Decreased Collagen Synthesis Research Articles

Effects of aging on the histology and biochemistry of rat tendon healing

IntroductionTendon diseases and injuries are a serious problem for the aged population, often leading to pain, disability and a significant decline in quality of life. The purpose of this study was to determine the influence of aging on biochemistry and histology during tendon healing and to provide a new strategy for improving tendon healing.MethodA total of 24 Sprague-Dawley rats were equally divided into a young and an aged group. A rat patellar tendon defect model was used in this study. Tendon samples were collected at weeks 2 and 4, and hematoxylin-eosin, alcian blue and immunofluorescence staining were performed for histological analysis. Meanwhile, reverse transcription-polymerase chain reaction (RT-PCR) and western blot were performed to evaluate the biochemical changes.ResultsThe histological scores in aged rats were significantly lower than those in young rats. At the protein level, collagen synthesis-related markers Col-3, Matrix metalloproteinase-1 and Metallopeptidase Inhibitor 1(TIMP-1) were decreased at week 4 in aged rats compared with those of young rats. Though there was a decrease in the expression of the chondrogenic marker aggrecan at the protein level in aged tendon, the Micro-CT results from weeks 4 samples showed no significant difference(p>0.05) on the ectopic ossification between groups. Moreover, we found more adipocytes accumulated in the aged tendon defect with the Oil Red O staining and at the gene and protein levels the markers related to adipogenic differentiation.ConclusionsOur findings indicate that tendon healing is impaired in aged rats and is characterized by a significantly lower histological score, decreased collagen synthesis and more adipocyte accumulation in patellar tendon after repair.

Polydeoxyribonucleotide: A Promising Biological Platform to Accelerate Impaired Skin Wound Healing.

The normal wound healing process is characterized by a complex, highly integrated cascade of events, requiring the interactions of many cell types, including inflammatory cells, fibroblasts, keratinocytes and endothelial cells, as well as the involvement of growth factors and enzymes. However, several diseases such as diabetes, thermal injury and ischemia could lead to an impaired wound healing process characterized by wound hypoxia, high levels of oxygen radicals, reduced angiogenesis, decreased collagen synthesis and organization. Polydeoxyribonucleotide (PDRN) has been used to improve wound healing through local and systemic administration thanks to its ability to promote cell migration and growth, angiogenesis, and to reduce inflammation on impaired wound healing models in vitro, in vivo and clinical studies. In light of all these observations, the aim of this review is to provide a full overview of PDRN applications on skin regeneration. We reviewed papers published in the last 25 years on PubMed, inserting “polydeoxyribonucleotide and wound healing” as the main search term. All data obtained proved the ability of PDRN in promoting physiological tissue repair through adenosine A2A receptor activation and salvage pathway suggesting that PDRN has proven encouraging results in terms of healing time, wound regeneration and absence of side effects.

A brief study of Nox 4 inhibitors in diabetic nephropathy

The purpose of the study was to find the merits and demerits of NADPH oxidase 4 (Nox 4) inhibitors. Nox inhibitors are tested from natural sources like green tea, plumbagin, Chinese formulas like Baoshenfang, Cudrania tricuspidata, Huangqi decoction and synthetic dual Nox inhibitors. Some of them activate and regulate AMP-activated protein kinase (AMPK). Some chunk the high glucose activated alleyway, dawdling the succession of diabetic nephropathy (DN). Overall, the benefits of NOX are: reducing oxidative damage, improving renal function, reducing podocyte injury, preventing interstitial fibrosis, regulating AMPK which inhibits reactive oxygen species (ROS) and transforming growth factor-beta (TGF-beta), decreasing inflammation due to high glucose, protecting mesangial cells, decreasing collagen synthesis, and reducing glomerular hypertrophy. While targeting NOX in renal impairment, off-target effects, especially cardiovascular effects, are one of the major hurdles since diabetes mellitus (DM) is associated with co-morbid cardiovascular problems.

