Effect of rhynchophylline on TGF-β1/Smad pathway for processing ventricular remodeling in spontaneously hypertensive rats

AIM: To investigate the effect of rhynchophylline (Rhy) on blood pressure, cardiac hypertrophy and myocardial fibrosis in spontaneously hypertensive rats (SHR). METHODS: Spontaneously hypertensive rats were randomly divided into model group, high dose (10 mg·kg^-1·d^-1) and low dose (2.5 mg·kg^-1·d^-1) group of rhynchophylline, captopril group (17.5 mg·kg^-1·d^-1). Wistar-Kyoto rats were used as normal control. Respectively, systolic blood pressure was measured by tail cuff every 2 weeks. After 10 weeks, heart weight index and left ventricular weight index were calculated. The myocardial hydroxyproline and plasma angiotensin Ⅱ were detected. Moreover, basic myocardial histopathological changes and myocardial collagen fibres were observed by HE staining and Masson staining, respectively. The protein expression of TGF-β₁ and Smad3 in the myocardium was measured by the methods of immunohistochemistry and Western blot. RESULTS: Compared with SHR model group, Rhy significantly reduced blood pressure (P<0.05), the levels of HYP in the myocardium (P<0.05) and the levels of AngⅡ in the plasma (P<0.01). The pathological damages of the myocardial tissues and collagen deposition were attenuated. The protein expression of TGF-β₁ and Smad3 was significantly reduced by the treatment with Rhy (P<0.01). CONCLUSION: Rhynchophylline reduces blood pressure and adjusts to improve ventricular remodeling of SHR. The mechanism may be involved in the TGF-β₁/Smad pathway and reducing AngⅡ content.     

Effects of Ginkgo biloba extract on myocardial TGF-β1 and collagen expression and interstitial fibrosis in type I diabetic cardiomyopathy rats

AIM:To study the effects of Ginkgo biloba extract (EGB) on myocardial TGF-β1 and collagen expression and interstitial fibrosis in type I diabetic cardiomyopathy rats. METHODS:Thirty male SD rats were randomly divided into normal control group (CON), diabetes mellitus group (DM) and EGB treatment group (EGB). Streptozocin was intraperitoneally injected into the animals in the latter 2 groups to induce type I diabetic rat model. The rats in EGB group were intraperitoneally injected with EGB. At the end of the 12th week, the body weight of each rat and its left ventri-cular weight, blood glucose, glycosylated hemoglobin and serum insulin concentration were measured. The left ventricular end-diastolic volume (LVEDV), the left ventricular end-systolic volume (LVESV), the left ventricular ejection fraction (LVEF) and the stroke volume (SV) were determined by echocardiography. The content of collagen in left ventricular myocardium, and the expression of transforming growth factor β1 (TGF-β1), procollagen type I and collagen type III were assayed by Sirius red staining, immunohistochemical staining and RT-PCR, respectively. Left ventricular myocardial cells of the neonatal SD rats were isolated and cultured in vitro with low-glucose culture medium (LG group), high-glucose culture medium (HG group) or high-glucose culture medium plus EGB (HG+EGB group). The mRNA levels of TGF-β1, procollagen type I and collagen type III were detected by RT-PCR. RESULTS:Compared with CON group, blood glucose, glycosylated hemoglobin, left ventricular weight index, the content of collagen, and the expression of TGF-β1, procollagen type I and collagen type III in left ventricular myocardial tissues of DM group were significantly increased, while the levels of blood insulin, LVEDV and SV were significantly decreased. However, compared with DM group, blood glucose, glycosylated hemoglobin, left ventricule weight index, the content of collagen, and the expression levels of TGF-β1, procollagen type I and collagen type III in the left ventricular myocardial tissues of EGB-treated rats were significantly decreased, while the levels of blood insulin, LVEDV and SV were significantly increased. Compared with LG group, the mRNA expression levels of TGF-β1, procollagen type I and collagen type III were significantly increased. However, compared with HG group, the mRNA expression levels of TGF-β1, procollagen type I and collagen type III were significantly decreased after treated with EGB. CONCLUSION: EGB retards the process of myocardial fibrosis and improves the cardiac functions in type I diabetic cardiomyopathy rats by down-regulating the expression of TGF-β1, reducing the synthesis and deposition of collagen type I and collagen type III.     

