Betanin protects against bleomycin-induced pulmonary fibrosis by regulating the NLRP3/IL-1ß/TGF-ß1 pathway-mediated epithelial-to-mesenchymal transition.

Idiopathic pulmonary fibrosis (IPF) is a life-threatening disease that leads to dyspnea and progressive loss of lung function. This study aimed to investigate the protective effect of betanin (BET), the major pigment in red beetroot, on pulmonary fibrosis induced by bleomycin (BLM) in rats and to assess the underlying mechanisms. In this view, total and differential cell counts and LDH activity in bronchoalveolar lavage fluid were estimated. Furthermore, MDA and GSH contents in the lungs were colorimetrically measured, while hydroxyproline, NLRP3, ASC, caspase-1, TGF-ß1, and vimentin levels in lung tissue were evaluated using the ELISA technique. Moreover, IL-1ß, E-cadherin, and α-SMA expressions were analyzed by immunostaining of lung specimens. BET treatment protects against pulmonary fibrosis as indicated by the reduction in total and differential cell counts, LDH activity, hydroxyproline, NLRP3, ASC, caspase-1, IL-1ß, and TGF-ß1 levels. MDA content was also decreased following BET administration, while GSH content was elevated. Additionally, BET suppressed the EMT process as evidenced by an increase in E-cadherin expression besides the reduction in vimentin and α-SMA expressions. To conclude, these results revealed the protective effect of BET against pulmonary fibrosis that might be attributed to the attenuation of the NLRP3/IL-1ß/TGF-ß1 signaling pathway and EMT process.

Low-Molecular-Weight Fish Collagen Peptide (Valine-Glycine-Proline-Hydroxyproline-Glycine-Proline-Alanine-Glycine) Prevents Osteoarthritis Symptoms in Chondrocytes and Monoiodoacetate-Injected Rats.

The objective of this study was to investigate the effect of low-molecular-weight fish collagen (valine-glycine-proline-hydroxyproline-glycine-proline-alanine-glycine; LMWCP) on H2O2- or LPS-treated primary chondrocytes and monoiodoacetate (MIA)-induced osteoarthritis rat models. Our findings indicated that LMWCP treatment exhibited protective effects by preventing chondrocyte death and reducing matrix degradation in both H2O2-treated primary chondrocytes and cartilage tissue from MIA-induced osteoarthritis rats. This was achieved by increasing the levels of aggrecan, collagen type I, collagen type II, TIMP-1, and TIMP-3, while simultaneously decreasing catabolic factors such as phosphorylation of Smad, MMP-3, and MMP-13. Additionally, LMWCP treatment effectively suppressed the activation of inflammation and apoptosis pathways in both LPS-treated primary chondrocytes and cartilage tissue from MIA-induced osteoarthritis rats. These results suggest that LMWCP supplementation ameliorates the progression of osteoarthritis through its direct impact on inflammation and apoptosis in chondrocytes.

The Wound-Healing Potential of the Endemic Plant Helianthemum canum (L.) Baumg: Preclinical Studies Supported with Phytochemical Profiling.

The study's objective is to clarify the probable mechanisms underlying the wound-healing properties of Helianthemum canum L. (Cistaceae), a traditional anti-inflammatory and wound-healing medicine. LC/MS-MS was used to perform phytochemical analyses on a 70 % methanol extract of the plant's aerial parts. In vivo, linear incision and circular excision models were used to evaluate the wound healing activity. For anti-inflammatory effect, in vivo acetic acid capillary permeability assay and in vitro Interleukin 1, Interleukin 6, and Interferon É£ levels in LPS-induced FR skin fibroblast cell line were also evaluated. The extract significantly improved wound healing in experimental models, with tensile strength values of 27.8 % and a contraction value of 35.09 %. Histopathological examinations, hydroxyproline estimation, hyaluronidase, collagenase, and elastase enzyme inhibitory assays confirmed wound healing potential. Inflammatory cytokines were significantly inhibited in the LPS-induced FR cell line, with the highest effect seen on IL-6 (34.5±2.12 pg/mL). This study offered the first concrete proof that H. canum can be used to treat wounds by suggesting that the myricetin and quinic acid content identified by LCMS-MS analysis may be accountable for the effect of H. canum on wound contraction and hydroxyproline production.

