Study on inhibitory effect and mechanism of hydrogen sulfide on the proliferation of cardiac fibroblasts / 中国药房

OBJECTIVE To investigate the inhibitory effect and the possible mechanism of hydrogen sulfide （H2S） on the proliferation of cardiac fibroblasts. METHODS The heart of neonatal SD rats was collected， and cardiac fibroblasts were separated with differential centrifugation. Using sodium hydrosulfide as the donor of H2S， the effects of H2S on the proliferation of cardiac fibroblasts induced by angiotensin Ⅱ （Ang Ⅱ）， hydroxyproline content and the expression of sirtuin 3 （SIRT3） protein were detected. After SIRT3 knockdown with siRNA technology， the effects of H2S on the proliferation of cardiac fibroblasts induced by Ang Ⅱ， hydroxyproline content， the expressions of collagen Ⅰ （Col Ⅰ）， collagen Ⅲ （Col Ⅲ ） and optic atrophy protein 1 （OPA1） were detected. RESULTS H2S could inhibit the proliferation of Ang Ⅱ-induced cardiac fibroblasts， reduce the content of hydroxyproline and increase the expression of SIRT3 （P＜0.05）. After down-regulating the expression of SIRT3 with siRNA technology， the inhibition of H2S on the proliferation of Ang Ⅱ-induced cardiac fibroblasts and the reduction of hydroxyproline content were both inhibited， and the effect of H2S on reducing the expression of Col Ⅰ and Col Ⅲ and enhancing the expression of OPA1 was also significantly weakened. CONCLUSIONS H2S inhibits the proliferation of Ang Ⅱ -induced cardiac fibroblasts through increasing the expression of SIRT3.

Experimental study of miR-26a regulating CTGF expression in radiation-induced heart disease / 中华放射肿瘤学杂志

Objective:To investigate the regulatory effect of miR-26a in radiation-induced heart disease (RIHD) mice.Methods:C57/BL6 mice were used to establish RIHD models. The cardiac function, fibrosis, the expression levels of collagen 1 (COL1) and connective tissue growth factor (CTGF), and miR-26a were detected in RIHD mice. Whether CTGF was the target gene of miR-26a was verified by dual luciferase kit. Moreover, cardiac fibroblasts were transfected with miR-26a up and miR-26a down lentivirus vectors to construct the miR-26a overexpression and underexpression cell models. The expression of CTGF, proliferation, and apoptosis of cardiac fibroblasts were detected.Results:In the RIHD mice, heart function was decreased, myocardial fibrosis was remodeled, the expression levels of COL1 and CTGF were up-regulated, and the expression level of miR-26a was down-regulated. Dual luciferase reporter assay confirmed that CTGF was the target gene regulated by miR-26a. Overexpression of miR-26a could inhibit the expression of CTGF, suppress the proliferation of cardiac fibroblasts, promote cell apoptosis and secrete collagen. Underexpression of miR-26a yielded the opposite results.Conclusion:MiR-26a affects the function of cardiac fibroblasts by targeting CTGF and probably mediates the process of radiation-induced myocardial fibrosis, which may become a new regulatory target of RIHD.

hsa-miR-4443 inhibits myocardial fibroblast proliferation by targeting THBS1 to regulate TGF-ß1/α-SMA/collagen signaling in atrial fibrillation

Fibrosis caused by the increase in extracellular matrix in cardiac fibroblasts plays an important role in the occurrence and development of atrial fibrillation (AF). The aim of this study was to investigate the role of hsa-miR-4443 in AF, human cardiac fibroblast (HCFB) proliferation, and extracellular matrix remodeling. TaqMan Stem-loop miRNA assay was used to measure hsa-miR-4443 expression in patients with persistent AF (n=123) and healthy controls (n=100). Patients with AF were confirmed to have atrial fibrosis by late gadolinium enhancement. At the cellular level, after hsa-miR-4443 mimic and inhibitor were transfected with HCFBs, proliferation, apoptosis, migration, and invasion were analyzed. Lastly, hsa-miR-4443-targeted gene and transforming growth factor (TGF)-β1/α-SMA/collagen pathway were evaluated by dual-luciferase reporter assay and western blot, respectively. In patients with AF, hsa-miR-4443 decreased significantly and collagen metabolism level increased significantly. Logistic regression analysis showed that low hsa-miR-4443 level was a risk factor of AF (P<0.001). The receiver operating characteristic curve revealed that hsa-miR-4443 was useful for predicting AF (area under the curve: 0.828, sensitivity: 0.71, specificity: 0.78, P<0.001). In HCFBs, hsa-miR-4443 targeted thrombospondin-1 (THBS1) and downregulated TGF-β1/α-SMA/collagen pathway. The inhibition of hsa-miR-4443 expression promoted HCFB proliferation, migration, invasion, myofibroblast differentiation, and collagen production. The significant reduction of hsa-miR-4443 can be used as a biomarker for AF. hsa-miR-4443 protected AF by targeting THBS1 and regulated TGF-β1/α-SMA/collagen pathway to inhibit HCFB proliferation and collagen synthesis.

