【作者】 刘爱国；

【导师】 崔连群；

【作者基本信息】 山东大学 ， 内科学（专业学位）， 2013， 博士

【摘要】 研究目的：本研究的目的为阐明一种特异性组织蛋白酶B抑制剂CA-074Me在心肌梗死大鼠后心功能障碍和心室重塑、纤维化中的作用及潜在的机制。方法：1)大鼠心梗模型的建立SD大鼠购自北京维通利华公司。所有动物按照标准条件饲养于我校动物实验中心。采用10周龄的SD大鼠,体重约280-320g,通过结扎大鼠冠状动脉左前降支建立心肌梗死模型。具体的步骤如下：首先采用异氟烷(5%诱导,2%维持)麻醉大鼠,随后给予机械通气。通过观察大鼠的反应是否丧失和肌肉松弛的程度判断麻醉的效果。在第5和第6肋间开胸,打开心包,暴露心脏,冠状动脉位于肺动脉圆锥和左心房边缘交界处下1-2mm,用5.0的丝线自肺动脉圆锥进针自左心房边缘处出针永久性结扎冠状动脉。放置心脏于原位,复张肺部后采用4.0丝线关胸。假手术组为过线不结扎2)治疗实验分为3组：假手术组(n=10),心肌梗死组(n=15),CA-074Me心肌梗死治疗组(n=15)。采用DMSO二甲基亚砜(Dimethyl sulfoxide或DMSO),溶解组织蛋白酶B抑制剂CA-074Me制备10mg/ml储存液,采用生理盐水按照1：10的体积比稀释,按照10mg/kg的剂量腹腔注射大鼠进行治疗。其中心肌梗死组采用10%的DMSO注射4周；CA-074Me心肌梗死治疗组每天按照10mg/kg的剂量腹腔注射,治疗4周。3)组织蛋白酶B活性检测组织蛋白酶B的活性检测采用合成荧光底物Z-Arg-Arg-NHMec进行检测。在需要分析的溶液中加入150μlM Z-Arg-Arg-NHMec (pH6.0),随后在激发波长360nm和发射波长465nm,30分钟内每间隔1分钟检测荧光3次。数据表示为相对荧光单位,以均数±标准差表示。重复3次实验。4)免疫印迹采用组织裂解液(Cell Signaling Technology)制备心脏组织匀浆提取总蛋白,储存于-20℃。采用BCA方法(Pierce).检测蛋白浓度。蛋白煮沸变性,在8-12%的聚丙酰胺凝胶中分离,随后转至PVDF膜,5%脱脂奶粉封闭,采用特异性一抗孵育：anti-NLRP3(1:2000; BD); anti-caspase-1p20(1:2000), anti-pro-IL-1β/IL-1β (1:2000), anti-pro-IL-18/IL-18(1:2000)(Cell Signaling Technology).最后通过化学发光的方法进行检测,蛋白的相对定量通过与看家基因β-actin的光密度校正进行半定量。5)免疫酶联吸附血清IL-1p和IL-18水平采用美国R&D公司提供的ELISA试剂盒进行测定,具体测定方法严格按照试剂盒说明书操作。6)心功能检测大鼠的心功能检测采用超声心动图法。具体如下：采用10-12MHz的线状探头(model21380A with HP SONOS5500imaging system; Agilent Technologies).舒张期和收缩期左心室的内径(LVIDD和LVIDS),左心室短轴缩短率(LVFS)在左心室乳头肌平面采用M型超声测得,所有的测量数据采用双盲法测得连续3个心动周期,取平均值。所有数据的获得和分析由单盲的观察者采用EchoPAC (GE Vingmed)程序脱机分析。7)心肌梗死面积光镜下观察Masson’s trichrome染色的切片,采用图像软件(AIS, Analytical imaging Station Version6.0, Ontario, Canada)进行分析.心肌梗死的面积按照心内膜和心外膜瘢痕的平均周长和左心室心内膜和心外膜的平均周长的比值进行计算。8)细胞外基质沉积光镜下观察Masson’s trichrome染色的切片分析细胞外基质沉积的情况。定量非梗死区的基质沉积,具体如下：摄取左心室中部的染色切片的图片,用于电脑分析。分析的时候除去梗死区边缘2mm的区域,以此将非梗死区自梗死区和交界区分开,余下的心肌为非梗死区,采用图像分析软件进行分析(Analytical imaging Station Version6.0, Canada)。全部的非梗死区均用于细胞外基质沉积的分析,以免因为选定区域而造成的偏倚。Masson’s trichrome染色切片的蓝色区域代表细胞外基质。对于假手术组大鼠,定量分析方法同上。9)组织化学和免疫荧光分析4周后采用10%中性福尔马林固定心梗后大鼠心脏1小时,随后用0.1M甘氨酸孵育1小时,在0.6M的蔗糖溶液4℃过夜。采用OCT包埋样本,制成4μm切片,保存于-200C。对于免疫组化实验,大鼠的心脏切片通透和1%BSA封闭后采用a-sarcomeric actin (1:50; Sigma), wheat germ hemagglutinin (WGA)(1:50), and anti-His (1:50)(from Invitrogen)孵育。二抗浓度为1：100,购自Invitrogen公司,室温孵育1小时.DAPI染细胞核.倒置荧光显微镜下观察切片和获取图像(Zeiss)。结果1. CA-074Me抑制蛋白酶B-NLRP3-IL-1β信号通路的活化我们证实了CA-074Me可以抑制组织蛋白酶B的活性。随后我们检测了CA-074Me治疗对血清促炎因子IL-1β和IL-18的影响。结果显示在溶剂治疗组的心肌梗死大鼠血清中上述因子较假手术组大鼠明显增加；而CA-074Me治疗组的大鼠上述因子水平较溶剂治疗组大鼠明显下降。免疫印迹结果证实溶剂治疗的大鼠心脏成熟IL-1p的表达明显上调,假手术组和CA-074Me治疗组的大鼠IL-1β无明显升高。对比假手术组大鼠,溶剂治疗组大鼠心脏NLRP3的蛋白表达水平明显升高,上述效应能被CA-074Me治疗抑制。随着细胞因子的成熟,在溶剂治疗组的心肌梗死大鼠可以观察到caspase-1的活化,p20亚单位的出现证实了caspase-1的活化,而这种效应可以被CA-074Me治疗有效抑制。2. CA-074Me治疗改善心功能基础状态,3组大鼠的心功能无差异。心肌梗死4周后CA-074Me治疗的心梗大鼠LVIDD, LVIDS, LVFS和射血分数(EF)均明显改善。3. CA-074Me治疗减少心肌梗死面积和心肌细胞面积对比溶剂治疗组的心梗大鼠,CA-074Me治疗组的心梗大鼠心肌梗死面积明显降低(22±2.8%vs38+5.4%,p<0.01)。采用横截面面积和周长的定量心肌细胞大小的结果显示CA-074Me治疗抑制心室重塑(P<0.01,n=15)。4. CA-074Me治疗减少心肌纤维化单纯溶剂治疗组的心梗大鼠非梗死区细胞外基质沉积是假手术组大鼠的7倍,CA-074Me治疗明显减少整个非梗死区的基质沉积,并与假手术组大鼠基质沉积的情况类似(0.26±0.07%versus0.19±0.07%)。结论我们的研究结果证实了抑制组织蛋白酶B可以抑制NLRP3-IL-1β信号通路的活化从而抑制炎症小体活化,抑制促炎因子释放,改善心功能和抑制心脏纤维化。此外,研究证实复杂的信号通路参与了心肌细胞肥大,我们的研究结果显示了抑制组织蛋白酶B可以抑制心肌细胞肥大,从而证实了NLRP3-IL-1β信号通路在心脏收缩和功能的有害作用。更多还原

