本文選題：丹酚酸B　切入點(diǎn)：心肌損傷　出處：《南京中醫(yī)藥大學(xué)》2017年碩士論文　論文類(lèi)型：學(xué)位論文

【摘要】：研究目的:觀(guān)察丹酚酸B(salvianolic acid B,Sal B)對(duì)大鼠心肌缺血損傷的保護(hù)作用。從NLRP3炎癥小體角度出發(fā)探討丹酚酸B保護(hù)損傷心肌細(xì)胞的可能機(jī)制。為靶向炎癥小體進(jìn)行疾病的治療及指導(dǎo)臨床提供重要的參考。研究方法:研究共分為三章:第一章、異丙腎腺素(isoproterenol,ISO)誘導(dǎo)Sprague-Dawley(SD)大鼠復(fù)制心肌缺血損傷模型。通過(guò)心電圖中T-波的變化值,大鼠血清心肌酶(CK、GOT和LDH)、氧化指標(biāo)(MDA和T-SOD)和炎癥因子IL-1β含量,心肌組織HE和TUNEL染色研究Sal B對(duì)缺血損傷心肌的保護(hù)作用;進(jìn)一步應(yīng)用免疫組化、western blot和real-time PCR檢測(cè)心肌組織中炎癥小體NLRP3相關(guān)蛋白和mRNA表達(dá);探討心肌缺血損傷與NLRP3炎癥小體的相關(guān)性。第二章、建立H_2O_2誘導(dǎo)H_9C_2細(xì)胞氧化應(yīng)激損傷模型和LPS+ATP聯(lián)合誘導(dǎo)H_9C_2細(xì)胞炎性損傷模型。應(yīng)用MTT法檢測(cè)細(xì)胞存活率,試劑盒檢測(cè)細(xì)胞上清液中氧化指標(biāo)和炎癥因子的含量觀(guān)察Sal B對(duì)心肌細(xì)胞損傷的保護(hù)作用;應(yīng)用western blot和免疫熒光研究Sal B對(duì)心肌細(xì)胞損傷過(guò)程中NLRP3炎癥小體的影響;進(jìn)一步應(yīng)用TUNEL和Calciem-AM染色研究Sal B對(duì)細(xì)胞焦亡的影響。第三章、利用LPS+ATP聯(lián)合誘導(dǎo)促進(jìn)H_9C_2細(xì)胞中NLRP3炎癥小體的激活,通過(guò)探針檢測(cè)ROS和線(xiàn)粒體膜電位,western bolt分析SIRT1、p-Foxo3a和線(xiàn)粒體自噬相關(guān)蛋白,研究Sal B抑制NLRP3炎癥小體的可能機(jī)制,進(jìn)一步通過(guò)自噬抑制劑3-MA驗(yàn)證自噬在NLRP3炎癥小體激活中的作用。研究結(jié)果:第一章結(jié)果顯示:Sal B可以明顯減輕心肌組織壞死、炎細(xì)胞浸潤(rùn)和減少DNA損傷,明顯降低心電圖中T波值(P0.05,P0.01)。與模型組比較,Sal B各劑量組的CK值、GOT值和IL-1β值,Sal B中、高劑量組的MDA值及中劑量組的LDH值均明顯降低(P0.05,P0.01);而Sal B中、高劑量組的T-SOD值明顯升高(P0.05,P0.01);Sal B給藥組的心臟組織中NLRP3、ASC、caspase-1 P20和IL-1β蛋白和mRNA表達(dá)明顯降低,呈一定的劑量依賴(lài)性(P0.05,P0.01)。第二章結(jié)果顯示:經(jīng)MTT法檢測(cè),確定H_2O_2合適的造模濃度為l00μM,時(shí)間為2h;SalB合適的預(yù)處理濃度為1、5、25μM,時(shí)間為24h。與H_2O_2模型組比較,給予不同劑量的SalB可以提高細(xì)胞的存活率,保持細(xì)胞形態(tài)的穩(wěn)定性。與模型組比較,給予SalB可以明顯降低H_2O_2刺激的細(xì)胞上清液中LDH、MDA水平,升高T-SOD的水平(P0.05,P0.01),呈一定的劑量依賴(lài)性。Western bolt、免疫熒光和Elisa結(jié)果顯示,Sal B可以抑制H_9C_2細(xì)胞氧化損傷和炎性損傷中NLRP3炎癥小體的激活和IL-lβ的表達(dá)和分泌。TUNEL和Calcein-AM染色結(jié)果表明,Sal B可以保護(hù)細(xì)胞膜的完整性,減輕DNA損傷,從而抑制細(xì)胞焦亡。第三章結(jié)果顯示:在LPS+ATP聯(lián)合誘導(dǎo)作用下,Sal B可以促進(jìn)H_9C_2細(xì)胞中SIRT1、p-Foxo3a和MnSOD表達(dá)的升高(P0.05,P0.01),抑制細(xì)胞ROS和線(xiàn)粒體膜電位的升高,促進(jìn)自噬標(biāo)志蛋白LC3Ⅱ和Beclin-1以及線(xiàn)粒體自噬調(diào)控蛋白Parkin的表達(dá),抑制P62和PINK1的表達(dá)(P0.05,P0.01)。在自噬抑制劑3-MA的作用下,Sal B(51μM)促進(jìn)H_9C_2細(xì)胞線(xiàn)粒體自噬的作用被減弱,抑制NLRP3炎癥小體激活的作用也明顯被減弱。但是3-MA并沒(méi)有影響Sal B對(duì)SIRT1和p-Foxo3a的促進(jìn)作用。研究結(jié)論:1.Sal B可以抑制缺血心臟組織中NLRP3炎癥小體激活,從而減輕炎癥反應(yīng)達(dá)到保護(hù)心臟的目的。2.心肌細(xì)胞氧化應(yīng)激損傷時(shí)會(huì)誘導(dǎo)NLRP3炎癥小體的激活,丹酚酸B可以保護(hù)H_9C_2細(xì)胞氧化應(yīng)激損傷,抑制NLRP3炎癥小體的激活。3.Sal B抑制H_9C_2細(xì)胞中NLRP3炎癥小體活化的可能機(jī)制是激活SIRTl-FOXO3a-Parkin軸,改善心肌細(xì)胞線(xiàn)粒體自噬,減少受損線(xiàn)粒體的積累,從而抑制NLRP3激活信號(hào)的釋放。
