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Objective To investigate the effect and mechanism of salvianolic acid B(SalB)on myocardial fibrosis in diabetic rats based on RhoA/ROCK1 signaling pathway.Methods Diabetic rat model was established by injecting streptozotocin into tail vein of healthy male SD rats.At the end of the 12th week, the collagen fiber expression in the left ventricular myocardium of rats was detected by Sirius Red reagent staining.The protein expressions of α-SMA, Collagen and Collagen III in left ventricular myocardium were detected by Western blot.Cardiac fibroblasts(CFs)were cultured in SD rats by differential adhesion, and CFs were pretreated with different concentrations of SalB(12.5, 25, 50 μmol·L-1)for 1 h, and the optimal dose was determined by high glucose induction.RhoA protein expression in CFs was detected by immunofluorescence; the protein expressions of α-SMA, Collagen , Collagen III, RhoA and ROCK1 in CFs were detected by Western blot.Results Compared with the normal control group, the expression of collagen in left ventricular myocardium of diabetic rats increased significantly, and the expression of α-SMA, Collagen and Collagen III increased significantly.After SalB intervention, the expression of collagen decreased significantly, and the expression of the above proteins decreased significantly(P<0.01).The expression of α-SMA, Collagen , Collagen III, RhoA and ROCK1 in CFs stimulated with 25 mmol·L-1 high glucose for 24 h was significantly increased.After pretreatment with SalB(25 μmol·L-1)and inhibitor Y-27632(10 μmol·L-1), the activity of CFs was significantly inhibited, and the expression of these proteins was significantly decreased(P<0.01).Conclusion SalB can improve myocardial fibrosis in diabetic rats, and has a certain role in protecting the myocardium of diabetic rats.The mechanism may be related to the inhibition of RhoA/ROCK1 signaling pathway.
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OBJECTIVES@#Silence of SET domain containing lysine methyltransferase 7 (SET7) alleviates myocardial tissue injury caused by ischemia-reperfusion. But the effects of SET7 on angiotensin II (Ang II)-induced myocardial fibroblast proliferation and the collagen synthesis are not clear. The purpose of this study was to explore the effect of SET7 on the proliferation and collagen synthesis of myocardial fibroblasts and its mechanisms.@*METHODS@#Myocardial fibroblasts were isolated and identified by immunofluorescence. Myocardial fibroblasts were randomly divided into 4 groups: a control group (cells were normally cultured), an Ang II group (cells were treated with 100 nmol/L Ang II for 24 h), a siCtrl group (cells were transfected with siRNA control and were then treated with 100 nmol/L Ang II for 24 h), and a siSET7 group (cells were transfected with siRNA SET7 and were then treated with 100 nmol/L Ang II for 24 h). Cell counting kit-8 (CCK-8) and 5-ethynyl-2'-deoxyuridine (EdU) assay were used to evaluate cell proliferation. Real-time PCR was used to detect the mRNA levels of SET7, collagen I, collagen III, and α-smooth muscle actin (α-SMA). Western blotting was used to detect the protein expression of SET7, collagen I, collagen III, α-SMA, sonic hedgehog (Shh), ptched1 (Ptch1), and glioma-associated oncogene homolog 1 (Gli1).@*RESULTS@#Fluorescence microscopy showed positive vimentin staining, and myocardial fibroblasts were in good condition. As compared to the control group, the mRNA and protein levels of SET7 in the Ang II group were significantly upregulated; cell proliferation rate and EdU fluorescence intensity in the Ang II group were significantly increased; the mRNA and protein levels of collagen I, collagen III, and α-SMA were significantly upregulated (all @*CONCLUSIONS@#Silence of SET7 gene inhibits Ang II-induced proliferation and collagen synthesis of myocardial fibroblasts. Shh signaling pathway may be involved in this process.
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Angiotensin II/pharmacology , Cell Proliferation , Cells, Cultured , Collagen/genetics , Fibroblasts , Hedgehog ProteinsABSTRACT
Aim To explore the effect of miR-152 on proliferation of cardiac fibroblasts ( CFS) in diabetic cardiomyopathy. Methods Diabetic cardiomyopathy model was established in SD rats by STZ injection, and CFS proliferation model was established by high glucose (33. 3 mmol • L ~1). HE and Masson staining were performed in paraformaldehyde fixed myocardium of rats. Western blot determined a-SMA and collagen I protein expression. qPCK detected gene expression of miR-152. MTT assay analyzed the proliferation of cells. Results HE and Masson staining showed the higher level of myocardial collagen in diabetic cardiomyopathy model. Furthermore, the myocardial myo-cytes lined up in disorder. Western blot showed that the expressions of a-SMA and collagen I were up-regulated in the diabetes mellitus ( DM ) group, while the expression of miR-152 was down-regulated. The result of the in vitro experiment showed that a-SMA and collagen I expressions were down-regulated after trans-fected miR-152 mimics. The proliferation of CFS was also down-regulated after transfected miR-152 mimics. Conclusions miR-152 plays an important role in the proliferation of CFS and may ameliorate diabetic cardiomyopathy.
