OBJECTIVETo investigate the effect of dracorhodin perchlorate (DP) on inhibiting high glucose-induced serum and glucocorticoid induced protein kinase 1 (SGK1) and fibronectin (FN) expression in human mesangial cells (HMC), and its mechanism of prevention and treatment on renal fibrosis in diabetic nephropathy (DN) .

METHODThe HMC were divided into normal glucose group (NG group, 5.5 mmol x L(-1) D-glucose), normal glucose +low DP group (NG + LDP group, 5.5 mmol x L(-1) D-glucose +7.5 micromol x L(-1) DP), normal glucose +high DP group (NG + HDP group, 5.5 mmol x L(-1) D-glucose + 15 micromol x L(-1) DP), high glucose group (HG group,25 mmol x L(-1) D-glucose), high glucose +low DP group (HG + LDP group, 25 mmol x L(-1) D-glucose + 7.5 micromol x L(-1) DP)and high glucose +high DP group (HG +HDP group, 25 mmol x L(-1) D-glucose + 15 micromol x L(-1) DP). Each group was examined at 24 hours. The levels of SGK1 and FN mRNA was detected by real-time fluorescence quantitative PCR,and the expression of SGK1 and FN protein was detected by Western blot or indirect immunofluorescence.

RESULTA basal level of SGK1 and FN in HMC were detected in NG group, and the level of SGK1 and FN mRNA and protein were not evidently different compared to that of NG group adding 7.5 micromol x L(-1) DP for 24 hours. On the other hand, the levels of SGK1 and FN mRNA and protein were obviously decreased by adding 15 micromol x L(-1) DP for 24 hours. Compared to NG group, the levels of SGK1 and FN mRNA and protein were increased in HG group after stimulating for 24 hours (P < 0.01). Compared to HG group, the level of SGK1 and FN mRNA and protein were evidently reduced in HG + LDP and HG + HDP groups by adding 7.5 micromol x L(-1) DP and 15 micromol x L(-1) DP for 24 hours (P < 0.01).

CONCLUSIONDracorhodin perchlorate can inhibit high glucose-induced serum and glucocorticoid induced protein kinase 1 (SGK1) and fibronectin(FN) expression in human mesangial cells, and this may be part of the mechanism of preventing and treating renal fibrosis of DN.

Benzopyrans ; pharmacology ; Cell Line ; Diabetic Nephropathies ; drug therapy ; enzymology ; genetics ; metabolism ; Down-Regulation ; drug effects ; Drugs, Chinese Herbal ; pharmacology ; Fibronectins ; biosynthesis ; genetics ; Gene Expression ; drug effects ; Glucose ; metabolism ; Humans ; Immediate-Early Proteins ; genetics ; metabolism ; Mesangial Cells ; drug effects ; enzymology ; metabolism ; Perchlorates ; pharmacology ; Protein-Serine-Threonine Kinases ; genetics ; metabolism

Co-infection of herpes simplex virus type 1 (HSV-1) and human cytomegalovirus (HCMV) is not uncommon in immunocompromised hosts. Importantly, organ transplant recipients concurrently infected with HSV-1 and HCMV have a worse clinical outcome than recipients infected with a single virus. However, factors regulating the pathologic response in HSV-1, HCMV co-infected tissues are unclear. We investigated the potential biologic role of HCMV gene product immediate early 1 (IE1) protein in HSV-1-induced syncytial formation in U373MG cells. We utilized a co-infection model by infecting HSV-1 to U373MG cells constitutively expressing HCMV IE1 protein, UMG1-2. Syncytial formation was assessed by enumerating nuclei number per syncytium and number of syncytia. HSV-1-induced syncytial formation was enhanced after 24 hr in UMG1-2 cells compared with U373MG controls. The amplified phenotype in UMG1-2 cells was effectively suppressed by roscovitine in addition to inhibitors of viral replication. This is the first study to provide histological evidence of the contribution of HCMV IE1 protein to enhanced cytopathogenic responses in active HSV-1 infection.
Cell Line, Tumor ; Giant Cells/*virology ; Herpesvirus 1, Human/*growth & development ; Humans ; Immediate-Early Proteins/biosynthesis/*metabolism ; Protein Kinase Inhibitors/pharmacology ; Purines/pharmacology ; Transfection ; Virus Replication/drug effects

OBJECTIVETo explore the role of exogenous connective tissue growth factor (CTGF) in the collagen III synthesis of human renal tubular epithelial cell line HK2 in vitro.

METHODSCultured HK2 cells were randomly assigned to three groups: placebo-control, low-dose CTGF-treated (2.5 ng/mL) and high-dose CTGF-treated groups (20 ng/mL). Cell morphological changes were observed under an inverted microscope. Collagen III alpha mRNA expression was detected using RT-PCR. Immunohistochemistry staining was used to assess the levels of intracellular collagen III alpha protein.

