Effect of β3-adrenoceptor activation on ventricle fibrillation threshold and effective refractory period in rats with heart failure

AIM: To observe the effect of β₃-adrenoceptor (AR) on ventricle fibrillation threshold (VFT) and effective refractory period (ERP) in rats with heart failure.METHODS: Rats were randomized into control group and heart failure group. The expression of β₃- AR mRNA was detected with RT-PCR. The VFT, ERP, left ventricle end-systolic pressure(LVESP)，left ventricle end-diastolic pressure(LVEDP), +dp/dtmax and -dp/dtmax were measured at the same time with administration of BRL37344 (β₃-AR agonist).RESULTS: ① Both the expression of β₃-AR mRNA and the proportion (β₃/β₁+β₂+β₃) were increased in failure rats comparied with those in control rats (0.028 vs 0.011 and 5.4% vs 1.2%, P<0.05). ② ERP was longer in rats with heart failure than that in control group (70.5 ms±5.5 ms vs 59.5 ms±6.4 ms, P<0.05). No difference in ERP in rats with heart failure was observed before and after administration of BRL37344 (73.0 ms±4.8 ms vs 70.5 ms±5.5 ms, P>0.05). ③ VFT was lower in rats with heart failure than that in control group (10.9 mV±0.8 mV vs 30.5 mV±1.3 mV, P<0.05) and decreased obviously in rats with heart failure after administration of BRL37344 (7.1 mV±0.6 mV vs 10.9 mV±0.8 mV, P<0.05). The decrease in VFT correlated with the effect of LVESP, +dp/dtmax, -dp/dtmax with BRL37344 and the expression of β₃-AR mRNA (correlation coefficient: 0.788, 0.708, 0.759, 0.787; P<0.05). CONCLUSION: The expression of β₃-AR mRNA in left ventricle is obviously increased in rats with heart failure. The activation of β₃-AR has no effect on ERP but can decrease VFT which correlates with the effect of β₃-AR on LVESP, +dp/dtmax, -dp/dtmax and the expression of β₃-AR mRNA.     

Effects of extract of ginkgo biloba on human tubular epithelial-mesenchymal transition induced by transforming growth factor-β1

AIM: to investigate the effects of extract of ginkgo biloba (EGB) on human tubular epithelial-mesenchymal transition induced by transforming growth factor-β₁.METHODS: HK2 cells were induced to epithelial-mesenchymal transition by transforming growth factor-β₁ (TGF-β₁, 10 μg/L). EGB was added into the medium of HK2 cells 2 h before TGF-β₁ was added. The expressions of E-cadherin, α-smooth muscle actin (α-SMA), NADPH oxidase p67phox and superoxide dismutase (SOD) were determined by Western blotting. Malondialdehyde (MDA) in the mediums of HK2 cells was detected. RESULTS: EGB significantly attenuated the downregulation of E-cadherin, the upregulation of α-SMA and p67phox, the downregulation of SOD and the upregulation of MDA in HK2 cells induced by TGF-β₁.CONCLUSION: EGB significantly attenuates human tubular epithelial-mesenchymal transition induced by TGF-β₁, and its underlying mechanism is that EGB attenuates the upregulation of p67phox and the downregulation of SOD induced by TGF-β₁.     