Specific Parameters of Extracellular Matrix Remodeling of Liver and Lungs of Mice with BCG Granulomatosis during Chronic Inflammation Depending on the Method of Administration of Liposomal Oxidized Dextran

The objective: to study the effect of liposomal oxidized dextran (LOD) on remodeling of the extracellular matrix of organs of mice during the period of chronic BCG granulomatosis and effects of LOD on this process depending on the route of administration. Subjects and Methods. The liver and lungs of mice with BCG granulomatosis were studied using different methods of LOD administration (intraperitoneally and by inhalation). The content of glycosaminoglycans, hydroxyproline fraction, the activity of matrix metalloproteinases (MMPs), hyaluronidases and a2-macroglobulin, and the content of tissue inhibitors of MMPs (TIMP-1 and -2) were evaluated. Results. The administration of LOD to mice infected with the BCG vaccine suffering from chronic granulomatosis with severe fibrosis (6 months after infection for 3 months) resulted in the aggravation of collagen degradation in the liver. In the lungs, along with increased collagen degradation, decreased collagen synthesis was observed. It was apparently due to suppressed activity of a2-macroglobulin and decreased content of TIMP-1 and -2. In the liver, with intraperitoneal administration of LOD, signs of suppressed fibrogenesis and fibrolysis were observed versus the data obtained for inhalation administration. There were no differences in the content of hydroxyproline fractions in the lungs depending on the method of LOD administration. Thus, administration of LOD to mice led to lower severity of fibrosis while the mechanisms of fibrolysis in the lungs and liver differed.

Dermal Olfactory Receptor OR51B5 Is Essential for Survival and Collagen Synthesis in Human Dermal Fibroblast (Hs68 Cells).

Skin dermis comprises extracellular matrix components, mainly collagen fibers. A decrease in collagen synthesis caused by several factors, including ultraviolet (UV) irradiation and stress, eventually causes extrinsic skin aging. Olfactory receptors (ORs) were initially considered to be specifically expressed in nasal tissue, but several ORs have been reported to be present in other tissues, and their biological roles have recently received increasing attention. In this study, we aimed to characterize the role of ORs in cell survival and collagen synthesis in dermal fibroblasts. We confirmed that UVB irradiation and dexamethasone exposure significantly decreased cell survival and collagen synthesis in Hs68 dermal fibroblasts. Moreover, we demonstrated that the mRNA expression of 10 ORs detectable in Hs68 cells was significantly downregulated in aged conditions compared with that in normal conditions. Thereafter, by individual knockdown of the 10 candidate ORs, we identified that only OR51B5 knockdown leads to a reduction of cell survival and collagen synthesis. OR51B5 knockdown decreased cAMP levels and dampened the downstream protein kinase A/cAMP-response element binding protein pathway, downregulating the survival- and collagen synthesis-related genes in the dermal fibroblasts. Therefore, OR51B5 may be an interesting candidate that plays a role in cell survival and collagen synthesis.

Simulated Microgravity Remodels Extracellular Matrix of Osteocommitted Mesenchymal Stromal Cells.

The extracellular matrix (ECM) is the principal structure of bone tissue. Long-term spaceflights lead to osteopenia, which may be a result of the changes in composition as well as remodeling of the ECM by osteogenic cells. To elucidate the cellular effects of microgravity, human mesenchymal stromal cells (MSCs) and their osteocommitted progeny were exposed to simulated microgravity (SMG) for 10 days using random positioning machine (RPM). After RPM exposure, an imbalance of MSC collagen/non-collagen ratio at the expense of a decreased level of collagenous proteins was detected. At the same time, the secretion of proteases (cathepsin A, cathepsin D, MMP3) was increased. No significant effects of SMG on the expression of stromal markers and cell adhesion molecules on the MSC surface were noted. Upregulation of COL11A1, CTNND1, TIMP3, and TNC and downregulation of HAS1, ITGA3, ITGB1, LAMA3, MMP1, and MMP11 were detected in RPM exposed MSCs. ECM-associated transcriptomic changes were more pronounced in osteocommitted progeny. Thus, 10 days of SMG provokes a decrease in the collagenous components of ECM, probably due to the decrease in collagen synthesis and activation of proteases. The presented data demonstrate that ECM-associated molecules of both native and osteocommitted MSCs may be involved in bone matrix reorganization during spaceflight.

Content of the Major Extracellular Matrix Components of the Liver and Lungs in Mice with Chronic BCG-Granulomatosis Treated with Liposome-Encapsulated Dextrazide.

