Abstract:Aim To investigate the effect of circ_0036167 on the fibrotic phenotype of cardiac fibroblasts and the potential mechanism involved. Methods Massons trichrome staining was performed in the myocardium of patients with heart failure (HF) and healthy organ donors. Levels of circ_0036167 and its host gene of MYO9A were determined by real-time quantitative polymerase chain reaction (RT-qPCR) in the myocardium of HF patients and healthy organ donors. Actinomycin D treatment and RNase R exonuclease digestion were used to test the stability of circ_0036167 in AC16 cells. The myocardial fibroblasts (mCF) of C57BL/6 mice were infected with recombinant circ_0036167 adenovirus, and the expression of fibrosis related genes COL1A1, COL3A1 and ACTA2 were detected at RNA and protein levels.EdU staining and transwell migration assay were performed to detect the effects of circ_0036167 on mCF proliferation and migration activities. According to the results of bioinformatic prediction, RNA binding protein immunoprecipitation (RIP) assay was performed to confirm the interaction between circ_0036167 and fused in sarcoma (FUS) protein. Effect of FUS knockdown on inhibition of fibrosis-related genes expression by circ_0036167 in mCF was determined. ResultsMassons trichrome staining showed that the cardiac fibrosis was significantly increased in the myocardium of HF patients (P＜0.001). Circ_0036167 was found markedly increased in the myocardium of HF patients (P＜0.05), with no significant difference in its host gene of MYO9A. In response to actinomycin D treatment and RNase R exonuclease digestion, circ_0036167 was more stable than MYO9A. Over-expression of circ_0036167 suppressed proliferation and migration of mCF, and inhibited RNA and protein expression of fibrosis-related genes in mCF. RIP assay revealed the interaction between circ_0036167 and FUS protein. Knock-down of FUS could increase fibrosis-related genes expression (P＜0.05 or P＜0.01), and significantly attenuated the inhibitory effect of circ_0036167 on fibrosis-related gene expression in mCF(P＜0.01 or P＜0.001). Conclusion FUS mediates the anti-fibrotic effect of circ_0036167 in mCF.

Abstract:Aim To investigate the effect of microRNA miR-25-3p on fibrosis-related gene expression in cardiac fibroblasts and mechanism involved. Methods A mouse model of cardiac fibrosis induced by angiotensin Ⅱ (AngⅡ) infusion was constructed, and the differentially expressed miRNAs in the fibrotic mouse myocardium were detected by miRNA chip array. Primary isolation and culture of C57BL/6 mouse cardiac fibroblasts (mCF) were performed, and a cell model of myocardial fibrosis was established based on AngⅡ-treated mCF. The effect of miR-25-3p mimic on the expression of fibrosis-related genes in mCF was studied. The dual luciferase reporter gene assay was performed to verify the binding of miR-25-3p on the 3′-untranslated region (3′UTR) of the B-cell translocation gene 2 (BTG2) gene. Actinomycin D experiment was performed to detect the effect of BTG2 on the stability of superoxide dismutase 2 (SOD2) mRNA in mCF. Results Expression of miR-25-3p was increased in the myocardium of AngⅡ-infused mice and AngⅡ-treated mCF. miR-25-3p could enhance the expression of fibrosis-related genes, including collagen type Ⅰ alpha 1 chain (COL1A1), collagen type Ⅲ alpha 1 chain(COL3A1) and α-smooth muscle actin(α-SMA) in mCF. BTG2 was confirmed to be a target gene of miR-25-3p, and BTG2 mediated the effect of miR-25-3p on promoting fibrosis-related gene expression in mCF. In addition, BTG2 could enhance the expression of SOD2 by increasing the stability of SOD2 mRNA in mCF. Conclusion MiR-25-3p inhibited the level of SOD2 through targeting BTG2 in mCF, resulting in enhancing fibrosis-related gene expression and promoting myocardial fibrosis.