Preventive effects of anti-IGFBPrP1 antibody on hepatic fibrosis in mice

AIM: To investigate the preventive effect and mechanism of anti-insulin-like growth factor binding protein related protein 1(IGFBPrP1) antibody on hepatic fibrosis induced by thioacetamide (TAA) in mice.METHODS: Twenty-four male C57BL/6 wild-type mice were randomly divided into 3 groups (n= 8 in each group): normal control group, TAA group (4 weeks) and TAA+anti-IGFBPrP1 antibody group (4 weeks). The morphological changes of liver tissues were observed. The expression levels of α-smooth muscle actin (α-SMA), transforming growth factor beta 1 (TGF-β₁), Smad3, phosphorylated Smad2/3 (p-Smad2/3), fibronectin (FN), collagen I, collagen Ⅲ and IGFBPrP1 were detected by the methods of immunohistochemistry and Western blotting.RESULTS: In TAA group (4 weeks), obvious injury of liver was observed, and the expression levels of α-SMA, TGF-β₁, Smad3, p-Smad2/3, FN, collagen Ⅰ, collagen Ⅲ and IGFBPrP1 were significantly increased as compared with normal control group (P<0.01). Compared with TAA group (4 weeks), the injury of the liver was alleviated and the expression levels of the proteins above were decreased in TAA+anti-IGFBPrP1 antibody group (4 weeks, P<0.01). IGFBPrP1 was positively correlated with TGF-β₁, Smad3, p-Smad2/3, FN and collagen I (P<0.01). CONCLUSION: Anti-IGFBPrP1 antibody prevents TAA-induced hepatic fibrosis in mice by inhibiting the activation of hepatic stellate cells, reducing the expression of p-Smad2/3 and inhibiting the TGF-β₁/ Smad3 signal transduction, thereby depressing the deposition of extracellular matrix in liver tissues.     

Recombinant human defensin α1 induces proliferation of rat glomerular mesangial cells in vitro

AIM: To study the effects and mechanism of recombinant human defensin α₁ on cell proliferation in cultured rat glomerular mesangial cells.METHODS: The influences of defensin α₁ at various concentrations on rat 1097 mesangial cell line cultured in vitro were evaluated with MTT assay.The different concentrations of U0126,signal-regulated protein kinase (MEK) inhibitor,were added into the culture mediums of mesangial cells to do blocking test.Incubated with a final concentration of 3 mg/L defensin α₁,the phosphorylation of extracellular signal regulated kinase (ERK)1/2 and type IV collagen of mesangial cells in different times were evaluated by Western blotting.RESULTS: Defensin α₁ at 3-20 mg/L enhanced proliferation of rat glomerular mesangial cells.The incubation times for the maximum effect on proliferation was 12 h (P<0.01),whereas defensin α₁ concentration >20 mg/L decreased cell proliferation.The cell proliferation induced by defensin α₁ was inhibited by U0126.Stimulation of the cells with defensin α₁ at concentration of 3 mg/L for 5 minutes induced a maximum effect on a ratio of phosphorylation of ERK1/2 to total ERK.After 12 h incubation with defensin α₁,an increase in type IV collagen was observed by Western blotting and continued to increase at 24 h and 48 h (P<0.01).CONCLUSION: Defensin α₁ enhances rat glomerular mesangial cell proliferation and induces type IV collagen production by MAPK signaling pathway.     

Harmful factors affect glycine receptor α1 subunit mRNA expression in the neonatal rat cardiomyocytes

AIM: To investigate the effects of lipopolysaccharide, hypoxia/reoxygenation,isoproterenol and high concentration of glucose on glycine receptor α₁ subunit mRNA expression in the neonatal rat cardiomyocytes. METHODS: Isolation of cardiomyocytes from Sprague-Dawley rats aging 1～3 d were performed. Cardiomyocytes （1×10⁵～5×10⁵ cells·L^-1）were cultured in DMEM medium containing 15% fetal bovine serum at 37 ℃ in 5%CO₂ atmosphere for 72 h. Then, cultured rat cardiomyocytes were treated with lipopolysaccharide, isoproterenol or high concentration of glucose for 24 h, respectively, or were exposed to hypoxia for 3 h followed by reoxygenation for 3 h. Subsequently, the cell survival rate was measured using CCK-8 reactant and RT-PCR was applied to monitor the expression of glycine receptor α₁ subunit mRNA. RESULTS: Compared with the control group, no significant difference in the cell survival rate was observed （P＞0.05）. The expression of glycine receptor α₁ subunit mRNA was increased （P＜0.01） in lipopolysaccharide（5,10,20,40,80 mg/L）,isoproterenol（20,100,500 μmol/L） or hypoxia/reoxygenation, hypoxia groups, but decreased（P＜0.01）in the group treated with high concentration of glucose（25, 50 mmol/L）. CONCLUSION: Lipopolysaccharide, isoproterenol, hypoxia/reoxygenation or hypoxia upregulates the expression of glycine receptor α₁ subunit mRNA,but high concentration of glucose down-regulates the expression of glycine receptor α₁ subunit mRNA in cultured neonatal rat cardiomyocytes.     