Administration of the liposome-encapsulated dextrazide (LEDZ) to mice 6 months after infection with mycobacterium BCG vaccine for 3 months modulated metabolism of collagens and the intensity of fibrosis of internal organs. In the liver, we observed a redistribution of glycosaminoglycans towards sulfated forms, a decrease in the content of hyaluronan, a change in the ratio of hydroxyproline fractions indicating a decrease in fibrosis via two mechanisms: suppression of synthesis and increased degradation of collagens. In the lungs, administration of LEDZ did not affect the content of sulfated glycosaminoglycans, but significantly reduced the content of hyaluronan, stimulated degradation of collagen, and reduced its synthesis, but these processes were insufficient for significant reduction of fibrotic complications in the lungs. In animals treated with LEDZ, the decrease in collagen synthesis in the liver was 2-fold more pronounced than in the lungs.

Human umbilical cord mesenchymal stem cells ameliorate skin fibrosis development in a mouse model of bleomycin-induced systemic sclerosis.

Mesenchymal stem cell (MSC) infusion has become a novel therapeutic strategy for complex autoimmune diseases; however, few detailed studies have been performed to investigate the benefit and mechanism of MSC treatment on systemic sclerosis (SSc). The present study aimed to evaluate the therapeutic effect of human umbilical cord derived-MSCs (UC-MSCs) on bleomycin-induced SSc in mice and explore the potential underlying mechanism. The murine SSc model was established by daily subcutaneous injection of bleomycin for 4 weeks, followed with two UC-MSC infusions every 7 days. Skin fibrosis was assessed by H&E and Masson staining. Flow cytometry was used to determine IL-17A, IFN-γ, tumor necrosis factor-β, IL-10 and IL-12 levels in serum samples and T cell subsets in murine spleen. Additionally, gene expression levels of cytokines and fibrosis markers in skin samples were measured by reverse transcription-quantitative PCR. Immunofluorescence staining was performed to track UC-MSC localization and lymphocyte cell infiltration in vivo. UC-MSC treatment exerted an anti-fibrotic role in bleomycin-induced SSc mice, as confirmed by histological improvement, decreased collagen synthesis, and reduced collagen-1α1, collagen-1α2, fibronectin-1 and α-smooth muscle actin gene expression levels. The results indicated that UC-MSC treatment only had a limited systematic effect on cytokine production in serum samples and T cell activation in the spleen. By contrast, T helper (Th)17 cell infiltration and activation in skin were efficiently inhibited after UC-MSC infusion, as evidenced by the decreased IL-17A and retinoic acid-related orphan receptor γt gene expression as well as IL-17A production. UC-MSC administration significantly ameliorated bleomycin-induced skin fibrosis and collagen formation primarily by eliminating local inflammation and Th17 cell activation in the skin; however, the systemic inhibitory effect of UM-MSCs on cytokines was less profound.

In Vivo Evaluation of Three-Dimensional Printed, Keratin-Based Hydrogels in a Porcine Thermal Burn Model.

Keratin is a natural material that can be derived from the cortex of human hair. Our group had previously presented a method for the printed, sequential production of three-dimensional (3D) keratin scaffolds. Using a riboflavin-sodium persulfate-hydroquinone (initiator-catalyst-inhibitor) photosensitive solution, we produced 3D keratin-based constructs through ultraviolet crosslinking in a lithography-based 3D printer. In this study, we have used this bioink to produce a keratin-based construct that is capable of delivering small molecules, providing an environment conducive to healing of dermal burn wounds in vivo, and maintaining stability in customized packaging. We characterized the effects of manufacturing steps, such as lyophilization and gamma irradiation sterilization on the properties of 3D printed keratin scaffolds prepared for in vivo testing. Keratin hydrogels are viable for the uptake and release of contracture-inhibiting Halofuginone, a collagen synthesis inhibitor that has been shown to decrease collagen synthesis in fibrosis cases. This small-molecule delivery provides a mechanism to reduce scarring of severe burn wounds in vitro. In vivo data show that the Halofuginone-laden printed keratin is noninferior to other similar approaches reported in literature. This is indicative that the use of 3D printed keratin is not inhibiting the healing processes, and the inclusion of Halofuginone induces a more organized dermal healing after a burn; in other words, this treatment is slower but improves healing. These studies are indicative of the potential of Halofuginone-laden keratin dressings in dermal wound healing. We aim to keep increasing the complexity of the 3D printed constructs toward the production of complex scaffolds for the treatment and topographical reconstruction of severe burn wounds to the face.

