白桦脂醇对受损心肌细胞的保护作用

发布时间：2018-05-25 09:56

本文选题：白桦脂醇 + 缺血再灌注损伤　；参考：《山东大学》2014年博士论文

【摘要】：心肌缺血再灌注损伤(MIRI)是指心肌血流中断或下降一段时间后恢复血流供应,造成功能障碍、结构破坏进一步加重的现象。其发生机制复杂,包括氧自由基、钙超载、炎症反应、细胞凋亡和线粒体功能障碍等,而不同路径又通过各种细胞因子形成网状交互作用,共同加重细胞损伤。近年来,心肌炎症反应在心肌缺血再灌注损伤发生过程中的作用越来越受到重视。多种因素或应激都可使心肌表达并分泌促炎性细胞因子,如肿瘤坏死因子-α(TNF-a),白细胞介素1(IL-1),白细胞介素6(IL-6),单核细胞趋化蛋白-1(MCP-1)、细胞粘附分子-1(ICAM-1)等,这些细胞因子通常会导致NF-κB信号传导途径的激活。核因子κB (NF-κB)是哺乳动物细胞内的炎症调控因子。在炎症反应过程中,NF-κB被激活后从细胞浆易位至细胞核内与特异性的DNA结合,继而诱导其下游多种炎症介质基因的表达,导致炎症反应加重,增加心肌损伤。因此,NF-κB信号传导途径的调控对于心肌细胞炎症反应有重要的意义。信号转导及转录激活因子3(STAT3)广泛参与了细胞应激、生长、增殖、分化和凋亡等多种生物学效应。多项研究表明,STAT3参与多种心脏生理、病理活动,如心肌细胞存活、心肌血管再生、线粒体能量代谢、细胞外基质变化及炎症反应等。STAT3的缺乏会引起心肌细胞炎症和心肌纤维化,增加心功能衰竭的发生。白桦脂醇(betulin)是从白桦树皮提取的一种天然有机化学药物,可以转化为白桦脂酸,具有多种生物活性如抗炎、抗菌、抗病毒、抗肿瘤、抗HIV等作用,引起了人们极大的研究兴趣。近期研究还发现白桦酯醇能通过抑制胆固醇调节元件结合蛋白(SREBP)同时降低胆固醇和三酰甘油,改善肥胖患者的高脂血症和胰岛素抵抗,对2型糖尿病的治疗效果明显。但目前对白桦脂醇对心肌细胞损伤的作用还缺乏研究。本研究的目的是探讨白桦脂醇对损伤心肌细胞的保护作用及其信号机制。鉴于炎症反应在心肌缺血再灌注损伤中的重要作用,课题主要集中于两部分实验。第一部分构建大鼠离体心脏灌流模型,探讨白桦脂醇预处理对缺血再灌注后心肌细胞炎症的抵抗作用；第二部分以人源心肌细胞AC16为实验对象,从炎症反应的角度探讨白桦脂醇对心肌细胞的保护作用及其分子信号机制。第一部分白桦脂醇抵抗心肌细胞炎症的作用目的探讨白桦脂醇预处理对大鼠缺血再灌注后心肌细胞炎症的抵抗作用。方法将50只大鼠随机分为5组,每组10只。麻醉后快速游离出心脏,置于Langendorff灌流架上,经主动脉插管进行灌流。①假缺血再灌注组(Sham):心脏用K-H液持续灌流120min。②缺血再灌注模型组(Model):心脏用K-H液平衡灌流50min后,停灌30min再重新灌流K-H液40min。③三个不同浓度的白桦脂醇(Betulin:25/50/100mg/L)预处理组：心脏用K-H液平衡灌流10—min后,分别用含有相应浓度白桦脂醇的K-H液灌流5min后,再用不含白桦脂醇的K-H液灌5min,如此反复,共4次,以后的处理同Model组。灌流结束后从心尖部剪下一小块心肌组织,立即置于10%的多聚甲醛溶液置于4℃冰箱中,后续进行心肌组织病理HE染色、TTC染色测定、TUNEL法检测细胞凋亡和免疫组化测定TNF-α、ICAM-1和NF-κB表达水平；取下剩余的大部分左心室心肌组织剔除结缔组织,制做成心肌匀浆,放入-20℃保存,采用相应试剂盒检测LDH、CK和MPO活力等生化指标。结果 1、Model组大鼠心肌组织中CK的活力为14.29±2.08kU/g蛋白,LDH活力为1027±66U/g蛋白,均较Sham组明显降低(P0.01)；Betulin组可以浓度依赖性抑制I/R损伤后大鼠心肌组织中CK和LDH漏出入血,尤以Betulin100mg/L组作用更为明显,CK和LDH活性较Model组明显增高(P0.01),为22.42±4-1.44kU/g蛋白和1691±79U/g蛋白。 2、与Sham组相比,Model组出现大面积的心肌梗死(IS/AAR%=42.13±6.27),白桦脂醇100mg/L预处理后心肌梗死的面积(IS/AAR%=18.54±4.92)较Model组明显减小(P0.01),心肌损伤减轻。 3、心肌组织HE染色镜下可见Model组中大鼠心肌纤维出现明显的波纹状变和心肌收缩带,部分纤维断裂、溶解；Betulin组损伤变化较轻,肌原纤维排列基本整齐,肌节基本完整,没有收缩带和波纹状变等特征性变化。 4、与Sham组相比,Model组TUNEL染色最强,而经过白桦脂醇预处理后,TUNEL染色强度呈现剂量依赖性减弱,表明白桦脂醇可显著抑制心肌I/R引起的细胞凋亡。 