Effect Of Qindan Capsule On TGF-?1/Smad Signaling Pathway In Adventitial Fibroblasts

BackgroundVascular remodeling runs through the process of various cardiovascular diseases, such as the restenosis after percutaneous transluminal coronary angioplasty (PTCA), atherosclerosis(AS) and hypertension. And vascular remodeling is considered the major pathological base for maintaining and worsening of hypertension. Recently the adventitia has attracted more and more attention for the importance during vascular remodeling after vascular injury. The adventitia is the most sensitive layer responding to elevated blood pressure compared with intimal and medial. Adventitial fibroblasts (AFs), the main adventitial cell type, drive remodeling and may initiate other changes subsequently, such as alterations in arrangements of neointimal proliferation, smooth muscle cells and extracellular matrix (ECM), which is expected to lead to vascular remodeling.Transforming growth factor?1 (TGF-?1), a pleiotropic and multifunctional cytokine, is known as the most important and direct one to regulate AFs bioactivity. The classical signal transduction pathway of the TGF-?family is through Smad, which is related to the fibrosis of many parts, such as heart, liver, lung and kidney. Smads are divided into three subfamilies:(1) receptor-activated Smads (R-Smads: Smad1, Smad2, Smad3, Smad5, Smad8), which become phosphorylated receptor I and play an important role in transforming growth factor-?(TGF-?) family; (2)common mediator Smads (Co-Smads:Smad4), which become phosphorylated and oligomerise with activated R-Smads to practice their intracellular effects; (3) inhibitory Smads (I-Smads:Smad6 and Smad7), which exert a negative feedback effect by competing with R-Smads.RNA interference (RNAi) is a new biotechnology, which was considered the top of the ten scientific achievements in 2002 by Science. RNAi is a sequence-specific gene silencing procedure started by double-stranded (dsRNA) RNA corresponding to target gene. Synthetical siRNA could inhibit the expression of gene, endogenic and ectogenic specifically in mammalian cells. And it could keep the target genes in scilence or resting state. The inhibition of genes started by siRNA is of high efficacy and specificity.In the present study, we observed the biological activity of AFs induced by TGF-?1 and tried to investigate the mechanism of TGF-?1/Smad signaling pathway on the migration, proliferation, transdifferentiation and ECM deposition of AFs. And moreover, the different roles of Smad2 and Smad3 were studied during the process.Aims1. To investigate the effect of TGF-?1 on the migration, proliferation, transdifferentiation and ECM deposition of AFs.2. To investigate the possible mechanism of TGF-?1/Smad signaling pathway during the activity of AFs induced by TGF-?1. And to study the different roles of Smad2 and Smad3 during the procedure.Method1. Primary cell culture and identification in vitroAFs were cultured by tissue explant in vitro. AFs were observed to grow out from the tissues 3-7 days later. The AF and its purity were identified by immunocytochemistry of Vimentin and a-smooth muscle actin (SMA). Cells of the 3-8 passages were used in assays.2. Synthesis of siRNA and selection of the effective concentrationThree specific Smad2-siRNAs and Smad3-siRNAs, one FAM-siRNA and one Smad2/3-negative siRNA were designed and chemically synthesized, so were Smad3 siRNAs. Transfection of FAM-siRNA was performed when the AFs were at 50%-70% density. Under light microscopy after transfection, the positive cells were observed. The best concentration of siRNA and Lipofectamine 2000 was chosen according to the transfection efficiency.3. Transfection and selection of the effective siRNAsThe transfection was performed according to the manufacturer's instructions. Western-blot was used to detect the efficiency of gene silence and the effective siRNAs4. MTT assay for proliferationTwenty-four h after transfection, AFs in the experiment of TGF-?1 were treated with TGF-?1 for 24 h.20?l MTT (5 mg/ml) was added, followed by incubation for 4 h at 37?. Finally, medium was removed and cells were lysed