【摘要】：目的糖尿病心肌病是在糖尿病患者尤其是2型糖尿病患者的基礎(chǔ)上發(fā)病的,且具有病情重、預(yù)后差和病死率高的特點(diǎn)。為尋求有效的治療方法,現(xiàn)本文就褪黑素(MLT)對長期患2型糖尿病(T2DM)大鼠心肌細(xì)胞凋亡的影響及其可能的機(jī)制進(jìn)行研究和探討。方法(1)T2DM大鼠模型:在使用基礎(chǔ)飼料喂養(yǎng)一周后,36只SPF級健康雄性SD大鼠隨機(jī)分為對照組(Control,n=12),模型組(DM,n=12),治療組(DM+MLT,n=12)。采用高脂飲食喂養(yǎng)聯(lián)合腹腔注射鏈脲佐菌素(STZ)25 mg/kg的方法誘導(dǎo)2型糖尿病模型。造模成功后,治療組給予MLT(10 mg/(kg·d))灌胃治療。24周后,動(dòng)脈插管檢測大鼠心功能;收集大鼠腹主動(dòng)脈血和心肌組織,并檢測大鼠空腹血糖(FBG),血脂(TC、LDL-c、TG)和胰島素水平;HE染色和Masson染色分別觀察大鼠心肌的形態(tài)結(jié)構(gòu)變化和膠原纖維的堆積情況;原位末端標(biāo)記法(TUNEL)觀察心肌組織細(xì)胞凋亡;免疫組化(IHC)檢測Caspase-3的表達(dá)水平。此外,為探討其可能的分子機(jī)制,Western blot法檢測內(nèi)質(zhì)網(wǎng)應(yīng)激相關(guān)蛋白(GRP78,CHOP,PERK,ATF-6α,IRE1α,Caspase-12),絲裂原活化蛋白激酶(MAPK)通路相關(guān)蛋白(JNK,p38,ERK)和凋亡相關(guān)蛋白(Caspase-9,Caspase-3,Bcl-2,Bax)的表達(dá)情況。(2)高糖培養(yǎng)大鼠心肌細(xì)胞H9C2為細(xì)胞模型:不同糖濃度培養(yǎng)基培養(yǎng)細(xì)胞48 h后,Hoechst 33258染色檢測細(xì)胞凋亡,western blot檢測MAPK通路相關(guān)蛋白JNK、p38和ERK的磷酸化水平。結(jié)果(1)T2DM大鼠模型:模型組大鼠的FBG為(19.27±1.03)mmol/L,胰島素水平為(34.79±7.97μIU/m L),均顯著高于對照組大鼠,且胰島素敏感性指數(shù)(ln ISI)為-6.48±0.23明顯比對照組大鼠低,表明存在胰島素抵抗(IR)。這些結(jié)果都說明已成功構(gòu)建T2DM大鼠模型。MLT不僅能降低FBG和血脂(TC、LDL-c、TG)水平,還能增強(qiáng)胰島素的敏感性。HE染色結(jié)果提示MLT能改善心肌組織形態(tài)的紊亂情況,Masson染色結(jié)果表明MLT能減少心肌組織膠原纖維的堆積。模型組大鼠的心肌細(xì)胞凋亡細(xì)胞明顯增多,且心功能嚴(yán)重受損,經(jīng)MLT治療后均明顯改善。IHC顯示MLT能降低T2DM大鼠Caspase-3的表達(dá)。Western blot結(jié)果顯示模型組大鼠心肌組織內(nèi)質(zhì)網(wǎng)應(yīng)激相關(guān)蛋白GRP78、CHOP、PERK、ATF-6α、IRE1α和Caspase-12的表達(dá)均明顯上升,且JNK的磷酸化水平也明顯上調(diào)。T2DM大鼠經(jīng)MLT治療后,內(nèi)質(zhì)網(wǎng)應(yīng)激相關(guān)蛋白表達(dá)和JNK的磷酸化水平均下調(diào)。(2)高糖培養(yǎng)H9C2細(xì)胞模型:Hoechst 33258染色顯示高糖能誘導(dǎo)H9C2細(xì)胞凋亡;Western blot結(jié)果顯示高糖能上調(diào)H9C2細(xì)胞中JNK的磷酸化水平,并具有一定的濃度依賴性。結(jié)論T2DM大鼠心肌細(xì)胞凋亡增多,其機(jī)制可能是血糖血脂升高、經(jīng)由內(nèi)質(zhì)網(wǎng)應(yīng)激介導(dǎo)的JNK通路活化調(diào)控下游相關(guān)凋亡蛋白所致;而MLT能通過降低血糖血脂、增強(qiáng)胰島素敏感性和降低心肌細(xì)胞凋亡對T2DM大鼠心臟有一定的保護(hù)作用。在H9C2細(xì)胞中,高糖能上調(diào)MAPK通路中JNK的磷酸化水平,誘導(dǎo)細(xì)胞凋亡,并具有一定的濃度依賴性。
[Abstract]:Objective Diabetic cardiomyopathy is developed on the basis of diabetes, especially type 2 diabetes, and has the characteristics of severe disease, poor prognosis and high mortality. In order to find an effective treatment method, the effect of melatonin (MLT) on cardiomyocyte apoptosis and its possible mechanism in chronic type 2 diabetes mellitus (T2DM) rats were studied and discussed. Methods (1) T2DM rat model: after feeding with basic diet for one week, 36 SPF grade healthy male SD rats were randomly divided into three groups: control group (Control,n=12), model group (DM,n=12) and treatment group (DM MLT,n=12). Type 2 diabetic model was induced by high fat diet and intraperitoneal injection of streptozotocin (STZ) 25 mg/kg. After the model was successfully established, the treatment group was given MLT (10 mg/ (kg d) intragastric administration for .24 weeks), the cardiac function was measured by arterial catheterization, and the blood and myocardial tissues of abdominal aorta were collected. Fasting blood glucose, (FBG), lipids (TC,LDL-c,TG) and insulin levels were detected by HE staining and Masson