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Objective To investigate the role of microRNA-1 (miR-1) in cardiac fibroblasts induced by high glucose in rats. Methods The primary fibroblasts were cultured from the apical tissue of 1-3 day-old Sprague-Dawley (SD) rats. The cells which were passaged to generation 3 or 4, were randomly divided into normal glucose+lentivector-vehicle group (CON+Lv-Vehicle group), normal glucose+lentivector-miR-1 group (CON+Lv-miR1 group), high glucose+lentivector-vehicle group (HG+Lv-Vehicle group), high glucose+lentivector-miR-1 group (HG+Lv-miR1 group), high glucose+Lv-Vehicle+inhibitor group (HG+Lv-Vehicle+CC group), and high glucose+lentivector-miR-1+inhibitor group (HG+Lv-miR1+CC group). The myocardial fibroblasts were cultured in the concentration of 5.5 mmol/L glucose (normal glucose) or 25.0 mmol/L (high glucose) DMEM medium. Then lentiviral vector containing miR-1 silent sequence or the same volume of lentiviral vector was inoculated into the cells. The AMP activated protein kinase (AMPK) inhibitor Compound C (20 μmol/L) was added to the medium at 12 hours before sampling in inhibitor groups. The expression of phosphorylation of AMPK (p-AMPK), collagenⅠandⅢ, matrix metalloproteinase (MMP-2, MMP-9), and autophagy flux related protein LC3B-Ⅱ and p62/SQSTM1 were measured by Western Blot. Results The purity of rat myocardial fibroblasts in vitro was 97%. Compared with CON+Lv-Vehicle group, there was no significant difference in the expression of p-AMPK in CON+Lv-miR1 group, the expression of p-AMPK in HG+Lv-Vehicle group was significantly decreased (p-AMPK/t-AMPK: 44.72±3.29 vs. 100.00±7.77, 1 < 0.01). The expression of p-AMPK in HG+Lv-miR1 group was higher than that in HG+Lv-Vehicle group (p-AMPK/t-AMPK:60.52±5.16 vs. 44.72±3.29, 1 < 0.05). Compared with HG+Lv-Vehicle group, the expressions of collagen, MMP, LC3B-Ⅱand p62/SQSTM1 in HG+Lv-miR1 group were significantly decreased; after the treatment with AMPK inhibitor, the expressions of collagen, MMP, LC3B-Ⅱ, p62/SQSTM1 were significantly increased (HG+Lv-Vehicle+CC group vs. HG+Lv-Vehicle group: collagen Ⅰ/β-actin: 158.74±13.21 vs. 100.00±7.64, collagenⅢ/β-actin: 177.38± 17.31 vs. 100.00±5.18, MMP-2/β-actin: 130.09±14.31 vs. 100.00±10.47, MMP-9/β-actin: 215.54±20.92 vs. 100.00±11.28, LC3B-Ⅱ/β-actin: 159.34±13.83 vs. 100.00±6.44, p62/SQSTM1/β-actin: 201.01±24.02 vs. 100.00±8.62; HG+Lv-miR1+CC group vs. HG+Lv-miR1 group: collagenⅠ/β-actin: 108.69±9.93 vs. 80.83±7.24, collagenⅢ/β-actin: 127.68±10.46 vs. 81.56±9.97, MMP-2/β-actin: 106.66±10.21 vs. 74.80±7.43, MMP-9/β-actin: 145.65±11.56 vs. 74.63±10.55, LC3B-Ⅱ/β-actin: 150.15±13.28 vs. 22.98±2.87, p62/SQSTM1/β-actin: 130.48±10.74 vs. 49.90±2.27, all 1 < 0.05). Conclusion miR-1 gene silencing inhibits myocardial fibrosis induced by high glucose, its mechanism may be related to the up-regulation of p-AMPK, which can recover autophagy flux.