The Protective Effect of SLNP on Hepatic Fibrosis Induced by Thioacetamide in Rats.

INTRODUCTION: The incidence of non-alcoholic fatty liver disease (NAFLD) has increased in recent years. Hepatic fibrosis (HF) is an important step in the progression of NAFLD to cirrhosis and even carcinoma and is also recognized as a possible reversal phase. AIMS: We previously found that the aqueous extract of Sedum Lineare Thunb. has hepatoprotective effects. This study investigated the hepatoprotective effect and mechanism of the Sedum Lineare Thunb. n-butanol phase (SLNP) on HF in rats. METHODS: Animals were intraperitoneally injected with thioacetamide solution twice a week for 8 weeks to prepare an HF model and were administered the corresponding drugs or an equal volume of normal saline by intragastric administration once a day for 8 weeks. Liver function, hydroxyproline and malondialdehyde (MDA) content, superoxide dismutase (SOD), Na+-K+-ATPase, and Ca2+-Mg2+-ATPase were analyzed using colorimetric methods. Moreover, mRNA expression and protein levels in the liver tissue were detected via quantitative polymerase chain reaction and western blotting, respectively. RESULTS: The results showed that SLNP could effectively improve the liver function of rats with HF and significantly reduce the content of hydroxyproline; the mRNA expression and protein levels of alpha-smooth muscle actin (α-SMA), collagen I, III, and IV, transforming growth factor beta 1 (TGF-ß1), Smad2/3, and Smad4 were also significantly reduced. Simultaneously, SLNP significantly increased the activities of SOD, Na+-K+- ATPase, and Ca2+-Mg2+-ATPase in the rat liver tissues, whereas it reduced the levels of MDA and SOD in the serum and liver tissues. CONCLUSION: This study revealed that SLNP elicits an anti-fibrotic effect by inhibiting oxidative stress and stellate cell activation, thereby reducing the formation and deposition of the extracellular matrix. The TGF-ß1/Smads signaling pathway may be involved in this process.

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%).

Therapeutic Effects of Albumin-Fused BMP7 on 2 Experimental Models of Liver Fibrosis.

Despite the fact that liver fibrosis is an intractable disease with a poor prognosis, effective therapeutic agents are not available. In this study, we focused on bone morphogenetic factor 7 (BMP7) that inhibits transforming growth factor (TGF)-ß signaling, which is involved in liver fibrosis. We prepared an albumin-fused BMP7 (HSA-BMP7) that is retained in the blood and evaluated its inhibitory effect on liver fibrosis. Bile duct ligated mice were used as an acute liver fibrosis model, and carbon tetrachloride-induced liver fibrosis mice were used as a chronic model. All mice were administered HSA-BMP7 once per week. In the mice with bile duct ligation, the administration of HSA-BMP7 significantly suppressed the infiltration of inflammatory cells, the area of fibrosis around the bile duct, and decreased in the level of hydroxyproline as compared with saline administration. The mRNA expression of TGF-ß and its downstream fibrosis-associated genes (α-SMA and Col1a2) were also suppressed by the administration of HSA-BMP7. In the carbon tetrachloride-induced liver fibrosis mice, the HSA-BMP7 administration significantly decreased the hepatic fibrosis area and the level of hydroxyproline. Based on these results, it appears that HSA-BMP7 has the potential for serving as a therapeutic agent for the treatment of liver fibrosis.

Synthesis of glycine from 4-hydroxyproline in tissues of neonatal pigs with intrauterine growth restriction.

This study tested the hypothesis that the synthesis of glycine from 4-hydroxyproline (an abundant amino acid in milk and neonatal blood) was impaired in tissues of piglets with intrauterine growth restriction (IUGR), thereby contributing to a severe glycine deficiency in these compromised neonates. At 0, 7, 14, and 21 days of age, IUGR piglets were euthanized, and tissues (liver, small intestine, kidney, pancreas, stomach, skeletal muscle, and heart) were obtained for metabolic studies, as well as the determination of enzymatic activities, cell-specific localization, and expression of mRNAs for glycine-synthetic enzymes. The results indicated relatively low enzymatic activities for 4-hydroxyproline oxidase (OH-POX), proline oxidase, serine hydroxymethyltransferase, threonine dehydrogenase (TDH), alanine: glyoxylate transaminase, and 4-hydroxy-2-oxoglutarate aldolase in the kidneys and liver from 0- to 21-day-old IUGR pigs, in the pancreas of 7- to 21-day-old IUGR pigs, and in the small intestine and skeletal muscle (except TDH) of 21-day-old IUGR pigs. Accordingly, the rates of conversion of 4-hydroxyproline into glycine were relatively low in tissues of IUGR piglets. The expression of mRNAs for glycine-synthetic enzymes followed the patterns of enzymatic activities and was also low. Immunohistochemical analyses revealed the relatively low abundance of OH-POX protein in the liver, kidney, and small intestine of IUGR piglets, and the lack of OH-POX zonation in their livers. These novel results provide a metabolic basis to explain why the endogenous synthesis of glycine is insufficient for optimum growth of IUGR piglets and have important implications for improving the nutrition and health of other mammalian neonates including humans with IUGR.