Sodium tanshinone IIA sulfonate prevents radiation-induced damage in primary rat cardiac fibroblasts / 中国天然药物

This study investigated the effects of X-ray irradiation on primary rat cardiac fibroblasts (CFs) and its potential mechanism, as well as whether sodium tanshinone IIA sulfonate (STS) has protective effect on CFs and its possible mechanism. Our data demonstrated that X-rays inhibited cell growth and increased oxidative stress in CFs, and STS mitigated X-ray-induced injury. Enzyme-linked immuno-sorbent assay showed that X-rays increased the levels of secreted angiotensin II (Ang II) and brain natriuretic peptide (BNP). STS inhibited the X-ray-induced increases in Ang II and BNP release. Apoptosis and cell cycle of CFs were analyzed using flow cytometry. X-rays induced apoptosis in CFs, whereas STS inhibited apoptosis in CFs after X-ray irradiation. X-rays induced S-phase cell cycle arrest in CFs, which could be reversed by STS. X-rays increased the expression of phosphorylated-P38/P38, cleaved caspase-3 and caspase-3 as well as decreased the expression of phosphorylated extracellular signal-regulated kinase 1/2 (ERK 1/2)/ERK 1/2 and B cell lymphoma 2 (Bcl-2)/Bcl-2 associated X protein (BAX) in CFs, as shown by Western blotting. STS mitigated the X-ray radiation-induced expression changes of these proteins. In conclusion, our results demonstrated that STS may potentially be developed as a medical countermeasure to mitigate radiation-induced cardiac damage.

Effects of C-C motif chemokine receptor 2 on phenotypic transformation of cardiac fibroblasts after hypoxia / 解放军医学杂志

Objective To investigate the effect of C-C motif chemokine receptor 2 (CCR2) on phenotypic transformation of cardiac fibroblasts after hypoxia. Methods The mouse myocardium primary fibroblasts were extracted by collagenase digestion. Cells were divided into control, hypoxia-24h and hypoxia-48h, the mRNA and protein expression of CCR2 was examined by real-time PCR and Western blotting. CCR2 low expression cell (si-CCR2) was established using by small interfering RNA. Cells were divided into four groups including si-control, si-CCR2, si-control+hypoxia, si-CCR2+hypoxia. The mRNA and protein expressions of CCR2, α smooth muscle actin (α-SMA) and Collagen 1A (Col 1A) were detected by real-time PCR and Western blotting, cell proliferation was detected by Cell Counting Kit-8 (CCK8) and Bromodeoxyuridine (BrdU). Results Compared with control, the mRNA and protein levels of CCR2 significantly increased in hypoxia-24h and hypoxia-48h group (P<0.01), however, no significance was found in these two time points. Compared with si-control group, the mRNA and protein expressions of CCR2, α-SMA and Col 1A not significantly changed in si-CCR2 group. Compared with si-control group, the mRNA and protein levels of CCR2, α-SMA and Col 1A significantly increased in si-control+hypoxia group (P<0.01 or P<0.05), and cell proliferation increased (P<0.01). Compared with si-control+hypoxia group, the protein expression of α-SMA and Col 1A decreased in si-CCR2+hypoxia group (P<0.05), and cell proliferation also decreased in si-CCR2+hypoxia group (P<0.05). Conclusions CCR2 could affect the phenotypic transformation of cardiac fibroblasts after hypoxia.