[Abstract]:Study objective: To observe the effect of salvianolic acid B (salvianolic acid B, Sal B) protective effect on myocardial ischemia reperfusion injury in rats. The NLRP3 inflammasome perspective to explore the possible mechanism of salvianolic acid B to protect the damaged myocardial cells. Provide important references for the treatment of disease and guide the clinical target to inflammasome research. Methods: the study is divided into three chapters: the first chapter, isoproterenol gland hormone (isoproterenol, ISO) induced by Sprague-Dawley (SD) copy the model of myocardial ischemia reperfusion injury in rats. The changes of T- wave in ECG, rat serum myocardial enzymes (CK, GOT and LDH), oxidation index (MDA and T-SOD) the content of IL-1 and inflammatory factor beta, myocardial tissue HE and TUNEL staining to study the protective effects of Sal B on myocardial ischemia injury; the further application of immunohistochemistry, the expression of inflammatory corpuscle NLRP3 related protein and mRNA detection of myocardial Western blot and real-time PCR; to investigate the myocardial ischemia damage Correlation between injury and NLRP3 inflammasome. In the second chapter, the establishment of H_2O_2 induced oxidative stress damage in H_9C_2 cells and LPS+ATP H_9C_2 cells model of inflammatory injury model induced by the combination. The cell viability was measured by MTT method, to observe the content of cell supernatant kit in the oxidation index of inflammatory factor and the protective effect of Sal B on myocardial cell injury; effect the application of Western blot and Sal B immunofluorescence studies on myocardial cell damage in the process of NLRP3 inflammasome; affect the further application of TUNEL and Calciem-AM staining of Sal B on pyroptosis. In the third chapter, the use of LPS +ATP to promote the activation of H_9C_2 cells induced by the combination of NLRP3 inflammasome, detected by ROS and mitochondrial membrane potential probe, Western bolt analysis of SIRT1, p-Foxo3a and mitochondrial autophagy related protein, study the possible mechanism of Sal B inhibiting NLRP3 inflammasome, further through autophagy inhibitors 3-MA楠岃瘉鑷櫖鍦∟LRP3鐐庣棁灝忎綋嬋，

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