本文選題：白樺脂醇 + 缺血再灌注損傷��； 參考：《山東大學》2014年博士論文

【摘要】：心肌缺血再灌注損傷(MIRI)是指心肌血流中斷或下降一段時間后恢復血流供應,造成功能障礙、結(jié)構破壞進一步加重的現(xiàn)象。其發(fā)生機制復雜,包括氧自由基、鈣超載、炎癥反應、細胞凋亡和線粒體功能障礙等,而不同路徑又通過各種細胞因子形成網(wǎng)狀交互作用,共同加重細胞損傷。近年來,心肌炎癥反應在心肌缺血再灌注損傷發(fā)生過程中的作用越來越受到重視。多種因素或應激都可使心肌表達并分泌促炎性細胞因子,如腫瘤壞死因子-α(TNF-a),白細胞介素1(IL-1),白細胞介素6(IL-6),單核細胞趨化蛋白-1(MCP-1)、細胞粘附分子-1(ICAM-1)等,這些細胞因子通常會導致NF-κB信號傳導途徑的激活。 核因子κB (NF-κB)是哺乳動物細胞內(nèi)的炎癥調(diào)控因子。在炎癥反應過程中,NF-κB被激活后從細胞漿易位至細胞核內(nèi)與特異性的DNA結(jié)合,繼而誘導其下游多種炎癥介質(zhì)基因的表達,導致炎癥反應加重,增加心肌損傷。因此,NF-κB信號傳導途徑的調(diào)控對于心肌細胞炎癥反應有重要的意義。 信號轉(zhuǎn)導及轉(zhuǎn)錄激活因子3(STAT3)廣泛參與了細胞應激、生長、增殖、分化和凋亡等多種生物學效應。多項研究表明,STAT3參與多種心臟生理、病理活動,如心肌細胞存活、心肌血管再生、線粒體能量代謝、細胞外基質(zhì)變化及炎癥反應等。STAT3的缺乏會引起心肌細胞炎癥和心肌纖維化,增加心功能衰竭的發(fā)生。 白樺脂醇(betulin)是從白樺樹皮提取的一種天然有機化學藥物,可以轉(zhuǎn)化為白樺脂酸,具有多種生物活性如抗炎、抗菌、抗病毒、抗腫瘤、抗HIV等作用,引起了人們極大的研究興趣。近期研究還發(fā)現(xiàn)白樺酯醇能通過抑制膽固醇調(diào)節(jié)元件結(jié)合蛋白(SREBP)同時降低膽固醇和三酰甘油,改善肥胖患者的高脂血癥和胰島素抵抗,對2型糖尿病的治療效果明顯。但目前對白樺脂醇對心肌細胞損傷的作用還缺乏研究。 本研究的目的是探討白樺脂醇對損傷心肌細胞的保護作用及其信號機制。鑒于炎癥反應在心肌缺血再灌注損傷中的重要作用,課題主要集中于兩部分實驗。第一部分構建大鼠離體心臟灌流模型,探討白樺脂醇預處理對缺血再灌注后心肌細胞炎癥的抵抗作用；第二部分以人源心肌細胞AC16為實驗對象,從炎癥反應的角度探討白樺脂醇對心肌細胞的保護作用及其分子信號機制。 第一部分白樺脂醇抵抗心肌細胞炎癥的作用 目的 探討白樺脂醇預處理對大鼠缺血再灌注后心肌細胞炎癥的抵抗作用。 方法 將50只大鼠隨機分為5組,每組10只。麻醉后快速游離出心臟,置于Langendorff灌流架上,經(jīng)主動脈插管進行灌流。①假缺血再灌注組(Sham):心臟用K-H液持續(xù)灌流120min。②缺血再灌注模型組(Model):心臟用K-H液平衡灌流50min后,停灌30min再重新灌流K-H液40min。③三個不同濃度的白樺脂醇(Betulin:25/50/100mg/L)預處理組：心臟用K-H液平衡灌流10—min后,分別用含有相應濃度白樺脂醇的K-H液灌流5min后,再用不含白樺脂醇的K-H液灌5min,如此反復,共4次,以后的處理同Model組。 灌流結(jié)束后從心尖部剪下一小塊心肌組織,立即置于10%的多聚甲醛溶液置于4℃冰箱中,后續(xù)進行心肌組織病理HE染色、TTC染色測定、TUNEL法檢測細胞凋亡和免疫組化測定TNF-α、ICAM-1和NF-κB表達水平；取下剩余的大部分左心室心肌組織剔除結(jié)締組織,制做成心肌勻漿,放入-20℃保存,采用相應試劑盒檢測LDH、CK和MPO活力等生化指標。 結(jié)果 1、Model組大鼠心肌組織中CK的活力為14.29±2.08kU/g蛋白,LDH活力為1027±66U/g蛋白,均較Sham組明顯降低(P0.01)；Betulin組可以濃度依賴性抑制I/R損傷后大鼠心肌組織中CK和LDH漏出入血,尤以Betulin100mg/L組作用更為明顯,CK和LDH活性較Model組明顯增高(P0.01),為22.42±4-1.44kU/g蛋白和1691±79U/g蛋白。 2、與Sham組相比,Model組出現(xiàn)大面積的心肌梗死(IS/AAR%=42.13±6.27),白樺脂醇100mg/L預處理后心肌梗死的面積(IS/AAR%=18.54±4.92)較Model組明顯減小(P0.01),心肌損傷減輕。 3、心肌組織HE染色鏡下可見Model組中大鼠心肌纖維出現(xiàn)明顯的波紋狀變和心肌收縮帶,部分纖維斷裂、溶解；Betulin組損傷變化較輕,肌原纖維排列基本整齊,肌節(jié)基本完整,沒有收縮帶和波紋狀變等特征性變化。 