β-adrenergic receptor activation promotes cardiac miR-21 expression by paracrine action of IL-6 from fibroblasts / 中国病理生理杂志

AIM:To investigate the regulation ofβ-adrenergic receptor (β-AR) agonist isoproterenol (ISO) on cardiac microRNA-21 (miR-21) expression.METHODS:The primary cultured mouse cardiomyocytes and cardiac fibroblasts were isolated by enzyme digestion and treated with ISO at 10μmol/L for 1, 6, 12, 24 and 48 h.The expression of miR-21 was detected by real-time PCR.The protein levels of p-STAT3 and STAT3 were determined by Western blot, and the concentration of interleukin-6 (IL-6) in the cultured supernatant was measured by ELISA.The cells were transfected with the luciferase reporter gene plasmid p GL3-21PPR containing the miR-21 promoter region, and the luciferase reporter gene assay was used to examine the effect of conditioned medium on the transcriptional activity of miR-21.RESULTS:The medium supernatant produced by ISO on cardiac fibroblasts was used as the conditioned medium, which increased the miR-21 expression in the cardiomyocytes in a time-dependent manner after fibroblasts was treated with ISO (P<0.05).The conditioned medium caused a significant increase in the transcriptional activity of miR-21 in the cardiomyocytes, while24 h and 48 h conditioned medium increased the transcriptional activity by 94.9％and 77.1％, respectively (P<0.01).The concentration of IL-6 in the conditioned medium was significantly increased, and the activity of transcriptional factor STAT3 was enhanced by paracrine action of IL-6 in the cardiomyocytes, which promoted the transcription and expression of miR-21.CONCLUSION:β-AR stimulation induces fibroblast synthesis and expression of IL-6 with paracrine effect on cardiomyocytes, up-regulates the expression of miR-21 in cardiomyocytes by IL-6/STAT3 pathway, and participates in the cardiac remodeling.

Osthole decreases collagen I/III contents and their ratio in TGF-β1-overexpressed mouse cardiac fibroblasts through regulating the TGF-β/Smad signaling pathway / 中国天然药物

The present study was designed to elucidate whether the mechanism by which osthole decreases collagenI/III contents and their ratio is regulating the TGF-β/Smad signaling pathway in TGF-β1-overexpressed mouse cardiac fibroblasts (CFs). These CFs were cultured and treated with different concentrations of osthole. Our results showed that the TGF-β1 expression in the CFs transfected with that the recombinant expression plasmids pcDNA3.1(+)-TGF-β1 was significantly enhanced. After the CFs were treated with 1.25-5 μg·mL of osthole for 24 h, the mRNA and protein expression levels of collagensIand III were reduced. The collagen I/III ratio was also reduced. The mRNA and protein expression levels of TGF-β1, TβRI, Smad2/3, P-Smad2/3, Smad4, and α-SMA were decreased, whereas the expression level of Smad7 was increased. These effects suggested that osthole could inhibit collagen I and III expression and reduce their ratio via the TGF-β/Smad signaling pathway in TGF-β1 overexpressed CFs. These effects of osthole may play beneficial roles in the prevention and treatment of myocardial fibrosis.

Osthole decreases collagen I/III contents and their ratio in TGF-β1-overexpressed mouse cardiac fibroblasts through regulating the TGF-β/Smad signaling pathway / 中国天然药物

The present study was designed to elucidate whether the mechanism by which osthole decreases collagenI/III contents and their ratio is regulating the TGF-β/Smad signaling pathway in TGF-β1-overexpressed mouse cardiac fibroblasts (CFs). These CFs were cultured and treated with different concentrations of osthole. Our results showed that the TGF-β1 expression in the CFs transfected with that the recombinant expression plasmids pcDNA3.1(+)-TGF-β1 was significantly enhanced. After the CFs were treated with 1.25-5 μg·mL of osthole for 24 h, the mRNA and protein expression levels of collagensIand III were reduced. The collagen I/III ratio was also reduced. The mRNA and protein expression levels of TGF-β1, TβRI, Smad2/3, P-Smad2/3, Smad4, and α-SMA were decreased, whereas the expression level of Smad7 was increased. These effects suggested that osthole could inhibit collagen I and III expression and reduce their ratio via the TGF-β/Smad signaling pathway in TGF-β1 overexpressed CFs. These effects of osthole may play beneficial roles in the prevention and treatment of myocardial fibrosis.

