Effects of MG132 on protein expression of SnoN and fibrosis-related indicators in NRK-52E cells after incubated with high concentration of glucose

AIM: To investigate the effects of proteasome inhibitor MG132 on the expression of SnoN in renal tubule epithelial cells incubated in high glucose, and to explore the possible mechanism and function that MG132 reduces or slows down renal tubular interstitial injury after incubated in high glucose. METHODS: The NRK-52E cells were divided into normal control group (NG), high glucose group (HG) and high glucose plus pretreatment with different doses of MG132 group (HG+MG132). The immunofluorescence staining was used to detect the protein expression of E-cadherin and α-smooth muscle actin (α-SMA) in NRK-52E cells under different conditions. The relative protein expression levels of SnoN, Smad ubiquitination regulatory factor 2 (Smurf2), Arkadia, E-cadherin, α-SMA and collagen type Ⅰ(Col-Ⅰ) were detected by Western blotting. RESULTS: Compared with NG group, the expression of E-cadherin and SnoN was decreased (P<0.05), while the expression of α-SMA, Col-Ⅰ, Smurf2 and Arkadia was increased (P<0.05). Compared with HG group, the protein expression of SnoN and E-cadherin was significantly up-regulated in HG+MG132 group (P<0.05), and the protein expression of α-SMA and Col-Ⅰ was significantly down-regulated in a dose-depended manner (P<0.05). However, no effect on the protein expression of Smurf2 and Arkadia was observed. CONCLUSION: MG132 inhibits the degradation of SnoN protein induced by high glucose, thus reducing the renal fibrosis.     

Effect of cyclopamine on aristolochic acid-induced phenotypic transformation and Hedgehog pathway in renal epithelial cells

AIM: To investigate the effect of cyclopamine on Hedgehog (HH) signaling, phenotypic transformation and matrix accumulation induced by aristolochic acid (AA) in renal tubular epithelial cell NRK-52E. METHODS: NRK-52E cells were randomly divided into control group (treated with solvent only), AA group (treated with AA at concentrations of 1, 5, 10 mg/L) and cyclopamine group (treated with AA at concentration of 10 mg/L plus cyclopamine at concentrations of 1, 5, 10 μmol/L). After cultured for 24 h, the mRNA expression of Ptch1, Smo, α-SMA, E-cadherin, ZO-1, BMP-7, type I collagen and type III collagen was quantified by real-time PCR. The protein levels of Shh and TGF-β1 were detected by ELISA. Immunofluorescence staining was used to evaluate the expression of Ptch1, Smo, α-SMA, E-cadherin and type III collagen in the NRK-52E cells. RESULTS: AA increased the expression of TGF-β1, α-SMA and type III collagen, decreased the expression of E-cadherin and ZO-1 protein, and down-regulated the expression of Ptch1, Shh and Smo mRNA in the NRK-52E cells, indicating that AA activated HH signaling, and phenotypic transformation and matrix accumulation occurred in AA-treated NRK-52E cells. Treatment with cyclopamine inhibited HH signaling by decreasing Smo expression and increasing Ptch1 expression. Moreover, cyclopamine also down-regulated the expression of TGF-β1, α-SMA, type I collagen and III collagen, and up-regulated the expression of BMP-7, ZO-1 and E-cadherin. CONCLUSION: AA induces phenotypic transformation and matrix accumulation in renal tubular epithelial cells, which can be inhibited by cyclopamine treatment. The possible mechanism is that cyclopamine suppresses the activation of HH signaling, resulting in the reduction of epithelial-to-mesenchymal transition and matrix deposition.     

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     

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.     

Role of ILK siRNA on expression of GSK-3β and β-catenin in human tubular epithelial cells stimulated by high glucose

AIM：To investigate the effects of siRNA targeting integrin-linked kinase (ILK) on the expression of glycogen synthase kinase 3β (GSK-3β) and β-catenin during epithelial-mesenchymal transition (EMT) in human kidney proximal tubular epithelial cell line HKC induced by high glucose. METHODS：HKC cells were divided into 4 groups:normal glucose (NG) group, high glucose (HG) group, HG+HK (a vector containing the non-specific siRNA designed as negative control) group and HG＋ILK siRNA group. The inverted fluorescence microscope was used to examine the expression of green fluorescent protein (GFP). The expression of ILK at mRNA and protein levels was detected by RT-PCR and Western blotting. The expression of p-GSK-3β and β-catenin was observed by immunocytochemical staining. The protein expression of total GSK-3β, p-GSK-3β, nuclear β-catenin, total β-catenin, E-cadherin and α-smooth muscle actin (α-SMA) was measured by Western blotting. RESULTS：GFP was observed in HKC cells, indicating that the transfection was successful. Both the protein and mRNA of ILK were down-regulated in HG＋ILK siRNA group compared with HG group and HG+HK group, but still higher than those in NG group. Silencing of ILK down-regulated the expression of p-GSK-3β and nuclear β-catenin. No difference of total GSK-3β or total β-catenin was observed among the 4 groups. CONCLUSION:These data support a functional role of ILK, GSK-3β and β-catenin in tubular EMT induced by high glucose. ILK may promote tubular EMT by regulating the activity of GSK-3β and β-catenin, the downstream effectors of the Wnt/β-catenin pathway.     