Effects of soluble transforming growth factor-β type Ⅱ receptor on cardiac functions after myocardial infarction in rats

AIM: To observe the effects of soluble transforming growth factor-β type Ⅱ receptor (sTGFβRⅡ) on cardiac functions after myocardial infarction (MI) in rats. METHODS: MI was induced in Sprague-Dawley (SD) rats by ligating the left anterior descending coronary artery. The rats surviving to the third day after MI were included in the study and randomly divided into MI group, pAd-sTGFβRⅡ group (transfected with recombinant adenovirus vector expressing the extracellular domain gene of TGF-βRⅡ), vector group and sham group. Four weeks later, the heart rate (HR), left ventricular end-diastolic dimension (LVEDD), left ventricular end-systolic dimension (LVESD) and ejection fraction (EF) were evaluated by echocardiograms. The expression of sTGFβRⅡ in myocardial tissues was observed under fluorescence microscope by frozen sectioning, and the expression of typeⅠ and Ⅲ collagens was observed by Sirius red-saturated picric acid staining. The expression of matrix metalloproteinase 9 (MMP-9) at mRNA and protein levels was determined by RT-PCR and immunohistochemical method. The activity of MMP-9 was assayed by gelatin zymography. RESULTS: Compared with sham group, HR, LVEDD, LVESD, typeⅠ and Ⅲ collagen, mRNA and protein of MMP-9, and the activity of MMP-9 increased significantly (P<0.01), and EF decreased (P<0.01) in MI group and vector group. Compared with MI group, EF was increased (P<0.01), but HR, LVEDD, LVESD, typeⅠ and Ⅲ collagen, mRNA and protein expression of MMP-9 and the activity of MMP-9 decreased significantly (P<0.01) in pAd-sTGFβRⅡ group, and all the parameters above were still higher than those in sham group. CONCLUSION: sTGFβRⅡ intervention improves the cardiac functions after MI by inhibiting TGF-β-mediated MMP-9 expression.     

Up-regulation of miR-133a expression attenuates myocardial fibrosis in spontaneously hypertensive rats

AIM: To investigate the effect of up-regulated expression of microRNA-133a (miR-133a) on myocardial fibrosis in spontaneously hypertensive rats (SHR). METHODS: Wistar-Kyoto (WKY) rats with homologous normal blood pressure served as the normal control group. SHR were divided into SHR group, SHR+ adeno-associated virus (AAV) group and SHR+miR-133a-AAV group randomly. miR-133a carried by miR-133a-AAV was transfected into SHR heart by coronary perfusion. The rat tail artery pressure was monitored. The myocardial collagen deposition was observed by Masson staining. The expression of miR-133a in myocardial tissue was detected by real-time PCR. The protein levels of transforming growth factor-β1 (TGF-β1) and connective tissue growth factor (CTGF) were determined by immunohistochemistry and Western blot. RESULTS: Compared with the WKY rats, the tail artery pressure of the SHR increased significantly. The expression of miR-133a in heart decreased, and the expression levels of TGF-β1 and CTGF increased (P<0.05), and myocardial fibrosis occurred. After up-regulating the expression level of miR-133a in the heart of SHR, the myocardial fibrosis was significantly reduced, and the expression levels of TGF-β1 and CTGF decreased (P<0.05). CONCLUSION: Up-regulation of the miR-133a expression improves myocardial fibrosis induced by hypertension, which may be related to inhibiting the protein expression of TGF-β1 and CTGF in myocardium.     

Role of canonical transient receptor potential and inflammation in left ventricular fibrosis induced by high salt in Wistar rats

AIM:To explore the role of transient receptor potential channels subfamily C (TRPCs) and inflammation in left ventricular fibrosis induced by high salt and the effect of telmisartan. METHODS:Wistar rats were randomly divided into 3 groups: normal control (C) group (n=13), high salt (8%) model group (HS, n=24) and high salt+telmisartan (T) group (n=12). Tail-cuff artery pressure was determined every 2 weeks. The interstitial collagen deposition and inflammation were observed by Masson and HE staining, respectively. The expression of TRPC1, TRPC3, TRPC6, calcineurin (CaN), nuclear factor-κB p65 (NF-κB p65), transforming growth factor β1 (TGF-β1), interleukin-1β (IL-1β), vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1) and monocyte chemoattractant protein-1 (MCP-1) were determined by real-time PCR or Western blotting. RESULTS:Masson staining showed that left ventricle in HS group exhibited severer myocardial interstitial fibrosis compared with C group. TRPC, CaN and NF-κB assays showed that high-salt diet increased the protein expression of TRPC1, TRPC3 and TRPC6, and activated CaN and NF-κB as compared with C group. The results of HE staining, real-time PCR and Western blotting showed that high salt-treated Wistar rats had enhanced cardiac infiltration of inflammatory cells, as well as increased cardiac levels of proinflammatory cytokines (TGF-β1, IL-1β, VCAM-1, ICAM-1 and MCP-1) as compared with C group. After treated with telmisartan, left ventricular mass index and collagen volume fraction became much lower, and the levels of TRPC1, TRPC3, TRPC6, CaN, NF-κB p65, TGF-β1, ICAM-1 and MCP-1 were significantly reduced. CONCLUSION: Inflammation is exacerbated in left ventricular fibrosis induced by high salt. The mechanism may be related to the up-regulation of TRPCs, CaN and NF-κB at mRNA and protein levels. Telmisartan inhibits the expression of TRPCs and NF-κB, and ameliorates the inflammatory responses in left ventricular fibrosis.     

