| BackgroundsMyocardial remodeling is the key factor that influences the prognosis of patients after myocardial infarction(MI). Several Pathophysiological processes are involved in myocardial remodeling which includes cell apoptosis and hypertrophy; angiogenesis; synthesis and deposition of extracellular matrix(ECM). In the early phase after MI, cardiac fibroblasts(CFs) are recruited to the infarcted area under the action of cytokines, and then synthesize and secrete ECM (mainly contains collagen) to replace the necrotic tissue in order to stabilize the normal structure and prevent cardiac rupture. However, excessive deposition of ECM will result incardiac fibrosis, which leads to decreased ventricular compliance, cardiac dysfunction and heart failure finally. Besides, cardiac fibrosis could also lead to the electrophysiological remodeling, which might induce arrhythmogenesis and result in a declined quality of life. So how to regulate cardiac fibrosis successfully is the key factor to improve the prognosis after myocardial infarction.Renin-angiotension-aldosterone system (RAAS) plays an important role in the myocardial remodeling after MI.Angiotensin Ⅱ (Ang Ⅱ), as the central product of the RAAS increases obviously in both serum and heart tissues after MI. Ang Ⅱ promotes cardiac fibrosis via inducing cardiac fibroblast (CF) proliferation and migration, collagen deposition, and extracellular matrix (ECM) degradation by activating MAPK and STAT3 pathway.Thymic stromal lymphopoietin (TSLP) is a kind of newly discovered cytokines, which is similar to IL-7. It is first discovered in thymic stromal cells. Its molecular weight is 29KD, which are principally expressed by epithelial cells, thymic stromal cells, mast cells and smooth muscle cells. In recent studies, TSLP was also found in fibroblasts and its expression in heart tissue is the most abundant compared to other organs. Under the action of external stimulation, the above cells synthesize and secrete TSLP, and exert biological effects through binding with a heterodimeric TSLP receptor (TSLPR, composed of TSLPR subunit and IL-7Ra subunit) on the cell surface. TSLPR is a kind of type I transmembrane protein, which belongs to hematopoietic cytokine receptor family. Recently, TSLP was found to have pro-fibrotic effects in systemic sclerosis, keloma and asthmatic airway remodelling. Previous studies reported that the level of TSLP in serum increased obviously in patients after MI. Ang II has been reported to have a potential role in promoting the expression of TSLP in vascular smooth muscle cells. However, whether TSLP is involved in the development of cardiac fibrosis after MI and its mechanism has not been investigated.The present study was designed to investigate the role and mechanism of TSLP in cardiac fibrosis after MI in MI mice and in CFs.Objectives1. To establish a MI model in mice and investigate the role and mechanism of TSLP in cardiac fibrosis after MI.2. To study the effects of TSLP on myocardial dysfunction following MI3. To investigate the role and mechanism of TSLP in Ang Ⅱ-induced CFs’ proliferation, migration and collagen synthesis.Methods1.Lentivirus vector RNA interferenceThree kinds of shTSLP lentivirus were synthesized and transduced into CFs.3 days after transduction, cells were harvested and the most effective shTSLP lentivirus was choosen according to the results from Western Blot. Lentivirus carrying nonsense RNA sequence was used as negative control.2.Animal model and RNA interferenceEight-week-old C57BL/6J wild-type male mice (weighing 20-25 g) were fed regular chow and maintained under standard conditions for one week. For MI model establishment, mice were anesthetized with 2% isoflurane inhalation. The chest was open at the 4th intercostal space. The heart was smoothly and gently extruded from the hole, and MI was created by permanent LAD ligation using a 7-0-suture line. Pallor and regional-wall motion abnormality of the left ventricle confirmed the success of the coronary occlusion. After LAD ligation, an amount of 1×107 UT/25 μL were injected at three sites around the ligated spot. Four weeks after MI induction, the transfection efficiency of lentivirus was evaluated by observation of green fluorescent protein(GFP) expression under fluorescence microscope and confirmed by western blot.3.Cardiac function evaluationFour weeks after surgery, transthoracic echocardiography was performed with the Vivo 770 imaging system.2D echocardiography and M-mode echocardiography were used to measure the diastolic and systolic left ventricular internal diameter (LVIDd and LVIDs). Left