MicroRNA-199a-3p enhances expressions of fibrosis-associated genes through targeting Smad1 in mouse cardiac fibroblasts / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the role of miR-199a-3p in cardiac fibrosis and the potential target of miR-199a-3p.</p><p><b>METHODS</b>Cardiac fibroblasts were isolated from C57BL/6 mice and cultured. The miR-199a-3p mimic and Smad1 siRNA were transiently transfected into the cardiac fibroblasts via liposome. Dual luciferase reporter assay was performed to confirm the interaction between miR-199a-3p and the 3'-UTR of Smad1. The expressions of Smad1 and fibrosis-related genes at the mRNA and protein levels in the cells after miR-199a-3p mimic transfection were determined using RT-qPCR and Western blotting, respectively. The expressions of Smad1, Smad3 and fibrosis-related genes at the protein level in cells transfected with miR-199a-3p mimic and Smad1 siRNA were detected using Western blotting.</p><p><b>RESULTS</b>Over-expression of miR-199a-3p significantly increased the expression of cardiac fibrosis-related genes in cultured mouse cardiac fibroblasts. Dual luciferase reporter assay revealed the interaction of miR-199a-3p with the 3'-UTR of Smad1. The results of RT-qPCR and Western blotting confirmed that miR-199a-3p inhibited Smad1 expression at the post- transcriptional level. Transfection with miR-199a-3p mimic and siRNA-mediated Smad1 silencing consistently activated the Smad3 signaling pathway and enhanced the expressions of cardiac fibrosis-related genes in the cardiac fibroblasts.</p><p><b>CONCLUSIONS</b>As the target gene of miR-199a-3p, Smad1 mediates the pro-fibrotic effect of miR-199a-3p by activating the Smad3 signaling in cultured mouse cardiac fibroblasts.</p>

Effect of astaxanthin on expression of type Ⅰ and type Ⅲ collagen in cardiac fibroblasts and its mechanism based on TGF-β1/Smad3 signaling pathway / 中国药理学通报

Aim To observe the effects of astaxanthin ( ASTX) on the expression of collegeⅠ( ColⅠ) and type Ⅲcollagen ( Col Ⅲ) of cardiac fibroblasts( CFs) which caused by transforming growth factor β1 ( TGF-β1) and to explore its mechanism of action. Methods CFs were induced by TGF-β1 , and then pretreated with different concentrations of ASTX ( 0 , 5 , 10 , 20 , 40, 80, 160 μmol·L-1) for 24 h. MTT assay was used to determine the activity of CFs. The activation of ROS in CFs cells was detected by DCFH-DA kit. Smad3 gene was silenced by siRNA technique, and re-al-time PCR was used to detect the expression of ColⅠ, Col Ⅲ mRNA before and after Smad3 silencing. Western blot was used to detect the expression of ColⅠ, Col Ⅲ and Smad3 protein levels before and after Smad3 silencing. Results ASTX had no obvious cyto-toxicity in the range of 0 ～20 μmol · L-1 , and could significantly reduce ROS production induced by TGF-β1 in CFs (P<0.05). In addition, ASTX significant-ly inhibited the expression of ColⅠand ColⅢmRNA and protein ( P<0.01 ) of TGF-β1-induced CFs in a concentration-dependent manner. Also, ASTX could significantly down-regulate phosphorylation of Smad3 in TGF-β1-induced CFs ( P <0.01 ) . The expression of Col Ⅰ and Col Ⅲ mRNA and protein was also signifi-cantly down-regulated by Smad3 gene silencing ( P <0.01 ) . Conclusions ASTX can effectively inhibit the expression of Col Ⅰ, Col Ⅲ mRNA and protein of TGF-β1-induced CFs, and the possible mechanism may involve the down-regulation of Smad3 phosphoryla-tion.

Effects of Tongxinluo on High Glucose-induced Proliferation and Apoptosis of Cardiac Fibroblasts / 中国医科大学学报

Objective This study investigated the effect of tongxinluo (TXL) on high glucose-induced proliferation and apoptosis of cardiac fibroblasts (CFs),to explore the possible mechanism by which TXL inhibits myocardial fibrosis. Methods Primary culture and subculture of neonatal SD rat CFs was carried out as follows. Immunofluorescence staining was performed to identify CFs. The CFs were divided into control group (cultured with low-glucose DMEM),model group (cultured with high-glucose DMEM),and TXL treatment groups (cultured with high-glucose DMEM+TXL at 20,80,and 320 μg/mL). The proliferation of CFs in each group was detected by MTT assay. The expression of collagen typesⅠand Ⅲ in each group was detected by ELISA. Western blotting was used to detect the expression of bax and bcl-2 in each group. Results CFs Proliferation and collagen typesⅠandⅢsecretion were higher in the model group than in the control group. The CFs proliferation and collagen content in the TXL treatment groups were significantly lower than those in the model group. bcl-2 expression was significantly increased and bax expression was decreased in the model group,compared with the corresponding values in the control group. In comparison with the model group,bcl-2 expression was downregulated and bax expression was upregulated in the TXL treatment groups. Conclusion TXL can reduce the CFs proliferation and inhibit their collagen secretion under high glucose conditions. TXL also induces the CFs apoptosis by reducing bcl-2 expression and increasing bax expression. Thus,TXL can play a role in the treatment of myocardial fibrosis.

