OBJECTIVE: To investigate the regulatory role of miRNA-26a in vascular smooth muscle cell (VSMC) calcification by regulating connective tissue growth factor (CTGF). METHODS: Rat thoracic aorta VSMCs (A7r5 cells) with induced calcification were treated with AR234960 agonist or transfected with miR-26a mimic, or with both treatments. Alizarin red staining was used to determine calcium deposition, and phosphatase (ALP) activity in the cells was measured. The mRNA and protein expressions of miR-26a, OPG, OPN, BMP-2 and collagen Ⅱ were detected using qPCR and Western blotting. The binding of miR-26a to CTGF was verified using dual luciferase reporter gene assay. RESULTS: After induced calcification, A7r5 cells showed gradually decreased miR-26a expression (P < 0.05) and progressively increased CTGF expression (P < 0.05) with the extension of induction time. Treatment of the cells with AR234960 obviously increased calcification in the cells, while transfection with miR-26a mimic significantly reduced cell calcification. The calcifying cells showed significantly increased ALP activity and expressions of OPN, BMP-2 and collagen Ⅱ (P < 0.05) and lowered OPG expression (P < 0.05), and treatment with AR234960 did not produce obvious effects on these changes (P > 0.05). Transfection with miR-26a mimic resulted in significantly decreased ALP activity and expressions OPN, BMP-2 and collagen Ⅱ expression (P < 0.05) and increased OPG expression (P < 0.05) in the calcifying cells. These effects of miR-26a mimic was significantly attenuated by treatment of the cells with AR234960 (P < 0.05). The result of luciferase reporter gene assay confirmed the binding of miR-26a to CTGF. CONCLUSION miRNA-26a can effectively alleviate vascular calcification by lowering the level of CTGF, reducing ALP activity and the expressions of OPN, BMP-2 and collagen Ⅱ, and increasing the expression of OPG.
Animals ; Calcium/metabolism* ; Cells, Cultured ; Connective Tissue Growth Factor/pharmacology* ; MicroRNAs/metabolism* ; Muscle, Smooth, Vascular ; Myocytes, Smooth Muscle ; Phosphoric Monoester Hydrolases/pharmacology* ; RNA, Messenger/metabolism* ; Rats ; Sulfones ; Vascular Calcification

OBJECTIVE: To study the preventive and therapeutic effects of safflower water extract on systemic scleroderma (SSc) in mice and its mechanism. METHODS: Sixty BALB/C mice were randomly divided into the control group, model group, prednisone group and safflower low, middle, high dose groups, 10 mice in each group.The control group was injected with normal saline, and the other five groups were subcutaneously injected with bleomycin hydrochloride with 100 μl at the concentration of 200 μg /ml on the back, once a day for 28 days to establish the SSc models.At the same time, the control group and model group were treated with normal saline (10 ml/kg), the prednisone group was treated with prednisone 4.5 mg/kg (10 ml/kg), and the low, middle, and high dose safflower groups were treated with safflower at the doses of 1.5, 3, 6 g/kg (10 ml/kg), and all groups were treated for 28 days.After 28 days, all mice were decapitated. The blood samples and back skin of the BLM injection part were collected.After that, all the tissue slices were taken to measure the dermal thickness, and the content of hydroxyproline (HYP) in the skin tissues was detected by hydrolysis method.The contents of tissue growth factor (CTGF) and transforming growth factor-β (TGF-β ) in the skin tissues and the levels of interleukin-6 (IL-6) and interleukin-17 (IL-17) in serum were determined by ELISA. RESULTS: Compared with the control group, the dermal thickness of the model group was increased(P＜0.05), the contents of CTGF, TGF-β and HYP in the skin tissues and the levels of IL-6 and IL-17 in the serum of the model group were increased(P＜0.05); compared with the model group, the dermal thickness in the prednisone group and safflower groups was decreased (P＜0.05), the levels of CTGF, TGF-β and HYP in the skin tissues and the serum levels of IL-6 and IL-17 in the prednisone group and safflower groups were decreased (P＜0.05). CONCLUSION Safflower water extract can improve skin condition (or dermal thickness) in SSc mice, and its mechanism may be related to reducing immune inflammatory response.
Animals ; Bleomycin ; Carthamus tinctorius ; chemistry ; Connective Tissue Growth Factor ; metabolism ; Disease Models, Animal ; Hydroxyproline ; analysis ; Interleukin-17 ; metabolism ; Interleukin-6 ; metabolism ; Mice ; Mice, Inbred BALB C ; Plant Extracts ; pharmacology ; Random Allocation ; Scleroderma, Systemic ; drug therapy ; Skin ; pathology ; Transforming Growth Factor beta1 ; metabolism

OBJECTIVETo investigate the effect of tranilast on myocardial fibrosis in mice with viral myocarditis (VMC).