Therapeutic effect of neuregulin-1β on pressure overload-induced myocardial hypertrophy in rats

AIM：To investigate the therapeutic effect and the mechanism of neuregulin-1β (NRG-1β) on the rat model of myocardial hypertrophy induced by pressure overload．METHODS：Eight weeks after coarctation of abdominal aorta, the Wistar rats were randomly divided into 4 groups: myocardial hypertrophy (model) group, sham operation (sham) group, NRG-1β treatment group (intravenous injection of NRG-1β at dose of 10 μg/kg daily for 7 d) and NRG-1β+Herceptin (HERCE) treatment group ［intravenous injection of NRG-1β (10 μg/kg) plus HERCE (10 μg/kg) daily for 7 d］. The characteristics of heart functions were evaluated by the methods of hemodynamics and echocardiography. Masson staining was employed to observe the pathological changes of myocardial tissues. The concentration of angiotensin II (Ang II) in myocardial tissues was measured by radioimmunoassay. The level of tumor necrosis factor α (TNF-α) in myocardial tissues was detected by ELISA. The mRNA expression of B-cell lymphoma/leukemia-2 (bcl-2) and bcl-2-associated X protein (bax) in the myocardium was determined by RT-PCR. RESULTS：The left ventricular ejection fraction (LVEF) and left ventricular fraction shortening (LVFS) were higher, while the left ventricular end-systolic diameter (LVESD) and left ventricular end-diastolic diameter (LVEDD) were smaller in NRG-1β group than those in model group. The left ventricular end-systolic pressure (LVESP) and maximal rate of increase/decrease in left ventricular pressure (±dp/dtmax) were higher, and left ventricular end-diastolic pressure (LVEDP) was significantly lower in NRG-1β group than those in model group. Compared with model group, treatment with NRG-1β decreased collagen volume fraction (CVF), reduced the Ang II and TNF-α, increased bcl-2 mRNA expression, and decreased bax mRNA expression in myocardial tissues. No difference of the above parameters between model group and NRG-1β+HERCE treatment group was observed. CONCLUSION：NRG-1 reduces the expression of Ang II and TNF-α in myocardial tissues in pressure-overload rats, thus reducing Ang II and TNF-α mediated myocardial interstitial remodeling. Increase in the mRNA expression of bcl-2 and decrease in the mRNA expression of bax by NRG-1 inhibit myocardial cell apoptosis, which is responsible for its role of improving cardiac function of myocardial hypertrophy induced by pressure overload.     

Relationship of microRNA-133a and TGF-β1 protein in myocardial tissues of spontaneously hypertensive rats

AIM：To observe the changes of microRNA-133a and transforming growth factor β1 (TGF-β1) protein in the myocardium of spontaneously hypertensive rats (SHR). METHODS：Male SHR (18 weeks old, n=12) and male Wistar-Kyoto rats (WKY, 18 weeks old, n=12) served as SHR group and control group, respectively. Caudal arterial blood pressure was detected by a noninvasive blood pressure measurement and analysis system. Myocardial collagen volume fraction (CVF) and perivascular collagen area ratio (PVCA) were determined by Masson staining. The level of miR-133a in the heart was detected by real-time quantitative PCR. The protein level of TGF-β1 in the heart was also analyzed by the methods of immunohistochemisty and Western blotting. RESULTS：Compared with control group, systolic and diastolic blood pressure, CVF and PVCA significantly increased, the expression of TGF-β1 protein was significantly up-regulated, and the level of miR-133a was significantly reduced in SHR group. In SHR group, the expression of miR-133a was decreased to (23.9±4.6)% in control group. A negative correlation between the levels of miR-133a and TGF-β1 protein in SHR group was observed (r=-0.791, P<0.01). CONCLUSION：The level of miR-133a is down-regulated along with the up-regulation of TGF-β1 protein expression and collagen synthesis in the myocardial tissues of SHR. miR-133a and TGF-β1 may be involved in myocardial fibrosis in SHR.     

Effect of plumbagin on levels of Nox4/ROS and α-SMA in human hepatic stellate cells

AIM: To observe the effect of plumbagin on the mRNA and protein expression of nicotinamide adenine dinucleotidephosphate oxidase 4 (Nox4), reactive oxygen species (ROS) level and protein expression of α-smooth muscle actin (α-SMA) in the HSC-LX2 cells stimulated with transforming growth factor β1 (TGF-β1) in vitro. METHODS: HSC-LX2 cells were cultured in vitro and divided into blank group, model group, high-, medium- and low-dose (2, 1.5 and 1 μmol/L) plumbagin groups. After incubated with each drug for 72 h, the mRNA expression of Nox4 was detected by RT-PCR. ROS levels were tested by in situ loading probe method. The protein contents of Nox4 and α-SMA were measured by Western blot. RESULTS: Compared with model group, after treated with plumbagin for 72 h, the mRNA expression of Nox4, ROS level and α-SMA protein were significantly decreased in high-and medium-dose plumbagin groups (P<0.01). CONCLUSION: Plumbagin inhibits the activation of HSC-LX2 cells via decreasing the expression of Nox4, thus decreasing ROS levels.     

