OBJECTIVE: Huangqi Decoction (HQD), a classical traditional Chinese medicine formula, has been used as a valid treatment for alleviating liver fibrosis; however, the underlying molecular mechanism is still unknown. Although our previous studies showed that microRNA-663a (miR-663a) suppresses the proliferation and activation of hepatic stellate cells (HSCs) and the transforming growth factor-β/small mothers against decapentaplegic (TGF-β/Smad) pathway, whether long noncoding RNAs (lncRNAs) are involved in HSC activation via the miR-663a/TGF-β/Smad signaling pathway has not yet reported. The present study aimed to investigate the roles of lncRNA lnc-C18orf26-1 in the activation of HSCs and the mechanism by which HQD inhibits hepatic fibrosis. METHODS: The expression levels of lnc-C18orf26-1, miR-663a and related genes were measured by quantitative reverse transcription-polymerase chain reaction. HSCs were transfected with the miR-663a mimic or inhibitor and lnc-C18orf26-1 small interfering RNAs. The water-soluble tetrazolium salt-1 assay was used to assess the proliferation rate of HSCs. Changes in lncRNA expression were evaluated in miR-663a-overexpressing HSCs by using microarray to identify miR-663a-regulated lncRNAs. RNA hybrid was used to predict the potential miR-663a binding sites on lncRNAs. Luciferase reporter assays further confirmed the interaction between miR-663a and the lncRNA. The expression levels of collagen α-2(I) chain (COL1A2), α-smooth muscle actin (α-SMA) and TGF-β/Smad signaling pathway-related proteins were determined using Western blotting. RESULTS: Lnc-C18orf26-1 was upregulated in TGF-β1-activated HSCs and competitively bound to miR-663a. Knockdown of lnc-C18orf26-1 inhibited HSC proliferation and activation, downregulated TGF-β1-stimulated α-SMA and COL1A2 expression, and inhibited the TGF-β1/Smad signaling pathway. HQD suppressed the proliferation and activation of HSCs. HQD increased miR-663a expression and decreased lnc-C18orf26-1 expression in HSCs. Further studies showed that HQD inhibited the expression of COL1A2, α-SMA, TGF-β1, TGF-β type I receptor (TGF-βRI) and phosphorylated Smad2 (p-Smad2) in HSCs, and these effects were reversed by miR-663a inhibitor treatment. CONCLUSION Our study identified lnc-C18orf26-1 and miR-663a as promising therapeutic targets for hepatic fibrosis. HQD inhibits HSC proliferation and activation at least partially by regulating the lnc-C18orf26-1/miR-663a/TGF-β1/TGF-βRI/p-Smad2 axis.
Humans ; Transforming Growth Factor beta/pharmacology* ; Transforming Growth Factor beta1/metabolism* ; RNA, Long Noncoding/pharmacology* ; Drugs, Chinese Herbal/pharmacology* ; MicroRNAs/genetics* ; Hepatic Stellate Cells/pathology* ; Liver Cirrhosis/metabolism* ; Cell Proliferation ; Transforming Growth Factors/pharmacology*