Abstract:Aim To investigate the effect of overexpression of methyltransferase-like 3 (METTL3) on myocardial fibrosis. Methods METTL3 protein expression was detected in the myocardium of patients with heart failure (HF) and the healthy donors by Western blot assay. A C57BL/6 mouse model of transverse aortic constriction (TAC) surgery-induced myocardial fibrosis was established, and METTL3 protein expression was detected in the myocardium of TAC mice and sham mice. A cell model of angiotensinⅡ(AngⅡ)-induced myocardial fibrosis in mouse cardiac fibroblasts (CF) was established and used to detect METTL3 expression by Western blot assay. Expression of fibrosis-related genes, including collagen type Ⅰα1 (COL1α1), collagen type Ⅲ α1 (COL3α1) and actin α2 (ACTα2), was detected in mouse CF with adenovirus-mediated overexpression of METTL3. Flow cytometry, EdU and Transwell migration assay were used to detect proliferation and migration activity of mouse CF, respectively. Effects of cardiac specific expression of METTL3 on cardiac function and fibrosis were explored in mice subjected to TAC surgery. Results Protein expression of METTL3 was significantly increased in the myocardium of HF patients (P＜0.05). Consistently, significant up-regulation of METTL3 was observed in the myocardium of TAC mice and AngⅡ-treated mouse CF (P＜0.05, respectively). Overexpression of METTL3 could markedly enhance mouse CF proliferation and migration activities, as well as expression of fibrosis-related genes in mouse CF. Compared with mice in the sham group, significant increase of fibrosis-related gene expression, cardiac fibrosis and cardiac function injury were observed in TAC-induced mice with cardiac specific overexpression of METTL3. Conclusion Overexpression of METTL3 promotes cardiac fibrosis in mice.

Abstract:Aim To explore possible action mechanism of mitogen-activated protein kinase (MAPK) signal transduction pathway on chemotaxis of cardiac fibroblasts (CF) induced by transforming growth factor-β1 (TGF-β1). Methods The CF of neonatal SD rats were cultured. Cell were randomly divided into blank control group, TGF-β1 group, c-Jun N-terminal kinase (JNK) inhibitor (SP600125 10 μmol/L) group, P38MAPK inhibitor (SB203580 10 μmol/L) group and extracellular signal-regulated protein kinase (ERK) inhibitor (U0126 10 μmol/L) group. Except blank control group, the other groups were given 10 μg/L TGF-β1 and corresponding inhibitors. Methyl thiazolyl tetrazolium (MTT) colorimetric assay was applied to detect cell viability of CF. Transwell chamber was applied to detect motor ability of CF. The collagen content was detected by hydroxyproline kit. The enzyme-linked immunosorbent assay (ELISA) was applied to detect levels of monocyte chemotactic protein-1 (MCP-1) and plasminogen activator inhibitor-1 (PAI-1) in CF. Western blot was applied to detect expression levels of α-smooth muscle actin (α-SMA), type Ⅰ collagen (Col-1) and matrix metalloproteinase-9 (MMP-9) in CF. Results TGF-β1 could significantly promote cell viability and chemotaxis of CF and collagen content. MAPK inhibitors could inhibit the proliferation and chemotaxis, and decrease collagen content (P＜0.05). ELISA results showed that MAPK signaling pathway inhibitors could significantly decrease increased MCP-1 and PAI-1 induced by TGF-β1 treatment (P＜0.05). Western blot results showed that MAPK signaling pathway inhibitors could significantly decrease increased expression levels of α-SMA, Col-1 and MMP-9 protein induced by TGF-β1 treatment (P＜0.05). Conclusion TGF-β1 may promote chemotaxis of CF by activating MAPK signaling pathway. The application of MAPK inhibitors can inhibit myocardial fibrosis to certain extent.

Abstract:Aim To investigate the effect of high salt on cardiac fibroblast proliferation and intracellular free Ca2＋ concentration and pH. Methods SD rat cardiac fibroblasts were cultured by adherent method, and vimentin in cardiac fibroblasts was identified by immunofluorescence staining. The third generation cells were divided into control group (Na＋ 122.27 mmol/L) and high salt group (Na＋ 161 mmol/L), treated for 48 hours. Cell proliferation was detected by EdU fluorescent staining, the treated cells were loaded with Fura2-AM and BCECF-AM fluorescent indicators to measure intracellular pH and free Ca2＋concentration. Results The proliferation of cardiac fibroblasts in the high salt group was significantly higher than that in the control group, and the intracellular pH and free Ca2＋ concentration in the high salt group were significantly higher than those in the control group as well. Conclusion High salt cloud induce cardiac fibroblast proliferation and lead to increase intracellular free Ca2＋ concentration and pH value.