Autologous Cell Therapy for Aged Human Skin: A Randomized, Placebo-Controlled, Phase-I Study

Introduction: Skin ageing involves senescent fibroblast accumulation, disturbance in extracellular matrix (ECM) homeostasis, and decreased collagen synthesis. Objective: to assess a cell therapy product for aged skin (RCS-01; verum) consisting of ~25 × 10⁶ cultured, autologous cells derived from anagen hair follicle non-bulbar dermal sheath (NBDS). Methods: For each subject in the verum group, 4 areas of buttock skin were injected intradermally 1 or 3 times at monthly intervals with RCS-01, cryomedium, or needle penetration without injection; in the placebo group RCS-01 was replaced by cryomedium. The primary endpoint was assessment of local adverse event profiles. As secondary endpoints, expression of genes related to ECM homeostasis was assessed in biopsies from randomly selected volunteers in the RCS-01 group taken 4 weeks after the last injection. ­Results: Injections were well tolerated with no severe adverse events reported 1 year after the first injection. When compared with placebo-treated skin, a single treatment with RCS-01 resulted in a significant upregulation of TGFβ1, CTGF, COL1A1, COL1A2, COL3A1, and lumican mRNA expression. Limitations: The cohort size was insufficient for dose ­ranging evaluation and subgroup analyses of efficacy. Conclusions: RCS-01 therapy is well tolerated and associated with a gene expression response consistent with an improvement of ECM homeostasis.

MicroRNA-448 overexpression inhibits fibroblast proliferation and collagen synthesis and promotes cell apoptosis via targeting ABCC3 through the JNK signaling pathway.

Idiopathic pulmonary fibrosis (IPF) is a condition that results in the progressive deterioration of lung function with poor prognosis. The current study is aimed at exploring how microRNA-448 (miR-448) targeting ABCC3 affects fibroblast proliferation, apoptosis, and collagen synthesis of mice with IPF via the Jun N-terminal kinase (JNK) signaling pathway. Bioinformatics and dual-luciferase polymerase chain reaction were used to predict the relationship of miR-448 and ABCC3. The expression of miR-448 and ABCC3 was detected in IPF tissues. Using IPF mouse models, lung fibroblasts for the experiments were treated with miR-448 mimic, miR-448 inhibitor, si-ABCC3, or SP600125 (inhibitor of JNK) to evaluate the cell proliferation and apoptosis in response to miR-448. Reverse transcription quantitative polymerase chain reaction and western blot analysis were used to identify the expression of miR-448, ABCC3, and the activation of the JNK signaling pathway. ABCC3 was targeted and downregulated by miR-448 based on bioinformatics prediction and dual-luciferase reporter gene assay. Additionally, miR-448 was found to be highly expressed in IPF lung tissues with low expression levels of ABCC3. In response to the treatment of miR-448 mimic or si-ABCC3, lung fibroblasts exhibited decreased cell proliferation and increased apoptotic rates, whereas the miR-448 inhibitor reversed the conditions. Notably, we also found that miR-448 mimic inhibited the JNK signaling pathway. In conclusion, by using miR-448 to target and downregulate ABCC3 to block the JNK signaling pathway in mice with IPF, we found an increase in fibroblast apoptosis, inhibited cell proliferation, and decreased collagen synthesis of fibroblasts.

Endothelial cell-derived small extracellular vesicles suppress cutaneous wound healing through regulating fibroblasts autophagy.

Diabetic foot ulcer is a life-threatening clinical problem in diabetic patients. Endothelial cell-derived small extracellular vesicles (sEVs) are important mediators of intercellular communication in the pathogenesis of several diseases. However, the exact mechanisms of wound healing mediated by endothelial cell-derived sEVs remain unclear. sEVs were isolated from human umbilical vein endothelial cells (HUVECs) pretreated with or without advanced glycation end products (AGEs). The roles of HUVEC-derived sEVs on the biological characteristics of skin fibroblasts were investigated both in vitro and in vivo We demonstrate that sEVs derived from AGEs-pretreated HUVECs(AGEs-sEVs) could inhibit collagen synthesis by activatingautophagy of human skin fibroblasts.Additionally, treatment with AGEs-sEVs could delay the wound healing process in Sprague-Dawley (SD) rats. Further analysis indicated that miR-106b-5p was up-regulated in AGEs-sEVsand importantly, in exudate-derived sEVsfrom patients with diabetic foot ulcer. Consequently, sEV-mediated uptake of miR-106b-5p inrecipient fibroblasts reduces expression of extracellular signal-regulated kinase 1/2 (ERK1/2), resultingin fibroblasts autophagy activation and subsequent collagen degradation. Collectively, our data demonstrate that miR-106b-5p could be enriched in AGEs-sEVs, then decreasescollagen synthesis and delayscutaneous wound healing by triggering fibroblasts autophagy through reducing ERK1/2 expression.