PURPOSE: Wound represents a major health challenge as they consume a large amount of healthcare resources to improve patient's quality of life. Many scientific studies have been conducted in search of ideal biomaterials with wound-healing activity for clinical use and collagen has been proven to be a suitable candidate biomaterial. This study intended to investigate the wound healing activity of collagen peptides derived from jellyfish following oral administration. METHODS: In this study, collagen was extracted from the jellyfish--Rhopilema esculentum using 1% pepsin. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and fourier transform infrared (FTIR) were used to identify and determine the molecular weight of the jellyfish collagen. Collagenase II, papain and alkaline proteinase were used to breakdown jellyfish collagen into collagen peptides. Wound scratch assay (in vitro) was done to determine migration potential of human umbilical vein endothelial cells (HUVEC) covering the artificial wound created on the cell monolayer following treatment with collagen peptides. In vivo studies were conducted to determine the effects of collagen peptides on wound healing by examining wound contraction, re-epithelialization, tissue regeneration and collagen deposition on the wounded skin of mice. Confidence level (p < 0.05) was considered significant using GraphPad Prism software. RESULTS: The yield of collagen was 4.31%. The SDS-PAGE and FTIR showed that extracted collagen from jellyfish was type I. Enzymatic hydrolysis of this collagen using collagenase II produced collagen peptides (CP) and hydrolysis with alkaline proteinase/papain resulted into collagen peptides (CP). Tricine SDS-PAGE revealed that collagen peptides consisted of protein fragments with molecular weight <25 kDa. Wound scratch assay showed that there were significant effects on the scratch closure on cells treated with collagen peptides at a concentration of 6.25 μg/mL for 48 h as compared to the vehicle treated cells. Overall treatment with collagen peptide on mice with full thickness excised wounds had a positive result in wound contraction as compared with the control. Histological assessment of peptides treated mice models showed remarkable sign of re-epithelialization, tissue regeneration and increased collagen deposition. Immunohistochemistry of the skin sections showed a significant increase in β-fibroblast growth factor (β-FGF) and the transforming growth factor-β (TGF-β) expression on collagen peptides treated group. CONCLUSION Collagen peptides derived from the jellyfish-Rhopilema esculentum can accelerate the wound healing process thus could be a therapeutic potential product that may be beneficial in wound clinics in the future.
Administration, Oral ; Animals ; Collagen ; administration & dosage ; isolation & purification ; metabolism ; pharmacology ; Fibroblast Growth Factors ; metabolism ; Human Umbilical Vein Endothelial Cells ; Humans ; Male ; Regeneration ; Scyphozoa ; chemistry ; Skin ; metabolism ; Skin Physiological Phenomena ; Stimulation, Chemical ; Transforming Growth Factor beta1 ; metabolism ; Wound Healing ; drug effects

OBJECTIVE: To investigate the effects of Yiqi Huayu Hutan decoction on pulmonary fibrosis of rats which induced by bleomycin. METHODS: The rat model of pulmonary fibrosis was induced by intratracheal injection of bleomycin hydrochloride (5 mg/kg). Sixty SD rats were randomly divided into the normal group (group N), the model group (group M), the positive control group (group Y), group of low concentration (group LC), group of medium concentration (group MC) and group of high concentration of Yiqi Huayu Hutan decoction (group HC). After 4 weeks, the experimental groups were treated with low concentration decoction, medium concentration decoction and high concentration decoction respectively, and the Y group was treated with hydrocortisone acetate, the Group N and group M were treated with saline by intragastric administration. Twelve weeks later, rats were killed and the pathomorphism of pulmonary tissues of each group was observed by HE staining and Masson staining. Further, the expressions of transforming growth factor-β1(TGF-β1), Snail1, E-cadherin and Fibronectin in pulmonary tissues of each group were detected by qTR-PCR and Western blot. RESULTS: Compared with the model group, the collagen sediment in the interstitial was reduced in the experimental groups, especially in the group of medium concentration, which was observed by HE staining and Masson staining .Compared with the model group, the expressions of TGF-β1, Snail1 and Fibronectin protein in pulmonary tissues of the treatment groups were decreased in the experimental group, especially in the group of medium concentration, which were detected by qRT-PCR and Western blot. CONCLUSION Yiqi Huayu Hutan decoction can significantly improve the pulmonary fibrosis which is induced by bleomycin, and the mechanism is related to the inhibition of the expression of TGF-β/Snail pathway of transcription TGF-β1.
Animals ; Bleomycin ; Cadherins ; metabolism ; Drugs, Chinese Herbal ; pharmacology ; Fibronectins ; metabolism ; Idiopathic Pulmonary Fibrosis ; chemically induced ; drug therapy ; Lung ; metabolism ; pathology ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Snail Family Transcription Factors ; metabolism ; Transforming Growth Factor beta1 ; metabolism