Effects of Scutellarin on the Proliferation of Cardiac Fibroblasts in Neonate Rats Induced by Angiotensin Ⅱ and Signal Pathway of ERK1/2 and p38 MAPK / 中国药房

OBJECTIVE: To study the effects of scutellarin on the proliferation of cardiac fibroblasts (CFs) in neonate rats induced by angiotensin Ⅱ (ANG Ⅱ) and signal pathway of extracellular regulated protein kinase (ERK1/2) and p38 mitogen activated protein kinase (p38 MAPK).METHODS: CFs of neonatal rat were isolated and cultured in vitro, and then divided into blank group (blank culture medium), Ang Ⅱ group (10-7 μmol/L), 50 μmol/L scutellarin group and Ang Ⅱ (10-7 μmol/L)+5, 10, 20, 50 μmol/L scutellarin groups. After cultured for 48 h, CCK-8 assay was used to detect the proliferation ability of cells. Other cells were selected and divided into blank group (blank culture medium), Ang Ⅱ group (10-7 μmol/L) and Ang Ⅱ +5, 10, 20, 50 μmol/L scutellarin groups. After cultured for 48 h, mRNA expression of Col I, Col Ⅲ and α-SMA in cells and hydroxyproline (HYP) content in cell culture fluid were detected; phosphorylation levels of ERK1/2 and p38 MAPK in cells were also detected. RESULTS: 5, 10, 20, 50 μmol/L scutellarin could significantly inhibit the proliferation of CFs induced by Ang Ⅱ (P<0. 05), and decreased mRNA expression of Col I, Col Ⅲ and α-SMA in CFs induced Ang Ⅱ (P<0. 05). 5, 10, 20, 50 μmol/L scutellarin could significantly inhibit the increase of HYP content and the phosphorylation of ERK1/2 and p38 MAPK after induced by Ang Ⅱ (P<0. 05), in dose-dependent manner. CONCLUSIONS: Scutellarin inhibits the proliferation of CFs induced by Ang Ⅱ, the mechanism of which may be associated with reduction of ERK1/2 and p38 MAPK phosphorylation.

Regulation of STAT3 signaling pathway by PTEN on proliferation of cardiac fibroblasts / 中国免疫学杂志

Objective:To investigate the effect of PTEN on proliferation of cardiac fibroblasts and its mechanism. Methods:Stimulation of cardiac fibroblasts by high glucose, the levels of PTEN in cells were detected by qRT-PCR and Western blot. Cell transfection of PTEN over expression vector,the levels of PTEN in transfected cells were detected by qRT-PCR and Western blot. High glucose stimulated transfection of PTEN overexpression vector into cardiac fibroblasts,cell proliferation was detected by MTT,the levels of p-STAT3 and STAT3 in cells were detected by Western blot,STAT3 pathway blocker AG490 was added into the cell culture medium to treat the cells, cell proliferation was detected by MTT, the levels of p-STAT3 and STAT3 in cells were detected by Western blot. Results:The levels of PTEN mRNA and protein in cardiac fibroblasts after high glucose treatment were significantly lower than those in normal culture ( P＜0. 05 ) . The expression of PTEN mRNA and protein in transfected PTEN overexpressing cells was significantly higher than that in non transfected cells( P＜0. 05) . The cell proliferation activity and p-STAT3 level were significantly higher than those of normal cells after high glucose(P＜0. 05). The expression of PTEN was increased after high glucose induction,the cell proliferation activity and p-STAT3 level were decreased, the proliferation of the cells treated with AG490 decreased further. Conclusion:PTEN slows down the proliferation of cardiac fibroblasts induced by high glucose by inhibiting STAT3 signaling pathway.

Effect of N-acetyl cysteine pathway on expression of intermediate conductance Ca2+-activated K+ channels in cardiac fibroblasts of AGT-REN double transgenic hypertension mice / 吉林大学学报(医学版)

