circRNA-42398 inhibits activation of hepatic stellate cells via TGF-?β1/Smads signaling pathway modulation

AIM To study the effect of mouse circular RNA-42398 (mmu_circ_42398) over-expression on the activation of hepatic stellate cells. METHODS Mouse hepatic stellate JS1 cells were cultured and randomly divided into control group, vector group and mmu_circ_42398 over-expression group.mmu_circ_42398 over-expression plasmid vector was constructed, and then transiently transfected into JS1 cells using Lipofectamine 2000. The cells were collected 48 h after transfection. Expression of mmu_circ_42398 was detected by RT-qPCR.The backsplice site of PCR products was verified by sequencing. The protein levels of α-smooth muscle actin (α-SMA), collagen type I （Col I), transforming growth factor β1(TGF-β1), Smad2, Smad3, p-Smad2 and p-Smad3 in the cells were determined by Western blot. RESULTS RT-qPCR results showed that the expression of mmu_circ_42398 was significantly increased after mmu_circ_42398 over-expression vector was transiently transfected into the JS1 cells (P<0.01). The protein expression levels of α-SMA and Col I were significantly decreased(P<0.01), and the phosphorylation levels of Smad2 and Smad3 were decreased significantly in mmu_circ_42398 over expression group (P<0.01). However, the protein expression levels of TGF-β1, Smad2 and Smad3 had no significant change (P>0.05). CONCLUSION mmu-circ-42398 inhibits the activation of hepatic stellate cells via TGF-β1/Smads signaling pathway modulation.     

Effect of c-SKI on coronary endothelial cell proliferation and endothelial-mesenchymal transition

AIM: To investigate the effect of cellular Sloan-Kettering Institute (c-SKI) on the proliferation and endothelial-mesenchymal transition of human coronary artery endothelial cells (HCAECs). METHODS: HCAECs were treated with transforming growth factor-β1 (TGF-β1) at varying concentrations for different time points. Western blot was used to test the expression of c-SKI and mesenchymal markers such as α-smooth muscle actin (α-SMA) and vimentin. Meanwhile, the endothelial marker E-cadherin was also detected. HCAECs were transfected with c-ski gene mediated by lentivirus (LV), the efficiency of LV-SKI transfection was detected by RT-qPCR. The HCAECs were divided into 4 groups:control group, TGF-β1 (5 μg/L) group, LV-SKI+ TGF-β1 group, LV-NC+ TGF-β1 group. The cell viability and colony formation were measured by MTT assay and colony formation assay. The protein levels of vimentin, α-SMA, E-cadherin, Smad2, Smad3, p-Smad2 and p-Smad3 were determined by Western blot. RESULTS: The expression of c-SKI was down-regulated in the HCAECs treated with TGF-β1 (P<0.01). Over-expression of c-SKI inhibited the proliferation of HCAECs (P<0.01). Compared with LV-NC group, over-expression of c-SKI down-regulated the expression of α-SMA and vimentin (P<0.01), up-regulated the expression of E-cadherin (P<0.01), and inhibited the protein phosphorylation of Smad2 and Smad3 (P<0.01), reversed the endothelial-mesenchymal transition induced by TGF-β1. CONCLUSION: The expression of c-SKI in the HCAECs is down-regulated in the process of endothelial-mesenchymal transition. Over-expression of c-SKI inhibits proliferation and endothelial-mesenchymal transition of HCAECs, the mechanism may be related to regulation of the TGF-β1/Smad signaling pathway.     

Up-regulation of miR-181a attenuates releases of CSE-induced pro-inflammatory factors and expression of collagen IV,fibronectin and α-SMA in human bronchial epithelial cells

AIM: To investigate the effect and potential mechanism of microRNA-181a (miR-181a) on cigarette smoke extract (CSE)-induced the productions of pro-inflammatory factors and the expression of collagen IV, fibronectin and α-smooth muscle actin (α-SMA) in human bronchial epithelial cells (HBECs). METHODS: CSE-induced miR-181a expression was detected by RT-qPCR in the HBECs. After tansfected with miR-181a mimic, the releases of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), IL-6 and transforming growth factor-β1 (TGF-β1) were measured by ELISA, the protein expression of collagen IV, fibronectin and α-SMA was determined by Western blot. The activation of NF-κB/TGF-β1/Smad3 pathway was also evaluated by Western blot. RESULTS: CSE increased the levels of TNF-α, IL-1β, IL-6 and TGF-β1 and the expression of collagen IV, fibronectin and α-SMA, and decreased the expression of miR-181a in the HBECs (P<0.05). However, transfected with miR-181a mimic partially prevented the releases of TNF-α, IL-1β, IL-6 and TGF-β1, and inhibited the expression of collagen IV, fibronectin and α-SMA (P<0.05). Additionally, the activation of NF-κB/TGF-β1/Smad3 evoked by CSE was attenuated after transfected with miR-181a mimic. CONCLUSION: Up-regulation of miR-181a prevents the releases of CSE-induced pro-inflammatory factors and expression of collagen IV, fibronectin and α-SMA in the HBECs, and its mechanism may be related to the inhibition of NF-κB/TGF-β1/Smad3 pathway.     