To investigate the effect of bromodomain and extra-terminal (BET) protein inhibitor JQ1 on expression of autoimmune-related genes in CD4+T cells from patients with systemic lupus erythematosus (SLE).
 Methods: Peripheral CD4+T cells were isolated by positive selection with CD4 microbeads. The percentage of CD4+T cells were detected by flow cytometry. CD4+T cells were treated by JQ1 at 100 nm/L for 6, 24, 48 h. The expression of T cell-related genes was measured by quantitative real-time PCR (qPCR). The secretion levels of cytokines in culture supernatant were measured by ELISA at 48 h.
 Results: The percentage of CD4+T cells isolated by CD4 microbeads is 97.2%. Compared with the control group, the mRNA expression levels of IFNG, IL-17F, IL-21, CXCR5 and FOXP3 were down-regulated at 6, 24 and 48 h (P<0.05), and IL-17A mRNA level was decreased at 6 and 24 h (P<0.01); while IL-4 mRNA level was up-regulated at 24, 48 h (P<0.01), and TGF-β1 mRNA level was up-regulated at 6 and 48 h (P<0.05) in SLE CD4+T cells treated with JQ1. The secretion levels of IFN-γ and IL-21 in JQ1-treated group were decreased significantly (P<0.05), while the secretion levels of IL-4 and TGF-β were up-regulated compared with control group (P<0.05).
 Conclusion: JQ1 can reverse the immune dysregulation and improve the immunity homeostasis in CD4+T cells from patients with SLE.
Azepines ; pharmacology ; CD4 Lymphocyte Count ; CD4-Positive T-Lymphocytes ; cytology ; drug effects ; metabolism ; Cytokines ; analysis ; metabolism ; Flow Cytometry ; Humans ; Interferon-gamma ; metabolism ; Lupus Erythematosus, Systemic ; immunology ; metabolism ; Proteins ; antagonists & inhibitors ; RNA, Messenger ; metabolism ; Time Factors ; Transforming Growth Factor beta1 ; Triazoles ; pharmacology

PURPOSE: The degradation of the extracellular matrix has been shown to play an important role in the treatment of hepatic cirrhosis. In this study, the effect of thalidomide on the degradation of extracellular matrix was evaluated in a rat model of hepatic cirrhosis. MATERIALS AND METHODS: Cirrhosis was induced in Wistar rats by intraperitoneal injection of carbon tetrachloride (CCl4) three times weekly for 8 weeks. Then CCl4 was discontinued and thalidomide (100 mg/kg) or its vehicle was administered daily by gavage for 6 weeks. Serum hyaluronic acid, laminin, procollagen type III, and collagen type IV were examined by using a radioimmunoassay. Matrix metalloproteinase-13 (MMP-13), tissue inhibitor of metalloproteinase-1 (TIMP-1), and alpha-smooth muscle actin (alpha-SMA) protein in the liver, transforming growth factor beta1 (TGF-beta1) protein in cytoplasm by using immunohistochemistry and Western blot analysis, and MMP-13, TIMP-1, and TGF-beta1 mRNA levels in the liver were studied using reverse transcriptase polymerase chain reaction. RESULTS: Liver histopathology was significantly better in rats given thalidomide than in the untreated model group. The levels of TIMP-1 and TGF-beta1 mRNA and protein expressions were decreased significantly and MMP-13 mRNA and protein in the liver were significantly elevated in the thalidomide-treated group. CONCLUSION: Thalidomide may exert its effects on the regulation of MMP-13 and TIMP-1 via inhibition of the TGF-beta1 signaling pathway, which enhances the degradation of extracellular matrix and accelerates the regression of hepatic cirrhosis in rats.
Actins ; Animals ; Carbon Tetrachloride/toxicity ; Collagen Type III/metabolism ; Down-Regulation ; Extracellular Matrix/metabolism ; Immunohistochemistry ; Immunosuppressive Agents/*pharmacology ; Liver Cirrhosis, Experimental/chemically induced/*metabolism/pathology/*prevention & control ; Male ; RNA, Messenger/analysis/metabolism ; Rats ; Rats, Wistar ; Thalidomide/*pharmacology ; Tissue Inhibitor of Metalloproteinase-1/biosynthesis/*drug effects ; Transcription Factor RelA/biosynthesis/drug effects ; Transforming Growth Factor beta1/biosynthesis/*drug effects ; Transforming Growth Factors/metabolism

OBJECTIVETo investigate the effect of nuclear factor I-C (NFI-C) on platelet-derived growth factor (PDGF)-induced up-regulation of TGF-β receptor II (TβRII) in dermal fibroblasts.

