Lung tissue was collected for histopathological examination. Compared with control group, collagen markedly increased and HYP content significantly increased on 7th day in model group (p<0.01). On 28th day, collagen was diffusely deposited, alveolar was destroyed, and HYP content significantly increased (p<0.01). Compared with model group, bleomycin-induced suffering injury caused MMP-9 expression levels to rapidly increase (7and 14 days, p<0.01). TIMP-1 markedly increased (7and 14 days, p<0.01) and stayed at a high level to28th day. Yangyinyiqi Mixture exerted an effect against pulmonary fibrosis, which could involved prevention of collagen deposition through inhibitingMMP-9 and TIMP-1 expression.

El trabajo investiga los efectos de la mezcla Yangyinyiqi sobre la fibrosis pulmonary causada por bleomicina. Ratas SD se dividieron aleatoriamente en: grupo modelo (agua destilada, 1 mL·0.1 kg-1), grupo acetate de dexametasona (acetate de dexametasona, la dosis se redujo gradualmente), grupo de dosis baja (mezcla Yangyinyiqi, 11 g·kg-1), grupo de dosis moderada (mezcla Yangyinyiqi, 22 g·kg-1), grupo de dosis alta (mezcla Yangyinyiqi, 44 g·kg-1) y grupo control (agua destilada, 1 Ml·0.1 kg-1). La mezcla de Yangyinyiqi y el acetate de dexametasona se administraron por vía intragástrica. Se recolectó tejido pulmonary para examen histopatológico. En comparación con el grupo control, el colágeno aumentó notablemente y el contenido de HYP aumentó significativamente el séptimo día en el grupo modelo (p<0.01). El día 28, el colágeno se depositó difusamente, se produjo destrucción alveolar y el contenido de HYP aumento significativamente (p<0.01). En comparación con el grupo modelo, la lesión inducida por bleomicina causó que los niveles de expression de MMP-9 aumentaron rápidamente (7 y 14 días, p<0.01). TIMP-1 aumentó notablemente (7 y 14 días, p<0.01) y se mantuvo en un nivel alto hasta el día 28. La mezcla Yangyinyiqi ejerció un efecto contra la fibrosis pulmonary, lo que podría implicar la prevención del deposito de colágenio mediante la inhibición de la expression de MMP-9 y TIMP-1.

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.

In-vivo evaluation of Alginate-Pectin hydrogel film loaded with Simvastatin for diabetic wound healing in Streptozotocin-induced diabetic rats.

Previously we developed and characterized a novel hydrogel film wound dressing containing Sodium Alginate and Pectin loaded with Simvastatin with multi-functional properties. This study investigated the in-vivo efficacy of the developed wound dressing on type I diabetic wound model. Experiments were performed on male Wistar rats for the period of 21-days. Animals developed diabetes after intraperitoneal injection (50 mg/kg) of Streptozotocin then randomly divided into different groups. On days 7, 14, and 21 of post-wounding, animals were euthanized and the wounds tissue were harvested for analysis. The wound healing rate, hematology and histological analysis, hydroxyproline assay, and Vascular Endothelial Growth Factor A measurements were noted. The results revealed that the wound dressing healed the wounded area significantly (p < 0.05) higher than the control after 21-day treatment and wound closure was ~99% without any adverse systemic reactions. Histological analysis qualitatively revealed an enhanced re-epithelialization and collagen deposition. Moreover, results also showed an improved rate of collagen synthesis and angiogenesis in the group treated with the hydrogel film loaded with Simvastatin. Thus, the present study demonstrated that developed film holds great potential for the acceleration of diabetic wound healing by its pro-angiogenic effect, faster re-epithelialization and increased collagen deposition.

IGF-1 Receptor Signaling Regulates Type II Pneumocyte Senescence and Resulting Macrophage Polarization in Lung Fibrosis.

