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.     

Effects of endothelin-1 on the induction of the transdifferentiation of human airway sub-epithelial fibroblasts

AIM: To explore the role of endothelin-1 (ET-1) in initiating transdifferentiation of sub-epithelial fibroblasts (SEFs) into myofibroblasts and its ionic and signal transduction mechanism.METHODS: Human SEFs or SEFs plated in collagen gels were co-cultured with human bronchial epithelial cells (16HBE) treated with lipopolysaccharide (LPS) plus mechanical scratch. ET receptor A inhibitor (BQ123) or the inhibitors specific for p38 MAPK, ERK1/2 were added, repectively. The expression of α-smooth muscle actin (α-SMA) in the SEFs and contractility of the collagen gels containing with SEFs as well as the effects of p38 MAPK or ERK1/2 on α-SMA expression were evaluated. Using Ca2+ sensitive Fluo-3/AM, dynamic changes of intracellular calcium concentration (［Ca²⁺］_i) were observed in the SEFs by laser confocal microscopy.RESULTS: Injured 16HBE induced the transdifferentiation of myofibroblasts, which expressd α-SMA and increased contractility. BQ123 blocked the induction to a certain extent. Injured 16HBE activated p38 MAPK and ERK1/2 pathways in SEFs, both inhibitors of p38 MAPK and ERK1/2 attenuated the induction of α-SMA by injured 16HBE. The addition of exogenous ET-1 enhanced α-SMA expression and activated p38 MAPK, ERK1/2 pathways in the SEFs. Additionally, ET-1 significantly facilitated Ca²⁺ inflow into the fibroblasts.CONCLUSION: Injured 16HBE induces the transdifferentiation of SEFs into myofibroblasts, which is involved in the activation of p38 MAPK and ERK1/2 pathways. The ET-induced influx of Ca²⁺ may be an early signal for initiating the myofibroblasts transdifferentiation.     

Signal mechanism of endothelin-1-mediated activation of airway fibroblasts induced by injured airway epithelial cells

AIM: To explore the effects of p38 mitogen-activated protein kinases (MAPK) and phosphoinositide 3-kinases (PI3K)/Akt on interleukin (IL)-6, the endothelin (ET)-1-mediated process of airway fibroblast activation induced by injured human bronchial epithelial cells (HBE). METHODS: Human primary cultured airway fibroblasts were co-cultured with HBE pre-treated with or without poly-L-arginine (PLA). The procedure was also performed in the presence or absence of p38 MAPK selective inhibitor SB203580, PI3K selective inhibitor LY294002 or ETA receptor blocker BQ123, respectively. Immunostaining, Western blotting or ELISA were used for detecting α-smooth muscle actin (α-SMA) expression, the activities of p38 MAPK and Akt in fibroblasts or IL-6 levels in supernatants of fibroblasts. In addition, fibroblasts were mixed with soluble collagen and cultured with HBE treated as the same mentioned above, the gel contraction was measured by serial area measurements. RESULTS: ET-1 and IL-6 levels ［(13.69±1.36) ng/L, (56.7±10.7) ng/L］ in the supernatants of fibroblasts cultured with injured HBE were significantly higher than those in the supernatants of fibroblasts cultured with HBE ［(3.79±0.64) ng/L, (15.5±3.2) ng/L］. BQ123, SB203580 or LY294002 decreased IL-6 levels ［(27.2±3.1) ng/L, (31.5±3.6) ng/L, (41.3±3.2) ng/L］ differently in the supernatants of fibroblasts induced by injured HBE. Activation of p38 MAPK preceded Akt in fibroblasts cultured with injured HBE. BQ123 reduced the phosphorylation levels of p38 MAPK and Akt. SB203580 concentration-dependently attenuated Akt phosphorylation, while LY294002 had little effect on p38 MAPK phosphorylation. Fibroblasts expressed more α-SMA after cultured with injured HBE and showed significant increase in the gel contraction compared to fibroblasts cultured with HBE ［percentage of gel contraction: (61.2±2.7)% vs (15.4±7.3)%］, all these effects were diminished or inhibited by BQ123, SB203580 or LY294002. Furthermore, the effects of BQ123 and SB203580 on decreased gel contraction were stronger than the effect of LY294002. CONCLUSION: ET-1 exerts a key role in the airway fibroblasts activation induced by injured HBE through activating p38 MAPK, PI3K/Akt signaling and promoting IL-6 expression.     