【Abstract】 Objective:This study aimed to elucidate the effects of a specific cathepsin B inhibitor CA-074Me on cardiac dysfunction and remodeling as well as fibrosis following myocardial infarction using an MI rat model, and to investigate its potential mechanisms of action.Methods:1) Induction of Rat MIMale Sprague-Dawley rats were purchased from Vital River Laboratories (Beijing, China). Animals were housed and maintained under standard conditions in our experimental animal center. All experiments conformed to the Guide for the Care and Use of Laboratory Animals by our Institute. MI was induced in male10-week old rats (weighing280-320g) by ligating the left anterior descending coronary artery as previously described. Briefly, anaesthesia with isoflurane (5%induction,2%maintenance) preceded intubation and ventilation. Adequacy of anaesthesia was monitored by loss of reflexes, and degree of muscle relaxation. Left-sided thoracotomy was performed between the fifth and sixth ribs. The pericardium was opened, and the heart exteriorized. The coronary artery was localized1-2mm below the junction of the pulmonary conus and the left atrial appendage. A5.0silk suture was used to permanently constrict the artery from the left border of the pulmonary conus to the right border of the left atrial appendage. The heart was returned to the chest, and lungs were re-expanded before closing the chest wall with4.0silk. Sham animals underwent every procedure except coronary artery ligation.2) Treatment Stock solutions of the cathepsin B inhibitor CA-074Me were made at a concentration of10mg/ml in dimethyl sulfoxide (DMSO). This was diluted1:10in saline and administered at a dose of10mg/kg by intraperitoneal injection according to previously validated protocols. All rats were randomly assigned to3groups. Group I (n=10):sham-operated rats as the normal control; Group II:MI rats with vehicle (10%DMSO)(n=15) for4weeks; Group III:MI rats with CA-074Me treatment at a dosage of10mg/kg/day (n=15) for4weeks.3) Cathepsin B activity assayCathepsin B activity was measured using the synthetic fluorometric substrate Z-Arg-Arg-NHMec. Briefly,150μM Z-Arg-Arg-NHMec (pH6.0) was added to the assay buffer and fluorescence was measured in triplicate, at one-minute intervals for30minutes, at an excitation of360nm and an emission of465nm. Data are represented as relative fluorescent units and presented as mean±standard deviation (SD) of three independent experiments.4) Western blotTotal protein extracts were prepared by homogenizing heart tissues in lysis buffer (Cell Signaling Technology) and stored at-20C. The protein content was determined using the bicinchoninic assay method (Pierce). Equal amounts of protein were boiled with SDS buffer, loaded on8-12%denaturing polyacrylamide gels, blotted onto PVDF membrane. After blocking with5%skimmed milk, specific primary antibodies were used as follows:anti-NLRP3(1:2000; BD); anti-caspase-lp20(1:2000), anti-pro-IL-1β/IL-1β (1:2000), anti-pro-IL-18/IL-18(1:2000)(all from Cell Signaling Technology). Immunoblots were visualized by enhanced chemiluminescence, and quantified for specific protein content by densitometry with normalization for housekeeping gene β-actin (1:2000; Cell Signaling Technology).5) ELISASerum IL-1β and