After the articular cartilage injury, the metabolic level is increased during the progressive degeneration, the chondrocytes secrete a variety of inflammatory factors, and the original cell phenotype is gradually changed. For a long time, a large number of researchers have done a lot of researches to promote anabolism of chondrocytes and to maintain the stability of chondrocyte phenotype. There are many molecular signaling pathways involved in the process of promoting cartilage repair. This review focuses on the key signaling molecules in articular cartilage repair, such as transforming growth factor-beta and bone morphogenetic protein, and reveals their roles in the process of cartilage injury and repair, so that researchers in related fields can understand the molecular mechanism of cartilage injury and repair widely and deeply. Based on this, they may find promising targets and biological methods for the treatment of cartilage injury.
Bone Morphogenetic Proteins ; physiology ; Cartilage, Articular ; growth & development ; injuries ; Chondrocytes ; physiology ; Humans ; Regeneration ; Signal Transduction ; Transforming Growth Factor beta ; physiology

OBJECTIVE: To investigate the effects of Ganoderma lucidum polysaccharides (GL-PS) on human fibroblasts and skin wound healing in Kunming male mice and to explore the putative molecular mechanism. METHODS: Primary human skin fibroblasts were cultured. The viability of fibroblasts treated with 0, 10, 20, 40, 80, and 160 μg/mL of GL-PS, respectively were detected by 3-4,5-dimethyl-2-thiazolyl-2,5-diphenyl-2-Htetrazolium bromide (MTT). The migration ability of fibroblasts treated with 0, 10, 20, and 40 μg/mL of GL-PS were measured by transwell assay. The secretion of the C-terminal peptide of procollagen type I (CICP) and transforming growth factor-β1 (TGF-β1) in the cell supernatant was tested by enzyme-linked immunosorbent assay. The expression of β-catenin was detected by Western blot. Furthermore, the Kunming mouse model with full-layer skin resection trauma was established, and was treated with 10, 20, and 40 mg/mL of GL-PS, respectively as external use. The size of the wound was measured daily, complete healing time in each group was recorded and the percentage of wound contraction was calculated. RESULTS: Compared with the control group, 10, 20, and 40 μg/mL of GL-PS significantly increased the viability of fibroblasts, promoted the migration ability of fibroblasts, and up-regulated the expressions of CICP and TGF-β1 in fibroblasts (Plt;0.05 or Plt;0.01). The expression of β-catenin in fibroblasts treated with 20 and 40 μg/mL of GL-PS was significantly higher than that of the control group (Plt;0.01). Furthermore, after external use of 10, 20, and 40 mg/mL of GL-PS, the rates of wound healing in mice were significantly higher and the wound healing time was significantly less than the control group (Plt;0.05 or Plt;0.01). CONCLUSION A certain concentration of GL-PS may promote wound healing via activation of the Wnt/β-catenin signaling pathway and up-regulation of TGF-β1, which might serve as a promising source of skin wound healing.
Animals ; Cell Movement ; drug effects ; Cell Survival ; drug effects ; Cells, Cultured ; Collagen Type I ; biosynthesis ; Fibroblasts ; drug effects ; Humans ; Male ; Mice ; Polysaccharides ; pharmacology ; Reishi ; chemistry ; Skin ; drug effects ; injuries ; Transforming Growth Factor beta1 ; physiology ; Wound Healing ; drug effects ; beta Catenin ; physiology