Effect of siRNA on myocarditis induced by MCMV in mice

AIM: To investigate the effect of siRNA on myocarditis induced by MCMV in BALB/c mice. METHODS: One handred and twenty male BALB/c mice (4 weeks old) were divided randomly into 6 groups with 20 mice each. Among these groups, one normal control group, one viral control group and 4 experimental groups were assigned. Mice in viral control and 4 experimental groups were inoculated intraperitoneally injection (ip) with 100 μL of DMEM medium containing TCID5010-4 MCMV, and followed by 200 μL of different doses of liposomally endocapsulated siRNA in 4 experimental groups and by 200 μL of vehicle solution of siRNA in viral control group 30 min later. Mice in normal control group were first inoculated ip 100 μL of DMEM medium and followed by 200 μL of vehicle solution of siRNA 30 min later. At day 7, 14, 21, 28, 35, 42, 49, 56, 63 and 70 of postinfection, all mice were killed and blood samples were collected for detecting serum zymogram (CK, CK-MB, GOT, LDH and LDH₁/LDH₂) and the anti-cardiac β₁ receptor autoantibody titer level. Hearts were removed aseptically for detecting cardiac histological lesions and ultrastructure changes. RESULTS: Decreased morbidity of myocarditis, lightened cardiac pathologic lesions and ultrastructural changes in 4 experimental groups were observed. Serum zymogram (CK, CK-MB, GOT, LDH and LDH₁/LDH₂) and β₁ autoantibody titer level in viral control group were significantly higher than those in normal control group, but zymogram and β₁ autoantibody titer levels in 4 experimental groups were between those in viral control group and normal control group. The activities of CK, CK-MB, GOT, LDH and LDH₁/LDH₂ and β₁ autoantibody titer level decreased accompanied with siRNA dose increasing. These effects were in a dose dependent manner. CONCLUSION: siRNA protects myocardial tissue against MCMV infection.     

Expression of TGF-β1 and apoptosis in myocardium of diabetic rats

AIM: To study the pathophysiological mechanism of cardiomyopathy, the expression of TGF-β₁ and apoptosis in myocardium of diabetic rats were investigated. METHODS:The diabetes models were established by single intravenous injection of streptozotocin (50 mg/kg) in rats. By the method of immunochemistry, the expression of TGF-β₁ in the cardiomyocytes was detected as the index to evaluate the degree of fibrosis. The method of TUNEL was used to measure the cardiomyocyte apoptosis as the index to explore its importance in process of diabetic cardiomyopathy. RESULTS:① The weight of diabetic rats was apparently lower than that in the rats before the diabetic model was built (P<0.01), and the increase in weight in diabetic rats within three month was less than that in normal group. ② Compared with control group, the concentration of blood glucose was continually elevated during the experiment. ③ The expression of TGF-β₁ in the diabetic cardiac muscle was much more than that in normal group (P<0.01). ④ The apoptosis of myocardium measured by the method of TUNEL was apparent in the diabetic groups than that in normal one (P<0.01). However, no significance was detected in the different courses of diabetic groups. CONCLUSIONS:The apoptosis might play an important role in leading the diabetic cardiomyopathy to heart failure. The expression of TGF-β₁ in the myocardium of diabetic rats was more than that in normal and had an increasing trend in the procession of diabetic cardiomyopathy. TGF-β₁ might be a significant factor in diabetic myocardium fibrosis. Apoptosis might play an important role in the initial stage of diabetes, which promotes the diabetic cardiomyopathy to heart failure.     