IL-18levels were determined by ELISA kits (R&D Systems) according to the manufacturer’s instructions.6) Cardiac function assessed by echocardiographyCardiac function of all rats was evaluated by noninvasive echocardiography. Briefly, images were recorded using a10-12MHz phased-array transducer (model21380A with HP SONOS5500imaging system; Agilent Technologies). Diastolic and systolic LV internal dimensions (LVIDD and LVIDS) and LV fractional shortening (LVFS) were measured with M-mode tracings from the short-axis view of the LV at the papillary muscle level. All measurements were performed in a blinded fashion according to the guidelines of the American Society for Echocardiology and averaged over3consecutive cardiac cycles. All data were acquired and analyzed by a single blinded observer using EchoPAC (GE Vingmed) off-line processing.7) Infarct sizeThe Masson’s trichrome-stained slides were examined under light microscopy and digitized, then analyzed using image analysis (AIS, Analytical imaging Station Version6.0, Ontario, Canada). Infarct size was assessed morphologically and calculated as the ratio of scar average circumferences of the endocardium and the epicardium to LV average circumferences of the endocardium and the epicardium.8) Extracellular matrix depositionSections were stained with Masson’s Trichrome to examine extracellular matrix (ECM) deposition as previously described. All tissues were assessed with the examiner masked to the experimental groups. The accumulation of matrix within the non-infarct zone (NIZ) was then quantified. Briefly, stained sections from the mid left ventricle were digitally captured in their entirety with a standard polarizing filter, and then loaded onto a Pentium III IBM computer. To isolate the NIZ from the infarct and the peri-infarct zone, the infarct and a2mm zone on either side of it were excluded from analysis. The remaining myocardium composed the NIZ, and was analyzed using computer-assisted image analysis using image analysis software (Analytical imaging Station Version6.0, Canada). The whole NIZ was used for quantification of ECM in order to prevent possible bias from using selected fields. An area of blue on a trichrome-stained section, representing ECM, was selected for its color range. For sham animals, the ECM content of the entire LV was quantitated by the same method, as described above.9) Histological and immunofluorescence analysis Rat hearts were collected at4weeks post-MI fixed with buffered10%PFA for1h, followed by1h incubation in0.1M glycine and overnight incubation at4℃in0.6M sucrose. Samples were embedded in OCT and sections (4μm) were cut and stored in-20℃. Sections for Masson-trichrome were processed as per manufacturer instructions (Sigma).In a separate set of IHC experiments, rat heart sections were permeabilized and blocked for1h in1%BSA and probed with primary polyclonal antibodies to a-sarcomeric actin (1:50; Sigma), wheat germ hemagglutinin (WGA)(1:50), and anti-His (1:50)(both from Invitrogen). Secondary antibodies (1:100; Invitrogen) were used at the recommended dilution and incubated on sections for1h at room temperature. Nuclei counterstained with (DAPI). Images