4、與Sham組相比,Model組TUNEL染色最強,而經(jīng)過白樺脂醇預處理后,TUNEL染色強度呈現(xiàn)劑量依賴性減弱,表明白樺脂醇可顯著抑制心肌I/R引起的細胞凋亡。 5、Model組炎癥因子NF-κB、TNF-α和ICAM-1的表達明顯增加,陽性反應的平均灰度值較Sham組增高顯著(P0.01)；和Model相比,Betulin組NF-κB、 TNF-a和ICAM-1的表達均有所降低,以100mg/L組作用更加明顯(P0.01)。 6、Sham組大鼠心肌組織勻漿中MPO的活力很低(0.15±0.007U/g蛋白),而Model組的MPO活力(0.87±0.012U/g蛋白)較Sham組明顯增高(P0.01)；而各Betulin組心肌組織中MPO的活力較Model降低(P0.05)。 結(jié)論 白樺脂醇預處理可以減輕大鼠心肌細胞炎癥反應,保護心肌組織缺血再灌注損傷。 目的 探討白樺脂醇改善人源心肌細胞AC16的炎癥反應及分子信號機制。 方法 ACl6細胞是購自ATCC從成人心室肌細胞衍生而來的細胞株,以低糖DMEM培養(yǎng)基培養(yǎng)。細胞生長融合后分瓶,漂洗、離心出細胞懸液,分裝于培養(yǎng)板中。當細胞密度達到70%-80%時進行分組實驗處理,然后收集細胞上清和細胞,于-80℃冰箱凍存檢測。 1、給予空白對照和白樺脂醇4小時后,加入TNF-α賦予,收集細胞上清,通過EILSA實驗檢測IL-6和MCP-1的蛋白濃度,收取細胞RNA并逆轉(zhuǎn)錄后進行Real-time PCR檢測IL-6、MCP-1、IL-1β等炎癥基因的表達。 2、在AC16細胞中轉(zhuǎn)染NF-κB報告基因質(zhì)粒,并給予TNF-a和白樺脂醇刺激。用熒光素酶報告和p65抗體ChIP實驗對NF-κB信號傳導的基因表達進行分析,以評估白樺脂醇對NF-κB在AC16細胞中的轉(zhuǎn)錄活性的影響。 3、白樺脂醇孵育AC16細胞后,對STAT3的磷酸化和其下游靶基因SOCS3、 BCL-xL的水平進行了檢測,以評估STAT3的活化。 4、通過加入STAT3通路的抑制劑AG490和小RNA干擾片段,以檢測STAT3通路是否是白樺脂醇的抗炎效應所必須的。 結(jié)果 1、TNF-α能顯著提高AC16細胞中IL-6,MCP-1和IL-lβ的mRNA表達水平,而白樺脂醇能顯著抑制TNF-a所誘導的該炎癥基因的表達,細胞中IL-6和MCP-1的蛋白濃度也低于對照組。 2、TNF-α能顯著激活NF-κB報告基因質(zhì)粒的轉(zhuǎn)錄活性,促進P65在核內(nèi)的積聚、磷酸化和乙�；�,而白樺脂醇則顯著抑制TNF-a的作用,阻斷NF-κB信號在ACl6細胞內(nèi)的活化。 3、白樺脂醇孵育AC16細胞后,細胞內(nèi)STAT3磷酸化增強,下游基因-SOCS3和BCL-xL基因的表達顯著增強,證實該過程中存在STAT3通路的激活。 4、給予STAT3抑制劑AG490后,白樺脂醇對促炎細胞因子IL-6和MCP-1的表達抑制作用減弱；以小RNA干擾片段敲除細胞內(nèi)源性STAT3的表達后,白樺脂醇的保護作用發(fā)生逆轉(zhuǎn),證明提示白樺脂醇的心肌保護作用依賴于STAT3通路的激活。 結(jié)論 白樺脂醇通過激活STAT3信號通路,抑制AC16心肌細胞炎癥通路NF-κB和炎癥相關基因的表達。
[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.
【學位授予單位】：山東大學
【學位級別】：博士
【學位授予年份】：2014
【分類號】：R542.2

【共引文獻】

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2 孫妍;韓秀珍;;心肌營養(yǎng)素-1與心肌保護[J];國際兒科學雜志;2006年03期

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5 何寧;歐陽元明;范存義;;血管緊張素Ⅱ與慢性炎癥性關節(jié)粘連[J];國際骨科學雜志;2014年02期

6 文亦磊;葉星江;;核因子-κB及Bcl-2在胃癌組織中的表達及意義[J];廣東醫(yī)學;2014年11期

7 張新軍,章茂順;心肌炎癥反應與心室重構[J];華西醫(yī)學;2005年04期

8 薛強;劉作金;楊慷;涂兵;丁雄;;N-乙酰半胱氨酸對急性梗阻性化膿性膽管炎時內(nèi)毒素性急性肝損傷的保護作用[J];解放軍醫(yī)學雜志;2012年06期

9 吳英;王力寧;李艷濃;張揚;;高糖培養(yǎng)下系膜細胞JAK_2/STAT_3信號轉(zhuǎn)導通路活性變化及與活性氧簇作用的研究[J];中國中西醫(yī)結(jié)合腎病雜志;2009年05期

10 劉廣鵬;黃山虎;劉志禮;羅，

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