The effect of astragalus membranaous on urotensinII-induced collagen synthesis and transforming growth factor-β1 scretion of cardiac fibroblasts
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Objective To observe the effect of astragalus membranaous on UrotensinⅡ(UⅡ)-induced collagen synthesis and transforming growth factor-β1 scretion of cardiac fibroblasts.Methods The neonatal cardiac fibroblasts were cultured and the forth generation cells were allocated into 3 groups:control group (cells were cultured under normal conditions),UⅡ groups(cells were cultured with 10～7mol/L UⅡ),astragalus membranaous groups(cells were cultured with 10～7mol/L UⅡ and 40mg/L astragalus membranaous)respectively.After certain periods of time,the mRNA expression of collagenⅠ,collagen Ⅲ and TGF-β1 were measured by real-time RT-PCR,collagen synthesis was measured by 3H-proline incorporation and TGF-β1 secretion was measured by ELISA.Results UⅡ significantly induced collagen synthesis and secretion of TGF-β1 in neonatal cardiac fibroblasts in vitro and astragalus membranous could reduce these effects of UⅡ.Conclusion Astragalus membranaous can delay the progression of cardiac fibrosis and cardiac remodeling by inhibiting the UⅡ-induced collagen synthesis and TGF-β1 secretion in cardiac fibroblasts.Keywords:
Cardiac Fibrosis
Astragalus
Urotensin II
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Objective To investigate the mechanism of TGF-β1 action in chronic renal fibrosis.Methods Human renal fibroblasts primarily cultured in vitro were divided into 5 groups:(1) the control group with 0ng/ml TGF-β1;(2) 0.1gng/ml TGF-β1 trial group;(3)1ng/ml TGF-β1 trial group;(4)5ng/ml TGF-β1 trial group;(5)10ng/ml TGF-β1 trial group.Expression of heat shock protein 47(HSP47),which in human renal fibroblasts in vitro under the effect of TGF-β1,was determined by immunocytochemistry.And expressions of HSP47 mRNAs and I collagen mRNAs were examined by semi-quantitive reverse transcriptase polymerase chain reaction(RT-PCR).Results Compared with the control group,expression of HSP47 and collagen I in cultured human renal fibroblasts significantly increased in the trial groups.TGFβ1 promoted expression of collagen I and HSP47 of renal fibroblast in dose-dependent manner range from 0.1ng/ml to 5ng/ml.Conclusion TGF-β1 is involved in occurrence and development of renal fibrosis partly by enhancing expression of HSP47 in renal fibroblasts and further increasing collagen synthesis.
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Objective To investigate the role of extracellular signal-regulated kinase1/2 (ERK1/2) pathway in collagen synthesis of rat cardiac fibroblasts (CFb) induced by platelet-derived growth factor (PDGF).Methods Neonatal rat cardiac fibroblasts were isolated and expressions of typeⅠand type Ⅲ collagen were measured by immunocytochemistry and western blot.Results Compared with control group,expressions of type Ⅰand type Ⅲ collagen in cultured rat cardiac fibroblasts induced by PDGF were increased,while the role of PDGF stimulation was inhibited by 25 μmol/L PD98059.Conclusion The collagen synthesis of cultured rat cardiac fibroblasts was regulated by PDGF through ERK1/2 pathway.
Platelet-derived growth factor
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The aim of the present study was to determine whether transforming growth factor-β1 (TGF-β1)-induced collagen production in cardiac fibroblasts is affected by reactive oxygen species (ROS). Cardiac fibroblasts (passage 2) from normal male adult rats were cultured to confluency and incubated in serum-free Dulbecco’s modified Eagle’s medium for 24 h. The cells were then preincubated with(out) the tested inhibitors for 1 h and further incubated with(out) TGF-β1 at various concentrations and for 1, 2, 4, 24 or 48 h. TGF-β1 induced a dose-dependent increase in the soluble collagen production in cardiac fibroblasts after 48 h of incubation. No significant effect of TGF-β1 (600 pmol/l) on collagen production and on α-smooth muscle actin (α-SMA) protein expression was found after 1, 2 and 4 h of incubation. After 24 and 48 h, TGF-β1 stimulated collagen production and α-SMA protein expression in cardiac fibroblasts. Intracellular ROS were not affected by TGF-β1 after 0.5 and 1 h of incubation, began to rise after 2 h, and reached a maximal increase after 4 h. The TGF-β1-stimulated ROS and collagen production is reduced by the ROS inhibitor diphenyleneiodonium chloride (DPI). DPI also decreased the TGF-β1-stimulated α-SMA protein expression in rat cardiac fibroblasts. Our data indicate that the TGF-β1-induced increase in ROS preceded the rise in collagen production and α-SMA protein expression and that ROS inhibition diminished the conversion of fibroblasts into myofibroblasts.