Role of EndoMT in HHPH based on TGF-β/Smads signaling pathway and regulated by Yiqi-Wenyang-Huoxue-Huatan formula

AIM: To investigate the relationship between transforming growth factor-β (TGF-β)/Smads signaling pathway and pulmonary arterial endothelial-mesenchymal transition (EndoMT) in hypoxia-hypercapnia pulmonary hypertension (HHPH) process and the regulatory effect of Yiqi-Wenyang-Huoxue-Huatan formula (YWHHF). METHODS: Healthy male SD rats were randomly divided into 5 groups:normal control (N) group, hypoxia-hypercapnia (HH) group, high-dose YWHHF (YH) group, middle-dose YWHHF (YM) group and low-dose YWHHF (YL) group. The rats in N group was housed in normoxic environment, and the rats in the other 4 groups were housed in hypoxia-hypercapnia environment (9%~11% O₂ and 5%~6% CO₂) for 4 weeks, 8 h/d, 6 d/week. The excess water vapor was absorbed by anhydrous CaCl₂, and CO₂ was absorbed by sodium hydroxide. The rats in YWHHF groups were put into the oxygen chamber before the same volume of YWHHF at different concentrations were given (200 g/L for YH group, 100 g/L for YM group and 50 g/L for YL group). The average pulmonary artery pressure and the average carotid artery pressure were measured during the operation. After operation, the right ventricular free wall and left ventricle plus interventricular septum were collected for determining the right ventricular hypertrophy index. Moreover, the morphological changes of the lung tissues were observed under light microscope. The mRNA and protein levels of α-smooth muscle actin (α-SMA), CD31, TGF-β1 and Smad2/3, and the protein level of p-Smad2/3 were detected by RT-PCR and Western blot. RESULTS: Compared with N group, the pulmonary artery mean pressure, the mRNA and protein expression of α-SMA, TGF-β1 and Smad2/3, and the protein level of p-Smad2/3 were increased, the levels of CD31 were decreased (P<0.05), and the lung tissue damage was observed in the other 4 groups. Compared with HH group, the pulmonary artery mean pressure, the mRNA and protein expression of α-SMA, TGF-β1 and Smad2/3, and the protein level of p-Smad2/3 were decreased, while the mRNA and protein levels of CD31 were increased. Moreover, the lung tissue damage was reduced in YH, YM and YL groups. CONCLUSION: TGF-β/Smads pathway may be involved in the process of EndoMT under hypoxia and hypercapnia condition, and YWHHF may reduce EndoMT by inhibiting the expression of TGF-β/Smads pathway-related molecules.     

Pycnogenol suppresses TGF-β1-induced hepatic stellate cell activation via ERK-mediated autophagy inhibition

AIM: To explore the effect of Pycnogenol on transforming growth factor-β1 (TGF-β1)-induced hepatic stellate cell activation. METHODS: Cultured LX-2 cells were treated with 5 μg/L TGF-β1 and different concentrations (0, 10, 25 and 50 mg/L) of Pycnogenol. The viability of the LX-2 cells under the conditions with or without autophagy inhibitor 3-MA and ERK inhibitor PD98059 was determined by MTT assay. The protein levels of α-SMA, ColⅠ, TIMP-1, LC3-Ⅱ/Ⅰ, beclin 1, p-ERK1/2 and ERK1/2 were detected by Western blot. RESULTS: Compared with control group, 5 μg/L TGF-β1 treatment elevated the cell viability, and increased the protein levels of α-SMA, ColⅠ, TIMP-1, LC3-Ⅱ/Ⅰ, beclin 1, p-ERK1/2, and ERK1/2 in the LX-2 cells (P<0.05). However, these effects were reversed by Pycnogenol pretreatment in a dose-dependent manner and the inhibitory effect of 50 mg/L Pycnogenol was the most significant in the LX-2 cells (P<0.05). Furthermore, compared with TGF-β1 group, pretreatment with 50 mg/L Pycnogenol, 5 mmol/L 3-MA or 20 μmol/L PD98059 downregulated TGF-β1-induced cell viability and the protein levels of α-SMA and LC3-Ⅱ/Ⅰ in the LX-2 cells (P<0.05). CONCLUSION: Pycnogenol suppresses TGF-β1-induced hepatic stellate cell activation via p-ERK and autophagy inhibition.     