Metabolomics analysis of human cumulus cells obtained from cumulus-oocyte complexes with different developmental potential.

STUDY QUESTION: Is the abundance of certain biochemical compounds in human cumulus cells (CCs) related to oocyte quality? SUMMARY ANSWER: Malonate, 5-oxyproline, and erythronate were positively associated with pregnancy potential. WHAT IS KNOWN ALREADY: CCs are removed and discarded prior to ICSI, thereby constituting an interesting biological material on which to perform molecular analysis aimed to predict oocyte developmental competence. Mitochondrial DNA content and transcriptional analyses in CC have been shown to provide a poor predictive value of oocyte competence, but the untargeted analysis of biochemical compounds (metabolomics) has been unexplored. STUDY DESIGN, SIZE, DURATION: CCs were obtained from three groups of cumulus-oocyte complexes (COCs) of known developmental potential: oocytes not developing to blastocyst following ICSI (Bl-); oocytes developing to blastocyst but failing to establish pregnancy following embryo transfer (P-); and oocytes developing to blastocyst able to establish a pregnancy (P+). Metabolomics analyses were performed on 12 samples per group, each sample comprising the CC recovered from a single COC. PARTICIPANTS/MATERIALS, SETTING, METHODS: Human CC samples were obtained from IVF treatments. Only unfrozen oocytes and embryos not submitted to preimplantation genetic testing were included in the analysis. Metabolomics analysis was performed by ultra-high performance liquid chromatography-tandem mass spectroscopy. MAIN RESULTS AND THE ROLE OF CHANCE: The analysis identified 98 compounds, five of which were differentially abundant (P < 0.05) between groups: asparagine, proline, and malonate were less abundant in P- compared to Bl-, malonate and 5-oxoproline were less abundant in P- group compared to P+, and erythronate was less abundant in Bl- group compared to P+. No significant association between the abundance of the compounds identified and donor age or BMI was noted. LIMITATIONS, REASONS FOR CAUTION: Data dispersion and the lack of coherence between developmental groups preclude the direct use of metabolic markers in clinical practice, where the uterine environment plays a major role in pregnancy outcome. The abundance of other compounds not detected by the analysis may be associated with oocyte competence. As donors were lean (only two with BMI > 30 kg/m2) and young (<34 years old), a possible effect of obesity or advanced age on the CC metabolome could not be determined. WIDER IMPLICATIONS OF THE FINDINGS: The abundance of malonate, 5-oxyproline, and erythronate in CC was significantly higher in COCs ultimately establishing pregnancy, providing clues on the pathways required for oocyte competence. The untargeted analysis uncovered the presence of compounds that were not expected in CC, such as ß-citrylglutamate and the neurotransmitter N-acetyl-aspartyl-glutamate, which may play roles in chromatin remodeling and signaling, respectively. STUDY FUNDING/COMPETING INTEREST(S): Research was supported by the Industrial Doctorate Project IND2017/BIO-7748 funded by Madrid Region Government. The authors declare no competing interest. TRIAL REGISTRATION NUMBER: N/A.

Synthesis of glycine from 4-hydroxyproline in tissues of neonatal pigs.