Effects of Nrf2 overexpression on proliferation,activation and collagen type I synthesis in cultured hepatic stellate cells stimulated by ethanol

AIM：To investigate the effects of nuclear factor E2-related factor 2 (Nrf2) overexpression on the proliferation, cell cycle distribution, collagen type I (Col I) synthesis and alpha-smooth muscle actin (α-SMA) expression in cultured hepatic stellate cell line HSC-T6 stimulated by ethanol. METHODS：Cultured HSC-T6 cells were transfected with pEGFP-Nrf2 or pEGFP-N1 (empty vector) plasmid by liposome transient transfection. The cells were divided into control group, ethanol group, ethanol+pEGFP-Nrf2 group and ethanol+pEGFP-N1 group. The mRNA expression of Nrf2, α-SMA and Col I was determined by RT-PCR, and their protein expression was detected by Western blotting. Cell proliferation was assessed by MTT assay, and cell cycle was detected by flow cytometry. RESULTS：The pEGFP-Nrf2 plasmid was successfully transfected into HSC-T6 cells, and the mRNA and protein expression of Nrf2 was higher than other three groups 48 h after transfection (P<0.05). Compared with control group, the cell proliferation and the mRNA and protein expression of α-SMA and Col I in ethanol group and ethanol+pEGFP-N1 group were significantly increased (P<0.05), and the numbers of HSC-T6 cells were decreased in G1 phase and increased in S phase (P<0.05), without significant differences between the two groups (P>0.05). Meanwhile, the cells in ethanol+pEGFP-Nrf2 group showed significantly decreased proliferation level, down-regulated mRNA and protein expression of α-SMA and Col I, higher numbers in G1 phase and lower numbers in S phase compared with ethanol group and ethanol+pEGFP-N1 group (P<0.05). CONCLUSION：Nrf2 overexpression could significantly down-regulate the expression of α-SMA and Col I and cause G1/S phase arrest in HSC-T6 cells cultured with ethanol, thus inhibiting the proliferation and activation of the cells.     

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.     

Effect of plumbagin on levels of Nox4/ROS and α-SMA in human hepatic stellate cells

AIM: To observe the effect of plumbagin on the mRNA and protein expression of nicotinamide adenine dinucleotidephosphate oxidase 4 (Nox4), reactive oxygen species (ROS) level and protein expression of α-smooth muscle actin (α-SMA) in the HSC-LX2 cells stimulated with transforming growth factor β1 (TGF-β1) in vitro. METHODS: HSC-LX2 cells were cultured in vitro and divided into blank group, model group, high-, medium- and low-dose (2, 1.5 and 1 μmol/L) plumbagin groups. After incubated with each drug for 72 h, the mRNA expression of Nox4 was detected by RT-PCR. ROS levels were tested by in situ loading probe method. The protein contents of Nox4 and α-SMA were measured by Western blot. RESULTS: Compared with model group, after treated with plumbagin for 72 h, the mRNA expression of Nox4, ROS level and α-SMA protein were significantly decreased in high-and medium-dose plumbagin groups (P<0.01). CONCLUSION: Plumbagin inhibits the activation of HSC-LX2 cells via decreasing the expression of Nox4, thus decreasing ROS levels.     

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.     

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.     