with DMSO. Absorbance of the samples was recorded.5. Transwell assay for migrationMigration of AFs was measured using transwell chamber apparatus in 6-well plates. AFs were grouped and treated as described above. Briefly, cells were trypsinized and counted,600?l cell suspension at 5×105/ml in DMEM was added to the upper compartment of the chamber, and 1.5 ml DMEM containing 10% FCS was added to the lower compartment. After incubation for 6 h, cells on the upper face of the membrane were removed with a cotton-tipped applicator carefully. Then the membranes were fixed in methanol and stained with hematoxylin and eosin (HE). The number of migrated cells was counted in 5 random fields in each membrane.6. Real-time RT-PCRAfter treatment, total RNA was isolated from AFs with use of Trizol Reagent according to the manufacturer's instructions. RNA was reversely transcribed by a standard protocol. The expression of Smad2, Smad3, Smad7,?-SMA, procollagen?and?mRNA was studied after treatment of siRNA and TGF-?1. The relative mRNA expression of the genes was determined by the 2-??Ct method.7. Western blot analysisCells were harvested after transfection for 24 h and stimulation by TGF-?1 for another 24 h. Protein was extracted and detected. Western-blot was used to detect the protein expression of p-Smad2, p-Smad3, Smad2, Smad3, Smad7,?-SMA, procollagen?.8. Statistical analysisThe data are presented as mean±SD and analyzed with SPSS 16.0. Comparisons among groups were analyzed by ANOVA. An unpaired Student's t-test was applied, when only two groups were compared. P< 0.05 was considered to be statistically significant.Results1. Appearance and identification of AFsAFs were observed to grow out from different tissues 3-7 days later. Immunocytochemistry analysis demonstrated that the multiclonal antibody staining for SMA was negative and the monoclonal antibody staining for vimentin was positive, suggesting 100% purity of AFs.2. siRNAs targeting Smad2 and Smad3 downregulation the expression of Smad2 and Smad3 efficientlyAfter transfection and stimulation by TGF-?1, the protein expression of Smad2 or Smad3 in siRNA-Smad2 or siRNA-Smad3 group was decreased more than 80% compared with TGF-?1 group. Moreover, the knockdown was specific and selective, as Smad3 protein levels were decreased by Smad3 siRNA only, so were Smad2 protein levels.3. Proliferation of AFs induced by TGF-?1 was inhibited by siRNA-Smad2 and-Smad3(1) Cell proliferation in TGF-?1 group was significantly increased compared with the control group (P<0.01). However, the proliferation decreased when Smad2 expression was knocked down by siRNA (P<0.01). Knockdown of Smad2 could inhibit the proliferation of AFs significantly.(2) The proliferation decreased when Smad3 expression was knocked down by siRNA (P<0.01). Knockdown of Smad3 by siRNA could inhibit the proliferation of AFs significantly.(3) SiRNA-Smad2 and siRNA-Smad3 could both inhibit the proliferation of AFs significantly, with no significant difference between Smad2 and Smad3 knockdown (P>0.05).4. Migration of AFs induced by TGF-?1 was inhibited by siRNA-Smad2 and-Smad3(1) After stimulation by TGF-?1 for 24 h, the migration of AFs was significantly increased compared with the control group(P<0.01). However, the blockade of Smad2 expression by siRNA could reduce the migration of AFs induced by TGF-?1 (P<0.01). Knockdown of Smad2 by siRNA could inhibit the migration of AFs significantly.(2) The migration decreased when Smad3 expression was knocked down by siRNA (P<0.01). Knockdown of Smad3 by siRNA could inhibit the migration of AFs significantly.(3) SiRNA-Smad2 and siRNA-Smad3 could both inhibit the migration of AFs significantly, with no significant difference between Smad2 and Smad3 knockdown (P>0.05).5. mRNA expression of the related gene induced by TGF-?1 was inhibited by siRNA-Smad2 and -Smad3(1) After stimulation by TGF-?1, the mRNA expression of Smad2, Smad3,?-SMA, procollagen?and?all increased compared with the control group (P<0.05 or P<0.01), but not Smad7 (P>0.05). Knockdown of Smad2 by siRNA, the mRNA expression of?