METHODSMale balb/c mice (n=72) were randomly divided into control, VMC and tranilast groups (n=24 each). In the VMC and tranilast groups, the mice were infected with Coxsackie virus B3 (CVB3) to prepare VMC model, while the control group was treated with Eagle's medium. After modeling, the tranilast group was administrated with tranilast [200 mg/(kg.d)] until the day before sampling. On days 7, 14 and 28 after CVB3 or Eagle's medium infection, heart specimens (n=8) were taken and examined after Toluidine blue staining and Nissl staining for counts of mast cells (MC), hematoxylin-eosin staining for myocardial pathological changes, and Masson staining for myocardial fibrosis. The expression of CTGF and type I collagen (Col I) in the myocardial tissue was measured by RT-PCR and Western blot. The correlations of CTGF mRNA expression with MC counts and Col I expression were analyzed.

RESULTSThe myocardial pathological changes and collagen volume fraction in the VMC group were significantly higher than in the control group at all three time points (P<0.05). Tranilast treatment significantly decreased the myocardial pathological changes and collagen volume fraction compared with the VMC group (P<0.05). The mRNA and protein expression of CTGF and Col I increased in the VMC group compared with the control group, and the increases were reduced with tranilast treatment (P<0.05). The number of MC was positively correlated to CTGF mRNA expression on the 7th day and 14th day (r=0.439, P=0.049) in the VMC group. There were positive correlations between the mRNA expression of Col I and CTGF on the 7th day and 14th day (r=0.646, P=0.007) and the 28th day (r=0.326, P=0.031).

CONCLUSIONSTranilast may inhibit the aggregation of MC and down-regulate the expression of CTGF, relieving myocardial fibrosis of mice with VMC.

Animals ; Collagen Type I ; genetics ; Connective Tissue Growth Factor ; genetics ; Coxsackievirus Infections ; drug therapy ; Enterovirus B, Human ; Fibrosis ; Male ; Mice ; Mice, Inbred BALB C ; Myocarditis ; drug therapy ; Myocardium ; pathology ; RNA, Messenger ; analysis ; ortho-Aminobenzoates ; pharmacology

To investigate the role of glucose transporter 1 (GLUT1) and sodium-glucose cotransporter 1 (SGLT1) in high glucose dialysate-induced peritoneal fibrosis.Thirty six male SD rats were randomly divided into 6 groups (6 in each):normal control group, sham operation group, peritoneal dialysis group (PD group), PD+phloretin group (PD+T group), PD+phlorizin group (PD+Z group), PD+phloretin+phlorizin group (PD+T+Z group). Rat model of uraemia was established using 5/6 nephrotomy, and 2.5% dextrose peritoneal dialysis solution was used in peritoneal dialysis. Peritoneal equilibration test was performed 24 h after dialysis to evaluate transport function of peritoneum in rats; HE staining was used to observe the morphology of peritoneal tissue; and immunohistochemistry was used to detect the expression of GLUT1, SGLT1, TGF-β1 and connective tissue growth factor (CTGF) in peritoneum. Human peritoneal microvascular endothelial cells (HPECs) were divided into 5 groups:normal control group, peritoneal dialysis group (PD group), PD+phloretin group (PD+T group), PD+phlorezin group (PD+Z group), and PD+phloretin+phlorezin group (PD+T+Z group). Real time PCR and Western blotting were used to detect mRNA and protein expressions of GLUT1, SGLT1, TGF-β1, CTGF in peritoneal membrane and HPECs., compared with sham operation group, rats in PD group had thickened peritoneum, higher ultrafiltration volume, and the mRNA and protein expressions of GLUT1, SGLT1, CTGF, TGF-β1 were significantly increased (all<0.05); compared with PD group, thickened peritoneum was attenuated, and the mRNA and protein expressions of GLUT1, SGLT1, CTGF, TGF-β1 were significantly decreased in PD+T, PD+Z and PD+T+Z groups (all<0.05). Pearson's correlation analysis showed that the expressions of GLUT1, SGLT1 in peritoneum were positively correlated with the expressions of TGF-β1 and CTGF (all<0.05)., the mRNA and protein expressions of GLUT1, SGLT1, TGF-β1, CTGF were significantly increased in HPECs of peritoneal dialysis group (all<0.05), and those in PD+T, PD+Z, and PD+T+Z groups were decreased (all<0.05). Pearson's correlation analysis showed that the expressions of GLUT1, SGLT1 in HPECs were positively correlated with the expressions of TGF-β1 and CTGF (all<0.05).High glucose peritoneal dialysis fluid may promote peritoneal fibrosis by upregulating the expressions of GLUT1 and SGLT1.
Animals ; Cells, Cultured ; Connective Tissue Growth Factor ; analysis ; drug effects ; Dialysis Solutions ; adverse effects ; chemistry ; pharmacology ; Gene Expression Regulation ; drug effects ; Glucose ; adverse effects ; pharmacology ; Glucose Transporter Type 1 ; analysis ; drug effects ; physiology ; Hemodiafiltration ; adverse effects ; methods ; Humans ; Male ; Peritoneal Dialysis ; adverse effects ; methods ; Peritoneal Fibrosis ; chemically induced ; genetics ; physiopathology ; Peritoneum ; chemistry ; drug effects ; pathology ; Phloretin ; Phlorhizin ; RNA, Messenger ; Rats ; Rats, Sprague-Dawley ; Sodium-Glucose Transporter 1 ; analysis ; drug effects ; physiology ; Transforming Growth Factor beta1 ; analysis ; drug effects ; Uremia ; chemically induced