Expression of ROCK I and TGF-β1 in pulmonary arterioles of rat with chronic thromboembolism pulmonary hypertension

AIM:To investigate the dynamic expression of Rho kinase (ROCK I) and transforming growth factor β₁ (TGF-β₁) in pulmonary arterioles of rat with chronic thromboembolic pulmonary hypertension. METHODS: Sixty-four male Wister rats were randomly divided into eight groups: beginning control group, embolism for 3 d, 1 week, 2 weeks, 4 weeks, 8 weeks, 12 weeks groups and end control group. The pulmonary thromboembolism (PTE) model was established by injecting thrombin into jugular vein two times in two weeks and each rat underwent peritoneal injection with tranexamic acid one time a day during experiment to prevent thrombolysis. The mean pulmonary artery pressure (mPAP), right ventricular hypertrophy index (RVHI), relative medial thickness of small pulmonary arteries (PAMT) and vessel wall area/total area (WA/TA) were measured. The levels of ROCK I mRNA and TGF-β₁ protein in rat pulmonary artery were determined by in situ hybridization, immunohistochemistry and image analysis, respectively. RESULTS: mPAP, PAMT and WA/TA were higher respectively in embolism from 4 weeks group to 12 weeks group than those in beginning control group (mPAP: all P<0.01, PAMT and WA/TA: 4 weeks group P<0.05, 8 weeks group and 12 weeks group P<0.01). RVHI was elevated in 8 weeks group P<0.05, in 12 weeks group P<0.01. ROCK I mRNA and TGF-β₁ protein in pulmonary arterioles got the enhanced positive signals of in situ hybridization or immunohistochemistry staining with prolonging the time of rats with pulmonary thromboembolism. ROCKⅠ mRNA: embolism from 3 d group to 2 weeks group P<0.05, 4 weeks group to 12 weeks group P<0.01, TGF-β₁ protein: 1 week group and 2 weeks group P<0.05, 4 weeks group to 12 weeks group P<0.01. Linear correlation analysis showed that ROCK I mRNA and TGF-β₁ protein were positively correlated with mPAP, RVHI and vessel remodeling index (all P<0.01), ROCK I mRNA were positively correlated with TGF-β₁ protein (P<0.01). CONCLUSION:ROCK I and TGF-β₁ play a role in the pathogenesis of chronic thromboembolic pulmonary hypertension and pulmonary vessel remodeling. TGF-β₁ produces biological effect by active ROCK signal pathway.     

Embryonic tissue extracts modulate cell-fate determination in human epidermal stem cells

AIM: To investigate the phenotypic changes and cell-fate determination of keratinocyte stem cells in vitro. METHODS: Improved collagen Ⅳ-coated adhesion method was used to isolate and culture the keratinocyte stem cells after neutron-protease selectively digested the dermo-epidermal junctions. Morphological features and identification of these immature cells were studied by immunochemistry and confocal microscopy analysis. After treated with extracts from 14-day-old mouse fetuses at the concentration of 10% (W/V), phenotypic changes and the cell-fate determination of keratinocyte stem cells (KSCs) were detected by flow FACS analysis and RT-PCR assays. RESULTS: Immunocytochemical analysis proved that KSCs and their subunits were positive for α₆ and β₁ integrin, keratin 19 and 14, p63, nestin and PCNA, yet negative for keratin 10 expressions. Double staining immunofluorescence demonstrated that markers such as α₆ integrin and CD71 was co-expressed in KSCs, and there were important regional differences in the distribution of α6 integrin and CD71 in KSCs and transit amplifying cells (TA cells). Meanwhile, two-color flow cytometric analysis of α₆ integrin and CD71 consistently revealed that after treated with an extract from mouse fetuses, the percent of α+6 CD71+ and CD71+ fraction increased and reached to 68.43% and 4.51% of the total isolated cells, respectively. However, the percent of α+6 CD71- fraction reduced from 22.49% to 10.92% determined by flow cytometry. Moreover, the expression levels of β₁ integrin and some putative biological markers in human epidermal cells were analyses by RT-PCR after fetus extract treatment. The relative gene expressions of β₁ integrin, K19 and K10 increased in KSCs, whereas the expression of K14 in keratinocyte stem cells was observed to be significantly suppressed when compared to the appropriate controls. CONCLUSION: The keratinocyte stem cells isolated by collagen Ⅳ-coated adhesion method have some characteristics of adult stem cells and perhaps the potentiality to differentiate and cross-differentiate. The regional differences in the distribution of α6 integrin and CD71 are one of the transition markers to discriminate the KSCs and TA cells. Furthermore, fetus extracts contribute to not only the proliferation and self-renewal of KSCs but also the dedifferentiation of TA cell subunits.     

