1.Inhibitory effect of retroviral vector containing anti-sense Smad4 gene on Ito cell line, LI90.
Xin-bao XU ; Xi-sheng LENG ; Zhen-ping HE ; Zhi-qing LIANG ; Kai LIN ; Yu-hua WEI ; Xin YU ; Ji-run PENG
Chinese Medical Journal 2004;117(8):1170-1177
BACKGROUNDTransforming growth factor-beta1 (TGF-beta1) exerts strong fibrogenic potential in culture-activated HSCs. Smad4 is a key intracellular mediator for the transforming growth factor-beta (TGF-beta) superfamily of growth factors. The aim of this study was to assess the effects of the antisense Smad4 gene on Ito cell line, LI90.
METHODSThe recombinant retroviral vector pLXSN-Smad4 was constructed by cloning the rat antisense Smad4 cDNA into the retroviral vector pLXSN. Retroviruses with or without the antisense gene were obtained by transfecting pLXSN-Smad4 and pLXSN vectors into PA317 cells. Human hepatic stellate cells (HSCs) LI90 were infected with these retroviruses followed by selection with G418. The expression of Smad4 was detected by Northern and Western blots. Cell biological characteristics, including cell growth curve, 3H-TdR and 3H-proline uptake by HSCs and the production of extracellular matrix were assessed.
RESULTSmRNA and protein expressions of Smad4 in LI90 cells transfected with retrovirus containing the antisense Smad4 gene were much lower than those in LI90 cells transfected with empty vector or parental LI90 cells. Cells hypoexpressing the Smad4 gene exhibited a slower rate of growth, a lower uptake of 3H-TdR and 3H-proline (P < 0.01), and smaller production of th extracellular matrix, compared with parental LI90 cells and cells transfected with empty retrovirus.
CONCLUSIONSThe antisense Smad4 gene can suppress the expression of the Smad4 gene, reduce endogenous production of Smad4 mRNA and protein, block TGF-beta1 signaling pathway, inhibit activation of Ito cells, obstruct the growth of Ito cells, decrease the production of the extracellular matrix (ECM). Our results may provide a basis for the development of antifibrotic gene therapy.
Cell Line ; DNA, Antisense ; pharmacology ; DNA-Binding Proteins ; antagonists & inhibitors ; genetics ; Genetic Therapy ; Genetic Vectors ; genetics ; Humans ; Liver Cirrhosis ; therapy ; Retroviridae ; genetics ; Smad4 Protein ; Trans-Activators ; antagonists & inhibitors ; genetics ; Transfection ; Transforming Growth Factor beta ; physiology ; Transforming Growth Factor beta1
2.Analysis of transforming growth factor β signaling in chronic rhinosinusitis.
Yun-Chuan LI ; Yun-Song AN ; Tong WANG ; Hong-Rui ZANG
Chinese Medical Journal 2013;126(17):3340-3343
BACKGROUNDIt has been reported that there is a significant difference in the local tissue concentration of transforming growth factor (TGF)-β1 between chronic rhinosinusitis without nasal polyps (CRSsNP) and chronic rhinosinusitis without nasal polyps (CRSwNP) patients. TGF-β has been reported to play an important role in regulating epithelial cell repair in lower airway remodeling and may be a critical factor involved in the remodeling process of chronic rhinosinusitis (CRS).
METHODSEthmoidal mucosal samples collected from CRS and healthy control patients were analyzed for TGF-β1, TGF-β receptor I, TGF-β receptor II, Smad3, phospho-Smad3, Smad7, and Smad anchor for receptor activation by Western blotting analysis. The proliferation of sinonasal epithelial cells at baseline and after TGF-β1 and/or EGF stimulation was evaluated by the MTT assay.
RESULTSIn CRSsNP, TGF-β1, TGF-β receptor I, TGF-β receptor II, and Smad3 protein levels were significantly higher than controls. In CRSwNP, TGF-β1, Smad3, and pSmad3 protein levels were significantly lower than controls. Smad7 protein was significantly higher in CRS than controls. In vitro experiments demonstrated that the baseline proliferation levels of sinonasal epithelial cells were lower in CRS than controls.