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     

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.     

RTN1A induces renal tubular epithelial cells to secrete VEGF and IL-8 and promotes diabetic nephropathy renal fibrosis via ERK signaling pathway

AIM:To investigate the effects of reticulon 1A (RTN1A) on the secretion of vascular endothelial growth facter (VEGF) and interleukin-8 (IL-8) in renal tubular epithelial cells, and on the diabetic nephropathy (DN) renal fibrosis, and to explore the underlying mechanism. METHODS:The mouse model of DN was established, and the blood glucose, kidney index, urine microalbumin (UMA) and creatinine clearance (CCr) were measured. The protein levels of RTN1A, p-ERK, ERK, VEGF, IL-8 and renal fibrosis markers α-smooth muscle actin (α-SMA) and fibronectin (FN) were determined by Western blot. Human renal tubular epithelial cell line HK-2 was treated with high glucose, and the ERK signaling proteins, fibrosis markers and secretion of cytokines were detected by Western blot and ELISA. The cells were treated with high glucose combined with RTN1A silencing or ERK inhibitor PD98059 for 24 h, and the ERK signaling proteins, fibrosis markers and secretion of cytokines were also detected by Western blot and ELISA. RESULTS:The blood glucose, kidney index, UMA and CCr in the DN mice were significantly higher than those in control group (P<0.05), suggesting that DN model was successfully constructed. The protein levels of RTN1A and its downstream protein p-ERK, the cytokines VEGF and IL-8, and the fibrosis markers α-SMA and FN were significantly increased in the DN model mice (P<0.05). The protein levels of RTN1A, p-ERK, VEGF, IL-8, α-SMA and FN were also significantly increased in the HK-2 cells after treated with high glucose for 24 h, while these proteins were significantly decreased after silencing of RTN1A expression. CONCLUSION:RTN1A may be associated with the occurrence and development of DN. Silencing of RTN1A expression inhibits DN renal inflammation and fibrosis through ERK signaling. RTN1A may be an effective therapeutic target.     

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.     

Erythropoietin inhibits adipogenesis of mouse bone marrow mesenchymal stem cells by activating ERK and p38 MAPK

AIM:To explore the role of erythropoietin (EPO) in the differentiation of mouse bone marrow-derived mesenchymal stem cells (MSCs) into adipocytes. METHODS:The mouse MSCs were cultured using routine methods. The cells were induced to differentiate by the cocktail medium containing 3-isobutyl-1-methylxanthine, insulin and dexamethasone, and the cells in the experiment group were treated with EPO. On the 20th day of induced differentiation, the cells were detected by oil red O staining. The mRNA expression of peroxisome proliferator-activated receptor γ (PPARγ), CCAAT/enhancer-binding protein α (C/EBPα), fatty acid binding protien 4 (FABP4) and adiponectin were determined by real-time fluorescence quantitative PCR. The phosphorylation levels of PPARγ, extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (p38 MAPK) were measured by Western blotting. MTT assay was adopted to detect the proliferation. RESULTS:During adipogenic induction, EPO decreased lipid accumulation, and inhibited the adipogenic differentiation of MSCs without cytotoxicity. The mRNA expression of PPARγ, C/EBPα, FABP4 and adiponectin was significantly inhibited in induced cells. Moreover, EPO enhanced the activity of both p38 MAPK and ERK, and increased PPARγ phosphorylation. CONCLUSION: EPO significantly inhibits differentiation of mouse bone marrow-derived MSCs into adipocytes in vitro via reducing the mRNA expression of PPARγ, C/EBPα, FABP4 and adiponectin, which may be mediated by the p38 MAPK and ERK signaling pathways.     