Inhibitors of CaMKⅡ suppress the expression of TNF-α, TGF-β1 and collagen I,III in cardiac fibroblasts treated with angiotensin II or electrical field stimulation

AIM: To observe the role of calcium/calmodulin-dependent protein kinase Ⅱ (CaMKⅡ) in the proliferation, released cytokines and expression of collagen Ⅰ and Ⅲ in rat cardiac fibroblasts induced by angiotensin Ⅱ (AngⅡ) or electrical field stimulation (EFS).METHODS: The cultured cardiac fibroblasts were isolated from the neonatal rats of 1-3 days and used in the 3rd passage. The cells were divided into 10 groups: control group, 0.1 μmol/L AngⅡ group, 0.1 μmol/L AngⅡ+0.5 μmol/L KN92 group, 0.1 μmol/L AngⅡ+0.5 μmol/L KN93 group, 0.1 μmol/L AngⅡ+0.5 μmol/L AIP group; 10V 1.0 Hz EFS group, 10 V 1.0 Hz EFS+0.5 μmol/L KN92 group, 10 V 1.0 Hz EFS+0.5 μmol/L KN93 group, 10 V 1.0 Hz EFS+0.5 μmol/L AIP group, 10 V 1.0 Hz EFS+0.1 μmol/L AngⅡ group.MTT was used to detect the proliferation of cardiac fibroblasts. The release of cytokines was measured by ELISA. The mRNA expression of TNF-α, TGF-β₁ and collagen Ⅰ, Ⅲ was determined by RT-PCR.RESULTS: CaMKⅡ inhibitors (0.5 μmol/L KN93 or 0.5 μmol/L AIP) prevented the proliferation and the increase in the expression of TGF-β₁ and TNF-α in cardiac fibroblasts induced by AngⅡ (0.1 μmol/L) or EFS (10 V 1.0 Hz). CaMKⅡ inhibitors (0.5 μmol/L AIP or 1.0 μmol/L AIP) also prevented the increase in mRNA expression of collagen Ⅰ and Ⅲ induced by 0.1 μmol/L AngⅡ. CONCLUSION: Inhibition of CaMKⅡ prevents the proliferation of cardiac fibroblasts induced by AngⅡ or EFS. The possible mechanism of CaMKⅡ inhibitors may be involved in preventing the mRNA expression and release of cytokines (TGF-β₁ and TNF-α), and regulating collagen I and III expression.     

Effects of Sini decoction on the expression of TGF-β1 in balloon injured iliac artery of rabbits

AIM:To investigate the effect of Sini decoction (SND) on vascular stenosis and the expression of transfoming growth factor-β1 (TGF-β₁) in iliac artery balloon injured rabbits. METHODS:24 male New Zealand albino rabbits were divided into three groups:control group, model group and SND treatment group. The iliac arteries were injured by balloon in model and SND groups. Four weeks later, serum TGF-β₁ level was assayed by ELISA. Endothelial hyperplasia, TGF-β₁ protein and mRNA expression were observed in injured iliac artery. RESULTS:Light microscope showed that the vascular lumina were narrower, intima was thicker in model group control and SND treatment group. The serum TGF-β₁ level was lower in control than model group and SND treatment group, and the serum TGF-β₁ level in SND treatment group was lower than that in model group. Immunohistochemistry and RT-PCR results showed that TGF-β₁ protein and mRNA expression was lower in rabbit iliac artery of control group than that in model group and SND treatment group, and the expression of TGF-β₁ protein and mRNA decreased significantly in SND treatment group compared with model group. CONCLUSION:SND could lessen intimal hyperplasia and vascular stenosis in balloon injured iliac artery, which might be related to decrease in TGF-β₁ protein and gene expression in iliac artery.      