OBJECTIVE: To investigate the effects of tetrandrine (Tet) on proliferation and activation of rat cardiac fibroblasts. METHODS: Firstly, the cell counting kit-8 (cck-8) assay was applied to detect the effects of Tet with different concentrations on proliferation of cardiac fibroblasts. Secondly, transforming growth factor (TGF-β)with a concentration of 5 μg/L was used to induce the cardiac fibroblast activation, and Western blot was performed to measure the expression variation of β-catenin, vimentin (Vm), fibronectin (Fn) and smooth muscle α-actin (SMA). At last, the real-time PCR was conducted to measure the expression change of collagen-1(Col-1) and collagen-3(Col-3). RESULTS: The cck-8 assay showed that the Tet with different concentrations respectively, which were 0.5 μmol/L, 1 μmol/L, 2 μmol/L, 4 μmol/L, and 8 μmol/L, significantly inhibited the proliferation of cardiac fibroblasts. The viability was decreased to 94.4%,84.9%,74.9%,63.8%and 50.3% respectively of the control group when the Tet concentration changed, and the difference was statistically significant, P=0.043, P<0.001, P<0.001, P<0.001, P<0.001 respectively. Western blot revealed that the expressions of β-catenin, Fn, SMA and Vm, were up-regulated by TGF-β(5 μg/L), the result showed that the difference was statistically significant, and the P values were 0.001,0.008,0.010,0.001 respectively. Then, the up-regulation of β-catenin, Fn and SMA was attenuated by pre-treatment of Tet, and the result also displayed that the difference was statistically significant, and the P values were 0.009, 0.005, 0.019,respectively. While there was no significant change in the expression of Vm, according to Western blotting, and P>0.05,at the same time, real-time PCR indicated that the up-regulations of Col-1 and Col-3 which were induced by TGF-β were blocked by pre-treatment of Tet, the result showed that the difference was statistically significant, P<0.001. CONCLUSION According to the experimental results, we can draw the conclusion that: the Tet can significantly inhibit the proliferation of cardiac fibroblasts, meanwhile, it can block the activation of cardiac fibroblasts, which is induced by TGF-β. It is supposed that the Tet may probably have anti myocardial fibrosis, which indicates that it may probably be a medicine which is used to block the cardiac remodeling.
Actins ; Animals ; Benzylisoquinolines/pharmacology* ; Blotting, Western ; Calcium Channel Blockers/pharmacology* ; Cell Proliferation ; Collagen ; Collagen Type I ; Fibroblasts/physiology* ; Fibrosis ; Myocardium/cytology* ; Neoplasm Proteins/metabolism* ; Rats ; Signal Transduction ; Transforming Growth Factor beta/metabolism* ; Transforming Growth Factor beta1

Transforming growth factor (TGF)-β regulates a wide variety of cellular responses, including cell growth arrest, apoptosis, cell differentiation, motility, invasion, extracellular matrix production, tissue fibrosis, angiogenesis, and immune function. Although tumor-suppressive roles of TGF-β have been extensively studied and well-characterized in many cancers, especially at early stages, accumulating evidence has revealed the critical roles of TGF-β as a pro-tumorigenic factor in various types of cancer. This review will focus on recent findings regarding epithelial-mesenchymal transition (EMT) induced by TGF-β, in relation to crosstalk with some other signaling pathways, and the roles of TGF-β in lung and pancreatic cancers, in which TGF-β has been shown to be involved in cancer progression. Recent findings also strongly suggested that targeting TGF-β signaling using specific inhibitors may be useful for the treatment of some cancers. TGF-β plays a pivotal role in the differentiation and function of regulatory T cells (Tregs). TGF-β is produced as latent high molecular weight complexes, and the latent TGF-β complex expressed on the surface of Tregs contains glycoprotein A repetitions predominant (GARP, also known as leucine-rich repeat containing 32 or LRRC32). Inhibition of the TGF-β activities through regulation of the latent TGF-β complex activation will be discussed.
Drug Discovery ; Humans ; Lung Neoplasms ; drug therapy ; immunology ; metabolism ; Membrane Proteins ; metabolism ; Pancreatic Neoplasms ; drug therapy ; immunology ; metabolism ; Signal Transduction ; drug effects ; physiology ; T-Lymphocytes, Regulatory ; metabolism ; Transforming Growth Factor beta ; antagonists & inhibitors ; immunology ; metabolism