Glycine from sow's milk only meets 20% of the requirement of suckling piglets. However, how glycine is synthesized endogenously in neonates is not known. This study determined glycine synthesis from 4-hydroxyproline (an abundant amino acid in milk and neonatal blood) in tissues of sow-reared piglets with normal birth weights. Piglets were euthanized at 0, 7, 14 and 21 days of age, and their tissues were used to determine glycine synthesis from 0 to 5 mM 4-hydroxyproline, activities and mRNA expression of key glycine-synthetic enzymes, and their cell-specific localization. Activities of 4-hydroxyproline oxidase (OH-POX), proline oxidase (POX), serine hydroxymethyltransferase (SHMT), threonine dehydrogenase (TDH), alanine:glyoxylate transaminase (AGT), and 4-hydroxy-2-oxoglutarate aldolase (HOA) occurred in the kidneys and liver from all age groups of piglets, and in the pancreas of 7- to 21-day-old piglets. Activities of OH-POX and HOA were absent from the small intestine of newborn pigs but present in the small intestine of 7- to 21-day-old piglets and in the skeletal muscle of 14- to 21-day-old piglets. Between days 0 and 21 of age, the enzymatic activities of OH-POX, AGT, and HOA decreased in the liver and kidneys but increased in the pancreas and small intestine with age. The mRNA levels of these three enzymes changed in a manner similar to their enzymatic activities. In contrast to OH-POX, AGT, and HOA, the enzymatic activities of POX, SHMT, and TDH were present in the kidneys, liver, and intestine of all age groups of piglets. Glycine was synthesized from 0.1 to 5 mM 4-hydroxyproline in the liver and kidney from 0- to 21-day-old piglets, as well as the pancreas, small intestine, and skeletal muscle from 14- to 21-day-old piglets in a concentration-dependent manner. Collectively, our findings indicate that 4-hydroxyproline is used for the synthesis of glycine in tissues of piglets to compensate for the deficiency of glycine in milk.

[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.

The mechanism of lung tissue YKL-40 promoting the interstitial transformation of alveolar epithelial cells and its effect on TGF-ß1 level in mice with idiopathic pulmonary fibrosis.

This study aimed to investigate the mechanism of lung tissue YKL-40 promoting the interstitial transformation of alveolar epithelial cells in mice with idiopathic pulmonary fibrosis and its effect on the level of TGF-ß1. For this purpose, Forty SPF SD mice were randomly divided into 4 groups. They were the blank control group (CK group), virus-negative control group (YKL-40-NC group), YKL-40 knockdown group (YKL-40-inhibitor group) and YKL-40 overexpression group (YKL-40-mimics group), respectively. The mRNA expressions of alveolar epithelial cell mesenchymal transformation-related proteins, pulmonary fibrosis-related factors and TGF-ß1-related pathway proteins in the above four groups of mice were compared to determine the mechanism of the promotion of alveolar epithelial cell mesenchymal transformation by YKL-40 in the lung tissues of mice with idiopathic pulmonary fibrosis and the effect of YKL-40 on the level of TGF-ß1. The results showed that in terms of lung wet/dry weight ratio, the YKL-40-NC group, YKL-40-inhibitor group and YKL-40-mimics group were significantly increased compared with the CK group (P<0.05). About YKL-40 protein expression, compared with the CK group, AOD value and YKL-40 protein expression in the YKL-40-NC group, YKL-40-inhibitor group and YKL-40-mimics group were significantly increased (P<0.05), and compared with YKL-40-NC group, The AOD value and YKL-40 protein expression in YKL-40-inhibitor group were significantly decreased, while the AOD value and YKL-40 protein expression in YKL-40-mimics group were significantly increased (P<0.05), suggesting successful lentivirus transfection. Compared with the CK group, ß-catenin and E-cadherin in the alveolar epithelial cells were significantly increased, while Pro-SPC was significantly decreased (P<0.05). The mRNA expression of pulmonary fibrosis-related factors showed that compared with the CK group, the mRNA expression of vimimin and hydroxyproline was significantly increased, while the mRNA expression of E-cadherin was decreased (P<0.05). However, the mRNA expressions of vimimin and hydroxyproline in the YKL-40-inhibitor group were significantly decreased, but the mRNA expression of E-cadherin was significantly increased. Compared with CK group, the protein expressions of TGF-ß1, Smad3, Smad7 and α-Sma in the CK group were significantly increased (P<0.05). The protein expressions of TGF-ß1, Smad3, Smad7 and α-SMA in the YKL-40-mimics group were significantly increased, but the protein expressions of TGF-ß1, Smad3, Smad7 and α-SMA in YKL-40-inhibitor group were significantly decreased (P<0.05). In general, overexpression of YKL-40 can promote the progression of pulmonary fibrosis and the interstitial transformation of alveolar epithelial cells in mice with idiopathic fibrosis.

