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.     

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.     

Effect of circRNA_000203 on fibrotic phenotypes in mouse cardiac fibroblasts

AIM: To determine circular RNA (circRNA) profiles in the diabetic mouse myocardium, and to investigate the effect of circRNA_000203 on fibrotic phenotypes in cardiac fibroblasts.METHODS: Masson trichrome staining was performed on the myocardium of the diabetic db/db mice and the non diabetic db/m control mice. circRNA expression profile in the diabetic myocardium was detected by circRNAs microarray. The expression of circRNA_000203 was determined by real time fluorescence quantitative PCR (RT-qPCR). Recombinant circRNA_000203 adenovirus was prepared for enforced the expression of circRNA_000203 in mouse cardiac fibroblasts. The expression of Col1a2, Col3a1and α-SMA was determined in circRNA_000203-modified cardiac fibroblasts, respectively. RESULTS: Masson trichrome staining showed that fibrosis was increased in the diabetic mouse myocardium. The results of circRNA array detection revealed that circRNAs were dysregulated in the diabetic myocardium. circRNA_000203 was up-regulated in the diabetic myocardium. Significant over-expression of circRNA_000203 was achieved in the cardiac fibroblasts after infection with the recombinant circRNA_000203 adenovirus. The mRNA and protein expression of Col1a2, Col3a1 and α-SMA was significantly increased in the cardiac fibroblasts with over-expression of circRNA_000203.CONCLUSION: circRNA_000203 is up-regulated in the diabetic mouse myocardium. It has pro-fibrotic effect on the cardiac fibroblasts.     

苹果乙烯响应因子基因MdERF11对脱落酸的敏感性分析

以‘嘎拉’苹果（Malus × domestica Borkh.）为试材,克隆了乙烯响应因子基因MdERF11（序列号 MDP0000756341）。测序发现,该基因包含全长为483 bp的完整开放阅读框,编码161个氨基酸。系统进化树分析表明,这一乙烯响应因子与拟南芥AtERF11蛋白同源序列相似性最高。利用PlantCare数据库进行基因启动子顺式作用元件预测,MdERF11启动子序列中含有与脱落酸（ABA）、乙烯及干旱信号相关的顺式作用元件。荧光定量PCR分析表明,MdERF11在苹果的各组织中均有表达,在叶柄和果实中表达量相对较高;并且MdERF11的表达明显受到ABA的诱导。在外源ABA的处理下,MdERF11过量表达的苹果愈伤组织的生长势明显比野生型强,表明MdERF11降低了苹果愈伤组织对ABA的敏感性。     

Role of miR-219 and TGFBR2 in pathogenesis of renal fibrosis

AIM:To study the role of microRNA-219 (miR-219) in regulation of transforming growth factor-β receptor type 2 (TGFBR2) in renal fibrosis. METHODS:The renal fibrosis patients (n=70) were selected in this stu-dy, and 20 cases of healthy people were selected as control group. RT-qPCR was used to detect the expression of miR-219 in the serum of the patients with renal fibrosis and control group, and the expression of miR-219 in NRK49F cells after stimulation with angiotensin Ⅱ(AngⅡ) was detected. The protein expression of α-smooth muscle actin (α-SMA) in the NRK49F cells transfected with miR-219 mimics after stimulation with AngⅡ was determined by Western blot. The potential target gene TGFBR2 of miR-219 was screened and verified by the method of luciferase reporter gene. RT-qPCR and Western blot were used to detected the effect of miR-219 mimics on the expression of TGFBR2 at mRNA and protein levels, and the mRNA expression of α-SMA, connective tissue growth factor (CTGF), type I collagen α1 (COL1A1) and COL3A1 in the NRK49F cells was also detected, respectively. The unilateral ureteral occlusion (UUO) mouse model was established and the expression of miR-219 in the renal tissue was monitored. The morphological change of renal fibrosis was observed in the UUO mice after injection of miR-219, and the mRNA expression levels of COL1A1 and COL3A1 were detected. RESULTS:The expression level of miR-219 in the patients with renal fibrosis was significantly lower than that in control group, and the expression of miR-219 in the UUO mice was decreased significantly (P<0.01). The expression level of miR-219 was significantly decreased in the NRK49F cells after AngⅡ stimulation, and miR-219 mimics inhibited the protein expression of α-SMA(P<0.01). miR-219 mimics had a targeted regulatory effect on TGFBR2 gene, which inhibited the mRNA and protein expression of TGFBR2. miR-219 mimics inhibited the mRNA expression of α-SMA, CTGF, COL1A1 and COL3A1. miR-219 also down-regulated the mRNA expression of COL1A1 and COL3A1 in the UUO mice and inhibited the process of renal fibrosis. CONCLUSION:miR-219 inhibits the development of renal fibrosis by inhibiting the expression of TGFBR2, which may become a new target for the diagnosis and treatment of renal fibrosis.     