5、Model组炎症因子NF-κB、TNF-α和ICAM-1的表达明显增加,阳性反应的平均灰度值较Sham组增高显著(P0.01)；和Model相比,Betulin组NF-κB、 TNF-a和ICAM-1的表达均有所降低,以100mg/L组作用更加明显(P0.01)。 6、Sham组大鼠心肌组织匀浆中MPO的活力很低(0.15±0.007U/g蛋白),而Model组的MPO活力(0.87±0.012U/g蛋白)较Sham组明显增高(P0.01)；而各Betulin组心肌组织中MPO的活力较Model降低(P0.05)。结论白桦脂醇预处理可以减轻大鼠心肌细胞炎症反应,保护心肌组织缺血再灌注损伤。目的探讨白桦脂醇改善人源心肌细胞AC16的炎症反应及分子信号机制。方法 ACl6细胞是购自ATCC从成人心室肌细胞衍生而来的细胞株,以低糖DMEM培养基培养。细胞生长融合后分瓶,漂洗、离心出细胞悬液,分装于培养板中。当细胞密度达到70%-80%时进行分组实验处理,然后收集细胞上清和细胞,于-80℃冰箱冻存检测。 1、给予空白对照和白桦脂醇4小时后,加入TNF-α赋予,收集细胞上清,通过EILSA实验检测IL-6和MCP-1的蛋白浓度,收取细胞RNA并逆转录后进行Real-time PCR检测IL-6、MCP-1、IL-1β等炎症基因的表达。 2、在AC16细胞中转染NF-κB报告基因质粒,并给予TNF-a和白桦脂醇刺激。用荧光素酶报告和p65抗体ChIP实验对NF-κB信号传导的基因表达进行分析,以评估白桦脂醇对NF-κB在AC16细胞中的转录活性的影响。 3、白桦脂醇孵育AC16细胞后,对STAT3的磷酸化和其下游靶基因SOCS3、 BCL-xL的水平进行了检测,以评估STAT3的活化。 4、通过加入STAT3通路的抑制剂AG490和小RNA干扰片段,以检测STAT3通路是否是白桦脂醇的抗炎效应所必须的。结果 1、TNF-α能显著提高AC16细胞中IL-6,MCP-1和IL-lβ的mRNA表达水平,而白桦脂醇能显著抑制TNF-a所诱导的该炎症基因的表达,细胞中IL-6和MCP-1的蛋白浓度也低于对照组。 2、TNF-α能显著激活NF-κB报告基因质粒的转录活性,促进P65在核内的积聚、磷酸化和乙酰化,而白桦脂醇则显著抑制TNF-a的作用,阻断NF-κB信号在ACl6细胞内的活化。 3、白桦脂醇孵育AC16细胞后,细胞内STAT3磷酸化增强,下游基因-SOCS3和BCL-xL基因的表达显著增强,证实该过程中存在STAT3通路的激活。 4、给予STAT3抑制剂AG490后,白桦脂醇对促炎细胞因子IL-6和MCP-1的表达抑制作用减弱；以小RNA干扰片段敲除细胞内源性STAT3的表达后,白桦脂醇的保护作用发生逆转,证明提示白桦脂醇的心肌保护作用依赖于STAT3通路的激活。结论白桦脂醇通过激活STAT3信号通路,抑制AC16心肌细胞炎症通路NF-κB和炎症相关基因的表达。
[Abstract]:Myocardial ischemia and reperfusion injury (MIRI) refers to the recovery of blood flow supply after the interruption or decline of myocardial blood flow, resulting in dysfunction and further aggravation of structural damage. Its mechanism is complex, including oxygen free radicals, calcium overload, inflammatory reaction, cell apoptosis and mitochondrial dysfunction, while different pathways pass through various cellular causes. In recent years, many factors or stress can make myocardium express and secrete proinflammatory cytokines, such as TNF-a, interleukin 1 (IL-1), interleukin and interleukin Element 6 (IL-6), monocyte chemoattractant protein -1 (MCP-1), cell adhesion molecule -1 (ICAM-1), etc. these cytokines usually lead to activation of NF- kappa B signal transduction pathway.