Low-dose vanadium pentoxide perturbed lung metabolism associated with inflammation and fibrosis signaling in male animal and in vitro models.

Vanadium is available as a dietary supplement and also is known to be toxic if inhaled, yet little information is available concerning the effects of vanadium on mammalian metabolism when concentrations found in food and water. Vanadium pentoxide (V+5) is representative of the most common dietary and environmental exposures, and prior research shows that low-dose V+5 exposure causes oxidative stress measured by glutathione oxidation and protein S-glutathionylation. We examined the metabolic impact of V+5 at relevant dietary and environmental doses (0.01, 0.1, and 1 ppm for 24 h) in human lung fibroblasts (HLFs) and male C57BL/6J mice (0.02, 0.2, and 2 ppm in drinking water for 7 mo). Untargeted metabolomics using liquid chromatography-high-resolution mass spectrometry (LC-HRMS) showed that V+5 induced significant metabolic perturbations in both HLF cells and mouse lungs. We noted 30% of the significantly altered pathways in HLF cells, including pyrimidines and aminosugars, fatty acids, mitochondrial and redox pathways, showed similar dose-dependent patterns in mouse lung tissues. Alterations in lipid metabolism included leukotrienes and prostaglandins involved in inflammatory signaling, which have been associated with the pathogenesis of idiopathic pulmonary fibrosis (IPF) and other disease processes. Elevated hydroxyproline levels and excessive collagen deposition were also present in lungs from V+5-treated mice. Taken together, these results show that oxidative stress from environmental V+5, ingested at low levels, could alter metabolism to contribute to common human lung diseases.NEW & NOTEWORTHY We used relevant dietary and environmental doses of Vanadium pentoxide (V+5) to examine its metabolic impact in vitro and in vivo. Using liquid chromatography-high-resolution mass spectrometry (LC-HRMS), we found significant metabolic perturbations, with similar dose-dependent patterns observed in human lung fibroblasts and male mouse lungs. Alterations in lipid metabolism included inflammatory signaling, elevated hydroxyproline levels, and excessive collagen deposition were present in V+5-treated lungs. Our findings suggest that low levels of V+5 could trigger pulmonary fibrotic signaling.

Collagen-Derived Dipeptides and Amino Acids Have Immunomodulatory Effects in M1-Differentiated RAW264.7 Cells and PBMC.

A number of food components, such as polyphenols and phytonutrients, have immunomodulatory effects. Collagen has various bioactivities, such as antioxidative effects, the promotion of wound healing, and relieving symptoms of bone/joint disease. Collagen is digested into dipeptides and amino acids in the gastrointestinal tract and subsequently absorbed. However, the difference in immunomodulatory effects between collagen-derived dipeptides and amino acids is unknown. To investigate such differences, we incubated M1 macrophages or peripheral blood mononuclear cells (PBMC) with collagen-derived dipeptides (hydroxyproline-glycine (Hyp-Gly) and proline-hydroxyproline (Pro-Hyp)) and amino acids (proline (Pro), hydroxyproline (Hyp), and glycine (Gly)). We first investigated the dose dependency of Hyp-Gly on cytokine secretion. Hyp-Gly modulates cytokine secretion from M1 macrophages at 100 µM, but not at 10 µM and 1 µM. We then compared immunomodulatory effects between dipeptides and mixtures of amino acids on M1 macrophages and PBMC. There was, however, no difference in cytokine secretion between dipeptides and their respective amino acids. We conclude that collagen-derived dipeptides and amino acids have immunomodulatory effects on M1-differentiated RAW264.7 cells and PBMC and that there is no difference in the immunomodulatory effects between dipeptides and amino acids.

Collagen constitutes about 12% in females and 17% in males of the total protein in mice.