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.     

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.     

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.     

Activation of macrophage peroxisome proliferator-activated receptor α inhibits macrophage inflammation-induced cardiac fibroblast activation and migration

AIM: To investigate the effect of macrophage peroxisome proliferator-activated receptor α (PPARα) activation on macrophage inflammation-induced activation and migration of cardiac fibroblasts. METHODS: Mouse bone marrow-derived macrophages were treated with vehicle, PPARα agonist WY14643 (10 μmol/L), angiotensin Ⅱ (Ang II; 1 μmol/L) or Ang II+WY14643 for 24 h, and the supernatants were collected as conditioned medium (CM) to stimulate cardiac fibroblasts for additional 24 h. The mRNA levels of PPARα, interleukin-6 (IL-6), IL-1β and tumor necrosis factor-α (TNF-α) in the macrophages as well as fibrotic markers collagen type Ⅰ alpha 2 chain (Col1a2), collagen alpha 1 chain (Col3a1) and actin alpha 2 (Acta2) in the cardiac fibroblasts were detected by RT-qPCR. The protein levels of IL-6 and IL-1β in the macrophages as well as collagen I, collagen III and α-smooth muscle actin (α-SMA; encoded by Acta2 gene) in the cardiac fibroblasts were determined by Western blot. Wound-healing assay was applied to eva-luate the migration ability of cardiac fibroblasts. RESULTS: Ang II significantly increased the mRNA levels of pro-inflammatory factors, such as IL-6, IL-1α and TNF-α, but decreased the mRNA level of PPARα in the macrophages. Administration of PPARα agonist WY14643 dramatically decreased Ang II-induced mRNA levels of IL-6, IL-1β and TNF-α in the macrophages, and significantly decreased Ang II-induced protein expression of IL-6 and pro-IL-1β in the macrophages. The CM from Ang II-treated macrophages significantly up-regulated the mRNA levels of Col1a2, Col3a1 and Acta2 in the cardiac fibroblasts, which were inhibited by the CM from WY14643-treated macrophages. The same results were observed in the protein levels of collagen I, collagen III and α-SMA in the cardiac fibroblasts. Moreover, the CM from Ang II-treated macrophages significantly promoted cardiac fibroblast migration, whereas the CM from WY14643-treated macrophages markedly inhibited macrophage inflammation-induced cardiac fibroblast migration. CONCLUSION: WY14643-activated PPARα inhibits activation and migration of cardiac fibroblasts by attenuating Ang II-induced macrophage inflammatory response.     

Hypoxia induces myofibroblast formation and stimulates production of collagen I in myofibroblasts through ERK1/2 pathway

