The protein expression of cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-activated Cl(-) channel, in ovarian stimulated premature female rat ovary during a cycle of follicle development and corpus luteum formation was investigated. Animals were injected with 10 U pregnant Mare's serum gonadotropin (PMSG) and subsequently 10 U hCG 48 h later. Time-dependent immunohistochemistry and Western blotting experiments were performed before and 24, 48, 72 h after hCG treatment. The immunohistochemistry revealed that administration of PMSG stimulated the CFTR expression in thecal cell layer and granulosa cell layer of mature follicles 48 h post injection, coincident with the PMSG-induced peak in follicular estradiol. However, the expression of CFTR in the granulose lutein cell layer and thecal lutein cell layer was time-dependently reduced following hCG injection, in accordance with the gradually increased progestogen level during luteum corpus formation. Western blotting analysis demonstrated that rat ovarian tissue expressed the special CFTR band at 170 kD. It is concluded that cAMP-dependent Cl(-) channels are involved in regulation of follicle development and luteum formation.
Connective Tissue Growth Factor/genetics ; Connective Tissue Growth Factor/*metabolism ; Connective Tissue Growth Factor/*physiology ; Corpus Luteum/growth & development ; Ovarian Follicle/growth & development ; Ovary/*metabolism ; Rats, Wistar

OBJECTIVETo investigate whether aristolochic acid can be transported into human kidney proximal tubular cell (HKC) and its potential mechanism.

METHODSIntracellular aristolochic acid was measured by liquid chromatography-tandem mass spectrometry. The release of lactate dehydrogenase (LDH) induced by aristolochic acid in the presence of organic anion transporter inhibitor (probenecid) or organic cation transporter inhibitor (tetraethylammonium) was evaluated. The effects of probenecid on aristolochic acid induced connective tissue growth factor (CTGF) mRNA and protein expression were also examined by real time polymerase chain reaction and Western blot, respectively.

RESULTSAristolochic acid was detected in the suspension of the denatured HKC after incubation with aristolochic acid sodium salt. The release of LDH from HKC, which was induced by 60 mg/L aristolochic acid sodium salt, was significantly inhibited by 1 mmol/L probenecid (P < 0.01), but not by 1 mmol/L tetraethylammonium. The increased CTGF mRNA and protein expression in HKC stimulated by 40 mg/L aristolochic acid sodium salt was significantly down-regulated by 1 mmol/L probenecid (P < 0.05), with an inhibition rate of 16% and 21%, respectively.

CONCLUSIONAristolochic acid can be transported into HKC by organic anion transport system, and then exerts its biological effects.

Aristolochic Acids ; metabolism ; Connective Tissue Growth Factor ; metabolism ; Epithelial Cells ; metabolism ; Humans ; Kidney ; physiology ; Organic Anion Transporters ; metabolism

Animals ; Arginine Vasopressin ; physiology ; Connective Tissue Growth Factor ; physiology ; Glomerular Mesangium ; pathology ; Humans ; Integrin alpha1beta1 ; physiology ; Kidney Diseases ; etiology ; Proto-Oncogene Protein c-ets-1 ; physiology

Liver fibrosis is a common pathological process for chronic liver injury caused by multiple etiological factors and an inevitable phase leading to liver cirrhosis. According to the previous studies, hesperidin (HDN) shows a very good protective effect on CCl4-induced chemical hepatic fibrosis in rats. In this experiment, based on the findings of the previous studies, a platelet-derived growth factor (PDGF)-induced HSC-T6 model was established to observe the inhibitory effect of HDN on HSC-T6 proliferation. The ELISA method was adopted to detect the content of collagen I in HSC-T6 supernatant. Transforming growth factor (TGF)-beta1, Smad2, Smad3, Smad7 and connective tissue growth factor (CTGF) mRNA expressions were measured by RT-PCR; TGF-beta1 and CT-GF protein expressions in HSC-T6 were determined by Western blot, in order to study HDN's effect on TGF-beta1 signaling pathway in HSC and its potential action mechanism. The results demonstrated that HDN could notably improve HSC-T6 proliferation, Collagen I growth and TGF-beta1, Smad2, Smad3 and CTGF mRNA.expressions. After being intervened with HDN, it could notably inhibit HSC-T6 proliferation and Collagen I growth, reduce TGF-beta1, Smad2, Smad3 and CTGF mRNA and TGF-beta1, CTGF protein expressions and increase Smad7 mRNA expression. HDN's antihepatic fibrosis effect may be related to the inhibition of HSC proliferation and activation by modulating TGF-beta/Smad signaling pathway.
Animals ; Cell Proliferation ; drug effects ; Cells, Cultured ; Connective Tissue Growth Factor ; physiology ; Hesperidin ; pharmacology ; Platelet-Derived Growth Factor ; pharmacology ; Rats ; Signal Transduction ; drug effects ; Smad Proteins ; physiology ; Transforming Growth Factor beta1 ; physiology