CONCLUSIONSCRSwNP is characterized by a lower level of TGF-signaling compared with the control. In CRSsNP, although the upstream signaling of TGF-β was enhanced, the high Smad7 protein expression may restrain the downstream signaling components (e.g., pSmad3) and the TGF-β antiproliferative effect on sinonasal epithelium. The difference in the local tissue concentration of TGF-β1 between CRSsNP and CRSwNP patients did not result in significant differences in epithelial proliferation.
Adult ; Aged ; Benzamides ; pharmacology ; Cells, Cultured ; Dioxoles ; pharmacology ; Female ; Humans ; Intracellular Signaling Peptides and Proteins ; metabolism ; Male ; Middle Aged ; Protein-Serine-Threonine Kinases ; antagonists & inhibitors ; metabolism ; Receptors, Transforming Growth Factor beta ; antagonists & inhibitors ; metabolism ; Serine Endopeptidases ; metabolism ; Signal Transduction ; drug effects ; Sinusitis ; metabolism ; Smad3 Protein ; metabolism ; Smad7 Protein ; metabolism ; Transforming Growth Factor beta ; metabolism ; Transforming Growth Factor beta1 ; antagonists & inhibitors ; metabolism ; Young Adult
3.Inhibiting effect of short hairpin RNA on expression of transforming growth factor-beta1 in human peritoneal mesothelial cells induced by peritoneal dialysis solution.
Fu-you LIU ; Guang-hui LING ; Hong LIU ; You-ming PENG ; Ying-hong LIU ; Shao-bin DUAN
Chinese Medical Journal 2005;118(18):1552-1556
Cell Line
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Dialysis Solutions
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adverse effects
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Epithelial Cells
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metabolism
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Fibrosis
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Humans
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Peritoneal Dialysis, Continuous Ambulatory
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adverse effects
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Peritoneum
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metabolism
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pathology
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RNA Interference
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RNA, Messenger
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analysis
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RNA, Small Interfering
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pharmacology
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Transforming Growth Factor beta
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antagonists & inhibitors
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genetics
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Transforming Growth Factor beta1
4.Inhibitory effects of antisense TGF beta1 on in vitro and in vivo proliferation of human bladder cancer cells.
Xin YAO ; Ji-wu CHANG ; Wen-lu LI ; Rui-fang NIU ; Bao-cun SUN ; Teng-xiang MA
Chinese Journal of Oncology 2004;26(1):18-21
OBJECTIVETo investigate the inhibitory effects of antisense TGF beta1 on proliferation of human bladder transitional cell carcinoma in vitro and in vivo.
METHODSHuman bladder carcinoma cell line EJ was transfected with pRevT beta-AS, a replication defective retroviral vector carried antisense TGF beta1 fragment. The growth of the transfected cells was observed in vitro and in vivo. TGF beta1 mRNA expression and protein expression were detected by RT-PCR and ELISA. The proliferative activity was evaluated by immunohistochemistry method. The ultrastructure of cells was observed by image analysis system and electron microscopy. Cell cycle was determined by flow cytometry.
RESULTSThe expression of TGF beta1 mRNA and protein in EJ cells was inhibited by pRevT beta-AS, G(1) to S transition was restrained in cell cycle and cell proliferation decreased in vitro. The tumorigenesis and growth of EJ cells and DNA heteroploidy were reduced by antisense TGF beta1 in vivo.
CONCLUSIONTGF beta1 plays a role in vitro proliferation and in vivo growth of bladder transitional cell carcinoma.
Animals ; Cell Division ; drug effects ; Cell Line, Tumor ; Female ; Humans ; Mice ; Mice, SCID ; RNA, Antisense ; therapeutic use ; Transforming Growth Factor beta ; antagonists & inhibitors ; genetics ; Transforming Growth Factor beta1 ; Urinary Bladder Neoplasms ; drug therapy ; pathology
5.Effect of Rhein on the development of hepatic fibrosis in rats.
Mei-zi GUO ; Xiao-sheng LI ; Ding-ming SHEN ; Xiao-qin GUAN ; Hai-rong XU ; Jian GAO
Chinese Journal of Hepatology 2003;11(1):26-29
OBJECTIVETo investigate the effect of rhein on the development of hepatic fibrosis.
