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.     

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.     

Effects of rosiglitazone on expressions of peroxisome proliferator-activated receptor gamma and TGF-β1 in rats with adriamycin nephrosis

AIM: To investigate the expression of renal peroxisome proliferator-activated receptor gamma (PPARγ) in rats with adrimycine nephrosis (ADR), and the effect of rosiglitazone on the activation of NF-κB p65 in renal tissue rats with ADR. METHODS: The rats were randomly assigned to following groups: control (CTR) group, adrimycine nephrosis (ADR) group, and ADR treated with rosiglitazone (5 mg·kg^-1·d^-1) group（RGL）. The levels of urinary protein, albumin, total cholesterol, triglyceride and renal function change in rats were measured after 12 weeks. The nuclear-translocation of cortical NF-κB p65 was detected by immunohistochemistry. The activity of cortical NF-κB p65 was measured by sandwich ELISA. The mRNA levels of cortical PPARγ and TGF-β₁ were detected by RT-PCR. The protein expressions of PPARγ and TGF-β₁ in the rat kidney tissues were detected by Western blotting. RESULTS: As compared to ADR group, the urinary protein excretion in RGL treatment group was decreased and the serum albumin levels were increased, but the serum total cholesterol and triglyceride were decreased and the renal pathological lesion was ameliorated. The activity of NF-κB p65 and the expressions of TGF-β₁ mRNA and protein were significantly decreased in rosiglitazone group, while the expression of PPARγ mRNA and protein was increased in RGL group (P<0.01). The correlation analysis was manifested: in ADR and RGL group, a negative correlation between the activity of NF-κB p65 and the expression of PPARγ in renal tissue (r=-0.8305, P<0.01) was observed. There was a negative correlation between the expression of TGF-β₁ and PPARγ in renal tissues (r=－0.7938, P<0.01). CONCLUSION: The expression of renal cortical PPARγ is up-regulated in rats with adrimycine nephrosis by rosiglitazone. Rosiglitazone inhibits the activation of renal cortical NF-κB p65 in part, so it inhibits the gene expression of renal TGF-β₁ and relieves the renal pathological lesion.     

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.     

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.     

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 IOX1 on proliferation,apoptosis and extracellular matrix-related protein expression in LX2 cells induced by TGF-β

AIM To investigate the effects of histone demethylase inhibitor IOX1 (5-carboxy-8-hydroxyquinoline) on the proliferation, apoptosis and extracellular matrix (ECM)-related protein expression in transforming growth factor-β (TGF-β)-induced human hepatic stellate LX2 cells. METHODS The proliferation and apoptosis of the LX2 cells were determined by real-time cell analysis and flow cytometry, respectively. The level of histone H3 lysine 9 dimethylation (H3K9me2) and the protein expression of ECM-related molecules [α-smooth muscle actin (α-SMA), collagen type I (Col I), matrix metalloproteinase-1 (MMP-1) and tissue inhibitor of metalloproteinase-1 (TIMP-1)] in the LX2 cells were detected by Western blot. RESULTS Treatment with IOX1 at 50~300 μmol/L significantly inhibited LX2 cell proliferation, and 300 μmol/L IOX1 significantly promoted the apoptosis of the LX2 cells. In addition, different concentrations of IOX1 increased the levels of H3K9me2 and MMP-1, and down-regulated the expression of α-SMA, Col I and TIMP-1 in TGF-β-induced LX2 cells (P<0.05). CONCLUSION Treatment with IOX1 inhibits the proliferation of LX2 cells induced by TGF-β, promotes the cell apoptosis, and regulates the synthesis and metabolism of ECM by elevating H3K9me2 level, thus attenuating hepatic fibrosis.     

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.     

Effects of Astragalus injection combined with puerarin injection on expression of BMP-7 and TGF-β1 in type 2 diabetic KKAy mouse kidney