Collagen has been postulated to be the most abundant protein in our body, making up one-third of the total protein content in mammals. However, a direct assessment of the total collagen levels of an entire mammal to confirm this estimate is missing. Here we measured hydroxyproline levels as a proxy for collagen content together with total protein levels of entire mice or of individual tissues. Collagen content normalized to the total protein is approximately 0.1% in the brain and liver, 1% in the heart and kidney, 4% in the muscle and lung, 6% in the colon, 20-40% in the skin, 25-35% in bones, and 40-50% in tendons of wild-type (CD1 and CB57BL/6) mice, consistent with previous reports. To our surprise, we find that collagen is approximately 12% in females and 17% in males of the total protein content of entire wild-type (CD1 and CB57BL/6) mice. Although collagen type I is the most abundant collagen, the most abundant proteins are albumin, hemoglobulin, histones, actin, serpina, and then collagen type I. Analyzing amino acid compositions of mice revealed glycine as the most abundant amino acid. Thus, we provide reference points for collagen, matrisome, protein, and amino acid composition of healthy wild-type mice.

The hydroxyproline O-arabinosyltransferase FIN4 is required for tomato pollen intine development.

The pollen grain cell wall is a highly specialized structure composed of distinct layers formed through complex developmental pathways. The production of the innermost intine layer, composed of cellulose, pectin and other polymers, is particularly poorly understood. Here we demonstrate an important and specific role for the hydroxyproline O-arabinosyltransferase (HPAT) FIN4 in tomato intine development. HPATs are plant-specific enzymes which initiate glycosylation of certain cell wall structural proteins and signaling peptides. FIN4 was expressed throughout pollen development in both the developing pollen and surrounding tapetal cells. A fin4 mutant with a partial deletion of the catalytic domain displayed significantly reduced male fertility in vivo and compromised pollen hydration and germination in vitro. However, fin4 pollen that successfully germinated formed morphologically normal pollen tubes with the same growth rate as the wild-type pollen. When we examined mature fin4 pollen, we found they were cytologically normal, and formed morphologically normal exine, but produced significantly thinner intine. During intine deposition at the late stages of pollen development we found fin4 pollen had altered polymer deposition, including reduced cellulose and increased detection of pectin, specifically homogalacturonan with both low and high degrees of methylesterification. Therefore, FIN4 plays an important role in intine formation and, in turn pollen hydration and germination and the process of intine formation involves dynamic changes in the developing pollen cell wall.

Type II arabinogalactans initiated by hydroxyproline-O-galactosyltransferases play important roles in pollen-pistil interactions.

Arabinogalactan-proteins (AGPs) are hydroxyproline-rich glycoproteins containing a high sugar content and are widely distributed in the plant kingdom. AGPs have long been suggested to play important roles in sexual plant reproduction. The synthesis of their complex carbohydrates is initiated by a family of hydroxyproline galactosyltransferase (Hyp-GALT) enzymes which add the first galactose to Hyp residues in the protein backbone. Eight Hyp-GALT enzymes have been identified so far, and in the present work a mutant affecting five of these enzymes (galt2galt5galt7galt8galt9) was analyzed regarding the reproductive process. The galt25789 mutant presented a low seed set, and reciprocal crosses indicated a significant female gametophytic contribution to this mutant phenotype. Mutant ovules revealed abnormal callose accumulation inside the embryo sac and integument defects at the micropylar region culminating in defects in pollen tube reception. In addition, immunolocalization and biochemical analyses allowed the detection of a reduction in the amount of glucuronic acid in mutant ovary AGPs. Dramatically low amounts of high-molecular-weight Hyp-O-glycosides obtained following size exclusion chromatography of base-hydrolyzed mutant AGPs compared to the wild type indicated the presence of underglycosylated AGPs in the galt25789 mutant, while the monosaccharide composition of these Hyp-O-glycosides displayed no significant changes compared to the wild-type Hyp-O-glycosides. The present work demonstrates the functional importance of the carbohydrate moieties of AGPs in ovule development and pollen-pistil interactions.

Synergistic Protective Effect of Interactions of Quercetin with Lycopene Against Ochratoxin A-Induced Ulcerative Colitis.