Xinshuaikang inhibits myocardial autophagy and MAPK/ERK1/2 signaling pathway in rats with chronic heart failure

AIM:To explore the effect of Xinshuaikang on myocardial autophagy in the rats with chronic heart failure and its relationship with the MAPK/ERK1/2 signaling pathway. METHODS:The rats were divided into sham group, model group (rat model of chronic heart failure was established by ligation of anterior descending branch of left coronary artery), low-, middle-, and high-dose Xinshuaikang treatment (TL, TM and TH) groups and captopril group (treated with captopril as positive control), with 12 in each group. Doppler echocardiography was used to evaluate the cardiac function. The morphological changes of the myocardium were observed by HE staining. TUNEL staining was used to detect cardiomyocyte apoptosis. The expression of microtubule-associated protein 1 light chain 3-Ⅱ (LC3-Ⅱ) in the myocardium was detected by immunofluorescence labeling. The protein levels of p-ERK, p-p38 MAPK, LC3-Ⅱ, beclin-1 and p62 in the myocardium were determined by Western blot. RESULTS:Compared with sham group, left ventricular end-diastolic dia-meter (LVEDD) and left ventricular end-systolic diameter (LVESD) in model group were increased, while left ventricular posterior wall thickness at end-diastole (LVPWTd), left ventricular posterior wall thickness at end-systole (LVPWTs), left ventricular ejection fraction (LVEF), cardiac output (CO), left ventricular diastolic pressure (LVDP), left ventricular systolic pressure (LVSP) and maximum rate of rise/decrease of left ventricular pressure (+dp/dt_max/-dp/dt_max) were decreased (P<0.05). The myocardial cells were deformed and necrotic, and the myocardial fibers were broken, with inflammatory cell infiltration. The apoptotic rate, the positive rate of LC3-Ⅱ, and the protein levels of p-ERK, p-p38 MAPK, LC3-Ⅱ/LC3-I and beclin-1 were increased, and the protein expression of p62 was decreased (P<0.05). Compared with model group, the levels of LVEDD and LVESD were decreased, LVPWTd, LVPWTs, LVEF, CO, LVSP, LVDP, +dp/dt_max and -dp/dt_max were increased in Xinshuaikang groups and captopril group (P<0.05). The morphological changes of myocardial cells were gradually returned to normal, and inflammatory cell infiltration, the apoptotic rate and the positive rate of LC3-Ⅱ were decreased. The protein levels of p-ERK, p-p38 MAPK, LC3-Ⅱ/LC3-I and beclin-1 were decreased, and the protein expression of p62 was increased (P<0.05). CONCLUSION:Xinshuaikang inhibits myocardial auto-phagy to play a role of cardiac protection in the rats with chronic heart failure, and its mechanism may be related to inhibition of MAPK/ERK1/2 signaling pathway.     

Zacopride attenuates pressure overload-induced ventricular remodeling in rats

AIM:To investigate the protective effect of zacopride (ZAC) on the pressure-overload left ventricular remodeling in the rats induced by coarctation of abdominal aorta. METHODS:Male Sprague-Dawley (SD) rats with pressure overload were induced by the coarctation of abdominal aorta. The model rats were intraperitoneally administered with ZAC, chloroquine (Chlor), and zacopride+chlorquine (ZAC+Chlor). The study duration was 8 weeks. The cardiac structure and function were assessed by echocardiography. The heart weight/body weight (HW/BW) ratio and the left ventricular weight/body weight (LVW/BW) ratio were calculated. The changes of structure and shape in myocardial tissue were observed with HE staining. The ultrastructure of the myocytes was observed under transmission electron microscope. The inward rectifier potassium channel (I_K1) protein expression was determined by Western blot. The mRNA expression of Kir2.1 was detected by RT-PCR. RESULTS:Compared with vehicle group, ZAC improved cardiac function, as indicated by the decreased left ventricular end-diastolic dimension (LVEDD) and left ventricular end systolic dimension (LVESD) (P<0.05), and the increased left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS) (P<0.01). The HW/BW and LVW/BW ratios were significantly decreased, and the cross-sectional area of the cardiomyocytes was significantly less in ZAC group than that in vehicle group (P<0.01). The ultrastructure of the myocytes was significantly improved. Chlor blocked the protective effect of zacopride on the pressure-overload left ventricular remodeling. The protein level of I_K1 and mRNA expression of Kir2.1 in the cardiac tissues in ZAC group were significantly increased compared with vehicle group (P<0.01). CONCLUSION:I_K1 agonist ZAC significantly attenuates pressure overload-induced ventricular remodeling in rats.     

