Collagen mRNA Levels Research Articles

Combination of Glycinamide and Ascorbic Acid Synergistically Promotes Collagen Production and Wound Healing in Human Dermal Fibroblasts.

The purpose of this study is to present a novel strategy to enhance collagen production in cells. To identify amino acid analogs with excellent collagen production-enhancing effects, human dermal fibroblasts (HDFs) were treated with 20 kinds of amidated amino acids and 20 kinds of free amino acids, individually at 1 mM. The results showed that glycinamide enhanced collagen production (secreted collagen level) most effectively. Glycine also enhanced collagen production to a lesser degree. However, other glycine derivatives, such as N-acetyl glycine, N-acetyl glycinamide, glycine methyl ester, glycine ethyl ester, and glycyl glycine, did not show such effects. Glycinamide increased type I and III collagen protein levels without affecting COL1A1 and COL3A1 mRNA levels, whereas transforming growth factor-β1 (TGF-β1, 10 ng mL−1) increased both mRNA and protein levels of collagens. Ascorbic acid (AA, 1 mM) increased COL1A1 and COL3A1 mRNA and collagen I protein levels. Unlike TGF-β1, AA and glycinamide did not increase the protein level of α-smooth muscle actin, a marker of differentiation of fibroblasts into myofibroblasts. The combination of AA and glycinamide synergistically enhanced collagen production and wound closure in HDFs to a level similar to that in cells treated with TGF-β1. AA derivatives, such as magnesium ascorbyl 3-phosphate (MAP), 3-O-ethyl ascorbic acid, ascorbyl 2-O-glucoside, and ascorbyl tetraisopalmitate, enhanced collagen production, and the mRNA and protein levels of collagens at 1 mM, and their effects were further enhanced when co-treated with glycinamide. Among AA derivatives, MAP had a similar effect to AA in enhancing wound closure, and its effect was further enhanced by glycinamide. Other AA derivatives had different effects on wound closure. This study provides a new strategy to enhance cell collagen production and wound healing using glycinamide in combination with AA.

Antifibrotics effects of nintedanib on lung fibroblasts derived from patients with progressive fibrosing intertitial lung diseases

A larger group of patients with differing clinical interstitial lung diseases (ILD) develop a progressive fibrosing (PF-ILD) phenotype during the course of their disease despite immunosuppressive therapy. The kinase inhibitor nintedanib is approved for the treatment of the most agressive fibrosing ILD, idiopathic pulmonary fibrosis (IPF), and was recently reported to exert positives effects on patients with PF-ILD. In order to highlight the in vitro anti-fibrotic properties of nintedanib in non-IFP PF-ILD, we investigated its effects on main functions of human lung fibroblasts (HLFs) isolated from patients with progressive-fibrosing phenotype like chronic fibrotic hypersensitivity pneumonitis, sarcoidosis, idiopathic nonspecific interstitial pneumonia and unclassifiable ILD. PF-ILD HLFs were pre-treated with nintedanib (0.01–1 μM) and stimulated with PDGF-BB (25–50 ng/ml) or TGF-β1 (1 ng/ml). PDGF receptor and ERK activation, proliferation, migration and differentiation of cells were assessed. Nintedanib in a range of 0.01 to 1 μM strongly blocked the phosphorylation of PDGF receptor and decreased ERK1/2 phosphorylation induced by PDGF-BB. The pro-proliferative and pro-migratory phenotype driven by PDGF-BB were also significantly reduced by nintedanib from 0.03 μM without affected the cell viability. Moreover, the drug also decreased, in a concentration dependant manner, TGF-β1-induced myofibroblastic differentiation by down-regulating mRNA and protein expression level of collagen, fibronectin, α-Smooth muscle actin and the phosphorylation of SMAD2/3 signalling pathway. Our data demonstrates that nintedanib, as described in IPF, counteracts the detrimental functions of PF-ILD fibroblasts and are in favor of the management of PF-ILD with this drug.

Extracellular Vesicles from miR-148a-5p-Enriched Bone Marrow Mesenchymal Stem Cells Relieve Hepatic Fibrosis by Targeting Smad4.