BACKGROUNDSmall noncoding microRNAs regulate gene expression in cardiac development and disease and have been implicated in the aging process and in the regulation of extracellular matrix proteins. However, their role in age-related cardiac remodeling and atrial fibrillation (AF) was not well understood. The present study was designed to decipher molecular mechanisms underlying age-related atrial structural remodeling and AF.

METHODSThree groups of dogs were studied: adult and aged dogs in sinus rhythm and with persistent AF induced by rapid atrial pacing. The expressions of microRNAs were measured by quantitative real-time polymerase chain reaction. Pathohistological and ultrastructural changes were tested by light and electron microscopy. Apoptosis index of myocytes was detected by TUNEL.

RESULTSSamples of atrial tissue showed the abnormal pathohistological and ultrastructural changes, the accelerated fibrosis, and apoptosis with aging and/or in AF dogs. Compared to the adult group, the expressions of microRNAs-21 and -29 were significantly increased, whereas the expressions of microRNAs-1 and -133 showed obvious downregulation tendency in the aged group. Compared to the aged group, the expressions of microRNAs-1, -21, and -29 was significantly increased in the old group in AF; contrastingly, the expressions of microRNA-133 showed obvious downregulation tendency.

CONCLUSIONThese multiple aberrantly expressed microRNAs may be responsible for modulating the transition from adaptation to pathological atrial remodeling with aging and/or in AF.

Age Factors ; Animals ; Apoptosis ; Atrial Fibrillation ; etiology ; Atrial Remodeling ; Connective Tissue Growth Factor ; physiology ; Dogs ; Electrocardiography ; Fibrosis ; In Situ Nick-End Labeling ; MicroRNAs ; analysis ; physiology ; Myocardium ; pathology ; ultrastructure

OBJECTIVELung fibrosis is the ultimate outcome of hyperoxia-induced chronic lung diseases (CLD) and connective tissue growth factor (CTGF) is correlated with fibrosis. This study investigated the role of CTGF in hyperoxia-induced CLD.

METHODSFifty premature rats were randomly exposed to hyperoxia (Model group) and to room air (Control group) (n = 25 each). CLD was induced by hyperoxia exposure. The expression of CTGF was detected by immunohistochemical method at 1, 3, 7, 14 and 21 days after exposure. The severity of pulmonary fibrosis was evaluated.

RESULTSIn the Control group there was a slight expression of CTGF in the bronchial epithelial cells and vascular endothelial cells. The intensity and range of CTGF expression in the Model group were similar to the Control group on days 1, 3 and 7 of exposure. On the 14th day, CTGF was expressed in some alveolar epithelial cells, fibroblasts and interstitial cells, and the intensity of CTGF expression increased significantly compared with the Control group, with the IODT of CTGF of 10.53 +/- 4.24 vs 5.58 +/- 1.18 (P < 0.01). On day 21, the expression intensity and range of CTGF in the Model group (IODT: 16.61 +/- 5.39) increased compared with that of Control group (P < 0.01). The expression of CTGF was correlated with the degree of fibrosis in the Model group on days 14 and 21 (r = 0.903, r = 0.926 respectively, P < 0.01).