Background: Follistatin-like 1 (FSTL1) is a novel profibrogenic factor that induces pulmonary fibrosis (PF) through the transforming growth factor-beta 1 (TGF-β1)/Smad signaling. Little is known about its effects on PF through the non-Smad signaling, like the mitogen-activated protein kinase (MAPK) pathway. Therefore, this study aimed to investigate the role of FSTL1 in PF through the MAPK signaling pathway and its mechanisms in lung fibrogenesis. Methods: PF was induced in Fstl1and wild-type (WT) C57BL/6 mice with bleomycin. After 14 days, the mice were sacrificed, and lung tissues were stained with hematoxylin and eosin; the hydroxyproline content was measured to confirm PF. The mRNA and protein level of FSTL1 and the change of MAPK phosphorylation were measured by quantitative polymerase chain reaction and Western blotting. The effect of Fstl1 deficiency on fibroblasts differentiation was measured by Western blotting and cell immunofluorescence. MAPK signaling activation was measured by Western blotting in Fstl1 and WT fibroblasts treated with recombinant human FSTL1 protein. We pretreated mouse lung fibroblast cells with inhibitors of the extracellular signal-regulated kinase (ERK), p38, and Jun N-terminal kinase (JNK) signaling and analyzed their differentiation, proliferation, migration, and invasion by Western blotting, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide analysis, and transwell assays. The Student's t-test was used to compare the differences between two groups. Results: Fstl1 deficiency attenuated phosphorylation of the ERK, p38, and JNK signaling in bleomycin-induced fibrotic lung tissue 14 days after injury (0.67 ± 0.05 vs. 1.22 ± 0.03, t = 14.92, P = 0.0001; 0.41 ± 0.01 vs. 1.15 ± 0.07; t = 11.19; P = 0.0004; and 0.41 ± 0.01 vs. 1.07 ± 0.07, t = 8.92, P = 0.0009; respectively), compared with WT lungs at the same time and in primary lung fibroblasts (0.82 ± 0.01 vs. 1.01 ± 0.04, t = 4.06, P = 0.0150; 1.04 ± 0.03 vs. 1.24 ± 0.03, t = 4.44, P = 0.0100; and 0.76 ± 0.05 vs. 0.99 ± 0.05, t = 4.48, P = 0.0100; respectively), compared with TGF-β1-stimulated WT group. Recombinant human FSTL1 protein in lung fibroblasts enhanced TGF-β1-mediated phosphorylation of the ERK (1.19 ± 0.08 vs. 0.55 ± 0.04, t = 6.99, P = 0.0020), p38 (1.18 ± 0.04 vs. 0.66 ± 0.03, t = 11.20, P = 0.0020), and JNK (1.11 ± 0.01 vs. 0.84 ± 0.04, t = 6.53, P = 0.0030), compared with the TGF-β1-stimulated WT group. Fstl1-deficient fibroblasts showed reduced alpha-smooth muscle actin (α-SMA) expression (0.70 ± 0.06 vs. 1.28 ± 0.11, t = 4.65, P = 0.0035, compared with the untreated WT group; 1.40 ± 0.05 vs. 1.76 ± 0.02, t = 6.31, P = 0.0007; compared with the TGF-β1-treated WT group). Compared with the corresponding condition in the control group, the TGF-β1/FSTL1-mediated α-SMA expression was significantly suppressed by pretreatment with an inhibitor of p38 (0.73 ± 0.01 vs. 1.13 ± 0.10, t = 3.92, P = 0.0078) and JNK (0.78 ± 0.03 vs. 1.08 ± 0.06, t = 4.40, P = 0.0046) signaling. The proliferation of mouse lung fibroblast cells (MLgs) significantly decreased after treatment of an inhibitor of p38 (0.30 ± 0.01 vs. 0.46 ± 0.03, t = 4.64, P = 0.0009), JNK (0.30 ± 0.01 vs. 0.49 ± 0.01, t = 12.84, P = 0.0001), and Smad2/3 (0.18 ± 0.02 vs. 0.46 ± 0.02, t = 12.69, P = 0.0001) signaling compared with the dimethylsulfoxide group. The migration and invasion cells of MLgs significantly decreased in medium pretreated with an inhibitor of p38 (70.17 ± 3.28 vs. 116.30 ± 7.11, t = 5.89, P = 0.0042 for the migratory cells; 19.87 ± 0.84 vs. 32.70 ± 0.95, t = 10.14, P = 0.0005 for the invasive cells), JNK (72.30 ± 3.85 vs. 116.30 ± 7.11, t = 5.44, P = 0.0056 for the migratory cells; 18.03 ± 0.94 vs. 32.70 ± 0.95, t = 11.00, P = 0.0004 for the invasive cells), and Smad2/3 (64.76 ± 1.41 vs. 116.30 ± 7.11, t = 7.11, P = 0.0021 for the migratory cells; 18.03 ± 0.94 vs. 32.70 ± 0.95, t = 13.29, P = 0.0002 for the invasive cells) signaling compared with the corresponding condition in the dimethylsulfoxide group. Conclusion FSTL1 affects lung fibroblast differentiation, proliferation, migration, and invasion through p38 and JNK signaling, and in this way, it might influence the development of PF.
Animals ; Antibiotics, Antineoplastic ; adverse effects ; Bleomycin ; adverse effects ; Cells, Cultured ; Fibroblasts ; Follistatin ; Follistatin-Related Proteins ; physiology ; Humans ; Mice ; Mice, Inbred C57BL ; Pulmonary Fibrosis ; chemically induced ; Transforming Growth Factor beta ; Transforming Growth Factor beta1 ; drug effects ; physiology ; p38 Mitogen-Activated Protein Kinases ; drug effects