-SMA, procollagen?and?was decreased significantly compared with TGF-?1 group (P< 0.05 or P<0.01). However, Smad7 mRNA expression did not differ from that of the control (P> 0.05).(2) The mRNA expression of?-SMA, procollagen?and?decreased when Smad3 expression was knocked down by siRNA compared with TGF-?1 group (P< 0.05 or P< 0.01), but not Smad7 (P> 0.05).(3) SiRNA-Smad2 and siRNA-Smad3 could both reduce the mRNA expression of?-SMA, procollagen?and?of AFs significantly, with no significant difference between Smad2 and Smad3 knockdown(P>0.05).6. Protein expression of the related gene induced by TGF-?1 was inhibited by siRNA-Smad2 and -Smad3 (1) After stimulation by TGF-?1, the protein expression of Smad2, Smad3,?-SMA, procollagen?was all increased compared with the control group (P<0.01), but not Smad7 (P>0.05). Knockdown of Smad2 by siRNA, the protein expression of?-SMA, procollagen?was decreased significantly compared with TGF-?1 group (P< 0.05 or P<0.01). However, Smad7 protein expression did not differ from that of the control (P>0.05).(2) The protein expression of?-SMA, procollagen?decreased when Smad3 expression was knocked down by siRNA compared with TGF-?1 group (P<0.01), but not Smad7 (P>0.05).(3) SiRNA-Smad2 and siRNA-Smad3 could both reduce the protein expression of?-SMA, procollagen?of AFs significantly, with no significant difference between Smad2 and Smad3 knockdown (P>0.05).Conclusion(1) SiRNA in the experiment could knockdown the expression of Smad2 and Smad3 effectively and specifically.(2) TGF-?1 could promote the proliferation of AFs both Smad2 and Smad3-dependently.(3) TGF-?1 could promote the migration of AFs both Smad2 and Smad3-dependently.(4) Both Smad2 and Smad3 work as mediators of phenotypic transition and collagen synthesis of AFs induced by TGF-?1 BackgroundHypertension is the familiar and risk factor for human health. The attack rate is growing year by year and now the rate is up to 18.8%. Blood pressure could be controlled effectively thanks to the intensive study of mechanism of hypertension and development of hypotensor. But the rate of morbidity, fatality and deformity is still very high. Vascular remodeling is the the adaptive reaction to the disorders in hypertension. Moreover, it is the major pathological base of deterioration in the development of hypertension. Therefore, it is very important to investigate the mechanism of vascular remodeling for blood pressure control, prevention and treatment of hypertension and the complications.Growing experimental evidence shows that the adventitia plays an critical role in vascular remodeling. High blood pressure is an important factor for adventitial fibroblasts (AFs) transdifferentiation to Myofibroblasts (MF). The migratory and proliferative responses of myofibroblasts, in addition to synthesis of ECM, lead to fibrosis and lumen stenosis, which play important roles during vascular remodeling.Transforming growth factor?1 (TGF-?1) is an important multifunctional cytokine that regulates fibrosis by activating a series intracellular signaling pathways, which might lead to the transcription of target genes. Smad is the classical one among the pathways activated by TGF-(31. And Smad signaling pathway is closely related to the fibrosis of cardiac, lung, renal and so on.Serum pharmacological method is a useful method for the research of traditional Chinese medicine. After fed with traditional Chinese medicine for a long time, the serum was get from the animals and was used in the experiment instead of drugs. This method presents the biotransformation in vivo and also overcomes confusion of other substances.Qindan capsule (QC), a traditional Chinese medicine presciptions, has been used as an anti-hypertensive drug in clinic by the tutor. It could calm the liver and dominate heat and remove blood stasis. Previous studies have shown that QC could improve the morphological index of the artery, downregulate volume fraction of collagen (VFC) in the media and inhibit the transformation of Smooth muscle cells (VSMCs). But its effect on TGF-?1 signaling pathway in AFs remains unclear. In the present study, we studied the effect of QC on the proliferation, migration, transdifferentiation and collagen synthesis induced by TGF-?1, and investigated the mechanism of QC on TGF-?1/Smad pathway in the process, which might provide more scientific evidence for the treatment of hypertensive vascular remodeling clinically.Aims1. To investigate the the effect of QC on the proliferation, migration, and the variation of alpha smooth muscle actin (?