Macrophage migration-inhibitory factor induces myofibroblastic phenotype transformation of human embryonic lung fibroblasts via Rho-kinase in vitro

AIM: To investigate the influence and the mechanism of recombinant macrophage migration-inhibitory factor (rMIF) on fibroblasts. METHODS: Cultured human embryonic lung fibroblasts (MRC-5 cells) were divided into 2 groups: the cells in treatment group were treated with rMIF (25~100 μg/L) for 24 h or 48 h, and the control cells were without rMIF treatment. The mRNA expression of α-SMA was examined by RT-PCR. The protein level of α-SMA induced by rMIF was quantified by Western blotting. Half an hour before 100 μg/L rMIF challenge, Y27632 was added to the cells of 2 groups. After challenged for 48 h, the total RNA and protein were extracted,and the expression of α-SMA at mRNA and protein levels was determined by means of RT-PCR and Western blotting. After challenged by 100 μg/L rMIF for 6 h, 12 h, 24 h and 48 h, the cell total protein was extracted, and the protein level of α-SMA induced by rMIF was quantified by Western blotting. RESULTS: After stimulation for 24 h, rMIF did not increase the α-SMA mRNA synthesis compared with control. After stimulation for 48 h, rMIF significantly increased the expression of α-SMA mRNA and protein in a dose-dependent manner compared with control (r=0.697 and r=0.957, both P<0.01). Y27632 pretreatment prevented the increase (P<0.01). The amount of phosphorylated MYPT1 increased at 6 h (P<0.01), reached the maximum at 12 h (P<0.01), and decreased but still higher than the control at 24 h and 48 h (P<0.01). CONCLUSION: rMIF increases the α-SMA synthesis in MRC-5 cells and Rho-kinase regulates this process, suggesting that MIF may play important roles in the pathogenesis of airway remodeling.     

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.     

Effect of angiotensin-(1-7) on angiotensin II-induced rat renal interstitial fibroblast activation

AIM: To explore the influence of angiotensin-(1-7) on angiotension II (Ang II)-induced activation and extracellular matrix secretion in rat renal interstitial fibroblasts (NRK-49F cells). METHODS: The NRK-49F cells were maintained and sub-cultured, then the cells were divided into control group, Ang II group, Ang-(1-7) group and Ang II+Ang-(1-7) group. The expression of α-smooth muscle actin(α-SMA),transforming growth factor β₁ (TGF-β₁) and insulin-like growth factor I(IGF-I) was detected by the method of immunocytochemistry when the cells were cultured for 72 h. The content of TGF-β₁, IGF-I and collagen type I(Col I) in the cultured supernatants were measured by ELISA. RESULTS: In control group and Ang-(1-7) group, only basic expression of α-SMA and almost no expression of TGF-β₁, IGF-I and Col I were observed. Compared with control group, the expression of α-SMA, TGF-β₁, IGF-I and Col I was increased in Ang II group. Compared with Ang II group, the expression of α-SMA, TGF-β₁, IGF-I and Col I was significantly decreased in Ang II+Ang-(1-7) group.CONCLUSION: Ang-(1-7) inhibits the activation of renal interstitial fibroblasts and decreases the Ang II induced secretion of Col I by suppressing TGF-β₁ and IGF-I expression.     

Snail1/IGF-1 pathway mediates high glucose-induced EMT in renal tubular epithelial cells

AIM: To observe the expression of Snail1 and insulin-like growth factor-1 (IGF-1) in NRK-52E cells induced by high glucose, and to investigate the relationship of Snail1 and IGF-1 in the mechanism of epithelial to mesenchymal transition (EMT) in diabetic kidney disease (DKD).METHODS: The NRK-52E cells were treated with Snail1 siRNA and IGF-1 siRNA after cultured with high glucose medium for 72 h, and divided into control group, high glucose group, non-targeting (NT) siRNA group, Snail1 RNAi group and IGF-1 RNAi group. The cells were harvested at 48 h and 72 h. Real-time PCR was used to detect the mRNA expression of Snail1, IGF-1, E-cadherin and fibronectin (FN), and the protein levels were determined by immunofluorescence staining.RESULTS: Compared with control group, the expression of E-cadherin at mRNA and protein levels declined after stimulation with high glucose (P<0.01), while that of FN was elevated (P<0.01). Meanwhile, the mRNA and protein levels of Snail1 and IGF-1 were markedly increased (P<0.01).The expression of E-cadherin at mRNA and protein levels was improved in Snail1 RNAi group as compared with high glucose group(P<0.01), while that of FN, IGF-1 and Snail1 was significantly down-regulated (P<0.01). The same changes were observed in IGF-1 RNAi group (P<0.01). The protein expression of each factor in NT group had no significant change as compared with high glucose group (P>0.05). Pearson correlation analysis showed a close positive relationship between the expression of Snail1 and IGF-1 protein (r=0.852, P<0.01).CONCLUSION: Snail1 may facilitate DKD development by regulating IGF-1 in the process of EMT.     