AIM: To investigate the effect of hypoxia on the myofibroblast transdifferentiation from fibroblasts,and associated signaling of hypoxia on the production of collagen I in cultured rat renal cortical myofibroblasts.METHODS: The study is composed of two relevant parts.In the first part,a normal rat renal interstitial fibroblast cell line NRK-49F was treated with hypoxia (1% O2) or normoxia (21% O2) for 6 h,12 h and 24 h.The expression of hypoxia inducible factor-1α (HIF-1α) was examined by Western blotting in order to make sure the hypoxic condition is reliable.The myofibroblast transformation from fibroblasts induced by hypoxia was assayed by detecting the protein levels of α-smooth muscle actin (α-SMA).In the second part,the object was done on the primary cultured rat renal cortical myofibroblasts.Myofibroblasts were subjected to hypoxic or normoxic conditions for variety of times.The levels of HIF-1α in cell lysates and collagen I protein in supernatant culture medium and the activation of extracellular signal-regulated kinase (ERK)1/2 MAPK pathway were analyzed by Western blotting.RT-PCR was carried out to measure the levels of collagen I mRNA at different time points (2 h,4 h and 6 h).The distribution of HIF-1α in myofibroblasts was demonstrated by immunocytochemistry.The changes of collagen I production were detected after PD98059,a specific inhibitor of ERK1/2 activation pretreatment and during the hypoxia incubation.The activity of gelatinase matrix metalloproteinase-2(MMP-2) and MMP-9 in the supernatant medium from the cultured cells were assayed by gelatin zymography.RESULTS: Significant increased levels of HIF-1α protein appeared in cell lysates under hypoxia for 6 h.Furthermore,HIF-1α was translocated into nuclei of myofibroblasts after 6 h exposure of myofibroblasts to hypoxia.The levels of α-SMA protein increased in NRK-49F under hypoxia for 12 h (187%±32%,P<0.05).The level of collagen I protein in culture medium was increased in hypoxia treated myofibroblasts at 6 h (171%±27%,P<0.05) and 12 h (256%±61%,P<0.05).Collagen I mRNA expression was increased in cells under hypoxia condition for 4 h (189%±28%,P<0.05) and 6 h (221%±44%,P<0.05).The activities of MMP-2 and MMP-9 in the supernatant medium were not significantly changed at different experimental time points between the normoxic and hypoxic conditions.Activation of ERK1 /2 occurred as early as 15 min,sustained the high level at 30 min and 60 min and was back to the baseline level at 2 h.Blockade of ERK activation with PD98059 abolished hypoxia-induced expressions of collagen I protein.CONCLUSION: Hypoxia contributes to the renal interstitial fibrosis through inducing formation of myofibroblasts and stimulating the production of collagen I in myofibroblasts.     

Pirfenidone inhibits TGF-β1-induced differentiation of myofibroblast in human lung fibroblast populations

ATM: To investigate the effect of pirfenidone on transforming growth factor-β1 (TGF-β1)-induced fibroblast-to-myofibroblast transition in vitro. METHODS: The cell viability was measured by MTT assay. The proliferation of human lung fibroblasts (HLFs) was detected by EdU incorporation. Migratory and invasive abilities were measured by Boyden chamber assay. The α-smooth muscle actin (α-SMA) protein expression was determined by Western blot and immunofluorescence. The mRNA expression of α-SMA and type Ⅰ and Ⅲ collagens was evaluated by RT-qPCR. RESULTS: Pirfenidone at different concentrations (0.1, 0.2, 0.3, 0.5 and 0.8 mg/L) had no cytotoxic effect on the HLFs, and pirfenidone at 0.2 mg/L was used for the intervention. Pretreatment of the HLFs with 0.2 mg/L pirfenidone prior to TGF-β1 not only markedly suppressed the changes of proliferation, migration, invasion and reorganization of actin cytoskeleton in the HLFs (P<0.01﹚,but also down-regulated the expression of α-SMA and type C and Ⅲ collagens triggered by TGF-β1 ﹙P<0.05﹚.CONCLUSION: Pirfenidone has an inhibitory effect on TGF-β1-induced activated cell functions and fibroblast-to-myofibroblast transition in HLFs.     

Role of TGF-β1-activated p38 MAPK in up-regulation of PAI-1 expression by TGF-β1 in human ovarian cancer cells

AIM: To investigate the relationship between up-regulation of plasminogen activator inhibitor-1(PAI-1) expression and activation of p38 mitogen-activated protein kinase(p38 MAPK) and extracellular signal-regulated kinase(ERK) pathways by TGF-β1 in human ovarian cancer cells. METHODS: PAI-1 expression in human ovarian cancer cells treated with TGF-β1(10 μg/L)was assayed by real-time PCR and Western blotting. The activation of p38 MAPK and ERK was determined by Western blotting using phosphorylated p38 MAPK and phosphorylated ERK antibodies. Specific p38 MAPK inhibitor(SB203580) or ERK inhibitor(PD98059) was used to inhibit their activation. RESULTS: TGF-β1 up-regulated the expression of PAI-1, and activated p38 MAPK and ERK pathways in the ovarian cancer cells. Inhibition of p38 MAPK activation by SB203580 resulted in significant inhibition of the mRNA expression of PAI-1 induced by TGF-β1. However, inhibition of ERK activation did not significantly alter TGF-β1-induced increase in PAI-1 mRNA level. CONCLUSION: TGF-β1-activated p38 MAPK pathway contributes to the up-regulation of PAI-1 expression by TGF-β1 in ovarian cancer cells.     