METHODSThe animal models were made with carbon tetrachloride (CCl(4)) mixed with vegetable oil (3/2, v/v), which was injected subcutaneously twice a week for 6 weeks, and with 5% ethanol for free drinking water. At the same time, Rhein was administrated at the dose of 25 mg/kg or 100 mg/kg once a day for 6 weeks. The changes of both biochemical markers, such as the levels of alanine aminotransferase (ALT), hyaluronic acid (HA), procollagen type III (PCIII) in serum and SOD, malondialdehyde (MDA) in liver, and related histopathological parametres were determined.
RESULTSCompared with the model group, there were three kinds of changes in the larger quantity of rhein treated group. (1) The levels of ALT, HA, PCIII in serum and MDA in liver homogenate were decreased significantly (from 150 U/L +/- 16 U/L to 78 U/L +/- 18 U/L, 321 microg/L +/- 97 microg/L to 217 microg/L +/- 75 microg/L, 31 microg/L +/- 14 microg/L to 16 microg/L +/- 6 microg/L and 3.67 nmol/mg +/- 0.68 nmol/mg to 1.88 nmol/mg +/- 0.34 nmol/mg, respectively, t > or 2.977, P<0.01). However the level of SOD in liver was increased (from 62.45 NU/mg +/- 8.74 NU/mg to 91.26 NU/mg +/- 14.04 NU/mg, t=4.453, P<0.01). (2) The expressions of transforming growth factor beta 1 (TGF-beta 1) and alpha-smooth muscle actin (alpha-SMA) in liver were markedly reduced (P<0.05 and P<0.01). (3) The collagen staining positive area was decreased and the grade of fibrosis was reduced significantly in liver (P<0.05 and P<0.01).
CONCLUSIONRhein can protect hepatocyte from injury and prevent the progress of hepatic fibrosis in rats, which may associate with that rhein plays a role in antioxidation, anti-inflammation, inhibiting the expression of TGF-beta1 and suppressing the activation of hepatic stellate cells (HSCs).
Animals ; Anthraquinones ; pharmacology ; therapeutic use ; Anti-Inflammatory Agents ; pharmacology ; Antioxidants ; pharmacology ; Collagen ; analysis ; Liver ; drug effects ; pathology ; Liver Cirrhosis, Experimental ; drug therapy ; metabolism ; pathology ; Male ; Rats ; Rats, Wistar ; Transforming Growth Factor beta ; antagonists & inhibitors ; Transforming Growth Factor beta1
6.Substance P up-regulates the TGF-beta 1 mRNA expression of human dermal fibroblasts in vitro.
Dahai HU ; Bi CHEN ; Xiongxinag ZHU ; Ke TAO ; Chaowu TANG ; Jianbo WANG
Chinese Journal of Plastic Surgery 2002;18(4):234-236
OBJECTIVETo investigate the role of substance P in the formation of hypertrophic scar.
METHODSDermal fibroblasts derived from human normal skin were cultured with substance P alone or together with selective non-peptide NK-1 tachykinin antagonist, L-703, 606 oxalate salt. The effect of substance P on proliferation of fibroblasts was measured by MTT assay. Furthermore, the TGF-beta 1 mRNA expression in the fibroblasts was determined by in situ hybridization and image analysis.
RESULTSSubstance P enhanced fibroblast proliferation dose-dependently, which showed the maximum rate when the concentration of substance P was 25 ng/ml or higher in the culture media. By 48 hours cultured with 25 ng/ml of substance P, the fibroblasts expressed TGF-beta 1 mRNA more significantly than the fibroblasts without substance P. The effects of substance P on both fibroblast proliferation and TGF-beta 1 mRNA expression could be antagonized by L-703, 606 oxalate salt.
CONCLUSIONThe results suggest that substance P may play an important role in phenotype changes of fibroblasts in skin scarring.
Cell Division ; Cells, Cultured ; Dermis ; cytology ; Fibroblasts ; cytology ; drug effects ; metabolism ; Gene Expression ; drug effects ; Neurokinin-1 Receptor Antagonists ; Quinuclidines ; pharmacology ; RNA, Messenger ; Substance P ; metabolism ; pharmacology ; Transforming Growth Factor beta ; genetics ; Transforming Growth Factor beta1 ; Up-Regulation
7.The experimental study on antisense TGF-beta 1 in inhibiting scar formation in III degree burn wound.