Protective effect of aFGF delivery by ultrasound-targeted microbubble destruction on left ventricular function in diabetic cardiomyopathy rats

AIM：To observe the protective effect of delivery of acidic fibroblast growth factor (aFGF) to myocardium by ultrasound-targeted microbubble destruction (UTMD) on left ventricular function in diabetic cardiomyopathy (DCM) rats and to investigate the possible mechanisms. METHODS：Twenty-four rats were intraperitoneally injected with streptozocin to induce DCM and were randomly divided into DCM group and aFGF treatment group. Twelve healthy rats served as normal controls. The rats in aFGF treatment group were infused with SonoVue-aFGF mixed fluid through tail vein and UTMD was simultaneously performed. Four weeks after intervention, all rats underwent cardiac catheterization to mea-sure left ventricular end-systolic pressure (LVESP), left ventricular end-diastolic pressure (LVEDP) and the maximal increase/decrease rate of left ventricular pressure (LV±dp/dtmax). The microvessel density (MVD) of rat myocardial tissues was measured by immunohistochemical staining for CD31. The myocardial collagen volume fraction (CVF) was determined by improved Masson staining. The apoptotic index (AI) was detected by TUNEL method. RESULTS：Four weeks after intervention, the LVESP and LV±dp/dtmax in aFGF treatment group were significantly increased compared with DCM group (P<0.01), while the LVEDP in aFGF treatment group was significantly lower than that in DCM group (P<0.01). The MVD in aFGF treatment group was significantly increased compared with DCM group (P<0.01), but the CVF and AI in aFGF treatment group were significantly lower than those in DCM group (P<0.01). CONCLUSION: Delivery of aFGF to diabetic myocardium by UTMD could improve the left ventricular function of DCM rats and may be a new feasible therapeutic method for DCM.     

Expression of programmed cell death factor 4 in pulmonary fibrosis model

AIM:To detect the expression of programmed cell death protein 4 (PDCD4) in pulmonary fibrosis model and its effect on cell viability and pulmonary fibrosis indicators, and to explore its mechanism. METHODS:The expression level of PDCD4, α-smooth muscle actin (α-SMA) and collagen type I (COL-I) in human embryonic lung fibroblasts cell line HFL-1 group and HFL-1+TGF-β1 group were detected by Western blot and RT-qPCR. The plasmid pEZ-M03-PDCD4 and empty vector pEZ-M03 were transfected into myofibroblasts (MB), and the protein expression level of PDCD4 was detected by Western blot. The protein levels of p-AKT and AKT in blank control group, pEZ-M03-PDCD4 group, pEZ-M03 group and LY294002 group and the expression of cell cycle-related proteins c-Myc and cyclin D1 were determined by Western blot. The effect of PDCD4 on the viability of MB was measured by CCK-8 assay. The hydroxyproline content in the culture supernatant of HFL-1 cells and transfected MB was detected by hydroxyproline digestion method. The expression of PDCD4 in the lung tissues of the mice in model group and control group was detected by Western blot. RESULTS:Compared with HFL-1 group, the expression of α-SMA and COL-I at mRNA and protein levels in HFL-1+TGF-β1 group was significantly increased (P<0.01), the mRNA expression of PDCD4 was not significantly changed, while PDCD4 protein was significantly down-regulated (P<0.01). Compared with blank control group and pEZ-M03 group, the protein expression of PDCD4 in pEZ-M03-PDCD4 group was significantly increased (P<0.05), the protein expression of c-Myc and cyclin D1 was significantly decreased (P<0.05), the cell viability was also significantly inhibited (P<0.01), and the content of hydroxyproline in the culture supernatant was significantly reduced (P<0.05). Compared with blank control group, the protein levels of p-AKT were significantly decreased in pEZ-M03-PDCD4 group and LY294002 group, and no significant difference between blank control group and pEZ-M03 control group was observed. Compared with control group, PDCD4 expression was decreased in model group (P<0.01).CONCLUSION:PDCD4 is low expressed in the process of pulmonary fibrosis. Over-expression of PDCD4 inhibits the viability of MB, decreases the content of hydroxyproline, and inhibits the PI3K/AKT signaling pathway.     

Effects and mechanism of fenofibrate and pioglitazone on ventricular remodelingin in pressure overload rats