-SM actin), procollagen?and?expression, and to study the effect of QC on the biological activity of AFs induced by TGF-?1.2. To investigate the the effect of QC on the variation of Smad2, Smad3, Smad7, phosphorylation-Smad2 (p-Smad2) and phosphorylation-Smad3 (p-Smad3) expression, and to study the possible mechanism of QC on TGF-?1/Smad signaling pathway in AFs.Methods1. Preparation of QC and research on QC quality standardHigh performance liquid chromatography (HPLC) and Thin-layer chromatography (TLC) methods were used for the research on quality standard, in order to ascertain the active principle in QC.2. Cell thawing and subcultureFreezing tubes were get out from the nitrogen canister and a thaw was taken as soon as possible. After suspension in culture medium and centrifugation, cells were placed on culture flasks and cultured. When it was 90% in density, cells were subcultured.3. Preparation of QC-containing serumSixty male WKY rats were divided into three groups randomly:the high dosage QC group (QCHD,750 mg/kg), the low dosage QC group (QCLD,150 mg/kg) and Losartan group (30 mg/kg). Rats were starved for 24 h and anaesthetised 1 h (Losartan group) and 2 h (QC groups) after administration of whole-day drugs on the last day. Blood was drawn from the abdominal aorta, separated on a centrifugation (3000 rpm for 15 min). And then mixed as in the same group. After being dried at -57?for 48 h, the serum was stored at-20?.4. MTT colorimetry assay for cell proliferationAFs were seeded in 96-well plates. AFs in the experiment were treated with QC containing serum of different doses for 48 h prior to stimulation by TGF-?1 for an additional 24 h.20?l MTT was added, followed by incubation for 4 h at 37?. Finally, cells were lysed with DMSO. Absorbance of the samples was recorded.5. Transwell assay for migrationAfter treatment, cells were trypsinized and 600?l cell suspension was added to the upper compartment of the chamber, while 1.5 ml culture medium containing 10% FCS was added to the lower. After 6 h incubation, the chambers were taken out. Cells on the upper face of the membrane were removed by a cotton-tipped applicator carefully. Membranes were then fixed and stained. Cells migrated were counted in each membrane.6. Real-time RT-PCR for mRNA expressionAFs were grouped and treated as described above. Total RNA was isolated from AFs with use of Trizol Reagent. The expression of Smad2, Smad3, Smad7,?-SMA, procollagen?and?mRNA was studied after treatment of drug-containing serum and TGF-?1. The relative mRNA expression of the genes was determined by the 2-??Ct method.7. Western blot analysis for protein expressionAFs were grouped and treated as described above. Cells were harvested. Protein was extracted and detected. Western-blot was used to detect the protein expression of p-Smad2, p-Smad3, Smad2, Smad3, Smad7,?-SMA, procollagen?.8. Statistical analysisThe data are presented as mean±SD and analyzed with SPSS 16.0. An unpaired Student's t-test was applied, when only two groups were compared. Comparisons among groups were analyzed by ANOVA. P<0.05 was considered to be statistically significant.Results1. Comparison of proliferation of AFs induced by TGF-?1The proliferation in the TGF-?1 group was higher than that in the contrl group (P<0.01). The proliferation in the treatment groups were decreased compared with that in TGF-?1 group (P<0.05 or P<0.01). And among the treatment groups, the Losartan group had the lowest proliferation (P<0.01). Then it was the QCHD group (P<0.01). The proliferation in the QCLD group was higher than that in the QCHD and Losartan groups (P<0.05).2. Comparison of migration of AFs induced by TGF-?1The mumber of cell migration in the TGF-?1 group was higher than that in the contrl group (P<0.01). The migration in the treatment groups were decreased compared with that in TGF-?1 group (P<0.05 or P<0.01).3. Comparison of Smad2, Smad3, Smad7,?