Inhibitory effect of oxymatrine on high glucose-induced rat renal tubular epithelial-mesenchymal transition

AIM:To investigate the inhibitory effect of oxymatrine (OM) on high glucose-induced rat renal tubular epithelial-mesenchymal transition (EMT). METHODS:The rat renal tubular epithelial NRK52E cells were cultured in vitro. The cells were divided into control group, high glucose group, high glucose+different concentrations of OM groups and high glucose+0.50 g/L OM dynamic observation group. The expression of TGF-β1, Smad7, α-SMA and E-cadherin at mRNA and protein levels was detected by real-time PCR and Western blotting. The viability of NRK52E cells was determined by MTT assay. RESULTS:(1) Compared with control group, the expression of TGF-β1 and α-SMA at mRNA and protein levels in high glucose group gradually increased, and Smad7 protein and E-cadherin mRNA and protein gradually reduced, but the mRNA expression of Smad7 gradually increased. (2) Compared with high glucose group, as increases in OM doses, the expression of TGF-β1 and α-SMA at mRNA and protein levels in high glucose+different concentrations of OM groups gradually reduced, and Smad7 protein and E-cadherin mRNA and protein gradually increased, but the mRNA expression of Smad7 had no significant change. (3) Compared with high glucose group, the expression of TGF-β1 and α-SMA at mRNA and protein levels was significantly reduced, the expression of E-cadherin at mRNA and protein levels significantly increased, and the protein expression of Smad7 significantly increased, but the mRNA expression of Smad7 had no significant change in high glucose+0.50 g/L OM dynamic observation group. CONCLUSION: In NRK52E cells, oxymatrine inhibits high glucose induced EMT by down-regulating TGF-β1 and up-regulating Smad7, thus preventing the fibrosis effect of TGF-β1/Smads signaling.     

Effect of salvianolic acid B on high glucose-induced phenotypic transition and extracellular matrix secretion in human glomerular mesangial cells

AIM:To investigate the effect of salvianolic acid B (Sal B) on high glucose-induced phenotypic transition and extracellular matrix (ECM) secretion in human glomerular mesangial cells (HGMCs) and the underlying mechanisms. METHODS:HGMCs were randomly divided into control group, high glucose group and high glucose plus high dose, medium dose and low dose of Sal B groups. The HGMCs except those in control group were exposed to high glucose (33.3 mmol/L) for 72 h, while those in Sal B groups were co-incubated with indicated concentrations of Sal B. The protein levels of α-smooth muscle actin (α-SMA), transforming growth factor-β₁ (TGF-β₁) and phosphorylated Smad2 and p38 mitogen-activated protein kinase (MAPK) were determined by Western blot. The secretion levels of collagen type I (Col I), collagen type Ⅲ (Col Ⅲ), fibronectin (FN) and laminin (LN) were measured by ELISA. RESULTS:Exposure to high glucose markedly increased the protein expression of α-SMA, TGF-β₁, Col I, Col Ⅲ, FN and LN in the HGMCs (P<0.01). The phosphorylation levels of Smad2 and p38 MAPK were also significantly increased (P<0.01). Co-incubation with Sal B evidently decreased the protein expression of α-SMA, TGF-β₁, Col I, Col Ⅲ, FN and LN in the HGMCs induced by high glucose (P<0.05 or P<0.01). The phosphorylated levels of Smad2 and p38 MAPK were also reduced noticeably (P<0.05 or P<0.01). CONCLUSION:Sal B significantly suppresses high glucose-induced phenotypic transition and ECM secretion in the HGMCs, which might be attributed, at least partly, to inhibition of TGF-β₁/Smad signaling pathway and p38 MAPK activation.     