Placental growth factor is involved in angiotensin II-induced cardiac fibroblast activation

AIM：To explore the role of placental growth factor (PLGF) in the process of angiotensin II (Ang II)-induced activation of cardiac fibroblasts (CFs). METHODS：Primary culture and identification of CFs from neonatal Sprague-Dawley rats were performed. The method of fluorescence immunocytochemistry was employed to observe the expression of alpha-smooth muscle actin (α-SMA). Real-time PCR and Western blotting were used to determine mRNA and protein levels. The cell proliferation was observed by WST-1 assay. RESULTS：Compared with control group, the PLGF expression at mRNA and protein levels in Ang II-treated CFs was significantly increased, whereas the mRNA expression of PLGF was decreased in the CFs treated with telmisartan and Ang II. Treatment with PLGF induced the proliferation of CFs and increased the protein expression of α-SMA. Treatment with PLGF for 60 min significantly increased the protein levels of p-ERK1/2 in the CFs. Compared with Ang II group, the proliferation of CFs was depressed and the protein expression of α-SMA was attenuated in Ang II+anti-PLGF group.The mRNA expression levels of type I and type III collagens were also down-regulated. CONCLUSION：PLGF might be involved in the process of Ang II-induced proliferation of CFs and fibrosis.     

Effects of epithelial cells treated with poly (I:C) on activation of fibroblasts and roles of EMMPRIN

AIM: To investigate the effects of epithelial cells treated with polyinosinic-polycytidylic acid [poly(I:C)] on the proliferation, transdifferentiation and signaling mechanisms of airway fibroblasts.METHODS:Human alveolar epithelial cells were treated with poly(I:C). The cell culture supernatants were used to stimulate the airway fibroblasts or the fibroblasts growing in collagen gels. The proliferation of the fibroblasts, the expression of a-smooth muscle actin (α-SMA) in the fibroblasts and the contractility of the collagen gels containing fibroblasts, as well as the expression of extracellular matrix metalloproteinase inducer (EMMPRIN) were observed. The proliferation of the fibroblasts and the expression levels of α-SMA, MMP-2 and MMP-9 in the fibroblasts stimulated with EMMPRIN were detected. The inhibitors specific for p38 MAPK or ERK1/2 were used to explore the effects on α-SMA expression and EMMPRIN secretion. RESULTS:The culture supernatants of the epithelial cells treated with poly(I:C) induced the proliferation, α-SMA expression and gel contraction as well as EMMPRIN secretion in the fibroblasts. EMMPRIN dose-dependently enhanced fibroblast proliferation, α-SMA expression and activity of MMP-2 and MMP-9. The supernatants of epithelial cells treated with poly(I:C) activated p38 MAPK and ERK1/2 signaling in the fibroblasts, as the specific inhibitors of MAPK or ERK1/2 signaling attenuated the expression of α-SMA and EMMPRIN in the fibroblasts. CONCLUSION: Poly(I:C) induces fibroblast proliferation, α-SMA expression and gel contraction by affecting the process mediated by p38 MAPK and ERK1/2 signaling pathways in epithelial cells. EMMPRIN may be the important media involved in this event.     

Regulatory effect of miR-199a-5p on cardiomyocyte hypertrophy in rat

AIM：To investigate the expression of miR-199a-5p in rat cardiomyocyte hypertrophy models. METHODS：The in vivo cardiomyocyte hypertrophy model was established by transverse abdominal aortic constriction (TAAC) and the in vitro model was induced by angiotensin II. The content of miR-199a-5p was detected by qRT-PCR in the plasma of the TAAC rats and in the cardiomyocytes (CM) of the newborn rats. The CM was isolated and transfected with miR-199a-5p mimic or inhibitor at concentration of 100 nmol/L by Lipofectamine RNAiMAX. The mRNA levels of atrial natriuretic factor (ANF) and β-myosin heavy chain (β-MHC) were detected by qRT-PCR. Tritium-labeled leucine incorporation was employed to determine the protein synthesis rate in the CM. The method of cyto-fluorescent staining was applied to measure the changes of the CM surface area. RESULTS：Compared with control group, the content of miR-199a-5p significantly increased in the TAAC rats and in the CM induced by angiotensin II. In addition, over-expression of miR-199a-5p in the CM up-regulated the mRNA expression of ANF and β-MHC, accelerated the protein synthesis rate and enlarged the CM surface area. In the CM transfected with miR-199a-5p inhibitor following induced by angiotensin II, the hypertrophy effect receded inversely (P<0.05). CONCLUSION:Over-expression of miR-199a-5p may promote cardiomyocyte hypertrophy, and repression of miR-199a-5p may inhibit cardiomyocyte hypertrophy in the CM.     

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.     