AIM: To study the effects and mechanism of peroxisome proliferator-activated receptors (PPARs) ligands,fenofibrate and pioglitazone,on ventricular remodeling in pressure overload rats.METHODS: A pressure overload model was established by the constriction of abdominal aorta in Wistar rats.The hemodynamics and ventricular remodeling parameters,plasma and myocardial renin activity,angiotensin Ⅱ and aldosteron,the mRNA expression of angiotensin Ⅱ type 1 receptor (AT1) were investigated in the constriction of abdominal aorta group (CAA group,n=7) at 12-week after operation and treated experimental groups in which rats were treated with fenofibrate (F group,n=8),pioglitazone (P group,n=7),concomitant fenofibrate and pioglitazone (F+P group,n=6) for 12 weeks since 2 days after operation.The sham-operated rats served as controls (n=8).RESULTS: The ratio of left ventricular weight to body weight,mean arterial pressure,left ventricular systolic pressure,left ventricular end diastolic pressure,left ventricular systolic pressure and heart rate were significantly lower,the maximum left ventricular pressure rising and declining rates(±dp/dtmax) were significantly higher in all treated experimental groups than those in CAA group.Fenofibrate or pioglitazone had no effect on plasma and myocardial levels of renin,angiotensin Ⅱand aldosteron.The mRNA expression of AT1 was downregulated in treated groups except F group.CONCLUSION: PPAR ligands have no effect on plasma and myocardial levels of renin,angiotensin Ⅱand aldosteron,but fenofibrate and pioglitazone inhibit ventricular remodeling,decrease preload and afterload,increase ±dp/dtmax in pressure overload rats.The expression of mRNA of AT1 is downregulated in myocardium of pressure overload rats by the PPARγ signaling pathway.     

Effect of CKLF1-C19 polypeptide on differentiation of human lung fibroblast into myofibroblast induced by TGF-β

AIM: To investigate the effect of CKLF1-C19 polypeptide (C19) on differentiation of human lung fibroblast (LFB) into myofibroblast (MFB) induced by TGF-β. METHODS: LFBs were cultured and identified. LFBs were treated with TGF-β (5 μg/L) to establish the cell model of LFB differentiate into MFB. The LFBs were divided into 6 experimental groups including control group, TGF-β group, and TGF-β plus different doses (1, 0.1, 0.01, 0.001 mg/L) C19 groups. The cell morphology, cell proliferation rate, and the expression of α smooth muscle actin (α-SMA) and collagen I were observed. RESULTS: Human primary LFB was successfully cultured and was confirmed by the method of immunofluorescence. TGF-β at 5 μg/L induced proliferation and differentiation of LFB. The mRNA levels of α-SMA and collagen I in TGF-β group were higher than that in control group (P<0.05).The cell proliferation rates, mRNA levels of α-SMA and collagen I, and the protein expression of α-SMA in 0.01 mg/L+TGF-β group and 0.001 mg/L+TGF-β group were markedly lower than those in TGF-β group (P<0.05). CONCLUSION: C19 at 0.01 mg/L and 0.001 mg/L effectively inhibits differentiation of LFB into MFB induced by TGF-β, thus inhibiting the process of airway remodeling and fibrosis to some extent.     

Role of EndoMT in HHPH based on TGF-β/Smads signaling pathway and regulated by Yiqi-Wenyang-Huoxue-Huatan formula

AIM: To investigate the relationship between transforming growth factor-β (TGF-β)/Smads signaling pathway and pulmonary arterial endothelial-mesenchymal transition (EndoMT) in hypoxia-hypercapnia pulmonary hypertension (HHPH) process and the regulatory effect of Yiqi-Wenyang-Huoxue-Huatan formula (YWHHF). METHODS: Healthy male SD rats were randomly divided into 5 groups:normal control (N) group, hypoxia-hypercapnia (HH) group, high-dose YWHHF (YH) group, middle-dose YWHHF (YM) group and low-dose YWHHF (YL) group. The rats in N group was housed in normoxic environment, and the rats in the other 4 groups were housed in hypoxia-hypercapnia environment (9%~11% O₂ and 5%~6% CO₂) for 4 weeks, 8 h/d, 6 d/week. The excess water vapor was absorbed by anhydrous CaCl₂, and CO₂ was absorbed by sodium hydroxide. The rats in YWHHF groups were put into the oxygen chamber before the same volume of YWHHF at different concentrations were given (200 g/L for YH group, 100 g/L for YM group and 50 g/L for YL group). The average pulmonary artery pressure and the average carotid artery pressure were measured during the operation. After operation, the right ventricular free wall and left ventricle plus interventricular septum were collected for determining the right ventricular hypertrophy index. Moreover, the morphological changes of the lung tissues were observed under light microscope. The mRNA and protein levels of α-smooth muscle actin (α-SMA), CD31, TGF-β1 and Smad2/3, and the protein level of p-Smad2/3 were detected by RT-PCR and Western blot. RESULTS: Compared with N group, the pulmonary artery mean pressure, the mRNA and protein expression of α-SMA, TGF-β1 and Smad2/3, and the protein level of p-Smad2/3 were increased, the levels of CD31 were decreased (P<0.05), and the lung tissue damage was observed in the other 4 groups. Compared with HH group, the pulmonary artery mean pressure, the mRNA and protein expression of α-SMA, TGF-β1 and Smad2/3, and the protein level of p-Smad2/3 were decreased, while the mRNA and protein levels of CD31 were increased. Moreover, the lung tissue damage was reduced in YH, YM and YL groups. CONCLUSION: TGF-β/Smads pathway may be involved in the process of EndoMT under hypoxia and hypercapnia condition, and YWHHF may reduce EndoMT by inhibiting the expression of TGF-β/Smads pathway-related molecules.     