Nuclear factor kappa B (NF- kappa B) is an inflammatory regulator within mammalian cells. In the process of inflammation, NF- kappa B is activated from cytoplasm to the nucleus with specific DNA, and then induces the expression of a variety of inflammatory mediators in the lower reaches of the cell, resulting in heavy inflammatory response and increased myocardial damage. Therefore, the NF- kappa B signal transduction pathway Regulation plays an important role in the inflammatory response of cardiac myocytes.
Signal transduction and transcription activator 3 (STAT3) are widely involved in many biological effects, such as cell stress, growth, proliferation, differentiation and apoptosis. A number of studies have shown that STAT3 participates in a variety of cardiac physiology and pathological activities, such as myocardial cell survival, myocardial angiogenesis, mitochondrial energy metabolism, extracellular matrix changes and inflammatory responses, such as.STAT3 deficiency. Deficiency can cause myocardial cell inflammation and myocardial fibrosis, and increase the occurrence of heart failure.
Betula alba (betulin) is a natural organic chemical extracted from the bark of Betula platyphylla. It can be converted to Betula alba. It has many biological activities, such as anti-inflammatory, antibacterial, antiviral, anti-tumor, anti HIV and so on. It has aroused great interest in research. Recent research also found that Betula aldiol can combine the cholesterol regulating element to combine the egg with the egg. White (SREBP) reduces cholesterol and three glycerol at the same time, improves hyperlipidemia and insulin resistance in obese patients, and has a significant effect on type 2 diabetes. However, the effect of Betula lipol on myocardial injury is still lacking.
The purpose of this study is to explore the protective effect of Betula platyphylol on damaged myocardial cells and its signal mechanism. In view of the important role of inflammatory reaction in myocardial ischemia reperfusion injury, the main focus of this topic is on the two part of the experiment. The resistance of myocyte inflammation; in the second part, the human cardiac myocyte AC16 was used as the experimental object to investigate the protective effect of Betula lipol on myocardial cells and its molecular signal mechanism from the angle of inflammatory reaction.
Part 1 Betula platyphylla resistance to inflammation of cardiac myocytes
objective
Objective to investigate the resistance of Betula platyphylla preconditioning to inflammation of myocardial cells after ischemia-reperfusion in rats.
Method
50 rats were randomly divided into 5 groups, 10 rats in each group. After anesthesia, the heart was quickly dissociated and placed on the Langendorff perfusion frame and perfusion through the aortic cannula. (1) false ischemia reperfusion group (Sham): K-H fluid continuous perfusion 120min. (Model) in the model group of ischemia reperfusion (Model): after the cardiac K-H fluid was perfused with 50min, 30min was reused again to reflow K- H solution 40min. (three different concentrations of Betula platyphylol (Betulin:25/50/100mg/L) pretreatment group: after the heart use K-H liquid balanced perfusion of 10 to min, respectively, using K-H liquid containing the corresponding concentration of Betula platyphylol, respectively, after 5min, then reused the K-H solution without Betula aliphatic alcohol 5min, so repeated, a total of 4 times, after the treatment of the same Model group.
After perfusion, a small block of myocardial tissue was cut from the apex of the heart and 10% of the poly Formaldehyde Solution was placed in the refrigerator at 4. The subsequent myocardial histopathological HE staining, TTC staining, and TUNEL assay were used to detect TNF- alpha, ICAM-1 and NF- kappa B, and the remaining left ventricular myocytes were removed. In addition to connective tissue, cardiac homogenate was prepared and stored at -20 C, and the biochemical indicators such as LDH, CK and MPO activity were detected by corresponding kit.
Result
1, the activity of CK in the myocardium of the Model group was 14.29 + 2.08kU/g protein, and the activity of LDH was 1027 + 66U/g protein, which was significantly lower than that in the Sham group (P0.01). The Betulin group could inhibit the CK and LDH in the myocardium of rats after the concentration dependent inhibition of I/R injury, especially in the Betulin100mg/L group. High (P0.01) was 22.42 + 4-1.44kU/g protein and 1691 + 79U/g protein.