PURPOSE: Type II pneumocyte (alveolar epithelial cells type II [AECII]) senescence has been implicated in the progression of lung fibrosis. The capacity of senescent cells to modulate pulmonary macrophages to drive fibrosis is unexplored. Insulin-like growth factor-1 receptor (IGF-1R) signaling has been implicated as a regulator of senescence and aging. METHODS AND MATERIALS: Mice with an AECII-specific deletion of IGF-1R received thoracic irradiation (n ≥ 5 per condition), and the effect of IGF-1R deficiency on radiation-induced AECII senescence and macrophage polarization to an alternatively activated phenotype (M2) was investigated. IGF-1R signaling, macrophage polarization, and senescence were evaluated in surgically resected human lung (n = 63). RESULTS: IGF-1R deficient mice demonstrated reduced AECII senescence (senescent AECII/field; intact: 7.25% ± 3.5% [mean ± SD], deficient: 2.75% ± 2.8%, P = .0001), reduced accumulation of M2 macrophages (intact: 24.7 ± 2.2 cells/field, deficient: 15.5 ± 1.2 cells/field, P = .0086), and fibrosis (hydroxyproline content; intact: 71.9 ± 21.7 µg/lung, deficient: 31.7 ± 7.9, P = .0485) after irradiation. Senescent AECII enhanced M2 polarization in a paracrine fashion (relative Arg1 mRNA, 0 Gy: 1.0 ± 0.4, 17.5 Gy: 7.34 ± 0.5, P < .0001). Evaluation of surgical samples from patients treated with chemoradiation demonstrated increased expression of IGF-1 (unirradiated: 10.2% ± 4.9% area, irradiated: 15.1% ± 11.5%, P = .0377), p21 (unirradiated: 0.013 ± 0.02 histoscore, irradiated: 0.084 ± 0.09 histoscore, P = .0002), IL-13 (unirradiated: 13.7% ± 2.8% area, irradiated: 21.7% ± 3.8%, P < .0001), and M2 macrophages in fibrotic regions relative to nonfibrotic regions (unirradiated: 11.4 ± 12.2 CD163 + cells/core, irradiated: 43.1 ± 40.9 cells/core, P = .0011), consistent with findings from animal models of lung fibrosis. CONCLUSIONS: This study demonstrates that senescent AECII are necessary for the progression of pulmonary fibrosis and serve as a targetable, chronic stimuli for macrophage activation in fibrotic lung.

Hydroxyproline determination for initial detection of halal-critical food ingredients (gelatin and collagen).

Gelatin and collagen are considered halal-critical ingredients as they are typically derived from either bovine or porcine animals. Current analytical methods for determining the sources of gelatin and collagen suffer from limitations in terms of robustness and false positives in peptide matching. Thus, the aim of this study was to investigate the utility of monitoring hydroxyproline, a signature amino acid for gelatin and collagen, for identifying potentially haram foodstuffs. To determine the hydroxyproline profiles among animal- and plant-based samples, one-way univariate analysis of variance followed by pair-wise comparison was used to establish statistical significance. Multivariate chemometric analysis through principal component analysis revealed a discrete distribution pattern among 59 samples due to hydroxyproline variability. Finally, inter- and intra-laboratory comparisons demonstrated the validity and robustness of hydroxyproline determination according to ISO 17025. Thus, this preliminary identification technique will aid the identification of potentially haram foodstuffs.

[Correlational analysis of alternation of clinical features of intrahepatic lymphocyte subsets with HBV virology and liver fibrosis in HBV-Tg composite CCl(4) mice model].