Role of TGFβ1/Smad3 signaling pathway-mediated lysine hydroxylase 2 activity in collagen deposition of pulmonary fibrosis

AIM: To investigate the effect of TGFβ1/Smad3 signaling pathway on the changes of lysyl hydro-xylase2 (LH2) activity, and to study the role in the relationship between LH2 and collagen deposition of pulmonary fibrosis. METHODS: Human lung fibroblast cell line HFL1 was cultured in F12 medium with 10% fetal bovine serum. The cells were divided into control group, TGFβ1 (10 μg/L) stimulation group, and minoxidil (5 μmol/L) intervention group. The cells in control group were treated with the equivalent volume of medium. The RNA and protein were collected after 48 h. The mRNA levels of PLOD2, α-SMA and COLⅠ were detected by RT-qPCR. The protein levels of LH2, total Smad3, phosphorylated Smad3, α-SMA, COLⅠ and COL Ⅳ were determined by Western blot. Hydroxylysylpyridinoline (HP) content was detected by ELISA. RESULTS: After stimulation with TGFβ1, the mRNA expression of PLOD2, α-SMA and COLⅠ was increased (P<0.01), and the protein levels of LH2, p-Smad3, α-SMA, COLⅠ and COL Ⅳ were also up-regulated, but the total Smad3 protein did not change. Treatment with minoxidil decreased the levels of above indexes (P<0.01). Compared with control group, stimulation with TGFβ1 increased the content of HP. However, treatment with minoxidil decreased the synthesis of HP (P<0.05). CONCLUTION: Activation of TGFβ1/Samd3 signaling pathway enhances LH2 expression. Minoxidil inhibits the TGFβ1/Samd3 signaling transduction, thereby reducing the expression of LH2 and the synthesis of hydroxylysyl collagen pyridine chain, and reducing pulmonary fibrosis.     

Effect of miR-29b-mediated TGF-β/Smad signaling pathway on progression of hepatic fibrosis in rats

AIM: To investigate the role of microRNA-29b (miR-29b)-mediated TGF-β/Smad signaling pathway in the activation of hepatic stellate cells (HSC) and its effect on the progression of hepatic fibrosis in rats.METHODS: Hepatic liver fibrosis rat model was established, and its HSC were isolated. Normal rat HSC were also obtained and identified in vitro. RT-qPCR and Western blot were used to detect the alterations of miR-29b, TGF-β/Smad signaling pathway-related proteins and liver fibrosis marker proteins in the acquired cells. Finally, the direct targeting binding of miR-29b to TGF-β1 was identified by dual-luciferase reporter assay system.RESULTS: With the activation of HSC, the expression of miR-29b gradually decreased (P<0.01), while the expression of collagen type I and α-smooth muscle actin gradually increased (P<0.01). At the same time, the expression of Smad2/3/4 was significantly increased, and the expression of Smad7 was significantly decreased (P<0.01). Dual-luciferase reporter assay showed that miR-29b bound directly to "UCUCUCCGU" in the 3'UTR of TGF-β1, indicating that TGF-β1 was a downstream target gene of miR-29b.CONCLUSION: miR-29b may be involved in the inhibition of HSC activation and migration, thereby inhibiting the process of liver fibrosis. The biological function of miR-29b may be through the direct targeting of TGF-β1, thus regulating and inhibiting the TGF-β/Smad signaling pathway.     

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.     

Inhibitory effect of metformin on alveolar epithelial-mesenchymal transition in lung tissue of rats with pulmonary fibrosis

AIM: To study the inhibitory effect of metformin on alveolar epithelial-mesenchymal transition (EMT) in rats with pulmonary fibrosis and the possible mechanism. METHODS: SD rats (n=48) were used, 12 of which were set up as normal control group, and 36 of which were induced by bleomycin (5 mg/kg) by tracheal instillation to establish pulmonary fibrosis. The pulmonary fibrosis rats were randomly divided into bleomycin group, low dose (100 mg/kg) of metformin group, and high dose (300 mg/kg) of metformin group. The rats in metformin groups were given the corresponding dose of metformin daily for 4 weeks. HE staining and Masson staining were used to observe the changes of lung histopathology and collagen deposition. Real-time PCR, Western blot and innunohistochemical staining were used to detect the mRNA and protein expression of α-smooth muscle actin (α-SMA), E-cadherin, vimentin, zonula occludens-1 (ZO-1), collagen I, collagen III and transforming growth factor-β₁ (TGF-β₁), and the protein phosphorylation levels of Smad2/3 and extracellular signal-regulated kinase 1/2 (ERK1/2) were also determined. RESULTS: Metformin up-regulated the expression of E-cadherin and ZO-1, down-regulated the expression of α-SMA, vimentin, collagen I and collagen III, and the protein phosphorylation levels of Smad2/3 and ERK1/2 were also decreased (P<0.05). CONCLUSION: Metformin inhibits alveolar EMT in the rats with pulmonary fibrosis, and its mechanism may be related to the inhibition of TGF-β₁ signal transduction pathway.     