CONCLUSIONSThe CTGF expression increased with the time of hyperoxia exposure and the development of fibrosis. CTGF is closely related to the development of hyperoxia-induced pulmonary fibrosis.

Animals ; Chronic Disease ; Connective Tissue Growth Factor ; Female ; Hyperoxia ; complications ; Immediate-Early Proteins ; analysis ; physiology ; Immunohistochemistry ; Intercellular Signaling Peptides and Proteins ; analysis ; physiology ; Lung ; chemistry ; pathology ; Pulmonary Fibrosis ; etiology ; pathology ; Rats ; Rats, Sprague-Dawley ; Transforming Growth Factor beta ; physiology

Animals ; Collagen Type I ; genetics ; Connective Tissue Growth Factor ; Corneal Injuries ; Fibronectins ; analysis ; Immediate-Early Proteins ; analysis ; genetics ; physiology ; Immunohistochemistry ; Intercellular Signaling Peptides and Proteins ; analysis ; genetics ; physiology ; RNA, Messenger ; analysis ; Rabbits ; Signal Transduction ; physiology ; Smad Proteins ; physiology ; Transforming Growth Factor beta ; analysis ; genetics ; physiology ; Transforming Growth Factor beta1 ; Wound Healing ; physiology

The role of serum and glucocorticoid-induced kinase 1 (SGK1) pathway in the connective tissue growth factor (CTGF) expression was investigated in cultured human mesangial cells (HMCs) under high glucose. By using RT-PCR and Western blot, the effect of SGK1 on the CTGF expression in HMCs under high glucose was examined. Overexpression of active SGK1 in HMCs transfected with pIRES2-EGFP-S422D hSGK1 (SD) could increase the expression of phosphorylated SGK1 and CTGF as compared with HMCs groups transfected with pIRES2-EGFP (FP) under high glucose or normal glucose. Overexpression of inactive SGK1 in HMCs transfected with pIRES2-EGFP-K127N hSGK1 (KN) could decrease phosphorylated SGK1 and CTGF expression as compared with HMCs groups transfected with FP under high glucose. In conclusion, these results suggest that high glucose-induced CTGF expression is mediated through the active SGK1 in HMCs.
Cells, Cultured ; Connective Tissue Growth Factor/genetics ; Connective Tissue Growth Factor/*metabolism ; Glucose/*pharmacology ; Immediate-Early Proteins/metabolism ; Immediate-Early Proteins/*physiology ; Mesangial Cells/cytology ; Mesangial Cells/*metabolism ; Protein-Serine-Threonine Kinases/metabolism ; Protein-Serine-Threonine Kinases/*physiology ; Signal Transduction/drug effects

OBJECTIVELipoxin A(4) is formed by the metabolism of arachidonic acid. Anti-inflammatory and anti-proliferative effect of lipoxin A(4) has been shown in many human diseases. Recently, as a novel high affinity receptor for ligand lipoxin A(4), Lipoxin A(4) receptor-like protein (LRLP) has been identified. Currently close attention is paid to the important contribution of connective tissue growth factor (CTGF) in lung fibrosis. The purpose of the study was to transfect LRLP gene into human lung fibroblasts and investigate the mechanism of its enhancing antagonistic effect of Lipoxin A(4) on human lung fibroblasts proliferation induced by connective tissue growth factor.

METHODSEukaryocytic expression vector pEGFP/LRLP which contained LRLP and green fluorescence protein fusion gene (GFP) was constructed and transfected into human lung fibroblasts (HLF). After selecting with G418, HLF/LRLP cell clone which stably expressed LRLP/GFP fusion protein was isolated and characterized by the laser scanning confocal microscope. Cultured HLF and HLF/LRLP were stimulated for 24 h with CTGF (1 microg/ml) in the presence and absence of pretreatment of Lipoxin A(4) (10.0 nmol/L) for 30 min. Inhibition of cell proliferation was determined by MTT assay. Cell cycle analysis was performed by flow cytometry. Western blot was used to detect the expression of cyclin D(1) protein. Electrophoretic mobility shift assay (EMSA) was employed to detect the DNA binding activity of STAT(3).