-SMA, procollagen?and?mRNA expression induced by TGF-?1(1) Comparison of Smad2 mRNA expression:The expression of Smad2 mRNA was higher in TGF-?1 group than that in the control group (P<0.01). And the mRNA expression in the treatment groups were decreased compared with that in TGF-?1 group (P<0.01). And among the treatment groups, the Losartan group had the lowest expression (P<0.01). Then it was the QCHD group (P<0.01). The Smad2 mRNA expression in the QCLD group was higher than that in the QCHD and Losartan groups (P<0.05).(2) Comparison of Smad3 mRNA expression:The expression of Smad3 mRNA was higher in TGF-?1 group than that in the control group (P<0.01). The expression of Smad3 mRNA was decreased significantly in the QCHD group and Losartan group compared with that in the TGF-?1 group (all P<0.05). There was no significant difference in comparison between the QCLD group and the TGF-?1 group (P>0.05). There was a significant difference between the QCLD group and the Losartan group (P<0.05).(3) Comparison of Smad7 mRNA expression:There was no significant difference in comparison between the TGF-?1 group and the control group (P>0.05). And the Smad7 mRNA expression in the treatment groups was increased compared with that in TGF-?1 group (P<0.01). The expression of Smd7 mRNA was inreased significantly in the QCHD group and the Losartan group compared with that in the QCLD group (P<0.05).(4) Comparison of?-SMA mRNA expression:The expression of?-SMA mRNA was higher in TGF-?1 group than that in the control group (P<0.01). And the mRNA expression in the treatment groups was decreased compared with that in TGF-?1 group (P<0.01). Among the treatment groups, the Losartan group had the lowest expression (P<0.01). Then it was the QCHD group (P<0.01). The?-SMA mRNA expression in the QCLD group was higher than that in the Losartan groups (P<0.05).(5) Comparison of procollagen?mRNA expression:The expression of procollagen?mRNA was higher in TGF-?1 group than that in the control group (P<0.01). The expression of procollagen?mRNA was decreased significantly in the QCHD group and Losartan group compared with that in the TGF-?1 group (P<0.05). There was no significant difference in comparison between the QCLD group and the TGF-?1 group (P>0.05).(6) Comparison of procollagen?mRNA expression:The expression of procollagen?mRNA was higher in TGF-?1 group than that in the control group (P<0.01). And the mRNA expression in the treatment groups was decreased compared with that in TGF-?1 group (P<0.05 or P<0.01). And among the treatment groups, the Losartan group had the lowest proliferation (P<0.01). Then it was the QCHD group (P<0.01). The procollagen?mRNA expression in the QCLD group was higher than that in the Losartan group (P<0.05). 3. Comparison of Smad2, Smad3, p-Smad2, p-Smad3,?-SMA, procollagen?, Smad7 protein expression induced by TGF-?1(1) Comparison of expression of Smad2 protein:The expression of Smad2 protein was higher in TGF-?1 group than that in the control group (P<0.01). And the protein expression in the treatment groups was decreased compared with that in TGF-?1 group (P<0.01). And among the treatment groups, the Losartan group had the lowest expression (P<0.01). And it was lower in the Losartan group than that in the QCHD and QCLD groups (P<0.05 or P<0.01).(2) Comparison of p-Smad2 protein expression:The expression of p-Smad2 protein was higher in TGF-?1 group than that in the control group (P<0.01). And the protein expression in the treatment groups was decreased compared with that in TGF-?1 group (P<0.01). And it was lower in the Losartan group and QCHD group than that in the QCLD group (P<0.05).(3) Comparison of Smad3 protein expression:The expression of Smad3 protein was higher in TGF-?1 group than that in the control group (P<0.01). The expression of Smad3 protein was decreased significantly in the QCHD group and Losartan group compared with that in the TGF-?1 group (all P<0.05). There was a significant difference between the QCLD group and the Losartan group (P<0.05).