Liver fibrosis is a hallmark feature of many chronic liver diseases, which is the leading cause of morbidity and mortality worldwide. Bone marrow mesenchymal stem cells (BMSCs)-derived extracellular vesicles have been applied in many diseases. In this study, we aimed to explore the specific mechanism of extracellular vesicles from BMSCs in liver fibrosis. Bioinformatics analysis was employed to screen miRNA and its target mRNA. Sirius Red staining was carried out to examine fibrosis in liver tissues. Extracellular vesicle morphology was assessed using Transmission Electron Microscopy. Quantitative real-time PCR (qRT-PCR) and western blotting analysis were performed to detect the expressions of miR-148a-5p, Smad4, transforming growth factor-β1 (TGF-β1), tissue inhibitor of metalloproteinase 1 (TIMP-1), Collagen I, α-smooth muscle actin (α-SMA), and extracellular vesicle markers CD9, TSG101, CD63, and calnexin. Dual-luciferase report gene assay was used for the luciferase activity analysis. Bioinformatics analysis revealed miR-148a-5p as a regulator in liver fibrosis. QRT-PCR results indicated that miR-148a-5p was lowly expressed in both thioacetamide (TAA)-induced mice and TGF-β1-activated hepatic stellate cells. Extracellular vesicles from miR-148a-5p enriched BMSCs downregulated the mRNA and protein levels of TGF-β1, TIMP-1, Collagen I,and α-SMA. Further bioinformatics analysis indicated that Smad4 was related to liver fibrosis. Furthermore, the dual-luciferase report gene assay confirmed the binding relationship between miR-148a-5p and Smad4. Extracellular vesicles from miR-148a-5p enriched BMSCs attenuated hepatic fibrosis in liver fibrosis by targeting Smad4.

Quercetin ameliorated cardiac injury via reducing inflammatory actions and the glycerophospholipid metabolism dysregulation in a diabetic cardiomyopathy mouse model.

Quercetin has multiple protective effects against cardiometabolic diseases, but the biological mechanisms underlying the benefits in diabetic cardiomyopathy (DCM) are unclear. A mouse DCM model was established by high-fat diet (HFD, 8 months) combined with streptozotocin injection (at the mid-feeding), and quercetin (100 mg per kg per day) was administrated orally after streptozotocin. Our major results indicated that the cardiac ejection fraction and fractional shortening, mRNA levels of Collagen I and CTGF, and the protein expression levels of NLRP3, caspase-1, IL-1β, and IL-18 were reduced (P < 0.05) in DCM mice in response to quercetin intervention. Serum metabolomic analyses identified glycerophospholipid metabolism as the main pathway implicated in the cardiac benefits of quercetin in DCM. Such findings showed that quercetin may ameliorate cardiac dysfunction and myocardial fibrosis via reducing inflammatory actions and the glycerophospholipid metabolism dysregulation in DCM.

Influence of photobiomodulation and tricalcium silicate-based cement on viability and dentinogenic activity of cultured human dental pulp stem cells

Objective: To assess the effect of application of Biodentine (BD), Photobiomodulation (PBM) using 810 nm diodelaser and both on the proliferation and odontogenic differentiation of human dental pulp stem cells (HDPSCs).Material and Methods: HDPSCs were collected, isolated, and characterized and then divided into six groups:groups 1, control; groups 2, biodentine (BD); group 3, irradiation at 1 J/cm2 of 810-nm diode laser; group 4,irradiation at 1 J/cm2 and culture with BD; group 5, irradiation at 2 J/cm2, and group 6, irradiation at 2 J/cm2and culture with BD. Viability assay was measured through MTT assay and Alkaline phosphatase (ALP) enzymeactivity and mRNA levels of RUNX2, collagen 1 (Col-1) and BMP2 were also assessed. Results: Photobiomodulationat 1 and 2 J/cm2 combined with biodentine significantly promoted HDPSCs proliferation (in MTT assay results)and odontogenic differentiation (through the gene expression of RUNX2, Col-1 and BMP2 levels (p &lt; 0.05).Conclusion: Photobiomodulation at 2 J/cm2 combined with biodentine enhanced proliferation and odontogenicdifferentiation of cultured HDPSCs and thus could further be beneficial for dentin regeneration.KEYWORDSHuman dental pulp stem cells (HDPSCs); PBM; BMP2; RUNX2; COL1.

MicroRNA-122-5p prevents proliferation and promotes apoptosis of hepatic stellate cells by suppressing the cellular-Abelsongene/histone deacetylases 2 pathway.

Liver fibrosis is a wound-healing response and the activation of the hepatic stellate cell (HSC) is the core of hepatic fibrosis. MicroRNAs (miRNAs) participate in the development of fibrosis. It is reported that histone deacetylases (HDAC2) tyrosine phosphorylation by cellular-Abelsongene (c-Abl) induces malignant growth of cells. Here, we investigated the effect of miR-122-5p on the proliferation and apoptosis of HSCs. Normal human HSC line LX-2 and LX-2 cells stimulated by transforming growth factor (TGF)-β1 for 24h were cultured and assigned into the blank group and the TGF-β1 group. The miR-122-5p inhibitor and its negative control were transfected into LX-2 cells and miR-122-5p mimic and its negative control were transfected into LX-2 cells stimulated by TGF-β1. The result showed that miR-122-5p expression was decreased in TGF-β1-stimulated LX-2 cells. miR-122-5p overexpression reduced the mRNA and protein levels of collagen I and α-smooth muscle actin, inhibited cell proliferation, and accelerated cell apoptosis. miR-122-5p targeted c-Abl. Meanwhile, miR-122-5p overexpression inhibited HSC activation by suppressing the c-Abl/HDAC2 pathway. In summary, miR-122-5p overexpression exerted anti-fibrosis effect and inhibited HSC activation by suppressing the c-Abl/HDAC2 pathway.