Objective:To investigate the relationship between the level of oxidative stress and the expression of intermediate conductance calcium activated potassium channel (KCa3.1) protein in the cardiac fibroblasts (CFs) during hypertension process,and to clarify the role of KCa3.1 in cardiac fibrosis and its mechanism.Methods:The CFs of male C57B6 and AGT-REN double transgenic hypertension (dTH) mice were cultured and the wild C57B6 mouse CFs were used as control group.The CFs of dTH mice were randomly divided into high blood pressure group (dTH) and N-acetyl cysteine group (NAC).The CFs were treated with different concentrations of NAC for 24 h.The cell proliferation was detected by MTT and double dichlorofluorescein (DCFH-DA) probe was used for the detection of cellular reactive oxygen species (ROS) expression;Western blotting was employed to detect the expressions of collagen Ⅰ,collagen Ⅲ,Kca3.1 channel protein and the changes of PI3K signaling pathway protein phosphorylation.Results:The ROS production and protein expression of Kca3.1 channel of the dTH mice on 4,8,12 months were increased compared with 2 months (P＜0.05 or P＜0.01);the results of MTT suggested that the proliferation rates of CFs were 165.9％,138.72％,110.92％ and 109.82％ after administration of 1×10-6,1× 10 5,1× 10 4 and 1 × 10-3 mol · L-1 NAC in the dTH mice,and 1 × 10-4 and 1 × 10 3 mol · L-1 NAC significantly inhibited the proliferation of CFs.Compared with control group,the secretion of collagen Ⅰ and Ⅲ of CFs in the TH mice was decreased in 1 × 10-4 mol · L-1 NAC group (P＜0.01).The results of Western blotting showed that compared with control group,the expression level of Kca3.1 channel protein in CFs of the TH mice in 1 × 10-4 mol · L-1 NAC group was decreased (P＜0.01).Compared with control group,the p-AKT/T-AKt in CFs of the dTH mice was increased (P＜0.01);but in NAC group,the p-AKT/T-AKt was lower than that in dTH group (P＜0.01).Conclusion:NAC can inhibit the expression of KCa3.1 channel protein in CFs of the dTH hypertensive mice,which may be related to increasing the phosphorylation of AKt/PI3K signaling pathway.

Study on the Mechanism of Anti-myocardial Fibrosis of Salvianolic Acid B / 中国药房

OBJECTIVE:To study the effects of salvianolic acid B(Sal B)on angiotensin Ⅱ(Ang Ⅱ)-induced cardiac fibro-blast proliferation,secretion of type Ⅲ collagen,protein expressions of matrix metalloproteinase 9 (MMP-9),Smad2/3,Smad7, and explore its mechanism of anti-myocardial fibrosis. METHODS:Cells were divided into blank control group(culture medium) Ang Ⅱ model group,Sal B low-dose,medium-dose,high-dose groups (12.5,25,50 μmol/L). After cultured 1 h by blank or drug-containing culture,except for blank control group,cells in other groups were added 1 μmol/L Ang Ⅱ to induce proliferation. for 24 h. MTT method and hematoxylin-eosin staining method were adopted investigate the effect of Sal B on proliferation. Western blot method was adopted to detect the effects of Sal B on protein expressions of type Ⅲ collagen,MMP-9,Smad2/3,Smad7. RE-SULTS:Compared with blank control group,cells in Ang Ⅱ model group were significantly proliferated,protein expressions of type Ⅲ collagen,MMP-9,Smad2/3 were obviously enhanced,protein expression of Smad7 was obviously weakened,with statisti-cal significances(P<0.05). Compared with Ang Ⅱ model group,the cell proliferation in Sal B groups were inhibited,protein ex-pressions of typeⅢcollagen,MMP-9,Smad2/3 were weakened,while protein expression of Smad7 was enhanced. Except the pro-tein expression of type Ⅲ collagen in Sal B low-dose and medium-dose groups,the protein expression of Smad2/3 in Sal B high-dose group did not change significantly,other indexes had statistical significances(P<0.05). CONCLUSIONS:The anti-myo-cardial fibrosis effect of Sal B may be associated with inhibiting the proliferation of cardiac fibroblasts,down-regulating protein ex-pressions of typeⅢcollagen,MMP-9,Smad2/3 and up-regulating protein expression of Smad7.

Effect of N-acetyl cysteine pathway on expression of intermediate conductance Ca2+-activated K+ channels in cardiac fibroblasts of AGT-REN double transgenic hypertension mice / 吉林大学学报(医学版)