RESULTSAfter 48 hrs of low- or high- dose CTGF treatment, the appearances of HK2 cells were changed from oval to fusiform. High-dose CTGF treatment increased collagen III alpha mRNA expression (0.4461+/-0.0274 vs 0.2999+/-0.0115; P<0.05) as well as the protein expression of collagen III alpha (0.4075+/-0.0071 vs 0.3503+/-0.0136; P<0.05) compared with the placebo-control group.

CONCLUSIONSCTGF can induce morphological changes of human renal tubular epithelial cells in vitro. High concentration of CTGF may increase the synthesis of collagen III alpha.

Cells, Cultured ; Collagen Type III ; biosynthesis ; genetics ; Connective Tissue Growth Factor ; Epithelial Cells ; drug effects ; metabolism ; Humans ; Immediate-Early Proteins ; pharmacology ; Immunohistochemistry ; Intercellular Signaling Peptides and Proteins ; pharmacology ; Kidney Tubules ; drug effects ; metabolism ; RNA, Messenger ; analysis

OBJECTIVE: To characterize the antisense c-fos oligonucleotides that control the expression of immediate-early gene c-fos in retina in order to better understand the mechanism by which antisense c-fos oligonucleotides induced myopia. In this study the signal transduction in the pathway linking visual experience and the regulation of the eye's growth was investigated. METHODS: Thirty-one 3-week guinea pigs were assigned into 3 groups: antisense and sense c-fos oligonucleotides were intravitreally injected every 3 days to the eyes of the experimental guinea pigs at different concentrations; and saline vehicle to control guinea pigs in the same way. The refraction and axial length of the eyes were measured before and after the treatment, and the immediate-early gene c-fos expression in the retina was quantified by immunohistochemistry and RT-PCR. RESULTS: The moderate myopia was induced in high (1 nmol) and low (0.1 nmol) level of antisense c-fos oligonucleotide intravitreous injection (-5.425 D and -5.575 D, respectively) compared with the control ateral eyes. The refraction and axial length of the treated eyes increased, and the expression of immediate-early gene c-fos decreased significantly in the antisense c-fos oligonucleotides intravitreously injected eyes compared with the sense c-fos oligonucleotide intravitreously and saline vehicle injected eyes (P<0.01). The refraction and axial length were of no statistically significant differences among the sense c-fos oligonucleotides-treated eyes and saline-treated eyes and non-treated eyes (P>0.05). CONCLUSION The obvious myopia can be induced by antisense c-fos oligonucleotides in guinea pigs; antisense c-fos oligonucleotides inhibit c-fos expression in the retina. Immediate-early gene c-fos may be a potential factor in the prevention of myopia and plays an important role in the signal transduction of the retina.
Animals ; Genes, Immediate-Early ; genetics ; Guinea Pigs ; Immunohistochemistry ; Microinjections ; Myopia ; chemically induced ; genetics ; physiopathology ; Oligonucleotides, Antisense ; administration & dosage ; genetics ; toxicity ; Proto-Oncogene Proteins c-fos ; biosynthesis ; genetics ; RNA, Messenger ; genetics ; metabolism ; Random Allocation ; Retina ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Signal Transduction ; physiology

OBJECTIVETo investigate the effects of recombinal human connective tissue growth factor (rhCTGF) stimulation on epithelial-myofibroblast transdifferentiation (EMT) and collagen-synthesis in human renal tubular epithelial cell line (HK2) in vitro.

METHODSThe cultured HK2 cells were stimulated with rhCTGF of 5 ng/mL. The morphological changes were observed under an inverted microscope. The cells were collected at 0, 3, 6, 12, 24 and 48 hrs after rhCTGF stimulation. The expression of E-cadherin,alpha-smooth muscle actin (alpha-SMA), collagen Ialpha1 (Col Ialpha1) and collagen IValpha1 (Col IValpha1) mRNAs were detected by RT-PCR.

RESULTSrhCTGF stimulation changed the HK2 cell appearance from oval to fusiformdown-regulated the E-cadherin mRNA expression and up-regulated alpha-SMA mRNA expression, but had no effects the Col Ialpha1 and Col IValpha1 mRNA expression.

CONCLUSIONSExogenous CTGF can mediate the EMT but has no collagen-synthesis effects on HK2 cells.