ventricular fractional shortening (%FS) and ejection fraction (%EF) were calculated by the echocardiography system.4.Histology and immunohistochemistryThe tissue was paraffin-embedded and sectioned (5 μm) for staining. HE、 Masson and Sinus red staining was used to examining the morphological structure, infarct size and collagen deposition. Immunohistochemistry was used to examining TSLP, collagen I, collagen Ⅲ and TGF-β1.5.Cell cultureCFs were isolated from 1-to 3-day-old mice and passage 2 or 3 was used for further experiment. AngⅡ, recombinant TSLP protein and TSLP antibody were used to treat cells according to the objects.6. Immunofluorescence microscopyImmunofluorescence analysis was used to evaluate the expression and distribution of TSLP, TSLPR and IL-7Rα.7. Western blot analysisProtein was extracted from cells and heart tissue of different groups. The expression of TSLP, collagen I, collagen Ⅲ, TGF-β1、STAT3、p-STAT3、ERK、 p-ERK、JNK、p-JNK、p38 and p-p38 were evaluated by Western blot.8. Enzyme linked immunosorbent assay(ELISA)ELISA analysis was used to detect TSLP in serum of mice and cell culture medium9. Evaluation of cell proliferation and migrationCell Counting Kit-8 (CCK-8) and EdU imaging Kits were used to examine cell proliferation and CFs migration assays were performed in Transwell chambers.10. Statistical analysisSPSS 18.0 was used for the statistical analyses. The data are expressed, as the mean ±SD.p<0.05 was considered statistically significant.Results1. MI increased the level of TSLP in heart tissueResults from Western blot and immunohistochemistry showed that MI increased the expression of TSLP in the peri-infarct heart tissue. Compared to the negative control group, shTSLP lentivirus significantly inhibited TSLP expression.2. TSLP inhibition improved myocardial dysfunction after MIFour weeks after MI establishment, expanded ventricular diameters and decreased LVEF and FS indicated ventricular dysfunction. ShTSLP lentivirus transduction attenuated MI-induced myocardial dysfunction.3. TSLP inhibition attenuated MI-induced cardiac fibrosisAccording to Masson and Sinus Red Staining, MI increased collagen deposition in intramyocardial areas and perivascular areas. Western blot and immunohistochemistry showed that MI increased the expression of collagen I, collagen Ⅲ and TGF-β1. TSLP inhibition obviously decreased cardiac fibrosis compared to negative lentivirus group.4. Recombinant TSLP protein increased CFs’ proliferation, migration and collagen synthesisRecombinant TSLP protein dose-dependently increased the protein level of collagen I, collagen Ⅲ and TGF-β1 with its maximum effect at 20ng/ml. CFs showed fast proliferation rate after stimulation with recombinant TSLP protein which was confirmed by CCK-8 assays and EdU assays. Migration assay was performed in Transwell chamber and data showed TSLP treatment dose-dependently promoted the migration rate in CFs.5. AngⅡ stimulated the expression of TSLP and TSLP inhibition attenuated AngⅡ-induced CFs’ proliferation, and collagen synthesisDifferent concentrations of AngⅡ (1,10,100nM) were used to treat CFs which dose-dependently increased the expression of TSLP. TSLP antibody pretreatment inhibited AngⅡ-induced CFs collagen synthesis, proliferation and migration.6. TSLP promoted the phosphorylation of STAT3 and MAPK (JNK, ERK and p38)AngⅡ increased the activation of STAT3, ERK, JNK and p38 in CFs, while TSLP recombinant protein has the similar effects. TSLP inhibition with TSLP antibody decreased the above effects, which indicated the activation of STAT3 and MAPK pathway were involved in TSLP mediated myocardial fibrosis.Conclusion1. MI induced the expression of TSLP. TSLP inhibition improved cardiac fibrosis and cardiac dysfunction.2. TSLP promoted CFs’ proliferation, migration and collagen synthesis3. AngⅡpromoted the synthesis and secretion of TSLP, TSLP inhibition attenuated AngⅡ induced myocardial fibrosis.4. STAT3 and MAPK signal pathway might be involved in TSLP mediated myocardial fibrosis following MI.BackgroundsMyocardial remodeling after myocardial infarction involves several pathophysiological processes, such as inflammation, cell apoptosis, angiogenesis, mesenchymal cells proliferation, migration, synthesis and deposition of extracellular matrix(ECM), which results in changes in ventricular morphology, structure and dysfunction.Inflammation is necessary for cardiac repair after myocardial infarction, however, excessive inflammatory response can induce myocardial apoptosis and promote cardiac pathological remodeling. The synthesis and deposition of ECM can replace