A Potential Research Target for Scleral Remodeling: Effect of MiR-29a on Scleral Fibroblasts

Introduction: The purpose of this study was to determine whether miR-29a regulates cell survival and apoptosis and the expression of phosphatase and tensin homolog deleted on chromosome 10 (PTEN), MMP-2, and collagen I in scleral fibroblasts. Methods: We transfected scleral fibroblasts with the miR-29a mimic and inhibitor. The effects of miR-29a on cell proliferation and apoptosis were determined using the CCK-8 assay and flow cytometry, respectively. Quantitative polymerase chain reaction (qPCR) was used to determine whether miR-29a regulates the mRNA levels of PTEN, MMP-2, and collagen I. The protein expression of PTEN, MMP-2, and collagen I was also assessed by western blot analysis. Results: The results of CCK-8 showed that, at 0, 24, 48, and 72 h after transfection, the relative optical density values in the mimic group were 0.233 ± 0.005, 0.380 ± 0.008, 0.650 ± 0.040, and 0.906 ± 0.032, and in the inhibitor group were 0.272 ± 0.011, 0.393 ± 0.029, 0.597 ± 0.059, and 0.950 ± 0.101, respectively. The flow cytometry results showed that the apoptosis rates of each group were as follows: the mimic group (0.043 ± 0.007), the NC group (0.040 ± 0.006), the inhibitor group (0.032 ± 0.003), the inhibitor NC group (0.027 ± 0.010), the lipofectamine group (0.027 ± 0.005), and the blank group (0.031 ± 0.009). The qPCR results indicated that in the mimic group, PTEN (0.795 ± 0.182, p = 0.2783), MMP-2 (0.621 ± 0.105, p = 0.0033), and COL1A1 (0.271 ± 0.100, p = 0.0002) expression decreased, whereas in the inhibitor group, PTEN (1.211 ± 0.100, p = 0.2614), MMP-2 (1.161 ± 0.053, p = 0.1190), and COL1A1 (1.7040 ± 0.093, p = 0.0003) increased. Western blot analysis showed that in the mimic group, the expression of PTEN (0.392 ± 0.039, p < 0.0001), MMP-2 (0.577 ± 0.017, p < 0.0001), and COL1A1 (0.072 ± 0.006, p < 0.0001) protein decreased, whereas in the inhibitor group, PTEN (1.043 ± 0.042, p = 0.9413), MMP-2 (1.397 ± 0.075, p = 0.0002), and COL1A1 (1.935 ± 0.081, p < 0.0001) expression increased. Conclusion: MiR-29a inhibits the expression of PTEN, MMP-2, and collagen I on scleral fibroblasts, which may provide a basis studies in sclera.

Tert-Butylhydroquinone Prevents Oxidative Stress-Mediated Apoptosis and Extracellular Matrix Degradation in Rat Chondrocytes

Oxidative stress-induced chondrocyte apoptosis and degradation of the extracellular matrix (ECM) play an important role in the progression of osteoarthritis (OA). In addition, tert-butylhydroquinone (TBHQ) is an activator of the nuclear factor erythroid derived-2-related factor 2 (Nrf2). The present study aimed to determine the effectiveness of TBHQ in preventing the apoptosis of chondrocytes and degradation of the extracellular matrix, induced by oxidative stress, in vitro. Therefore, rat chondrocytes were exposed to 20 μM tert-butyl hydroperoxide (TBHP) for 24 h to establish an oxidative damage model, in vitro. Thereafter, cell viability was evaluated using the Cell Counting Kit-8 assay. Moreover, the level of ROS was determined through 2′,7′-dichlorofluorescein diacetate staining. The mitochondrial membrane potential of chondrocytes was also measured using JC-1. Furthermore, cell apoptosis was assessed through Annexin V-fluorescein isothiocyanate/propidium iodide staining. The study also performed Western blotting and qPCR to evaluate the expression of extracellular matrix components, matrix catabolic enzymes, and changes in signalling pathways. The results showed that 2.5 and 5 μM of TBHQ reduced the TBHP-induced generation of excessive ROS and improved cell viability. Additionally, 2.5 and 5 μM of TBHQ prevented mitochondrial damage and apoptosis in rat chondrocytes. Treatment with TBHQ also increased the mRNA and protein expression levels of aggrecan and collagen II. However, TBHQ reduced the mRNA and protein expression levels of matrix metalloproteinase 3 (MMP3) and matrix metalloproteinase 13 (MMP13) in rat chondrocytes. In addition, treatment with TBHQ enhanced the protein expression levels of Nrf2, NADPH quinone oxidoreductase 1 (NQO-1), and hemeoxygenase-1 (HO-1) in rat chondrocytes. The current study showed that TBHQ was not only effective in protecting against TBHP-induced oxidative stress but also inhibited the apoptosis of rat chondrocytes and degradation of the ECM by activating the Nrf2 pathway. The results therefore suggest that TBHQ holds potential for use in the treatment of OA.

