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.     

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.     

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.     

Effects of arsenic trioxide on activities of MMPs and expression of TIMP-1 and TGF-β1 in skin fibroblasts and polymorphonuclear neutrophils

AIM: To observe the effects of arsenic trioxide (As₂O₃) on activities of matrix metalloproteinases (MMPs), expression of tissue inhibitor of metalloproteinase-1 (TIMP-1) and transforming growth factor beta1 (TGF-β₁) in human fibroblast (hFb), and to discuss weather As₂O₃ promotes the healing of chronic skin ulcer through regulating collagen metabolism. METHODS: Zymography was used for testing activity of MMP-9 deriving from rat polymorphonuclear neutrophils (PMNs) and activities of MMP-1, MMP-2 secreted by hFb. Immunocytochemical method was used to determine the expressions of TIMP-1 and TGF-β₁. RESULTS: At the concentration of 50 mg/L, As₂O₃ elevated the activity of MMP-9 (P<0.01). At the concentration of 0.8 mg/L, As₂O₃ increased the activities of MMP-1 and MMP-2 (P<0.01, respectively). After hFb was cultured with As₂O₃ for 6 h, 12 h and 18 h, the expressions of TIMP-1 and TGF-β₁ decreased continuously (P<0.01). CONCLUSION: As₂O₃ elevates the activities of MMP-1, MMP -2 and MMP-9, also inhibits the expressions of TIMP-1 and TGF-β₁, suggesting that arsenic preparation may exert positive effect on healing chronic skin ulcer through regulating collagen metabolism.     

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.     

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 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.     

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 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.     

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 icariin on expression of transforming growth factor β1 and type Ⅳ collagen in diabetic rat testis

AIM: To determine the beneficial effects of icariin on streptozotocin (STZ)-induced diabetic testopathy in rats. METHODS: The diabetic animal model was induced in male Sprague-Dawley rats by an injection of streptozotocin (40 mg/kg, iv). The rats were randomly divided into 3 groups: control group, model group and icariin (80 mg/kg, ig) group. Twelve weeks after injected with streptozotocin, all rats were anaesthetized and killed to remove the testes from scrotum. Serum concentrations of glucose and testosterone, and the levels of succinate dehydrogenase (SDH), acid phosphatase (ACP), γ-glutamyl transpeptidase (γ-GT) and lactate dehydrogenase (LDH) in testes were measured. The morphology of the testicular tissues was observed under light microscope. Immunohistochemistry was employed to determine the protein levels of TGF-β₁ and type Ⅳ collagen. RESULTS: Compared with control group, the content of serum glucose increased while the serum level of testosterone and the activitiy of SDH, ACP, γ-GT and LDH in testis decreased in model group (P<0.01). The histopathological examination showed that the diameters of seminiferous tubules and various grades of spermatocytes in the testis were markedly decreased. Compared with control group, the expression of TGF-β₁ and collagen Ⅳ was significantly increased in model group. These alterations were significantly attenuated in icariin group (P<0.01). CONCLUSION: Icariin evidently relieves testicular damage in rats with diabetic testopathy by improving the secretion of testosterone and reducing the expression of TGF-β₁ and collagen Ⅳ at protein level.     

Inhibitory effect of ground dragon on the expression of α-SMA and FN in the lung tissue of mouse with asthma

AIM: To investigate the inhibitory effect of ground dragon on the expression of α-SMA and FN in the lung tissue with asthma. METHODS: The BALB/c mice were divided into four groups: control group (group A, n=20), asthmatic model group (group B, n=20), large-dose ground dragon treatment group (group C, n=20) and low-dose ground dragon treatment group (group D, n=20). To establish a mouse model of chronic asthma, we sensitized the mouse with 0.02% ovalbumin (OVA) by intraperitoneal injection, and stimulated the mice with 1% OVA by atomization. The treatment groups were given ground dragon before stimulation every time. After the last time of stimulation, the mice were subjected to laboratory tests. Inflammatory cells in bronchoalveolar lavage fluid were counted. Total level of IgE in serum was detected by ELISA. FN mRNA and α-SMA mRNA in the lung tissue were measured by RT-PCR and AlphaImager 2200 semi-quantitation analysis system. Expressions of FN and α-SMA were measured by the method of two-step immunohistochemistry and leica QWIN V3 analysis system. RESULTS: (1) Compared with those in group A, the expressions of α-SMA and FN in group B were significantly increased (P<0.01). Compared with group B, those in group C were significantly decreased (P<0.01), while those in group D were slightly decreased (P>0.05). (2) Compared with those in group A, the expression levels of α-SMA mRNA and FN mRNA in group B had a great increase (P<0.01). There was a notably decreases of α-SMA mRNA and FN mRNA levels in group C, compared with group B (P<0.01). However, α-SMA and FN mRNA level in group D was just a slightly decreased, compared with group B (P>0.05). CONCLUSION: The ground dragon inhibits α-SMA and FN expression in the lung tissue of mice with chronic asthma, indicating that ground dragon may inhibit airway remodeling in asthma through the inhibition of α-SMA and FN expressions.     

Preventive effect of Sini decoction on expression of TGF-β1 in rat myocardiac fibrosis induced by isoproterenol

AIM: To explore the protective effects of Sini decoction (SD) on myocardial fibrosis induced by isoproterenol (Iso) in rats.METHODS: Nineteen Wistar rats were divided into Iso group, SD treatment group and control group. The rats in Iso group were injected with Iso and were then fed with saline. The rats in SD treatment group were injected with Iso and were then fed with SD. The rats in control group were injected with saline and were then fed with saline. The level of hydroxyproline (Hyp), the contents of angiotensin Ⅱ (AngⅡ) and transforming growth factor beta-1 (TGF-β1) in plasma were measured 4 weeks after administration. TGF-β1 at mRNA and protein levels were measured by the techniques of ELISA and RT-PCR. RESULTS: The plasma levels of TGF-β1 and AngⅡ were lower in control group than those in Iso group and SD treatment group (P<0.05). The plasma levels of TGF-β1 and AngⅡ in SD treatment group were lower than those in Iso group (P<0.05). Compared to Iso group, the cardiac diastolic function was significantly improved in SD treatment group (P<0.05). The results of immunohistochemistry and RT-PCR showed that the mRNA and protein expressions of TGF-β1 were lower in SD group than those in Iso group (P<0.05). CONCLUSION: SD alleviates myocardial fibrosis induced by Iso in rats by decreasing TGF-β1 expression at mRNA and protein levels.