the necrotic myocardial tissue, stabilize the structure of the heart and prevent heart rupturein the early stage of MI. However, excessive deposition of ECM can promote the formation of myocardial fibrosis, result in decreased ventricular compliance and cause cardiac dysfunctionand ultimately lead to heart failure in the long term. So how to regulate myocardial remodeling after MI is the key factor to improve the prognosis after MI.SIRT1 is a member of the SIRT family of class III histone deacetylases (SIRT1-SIRT7) that regulates various cellular processes, including oxidative stress, cellular proliferation, apoptosis, inflammation and fibrosisvia deacetylation of histone and transcriptional factors. It has been reported that SIRT1 plays very important role in cardiovascular diseases. SIRT1 activation improves myocardial ischemia-reperfusion injury via attenuating oxidative stress. However, the role and the mechanism of SIRT1 in myocardial remodeling after MI are still lacking systematic research.Curcumin is a natural phytochemical compound extracted from the rhizomes ofthe plant Curcuma longa. Considerable evidence has demonstrated that curcumin has anti-inflammatory, anti-proliferative, anti-apoptosis and antioxidant properties. The protective effects of curcumin on the cardiovascular system have been confirmed in several diseases. However, deficiency inthe study of curcumin’s protective mechanismis the bottleneckto limit its clinical application. It has been demonstrated that curcumin could regulate the activity of SIRT1, however whether the protection of curcumin against myocardial remodeling after MI is mediated by SIRT1 activation has not been investigated yet. Thus, the present study was designed to investigate the role and mechanism of SIRT1 activation in curcumin’s protection against myocardial remodeling after MI in MI mice and in Ang Ⅱ-stimulated H9C2 cardiomyocytes and cardiac fibroblasts.Objectives1. To investigate the role and mechanism of curcuminonmyocardial remodeling and to study the role of SIRT1 activation in Curcumin’s protection against MI.2. To study the effects of SIRT1 on apoptosis and fibrosis in vitro study and investigate whether SIRT1 activation was involved in curcumin’s protection against MI.Methods1.Animal modelEight-week-old male C57BL/6J wild-type mice (weighing 20-25 g)were fed regular chow and maintained under normal conditions.After 1 week of acclimatization,mice were randomly divided into 4 groups (n=12 each) for treatment: Sham, Sham+Cur, MI, and MI+Cur. The Sham+Cur and MI+Cur groups received curcumin (100 mg/kg/day orally) in distilled water (containing a small amount of DMSO for better dissolution) by gastric gavage, andthe2 other groups received only distilled water (containing an equal amount of DMSO). For MI model establishment, mice were anesthetized with 2% isoflurane inhalation. The chest was open at the 4th intercostal space. MI was created by permanent LAD ligation. Pallor and regional-wall motion abnormality of the left ventricle confirmed the success of the coronary occlusion.2.Echocardiography2D echocardiography and M-mode echocardiography were used to measure the diastolic and systolic left ventricular internal diameter (LVIDd and LVIDs), Left ventricular fractional shortening (%FS) and ejection fraction (%EF) at the papillary muscle level from the short-axis view.3.Histology and immunohistochemistryThe tissue was paraffin-embedded and sectioned (5 μm) for staining. The mean cardiomyocyte width as the shortest dimension per cardiomyocyte was obtained from the H&E-stained sections at×400 magnification by microscopy. The ratio of the heart weight to body weight (HW/BW) was evaluated four weeks after MI surgery. The heart sections were stained with Masson’s trichrome and Sirius red to evaluate the interstitial collagen deposition. Immunohistochemistry was used to examining SIRT1, collagen I, collagen Ⅲ, a-SMA and TGF-β1.4.TUNEL AssayApoptotic cells in the myocardiumwere assessed by the In Situ Cell Death Detection Kit.5.Cell cultureH9C2 cardiomyocytes and cardiac fibroblasts isolated from 1-to 3-day-old Wistar rats were used in the study. AngⅡstimulation was used to mimic the MI status and investigate the protective role of curcumin on apoptosis and fibrosis. Pretreatment of cells with SIRT1 siRNA and SIRT1 inhibitor sirtinol separately and investigate the role of SIRT1 in curcumin’s protection against apoptosis and fibrosis induced by AngⅡ.6.Western blot analysisProtein was extracted from cells and heart tissue of different groups. The expression of Bax, Bcl-2, caspase3、TNF-α、IL-6、Ac-FOXO1、SIRT1、collagenI、 