Myofibroblast
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Objective To investigate the effect of TGF β 1 on the proliferation, collagen synthesis and collagen mRNA type Ⅰand type Ⅲ expression of renal interstitial fibroblasts in vitro. Methods Fibroblasts were isolated from rat renal medulla. Collagen synthesis and expression of collagen type Ⅰand type Ⅲ mRNA of renal interstitial fibroblasts were investigated by L 3 H proline incorporation and RT PCR, respectively. Results TGF β 1 had a dose dependent stimulating effect on the collagen synthesis of fibroblasts with or without the presence of serum (r= 0 9822, P0 05 and r = 0 9597, P0 05, respectively). TGF β 1 could stimulate the proliferation and collagen synthesis of renal interstitial fibroblasts. By the RT PCR method,TGF β 1 could increase the expression of collagen type Ⅰ and type Ⅲ mRNA of fibroblasts with dose dependent (from 2 to 10 ng/ml) and time dependent effects (from 6 to 48 hours with the concentration of TGF β 1 of 5 ng/ml).Conclusion TGF β 1 at a low concentration may be involved in renal interstitial fibrosis by promoting the collagen synthesis and expression of fibroblasts.
Type I collagen
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Objective To obser ve the effect of transforming grow th factor beta 1(TGF-β1) on the ex pression of collagen in rat atrial and ventricular fibroblasts, and to investigate its specific molecular mechanisms. Methods Tissue explant attachment was used to culture fibroblasts obtained from the atrium and ventricle of rat heart, and they were identified with SABC immunocytochemical staining, and then the following experiments were carried out.(1) Hydroxyproline digestion was performed to study the effects of TGF-β1, within different concentrations(0, 5, 10ng/ml) and different action time(6, 12, 24, 48h) on the content of hydroxyproline in rat's atrial and ventricular fibroblasts.(2) Rat's atrial and ventricular fibroblasts were stimulated with TGF-β1 in optimal concentration and action time, the expression of α-smooth muscle actin(α-SMA) was determined with Western blotting, and the expressions of typeⅠ and Ⅲ collagen m RNA were evaluated with reverse-transcription PCR. The contents of hydroxyproline in the respective cells were measured with hydroxyproline determination. Western blotting was used to measure the protein expression of Smad2/3, p-Smad2/3 and Smad7. Results(1) TGF-β1 was shown to stimulate the collagen synthesis in rat's atrial and ventricular fibroblasts, and the optimal stimulus was TGF-β1 concentration 5ng/ml with action time of 24 h.(2) After being stimulated by optimal stimulation effect of TGF-β1, the expression of typeⅠ and Ⅲ collagen and p-Smad2/3 increased, while that of Smad7 decreased significantly only in atrial fibroblasts(P0.05), but not in ventricular fibroblasts. No statistical difference was found in the expression of Smad2/3 between the atrial and ventricular fibroblasts after being stimulated by TGF-β1under optimal stimulating conditions. Conclusion TGF-1 can induce dysbolism of collagen of cardiac fibroblasts with abnormal expression of cytoskeletal protein, which may occur more obviously in rat's atrial fibroblasts than in ventricular fibroblasts, and its mechanism may be related with TGF-β1/SMAD pathway.
Hydroxyproline
Atrium (architecture)
Type I collagen
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The aim of the present study was to elucidate how transforming growth factor-β 1 (TGF-β 1 ) can stimulate collagen deposition in cardiac tissue by interstitial cells via stimulation of fibroblasts, via myofibroblasts, or via differentiation of fibroblasts to myofibroblasts. The dose- and time-dependent stimulation of collagen production and of expression of α-smooth muscle actin (α-SMA), a marker of myofibroblasts, was studied in cultures of second-passage adult rat cardiac fibroblasts. The TGF-β 1 -stimulated collagen production is positively correlated ( r =0.68, P <0.001) with the appearance of α-SMA. Only at high concentrations (40 to 600 pmol/L) and after a long time (24 to 48 hours) of incubation, TGF-β 1 increases the collagen production and stimulates the differentiation of fibroblasts to myofibroblasts. The maximal stimulation of the collagen production (2-fold, P <0.001) observed after incubation of cultures of fibroblasts with 600 pmol/L TGF-β 1 for 48 hours is accompanied by a maximal stimulation of α-SMA expression (3.5-fold, P <0.001), when cultures consist mainly of myofibroblasts. The stimulation of collagen production cannot be reversed either after additional incubation of TGF-β 1 -stimulated second-passage cultures for 2 days or in their offspring in the next third passage after incubation for 7 days without TGF-β 1 . The increased collagen production in these third-passage cultures cannot be further stimulated by TGF-β 1 . Our data suggest that TGF-β 1 -stimulated collagen production in cultures of adult rat cardiac ventricular fibroblasts cannot be explained by a direct stimulation of the collagen production either in fibroblasts or in myofibroblasts. Instead, TGF-β 1 induces the differentiation of fibroblasts to myofibroblasts, which have a higher activity for collagen production than fibroblasts.
Myofibroblast
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The aim of this study was to explore the possible molecular mechanisms of paeonol in preventing ventricular remodeling. The cell viability of neonatal rat cardiac fibroblasts was detected by the method of MTT. RT-PCR and Western blot were used to measure the expression of TGF-β1, type I collagen and type III collagen. After treating the cardiac fibroblasts with paeonol, the cell viability decreased (p<0.01), and the expression of TGF-β1, type I collagen and types III collagen was significantly reduced (p<0.01). Thus, paeonol can inhibit the proliferation of fibroblast cells induced by aldosterone. The molecular mechanism is related to the down-regulation of TGF-β1 and type I and III collagen gene expression.
