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

【摘要】：研究目的:觀察丹酚酸B(salvianolic acid B,Sal B)對大鼠心肌缺血損傷的保護作用。從NLRP3炎癥小體角度出發(fā)探討丹酚酸B保護損傷心肌細胞的可能機制。為靶向炎癥小體進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(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對缺血損傷心肌的保護作用;進一步應(yīng)用免疫組化、western blot和real-time PCR檢測心肌組織中炎癥小體NLRP3相關(guān)蛋白和mRNA表達;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細胞氧化應(yīng)激損傷模型和LPS+ATP聯(lián)合誘導(dǎo)H_9C_2細胞炎性損傷模型。應(yīng)用MTT法檢測細胞存活率,試劑盒檢測細胞上清液中氧化指標和炎癥因子的含量觀察Sal B對心肌細胞損傷的保護作用;應(yīng)用western blot和免疫熒光研究Sal B對心肌細胞損傷過程中NLRP3炎癥小體的影響;進一步應(yīng)用TUNEL和Calciem-AM染色研究Sal B對細胞焦亡的影響。第三章、利用LPS+ATP聯(lián)合誘導(dǎo)促進H_9C_2細胞中NLRP3炎癥小體的激活,通過探針檢測ROS和線粒體膜電位,western bolt分析SIRT1、p-Foxo3a和線粒體自噬相關(guān)蛋白,研究Sal B抑制NLRP3炎癥小體的可能機制,進一步通過自噬抑制劑3-MA驗證自噬在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、炎細胞浸潤和減少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表達明顯降低,呈一定的劑量依賴性(P0.05,P0.01)。第二章結(jié)果顯示:經(jīng)MTT法檢測,確定H_2O_2合適的造模濃度為l00μM,時間為2h;SalB合適的預(yù)處理濃度為1、5、25μM,時間為24h。與H_2O_2模型組比較,給予不同劑量的SalB可以提高細胞的存活率,保持細胞形態(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),呈一定的劑量依賴性。Western bolt、免疫熒光和Elisa結(jié)果顯示,Sal B可以抑制H_9C_2細胞氧化損傷和炎性損傷中NLRP3炎癥小體的激活和IL-lβ的表達和分泌。TUNEL和Calcein-AM染色結(jié)果表明,Sal B可以保護細胞膜的完整性,減輕DNA損傷,從而抑制細胞焦亡。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H_9C_2細胞中SIRT1、p-Foxo3a和MnSOD表達的升高(P0.05,P0.01),抑制細胞ROS和線粒體膜電位的升高,促進自噬標志蛋白LC3Ⅱ和Beclin-1以及線粒體自噬調(diào)控蛋白Parkin的表達,抑制P62和PINK1的表達(P0.05,P0.01)。在自噬抑制劑3-MA的作用下,Sal B(51μM)促進H_9C_2細胞線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并沒有影響Sal B對SIRT1和p-Foxo3a的促進作用。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)達到保護心臟的目的。2.心肌細胞氧化應(yīng)激損傷時會誘導(dǎo)NLRP3炎癥小體的激活,丹酚酸B可以保護H_9C_2細胞氧化應(yīng)激損傷,抑制NLRP3炎癥小體的激活。3.Sal B抑制H_9C_2細胞中NLRP3炎癥小體活化的可能機制是激活SIRTl-FOXO3a-Parkin軸,改善心肌細胞線粒體自噬,減少受損線粒體的積累,從而抑制NLRP3激活信號的釋放。
[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，

本文編號：1578688