Objective: To observe the alteration of clinical features of intrahepatic lymphocyte subsets in C57BL/6N-TG (1.28HBV)/Vst hepatitis B virus (HBV) transgenic mice composite carbon tetrachloride (CCl(4)) with intraperitoneal injection under the background of hepatitis B to induce liver fibrosis mice model, and analyze their correlation with serum HBV DNA and liver tissue hydroxyproline (Hyp) content. Methods: HBV-Tg mice were intraperitoneally injected with 10% CCl(4) to induce the rapid formation of hepatic fibrosis. Serum HBV DNA, HBsAg, HBeAg levels and liver tissue HBsAg expressional conditions were used to evaluate the virological characteristics of mice model. The degree of hepatic inflammation and fibrosis in mice were observed by HE, Sirius Red staining and liver tissue hydroxyproline (Hyp) content. Intrahepatic T lymphocyte, B lymphocyte, CD4+T lymphocyte, CD8+T lymphocyte, natural killer (NK) cell and natural killer T (NKT) cells distribution were observed by flow cytometry. One-way analysis of variance was used for intergroup data comparison, and LSD was used for pairwise comparison. Pearson's correlation analysis was used to analyze the correlation between the above lymphocyte subsets and serum HBV DNA and liver tissue Hyp content. Results: Serum HBsAg, HBeAg and liver tissue HBsAg had equal positive expression in the HBV-Tg composite CCl(4) mice model group, and the serum HBV DNA load was > 1 × 10(6) IU / ml. Compared with the wild-type control group, liver tissue Hyp content of the composite model group was significantly higher [(196.39 ± 38.14) µg /g and (347.67 ± 59.53) µ g/g, P < 0.01). The degree of inflammation and fibrosis in liver tissues was aggravated, and the proportion of all intrahepatic CD4+T, NK and NKT cells was significantly reduced (P < 0.01), while the proportion of CD8+T lymphocytes (30.58% ± 2.89% vs. 46.50% ± 2.24%, P < 0.01) and B lymphocytes (28.82% ± 2.24% vs. 37.10% ± 8.59%, P < 0.05) was significantly increased. Serum HBV DNA level was positively correlated with the proportion of intrahepatic T lymphocytes (r = 0.413, P < 0.05), and negatively correlated with the proportion of NK cells (r = -0.419, P < 0.05). Liver tissue Hyp content was negatively correlated with the proportion of all CD4+T lymphocytes (r = -0.871), NK cells (r = -0.716), and NKT cells (r = -0.876) (all P < 0.01), and positively correlated with the proportion of all CD8 + T lymphocytes (r = 0.852), and B lymphocytes (r = 0.593) (all P < 0.01). Conclusion: HBV-Tg composite CCl4 mice model can induce positive HBV virological indicators, liver inflammation and fibrosis in mice model of hepatitis B coexisting with fibrosis. This model has the features of immune disorder of liver lymphocyte similar to human disease, and the immune disorder of intrahepatic lymphocytes is correlated with HBV viral load and liver fibrosis degree.

Peculiarities of Collagen Turnover in Aging BALB/c Mice.

Examination of collagen turnover in the liver, lungs, and spleen of BALB/c mice revealed the age-related differences in the levels of hydroxyproline and its fractions. The highest level of total hydroxyproline was observed in the lungs and spleen of young (2-month-old) mice. In the liver, this level attained maximum at the age of 6 months at the expense of elevation of protein-bound hydroxyproline relatively its level in 2-month-old mice. At the age of 12 months, the levels of total hydroxyproline in the liver and spleen were lower than in 6-month-old mice. The decrease in the collagen turnover rate in the liver of 12-month-old mice reflected lower levels of hydroxyproline fractions in comparison with the corresponding values in 6-month-old mice. The rates of collagen turnover in organs differed in mice of different ages: it was maximum in the lungs and spleen of young animals and in the liver of middle-aged mice.

Quantitative Analysis of the Positional Distribution of Hydroxyproline in Collagenous Gly-Xaa-Yaa Sequences by LC-MS with Partial Acid Hydrolysis and Precolumn Derivatization.

Collagen is extensively modified by various enzymes, including prolyl hydroxylases. Pro residues at the Yaa position of repeating Gly-Xaa-Yaa amino acid sequences are mostly hydroxylated to 4-hydroxyproline (4Hyp), which is essential for the thermal stability of collagen triple helix. In contrast, Pro residues at the Xaa position are rarely modified to 3Hyp and 4Hyp, the biological function of which is poorly understood. Overall estimation of prolyl hydroxylation with discrimination of the position (Xaa or Yaa) and hydroxylation type (4Hyp or 3Hyp) has been difficult to perform using traditional methods. In the present study, we developed a novel position-specific analytical method featuring LC-MS detection of collagenous Gly-containing dipeptides, including Gly-Pro, Pro-Gly, Gly-4Hyp, Gly-3Hyp, and 4Hyp-Gly, after partial acid hydrolysis and precolumn derivatization using 3-aminopyridyl-N-hydroxysuccinimidyl carbamate (APDS). We performed acid hydrolysis at 55 °C with HCl/trifluoroacetic acid/water (2:1:1, v/v) to avoid peptide inversion and imbalanced peptide generation observed for collagenous model peptides. The positional distribution of Pro, 4Hyp, and 3Hyp can be calculated from the relative concentrations of the APDS-derivatized dipeptides, and in combination with amino acid analysis, we can determine their absolute contents at the Xaa and Yaa positions. Bovine type I, III, and V collagens were analyzed by the established method, and the amount of 4Hyp was higher than that of 3Hyp at the Xaa position in type I and III collagens. In addition, we clearly showed that collagen extracted from earthworm cuticles has an extremely high content of Xaa position 4Hyp, reaching over 10% of the total amino acids.