Xiaoping LUO ; Hanfang ZHAO ; Youzhang ZHANG
Chinese Journal of Plastic Surgery 2002;18(2):89-91
OBJECTIVETo investigate the effect of antisense TGF-beta 1 in inhibiting scar formation during wound healing.
METHODSSD rats were divided into three groups after skin burn: group one was treated with antisense TGF-beta 1 oligonucleotide; group two was treated with antisense TGF-beta 1 recombinant plasmid and the control group. In different time, RT-PCR and immunohistochemistry were used to verify the expression of TGB beta 1 mRNA and protein. Type I Collagen mRNA expression was determined by hybridization in situ. Inflammatory reaction and collagen distribution were observed pathologically.
RESULTSIn the groups received antisense ODN and recombinant plasmid, the expression of TGF-beta 1 mRNA and protein reduced during 14 days after burn. In the control group, type I collagen mRNA began to express at the second week and reached a peak at the fourth week, while the antisense groups kept low expression. The antisense group also showed mild inflammatory reaction and less synthesis of collagen.
CONCLUSIONAntisense TGF-beta 1 could prevent the scar formation during wound healing.
Animals ; Burns ; drug therapy ; physiopathology ; Cicatrix ; prevention & control ; Male ; Oligonucleotides, Antisense ; therapeutic use ; Rats ; Rats, Sprague-Dawley ; Transforming Growth Factor beta ; antagonists & inhibitors ; Transforming Growth Factor beta1 ; Wound Healing ; physiology
8.Expression of 5-hydroxytamine receptors in hepatic stellate cell and action of 5-hydroxytamine on biological characteristics of hepatic stellate cell.
Tao LI ; Xi-sheng LENG ; Shan-geng WENG ; Ji-run PENG ; Yu-hua WEI ; Dong-cheng MOU ; Wan-xiang WANG ; Ji-ye ZHU
Chinese Journal of Surgery 2003;41(3):175-179
OBJECTIVETo investigate the expression of 5-hydroxytamine receptors in hepatic stellate cells HSCs and action of 5-hydroxytamine on biological characteristics of HSC.
METHODSLiver ex vivo perfusion of collagenase and density gradient centrifugation were used to isolate hepatic stellate cell. RT-PCR was used to detect the expression of 5-hydroxytamine receptor subtypes 1A, 2A, 2B and 3. Western blot hybridization was used to elucidate the effect of 5-hydroxytamine and its 2A receptor antagonist ketanserin and 3 receptor antagonist ondanosetron on expression of transforming growth factor-beta1 (TGF-beta1) and Smad4 in HSC. HSCs were cultured on silicone membrane. The effect of 5-hydroxytamine, ketanserin and ondanosetron on cell contraction were studied.
RESULTSHSC expressed 5-hydroxytamine receptors subtypes 1A, 2A and 2B. 5-hydroxytamine significantly increased the expression of TGF-beta1 and Smad4 in HSC (P < 0.05). This was antagonized by ketanserin, not by ondanosetron. 5-hydroxytamine induced cell contraction in a dose-dependant manner. Ketanserin antagonized this action, but ondanosetron did not.
CONCLUSIONSHSCs express 5-hydroxytamine receptors. 5-hydroxytamine could affect the biological characteristics of HSC through its receptor mediation, and may play a role in the pathogenesis of liver cirrhosis and portal hypertension.
Animals ; Cells, Cultured ; Hypertension, Portal ; etiology ; Liver ; chemistry ; cytology ; Liver Cirrhosis ; etiology ; Male ; Rats ; Rats, Wistar ; Receptors, Serotonin ; analysis ; physiology ; Serotonin ; pharmacology ; Serotonin Antagonists ; pharmacology ; Transforming Growth Factor beta ; physiology ; Transforming Growth Factor beta1
9.Modifiers of TGF-beta1 effector function as novel therapeutic targets of pulmonary fibrosis.