Abstract:Aim To investigate the mechanism of high sodium salt induced proliferation of cardiac fibroblasts (CFs) and the intervention effect of dendrobine. Methods Rat CFs were cultured with tissue explant method in vitro.The experiments were divided into 3 groups:control group (containing 139 mmol/L Na＋), high sodium salt group (containing 161 mmol/L Na＋), dendrobine group (161 mmol/L Na＋＋10－5 mol/L dendrobine), and cells were cultured for 48 hours. CCK-8 cell proliferation kit and MTT colorimetric assay were used to detect the proliferation of cells in each group.The protein expressions of proliferating cell nuclear antigen (PCNA), phosphorylated extracellular regulated protein kinase 1/2 (p-ERK1/2) and phosphorylated nuclear factor κB p65 (p-NF-κB p65) were detected by Western blot assay. The mRNA expressions of monocyte chemotactic protein 1 (MCP-1), vascular cell adhesion molecule 1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) were detected by real-time fluorescence quantitative PCR. Results Compared with the control group, the proliferations of CFs in the high sodium salt group were significantly increased after 48 hours culture. Compared with the high sodium salt group, 10－5 mol/L dendrobine could significantly inhibit the proliferation of CFs. Compared with the control group, the protein expressions of p-ERK1/2 and p-NF-κB p65 were increased, inflammatory factors MCP-1, VCAM-1 and ICAM-1 mRNA expressions were increased in high sodium salt group. Compared with the high sodium salt group, dendrobine could significantly reduce the expressions of PCNA and p-NF-κB p65 proteins and the expression of MCP-1 mRNA, but had no significant effect on the expressions of p-ERK1/2, VCAM-1 and ICAM-1. Conclusion High sodium salt induces the proliferation of CFs and the expression of inflammatory factors, and its mechanism is related to the upregulation of p-ERK1/2 and p-NF-κB p65 expressions. Dendrobine inhibits these effects and its mechanism is related to the inhibition of p-NF-κB p65.

Abstract:Aim To optimize the conditions and methods of SD rat cardiomyocytes and cardiac fibroblasts in vitro culture. Method Digested ventricular tissue with trypsin first, and then collagenaseⅡ. Collected and cultured neonatal rat cardiac myocytes and fibroblasts through the way of differential adhesion for two times. The basic shape change of cardiac myocytes and cardiac fibroblasts were observed under the opitical microscope. Cardiac myocytes were assayed by cardiac troponin immunofluorescenee and the second generation cardiac fibroblasts were assayed by vimentin immunofluorescence. Results Cardiomyocytes began to adhere and grow after 2～4 hours The adherent rate increased substantially and the cells beated spontaneously after 12～24 hours 48 ～72 hours later cardiomyocytes adhered to be clustered Both of the cells’ survival rate and purity reached more than 95%. The second generation to the fourth generation cardiac fibroblasts grew wel1 and its purity was as high as 98%. Conclusion A great quantity, purity and high survival rate of cardiocytes and cardiac fibroblasts can be effectively cultured by this method. And it provides an ideal experimental model for the cardiovascular disease and clinical studies.

Abstract:Aim To investigate the effect of metformin on angiotensin Ⅱ （AngⅡ） -induced proliferation of adult rat cardiac fibroblasts （CF） and its underlying mechanism. Methods The adult rat CF was isolated by a combination of trypsin and collagenase Ⅱ digestion.The cell proliferation was induced by AngⅡ （100 nmol/L） stimulation,and the CF was treated with metformin in different concentrations （10,50,and 200 μmol/L）.The proliferation of CF was evaluated by MTT assay,and the DNA synthesis was detected by EdU incorporation.The expression of endothelial nitric oxide synthase （eNOS） and phosphorylated endothelial nitric oxide synthase （p-eNOS） were detected by Western blot.The level of NO in CF culture supernatant fluids was measured by nitrate reductase method. Results Stimulation with AngⅡ for 48 h induced the proliferation of adult rat CF,and this effect was inhibited by pretreatment of CF with metformin in a concentration-dependent manner.Metformin significantly increased the phosphorylation level of eNOS in CF as well as the level of NO in cell culture supernatant fluids in a concentration-dependent manner.In addition,pretreatment of CF with eNOS inhibitor L-NAME markedly attenuated the inhibitory effect of metformin on AngⅡ-induced cell proliferation. Conclusion Metformin inhibits AngⅡ-induced proliferation of adult rat CF,and this effect may be associated with the activation of the eNOS/NO pathway.