(4) Comparison of p-Smad3 protein expression:The expression of p-Smad3 protein was higher in TGF-?1 group than that in the control group (P<0.01). And the protein expression in the treatment groups was decreased compared with that in TGF-?1 group (P<0.01). And it was lower in the Losartan group than that in the QCLD group (P<0.05).(5) Comparison of expression of?-SMA protein:The expression of a-SMA protein was higher in TGF-?1 group than that in the control group (P<0.01). And the protein expression in the treatment groups was decreased compared with that in TGF-?1 group. Among the treatment groups, the Losartan group had the lowest expression (P<0.01). And then it was QCHD group (P<0.01). It was lower in the Losartan group than that in the QCLD group (P<0.01).(6) Comparison of expression of procollagen?protein:The expression of procollagen?protein was higher in TGF-?1 group than that in the control group (P<0.01). And the protein expression in the treatment groups was decreased compared with that in TGF-?1 group (P<0.01). It was lower in the Losartan group than that in the QCLD group (P<0.05).(7) Comparison of Smad7 protein expression:There was no significant difference in comparison between the TGF-?1 group and the control group (P>0.05). And the Smad7 protein expression in the treatment groups was increased compared with that in TGF-?1 group (P<0.01). And among the treatment groups, the Losartan group had the highest expression (P<0.01). And then it was QCHD group(P<0.01).Conclusion(1) QC could inhibit the proliferation and migration induced by TGF-?1 in AFs through TGF-?1/Smad signaling pathway.(2) QC could inhibit the transdifferentiation induced by TGF-?1 in AFs, and the mechanisms might be related to the down-regulation of the mRNA and protein expression of Smad2, Smad3, p-Smad2, p-Smad3 and up-regulation of the mRNA and protein expression of Smad7.(3) QC could inhibit the extracellular matrix synthesis induced by TGF-?1 in AFs, and the mechanisms might be related to the down-regulation of the mRNA and protein expression of Smad2, Smad3, p-Smad2, p-Smad3 and up-regulation of the mRNA and protein expression of Smad7. BackgroundHypertensive vascular remodeling is the major adverse cardiac event. Transforming growth factor?1 (TGF-?1) can stimulate the proliferation, migration, extracellular matrix production, and induce the transdifferentiation of AF into myofibroblasts during hypertensive vascular remodeling.Quercetin (3,3',4',5,7-pentahydroxyflavone, Que), one of the most widely distributed bioflavonoids in the nature, has been reported to possess multiple properties in cardiovascular diseases, such as anti-hypertensive, anti-oxidative, anti-inflammatory, anti-coagulative and attenuating cardiac hypertrophy. Moreover, Que is one of the active principles in QC. In the present study, the effect of Que on the proliferation of AFs stimulated by TGF-?1 was observed. And the variation of Smad2,?-SMA and precollagen?and?mRNA expression was detected by real-time RT-PCR, while the protein expression of Smad2 and p-Smad2 was detected by western blot. The possible mechanism of Que in reversing biological function of AFs induced by TGF-?1 was investigated, which might provide scientific evidence for the treatment of vascular remodeling in clinic.AimsTo investigate the effects of Que on the proliferation and the variation of Smad2,?-SMA, precollagen?and?, p-Smad2 mRNA and protein expression. And to investigate the possible mechanism of Que in reversing the biological function of AFs induced by TGF-?1.Methods1. Cell thawing and subculture Freezing tubes were taken out from the nitrogen canister. After thaw, suspension and centrifugation, cells were placed on culture flasks. When the density reached 90%, cells were subcultured.2. MTT assay was used to detect the proliferationAFs (2×104) were seeded in 96-well plate and incubated overnight. Then cells were serum-starved and synchronized for 4 h. AFs in the experiment were treated with (6.25?mol/L,12.5?mol/L and 25?mol/L) Que for 24 h, then stimulated by TGF-?1 for an additional 24 h.20?l MTT was added and incubated for 4 h prior to harvest. Supernatant was removed and the DMSO was added. Absorbance of the samples was recorded by a microplate reader.3. Real-time RT-PCR analysisAfter starvation and synchronization for 4 h, cells were treated with Que of different doses for 24 h prior to induction by TGF-?1 for another 24 h. Total RNA was isolated using Trizol Reagent and reverse transcribed. The mRNA expression of Smad2,?