Effect of inhaled nitric oxide on aquaporin expression in newborn rat lung in acute hyperoxic lung injury

AIM:To investigate the effect of inhaled nitric oxide on aquaporin expression and alveolar epithelial fluid transport in newborn rats with acute hyperoxic lung injury. METHODS:32 newborn SD rats were randomized to breathe for 48 h room air (C), >95%O₂ (O), >95%O₂+10^-5 NO (NO only in the first 24 h, ONO), room air + NO (CN). Then, the rats were killed, the lung wet-to-dry weight ratio (Q_W/Q_D), the histology, and AQP1, AQP5, α₁-NKA, α-ENaC mRNA expressions in the lungs were measured. RESULTS:Compared with C group, the Q_W/Q_D in O group significantly increased (P<0.01), and AQP1 mRNA expression decreased significantly (P<0.01). Compared with O group, ONO group had a lower level of Q_W/Q_D (P<0.05), and AQP1 mRNA expression increased (P<0.05). AQP5 mRNA expression in all groups remained unchanged. CONCLUSION:In newborn rats with acute hyperoxic lung injury, inhaled 10-5 nitric oxide for 24 h may attenuate lung edema and increase AQP1 mRNA expression, suggesting that inhaled 10^-5 nitric oxide for 24 h may promote the AQP1 expression in lung in this model of acute lung injury.     

Effect of salvianolic acid B on expression of TGF-β1 receptors and Smad2 in activated mesangial cells

AIM: To study the mechanisms of salvianolic acid B (Sal B)antagonizing mesangial cell activation and kidney fibrosis through investigating the effect of Sal B on expression of transforming growth factor-β₁ (TGF-β₁) receptors and Smad2 in TGF-β₁-stimulated renal mesangial cell activation. METHODS: Mesangial cells was isolated and purified from rat kidney. TGF-β₁ was used to establish rat primary mesangial cell activation model and Smad2,Smad7 protein expression was detected. Sal B (10^-6 mol/L and 10^-5 mol/L) was employed to treat the cells; α-smooth muscle actin(α-SMA) expression was analyzed by immunofluorescence staining and Western blotting. Mesangial cells were treated with Sal B alone or additional with TGF-β₁,and TGF-β₁ receptor Ⅰ (TβRⅠ),TGF-β₁ receptorⅡ (TβRⅡ),Smad2 phosphorylation and Smad2 protein expression was determined by Western blotting. RESULTS: Cell ular model was established by incubating with 5 μg/L TGF-β₁ for 24 h,and in early stage Smad2 was significantly phosphorylated. Sal B (10^-6 mol/L and 10^-5 mol/L) could inhibit α-SMA expression,which was the biomarker of activated mesangial cells. In addition,in Sal B group,the protein expression of TβRⅠand TβRⅡ was significantly down-regulated while Smad2 phosphorylation in mesangial cells was inhibited. CONCLUSION: Sal B inhibits the TGF-β₁-Smad pathway,the protein expression of TβRⅠ,TβRⅡ and Smad2 phosphorylation in mesangial cells,which is probably one of the mechanisms of Sal B alleviating kidney fibrosis.     