AIM: To observe the effects of Astragalus injection combined with puerarin injection on the expression of transforming growth factor beta 1 (TGF-β₁) and bone morphogenetic protein 7 (BMP-7) in the kidney of type 2 diabetic KKA^y mouse. METHODS: The male KKA^y mice of 14 weeks old were randomly divided into model group and Astragalus injection combined with puerarin injection treatment (Astragalus+puerarin) group. The age-matched male C57BL/6J mice were selected as normal group. The general conditions and body weight of the mice were observed. Blood glucose (BG), triglyceride (TG), cholesterol (TC) and serum creatinine (SCr) were examined at the 20th, 24th and 28th week. The protein expression of renal TGF-β₁ was determined by immunohistochemical method. The mRNA expression of BMP-7 and TGF-β₁ was detected by RT-PCR. RESULTS: Compared with normal group, the body weight, BG, TG, TC and SCr increased significantly in model group. TGF-β₁ expression at protein and mRNA levels was increased, while mRNA expression of BMP-7 was decreased in KKA^y mice. Compared with model group, the body weight, BG, TG, TC and SCr reduced in Astragalus+puerarin group. The mRNA expression of BMP-7 in the renal tissues was higher, and TGF-β₁ expression at mRNA and protein levels was significantly lower in Astragalus+puerarin group than those in model group. CONCLUSION: Astragalus injection combined with puerarin injection has renal protective effects on type 2 diabetic KKA^y mice. The mechanism may be related to restoring BMP-7 expression and reducing the overexpression of TGF-β₁ in renal tissues.     

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.     

Influence of losartan potassium on renal expression of TGF-β1, CD68 and MCP-1 in type 2 diabetic nephropathy rats

AIM: To observe the effects of losartan potassium on renal expression of transforming growth factor beta 1(TGF-β₁), CD68 and monocyte chemoattractant protein-1 (MCP-1) in type 2 diabetic nephropathy rats for exploring the protective mechanism of losartan potassium on type 2 diabetic rat kidney. METHODS: Thirty Sprague-Dawley rats were randomized into 3 groups: normal control group, model group and treatment group. The morphology of kidney tissues, the renal function, and the change of 24 h urinary protein quantitative index were measured after 15 weeks of treatment, while TGF-β₁, CD68 and MCP-1 expression in kidney cortex was observed by immunohistochemistry. RESULTS: Compared with the normal control rats, the body weight of the rats was lower in other groups, but the levels of blood glucose, triglyceride and cholesterol were higher.The expression of CD68, MCP-1 and TGF-β₁, 24 h urinary protein quantitative index and serum creatinine were higher in model group than those in normal control rats. However, compared with model group, serum creatinine, 24 h urinary protein quantitative index and the expression of CD68, MCP-1 and TGF-β₁ were decreased in treatment group. CONCLUSION: Losartan potassium protects the kidney of diabetic nephropathy rats through inhibiting the expression of TGF-β₁ and MCP-1 in the kidney and restraining macrophage infiltration.     

Role of TGF-β1/Smads and ERK expression in vascular remodeling induced by high-salt diet in Wistar rats

AIM: To investigate the role of transforming growth factor β₁ (TGF-β₁)/Smads and extracellular signal-regulated kinase(ERK) expression in vascular remodeling induced by high-salt diet in Wistar rats. METHODS: Wistar rats were randomly divided into 3 groups: normal control group (n=13), high salt (8%) model group and high salt+telmisartan group (n=13). Tail-cuff arterial pressure was determined every 2 weeks. After 24 weeks, the rats in high salt model group were divided into model animals with hypertension group (MH, n=12) and model animals without hypertension group (MN, n=12). The remodeling of aorta and mesenteric artery was observed by HE and Masson staining. In addition, the techniques of immunohistochemistry and real-time PCR were applied to detect the expression of proliferating cell nuclear antigen (PCNA), TGF-β₁, p-Smad2/3, p-ERK1/2 and Smad7 at both protein and mRNA levels. RESULTS: Compared with normal control group, blood pressure in MH group was much higher, and media thickness (MT) and collagen volume fraction (CVF) of arteries in MH and MN groups were higher.The mRNA expression of TGF-β₁, Smad2 and Smad7 in the aorta was significantly increased, and the protein levels of PCNA, p-ERK1/2, TGF-β₁ and p-Smad2/3 in the aorta and mesenteric artery media were elevated, but Smad7 decreased. After telmisartan treatment, MT and CVF were much lower,and the protein levels of PCNA, TGF-β₁, p-Smad2/3 and p-ERK1/2 were significantly reduced, whereas Smad7 was increased. CONCLUSION: The abnormal expression of TGF-β₁/Smads and ERK may be involved in the mechanism of remodeling of aorta and mesenteric artery induced by high-salt diet. Telmisartan prevents the vascular remodeling via regulating TGF-β₁/Smads and ERK signal pathways mediated by angiotensinⅡ type 1 (AT₁) receptor, at least in part.     

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.