METHODSA lentiviral vector containing NFI-C sequence (Lenti-GFP-NFI-C) was transfected into a human foreskin fibroblast cell line (HFF-1). Cultured HFF-1 cells, cells transfected with Lenti-GFP-NFI-C, and cells transfected with a negative virus were stimulated with PDGF-BB, and Western blotting and RT-qPCR were used to detect the expression levels of TβRII in the treated cells.

RESULTSPDGF treatment significantly increased the expression level of TβRII in HFF-1 cells (P<0.05). The cells transfected with Lenti-GFP-NFI-C expressed a significantly lower level of TβRII than non-transfected cells in response to PDGF stimulation (P<0.05), but the negative virus showed no such inhibitory effect (P>0.05). No significant difference was found in the expression level of TβRII protein between cells transfected with Lenti-GFP-NFI-C-transfection before PDGF stimulation and the blank control cells.

CONCLUSIONNFI-C can inhibit PDGF-induced up-regulation of TβRII and thus reduce the sensitivity of the dermal fibroblasts to TGF-β.

Cell Line ; Fibroblasts ; drug effects ; Genetic Vectors ; Humans ; Lentivirus ; NFI Transcription Factors ; genetics ; Platelet-Derived Growth Factor ; pharmacology ; Protein-Serine-Threonine Kinases ; metabolism ; Proto-Oncogene Proteins c-sis ; Receptors, Transforming Growth Factor beta ; metabolism ; Transfection ; Transforming Growth Factor beta ; pharmacology ; Up-Regulation

OBJECTIVETo construct and characterize the TGF-β1, induced epithelial-mesenchymal transition (EMT) model of keloid epithelial cells in vitro, and to investigate the expression of epithelial stem cells related surface markers in keloid epithelial cells during EMT induction.

METHODSThe epithelial cells from 3 keloid samples of ears were cultured in vitro and induced by transforming growth factor betal (TGF-β1, 1 ng/ml) for 5 days, which was the experimental group, the same cells untreated were considered as the negative control group. The expressions of EMT-associated markers and regulative genes were detected using immunofluorescence staining, real-time PCR and western blot analysis. Then the surface markers of epithelial stem cells were detected using real-time PCR. Statistical significance was determined using Independent-Samples t Test, a p value less than 0. 05 was considered statistically significant.

RESULTSThe mRNA expression of transcription factor snail2 and mesenchymal-specific marker vimentin increased significantly in TGF-β1, induced keloid epithelial cells (P < 0. 05), in which snail2 increasing from 0. 91 ± 0. 23 to 1. 69 ± 0. 10, and vimentin from 5. 86 ± 2. 07 to 24. 29 ± 5. 39. Whereas the mRNA expression of epithelial-specific marker E-cadherin decreased from 1. 06 ± 0. 19 to 0. 65 ± 0. 09. The mRNA expression of CD29 and Lgr6, two surface markers of epithelial stem cells, significantly increased after induction of the TGF-β1, (P < 0. 05), from 0. 55 ± 0. 14 and 1. 61 ± 0. 31 to 1. 19 ± 0. 12 and 3. 84 t 0. 62 respectively. In induced cells, the immunofluorescence results showed staining of E- cadherin became faint, but the number of positive staining cells of vimentin increased. Western blot confirmed the protein expression of E-cadherin weakened, and the vimentin and p-Smad3 enhanced (P < 0. 05).

CONCLUSIONSTGF-β1, initiated EMT in keloid epithelial cells by inducing the up-regulation of snail2, and TGF-β1,/Smad3 signaling pathway was involved in EMT. EMT could change the phenotype of epithelial stem cells in keloid.