Recent studies have shown that diet can affect the body's immunity. Roughage of dairy cows consists of a variety of plant materials which make different contributions to health. This study investigated the effect of different roughages on the immunity of dairy cows. Serum, peripheral blood mononuclear cells (PBMCs), and milk samples were collected from 20 multiparous mid-lactation cows fed mixed forage (MF)- or corn straw (CS)-based diets. Expression profile analysis was used to detect the differentially expressed genes (DEGs) from PBMCs. The results showed that milk protein in the MF group increased to 3.22 g/100 ml, while that of the CS group milk was 2.96 g/100 ml; by RNA sequencing, it was found that 1615 genes were differentially expressed between the CS group and the MF group among the 24 027 analyzed probes. Gene ontology (GO) and pathway analysis of DEGs suggested that these genes (especially genes coding cytokines, chemokine and its receptors) are involved in the immune response. Results were confirmed at the protein level via detecting the levels of interleukin-2 (IL-2), IL-6, IL-10, IL-12, leptin (LEP), interferon-γ (IFN-γ), transforming growth factor-β1 (TGF-β1), and tumor necrosis factor-α (TNF-α) in peripheral blood by enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay analysis. Our data supported the conclusions that the protein content in milk of the MF group was higher than that of the CS group, the CS-based diets induced more release of cytokines than the MF-based diets in dairy cows' PBMCs, and milk protein content may be affected by cytokines.
Animals ; Cattle/immunology* ; Cytokines/physiology* ; Diet ; Female ; Gene Ontology ; Leukocytes, Mononuclear/immunology* ; Milk/chemistry* ; Transforming Growth Factor beta/physiology* ; Zea mays

OBJECTIVEThis study investigated the effects of hydroxyapatite (HA)/chitosan (CS)-transforming growth factor-&beta;1 (TGF-&beta;1) composite coatings on titanium surfaces, as well as on the attachment and proliferation of osteoblasts.

METHODSHA/CS-TGF-&beta;1 composite coatings were prepared on titanium surfaces by physical, chemical, and biological modifications. Scanning electron microscopy (SEM), X-ray diffraction (XRD), Fourier transform-infrared spectroscopy (FTIR), and other methods were employed to analyze the chemical composition and surface topography of the composite coatings. CCK-8 and immunofluorescence assays were used to analyze the effects of the coatings on the attachment and proliferation of osteoblasts.

RESULTSHA/CS-TGF-&beta;1 composite coatings were successfully prepared. Their contact angle was almost zero. These composite coatings were applied in vitro, with a drug released early and a burst release effect. The growth of osteoblasts was not inhibited on it and it had obvious promoting effect on the adhesion and early proliferation of osteoblasts.

CONCLUSIONThe composite coatings significantly promote the adhesion and early proliferation of osteoblasts in vitro. This finding shows that the proposed method demonstrates a good prospective application in surface modification of titanium.

Cell Proliferation ; Chitosan ; chemistry ; Dental Bonding ; methods ; Durapatite ; chemistry ; Microscopy, Electron, Scanning ; Osteoblasts ; physiology ; Prospective Studies ; Spectroscopy, Fourier Transform Infrared ; Surface Properties ; Titanium ; chemistry ; Transforming Growth Factor beta ; chemistry ; Transforming Growth Factors ; X-Ray Diffraction

BACKGROUNDMesenchymal stem cells (MSCs) are bone marrow stem cells which play an important role in tissue repair. The treatment with MSCs will be likely to aggravate the degree of fibrosis. The Wnt/&beta;-catenin signaling pathway is involved in developmental and physiological processes, such as fibrosis. Dickkopfs (DKKs) are considered as an antagonist to block Wnt/&beta;-catenin signaling pathway by binding the receptor of receptor-related protein (LRP5/6). DKK1 was chosen in attempt to inhibit fibrosis of MSCs by lowering activity of Wnt/&beta;-catenin signaling pathway.

METHODSStable MSCs were randomly divided into four groups: MSCs control, MSCs + transforming growth factor-&beta; (TGF-&beta;), MSCs + DKK1, and MSCs + TGF-&beta; + DKK1. Flow cytometry was used to identify MSCs. Cell viability was evaluated by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide test. Immunofluorescence was used to detect protein expression in the Wnt/&beta;-catenin signaling pathways. Western blotting analysis was employed to test expression of fibroblast surface markers and, finally, real-time reverse transcription polymerase chain reaction was employed to test mRNA expression of fibroblast surface markers and Wnt/&beta;-catenin signaling proteins.