Autophagy of hepatic stellate cell induced by Clonorchis sinensis.

Clonorchis sinensis was a food-borne zoonotic parasite in the worldwide and also an important risk factor of hepatic fibrosis. Excretory/secretion products of C. sinensis (CsESPs) are involved in parasite-host interactions and contribute to the development of hepatic damage. The aim of the present study was to investigate whether CsESPs and CsTP (adult protein) could induce autophagy of hepatic stellate cells (HSCs) and further activate HSCs so as to participate in the pathogenesis of hepatic fibrosis. The human hepatic stellate cell line LX-2 was stimulated by CsESPs and CsTP. CsESPs showed the effect on cell proliferation in methyl thiazolyl tetrazolium (MTT) assay while CsTP failed. Autophagosomes and autolysosomes were observed after the transmission mRFP-EGFP-LC3 plasmid into the LX-2 cells. CsESPs had more powerful to induce the accumulation of autophagosomes and autolysosomes to enhance autophagic flux compared with CsTP. Western-blotting analysis confirmed that the ratio of LC3-II/I in LX-2 cells was up-regulated after CsESPs treatment for 6h, which further proved that CsESPs could induce autophagy in LX-2 cells. Meanwhile, q-PCR results showed that the mRNA levels of collagen I, collagen III and α-SMA decreased in LX-2 cells after treatment with autophagy inhibitor chloroquine, whereas they increased when combination with CsESPs. These results suggested that CsESPs-induced autophagy might be involved in the activation of HSCs, and consequently participate in the pathogenesis of hepatic fibrosis caused by C. sinensis infection.

Astragalus Polysaccharide Reduces Blood Pressure, Renal Damage, and Dysfunction Through the TGF-β1-ILK Pathway.

Background: Astragalus polysaccharide extract (APS) has been shown to exhibit antioxidant and anti-inflammatory potential in the treatment of several diseases. However, whether APS could protect against renal damage in hypertensive mice is unknown. Methods: Hematoxylin and eosin staining, immunohistochemistry, real-time polymerase chain reaction, and Western blotting were used to investigate the effect of APS on the renal damage in deoxycorticosterone acetate- (DOCA) salt- and angiotensin II- (Ang II-) induced hypertensive mice and to elucidate the underlying mechanisms. Results: Our data demonstrated that APS significantly reduced blood pressure in DOCA-salt- and Ang II-treated mice. Furthermore, APS reduced the inflammatory response and renal fibrosis, thereby improving renal function. Furthermore, the levels of serum creatinine, urea nitrogen, and uric acid increased in DOCA-salt-treated mice, alleviated by APS administration. At the molecular level, DOCA-salt and Ang II increased the mRNA levels of IL-1β, IL-6, α-SMA, collagen I, and collagen III, while APS significantly inhibited these effects. APS inhibited the TGF-β1/ILK signaling pathway, which was activated in hypertensive mice due to the administration of DOCA-salt. Conclusion: Our results suggest that APS plays a beneficial role in improving renal dysfunction in hypertensive mice.

Paternal Resistance Exercise Modulates Skeletal Muscle Remodeling Pathways in Fathers and Male Offspring Submitted to a High-Fat Diet.