2, compared with the Sham group, there was a large area of myocardial infarction in group Model (IS/AAR%=42.13 + 6.27). The area of myocardial infarction (IS/AAR%=18.54 + 4.92) after Betula lipol 100mg/L preconditioning was significantly lower than that in the Model group (P0.01), and the myocardial injury was reduced.
3, under the HE staining microscope, there was obvious ripple change and myocardial contractile zone in group Model, some fibers were broken and dissolved, and the damage of group Betulin was lighter, myofibrils were arranged basically and neatly, the myofibrils were basically complete, without characteristic changes such as contraction band and ripple change.
4, compared with the Sham group, the TUNEL staining in group Model was the strongest, but after Betula platyphylol pretreatment, the intensity of TUNEL staining showed a dose-dependent decrease, indicating that Betula lipol significantly inhibited the apoptosis induced by I/R in the myocardium.
5, the expression of NF- kappa B, TNF- alpha and ICAM-1 increased significantly in the Model group, and the average gray value of the positive reaction was significantly higher than that in the Sham group (P0.01). Compared with Model, the Betulin group NF- kappa B, TNF-a and the expressions were reduced.
6, the activity of MPO in the myocardial homogenate of the Sham group was very low (0.15 + 0.007U/g protein), while the MPO activity (0.87 + 0.012U/g) in the group Model was significantly higher than that in the Sham group (P0.01), while the activity of MPO in the myocardium tissues of each group was lower than that of Model (P0.05).
conclusion
Betula alcohol pretreatment can reduce the inflammatory reaction of myocardial cells and protect myocardial ischemia reperfusion injury in rats.
objective
Objective to investigate the effects of Betula alcohol on the inflammatory response and molecular signaling mechanism of human cardiomyocytes AC16.
Method
ACl6 cells are derived from ATCC cells derived from adult ventricular myocytes and are cultured in a low sugar DMEM medium. Cells grow and fuse in a bottle, rinse, and centrifuge the cell suspension. When the cell density reaches 70%-80%, the cells are divided into groups, then the cell supernatant and cells are collected and stored at -80 at the temperature of the refrigerator.
1, after giving blank control and Betula Alba for 4 hours, TNF- alpha was added to the cell supernatant, the protein concentration of IL-6 and MCP-1 was detected by EILSA, and RNA was collected and Real-time PCR was used to detect the expression of IL-6, MCP-1, IL-1 beta and other inflammatory genes.
2, NF- kappa B was transfected in AC16 cells to report gene plasmids and stimulated by TNF-a and Betula lipol. The gene expression of NF- kappa B signal transduction was analyzed with Luciferase Report and p65 antibody ChIP test to evaluate the effect of Betula lipol on the transcriptional activity of NF- kappa B in AC16 cells.
3, after the AC16 cells were incubated with Betula platyphylla, the phosphorylation of STAT3 and the downstream target genes SOCS3 and BCL-xL levels were detected to evaluate the activation of STAT3.
4, by adding inhibitors AG490 and small RNA interference fragments of STAT3 pathway, it is necessary to detect whether STAT3 pathway is the anti-inflammatory effect of Betula.
Result
1, TNF- alpha could significantly increase the level of mRNA expression of IL-6, MCP-1 and IL-l beta in AC16 cells, while Betula Alba significantly inhibited the expression of the inflammatory gene induced by TNF-a, and the protein concentration of IL-6 and MCP-1 in the cells was also lower than that of the control group.
2, TNF- alpha significantly activates the transcriptional activity of the NF- kappa B reporter gene plasmid, promotes the accumulation, phosphorylation and acetylation of P65 in the nucleus, while Betula Alba significantly inhibits the effect of TNF-a and blocks the activation of NF- kappa B signal in ACl6 cells.
3, after Betula platyphylol incubates AC16 cells, the STAT3 phosphorylation in cells is enhanced and the expression of -SOCS3 and BCL-xL genes in the downstream genes are significantly enhanced. It is proved that the activation of STAT3 pathway exists in the process.
4, after the STAT3 inhibitor AG4 90, the inhibitory effect of Betula lipol on the expression of pro-inflammatory cytokines IL-6 and MCP-1 was weakened, and the protective effect of Betula lipol was reversed after the expression of endogenous STAT3 in the small RNA interfering fragment. It was suggested that the myocardial protection of Betula lipol was dependent on the activation of STAT3 pathway.
conclusion
Betula alcohol inhibited the expression of NF- B and inflammation related genes in inflammatory cells of AC16 cardiomyocytes by activating STAT3 signaling pathway.
【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R542.2

【共引文献】