Objective:To investigate the relationship between the level of oxidative stress and the expression of intermediate conductance calcium activated potassium channel (KCa3.1) protein in the cardiac fibroblasts (CFs) during hypertension process,and to clarify the role of KCa3.1 in cardiac fibrosis and its mechanism.Methods:The CFs of male C57B6 and AGT-REN double transgenic hypertension (dTH) mice were cultured and the wild C57B6 mouse CFs were used as control group.The CFs of dTH mice were randomly divided into high blood pressure group (dTH) and N-acetyl cysteine group (NAC).The CFs were treated with different concentrations of NAC for 24 h.The cell proliferation was detected by MTT and double dichlorofluorescein (DCFH-DA) probe was used for the detection of cellular reactive oxygen species (ROS) expression;Western blotting was employed to detect the expressions of collagen Ⅰ,collagen Ⅲ,Kca3.1 channel protein and the changes of PI3K signaling pathway protein phosphorylation.Results:The ROS production and protein expression of Kca3.1 channel of the dTH mice on 4,8,12 months were increased compared with 2 months (P＜0.05 or P＜0.01);the results of MTT suggested that the proliferation rates of CFs were 165.9％,138.72％,110.92％ and 109.82％ after administration of 1×10-6,1× 10 5,1× 10 4 and 1 × 10-3 mol · L-1 NAC in the dTH mice,and 1 × 10-4 and 1 × 10 3 mol · L-1 NAC significantly inhibited the proliferation of CFs.Compared with control group,the secretion of collagen Ⅰ and Ⅲ of CFs in the TH mice was decreased in 1 × 10-4 mol · L-1 NAC group (P＜0.01).The results of Western blotting showed that compared with control group,the expression level of Kca3.1 channel protein in CFs of the TH mice in 1 × 10-4 mol · L-1 NAC group was decreased (P＜0.01).Compared with control group,the p-AKT/T-AKt in CFs of the dTH mice was increased (P＜0.01);but in NAC group,the p-AKT/T-AKt was lower than that in dTH group (P＜0.01).Conclusion:NAC can inhibit the expression of KCa3.1 channel protein in CFs of the dTH hypertensive mice,which may be related to increasing the phosphorylation of AKt/PI3K signaling pathway.

Study on the Mechanism of Anti-myocardial Fibrosis of Salvianolic Acid B / 中国药房

OBJECTIVE:To study the effects of salvianolic acid B(Sal B)on angiotensin Ⅱ(Ang Ⅱ)-induced cardiac fibro-blast proliferation,secretion of type Ⅲ collagen,protein expressions of matrix metalloproteinase 9 (MMP-9),Smad2/3,Smad7, and explore its mechanism of anti-myocardial fibrosis. METHODS:Cells were divided into blank control group(culture medium) Ang Ⅱ model group,Sal B low-dose,medium-dose,high-dose groups (12.5,25,50 μmol/L). After cultured 1 h by blank or drug-containing culture,except for blank control group,cells in other groups were added 1 μmol/L Ang Ⅱ to induce proliferation. for 24 h. MTT method and hematoxylin-eosin staining method were adopted investigate the effect of Sal B on proliferation. Western blot method was adopted to detect the effects of Sal B on protein expressions of type Ⅲ collagen,MMP-9,Smad2/3,Smad7. RE-SULTS:Compared with blank control group,cells in Ang Ⅱ model group were significantly proliferated,protein expressions of type Ⅲ collagen,MMP-9,Smad2/3 were obviously enhanced,protein expression of Smad7 was obviously weakened,with statisti-cal significances(P<0.05). Compared with Ang Ⅱ model group,the cell proliferation in Sal B groups were inhibited,protein ex-pressions of typeⅢcollagen,MMP-9,Smad2/3 were weakened,while protein expression of Smad7 was enhanced. Except the pro-tein expression of type Ⅲ collagen in Sal B low-dose and medium-dose groups,the protein expression of Smad2/3 in Sal B high-dose group did not change significantly,other indexes had statistical significances(P<0.05). CONCLUSIONS:The anti-myo-cardial fibrosis effect of Sal B may be associated with inhibiting the proliferation of cardiac fibroblasts,down-regulating protein ex-pressions of typeⅢcollagen,MMP-9,Smad2/3 and up-regulating protein expression of Smad7.

Effects of short-chain acyl-CoA dehydrogenase on collagen expression and proliferation of rat cardiac fibroblasts / 中国病理生理杂志

AIM:To investigate the effect of short-chain acyl-CoA dehydrogenase ( SCAD) on collagen expres-sion and proliferation of rat cardiac fibroblasts and to explore the relationship between SCAD and cardiac fibrosis . METHODS:The model of proliferation and collagen expression of rat cardiac fibroblasts induced by angiotensin II was es -tablished.After treatment with siRNA-1186, the expression of SCAD at mRNA and protein levels , fatty acids beta oxida-tion rate, ATP, the enzyme activity of SCAD and free fatty acids in the rat cardiac fibroblasts were determined . RESULTS:The mRNA and protein expression of SCAD was decreased in the rat cardiac fibroblasts induced by angiotensin II compared with the control cells , and the expression of collagen I and collagen III was significantly upregulated .Com-pared with negative control group , SCAD expression and activity , fatty acid beta-oxidation rate and ATP significantly de-creased in siRNA-1186 group, but the content of free fatty acids were obviously increased in the rat cardiac fibroblasts , and the expression of collagen I and collagen III was significantly up-regulated.CONCLUSION:The expression and synthesis disorder of collagen may be triggered by down-regulation of SCAD .SCAD may be a promising therapeutic target for myocar-dial fibrosis .