Effects of several harmful factors on expression of glycine receptor α1 subunit in neonatal rat myocardial cells

AIM: To study the expression of glycine receptor α₁ subunit in neonatal rat myocardial cells and to investigate the effect of lipopolysaccharide (LPS), hypoxia/reoxygenation, isoproterenol (ISO) and high concentration of glucose (HG) on the expression of glycine receptor α₁ subunit in the neonatal rat myocardial cells. METHODS: Neonatal rat myocardial cells were cultured in vitro. The expression of glycine receptor α₁ subunit was detected by Western blotting. The neonatal rat myocardial cells were treated with LPS (20 mg/L), ISO (100μmol/L) or high concentration of glucose (25 mmol/L) for 24 h, or were exposed to hypoxia for 3 h followed by reoxygenation for 3 h. Subsequently, the cell viability was measured by CCK-8 assay, and the expression of glycine receptor α₁ subunit was determined by Western blotting. RESULTS: The expression of glycine receptor α₁ subunit in the neonatal rat myocardial cells was positively detectable by Western blotting. Compared with control group, no significant difference of the cell viability (P>0.05) in LPS group, ISO group, hypoxia/reoxygenation group and HG group was observed. The expression of glycine receptor α₁ subunit was increased (P<0.01) in LPS group, ISO group and hypoxia/reoxygenatio group, but decreased (P<0.01) in HG group. CONCLUSION: Glycine receptor α₁ subunit exists in the neonatal rat myocardial cells. A certain concentration of LPS or ISO, or hypoxia/reoxygenation for a certain period upregulate the expression of glycine receptor α₁ subunit, but HG downregulates the expression of glycine receptor α₁ subunit in cultured neonatal rat myocardial cells.     

Role of Snail in the renal tubular epithelial-mesenchymal- transition mediated by activator protein-1

AIM: To study the role of Snail in the renal tubular epithelial-myofibroblast transdifferentiation and fibronectin synthesis mediated by activator protein-1. METHODS: The cultured HK2 cells were divided into three groups: normal glucose group (NG), high glucose group (HG) and activator protein-1 (AP-1) inhibited group (AG). Concentration of fibronectin into the culture media was determined by ELISA. The activity of activator protein-1 was assessed with electrophoretic mobility shift assay (EMSA). The protein of E-cadherin and vimentin was determined by immunocytochemistry. RT-PCR was used to detect the mRNA expression of vimentin and Snail. Western blotting was used to detect the protein expression of E-cadherin.RESULTS: Secreted FN level was significantly up-regulated by the stimulation of high glucose (P<0.05), but the level significantly decreased in AP-1 inhibited group than that in high glucose group (P<0.05). AP-1 binding activity was significantly stimulated by high glucose and the inhibitor of AP-1 inhibited high glucose induced AP-1 activation in HK-2. High glucose induced Snail mRNA expression, while the level significantly decreased in AP-1 inhibited group than that in high glucose group (P<0.05). Upon the stimulation with high glucose, the expression of E-cadherin protein decreased markedly (P<0.05), while the level was higher in AP-1 inhibited group than that in high glucose group (P<0.05). Cultured with high glucose, the expression of vimentin mRNA and protein significantly increased (P<0.05), but the level significantly decreased in AP-1 inhibited group than that in high glucose group (P<0.05). CONCLUSION: High glucose induces the expression of Snail through the activation of AP-1. The expression of Snail downregulates E-cadherin expression and induces transdifferentiation of renal tubular cells characterized by vimentin expression and fibronectin synthesis.     

VPA alleviates bleomycin-induced pulmonary fibrosis in rats

AIM: To investigate the effect of sodium valproate (VPA) on bleomycin-induced pulmonary fibrosis (PF) and its mechanism. METHODS: SD rats (n=42) were randomly divided into model group and treatment group. Bleomycin at dose of 5 mg/kg was intratracheally injected to establish a rat PF model. The rats in treatment group were given normal saline (NS, 0.5 mL/d), VPA (300 mg·kg^-1·d^-1) or dexamethasone (DEX, 0.6 mg·kg^-1·d^-1) via intraperitoneal injection from the 14th day to the 28th day after modeling. The rats in model group were sacrificed on 0 d, 14 day and 28 d after modeling . The rats in treatment group were killed at 14th day after treatment. The effects of VPA on PF were evaluated by HE and Masson staining. The hydroxyproline (HYP) content in the rat lung tissues was detected, and the expression of α-smooth muscle actin (α-SMA) and E-cadherin was determined by Western blotting. RESULTS: HE staining showed that the alveolar structure and interstitial morphology in VPA group were better than those in NS group and DEX group. The level of collagen in VPA group was significantly lower than that in DEX group and NS group by determining the HYP content and Masson staining. VPA reduced the expression of α-SMA, a mesenchymal marker protein of PF, while increased the expression of epithelial marker protein E-cadherin. CONCLUSION: VPA reduces collagen content and distribution, and up-regulates the expression of the epithelial marker protein E-cadherin, while down-regulates mesenchymal marker protein α-SMA, thereby preventing the rat lung tissues from bleomycin-induced PF.     