PAX6 inhibits angiotensin II-induced cardiac fibroblast transdifferentiation

AIM To investigate the effects of paired box 6 （PAX6） on angiotensin II （Ang II）-induced transdifferentiation of cardiac fibroblasts （CFs） and its underlying mechanisms. METHODS Primary CFs were isolated from the hearts of adult mice, and Ang II was used to induce the transdifferentiation of CFs. The adenovirus vector carrying PAX6 was constructed and transfected into the CFs. The cells were divided into Ad-GFP+Ctrl group （transfected with control adenovirus vector）, Ad-GFP+Ang II group （transfected with control adenovirus vector and treated with Ang II）, Ad-PAX6+Ctrl group （transfected with adenovirus vector carrying PAX6） and Ad-PAX6+Ang II group （transfected with adenovirus vector carrying PAX6 and treated with Ang II）. The fluorescence expressed by transfected CFs was observed under an inverted fluorescence microscope. The protein levels of PAX6, α-smooth muscle actin （α-SMA）, collagen type I （Col I）, fibronectin （FN） and transforming growth factor β1 （TGFβ1） were detected by Western blot. The expression and distribution of α-SMA, Col I and FN were measured by immunofluorescence staining. The mRNA levels of PAX6 and TGFβ1 were determined by qPCR. RESULTS The fluorescence observed by inverted fluorescence microscopy confirmed the successful transfection of adenovirus vector into the CFs. qPCR and Western blot showed successful PAX6 overexpression in the CFs （P<0.01）. Ang II increased the myofibroblast marker α-SMA in the CFs, while overexpression of PAX6 significantly inhibited the expression of α-SMA induced by Ang II （P<0.01）. In addition, PAX6 overexpression significantly inhibited Ang II-induced synthesis of extracellular matrix （ECM） proteins Col I and FN （P<0.05）. Furthermore, Ang II treatment upregulated TGFβ1 mRNA and protein levels, while overexpression of PAX6 significantly inhibited TGFβ1 mRNA and protein expression induced by Ang II （P<0.05）. CONCLUSION PAX6 inhibits Ang II-induced CF transdifferentiation and ECM protein synthesis via inhibiting TGFβ1 expression.     

miR-125a-5p reverses cisplatin resistance of non-small-cell lung cancer A549/DDP cells by targeting LIMK1

AIM:To explore the effect of microRNA-125a-5p (miR-125a-5p) on cisplatin (DDP) resistance of non-small-cell lung cancer A549/DDP cells and its related mechanisms. METHODS:The expression levels of miR-125a-5p and LIM kinase 1 (LIMK1) in non-small-cell lung cancer tissues, A549 cells and A549/DDP cells were detected by RT-qPCR. The A549/DDP cell viability, apoptotic rate and expression of drug resistance-related proteins after over-expression or knockdown of miR-125a-5p and/or LIMK1 expression were detected by MTT assay, flow cytometry and Western blot, respectively. The targeting relationship between miR-125a-5p and LIMK1 was verified by TargetScan online prediction and dual-luciferase reporter system. The cell viability, apoptotic rate and expression of drug resistance-related proteins after co-expression of miR-125a-5p and LIMK1 were also determined. RESULTS:The expression level of miR-125a-5p was down-regulated and LIMK1 expression was up-regulated in non-small-cell lung cancer tissues and cell lines (P<0.05). The results of dual-luciferase assay indicated that miR-125a-5p negatively regulated the expression of LIMK1. The expression of drug resistance-related proteins and the viability of A549/DDP cells were inhibited after over-expression of miR-125a-5p or knockdown of LIMK1, while the apoptosis was enhanced. Over-expression of LIMK1 attenuated the inhibitory effect of miR-125a-5p on A549/DDP cell viability and drug resistance-related protein expression (P<0.05). CONCLUSION:miR-125a-5p reverses the resistance of A549/DDP cells to DDP by inhibiting the expression of LIMK1 and drug resistance-related proteins.     

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.     

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.     

miR-301a-3p regulates connexin 43 expression in rat astrocytes

AIM: To explore the mechanism of microRNA-301a-3p (miR-301a-3p) regulating connexin 43 (Cx43) expression in the rat astrocytes. METHODS: miR-301a-3p agomir, miR-301a-3p antagomir and microRNA negative control (miR-NC) were synthesized and transfected into the astrocytes. The protein expression of Cx43 was determined by Western blot. The recombinant vectors wt-pEZX-MT05-Cx43 and mut-pEZX-MT05-Cx43 were constructed. The target gene of miR-301a-3p was validated by dual-luciferase reporter assay. The expressional vector pcDNA3.1-Cx43 was constructed and the biological activity regulation of miR-301a-3p on astrocytes was analyzed by restore experiment. RESULTS: The results of Western blot showed that the Cx43 expression was inhibited by miR-301a-3p agomir transfection (P<0.05). The results of dual-luciferase reporter assay showed that miR-301a-3p bound to 3′-UTR of Cx43, thus regulating expression of Cx43 negatively. Transfection with recombinant vector pcDNA3.1-Cx43 without 3′-UTR of Cx43 into astrocytes resulted in the restoration of the negative function of miR-301a-3p on Cx43 expression, also induced the apoptosis of astrocytes. CONCLUSION: miR-301a-3p binds to 3′-UTR of Cx43 mRNA in the rat astrocytes, thus regulating expression of Cx43 negatively.     

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.