Retinoid X receptor agonist inhibits TGF-β1-induced collagen synthesis in cardiac fibroblasts by repressing Smad2 activation / 中国病理生理杂志

AIM:To investigate the effect of activation of retinoid X receptor (RXR) on transforming growth factor β1 (TGF-β1) induced collagen synthesis under hypoxic environment in rat cardiac fibroblasts (CFs) and underlying molecular mechanisms .METHODS: CFs were cultured using myocardial tissue with dry method .Hypoxic environment was established for CFs by continuous nitrogen supplement .Type I and type III collagens in supernatants were detected by ELISA.Nuclear and cytoplasmic extractions were prepared using NE-PER nuclear and cytoplasmic extraction reagents .The protein levels of Smad2 and p-Smad2 were determined by Western blot and immunocytochemical staining .RESULTS:Un-der hypoxic condition , TGF-β1 (0.01~10 μg/L) increased the synthesis of type I and type III collagens in a dose-de-pendent manner in the CFs .At the concentration of 5μg/L, the synthesis of collagen I and III was significantly increased as compared with control group (P<0.01).RXR agonist 9-cis-retinoic acid (9-cis-RA;10 -9 ~10 -6 mol/L) decreased TGF-β1 (5μg/L)-induced synthesis of type I and III collagens in a dose-dependent manner in the CFs under hypoxic con-dition.The synthesis of type I and type III collagens was significantly inhibited by 9-cis-RA (P<0.01).Smad2 inhibitor ( 20 nmol/L) showed similar inhibitory effect on the synthesis of type I and III collagens induced by TGF -β1 under hypoxic condition.Compared with TGF-β1 intervention group, the cytoplasmic level of p-Smad2 in the CFs was significantly in-creased in TGF-β1+9-cis-RA group, but the nuclear p-Smad2 level was significantly decreased (P<0.05).CONCLU-SION:Retinoid X receptor agonist 9-cis-RA inhibits TGF-β1-induced synthesis of type I and type III collagens in the CFs by repressing p-Smad2 nuclear translocation under hypoxic condition .

Effect of tranilast on cardiac fibroblasts proliferation and phenotype transformation incubated with high concentration of glucose / 中国药理学与毒理学杂志

OBJECTIVE To investigate the effect of tranilast on cardiac fibroblasts proliferation and phenotype transformation incubated with a high concentration of glucose and the possible mechanism. METHODS The cardiac fibroblasts were divided into seven groups in accordance with different nutrient solutions:normal control group(5.5 mmol · L-1 glucose),hypertonic group(5.5 mmol · L-1 glucose+mannitol 25 mmol · L- 1),high glucose group (25 mmol · L- 1 glucose),tranilast intervention groups (25 mmol·L-1 glucose+tranilast 50,100 and 200μmol·L-1),and activin receptor-like kinase 7(ALK7) inhibitor group(25 mmol · L-1 glucose+10μmol · L-1 SB431542). The cell proliferation was detected by MTT method. The transformation of cardiac fibroblasts was determined by immunfluorescence staining. The expression of fibroblast specific protein 1 (FSP-1),α-smooth muscle actin (α-SMA),transforming growth factor-β1(TGF-β1)and ALK7 was assessed by Western blotting. RESULTS Compared with nor?mal control group,A492 nm of cells in high glucose group was significantly increased(P<0.01),while the expression of α-SMA,TGF-β1 and ALK7 protein in high glucose group was significantly increased(P<0.01),but FSP-1 protein was significantly decreased(P<0.01). There was no difference between normal control group and hypertonic group. Compared with high glucose group,A492 nm of cells in tranilast or SB431542 intervention groups were decreased(P<0.05),and the expression of α-SMA,TGF-β1 and ALK7 protein was significantly decreased(P<0.05),but the expression of FSP-1 protein was increased (P<0.05)in tranilast or SB431542 intervention groups. CONCLUSION Tranilast can inhibit the proliferation and phenotype transformation of cardiac fibroblasts induced by high glucose,which may be related to down-regulation of the expression of ALK7.