Chronic alcohol intake-induced epithelial-mesenchymal transition contri-butes to hepatic fibrogenesis in mice

AIM：To evaluate the effect of chronic alcohol intake on the histopathological changes of the liver and to determine the contribution of epithelial-mesenchymal transition (EMT) to hepatic fibrogenesis. METHODS：Thirty male C57BL/6 mice were randomly divided into 3 groups as following: the mice in control group was given (ig) water; the mice in low-dose alcohol group (2.0 g·kg -1·d -1) and high-dose alcohol group (4.0 g·kg -1·d -1) were given (ig) alcohol for 5 months. Alcohol-induced histopathological changes of the liver or development of hepatic fibrosis were evaluated using the histological methods with HE and Masson trichrome staining. The apoptosis of the liver was detected by TUNEL fluorometric staining (counterstained with DAPI). The activity of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) was measured by an automated biochemical analyzer. The expression of fibroblast-specific protein 1 (FSP-1), α-smooth muscle actin (α-SMA) and E-cadherin in the hepatic tissues was detected by immunofluorescence examination. The protein levels of E-cadherin, α-SMA, FSP-1, transforming growth factor β 1 (TGF-β 1) and hypoxia-inducible factor 1α (HIF-1α) were analyzed by Western blotting. RESULTS：Compared with control, the activity of serum ALT and AST, and apoptotic index of liver tissues were increased in the mice treated with alcohol for 5 months. The histopathological changes of the livers in the mice of low-dose alcohol group included steatosis and mild liver fibrosis, while severe liver fibrosis was observed in the high-dose alcohol-treated mice. Chronic alcohol consumption induced the increase in malondialdehyde (MDA) level, and the decreases in the activity of superoxide dismutase (SOD) and catalase (CAT) in the livers. It also reduced E-cadherin expression and increased α-SMA expression. FSP-1 immunostaining and albumn immunostaining positive cells were co-localized in the hepatocytes of low-dose alcohol group, but only FSP-1 positive hepatocytes were observed in high-dose alcohol group. Chronic alcohol consumption decreased E-cadherin expression and increased α-SMA, FSP-1, TGF-β 1 and HIF-1α expression in a dose-dependent manner, but the HIF-1α expression was not altered between the 2 alcohol-treated groups. CONCLUSION：Chronic alcohol intake induces the progression of hepatic fibrosis. Some fibroblasts derive from hepatocytes in liver fibrosis via EMT. The underlying mechanism is associated with the changes of the redox state, and increased TGF-β 1 generation and HIF-1α expression.     

Effect of extracellular HSP70/HSP70-PCs on epithelial-mesenchymal transition of human hepatocellular carcinoma HepG2 cells

AIM：To investigate the effect of extracellular heat-shock protein 70 (HSP70)/HSP70-peptide complexes (HSP70-PCs) on epithelial-mesenchymal transition (EMT) of human hepatocellular carcinoma HepG2 cells and its probable mechanism. METHODS：HepG2 cells were divided into 3 groups: control group, HSP70/HSP70-PCs (2 mg/L) group and LY294002+HSP70/HSP70-PCs group. The mRNA and protein expression of epithelial cell surface marker E-cadherin, mesenchymal cell surface marker α-smooth muscle actin (α-SMA), phosphatidylinositol 3-kinase (PI3K) and hypoxia-inducible factor 1α (HIF-1α) was examined by real-time RT-PCR and Western blotting. RESULTS：Extracellular HSP70/HSP70-PCs promoted the initiation of EMT of HepG2 cells. The expression of HIF-1α and PI3K significantly increased in the process of EMT of HepG2 cells. After PI3K was blocked by LY294002, EMT did not occur and HIF-1α was not up-regulated in HepG2 cells. CONCLUSION：Extracellular HSP70/HSP70-PCs may promote EMT of hepatocellular carcinoma cells via PI3K/HIF-1α signaling pathway.