Ulcerative colitis is a type of inflammatory bowel disease responsible for the inflammation of the innermost lining of the colon and rectum. The present study's objective is to determine the potential synergistic impact of quercetin (QR) and lycopene (LP) in ulcerative colitis (UC) induced in rats by ochratoxin A (OTA) by biochemical and morphological alterations. QR and LP were administered alone and in combination with the OTA for 7 days. OTA administration caused UC generation, resulting in significant changes in body weight percentage, disease activity index (DAI), macroscopic evaluation, colon weight/length ratio, and histological score. In addition to the above parameters, it also leads to elevated oxidative stress, i.e. increased malondialdehyde (MDA), nitric oxide (NO), myeloperoxidase (MPO), and hydroxyproline levels and decreased superoxide dismutase (SOD) and reduced glutathione (GSH) levels. Histological changes in the colon architecture were also observed suggestive of extensive mucosal damage. In addition, a high level of matrix metalloproteinase 7 (MMP7) was observed in immunohistochemistry, and a high level of gene expression of osteopontin (OPN), runt-related transcription factor 2 (RUNX2), MMP-7, and interleukin-6 (IL-6) was observed in OTA administered animals. The combination of QR and LP significantly restored the per cent body weight loss and DAI score and improved macroscopic and histological changes, colon weight/length ratio, and macroscopic damages. It also improved the biochemical parameters to near-normal levels, i.e. reduced MDA, NO, MPO, and hydroxyproline levels and increased SOD and GSH levels. In addition, OPN, Runx2, MMP-7, and IL-6 gene expression decreased compared to the OTA-induced UC group. Outcomes of the present study indicate the potential of QR + LP as anti-inflammatory and immunomodulatory agents against OTA-induced UC in rats.

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.

Is nanoparaquat safer than bulk paraquat? An in vitro and in vivo evaluation.

Paraquat (PQ) is an herbicide which has brought some health problems through the production of reactive oxygen species. The increasing interest in the novel formulation of agrochemicals has been aiming to provide safety for non-target organisms. Chitosan is a well-known non-toxic polymer, commonly used in preparing particles via ionotropic gelation. In this study, we prepared PQ nanoparticles (PQNPs) and evaluated their toxicity in vivo and in vitro. PQNPs were prepared and characterized in two forms, with and without the utilization of chitosan. Relative cell survival of PQNPs were studied against bulk PQ in HEK-293. Also, the acute lung injury of PQNP was assessed against treatment with acetylcysteine. Total antioxidant capacity (TAC), lipid peroxidation (LPO), total thiol groups (TTG), and hydroxyproline, along with histological changes were assessed in the lungs. The size, zeta potential, and polydispersity index of the optimum particles were about 157.7 ± 7.03, 22.25 ± 4.52, and 0.701, respectively. The encapsulation efficiency was 65.11 ± 10.45, and the loading percent of PQ was 58.57 ± 2.37. PQNPs showed an initial burst of PQ release followed by a zero-degree pattern. PQNPs displayed lower cell cytotoxicity compared to bulk PQ. LPO, TAC, TTG, and hydroxyproline levels in lungs generally showed more satisfying status in PQNPQs as well. The levels of oxidative status markers indicate lower oxidative damage in lungs and a more desirable response to acetylcysteine treatment, in line with histological changes. PQ loaded in chitosan-alginate particles offers safer characteristics compared with bulk PQ.

Automated whole slide image analysis for a translational quantification of liver fibrosis.

Current literature highlights the need for precise histological quantitative assessment of fibrosis which cannot be achieved by conventional scoring systems, inherent to their discontinuous values and reader-dependent variability. Here we used an automated image analysis software to measure fibrosis deposition in two relevant preclinical models of liver fibrosis, and established correlation with other quantitative fibrosis descriptors. Longitudinal quantification of liver fibrosis was carried out during progression of post-necrotic (CCl4-induced) and metabolic (HF-CDAA feeding) models of chronic liver disease in mice. Whole slide images of picrosirius red-stained liver sections were analyzed using a fully automated, unsupervised software. Fibrosis was characterized by a significant increase of collagen proportionate area (CPA) at weeks 3 (CCl4) and 8 (HF-CDAA) with a progressive increase up to week 18 and 24, respectively. CPA was compared to collagen content assessed biochemically by hydroxyproline assay (HYP) and by standard histological staging systems. CPA showed a high correlation with HYP content for CCl4 (r = 0.8268) and HF-CDAA (r = 0.6799) models. High correlations were also found with Ishak score or its modified version (r = 0.9705) for CCl4 and HF-CDAA (r = 0.9062) as well as with NASH CRN for HF-CDAA (r = 0.7937). Such correlations support the use of automated digital analysis as a reliable tool to evaluate the dynamics of liver fibrosis and efficacy of antifibrotic drug candidates in preclinical models.