TAK1 regulates fibrosis of renal tubular epithelial cells by regulating p38 MAPK signaling pathway

AIM:To investigate the effect of transforming growth factor-β (TGF-β) activated kinase 1(TAK1) on renal tubular epithelial fibrosis. METHODS:The renal tubular epithelial cell line HK-2 was used as the research object. After induced by TGF-β1, real-time PCR and Western blot were used to detect the expression of TAK1 in the HK-2 cells. TAK1 shRNA lentivirus was used to infect HK-2 cells, real-time PCR and Western blot were used to determine the interference effect on TAK1 expression in the HK-2 cells with TGF-β1 stimulation. Under the condition of treating with p38 MAPK activator anisomycin, the levels of type I collagen and type Ⅲ collagen in the supernatant, and the protein levels of α-smooth muscle actin (α-SMA), connective tissue growth factor (CTGF) and p-p38MAPK^{Thr 180/Tyr 182} in the HK-2 cells with TAK1 knock-down were determined by ELISA and Western blot, respectively. RESULTS:TGF-β1 significantly increased the expression of TAK1 in the HK-2 cells(P<0.05). TAK1 shRNA significantly decreased the expression of TAK1 in the HK-2 cells with TGF-β1 stimulation. Type I collagen and type Ⅲ collagen secreted by the HK-2 cells after treatment with TGF-β1 were increased, the protein levels of α-SMA, CTGF and p-p38MAPK^{Thr 180/Tyr 182} were also increased(P<0.05). Knock-down of TAK1 expression significantly inhibited the secretion of type I and type Ⅲ collagen, reduced the protein levels of α-SMA, CTGF and p-p38MAPK^{Thr 180/Tyr 182} in the TGF-β1-induced HK-2 cells(P<0.05). Treatment with p38 MAPK activator reversed the inhibitory effect of TAK1 knock-down on the secretion of type I and type Ⅲ collagens, and the protein levels of α-SMA, CTGF and p-p38 MAPK^{Thr 180/Tyr 182} in the HK-2 cells(P<0.05). CONCLUSION:Knock-down of TAK1 expression attenuates the TGF-β1 induced fibrosis of renal tubular epithelial cells by inhibiting p38 MAPK signaling pathway.     

Mollugin inhibits viability and collagen synthesis of rat CFSC-2G cells

AIM: To investigate the effects of mollugin on the viability and collagen synthesis of rat hepatic stellate cell line CFSC-2G. METHODS: The activation of CFSC-2G cells was induced with low concentration (10 μmol/L) of hydrogen peroxide (H₂O₂) for 30 min in the experiment. The viability of the CFSC-2G cells after exposed to mollugin at different concentrations (0, 20, 40, 60 and 120 μmol/L) was detected by MTT assay. The mRNA and protein expression levels of nuclear factor E2-related factor 2 (Nrf2), heme oxygenase-1 (HO-1), nuclear factor-κB (NF-κB) p65, Bcl-2, Bcl-xL, Bax, and hepatic stellate cell activation markers α-smooth muscle actin (α-SMA) and collagen type I (Col Ⅰ) were detected by real-time PCR and Western blot. The phosphorylation level of p38 mitogen-activated protein kinase (p38 MAPK) was determined by Western blot. RESULTS: Mollugin significantly inhibited the viability and collagen synthesis of activated CSFC-2G cells induced by H₂O₂. The expression of Nrf2, HO-1 and Bax at mRNA and protein levels, and the phosphorylation level of p38 MAPK were promoted, while the levels of NF-κB p65, Bcl-2, Bcl-xL, α-SMA and ColⅠwere inhibited by mollugin (P<0.05). CONCLUSION: Mollugin may inhibit H₂O₂-induced viability and collagen synthesis of the CSFC-2G cells by activating Nrf2 and HO-1, and blocking the NF-κB p65 and Bcl-2 expression.     