Biomarkers ; metabolism ; Cadherins ; genetics ; metabolism ; Epithelial Cells ; drug effects ; physiology ; Epithelial-Mesenchymal Transition ; drug effects ; physiology ; Humans ; In Vitro Techniques ; Keloid ; pathology ; RNA, Messenger ; metabolism ; Signal Transduction ; Smad3 Protein ; genetics ; metabolism ; Snail Family Transcription Factors ; Transcription Factors ; genetics ; metabolism ; Transforming Growth Factor beta1 ; metabolism ; pharmacology ; Up-Regulation ; Vimentin ; genetics ; metabolism

α-smooth muscle actin (α-SMA) and tenascin-C are stress-induced phenotypic features of myofibroblasts. The expression levels of these two proteins closely correlate with the extracellular mechanical microenvironment. We investigated how the expression of α-SMA and tenascin-C was altered in the periodontal ligament (PDL) under orthodontic loading to indirectly reveal the intrinsic mechanical microenvironment in the PDL. In this study, we demonstrated the synergistic effects of transforming growth factor-β1 (TGF-β1) and mechanical tensile or compressive stress on myofibroblast differentiation from human periodontal ligament cells (hPDLCs). The hPDLCs under higher tensile or compressive stress significantly increased their levels of α-SMA and tenascin-C compared with those under lower tensile or compressive stress. A similar trend was observed in the tension and compression areas of the PDL under continuous light or heavy orthodontic load in rats. During the time-course analysis of expression, we observed that an increase in α-SMA levels was matched by an increase in tenascin-C levels in the PDL under orthodontic load in vivo. The time-dependent variation of α-SMA and tenascin-C expression in the PDL may indicate the time-dependent variation of intrinsic stress under constant extrinsic loading.
Actins ; analysis ; drug effects ; Adult ; Animals ; Biomechanical Phenomena ; Cell Culture Techniques ; Cell Differentiation ; physiology ; Cells, Cultured ; Cellular Microenvironment ; physiology ; Humans ; Male ; Myofibroblasts ; physiology ; Orthodontic Wires ; Periodontal Ligament ; chemistry ; cytology ; Pressure ; Rats ; Rats, Sprague-Dawley ; Stress, Mechanical ; Tenascin ; analysis ; drug effects ; Time Factors ; Tooth Movement Techniques ; instrumentation ; Transforming Growth Factor beta1 ; pharmacology

OBJECTIVETo investigate the effect of sunitinib on the migration of ovarian cells and its mechanism of the negative regulation TGF-β mediated of epithelial-mesenchymal transition(EMT) by sunitinib to inhibit ovarian cancer metastasis.

METHODSThe migration of human ovarian cancer cells SKOV3 was evaluated by wound-healing and transwell assays. The effects of sunitinib on TGF-β-induced E-cadherin expression was assessed by Western-blotting, real time RT-PCR and immunofluorescence assay. The protein levels of Snail and the transcriptional activity of Smad in sunitinib-treated cells were examined by Western-blotting and SBE-luciferase assay.

RESULTSSunitinib suppressed the migration of SKOV3 cells in a concentration-dependent manner. TGF-β stimulation reduced E-cadherin protein level, which was attenuated by sunitinib. Sunitinib inhibited the up-regulation of Snail protein level induced by TGF-β treatment. The SBE reporter was constructed by linking the Smad-binding elements promoter upstream of luciferase reporter gene. A remarkable increment of transcriptional activity of Smads complexes was observed in SKOV3 cells exposed to TGF-β, which was significantly prohibited by sunitinib.

CONCLUSIONSunitinib can inhibit the migration of SKOV3 cells and attenuate the down-regulation of E-cadherin protein level induced by TGF-β. Sunitinib can abolish TGF-β-induced up-regulation of Snail protein and decrease the transcriptional activity of Smad complexes. The results indicate that sunitinib suppresses migration of ovarian cancer cells through negative modulation of TGF-β-mediated epithelial-mesenchymal transition.

Cadherins ; metabolism ; Cell Line, Tumor ; drug effects ; Cell Movement ; drug effects ; Down-Regulation ; Epithelial-Mesenchymal Transition ; Female ; Humans ; Indoles ; pharmacology ; Ovarian Neoplasms ; pathology ; Pyrroles ; pharmacology ; Smad Proteins ; metabolism ; Snail Family Transcription Factors ; Transcription Factors ; metabolism ; Transforming Growth Factor beta ; pharmacology ; Up-Regulation

OBJECTIVETo explore the effects of blocking two sites of TGF-β/Smads signaling on the formation of scar-related proteins in human skin fibroblasts.