Capsular fibrosis and contracture occurs in most breast reconstruction patients who undergo radiotherapy, and there is no definitive solution for its prevention. Simvastatin was effective at reducing fibrosis in various models. Peri-implant capsular formation is the result of tissue fibrosis development in irradiated breasts. The purpose of this study was to examine the effect of simvastatin on peri-implant fibrosis in rats. Eighteen male Sprague-Dawley rats were allocated to an experimental group (9 rats, 18 implants) or a control group (9 rats, 18 implants). Two hemispherical silicone implants, 10 mm in diameter, were inserted in subpanniculus pockets in each rat. The next day, 10-Gy of radiation from a clinical accelerator was targeted at the implants. Simvastatin (15 mg/kg/day) was administered by oral gavage in the experimental group, while animals in the control group received water. At 12 weeks post-implantation, peri-implant capsules were harvested and examined histologically and by real-time polymerase chain reaction. The average capsular thickness was 371.2 μm in the simvastatin group and 491.2 μm in the control group. The fibrosis ratio was significantly different, with 32.33% in the simvastatin group and 58.44% in the control group (P < 0.001). Connective tissue growth factor (CTGF) and transforming growth factor (TGF)-β1 gene expression decreased significantly in the simvastatin group compared to the control group (P < 0.001). This study shows that simvastatin reduces radiation-induced capsular fibrosis around silicone implants in rats. This finding offers an alternative therapeutic strategy for reducing capsular fibrosis and contracture after implant-based breast reconstruction.
Administration, Oral ; Animals ; Breast/*drug effects/metabolism/pathology/radiation effects ; *Breast Implants ; Connective Tissue Growth Factor/genetics/metabolism ; Fibrosis ; Gamma Rays ; Male ; Rats ; Rats, Sprague-Dawley ; Real-Time Polymerase Chain Reaction ; Silicone Gels/*chemistry ; Simvastatin/*pharmacology ; Transforming Growth Factor beta1/metabolism

Capsular fibrosis and contracture occurs in most breast reconstruction patients who undergo radiotherapy, and there is no definitive solution for its prevention. Simvastatin was effective at reducing fibrosis in various models. Peri-implant capsular formation is the result of tissue fibrosis development in irradiated breasts. The purpose of this study was to examine the effect of simvastatin on peri-implant fibrosis in rats. Eighteen male Sprague-Dawley rats were allocated to an experimental group (9 rats, 18 implants) or a control group (9 rats, 18 implants). Two hemispherical silicone implants, 10 mm in diameter, were inserted in subpanniculus pockets in each rat. The next day, 10-Gy of radiation from a clinical accelerator was targeted at the implants. Simvastatin (15 mg/kg/day) was administered by oral gavage in the experimental group, while animals in the control group received water. At 12 weeks post-implantation, peri-implant capsules were harvested and examined histologically and by real-time polymerase chain reaction. The average capsular thickness was 371.2 μm in the simvastatin group and 491.2 μm in the control group. The fibrosis ratio was significantly different, with 32.33% in the simvastatin group and 58.44% in the control group (P < 0.001). Connective tissue growth factor (CTGF) and transforming growth factor (TGF)-β1 gene expression decreased significantly in the simvastatin group compared to the control group (P < 0.001). This study shows that simvastatin reduces radiation-induced capsular fibrosis around silicone implants in rats. This finding offers an alternative therapeutic strategy for reducing capsular fibrosis and contracture after implant-based breast reconstruction.
Administration, Oral ; Animals ; Breast/*drug effects/metabolism/pathology/radiation effects ; *Breast Implants ; Connective Tissue Growth Factor/genetics/metabolism ; Fibrosis ; Gamma Rays ; Male ; Rats ; Rats, Sprague-Dawley ; Real-Time Polymerase Chain Reaction ; Silicone Gels/*chemistry ; Simvastatin/*pharmacology ; Transforming Growth Factor beta1/metabolism