Effect of chelerythrine on TGF-β/Smads signaling pathway in mouse livers with hepatic fibrosis

AIM:To investigate the anti-hepatic fibrosis effect of chelerythrine on mice and the regulation of transforming growth factor-β (TGF-β)/Smads signaling pathway. METHODS:C57BL/6N mice (n=50) were randomly divided into control group, model group and chelerythrine groups (10 mg·kg^-1·d^-1, 20 mg·kg^-1·d^-1 and 40 mg·kg^-1·d^-1, ig). The mouse model of hepatic fibrosis was established by intraperitoneal injection of carbon tetrachloride (CCl₄) in combination with the olive oil for 8 weeks. At the 5th week, different doses of chelerythrine was used to treat hepatic fibrosis in the mice. At the 14th week, hepatic index was detected. Histopathological changes and the degree of hepatic fibrosis were observed by hematoxylin-eosin staining and Van Gieson staining. The serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and hyaluronic acid (HA), and hepatic hydroxyproline (Hyp) content were assayed by spectrophotometry and ELISA. The mRNA expression of TGF-β₁, Smad3, Smad4 and Smad7 in the liver was detected by RT-qPCR, and the protein expression of TGF-β₁, Smad4 and Smad7 was determined by Western blot. RESULTS:The degree of hepatic fibrosis changed markedly in model group compared with control group. The hepatic index, the serum levels of ALT and AST, and the contents of HA and Hyp were significantly increased (P<0.05). The mRNA expression of TGF-β₁, Smad3 and Smad4 was significantly up-regulated, while the mRNA expression of Smad7 was significantly down-regulated (P<0.05). The protein expression of TGF-β₁ and Smad4 was significantly up-regulated, while the protein expression of Smad7 was significantly down-regulated (P<0.05). Compared with model group, the changes of the above indexes in chelerythrine groups were inhibited. CONCLUSION:Chelerythrine protects the mouse liver from CCl₄-induced fibrogenesis injury by regulating TGF-β/Smads signaling pathway.     

Effects of miR-124 over-expression on right ventricular remodeling in rats with pulmonary hypertension

AIM: To investigate the effect of microRNA-124 (miR-124) over-expression mediated by adeno-associated virus (AAV) on right ventricular remodeling in rats with pulmonary arterial hypertension (PAH) induced by monocrotaline (MCT). METHODS: Male SD rats (n=32) were randomly divided into 4 groups:normal control (control) group, MCT+normal saline (NS) group, MCT+AAV-GFP (MCT+GFP) group and MCT+AAV-miR-124 (MCT+miR-124) group. The rats in the latter 3 groups were instilled slowly with 100 μL NS, AAV-GFP and AAV-miR-124 by orotracheal instillation after anesthesia, respectively. Three weeks later, MCT (60 mg/kg) was intraperitoneally injected to establish the PAH model. Right ventricular systolic blood pressure (RVSP) and mean arterial pressure of the rats were measured, and right ventricular hypertrophy index (RVHI) and right ventricular weight index (RVWI) were calculated. The pathological sections of the right heart were stained with Sirius red, and the pathological changes of myocardium were observed under a microscope. The expression of miR-124 in the lung tissues was detected by RT-qPCR. The protein levels of transforming growth factor-β₁(TGF-β₁) and p-Smad2 in right heart tissues were determined by Western blot. RESULTS: Compared with control group, RVSP, RVHI, RVWI and the protein levels of TGF-β₁ and p-Smad2 in MCT+NS group and MCT+GFP group were significantly increased (P<0.05), the right ventricular myocytes were significantly enlarged, and collagen deposition was significantly increased. However, compared with MCT+GFP group, RVSP, RVHI, RVWI and the protein levels of TGF-β₁ and p-Smad2 in MCT+miR-124 group were significantly decreased (P<0.05), the degree of right ventricular myocyte hypertrophy was significantly reduced, and collagen deposition was significantly reduced. CONCLUSION: Over-expression of miR-124 obviously reduces RVSP of rats induced by MCT and relieves myocardial remodeling, which may be related to the down-regulation of TGF-β₁ and p-Smad2.     

Evaluation of left ventricular diastolic dysfunction at the early stage of pressure overload-induced myocardial remodeling in rats