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Objective The mechanisms of methylation acting on myocardial fibrosis are not yet clear at present. The aim of this study was to investigate the role of DNA methyltransferase 3A (DNMT3A) in regulating the expressions of collagens during the activation of cardiac fibroblasts.Methods Cardiac fibroblasts were obtained from 50 neonatal mice and divided into three groups: blank control, DNMT3A overexpression plasmid (mDNMT3A-pEGFP-C3) and small interference DNMT3A siRNA. The contents of collagens in the cell supernatant were detected by ELISA. The mRNA and protein expressions of type I collagen (Col Ⅰ), type Ⅲ collagen (Col Ⅲ) and DNMT3A in the cardiac fibroblasts were determined by real-time quantitative PCR and Western blot respectively and the proliferative activity of the cardiac fibroblasts measured by CCK8 assay.Results The contents of Col I and Col Ⅲ in the cell supernatant were significantly increased in the DNMT3A overexpression plasmid group but decreased in the DNMT3A siRNA group as compared with those in the blank control (P<0.05). The expressions of Col Ⅰ, Col Ⅲ and DNMT3A were remarkably higher in the DNMT3A overexpression plasmid group but lower in the DNMT3A siRNA group than in the blank control (P<0.05). The cell activity was markedly higher in the DNMT3A overexpression plasmid group than in the empty vector plasmid and control groups (2.087±0.317 vs 1.063±0.223 and 1.082±0.207, P<0.05) but lower in the DNMT3A siRNA group (0.463±0.087) than in the latter two (P<0.05).Conclusion DNMT3A can increase the proliferation and activation of cardiac fibroblasts, upregulate the expressions of collagens and thus promote myocardial fibrosis.
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Objective To study the expression of osteopontin (OPN) and collagen type Ⅰ in the rat myocardial fibroblasts infected by coxsackievirus B3 (CVB3) and to explore the possible pathogenesis of OPN on viral myocarditis.Methods Cardiac fibroblasts were isolated from neonatal rat and cultured with an traditional method.The primary cultured rat myocardial fibroblasts were infected by CVB3 which multiplicity of infection (MOI) was 0.5 PFU/cell.The myocardial fibroblasts were divided into control group 12 h and CVB3 groups (infected after 12 h,1 d,2 d,3 d).The expression of OPN and collagen type Ⅰ were detected by RT-PCR,Western Blotting and immunohistochemistry.And the linear correlation between the expression of OPN and the expression of collagen type Ⅰ was analyzed.Results ( 1 ) The gray scale values of the OPN/β-actin in control group(12 h)and viral groups (12 h,1 d,2 d,3 d) were 0.38 ± 0.06,0.56 ± 0.06,0.72 ± 0.05,0.98 ± 0.06,0.86 ± 0.02 respectively with RT-PCR,and were 0.26 ± 0.03,0.36 ± 0.03,0.52 ± 0.04,0.76 ± 0.05,0.62 ± 0.02 respectively with Western Blotting.The expression of OPN was found to be increased after 12 h infection,reached to the maximum after 2 d infection and displayed a decreased tendency after 3 d infection.There was significant difference on the gray scale values of the OPN/β3-actin ( F=74.965,53.004,respectively,P ＜ 0.05 ).(2) The gray scale values of the collagen type Ⅰ/β-actin in control group (12 h)and viral groups (12 h,1 d,2d,3 d) were 1.12 ± 0.03,1.18 ± 0.01,1.22 ± 0.02,1.33 ± 0.02,1.28 ± 0.03,respectively with RTPCR.These results suggested that the collagen type Ⅰ expression started to increase at 12 h when infected by CVB3,reached to the maximum at 2 d,and then decreased after 3 d infection,the difference was significant ( F =38.241,P ＜ 0.05).(3) The expression of OPN was positively correlated with the expression of collagen type Ⅰ ( r=0.948,P ＜ 0.001 ).Conclusion CVB3 can induce the expression of OPN and collagen type Ⅰ in the rat myocardial fibroblasts and the expression of OPN and collagen type Ⅰ displays positive correlation.It suggests that OPN can promote the expression of collagen type Ⅰ in myocardial fibroblasts,and may play an important role in the pathogenesis of viral myocarditis.
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Objective To study the effect of angiotensin Ⅱ(AngⅡ) on myocardial fibroblasts(MFs) proliferation,the expression and transposition of protein kinase C epsilon(PKC?) and alpha(PKC?),and to find out the mechanism of AngⅡpromoting proliferation and signal trarsduction.Methods The primary culture neonate rat's MFs was used depending on the different time of cell adherence,by the method of immunohistochemical method identifying MFs,2-4 generations MFs were divided into experimental group and control group,experimental group was added with AngⅡ 10-6 mol/L,and nothing was added to control group.Colorimetric method of metrazolium salt(MTT) was used to detect the MFs proliferation; indirect immunofluorescence was used to detect the distribution and location of PKC? and PKC?,then Image-Pro-Plus 4.0 was used to add up fluorescence intensity.Results 1.The number of MFs in experimental group increased much more than that in control group and there was obviously statistical significance(P