Regulation of rat pulmonary fibroblasts differentiation into myofibroblasts though RhoA/ROCK pathway mediated by TGF-β1

AIM：To investigate the regulatory effect of RhoA/Rho-associated coiled-coil-forming protein kinase (ROCK) pathway mediated by transforming growth factor β1 (TGF-β1) on the differentiation of pulmonary fibroblasts into myofibroblasts. METHODS：Primarily cultured fibroblasts were obtained by trypsin digestion from the lung of neonatal rats. The fibroblasts were stimulated with TGF-β1 for different durations and were divided into control group, TGF-β1 induction group and Y-27632 treatment group. The distribution and expression of p-RhoA, ROCK, phosphorylated myosin binding subunit of myosin light chain phosphatase (p-MBS), serum response factor (SRF), α-smooth muscle actin (α-SMA),type I collagen and type Ⅲ collagen in the cells were detected by the methods of immunocytochemistry and Western blotting. RESULTS：A lot of parallel and cross arranged filaments labeled by α-SMA antibody appeared in the cells after TGF-β1 stimulation. The cultured cells stimulated with TGF-β1 were all myofibroblasts at 24 h determined by immunocytochemistry. The expression levels of p-RhoA, ROCK, p-MBS, SRF, α-SMA and type I and type III collagens were increased gradually with the extension of TGF-β1 stimulation time. The expression of RhoA/ROCK signaling protein in the cells stimulated with TGF-β1 (peaking at 6 h of exposure) was 2.96 folds higher as compared with the non-stimulated cells. The expression of SRF protein (peaking at 12 h of TGF-β1 exposure) was 4.55 folds higher as compared with the non-sti-mulated cells. The expression levels of α-SMA and type I and type III collagens (peaking at 24 h of TGF-β1 exposure) were 4.06 folds, 2.19 folds and 3.04 folds higher as compared with the non-stimulated cells, respectively. Compared with TGF-β1 induction group, the protein expression levels of ROCK, p-MBS, SRF, α-SMA and type I and type III collagens were significantly decreased at the corresponding time points in Y-27632 treatment group. CONCLUSION：TGF-β1 induces the differentiation of pulmonary fibroblasts into myofibroblasts, and then promotes the synthesis of collagen through the activation of ROCK pathway, which possibly plays an important role in the formation of pulmonary fibrosis.     

Effect of blood glucose control on expression of Smad7 and renal fibrosis in diabetic rats

AIM： To verify the hypothesis that treatment with insulin to control the blood glucose (BG) may relieve or slow down the development of diabetic nephropathy (DN) in diabetic rats by increasing the expression of Smad7. METHODS：The diabetic rat model was established by tail-vein injection of streptozotocin. Sixteen rats were divided into 2 groups. Eight of these animals in diabetes mellitus (DM) group had no treatment. The remaining eight of them in insulin treatment (INS) group were injected with insulin. After 13 weeks, the rats in INS group were given individual treatment with insulin to let the blood glucose level keep within 4 to 7 mmol/L. Meanwhile, 8 rats were used for normal control (NC group). After 16 weeks, the rats were sacrificed to detect the relevant biochemical parameters, and to observe the histophathological changes of the kidney and pancreas. In addition, immunohistochemical staining and Western blotting were employed to detect the protein expression of transforming growth factor β1 (TGF-β1), Smad ubiquitin regulatory factor 2 (Smurf2), Smad7, E-cadherin, α-sooth muscle actin (α-SMA), fibronectin (FN) and collagen I. RESULTS：Compared with NC group, the body weight was significantly reduced in DM group, whereas the body weight in INS group increased gradually. Compared with NC group, the levels of 24 h urine protein (24 h UP), BG and triglyceride (TG) were remarkably increased in DM group. Pathological detection on pancreas indicated that the islet was destroyed. The levels of TGF-β1, Smurf2, α-SMA, FN and collagenⅠ in the kidneys were increased in DM group, and the expression of Smad7 and E-cadherin, which were mainly located in renal tubular epithelial cells, was significantly reduced. Compared with DM group, the levels of 24 h UP and BG were significantly reduced in INS group, and the alleviated renal fibrosis was observed under light microscope. In addition, the protein levels of TGF-β1, Smurf2, α-SMA, FN and collagenⅠ in INS group were decreased compared with DM group, and the expression of Smad7 and E-cadherin was increased significantly. CONCLUSION：Target glucose control with insulin treatment restores the protein expression of Smad7 in the kidney of diabetic rats, reduces the accumulation of extracellular matrix and slows down DN progress. The decrease in TGF-β1 and Smurf2 expression, and the attenuation of Smad7 ubiquitination in renal tissues are the crucial parts in this process.     