Age-associated alterations in murine dermis through inflammatory response with mitochondrial DNA deletions.

Skin aging is caused by intrinsic and extrinsic mechanisms. Because it is difficult to distinguish intrinsic mechanisms from extrinsic skin aging, the mechanisms of intrinsic skin aging remain unclear. The present study aimed to characterize age-associated alterations in murine skin and investigate the mechanisms of intrinsic skin aging. We measured morphological changes in dorsal skin from young (aged 2 months) and old (aged 22-24 months) mice by histological analysis. Age-associated alteration of gene expression patterns was determined by quantitative polymerase chain reaction and immunohistochemistry. Reactive oxygen species production in mouse dorsal skin was detected by confocal laser scanning microscopy. Mitochondrial DNA deletions were detected by conventional polymerase chain reaction and quantitative polymerase chain reaction analyses. Chronologically aged skin had dermal atrophy caused by increased matrix-degrading enzymes and decreased collagen synthesis. Chronologically aged skin samples also had increased senescence-associated secretory phenotype factors, elevated reactive oxygen species production and a higher frequency of the mitochondrial DNA common deletion. These observations suggest that chronological skin aging is associated with increased frequency of the mitochondrial DNA common deletion and chronic inflammation through the reactive oxygen species-senescence-associated secretory phenotype axis. Geriatr Gerontol Int 2019; 19: 451-457.

Pentoxifylline decreases post-operative intra-abdominal adhesion formation in an animal model.

BackgroundIntra-abdominal adhesions develop after nearly every abdominal surgery, commonly causing female infertility, chronic pelvic pain, and small bowel obstruction. Pentoxifylline (PTX) is a methylxanthine compound with immunomodulatory and antifibrotic properties. The aim of this study was to investigate whether PTX can reduce post-operative intra-abdominal adhesion formation via collagen deposition, tissue plasminogen activator (tPA) level, inflammation, angiogenesis, and fibrosis.MethodsSeventy male BALB/c mice were randomized into one of three groups: (1) sham group without peritoneal adhesion model; (2) peritoneal adhesion model (PA group); (3) peritoneal adhesion model with PTX (100 mg/kg/day i.p.) administration was started on preoperative day 2 and continued daily (PA + PTX group). On postoperative day 3 and day 7, adhesions were assessed using the Lauder scoring system. Parietal peritoneum was obtained for histological evaluation with hematoxylin and eosin (HE) and picrosirius red staining. Fibrinolysis was analyzed by tPA protein levels in the peritoneum by ELISA. Immunohistological analysis was also conducted using markers for angiogenesis (ki67+/CD31+), inflammation (F4/80+) and fibrosis (FSP-1+ and α-SMA+). All the comparisons were made by comparing the PA group with the PTX treated PA group, and p < 0.05 was considered statistically significant.ResultsIntra-abdominal adhesions were markedly reduced by PTX treatment. Compared with the PA group, PTX treatment had lower adhesion scores than the PA group on both day 3 and day 7 (p < 0.05). Histological evaluations found that PTX treatment reduced collagen deposition and adhesion thickening. ELISA analysis showed that PTX treatment significantly increased the level of tPA in the peritoneum. In addition, in the immunohistological analysis, PTX treatment was found to significantly decrease the number of ki67+/CD31+ cells at the site of adhesion. Finally, we also observed that in the PTX treated group, there was a reduction in the expression of F4/80+, FSP-1+, and α-SMA+ cells at the site of adhesion.ConclusionPTX may decrease intra-abdominal adhesion formation via increasing peritoneal fibrinolytic activity, suppressing angiogenesis, decreasing collagen synthesis, and reducing peritoneal fibrosis. Our findings suggest that PTX can be used to decrease post-operative intra-abdominal adhesion formation.