Although some studies have shown that a high-fat diet (HFD) adversely affects muscle extracellular matrix remodeling, the mechanisms involved in muscle trophism, inflammation, and adipogenesis have not been fully investigated. Thus, we investigated the effects of 8 weeks of paternal resistance training (RT) on gene and protein expression/activity of critical factors involved in muscle inflammation and remodeling of fathers and offspring (offspring exposed to standard chow or HFD). Animals were randomly distributed to constitute sedentary fathers (SF; n = 7; did not perform RT) or trained fathers (TF n = 7; performed RT), with offspring from mating with sedentary females. After birth, 28 male pups were divided into four groups (n = 7 per group): offspring from sedentary father submitted either to control diet (SFO-C) or high-fat diet (SFO-HF) and offspring from trained father submitted to control diet (TFO-C) or high-fat diet (TFO-HF). Our results show that an HFD downregulated collagen mRNA levels and upregulated inflammatory and atrophy pathways and adipogenic transcription factor mRNA levels in offspring gastrocnemius muscle. In contrast, paternal RT increased MMP-2 activity and decreased IL-6 levels in offspring exposed to a control diet. Paternal RT upregulated P70s6k and Ppara mRNA levels and downregulated Atrogin1 mRNA levels, while decreasing NFκ-B, IL-1β, and IL-8 protein levels in offspring exposed to an HFD. Paternal physical training influences key skeletal muscle remodeling pathways and inflammatory profiles relevant for muscle homeostasis maintenance in offspring submitted to different diets.

ANGPTL4 Attenuates Ang II-Induced Atrial Fibrillation and Fibrosis in Mice via PPAR Pathway.

Atrial fibrillation (AF) is the more significant portion of arrhythmia in clinical practice, with inflammation and fibrosis as its central pathological mechanisms. This study aimed to investigate angiopoietin-like 4 (ANGPTL4) effects on angiotensin II- (Ang II-) induced AF and its related pathophysiological mechanisms. C57BL/6J mice were randomized and divided into three groups: the control group, the Ang II group, and the ANGPTL4 group (Ang II with ANGPTL4 treatment). Mice were infused with Ang II (2000 ng/kg/min) and were administrated with recombinant human ANGPTL4 (rhANGPTL4, 20 μg/kg/day) for 3 weeks. The fibrosis was evaluated with Masson's trichrome staining in the atrial myocardium. mRNA levels of IL-1β, IL-6, collagen I, and collagen III were measured using real-time qRT-PCR. Protein levels of PPARα, PPARγ, CPT-1, and SIRT3 were measured using Western blotting. Compared to the control group, the mice infused with Ang II showed electrocardiogram characteristics of AF, and this effect was markedly attenuated in ANGPTL4-treated mice. ANGPTL4 also reversed the increase in cardiomyocyte apoptosis, inflammation, interstitial collagen fraction, and collagen gene expression in mice with Ang II. Mechanistically, ANGPTL4 inhibited the activation of several fatty acid metabolism-related proteins, including PPARα, PPARγ, and CPT-1, and the expression of SIRT3 protein in atrial tissues. In conclusion, ANGPTL4 attenuates Ang II-induced AF and atrial fibrosis by modulation in the SIRT3, PPARα, and PPARγ signaling pathways.

Differentiation of human adipose-derived mesenchymal stem cells toward tenocyte by platelet-derived growth factor-BB and growth differentiation factor-6

Mesenchymal Stem Cells (MSCs) are important in regenerative medicine and tissue engineering and will be a very sensible choice for repair and regeneration of tendon. New biological practices, such as cellular therapy using stem cells, are promising for facilitating or expediting tendon therapy. Before using these cells clinically, it is best to check and confirm the optimal conditions for differentiation of these cells in the laboratory. Hence, in the present study, the impacts of PDGF-BB and GDF-6 supplementation on adipose-derived MSCs (ASCs) culture were studied. The frozen ASC were recovered and expanded in basic culture medium (DMEM with 10%FBS). The cells after passage five (P5) were treated with basic medium containing L-Prolin, Ascorbic Acid and only PDGF-BB or GDF-6 (20ng/ml) or both of them (mix) as 3 groups for 14days to investigate efficiency of ASCs differentiation towards tenocytes. The cells culturing in basic medium were used as control group. To validate tenogenic differentiation, H&E and Sirius Red staining were used to assess cell morphology and collagen production, respectively. In addition, mRNA levels of collagen I and III, Scleraxis and Tenomodulin as tenogenic markers were analyzed using qPCR. In all test groups, cells appeared slenderer, elongated cytoplasmic attributes compared to the control cells. The intensity of Sirius Red staining was significantly higher in GDF-6, PDGF-BB alone, than in group without supplements. The optical density was higher in the GDF-6 than PDGF-BB and mix-group. QPCR results showed that Col I and III gene expression was increased in all groups compared to the control. SCX expression was significantly increased only in the PDGF-BB group. TNMD mRNA expression was not significant among groups. In this study, we have corroborated that human ASCs are reactionary to tenogenic induction by GDF-6 and PDGF-BB alone or in combination. These outcomes will help greater insight into GDF-6 and PDGF-BB driven tenogenesis of ASCs and new directions of discovery in the design of ASC-based treatments for tendon healing.