Paeonol
Viability assay
Type I collagen
MTT assay
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This study was to investigate the effects of transforming growth factor β1(TGF—β1) and platelets derived growth factor (PDGF) on the collagen synthesis in cultured rat cardiac fibroblasts (CFb) derived from SHR (CFb SHR) and WKY (CFbWKY). CFb derived from 12-week-old SHR and WKY were cultured by outgrowth of tissue block. Cell proliferation of CFb was measured by direct cell counting. Collagen synthesis was determined by 3H-proline incorporation. Data expressed as mean±SD of (n) determinations performed in triplicate. It was showed that proliferation of CFb was significantly promoted by PDGF in a dose dependent manner from a concentration range of 3-30ng/ml. Similarly, 1 to 10 ng/ml TGF-β1 stimulated a markedly dose dependent increase in cell number. TGF-β1 at concentration of 10 ng/ml provoked a 152% and 143% increase in cell number of CFbSHR and CFbWKY respectively in the medium containing 0.4% FCS. 3H-proline incorporation of CFb was promoted by 1-10 ng/ml TGF-β1 and 3-30ng/ml in a concentration dependent manner in both CFb SHR and CFbWKY. It is concluded that cell proliferation and collagen synthesis of CFb was promoted by TGF—β1 and PDGF.
Platelet-derived growth factor
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Objective: To explore the influence of angiotensin Ⅱ(AngⅡ) on cardiac fibroblasts (cfbs) collagen synthesis as well as the activity of precollagenase secreted by cfbs and to clarify whether AngⅡ was mitogenic in cultured cfbs.Method: Sirus Red method was employed to evaluate cultured cardiac fibroblasts collagen synthesis;type Ⅰ collagen was measured by competitive ELISA; collagenase activity was tested by fluorescence spectrophotometry. Immunohistochemical method was employed to evaluate the manifestation of proliferative cellular nuclear antigen (PCNA) and c myc, the proliferation related gene, transcription level were tested by in situ hybridization.Result: At the doses of 10 -9 ～10 -7 mol/L, AngⅡ could enhance gross collagen production in both 24 h and 48 h and type Ⅰ collagen in medium in 24 h in a dose dependent manner.Collagenase activity decreased with concentration of AngⅡ increasing. These effects of AngⅡ could be completely abolished by its receptor antagonist, saralasin. AngⅡ did not increase PCNA expression and c myc gene transcription.Conclusion: AngⅡ enhances collagen synthesis of cardiac fibroblasts and inhibites collagenase activity, thus increasing net collagen accumulation and accelerating cardiac fibrosis. AngⅡ has no direct effect on cardiac fibroblasts proliferation in vitro.
Cardiac Fibrosis
Saralasin
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To investigate the effects of tetrandrine (Tet) on proliferation and activation of rat cardiac fibroblasts.Firstly, the cell counting kit-8 (cck-8) assay was applied to detect the effects of Tet with different concentrations on proliferation of cardiac fibroblasts. Secondly, transforming growth factor (TGF-β)with a concentration of 5 μg/L was used to induce the cardiac fibroblast activation, and Western blot was performed to measure the expression variation of β-catenin, vimentin (Vm), fibronectin (Fn) and smooth muscle α-actin (SMA). At last, the real-time PCR was conducted to measure the expression change of collagen-1(Col-1) and collagen-3(Col-3).The cck-8 assay showed that the Tet with different concentrations respectively, which were 0.5 μmol/L, 1 μmol/L, 2 μmol/L, 4 μmol/L, and 8 μmol/L, significantly inhibited the proliferation of cardiac fibroblasts. The viability was decreased to 94.4%,84.9%,74.9%,63.8%and 50.3% respectively of the control group when the Tet concentration changed, and the difference was statistically significant, P=0.043, P<0.001, P<0.001, P<0.001, P<0.001 respectively. Western blot revealed that the expressions of β-catenin, Fn, SMA and Vm, were up-regulated by TGF-β(5 μg/L), the result showed that the difference was statistically significant, and the P values were 0.001,0.008,0.010,0.001 respectively. Then, the up-regulation of β-catenin, Fn and SMA was attenuated by pre-treatment of Tet, and the result also displayed that the difference was statistically significant, and the P values were 0.009, 0.005, 0.019,respectively. While there was no significant change in the expression of Vm, according to Western blotting, and P>0.05,at the same time, real-time PCR indicated that the up-regulations of Col-1 and Col-3 which were induced by TGF-β were blocked by pre-treatment of Tet, the result showed that the difference was statistically significant, P<0.001.According to the experimental results, we can draw the conclusion that: the Tet can significantly inhibit the proliferation of cardiac fibroblasts, meanwhile, it can block the activation of cardiac fibroblasts, which is induced by TGF-β. It is supposed that the Tet may probably have anti myocardial fibrosis, which indicates that it may probably be a medicine which is used to block the cardiac remodeling.
Tetrandrine
Cell counting
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