Effects of TNF-α in rheumatoid arthritis via attenuating α1 (I) collagen promoter.

To explore the role of TNF-α in the peripheral blood of patients with rheumatoid arthritis (RA) and its underlying mechanism. 32 patients diagnosed with RA in our hospital from July 2016 to March 2017 were selected in the experimental group. Meanwhile, 32 normal healthy people were selected in the healthy control group and 21 patients with other autoimmune diseases in the same period were selected in the disease control group. Serum samples of the subjects in the experimental group and the control group were collected. The content of serum tumor necrosis factor-α (TNF-α) was detected by enzyme-linked immunosorbent assay (ELISA), and the correlation between TNF-α and RA activity was analyzed. We then constructed rat RA model. The effect of different doses of TNF-α on the RA progression was evaluated by measuring the foot paw thickness of both hind limbs of rats. Fibroblast-like synoviocytes (FLS) were treated with different concentrations of TNF-α cytokine in vitro. Cell counting kit-8 (CCK-8) assay was carried out to detect the cell viability after TNF-α treatment. Serum levels of VEGF (vascular endothelial growth factor) and hydroxyproline were detected. Moreover, the α1 (I) collagen overexpression recombinant was constructed and transfected into MH7A cells. The activation of α1 (I) collagen promoter was reflected by the CAT reporter gene activity. ELISA results showed higher content of TNF-α in the peripheral blood of the experimental group than that of the control group. In the RA rat model, the foot paw thickness of the hind limbs was increased with the increase of TNF-α concentration. CCK-8 and colony formation assay demonstrated that the proliferation of MH7A cells was elevated after TNF-α treatment. Higher levels of VEGF and IL-6 secreted by FLS and decreased collagen synthesis ability of MH7A cells were found after TNF-α treatment. Transfection of the α1 (I) collagen overexpression recombinant in MH7A cells led to the reduced activity of CAT after TNF-α treatment, suggesting that the activation of α1 (I) collagen promoter was inhibited. TNF-α participates in RA by inhibiting the activation of the promoter of α1 (I) collagen, as well as enhancing the secretion of VEGF and IL-6 in MH7A cells.

Beneficial effects of naringenin in liver diseases: Molecular mechanisms.

Liver diseases are caused by different etiological agents, mainly alcohol consumption, viruses, drug intoxication or malnutrition. Frequently, liver diseases are initiated by oxidative stress and inflammation that lead to the excessive production of extracellular matrix (ECM), followed by a progression to fibrosis, cirrhosis and hepatocellular carcinoma (HCC). It has been reported that some natural products display hepatoprotective properties. Naringenin is a flavonoid with antioxidant, antifibrogenic, anti-inflammatory and anticancer properties that is capable of preventing liver damage caused by different agents. The main protective effects of naringenin in liver diseases are the inhibition of oxidative stress, transforming growth factor (TGF-β) pathway and the prevention of the transdifferentiation of hepatic stellate cells (HSC), leading to decreased collagen synthesis. Other effects include the inhibition of the mitogen activated protein kinase (MAPK), toll-like receptor (TLR) and TGF-β non-canonical pathways, the inhibition of which further results in a strong reduction in ECM synthesis and deposition. In addition, naringenin has shown beneficial effects on nonalcoholic fatty liver disease (NAFLD) through the regulation of lipid metabolism, modulating the synthesis and oxidation of lipids and cholesterol. Moreover, naringenin protects from HCC, since it inhibits growth factors such as TGF-β and vascular endothelial growth factor (VEGF), inducing apoptosis and regulating MAPK pathways. Naringenin is safe and acts by targeting multiple proteins. However, it possesses low bioavailability and high intestinal metabolism. In this regard, formulations, such as nanoparticles or liposomes, have been developed to improve naringenin bioavailability. We conclude that naringenin should be considered in the future as an important candidate in the treatment of different liver diseases.