RESULTSCultivated MSCs were found to conform to the characteristics of standard MSCs: expression of cluster of differentiation (CD) 73, 90, and 105, not expression of 34, 45, and 79. We found that DKK1 could maintain the normal cell morphology of MSCs. Western blotting analysis showed that fibroblast surface markers were expressed in high quantities in the group MSCs + TGF-&beta;. However, the expression was lower in the MSCs + TGF-&beta; + DKK1. Immunofluorescence showed high expression of all Wnt/&beta;-catnin molecules in the MSCs + TGF-&beta; group but expressed in lower quantities in MSCs + TGF-&beta; + DKK1 group. Finally, mRNA expression of fibroblast markers vimentin, α-smooth muscle actin and Wnt/&beta;-catenin signaling proteins &beta;-catenin, T-cell factor, and glycogen synthase kinase-3&beta; was significantly increased in MSCs + TGF-&beta; group compared to control (P < 0.05). Expression of the same fibroblast markers and Wnt/&beta;-catenin was decreased to regular quantities in the MSCs + TGF-&beta; + DKK1 group.

CONCLUSIONSDKK1, Wnt/&beta;-catenin inhibitors, blocks the Wnt/&beta;-catenin signaling pathway to inhibit the process of MSCs fibrosis. It might provide some new ways for clinical treatment of certain diseases.

Animals ; Cell Differentiation ; physiology ; Cells, Cultured ; Female ; Fibroblasts ; cytology ; metabolism ; Intercellular Signaling Peptides and Proteins ; genetics ; metabolism ; Mesenchymal Stromal Cells ; cytology ; metabolism ; Mice ; Rats ; Transforming Growth Factor beta ; genetics ; metabolism

OBJECTIVETo predict the target genes of rno-microRNA-296-5p (miR-296) using bioinformatics software and databases, and to provide a theoretical basis for further studies of biological effects of miR-296 in fetal lung development.

METHODSPubMed and Google were used to search for all reported literature on miR-296. The miRBase database was used to determine the sequence and evolutionary conservatism of miR-296. The TargetScans database was used to predict the target genes of miR-296. The DAVID Bioinformatics Resources 6.8 database was used for the functional enrichment analysis of the target genes. The KEGG database was used to analyze the signaling pathways of target genes.

RESULTSmiR-296 was reported to play important roles in many biological processes and have a high degree of sequence conservation among species. The target genes of miR-296 were involved in biological processes, cell components, and molecular function. Those target genes were significantly enriched in the mitogen-activated protein kinase signaling pathway, Wnt signaling pathway, and transforming growth factor-&beta; signaling pathway (p<0.05).

CONCLUSIONSThe bioinformatics analysis of the target genes of miR-296 provides a basis for studying biological effects and mechanism of action of miR-296 in lung development.

Animals ; Computational Biology ; Humans ; Lung ; embryology ; MAP Kinase Signaling System ; physiology ; MicroRNAs ; physiology ; Transforming Growth Factor beta ; physiology ; Wnt Signaling Pathway ; physiology

Using forward and reverse genetics and global gene expression analyses, we explored the crosstalk between the IκB kinase β (IKKβ) and the transforming growth factor β (TGFβ) signaling pathways. We show that in vitro ablation of Ikkβ in fibroblasts led to progressive ROS accumulation and TGFβ activation, and ultimately accelerated cell migration, fibroblast-myofibroblast transformation and senescence. Mechanistically, the basal IKKβ activity was required for anti-oxidant gene expression and redox homeostasis. Lacking this activity, IKKβ-null cells showed ROS accumulation and activation of stress-sensitive transcription factor AP-1/c-Jun. AP-1/c-Jun activation led to up-regulation of the Tgfβ2 promoter, which in turn further potentiated intracellular ROS through the induction of NADPH oxidase (NOX). These data suggest that by blocking the autocrine amplification of a ROS-TGFβ loop IKKβ plays a crucial role in the prevention of fibroblast-myofibroblast transformation and senescence.
Adenoviridae ; genetics ; Animals ; Autocrine Communication ; physiology ; Cell Line ; Cell Movement ; Cellular Senescence ; Genetic Vectors ; genetics ; metabolism ; I-kappa B Kinase ; deficiency ; genetics ; metabolism ; JNK Mitogen-Activated Protein Kinases ; metabolism ; Mice ; Myofibroblasts ; cytology ; metabolism ; NADPH Oxidases ; metabolism ; Oxidative Stress ; Promoter Regions, Genetic ; Reactive Oxygen Species ; metabolism ; Signal Transduction ; Superoxide Dismutase ; genetics ; metabolism ; Transcription Factor AP-1 ; metabolism ; Transforming Growth Factor beta ; genetics ; metabolism ; Up-Regulation