Bone marrow-derived mesenchymal stem cells attenuate silica-induced pulmonary fibrosis by inhibiting apoptosis and pyroptosis but not autophagy in rats

This study aimed to investigate the effect of bone marrow-derived mesenchymal stem cells (BMSCs) on silica-induced lung fibrosis in a rat model. Thirty SD rats were randomly divided into three groups: control group, silica group, and BMSC group (n = 10 rats per group). BMSCs were injected successively into rats on the 14th, 28th, and 42nd days after silica exposure. All rats were sacrificed 56 days after silica exposure. We detected the pathological and fibrotic changes, apoptosis, autophagy, and pyroptosis in their lung tissue by histopathological examination, hydroxyproline content assays, real-time quantitative polymerase chain reactions, western blot assays, immunohistochemistry staining, immunofluorescence staining, and enzyme-linked immunosorbent assays. We found that BMSCs significantly relieved lung inflammatory infiltrates, collagen deposition, hydroxyproline content, and the mRNA and protein levels of collagen 1 and fibronectin. Compared to the silica group, in the BMSC group, apoptosis-associated proteins, including cleaved caspase 3 and Bax, were significantly downregulated, and Bcl-2/Bax was significantly upregulated; pyroptosis-related proteins, including Nlrp3, cleaved caspase 1, IL-1β, and IL-18, were significantly reduced. However, the BMSCs had no significant impact on autophagy-related proteins, including Beclin 1, P62, and LC3. In summary, BMSCs protected lung tissue against severe fibrosis by inhibiting apoptosis and pyroptosis but not autophagy.

Effects of SET7 on angiotensin II-mediated proliferation and collagen synthesis of myocardial fibroblasts and its mechanisms.

Silence of SET domain containing lysine methyltransferase 7 (SET7) alleviates myocardial tissue injury caused by ischemia-reperfusion. But the effects of SET7 on angiotensin II (Ang II)-induced myocardial fibroblast proliferation and the collagen synthesis are not clear. The purpose of this study was to explore the effect of SET7 on the proliferation and collagen synthesis of myocardial fibroblasts and its mechanisms. Myocardial fibroblasts were isolated and identified by immunofluorescence. Myocardial fibroblasts were randomly divided into 4 groups: a control group (cells were normally cultured), an Ang II group (cells were treated with 100 nmol/L Ang II for 24 h), a siCtrl group (cells were transfected with siRNA control and were then treated with 100 nmol/L Ang II for 24 h), and a siSET7 group (cells were transfected with siRNA SET7 and were then treated with 100 nmol/L Ang II for 24 h). Cell counting kit-8 (CCK-8) and 5-ethynyl-2'-deoxyuridine (EdU) assay were used to evaluate cell proliferation. Real-time PCR was used to detect the mRNA levels of SET7, collagen I, collagen III, and α-smooth muscle actin (α-SMA). Western blotting was used to detect the protein expression of SET7, collagen I, collagen III, α-SMA, sonic hedgehog (Shh), ptched1 (Ptch1), and glioma-associated oncogene homolog 1 (Gli1). Fluorescence microscopy showed positive vimentin staining, and myocardial fibroblasts were in good condition. As compared to the control group, the mRNA and protein levels of SET7 in the Ang II group were significantly upregulated; cell proliferation rate and EdU fluorescence intensity in the Ang II group were significantly increased; the mRNA and protein levels of collagen I, collagen III, and α-SMA were significantly upregulated (all P<0.05). As compared to the siCtrl group, the mRNA and protein levels of SET7 in the siSET7 group were significantly downregulated; cell proliferation rate and EdU fluorescence intensity in the siSET7 group were significantly decreased; the mRNA and protein levels of collagen I, collagen III, and α-SMA in the siSET7 group were significantly downregulated (all P<0.05). Silence of SET7 gene inhibits Ang II-induced proliferation and collagen synthesis of myocardial fibroblasts. Shh signaling pathway may be involved in this process.

Heightened Crescentic Glomerulonephritis in Immune Challenged 129sv Mice Is TGF-β/Smad3 Dependent.