Effect of Kang Xianling decoction on TGF-β1-Smad pathway in a unilateral ureteral obstruction rat model

AIM: To study the effect of Kang Xianling decoction,comprised of dahuang,danshen,taoren,niuxi and danggui,on TGF-β₁-Smad pathway in unilateral ureteral obstruction rat model.METHODS: Eighteen male SD rats were divided into 3 groups,sham group,model group and model group treated with Kang Xianling decoction randomly.Renal interstitial fibrosis model was established in rats by unilateral ureteral obstruction (UUO).After treatment for additional 14 d,parameters of hydroxyproline in obstructed kidney from 3 groups were analyzed.Rats were sacrificed and the pathological statuses of their kidneys were checked by HE staining and electron microscopy.Transforming growth factor-β₁ (TGF-β₁) mRNA in kidney tissue was determined by RT-PCR.TGF-β₁ receptor Ⅰ (TβRⅠ),TGF-β₁ receptorⅡ (TβRⅡ),phosphorylated Smad2 and Smad2 protein were determined by Western blotting.RESULTS: Parameters of hydroxyproline in animals of model group were significantly increased than those in sham operation group (P<0.05).The mRNA expression of TGF-β₁ and the protein expression of TβRⅠ,TβRⅡ,phosphorylated Smad2 and Smad2 in kidney tissue of animals in model group were significantly up-regulated.After intervention with Kang Xianling decoction,the above-mentioned up-regulated parameters,except TGF-β₁,were all significantly inhibited.Compared to model group,the pathological changes in renal tissues in treatment group were remarkable improved.CONCLUSION: Kang Xianling decoction inhibits the TGF-β₁-Smad pathway and the protein expression of TβRⅠ,TβRⅡ,phosphorylated Smad2 and Smad2,so as to decrease the level of collagen in obstructed kidney and to alleviate the renal interstitial fibrosis in UUO rats.     

The role of TGF-β signal protein Smad2/3 in tubulointerstitial fibrosis associated with unilateral ureteral obstruction in rats

AIM:To investigate the functional role of TGF-β₁ signal protein Smad2/3 in tubulointerstitial fibrosis associated with unilateral ureteral obstruction in rats. METHODS:The unilateral ureteral obstruction (UUO) model was induced by the ligation of left ureter. Rats were sacrificed at 1, 3, 7, 14, 21, and 28 days after UUO was initiated. TGFβ₁ protein, phosphorylated Smad2/3 and interstitial α-smooth muscle actin (α-SMA) expression were assayed by immunohistochemical staining. TGF-β1 mRNA in the obstructed kidney was analyzed with in situ hybridration. HE and Masson staining were used for histological and morphometric studies of the pathological change in obstructed kidney. RESULTS:The results showed that upregulation of TGF-β₁ in tubulointerstitium of both cortex and medulla at day 3 (a 3.1 fold increase vs control, P<0.05) when interstitial volume started to increase significantly. The highest expression of TGF-β1 was detected at day 7 (6.2 folds vs control, P<0.01). Phosphorylated Smad2/3, mainly detected in the nucleus of tubular cells, were also markedly upregulated at day 3 (a 3.5 fold increase vs control, P<0.05), and this was steadily increased by day 7 (7.8 folds vs control, P<0.01). The expression of interstitial α-SMA in both cortex and medulla was evident at day 3 (a 3.8 fold increase vs control, P<0.05) and peaked by day 7 (9.2 folds vs control, P<0.01). The deposit of extracellular matrix (ECM) and interstitial volume in renal cortex and medulla continued to increase until day 28 in obstructed kidney. CONCLUSION:These findings suggest that TGF-β₁ signal protein Smad2/3 may play an important role in tubulointerstitial fibrosis associated with unilateral ureteral obstruction in rats.     