Actins ; genetics ; Cadherins ; genetics ; Cell Differentiation ; drug effects ; Cell Line ; Collagen ; biosynthesis ; genetics ; Connective Tissue Growth Factor ; Epithelial Cells ; cytology ; drug effects ; Humans ; Immediate-Early Proteins ; pharmacology ; Intercellular Signaling Peptides and Proteins ; pharmacology ; Kidney Tubules ; cytology ; drug effects ; metabolism ; RNA, Messenger ; analysis ; Recombinant Proteins ; pharmacology

OBJECTIVETo investigate the effect of cytomegalovirus (CMV) infection on actin and microfilament in human embryo fibroblast cells (HF) and its relationship with CMV replication.

METHODSCell morphology was observed after the infection of CMV. Western-blot was used to measure the expression levels of beta-actin, G-actin and F-actin proteins. CMV immediately early antigen (CMV IE) in HF cells was analyzed by indirect immunofluorescence assay. Microfilament alteration was determined by cytoskeleton fluorescence probe.

RESULTCMV IE was demonstrated in more than 95% of HF cells after infection, which was primarily located in nucleus. The shape of HF cells changed from thin shuttle like to round and thick ball like, even escaping from wall after infection by CMV. Compared with control group, the expression of G-actin protein increased at 24 h of CMV infection (0.941 +/-0.061 compared with 0.714 +/-0.119, P <0.05), then decreased at 72 h, 96 h respectively(0.218 +/-.035, 0.230 +/-0.055 compared with 0.714 +/-0.119, P <0.05). The levels of F-actin in infected cells gradually decreased at 24 h, 72 h and 96 h compared with control HF cells (0.256 +/-0.021, 0.127 +/-0.032, 0.026 +/-0.008 compared with 0.373 +/-0.050, P<0.05). In infected HF cells, microfilaments were found ruptured, arranged turbulently. Cells fused and fluorescence density of microfilament markedly reduced.

CONCLUSIONCytomegalovirus can induce alteration of actins and microfilament, which may be associated with its infection, replication and reactivity in host cells.

Actin Cytoskeleton ; metabolism ; Actins ; biosynthesis ; genetics ; Antigens, Viral ; analysis ; Cells, Cultured ; Cytomegalovirus ; Cytoskeleton ; metabolism ; Embryo, Mammalian ; Fibroblasts ; metabolism ; ultrastructure ; virology ; Humans ; Immediate-Early Proteins ; analysis

OBJECTIVETo investigate the expression of CYR61 and VEGF in extranodal nasal-type NK/T cell lymphoma and its significance.

METHODSCYR61 mRNA and VEGF mRNA were detected by real-time fluorescence quantitative PCR method in 20 cases of extranodal nasal-type NK/T cell lymphoma. Expressions of CYR61 and VEGF were studied by immunohistochemistry in 40 cases of the tumor.

RESULTS(1) Over-expression of CYR61 mRNA and VEGF mRNA was found in 19/20(95.0% ) and 15/20(75.0% ) cases, respectively. (2)Tumor cells expressing CYR61 protein and VEGF protein were detected in 38(95.0% ) and 25 (62. 5% ) of the 40 cases respectively, being no significant difference from the control. Co-expression of CYR61 and VEGF at both the mRNA and protein levels was 95.0% and 65.0% , respectively. Over-expression of CYR61 and VEGF at both mRNA and protein levels was found in 8 of the 40 cases. (3) The prognosis of the patients over-expressing CYR61 and VEGF was worse.

CONCLUSIONIn extranodal nasal-type NK/T cell lymphoma, the expression level of CYR61 and VEGF was changed and it may be of prognostic implication of

Adolescent ; Adult ; Aged ; Cysteine-Rich Protein 61 ; Female ; Humans ; Immediate-Early Proteins ; biosynthesis ; genetics ; Intercellular Signaling Peptides and Proteins ; biosynthesis ; genetics ; Killer Cells, Natural ; Lymphoma, T-Cell ; metabolism ; pathology ; Male ; Middle Aged ; Nose Neoplasms ; metabolism ; pathology ; Polymerase Chain Reaction ; RNA, Messenger ; biosynthesis ; Vascular Endothelial Growth Factor A ; biosynthesis ; genetics

OBJECTIVETo observe the expression of connective tissue growth factor (CTGF) in the tubulointerstitium in type 2 diabetic KKA(y) mice and the effect of rosiglitazone on it.

METHODSKKA(y) and C57 BL/6 mice aged 16 weeks ( n = 5 in each group) were sacrificed as controls before treatment. Another 20 KKA(y) mice were treated with rosiglitazone (30 mg x kg (-1) d (-1), n = 10) or placebo (n = 10). The mice were sacrificed at 20 and 24-week-age (n = 5 at each time point). Protein expression of transforming growth factor-beta1 (TGF-beta1 ), CTGF, peroxisome proliferator-activated receptor-gamma (PPARgamma) , and fibronectin were assayed by Western blot, while protein CTGF, PPARgamma, and alpha-smooth muscle actin ( alpha-SMA) were assayed by immunohistochemistry in kidney tissue sections.