Mammalian pancreatic β-cells play a pivotal role in development and glucose homeostasis through the production and secretion of insulin. Functional failure or decrease in β-cell number leads to type 2 diabetes (T2D). Despite the physiological importance of β-cells, the viability of β-cells is often challenged mainly due to its poor ability to adapt to their changing microenvironment. One of the factors that negatively affect β-cell viability is high concentration of free fatty acids (FFAs) such as palmitate. In this work, we demonstrated that Yes-associated protein (Yap1) is activated when β-cells are treated with palmitate. Our loss- and gain-of-function analyses using rodent insulinoma cell lines revealed that Yap1 suppresses palmitate-induced apoptosis in β-cells without regulating their proliferation. We also found that upon palmitate treatment, re-arrangement of F-actin mediates Yap1 activation. Palmitate treatment increases expression of one of the Yap1 target genes, connective tissue growth factor (CTGF). Our gain-of-function analysis with CTGF suggests CTGF may be the downstream factor of Yap1 in the protective mechanism against FFA-induced apoptosis.
Actins ; metabolism ; Adaptor Proteins, Signal Transducing ; antagonists & inhibitors ; genetics ; metabolism ; Animals ; Apoptosis ; drug effects ; physiology ; Bridged Bicyclo Compounds, Heterocyclic ; pharmacology ; Cell Line, Tumor ; Connective Tissue Growth Factor ; genetics ; metabolism ; pharmacology ; Cytochalasin D ; pharmacology ; Fatty Acids, Nonesterified ; pharmacology ; HEK293 Cells ; Humans ; Immunohistochemistry ; Insulin-Secreting Cells ; cytology ; drug effects ; metabolism ; Mice ; Microscopy, Fluorescence ; Palmitic Acid ; pharmacology ; Phosphoproteins ; antagonists & inhibitors ; genetics ; metabolism ; RNA Interference ; RNA, Small Interfering ; metabolism ; Rats ; Recombinant Proteins ; genetics ; metabolism ; pharmacology ; Thiazolidines ; pharmacology

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

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

OBJECTIVE: To explore the eff ect of Qiluxiaobai (QLXB) decoction on rats with adriamycin (ADR)- induced focal segmental glomerular sclerosis (FSGS) nephropathy (ADN). METHODS: Adriamycin was injected into tail vein at total dose of 7.5 mg/kg for twice per week. According to random number table, rats were divided into 4 groups: the control group, the ADN group, the Losartan group [intragastric, 5.19 mg/(kg.d)], and the QLXB group [intragastric,134.40 mg/(kg.d)]. Eight weeks later, serum creatinine (SCr), blood urea nitrogen (BUN), serum cholesterol (CHO), serum triglycerides (TG) and albuminuria (ALB) were measured by routine biochemical methods. Pathological changes in the rat kidneys were observed under light microscopes. Connective tissue growth factor (CTGF), α-smooth muscle actin (α-SMA) and fibronectin (FN) mRNA and protein expression levels were measured by real-time PCR and Western blot, respectively. RESULTS: In the ADN group, SCr, BUN, CHO, TG was increased (P<0.05) while ALB was decreased (P<0.05), ALB was decreased (P<0.05) compared to the control group. In the QLXB and Losartan group, SCr, BUN, CHO, TG and ALB was improved compared to the ADN group (P<0.05). CTGF, FN, α-SMA mRNA and protein expression was decreased in QLXB group compared to ADN group (P<0.05). CONCLUSION QLXB could partly improve glomerular sclerosis in adriamycin-induced nephropathy, which was related to inhibition of CTGF, FN and α-SMA expression.
Actins ; metabolism ; Animals ; Connective Tissue Growth Factor ; metabolism ; Doxorubicin ; adverse effects ; Drugs, Chinese Herbal ; pharmacology ; Fibronectins ; metabolism ; Glomerulosclerosis, Focal Segmental ; chemically induced ; drug therapy ; Kidney ; pathology ; Rats ; Real-Time Polymerase Chain Reaction ; Sclerosis