Abstract:Aim To explore the effect of erythropoietin （EPO） on proliferation and phenotypic transformation of neonatal rat cardiac fibroblast （CF） induced by high glucose in vitro. Methods CF cells were cultured from neonatal rat primary cells. In the process of promoting the phenotypic transformation of CF cells,high glucose medium was selected for induction,and EPO was added for intervention. The same batch of CF cells were randomly divided into three groups: control group （5.5 mmol/L glucose）,high glucose group （25 mmol/L glucose）,and high glucose＋EPO group （25 mmol/L glucose with 20 kU/L EPO）. CF cells were identified by using immunohistochemistry Levels of type Ⅰ and Ⅲ collagens were detected by enzyme-linked immunosorbent assay （ELISA） Expression of fibrosis marker α-smooth muscle actin （α-SMA） was detected by Western blot. Results Immunohistochemistry staining showed that fibronectin,vimentin were positive,and α-actin was negative So,the cultured cells were CF cells. Compared with the control group,the expressions of type Ⅰ and Ⅲ collagens were significantly increased in high glucose group （P<0.01）. The expressions of type Ⅰ and Ⅲ collagens in high glucose＋EPO group were significantly lower than those in high glucose group （P<0.01）. The expression of α-SMA in high glucose group was significantly higher than that in control group （P<0.05）. The expression of α-SMA in high glucose＋EPO group was significantly lower than that in high glucose group （P<0.05）. Conclusions High glucose can effectively induce the phenotypic transformation of CF cells. EPO can reverse myocardial fibrosis induced by high glucose environment.

Abstract:Aim To investigate the different serum levels of tumor necrosis factor-alpha（TNF-α） and transforming grouth factor-beta（TGF-β） between atrial fibrilliation and other control groups, and its chemotatic effects on cardiac fubroblast. Methods Serum concentrations of TNF-α and TGF-β were measured by enzyme-linked immune-absorbent assay（ELISA）Cardiac fibroblast were isolated from the venticles of 1～3 days Sprague-Dawley neonatal rats and passaged three to four generation, the transwell chamber assay were used to test the different chemotatic effects of the cardiac fibroblasts with the different concentration （1 μg/L, 10 μg/L, 50 μg/L） of TNF-α and TGF-β. Results Compared with control group, the serum of TNF-α and TGF-β in other groups were obviously increased. The group of persistent atrial fibrillation （per-Af） was the highest, the serum of TNF-α and TGF-β in non-Af atrial arrhythmia（AA） was higher than paroxysmal Af（per-Af）, the control group was the least. In different concentration （1 μg/L,10 μg/L, 50 μg/L） of TNF-α and TGF-β chemotaxis induced, the number of cells that migrated through the polycarbonate membrane were different, 50 μg/L group were significantly increased compared with other concentrations, the number of migrated cell in 10 μg/L group was higher than 1 μg/L group. The multiple Logistic regression analysis showed that the migrated cells in the lower surface were related to the concentration of TNF-α and TGF-β. Conclusions The different concentration of TNF-α and TGF-β among each group show that the occurrence of atrial fibrillation is associated with the potential inflammatory state. TNF-α and TGF-β have chemotaxis effect on cardiac fibroblast,its level is correlated with chemotatic movement. The chemotaxis of the serum with atrial fibrillation patients is partly mediated by TNF-α and TGF-β and it plays an important role in atrial fibrosis. The concentration of TNF-α and TGF-β can be used to reflect indirectly the presence, severity, extent of the myocardial damage.