The 129sv mouse strain is particularly sensitive to experimental immune-mediated nephritis. Previous studies have indicated that transforming growth factor-β (TGF-β) plays a critical role in both immune modulation and tissue fibrogenesis in various diseases and that its biological activities are exerted via the SMAD family. In this study, we aimed to determine whether TGF-β/SMAD signaling is essential for the development of immune-mediated nephritis in 129sv mice. Relative to C57BL/6J control mice with anti-glomeruli basement membrane (GBM) nephritis, 129sv mice with anti-GBM nephritis exhibited increased renal collagen deposition. Additionally, higher mRNA levels of pro-collagen and collagen IV, higher serum levels of active and total TGF-β1, and increased TGF-β1, TGF-βIIR, and phosphorylated SMAD expression were detected in these mice. Deletion of Smad3 in 129sv mice ameliorated anti-GBM induced nephritis, including crescentic glomerulonephritis. Collectively, these findings indicate that the heightened experimental nephritis and fibrotic disease in the 129sv strain of mice are regulated by SMAD3, which could be a potential therapeutic target for immune-mediated nephritis.

Expression and role of specificity protein 1 and collagen I in recurrent pterygial tissues

To investigate the expression profiles of the transcription factor specificity protein 1 (Sp1) and collagen I in recurrent pterygial tissues. What is more, to compare the changes of Sp1 and collagen I among primary pterygial tissue, recurrent pterygial tissue and conjunctival tissue. In the prospective study, we collected the pterygial tissues of 40 patients who underwent resection of primary pterygial tissue and recurrent pterygial tissue, and the conjunctival tissues of 10 patients with enucleation due to trauma. The relative expression levels of Sp1 and collagen I were analyzed by reverse transcription quantitative-polymerase chain reaction and Western blot. Paired t-test was performed to compare the Sp1 and collagen I of recurrent pterygial tissues, as well as the primary pterygial tissues and conjunctival tissues. In further, Pearson's hypothesis testing of correlation coefficients was used to compare the correlations of Sp1 and Collagen I. The content of Sp1 and collagen I mRNA and protein was significantly greater in recurrent pterygial tissue than that was in primary and conjunctival tissue (P<0.05). There was a positive correlation between the mRNA and protein levels of Sp1 and collagen I in recurrent pterygial tissues (protein: r=0.913, P<0.05; mRNA: r=0.945, P<0.05). Sp1 and collagen I are expressed in normal conjunctival, primary, and recurrent pterygial tissues, but expression is significantly greater in the latter. Sp1 and collagen I may be involved in the regulation of the development of recurrent pterygium.

Chrysin inhibited epithelial-mesenchymal transition of type Ⅱ alveolar epithelial cell by regulating NF-κB/Twist 1 signaling pathway

This study aimed to assess whether chrysin(ChR) can inhibit epithelial-mesenchymal transition(EMT) of type Ⅱ alveolar epithelial cell and produce anti-pulmonary fibrosis effect by regulating the NF-κB/Twist 1 signaling pathway. Sixty rats were randomly divided into the control group, the bleomycin(BLC) group, BLC+ChR(50 mg·kg~(-1)) group and BLC+ChR(100 mg·kg~(-1)) group, with 15 rats in each group. The pulmonary fibrosis model was induced by intratracheal injection of BLC(7 500 U·kg~(-1)). Rats were orally administered with different doses of ChR after BLC injection for 28 days. The cells were divided into control group, TGF-β1 group(5 ng·mL~(-1)), and TGF-β1+ChR(1, 10, 100 μmol·L~(-1)) groups. The type Ⅱ alveolar epithelial cells were treated with TGF-β1 for 24 h, and then treated with TGF-β1 for 48 h in the presence or absence of different doses of ChR(1, 10 and 100 μmol·L~(-1)). The morphological changes and collagen deposition in lung tissues were analyzed by HE staining, Masson staining and immunohistochemistry. The mRNA and protein expression levels of collagen Ⅰ, E-cadherin, zonula occludens-1(ZO-1), vimentin, alpha smooth muscle actin(α-SMA), inhibitor of nuclear factor kappa B alpha(IκBα), nuclear factor-kappa B p65(NF-κB p65), phospho-NF-κB p65(p-p65) and Twist 1 in lung tissues and cells were detected by qPCR and Western blot, respectively. The animal experiment results showed that as compared with the BLC group, after administration of ChR for 28 days, bleomycin-induced pulmonary fibrosis in rats was significantly relieved, collagen Ⅰ expression in lung tissues was significantly reduced(P&lt;0.05 or P&lt;0.01), and EMT of alveolar epithelial cells was obviously inhibited [the expression levels of E-cadherin and ZO-1 were increased and the expression levels of vimentin and α-SMA were decreased(P&lt;0.05 or P&lt;0.01)], concomitantly with significantly reduced IκBα and p65 phosphorylation level in cytoplasm and decreased NF-κB p65 and Twist 1 expression in nucleus(P&lt;0.05 or P&lt;0.01). The cell experiment results showed that different doses of ChR(1, 10 and 100 μmol·L~(-1)) significantly reduced TGF-β1-induced collagen Ⅰ expression(P&lt;0.05 or P&lt;0.01), significantly inhibited EMT of type Ⅱ alveolar epithelial cells[the expression levels of E-cadherin and ZO-1 were increased and the expression levels of vimentin and α-SMA were decreased(P&lt;0.05 or P&lt;0.01)], and inhibited IκBα and p65 phosphorylation in cytoplasm and down-regulated NF-κB p65 and Twist 1 expression in nucleus induced by TGF-β1(P&lt;0.05 or P&lt;0.01). The results suggest that ChR can reverse EMT of type Ⅱ alveolar epithelial cell and alleviate pulmonary fibrosis in rats, and its mechanism may be associated with reducing IκBα phosphorylation and inhibiting NF-κB p65 phosphorylation and nuclear transfer, thus down-regulating Twist 1 expression.