collagenⅢ、TGF-β1、MMP2、MMP9 were evaluated by Western blot.7.Gelatin zymographyThe activity of MMP2 and MMP9 were determined by Gelatin zymography.8.Acessment of cell proliferation and migrationCell Counting Kit-8 (CCK-8) was used to examine cell proliferation and Transwell chambers was used to examine cell migration.9.Statistical analysisSPSS 18.0 was used for the statistical analyses. Data are expressed as the mean± SD.Results1. Curcumin attenuated myocardial remodeling after MICompared to sham group, mice showed obvious changes in cardiac morphology. MI increased heart weight/body weight (HW/BW) and caused obvious left ventricular dilatation. HE staining showed hypertrophy of cardiac myocytes.Masson’s trichrome and Picrosirius red staining of heart sections revealed greater fibrosis in the interstitial regions of the MI group compared withthesham group which are confirmed by the immunohistochemical staining results for collagen I, collagen Ⅲ, TGF-β1 and a-SMA. We also evaluated the levels of inflammation and apoptosis related protein by Western blot and immunohistochemical staining. Data showed that the expression of IL-6, TNF-a, Bax/Bcl-2 and caspase3 increased obviously. TUNEL staining revealed that MI increased the number of TUNEL-positive cardiomyocytes compared with sham group. However, curcumin pretreatment before MI improved myocardial remodeling significantly. Interestingly, curcumin also increased the level of SIRT1, which was confirmed by western blot and immunohistochemical staining.2. Curcumin improvedmyocardial dysfunction induced by MICardiac function was evaluated 4 weeks after the induction of the MI model. MI mice showed cardiac dysfunction compared with Sham mice, which indicated a successful MI model. The left ventricular function was preserved with MI+Cur compared with MI alone, as assessed by LVEF and FS. MI mice showed more serious3. Curcumin reversed Ang Ⅱ-induced apoptosis in H9C2 cellsH9C2 cardiomyocytes were exposed to angiotensin II (100 nmol/L) for 48 h; Ang II significantly increased the expression of apoptosis-related molecules (cleaved caspase-3, Bax), which were dose-dependently reduced by curcumin. The expression of SIRT1 was also upregulated with downregulation of Ac-FOXO1 after pretreatment of curcumin.4. SIRT1 inhibition attenuated curcumin’s protection against Ang Ⅱ-induced apoptosis in H9C2 cellsTo further explore the role of SIRT1 involvement in curcumin treatment, both a SIRT1 inhibitor (sirtinol) and SIRT1 siRNA were used in our study. Compared to Ang Ⅱ stimulation alone, SIRT1 inhibition significantly increased the expression of caspase3 and the ratio of Bax/Bcl-2, which indicated the protective role of SIRT1 in Ang Ⅱ-induced apoptosis. The data showed that sirtinol or genetic silencing of SIRT1 eliminated the protective effects of curcumin pretreatment compared to the control group.5. Pretreatment of curcumin improved Ang Ⅱ-induced fibrosis in CFsWe tested the Ang II dose-response using concentrations of 1 nM,10 nM, and 100 nM for 48 h, and found that the expression of collagen Ⅰ, collagen Ⅲ, and TGF-β1 increased gradually in a dose-dependent manner. We also tested the time-dependent manner for Ang II-induced fibrosis. Finally, we chose to treat CFs with Ang Ⅱ (100 nM) for 24 h as the subsequent experiments condition. Pretreatment of curcumin significantly decreased the synthesis of collagen and ECM degradation. Results of western blot showed that curcumin decreased the expresssion of collagen I, collagen Ⅲ, TGF-β1 with the upregulation of SIRT1. The expression and activity of MMP2 and MMP9 was downregulated after curcumin pretreatment. Data revealed byCCK-8 assays and transwell assays indicated that curcumin strongly inhibited the preliferation and migration of CFs.6. SIRT1 inhibition blocked the protective effects of curcumin in in Ang Ⅱ-induced myocardial fibrosisWe investigated the role of SIRT1 in the protective action of curcumin against myocardial fibrosis using SIRT1 siRNA-mediated knockdown. SIRT1 inhibition increased the collagen deposition and ECM degradation. The proliferation and migration of CFs was also upragulated by SIRT1 inhibition. Above all, the protective role of curcumin was almost blocked by SIRT1 inhibition.Conclusion1. Curcumin improved myocardial remodeling and cardiac dysfunction via SIRT1 activation after MI.2. Curcumin attenuated AngⅡ-induced apoptosisvia SIRT1 activation in H9C2 cardiomyocytes.3.Curcumin inhibited Angll-induced CFs’ proliferation, migration and collagen synthesis via SIRT1 activation. |
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