Chang Min LEE ; Jin Wook PARK ; Won Kyung CHO ; Yang ZHOU ; Boram HAN ; Pyoung Oh YOON ; Jeiwook CHAE ; Jack A ELIAS ; Chun Geun LEE
The Korean Journal of Internal Medicine 2014;29(3):281-290
Pulmonary fibrosis is a fatal progressive disease with no effective therapy. Transforming growth factor (TGF)-beta1 has long been regarded as a central mediator of tissue fibrosis that involves multiple organs including skin, liver, kidney, and lung. Thus, TGF-beta1 and its signaling pathways have been attractive therapeutic targets for the development of antifibrotic drugs. However, the essential biological functions of TGF-beta1 in maintaining normal immune and cellular homeostasis significantly limit the effectiveness of TGF-beta1-directed therapeutic approaches. Thus, targeting downstream mediators or signaling molecules of TGF-beta1 could be an alternative approach that selectively inhibits TGF-beta1-stimulated fibrotic tissue response while preserving major physiological function of TGF-beta1. Recent studies from our laboratory revealed that TGF-beta1 crosstalk with epidermal growth factor receptor (EGFR) signaling by induction of amphiregulin, a ligand of EGFR, plays a critical role in the development or progression of pulmonary fibrosis. In addition, chitotriosidase, a true chitinase in humans, has been identified to have modulating capacity of TGF-beta1 signaling as a new biomarker and therapeutic target of scleroderma-associated pulmonary fibrosis. These newly identified modifiers of TGF-beta1 effector function significantly enhance the effectiveness and flexibility in targeting pulmonary fibrosis in which TGF-beta1 plays a significant role.
Animals
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Drug Design
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Hexosaminidases/antagonists & inhibitors/metabolism
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Humans
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Lung/*drug effects/metabolism/pathology
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Molecular Targeted Therapy
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Pulmonary Fibrosis/*drug therapy/metabolism/pathology
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Receptor Cross-Talk
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Receptor, Epidermal Growth Factor/antagonists & inhibitors/metabolism
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Receptors, Transforming Growth Factor beta/antagonists & inhibitors/metabolism
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Signal Transduction
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Transforming Growth Factor beta1/*antagonists & inhibitors/metabolism
10.Effects of pioglitazone on TGFbeta1 expression in ischemia/reperfusion injury myocardium of rats.
Hao WANG ; Ping YE ; Yang LI ; Zong-Bin LI ; Lin WANG
Chinese Journal of Applied Physiology 2013;29(1):1-4
OBJECTIVETo investigate the effects of pioglitazone on transforming growth factor beta1 (TGFbeta1) expression in ischemia/reperfusion injury myocardium of rats.
METHODSThirty SD rats were randomly divided into five groups (n = 6): ischemia/reperfusion group, pioglitazone 5 mg/(kg x d) group, pioglitazone 10 mg/(kg x d) group, pioglitazone 20 mg/(kg x d) group and pioglitazone 20 mg/(kg x d) + peroxisome proliferator-activated receptor gamma (PPARgamma) specific antagonist GW9662 group. Left anterior descending coronary artery of rats were ligated for 30 min and reperfused for 120 min to establish the model of ischemia/reperfusion in vivo. RT-PCR was performed to detect the expression of TGFbeta1 mRNA. Western blot was performed to detect the expression of TGFbeta1 protein.
RESULTSMyocardial apoptosis was significantly suppressed by pioglitazone. Pioglitazone upregulated TGFPbeta1 expression both in mRNA and protein level. GW9662 reversed the inhibition of myocardial apoptosis and the upregulation of TGFbeta1 expression by pioglitazone.
CONCLUSIONPioglitazone can inhibit the myocardial apoptosis induced by ischemia/reperfusion. Pioglitazone may protect the myocardium from ischemia/reperfusion via upregulation of TGFbeta1. This protection may be mediated by PPARgamma.
Anilides ; pharmacology ; Animals ; Apoptosis ; Male ; Myocardial Reperfusion Injury ; metabolism ; Myocardium ; metabolism ; PPAR gamma ; antagonists & inhibitors ; Rats ; Rats, Sprague-Dawley ; Thiazolidinediones ; pharmacology ; Transforming Growth Factor beta1 ; metabolism