Effects of Danshensu on TGF-β1/Smads signaling pathways in rats with pulmonary fibrosis

AIM: To study the preventive and curative roles of Danshensu (DA) in bleomycin (BLM)-induced pulmonary fibrosis in rats. METHODS: Pulmonary fibrosis was induced in SD rats by intratracheal instillation of BLM. The rats were intraperitoneally injected with dexamethasone (1 mg·kg-1·d-1, DXM group), DA (15 mg·kg-1·d-1, DA group), or physiological saline (2 mL·d-1, BLM group). Normal controls (NC group) received physiological saline both intratracheally and intraperitoneally. At the 28th day after modeling, the histological changes of the lungs were evaluated by hematoxylin-eosin (HE) and Masson’s trichrome staining. The protein levels of α-smooth muscle actin (α-SMA) in the lung tissues were detected by the method of immunohistochemistry. The mRNA expression of transforming growth factor beta 1 (TGF-β1), Smad3 and Smad7 was assessed by real-time fluorescence quantitative PCR. RESULTS: Compared with BLM group, the degree of inflammation and fibrosis of the lung in DA group was obviously reduced, and so was the expression of α-SMA in the lung tissues. The mRNA expression of TGF-β1 and Smad3 in the lung tissues of the rats decreased and the mRNA expression of Smad7 increased. CONCLUSION: DA alleviates BLM-induced pulmonary fibrosis in rats in the early stage by inhibiting the expression of TGF-β1/Smad3 and stimulating the expression of Smad7 in the lung tissues.     

Sedum sarmentosum Bunge extract inhibits epithelial-mesenchymal transition and collagen accumulation in aristolochic acid-treated rat renal tubular epithelial cells

AIM:To investigate the effect of Sedum sarmentosum Bunge (SSB) extract on epithelial-mesenchymal transition (EMT) and collagen accumulation induced by aristolochic acid (AA) in renal tubular epithelial cells. METHODS:Rat renal tubular epithelial NRK-52E cells were randomly divided into 3 groups, including control group (only treated with solvent), AA group (treated with AA at concentrations ranging from 1 to 100 mg/L) and SSB group (treated with AA at a concentration of 10 mg/L plus SSB extract at concentrations ranging from 10 to 2 000 mg/L). After cultured for 24 h, the morphology of the NRK-52E cells was observed under inverted phase-contrast microscope. The level of transforming growth factor β1 (TGF-β1) in the culture supernatant was measured by ELISA. Immunofluorescent analysis was performed to detect the expression of epithelial marker α-smooth muscle actin (α-SMA), mesenchymal marker E-cadherin, and extracellular cell matrix component type III collagen. The mRNA expression of E-cadherin, α-SMA, bone morphogenetic protein 7 (BMP-7) and type I collagen was also quantified by real-time PCR. RESULTS: Fibrosis-like reaction observed under microscope was obviously increased in AA-treated NRK-52E cells, and aggravated as the increase in the concentration of AA. AA at concentrations of 1 and 10 mg/L increased the expression of α-SMA, type I and type III collagens, and decreased the expression of E-cadherin. With SSB extract treatment, fibrosis in NRK-52E cells was alleviated, accompanied with the decreasing expression of α-SMA, type I and type III collagen, and the enhancing expression of E-cadherin and BMP-7.Moreover, SSB extract down-regulated TGF-β1 level in a concentration-dependent manner. CONCLUSION: AA-induced fibrosis-like reaction in renal tubular epithelial cells is reduced by the treatment with SSB extract. The possible mechanism is that SSB extract decreases TGF-β1 level, and inhibits renal EMT and collagen accumulation induced by AA.［KEY WORDS］Sedum sarmentosum Bunge|Aristolochic acid|Transforming growth factor β1|Epithelial-mesenchymal transition|Collagen     

Retinoid X receptor agonist inhibits TGF-β1-induced collagen synthesis in cardiac fibroblasts by repressing Smad2 activation