Histopathological and Immunohistochemical Evaluation of Antifibrogenic Effect of Grape Seed Extract on CCl4 - Induced Model of Hepatic Fibrosis

Introduction: Liver fibrosis represent a worldwide challenge of clinical importance, results from chronic damage of liver, and evidenced by build up of excessive extracellular matrix proteins.. The present study was carried out to evaluate the antifibrogenic effect of grape seed extract (GSE) against hepatic fibrosis induced by CCl4 in mice. Experimental Design: forty adult male albino mice were divided into four equal groups; first (control) in which mice were injected IP with olive oil as vehicle. In the second group (GSE) mice were received GSE orally at a dose of 200mg/kg/day for 8 weeks while in the third group (CCl4) mice were injected IP with CCl4 (0.4ml/kg / twice weekly) for 8 weeks . In the fourth (GSE+ CCl4) group mice were injected IP with CCl4 and co-treated with GSE orally as in previous treated-groups. At the end of the experiment, animals were sacrificed and blood samples and liver tissue specimens were collected. Results: the examined liver of CCl4-intoxicated group revealed marked hepatic fibrotic lesions confirmed by Masson’s trichrome stain and associated with the presence of intensely stained α -SMA-positive hepatic stellate cells (HSCs) in entire of the hepatic lobules and in the vicinity of bridging fibrotic septa. Hepatic degeneration and necrosis were also seen. This hepatic damage was associated with significant increases in AST and ALT activities with low albumin levels and hypoproteinemia. Co-administration of GSE with CCl4 improved the microscopic picture of liver where scanty fibrotic lesions and mild degeneration of some hepatic cells were recorded. Less intensely stain ed α -SMA-immunopositive cells were observed. Serum AST , ALT, albumin and total protein values were more or less within the ranges of these parameters in the control non-intoxicated group. Conclusion: GSE has potent antifibrogenic effect on CCl4-induced hepatic fibrosis by inhibiting HSCs activation, decreasing collagen synthesis and improving hepatic regenerative capability through its powerful antioxidant and anti-inflammatory properties.

Dietary Lecithin Decreases Skeletal Muscle COL1A1 and COL3A1 Gene Expression in Finisher Gilts.

Simple SummaryIn this study, the effect of dietary lecithin on skeletal muscle gene expression of collagen precursors and enzymes was investigated in gilts. Thirty-six finisher gilts were fed with diets containing either 0, 4, 20 or 80 g/kg soybean lecithin for six weeks. Then, rectus abdominis muscle was sampled and analyzed for eight genes involved in collagen synthesis and degradation (COL1A1, COL3A1, MMP-1, MMP-13, TIMP-1, TIMP-3, lysyl oxidase and α-subunit P4H) using quantitative real-time PCR. The results showed that lecithin down-regulated COL1A1 and COL3A1 as well as tended to down-regulate α-subunit P4H expression.The purpose of this study was to investigate the effect of dietary lecithin on skeletal muscle gene expression of collagen precursors and enzymes involved in collagen synthesis and degradation. Finisher gilts with an average start weight of 55.9 ± 2.22 kg were fed diets containing either 0, 4, 20 or 80 g/kg soybean lecithin prior to harvest for six weeks and the rectus abdominis muscle gene expression profile was analyzed by quantitative real-time PCR. Lecithin treatment down-regulated Type I (α1) procollagen (COL1A1) and Type III (α1) procollagen (COL3A1) mRNA expression (p < 0.05, respectively), indicating a decrease in the precursors for collagen synthesis. The α-subunit of prolyl 4-hydroxylase (P4H) mRNA expression also tended to be down-regulated (p = 0.056), indicating a decrease in collagen synthesis. Decreased matrix metalloproteinase-1 (MMP-1) mRNA expression may reflect a positive regulatory response to the reduced collagen synthesis in muscle from the pigs fed lecithin (p = 0.035). Lecithin had no effect on tissue inhibitor metalloproteinase-1 (TIMP-1), matrix metalloproteinase-13 (MMP-13) and lysyl oxidase mRNA expression. In conclusion, lecithin down-regulated COL1A1 and COL3A1 as well as tended to down-regulate α-subunit P4H expression. However, determination of muscle collagen content and solubility are required to support the gene functions.

Intermittent energy restriction induces changes in breast gene expression and systemic metabolism.