were acquired on an inverted epifluorescent microscope (Zeiss).Results:1. CA-074Me inhibited activation of cathepsin B-NLRP3-IL-1β pathwayFirst of all, we confirmed that cathepsin B activity was inhibited by CA-074Me treatment. Further, we examined whether CA-074Me treatment affected serum levels of the proinflammatory cytokines IL-1β and IL-18by ELISA. A marked increase in serum levels of these cytokines was seen in vehicle-treated rats compared to the low baseline levels observed in sham control rats, and levels in CA-074Me-treated rats were significantly lower than those in vehicle-treated animals.As demonstrated by Western blot analysis, increasing amounts of mature IL-1β were observed in lysates of vehicle-treated hearts, but not CA-074Me-treated or sham control hearts. We then measured whether inflammasome activation was suppressed by CA-074Me administration. We then measured NLRP3protein levels in heart tissues using. In vehicle-treated rats, levels of NLRP3protein were significantly increased compared to sham controls and these effects were inhibited by CA-074Me treatment. Consistent with cytokine maturation, caspase-1activation was also seen in vehicle-treated hearts, as demonstrated by the appearance of the p20subunit, and this effect was significantly inhibited by CA-074Me treatment.2. Treatment with CA-074Me improves cardiac function Echocardiography was used to examine cardiac structure and function in rats. At baseline, no differences between the3groups were observed. CA-074Me treatment for4weeks contributed to significant improvements in LVIDD, LVIDS, LVFS and ejection fraction (EF).3. Infarct size and LV cardiomyocyte size were reduced by CA-074MeTreatment with CA-074Me significantly reduced the infarct size compared with vehicle-treated MI groups (22±2.8%vs38±5.4%, p<0.01). The cardiomyocyte size was assessed using high magnification microscopy to quantify circumference and the cross-sectional area. These data showed that treatment with CA-074Me prevented cardiac remodeling when compared with vehicle and sham (P<0.01for all comparisons, n=15).4. CA-074Me treatment reduced cardiac fibrosisThe extent of cardiac fibrosis was evaluated by Masson-trichrome staining at4weeks post-MI. On trichrome-stained sections, ECM deposition in the NIZ was more than sevenfold higher in MI animals compared with sham animals that were especially pronounced in the subendocardial region of the NIZ. Treatment with CA-074Me resulted in a reduction in ECM throughout the NIZ to levels similar to those seen in sham animals. Non-infarcted animals treated with CA-074Me had similar levels of ECM compared to the untreated sham-operated animals (0.26±0.07%versus0.19±0.07%, respectively).Conclusion:Our study provides additional data that inhibition of cathepsin B induced a significant decrease in NLRP3-IL-1β activation, resulting in improved cardiac function and less fibrosis. In addition, complex signaling pathways are involved in myocyte hypertrophy, and our findings showed that cardiomyocyte size was as also attenuated by such inhibition, which supports an adverse effect of the cathepsin B-NLRP3-IL-1β pathway on cardiac contractility and function.更多还原