RESULTS(1) HLF/LRLP cell clone which stably expressed LRLP and GFP fusion protein was successfully obtained. (2) Proliferation of HLF and HLF/LRLP was induced by 1 microg/ml CTGF. Pretreatment with 10 nm Lipoxin A(4) inhibited the proliferation of HLF and HLF/LRLP. And the inhibitory rate of HLF/LRLP was significantly higher than that of HLF [(54.1 +/- 4.2)%, (21.2 +/- 3.7)%, P < 0.05]. (3) The flow cytometry analysis showed that compared with HLF, more HLF/LRLP were arrested at G(0)/G(1) phase in the presence of pretreatment of Lipoxin A(4). [(76.3 +/- 3.5)%, (60.8 +/- 2.0)%, P < 0.05]. (4) Ten nmol/L Lipoxin A(4) antagonized CTGF induced increase of cyclin D(1) protein expression in HLF and HLF/LRLP. And its antagonistic effect on HLR/LRLP was stronger than that on HLF (P < 0.05). (5) Ten nmol/L Lipoxin A(4) antagonized CTGF induced increase of STAT(3) DNA binding activity, and its antagonistic effect on HLF/LRLP was more powerful than that on HLF (P < 0.05).

CONCLUSIONSTransfection of Lipoxin A(4) receptor-like protein gene enhanced the inhibitory effect of Lipoxin A(4) on human lung fibroblasts proliferation induced by CTGF. Its mechanism might be related to regulation of cyclin D(1) protein expression and STAT(3) DNA binding activity.

Connective Tissue Growth Factor ; antagonists & inhibitors ; Cyclin D1 ; analysis ; DNA ; metabolism ; Fibroblasts ; cytology ; drug effects ; Humans ; Lipoxins ; pharmacology ; Lung ; cytology ; drug effects ; Receptors, Formyl Peptide ; genetics ; physiology ; Receptors, Lipoxin ; genetics ; physiology ; STAT3 Transcription Factor ; metabolism ; Transfection

BACKGROUND/AIMS: The study of liver fibrogenesis by hepatitis C virus (HCV) has been limited due to the lack of an efficiency in vitro culture systems. In the present study, we investigated whether or not HCV core protein is directly related to liver fibrogenesis through stimulation of hepatic stellate cells (HSC). METHODS: Human and rat HSC were isolated and we established an in vitro co-culture system of a stable HepG2-HCV core cell line which was transfected with HCV core gene and primary HSC. We performed immunocytochemical staining and Western and Northern blot analysis in the stimulated HSC by HCV ocre protein to identify the expression of transforming growth factor beta1 (TGF-beta1), transforming growth factor beta receptor II (TGFbeta R II), alpha-smooth muscle actin (alpha-SMA) and connective tissue growth factor (CTGF). The expression of matrix metaloprotinase-2 (MMP-2) and collagen type I (Col I) in the culture media were measured by zymogram and ELISA, respectively. RESULTS: The expression of TGF-beta1 and CTGF was significantly higher in the stable HepG2-HCV core cell line than in HepG2 cells. Furthermore, the makers related to fibrosis such as alpha-SMA, TGF-beta1, Col I, TGFRII and MMP-2 were highly experssed in the co-culture of stable HepG2-HCV core with HSC. CONCLUSIONS: HCV core protein may play a direct role in the fibrogenesis of chronic liver disease with HCV infection.
Actins/metabolism ; Animals ; Cell Line, Tumor ; Coculture Techniques ; Connective Tissue Growth Factor ; Fibrosis ; Hepatitis C Antigens/*physiology ; Humans ; Immediate-Early Proteins/metabolism ; Immunoblotting ; Immunohistochemistry ; Intercellular Signaling Peptides and Proteins/metabolism ; Liver/metabolism/*pathology ; Protein-Serine-Threonine Kinases ; Rats ; Rats, Sprague-Dawley ; Receptors, Transforming Growth Factor beta/metabolism ; Transforming Growth Factor beta/metabolism ; Transforming Growth Factor beta1 ; Viral Core Proteins/*physiology