BackgroundObservational studies suggest weight loss and energy restriction reduce breast cancer risk. Intermittent energy restriction (IER) reduces weight to the same extent as, or more than equivalent continuous energy restriction (CER) but the effects of IER on normal breast tissue and systemic metabolism as indicators of breast cancer risk are unknown.MethodsWe assessed the effect of IER (two days of 65 % energy restriction per week) for one menstrual cycle on breast tissue gene expression using Affymetrix GeneChips, adipocyte size by morphometry, and systemic metabolism (insulin resistance, lipids, serum and urine metabolites, lymphocyte gene expression) in 23 overweight premenopausal women at high risk of breast cancer. Unsupervised and supervised analyses of matched pre and post IER biopsies in 20 subjects were performed, whilst liquid and gas chromatography mass spectrometry assessed corresponding changes in serum and urine metabolites in all subjects after the two restricted and five unrestricted days of the IER.ResultsWomen lost 4.8 % (±2.0 %) of body weight and 8.0 % (±5.0 %) of total body fat. Insulin resistance (homeostatic model assessment (HOMA)) reduced by 29.8 % (±17.8 %) on the restricted days and by 11 % (±34 %) on the unrestricted days of the IER. Five hundred and twenty-seven metabolites significantly increased or decreased during the two restricted days of IER. Ninety-one percent of these returned to baseline after 5 days of normal eating. Eleven subjects (55 %) displayed reductions in energy restriction-associated metabolic gene pathways including lipid synthesis, gluconeogenesis and glycogen synthesis. Some of these women also had increases in genes associated with breast epithelial cell differentiation (secretoglobulins, milk proteins and mucins) and decreased collagen synthesis (TNMD, PCOLCE2, TIMP4). There was no appreciable effect of IER on breast gene expression in the other nine subjects. These groups did not differ in the degree of changes in weight, total body fat, fat cell size or serum or urine metabolomic markers. Corresponding gene changes were not seen in peripheral blood lymphocytes.ConclusionThe transcriptional response to IER is variable in breast tissue, which was not reflected in the systemic response, which occurred in all subjects. The mechanisms of breast responsiveness/non-responsiveness require further investigation.Trial registrationISRCTN77916487 31/07/2012.Electronic supplementary materialThe online version of this article (doi:10.1186/s13058-016-0714-4) contains supplementary material, which is available to authorized users.

Local application of rapamycin reduces epidural fibrosis after laminectomy via inhibiting fibroblast proliferation and prompting apoptosis.

BackgroundEpidural fibrosis is a common complication after laminectomy. It is associated with intractable lower back pain and additional complications. To date, no study has evaluated whether the local application of rapamycin (RAPA) can inhibit fibroblast proliferation and reduce epidural scar adhesion after laminectomy. The results of the present study showed that the local application of RAPA reduces epidural fibrosis after laminectomy in rats.MethodsIn this study, 32 male Sprague-Dawley rats were randomly divided into four groups (0.2 mg/ml RAPA-treated group, 0.1 mg/ml RAPA-treated group, 0.05 mg/ml RAPA-treated group and physiological saline group). Laminectomy was performed at the level of lumbar segment 1 to 2, and different concentrations of RAPA or saline were applied to the laminectomy sites for 10 min. Four weeks after laminectomy, the rats were sacrificed, and the degrees of epidural adhesion in each group were evaluated. Macroscopic assessment, analysis of hydroxyproline content, and histological analysis were used to determine the therapeutic effect of the local application of RAPA on the inhibition of fibroblast proliferation and the reduction of epidural fibrosis after laminectomy. Next, we cultured fibroblasts from epidural scar tissues of rats that had undergone laminectomy. Fibroblasts were exposed to the indicated concentrations of RAPA, and western blotting and TUNEL assays were used to assess the effects of RAPA on inhibiting fibroblasts proliferation and promoting fibroblast apoptosis.ResultsThe results of macroscopic assessments, analysis of hydroxyproline content, and histological analyses indicated that RAPA significantly inhibited fibroblast proliferation and reduced epidural fibrosis in the treated groups in the rat model. The western blotting results indicated that the expression levels of the pro-apoptotic proteins cleaved-PARP and Bax were up-regulated, whereas those of Bcl-2 were reduced. TUNEL assay indicated that the apoptosis rates of fibroblasts were significantly increased after exposure to the indicated concentrations of RAPA.ConclusionsThe local application of RAPA reduced epidural fibrosis after laminectomy by inhibiting the proliferation of fibroblasts, stimulating their apoptosis, and decreasing collagen synthesis. This protocol may be used in new clinical treatment strategies to reduce epidural fibrosis after laminectomy.