Effects of thrombospondin 1 on rat cardiac fibroblasts induced by transforming growth factor β1

AIM:To investigate the effects of thrombospondin 1 on transforming growth factor β₁ induced rat cardiac fibroblasts (CFs). METHODS: CFs of neonatal Sprague -Dawley (SD) rats were isolated with the method of digestion and differential anchoring velocity. The proliferation and collagen synthesis of rat CFs were observed with MTT and hydroxyproline. The expression of TSP-1mRNA was analyzed by RT-PCR.RESULTS: The dose and time-dependent effects of TGF-β₁ were observed. Expression of TSP-1 was increased significantly (P<0.01). Stimulation of CFs with TGF-β₁ (20 μg/L, 24 h) significantly increased CFs proliferation and collagen synthesis (P<0.01). TSP-1 antisense oligonucleotide effectively inhibited TGF-β₁ induced CFs proliferation and collagen synthesis (P<0.01).CONCLUSION:The proliferation and collagen synthesis of CFs induced by TGF-β₁ are inhibited by TSP-1 antisense oligonucleotide, which may exert helpful effect on anti-fibrosis.     

Effect of angelica on myocardial fibrosis post myocardial infarction in rats

AIM: To investigate 1) the role of transforming growth factor-β₁ (TGF-β₁) and macrophage infiltration during the development of myocardial fibrosis (MF) in rats after myocardial infarction (MI);and 2) mechanisms of MF post-MI and the inhibitory effect of angelica.METHODS: Sprague-Dawley (SD) rats were subjected to MI by ligating the left anterior descending coronary artery.The animals were randomly divided into three groups: sham, MI and MI+angelica.After 24 hours of ligation, rats received angelica (20 mL·kg^-1·d^-1, ip) or saline.Left ventricular hemodynamics were measured and rats were killed at week 1, week 2 and week 4, respectively.Collagen content, macrophage infiltration and TGF-β₁ expression were examined in the non-infarcted area.RESULTS: ① In MI group, the numbers of macrophage and TGF-β₁ expression were significantly upregulated compared to sham at week 1 post-MI and remained elevated at week 4 (P<0.01).Angelica significantly decreased macrophage infiltration and TGF-β₁ expression (P<0.01 vs MI).② Collagen content was increased significantly in MI group compared to sham at week 2 and week 4 (P<0.01), and decreased in MI+angelica group (P<0.05 vs MI).③ Cardiac function was markedly decreased post-MI in MI group (P<0.01), and improved at week 4 in MI+angelica group (P<0.05).CONCLUSION: In MF post-MI, angelica may have an antifibrotic effect by decreasing macrophage infiltration and TGF-β₁ expression, by which reactive myocardial fibrosis is reduced, and cardiac function is improved.     

Effects of rhTGF-β1 and TGF-β1 gene transfection on the proliferation of cultured rabbit corneal endothelial cells in vitro

AIM: To investigate the effects of rhTGF-β₁ and TGF-β₁ gene transfection on the proliferation of cultured rabbit corneal endothelial cells in vitro. METHODS: Cell growth induced by various concentrations of rhTGF-β₁ was determined by MTT proliferation assay. Under the induction of liposomes, recombinant pSecTag2-TGF-β₁MP vectors were transferred into the corneal endothelial cells. Morphological changes of transfected cells were observed by HE staining. The expression levels of TGF-β₁ were assessed by ELISA. Cell cycle analysis was assessed by flow cytometry. DNA fragment analysis was used to confirm the presence of apoptosis. RESULTS: rhTGF-β₁ in concentrations of 5-20 μg/L showed a significant suppressive effect on the proliferation of corneal endothelial cells, 0.5-1 μg/L had no effect, 0.05-0.1 μg/L facilitated cell growth, as compared with negative controls. The morphous of transfected corneal cells had no significant abnormality compared with normal cells. According to the result of ELISA, the concentration of TGF-β₁ in the supernatant was calculated to be (98±3) ng/L. Flow cytometry assay showed that S and G₂/M phase of transfected cells decreased significantly compared with that of control group, but the cell cycle recovered normally after adding 10 μg/L EGF into the culture medium. Agarose electrophoresis didn′t show marked ladders in transfected group. CONCLUSION: Effects of rhTGF-β₁ on the proliferation of corneal endothelial cells are different with various concentrations. TGF-β₁ gene transfection shows suppressive effect on the proliferation of cultured corneal endothelial cells, but does not induce cell apoptosis. EGF is the antagonist of this suppressive effect.