AIM: To investigate the evaluation method of left ventricular diastolic function and myocardial hypertrophy induced by pressure overload in rats.METHODS: Male Sprague-Dawley rats were subject to left ventricular pressure overload by transverse aortic constriction (TAC).Cardiac structure and diastolic function were evaluated by echocardiography, hemodynamic analysis and examination of hydroxyproline concentration in the myocardial tissues.RESULTS: Compared with the sham-operated controls, left ventricular wall dimension in diastole significantly increased in the rats 3 weeks after TAC .Left ventricular early diastolic posterior wall motion velocity (E') significantly decreased in the rats 3 weeks after TAC , and was much lower than that in the rats 6 weeks after TAC.Left ventricular mass to tibia length in TAC rats was much higher than that in sham-operated controls .The ratio of maximum rate of degression of left ventricular pressure (dp/dt_min) to left ventricular systolic pressure (dp/dt_min/LVSP) started to decrease in TAC rats in the 3rd week (48.9±5.9 vs 63.5±9.9) and significantly decreased in TAC rats in the 6th week as compared with sham-operated controls (35.4±4.0 vs 54.4±2.9, P<0.01).Sirius red-stained collagen in cardiac interstitium, especially around the blood vessels, was increased in TAC rats.Six weeks after TAC, a significant increase in the content of myocardial hydroxyproline was observed.CONCLUSION: The early diastolic posterior wall motion velocity (E') detected by tissue Doppler imaging is a sensitive indicator of diastolic dysfunction at the early stage of myocardial remodeling induced by pressure overload in rats.     

Effect of leonurine on expression of miR-1 in rats with myocardial fibrosis induced by isoproterenol

AIM: To investigate effect of leonurine on the expression of microRNA-1 (miR-1) in rats with myocardial fibrosis induced by isoproterenol (ISO). METHODS: SD rats (n=10) were used as normal control group, and 80 rats were given ISO by intraperitoneal injection daily for 2 weeks to establish the model of myocardial fibrosis. The model rats were randomly divided into 5 groups:model group, low-dose (7.5 mg·kg^-1·d^-1) leonurine group, middle-dose (15 mg·kg^-1·d^-1) leonurine group, high-dose (30 mg·kg^-1·d^-1) leonurine group and p38 mitogen-activated protein kinase (p38 MAPK) inhibitor (0.3 mg·kg^-1·d^-1) group. After the treatment for 2 weeks, the ultrastructure of left ventricular myocardial tissues was observed under electron microscope. Masson staining was used to detect collagen fibrosis, and the expression of collagen I and collagen Ⅲ was determined by the method of immunohistochemistry. The contents of endothelin-1 (ET-1) and angiotensin Ⅱ (Ang Ⅱ) were measured by ELISA. The expression of miR-1 and ET-1 mRNA was detected by real-time PCR, and the protein expression of p38 MAPK, β-myosin heavy chain (MHC) and α-MHC was determined by Western blot. RESULTS: Compared with model group, the ultrastructure of left ventricular myocardial tissues in high-dose leonurine group was attenuated, and the expression of miR-1 and the protein expression of α-MHC in left ventricular myocardial tissues of high-dose leonurine group were increased (P<0.05). Collagen volume fraction, collagen I, collagen Ⅲ, the ratio of collagen Ⅰ/collagen Ⅲ, the contents of ET-1 and Ang Ⅱ, the mRNA expression of ET-1, and the protein expression of p38 MAPK and β-MHC in high-dose leonurine group were lower than those in model group (P<0.05). CONCLUSION: Leonurine attenuates myocardial fibrosis in the rats induced by ISO, and it is potentially associated with affecting the expression of miR-1, and inhibiting ET-1/p38 MAPK signaling pathway.     

Influence of paricalcitol on renal tubulointerstitial fibrosis in diabetic nephropathy

AIM: To investigate the effect of paricalcitol (P) on renal tubulointerstitial fibrosis and the underlying mechanisms in diabetic nephropathy (DN).METHODS: DN rat model was induced by a single intraperitoneal injection of streptozotocin after fasting. The animals were randomly divided into 2 groups:the DN rats in paricalcitol-intervened group (group P) were injected intraperitoneally with paricalcitol dissolved in propylene glycol after the day when the model was induced successfully at a dose of 0.4 μg/kg (3 times a week); the DN rats in DN group (group D) were given isopyknic propylene glycol. Normal control group (group C) was also set up. The samples of blood, urine and renal tissue were collected after intervention of paricalcitol for 12 weeks. The biochemical indexes were measured. The renal tissues were used for pathologic observation and determining the expression of transforming growth factor-β1 (TGF-β1), Wnt-4, β-catenin and Klotho by immunohistochemistry and Western blotting. In addition, the correlation among the above indexes was analyzed.RESULTS: (1) Scr, BUN and 24 h urine protein increased significantly in group D compared with group C, while decreased in group P compared with group D (P<0.05). (2) The area of renal tubulointerstitial fibrosis increased in group D compared with group C, while decreased in group P compared with group D (P<0.05). (3) The expression of Klotho decreased, while the expression of TGF-β1, Wnt-4 and β-catenin increased in group D compared with group C (P<0.05). Compared with group D, the expression of Klotho increased, while the expression of TGF-β1, Wnt-4 and β-catenin decreased in group P (P<0.05). (4) The expression of Klotho was negatively correlated with the fibrosis area, TGF-β1, Wnt-4 and β-catenin (P<0.05).CONCLUSION: Paricalcitol inhibits renal tubulointerstitial fibrosis in DN by promoting the expression of renal Klotho, and inhibiting Wnt/β-catenin signaling pathway activation and TGF-β1 synthesis.