AIM: To investigate the effect of activation of retinoid X receptor (RXR) on transforming growth factor β1 (TGF-β1) induced collagen synthesis under hypoxic environment in rat cardiac fibroblasts (CFs) and underlying molecular mechanisms. METHODS: CFs were cultured using myocardial tissue with dry method. Hypoxic environment was established for CFs by continuous nitrogen supplement. Type I and type III collagens in supernatants were detected by ELISA. Nuclear and cytoplasmic extractions were prepared using NE-PER nuclear and cytoplasmic extraction reagents. The protein levels of Smad2 and p-Smad2 were determined by Western blot and immunocytochemical staining. RESULTS: Under hypoxic condition, TGF-β1 (0.01~10 μg/L) increased the synthesis of type I and type III collagens in a dose-dependent manner in the CFs. At the concentration of 5 μg/L, the synthesis of collagen I and III was significantly increased as compared with control group (P<0.01). RXR agonist 9-cis-retinoic acid (9-cis-RA; 10^-9~10^-6mol/L) decreased TGF-β1 (5 μg/L)-induced synthesis of type I and III collagens in a dose-dependent manner in the CFs under hypoxic condition. The synthesis of type I and type III collagens was significantly inhibited by 9-cis-RA (P<0.01). Smad2 inhibitor (20 nmol/L) showed similar inhibitory effect on the synthesis of type I and III collagens induced by TGF-β1 under hypoxic condition. Compared with TGF-β1 intervention group, the cytoplasmic level of p-Smad2 in the CFs was significantly increased in TGF-β1+9-cis-RA group, but the nuclear p-Smad2 level was significantly decreased (P<0.05). CONCLUSION: Retinoid X receptor agonist 9-cis-RA inhibits TGF-β1-induced synthesis of type I and type III collagens in the CFs by repressing p-Smad2 nuclear translocation under hypoxic condition.     

Effect of alpha-lipoic acid on expression of miR-21/Smad7 and fibrosis in kidney of diabetic rats

AIM: To investigate the protective effect of alpha-lipoic acid (ALA) on the kidney of the rats with diabetes mellitus (DM), and to discuss the mechanism. METHODS: The DM rats were divided into normal control (NC) group, DM group and ALA group. After treated with ALA for 6 weeks, the rats were sacrificed to detect the relevant biochemical parameters, and the pathological changes of the kidney tissues were observed by HE staining and Masson staining. The protein levels of transforming growth factor-β1 (TGF-1), p-Smad2/3, Smad7, collagen I and collagen Ⅲ were determined by Western blot. In addition, the expression of microRNA-21 (miR-21) was detected by RT-qPCR. RESULTS: Compared with NC group, the kidney weight/body weight, blood glucose (BG), total cholesterol, triglyceride and 24-h urine protein were remarkably increased in DM group (P<0.05). The pathological observation of the kidney tissues showed fibrosis changes in DM group. The level of Smad7 was reduced in DM group, while the levels of TGF-β1, p-Smad2/3, collagen I, collagen Ⅲ and miR-21 in the kidney tissues were increased (P<0.05). After treatment with ALA for 6 weeks, all the relevant biochemical parameters were reduced except BG, and the renal fibrosis lesions were obviously alleviated. Compared with DM group, the levels of TGF-1, p-Smad2/3, collagen I, collagen Ⅲ and miR-21 in the kidney tissues were reduced in ALA group, while the level of Smad7 was increased (P<0.05).CONCLUSION: ALA may prevent the development of renal fibrosis in rats through restraining the expression of TGF-β1 and miR-21, increasing the levels of Smad7 protein, and reducing the deposition of extra cellular matrix.     

Effect of CKLF1-C19 polypeptide on differentiation of human lung fibroblast into myofibroblast induced by TGF-β

AIM: To investigate the effect of CKLF1-C19 polypeptide (C19) on differentiation of human lung fibroblast (LFB) into myofibroblast (MFB) induced by TGF-β. METHODS: LFBs were cultured and identified. LFBs were treated with TGF-β (5 μg/L) to establish the cell model of LFB differentiate into MFB. The LFBs were divided into 6 experimental groups including control group, TGF-β group, and TGF-β plus different doses (1, 0.1, 0.01, 0.001 mg/L) C19 groups. The cell morphology, cell proliferation rate, and the expression of α smooth muscle actin (α-SMA) and collagen I were observed. RESULTS: Human primary LFB was successfully cultured and was confirmed by the method of immunofluorescence. TGF-β at 5 μg/L induced proliferation and differentiation of LFB. The mRNA levels of α-SMA and collagen I in TGF-β group were higher than that in control group (P<0.05).The cell proliferation rates, mRNA levels of α-SMA and collagen I, and the protein expression of α-SMA in 0.01 mg/L+TGF-β group and 0.001 mg/L+TGF-β group were markedly lower than those in TGF-β group (P<0.05). CONCLUSION: C19 at 0.01 mg/L and 0.001 mg/L effectively inhibits differentiation of LFB into MFB induced by TGF-β, thus inhibiting the process of airway remodeling and fibrosis to some extent.