RESULTSProteinuria was significantly decreased in mice aged 24 weeks treated by rosiglitazone than same-aged mice treated with placebo [ (44. 53+/-1. 96) vs (63. 66 +/-5. 57) microg/24 h, P < 0. 05 ]. The expressions of TGF-beta1, CTGF, and fibronectin in mice aged 20 weeks treated with rosiglitazone decreased by 37% , 21% , and 52% than same-aged control (P <0. 01) , and those were decreased by 61% , 50% , and 51% in mice aged 24 weeks treated with rosiglitazone compared with same-aged control mice (P < 0. 01). CTGF in the tubulointerstitium were respectively downregulated by 25% and 44. 9% in treated mice aged 20 weeks and 24 weeks compared with the same-aged control mice ( P < 0. 01). The PPARgamma appeared in diabetic mice and increased by 18. 1% in mice aged 24 weeks and treated with rosiglitazone than the same-aged control mice (P <0. 05).

CONCLUSIONHeterogeneous rosiglitazone may upregulate the expression of PPARgamma in renal cortex, and remarkably inhibit the expressions of CTGF in the tubulointerstitium and renal cortex in diabetic KKA(y) mice.

Animals ; Connective Tissue Growth Factor ; Diabetes Mellitus, Experimental ; drug therapy ; metabolism ; Diabetes Mellitus, Type 2 ; drug therapy ; metabolism ; Down-Regulation ; Female ; Fibronectins ; biosynthesis ; Immediate-Early Proteins ; biosynthesis ; Intercellular Signaling Peptides and Proteins ; biosynthesis ; Kidney Cortex ; metabolism ; Kidney Tubules ; metabolism ; Mice ; Mice, Inbred C57BL ; Mice, Mutant Strains ; PPAR gamma ; agonists ; biosynthesis ; Thiazolidinediones ; pharmacology ; Transforming Growth Factor beta1 ; biosynthesis ; Up-Regulation

Animals ; Cells, Cultured ; Connective Tissue Growth Factor ; Hepatocytes ; cytology ; metabolism ; Immediate-Early Proteins ; biosynthesis ; genetics ; Intercellular Signaling Peptides and Proteins ; biosynthesis ; genetics ; Male ; Plasmids ; genetics ; RNA Interference ; RNA, Small Interfering ; genetics ; Rats ; Transfection

Liver cirrhosis is one of the major complications of hepatitis C virus (HCV) infection, but the mechanisms underlying HCV-related fibrogenesis are still not clear. Although the roles of HCV core protein remain poorly understood, it is supposed to play an important role in the regulation of cellular growth and hepatocarcinogenesis. The aim of this study was to examine the role of HCV core protein on the hepatic fibrogenesis. We established an in vitro co-culture system with primary hepatic stellate cell (HSC) isolated from rats, and a stable HepG2-HCV core cell line which had been transfected with HCV core gene. The expressions of fibrosis-related molecules transforming growth factor beta1 (TGF-beta1), transforming growth factor b receptor II (TGF beta RII), alpha-smooth muscle actin (alpha-SMA) and connective tissue growth factor (CTGF) were analyzed via histological or molecular methods. In addition, the expression levels of matrix metaloprotinase-2 (MMP-2) and collagen type I (Col I) from the co-cultured media were measured by zymogram and ELISA, respectively. The expressions of alpha-SMA, TGF-beta1, Col I, TGF beta RII and MMP-2 were significantly increased in the co-culture of stable HepG2-HCV core with HSC. Moreover, the significant increases of CTGF and TGF-beta1 in the HCV core-expressing cells were observed by either Northern or Western blot analysis. These results suggest that HCV core protein may contribute to the hepatic fibrogenesis via up-regulation of CTGF and TGF-beta1.
Actins/metabolism ; Animals ; Cell Line, Tumor ; Cells, Cultured ; Coculture Techniques ; Collagen Type I/metabolism ; Gelatinase A/metabolism ; Immediate-Early Proteins/*biosynthesis ; Intercellular Signaling Peptides and Proteins/*biosynthesis ; Liver/metabolism/*pathology ; Liver Cirrhosis/*metabolism ; Male ; Rats ; Rats, Sprague-Dawley ; Receptors, Transforming Growth Factor beta/metabolism ; Research Support, Non-U.S. Gov't ; Transforming Growth Factor beta/*metabolism ; Up-Regulation ; Viral Core Proteins/genetics/*metabolism