-SMA, procollagen?and?was detected.4. Western blot analysisAFs were grouped and treated as described above. Protein was extracted and detected. Western-blot was used to detect the protein expression of p-Smad2 and Smad2.5. Statistical analysisData are expressed as mean±SD. Independent-samples t-test and ANOVA was used with SPSS 6.0. A P-value of less than 0.05 was considered statistical significantly.Results1. Comparison of proliferation of AFs induced by TGF-?1The proliferation in the TGF-?1 group was higher than that in the contrl group (P<0.01). The proliferation in the high and medium dose groups was decreased compared with that in TGF-?1 group (P<0.01). And the high dose group had the lowest proliferation (P<0.01). Then it was the medium dose group (P<0.01). There was no significant difference in comparison between the low dose group and the TGF-?1 group (P>0.05).2. Comparison of Smad2,?-SMA, procollagen?and?mRNA expression induced by TGF-?1(1) Comparison of Smad2 mRNA expression:The expression of Smad2 mRNA was higher in TGF-?1 group than that in the control group (P<0.01). And the mRNA expression in the high dose and medium dose groups was decreased compared with that in TGF-?1 group (P<0.01). The high dose group had the lowest expression (P<0.01). Then it was the medium dose group (P<0.01). There was no significant difference in comparison between the low dose group and the TGF-?1 group (P>0.05).(2) Comparison of a-SMA mRNA expression:The expression of a-SMA mRNA was higher in TGF-?1 group than that in the control group (P<0.01). And the mRNA expression in the high dose and medium dose groups was decreased compared with that in TGF-?1 group (P<0.05 or P<0.01). No significant difference was shown in comparison between the low dose group and the TGF-?1 group (P>0.05).(3) Comparison of procollagen?mRNA expression:The expression of procollagen?mRNA was higher in TGF-?1 group than that in the control group (P<0.01). And the mRNA expression in the high dose and medium dose groups was decreased compared with that in TGF-?1 group (P<0.05). There was no significant difference in comparison between the low dose group and the TGF-?1 group (P>0.05).(4) Comparison of procollagen?mRNA expression:The expression of procollagen?mRNA was higher in TGF-?1 group than that in the control group (P<0.01). And the mRNA expression in the high dose and medium dose groups was decreased compared with that in TGF-?1 group (P<0.05). The high dose group had the lowest expression (P<0.05). Then it was the medium dose group (P<0.05). There was no significant difference in comparison between the low dose group and the TGF-?1 group (P>0.05).3. Comparison of Smad2 and p-Smad2 protein expression induced by TGF-?1(1) Comparison of expression of Smad2 protein:The expression of Smad2 protein was higher in TGF-?1 group than that in the control group (P<0.01). And the protein expression in high dose and medium dose groups was decreased compared with that in TGF-?1 group (all P<0.01). There was no significant difference in comparison between the low dose group and the TGF-?1 group (P>0.05).(2) Comparison of p-Smad2 protein expression:The expression of p-Smad2 protein was higher in TGF-?1 group than that in the control group (P<0.01). And the protein expression in the high dose and medium dose groups was decreased compared with that in TGF-?1 group (P<0.01 or P<0.05). No significant difference was shown in comparison between the lowdose group and the TGF-?1 group (P>0.05).Conclusion(1) Que could inhibit the proliferation stimulated by TGF-?1 in AFs.(2) Que could down-regulate the mRNA expression of Smad2,?-SMA and procollagen?and?, as well as the protein expression of Smad2 and p-Smad2.(3) Que could improve the harmful biological activity of AFs induced by TGF-?1 through TGF-?1/Smad2 signaling pathway.