METHODSTwo lentivirus vectors encoding soluble TGF-β receptor II (sTβRII) and mutant Smad 4-Smad 4ΔM4 were respectively transfected into human skin fibroblast cell line human foreskin fibroblast 1 (HFF-1) cells with the optimum multiplicity of infection (MOI) of 50. The protein expressions of sTβRII and Smad 4ΔM4 of the two types of transfected cells were determined by Western blotting so as to compare with those of the untransfected cells. The HFF-1 cells were divided into 6 groups as named below according to the random number table, with 6 dishes in each group, 1×10(4) cells per dish. Co-transfection group, transfected with the two previous lentivirus vectors, mixed with the ratio of 1:1 and MOI of 50, and then stimulated with 5 ng/mL TGF-β1 for 72 h; sTβRII group, transfected with lenti-sTβRII with MOI of 50, with the other treatment as above; Smad 4ΔM4 group, transfected with lenti-Smad 4ΔM4 with MOI of 50, with the other treatment as above; negative virus group, transfected with empty lentivirus vector, with the other treatment as above; positive control group, stimulated with 5 ng/mL TGF-β1 for 72 h; and blank control group, conventionally cultured without any other treatment. After stimulation, Western blotting and real-time fluorescent quantitative RT-PCR were respectively used to determine the protein and mRNA expressions of fibronectin in cells of each group. ELISA and Sircol collagen assay were respectively used to determine the protein expressions of connective tissue growth factor (CTGF) and total collagen in the cell culture supernate of each group. Data were processed with one-way analysis of variance and SNK-(q test).

RESULTS(1) HFF-1 cells transfected with lenti-sTβRII and HFF-1 cells transfected with lenti-Smad 4ΔM4 respectively expressed higher levels of sTβRII protein and Smad 4ΔM4 protein compared with those of untransfected cells, confirming that HFF-1 cells transfected with the two lentivirus vectors can efficiently express the target proteins. (2) There were statistically significant differences in the protein and mRNA expressions of fibronectin in cells of the 6 groups (with F values respectively 53.536 and 24.365, P values below 0.001). The protein and mRNA expressions of fibronectin in cells of positive control group (respectively 1.60 ± 0.18 and 1.99 ± 0.40) were similar with those of negative virus group (respectively 1.60 ± 0.15 and 1.94 ± 0.28, with q values respectively 0.091 and 0.419, P values above 0.05), and they were significantly higher than those of the rest 4 groups (with q values from 5.245 to 18.228, P values below 0.05). The protein and mRNA expressions of fibronectin in cells of co-transfection group (respectively 0.60 ± 0.05 and 0.70 ± 0.11) were significantly lower than those of sTβRII group (respectively 0.89 ± 0.13 and 1.24 ± 0.17) and Smad 4ΔM4 group (respectively 0.91 ± 0.14 and 1.28 ± 0.19, with q values from 3.964 to 4.294, P values below 0.05). (3) There were statistically significant differences in the protein expressions of CTGF and total collagen in the cell culture supernate of the 6 groups (with F values respectively 107.680 and 38.347, P values below 0.001). The protein expressions of CTGF and total collagen in the cell culture supernate of positive control group were similar with those of negative virus group (with q values respectively 1.106 and 0.491, P values above 0.05), and they were significantly higher than those of the rest 4 groups (with q values from 6.414 to 26.420, P values below 0.05). The protein expressions of CTGF and total collagen in the cell culture supernate of co-transfection group were significantly lower than those of sTβRII group and Smad 4ΔM4 group (with q values from 3.424 to 7.143, P values below 0.05).

CONCLUSIONSIn human skin fibroblasts, blockage of two sites of TGF-β/Smad signaling can reduce the expression of scar related proteins which are up-regulated by TGF-β1 to a greater extent than that of blocking one single site.

Cicatrix ; Connective Tissue Growth Factor ; Fibroblasts ; metabolism ; Genetic Vectors ; Humans ; Lentivirus ; genetics ; Protein-Serine-Threonine Kinases ; RNA, Messenger ; genetics ; Receptors, Transforming Growth Factor beta ; Signal Transduction ; drug effects ; Smad Proteins ; genetics ; metabolism ; Smad Proteins, Inhibitory ; genetics ; Transfection ; Transforming Growth Factor beta ; pharmacology ; Transforming Growth Factors