Plumbagin attenuates traumatic tracheal stenosis in rats and inhibits lung fibroblast proliferation and differentiation via TGF-β1/Smad and Akt/mTOR pathways

ABSTRACT Traumatic tracheal stenosis (TS) is a serious respiratory disease characterized by hyperplasia of airway granulation. Plumbagin (PLB) is a natural naphthoquinone component with anti-fibrotic properties. This research aimed to explore the roles of PLB in alleviating TS and the underlying mechanisms. For in vitro studies, lung fibroblasts (IMR-90 cells), with/without PLB treatment or TGF-β1 induction, were used. The viability and proliferation of IMR-90 cells were examined by CCK-8 and EdU incorporation assays. The differentiation of IMR-90 cells was assessed by detecting the mRNA and protein expression levels of collagen (COL)-1 and alpha-smooth muscle actin (α-SMA). Besides, immunofluorescence assay was conducted to evaluate the localization of α-SMA in TGF-β1-induced IMR-90 cells. Moreover, the combination of PLB with/without TβRI (SB-431,542), PI3K/Akt (Ly294002) or mTOR (rapamycin) inhibitor was pretreated on IMR-90 cells after TGF-β1 induction. For in vivo studies, a rat model of TS was established. The pathological features and severity of TS were determined by hematoxylin and eosin staining. The protein levels of TGF-β1/Smad and Akt/mTOR pathways were detected for both in vitro and in vivo models. PLB effectively inhibited the proliferation and differentiation of TGF-β1-induced IMR-90 cells, and suppressed TGF-β1/Smad and Akt/mTOR signaling pathways both in vivo and in vitro. Furthermore, PLB reduced the degree of TS in rats. Taken together, our results indicate that PLB regulates lung fibroblast activity and attenuates TS in rats by inhibiting TGF-β1/Smad and Akt/mTOR signaling pathways. In conclusion, this study implies that PLB may serve as a promising therapeutic compound for TS.

Deacetylation of FOXO4 by Sirt1 stabilizes chondrocyte extracellular matrix upon activating SOX9.

FOXO4 has essential roles in cellular metabolism and prevents cartilage degeneration in osteoarthritis (OA). Here we aim to provide evidence that deacetylated-FOXO4 stabilizes chondrocyte (CH) extracellular matrix (ECM) related to SOX9 activation. We used Chromatin immunoprecipitation (ChIP) and Dual-Luciferase reporter assay to verify that the FOXO4 protein activates SOX9 by binding to its promoter. We cultured human CHs with IL-1β to cause degeneration and supplied Sirt1 protein to deacetylate FOXO4. To confirm the function of FOXO4 and SOX9 during CHs degeneration, we also used the FOXO4 and SOX9 silenced CHs by siRNA transfection as a comparison. Western blot assay was used to analyze the protein level of Sirt1, SOX9, and the acetylated condition of FOXO4. Besides, RT-PCR was used to measure the mRNA level of collagen I/II/X, aggrecan, MMP-13, and ADAMTS-5 for determining the ECM states. FOXO4 protein transcriptionally activates SOX9 expression by binding to its promoter. Under the IL-1β stimulation, FOXO4 acetyl-lysine rate increased, and the SOX9 protein expression decreased, which was alleviated after the supplement of exogenic Sirt1 protein. Meanwhile, Sirt1 overexpression increased the collagen II and aggrecan and reduced the collagen I, collagen X, MMP-13, and ADAMTS-5 mRNA expression. However, the silencing of FOXO4 abolished the Sirt1 induced SOX9 expression and weakened the ECM production stability. Additionally, SOX9 silencing also alleviated the effect of the Sirt1 supplement on the degenerated CHs, though the FOXO4 was highly deacetylated. FOXO4 acetylation aggravates during the degeneration of CHs, and the deacetylation of FOXO4 by Sirt1 could activate the SOX9 expression and result in maintaining the ECM stability of cartilage.