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 all-trans-retinoic acid on the proliferation and expression of α-SMA in human embryonic lung fibroblasts

AIM: To investigate the effects of all-trans-retinoic acid (ATRA) on the proliferation and differentiation of transforming growth factor β₁(TGF-β₁)-stimulated human embryonic lung fibroblasts (HFL-I).METHODS: The HFL-I cells were cultured in vitro and were pretreated with ATRA for 3 days at the concentrations of 0.1 μmol/L, 1 μmol/L and 10 μmol/L. The proliferation of HFL-1 cells was detected by MTT method. The mRNA expression of α-smooth muscle actin(α-SMA) in HFL-I cells stimulated with TGF-β₁ for 0 h, 6 h, 12 h, 24 h, 48 h and 72 h was detected by RT-PCR and the protein expression of α-SMA at the time points of 1,3 and 5 days was detected by Western blotting. The mRNA expression of α-SMA in HFL-I cells pretreated with different concentrations of ATRA for 24 h was detected the by RT-PCR and the protein expression at time point of 3rd day was detected by Western blotting. RESULTS: Different concentration of ATRA inhibited the proliferation of HFL-I in a dose-dependent manner (P<0.05). Both mRNA and protein expression of α-SMA in HFL-I cells pretreated with TGF-β₁ was up-regulated (P<0.05). ATRA down-regulated the mRNA and protein expression of α-SMA induced by TGF-β₁ in a dose-dependent manner (P<0.05). CONCLUSION: ATRA inhibits the proliferation and TGF-β₁-stimulated differentiation in HFL-I cells by down-regulating the mRNA and protein expression of α-SMA.     

αvβ6-ERK pathway participates in norcantharidin-induced apoptosis of HT-29 colon cancer cells

AIM: To investigate the pharmacological mechanism of norcantharidin (NCTD)-induced apoptosis of HT-29 colon cancer cells. METHODS: Hoechst 33258 staining was used to analyze the apoptosis of HT-29 cells treated with NCTD. The effects of NCTD on the expression of integrin in HT-29 cells were determined by flow cytometry. The effects of several functional blocking antibodies on HT-29 cells were detected by MTT method. The expression and the phosphorylation of mitogen activated protein kinases (MAPKs) in HT-29 cells were measured by Western blotting. Co-immunoprecipitation assay was used to detect the activity of αvβ6-extracellular signal-regulated kinase (ERK) direct linkage in HT-29 cells.RESULTS: NCTD induced the apoptosis of HT-29 colon cancer cells. The expression of integrin αvβ6 in HT-29 cells treated with NCTD was reduced, but the expression of αvβ3 and αvβ5 was not changed. A function-blocking antibody to αvβ6,10D5,strengthened the growth inhibitory effect of NCTD on HT-29 cells ,but LM609 (a function-blocking antibody to αvβ3) and P1F6 (a function-blocking antibody to αvβ5) did not. The level of phosphorylated ERK (p-ERK) was decreased substantially after treated with NCTD in a dose-and time-dependent manner. NCTD also affected the association of αvβ6 and ERK. CONCLUSION: NCTD decreases the expression of integrin αvβ6 and interferes with the phosphorylation of ERK. As a result, the formation of αvβ6-ERK direct linkage is affected and the signal transduction mediated by αvβ6 is disturbed. The mechanism of NCTD-induced HT-29 cell apoptosis is involved in the αvβ6-ERK 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.