staining respectively. The expression of Caspase-3 was detected by immunohistochemical (IHC). In addition, In order to investigate its possible molecular mechanism, the expression of endoplasmic reticulum stress-related protein (GRP78,CHOP,PERK,ATF-6 偽), mitogen-activated protein kinase (MAPK) pathway protein (JNK,p38,ERK) and apoptosis-related protein (Caspase-9,Caspase-3,Bcl-2,Bax) was detected by Western blot method. (2) the expression of H9C2 in rat cardiomyocytes cultured with high glucose was fine. Cell model: after cultured in different glucose concentration medium for 48 h, Hoechst 33258 staining was used to detect the phosphorylation of MAPK pathway related proteins JNK,p38 and ERK by western blot. Results (1) T2DM rat model: FBG of the model group was (19.27 鹵1.03) mmol/L, insulin level was significantly higher than that of the control group (34.79 鹵7.97 渭 IU/m L), and the insulin sensitivity index (ln ISI) was -6.48 鹵0.23 significantly lower than that of the control group, indicating the existence of insulin resistance (IR). These results suggest that the successful establishment of T2DM rat model. MLT can not only reduce the levels of FBG and TC,LDL-c,TG. The results of HE staining showed that MLT could improve the disorder of myocardial morphology. The results showed that MLT could reduce the accumulation of collagen fibers in myocardial tissue. In the model group, the number of cardiomyocyte apoptosis was significantly increased, and the cardiac function was seriously damaged. After treatment with MLT, the results showed that MLT could decrease the expression of Caspase-3 in T2DM rats. Western blot showed that the expression of ER stress-related protein GRP78,CHOP,PERK,ATF-6 偽, IRE1 偽 and Caspase-12 in myocardial tissue of the model group were significantly increased. Moreover, the phosphorylation level of JNK was also up-regulated in rats treated with MLT. The expression of ER stress-related protein and the phosphorylation level of JNK were down-regulated. (2) in high glucose culture H9C2 cell model: Hoechst 33258 staining showed that high glucose could induce apoptosis of H9C2 cells. Western blot results showed that high glucose could up-regulate the phosphorylation of JNK in H9C2 cells. And it has a certain concentration dependence. Conclusion the increase of cardiomyocyte apoptosis in T2DM rats may be due to the elevation of blood glucose and lipids, which may be caused by activation of JNK pathway mediated by endoplasmic reticulum stress to regulate downstream apoptotic proteins, while MLT can reduce blood glucose and blood lipid. Increasing insulin sensitivity and decreasing cardiac myocyte apoptosis may protect the heart of T2DM rats. In H9C2 cells, high glucose can up-regulate the level of JNK phosphorylation in MAPK pathway and induce apoptosis in a dose-dependent manner.
【學(xué)位授予單位】：安徽醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R587.2;R542.2

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相關(guān)期刊論文 前1條

1 魏娜;賀海波;張長城;袁丁;王婷;;JNK信號通路與細(xì)胞凋亡關(guān)系的研究進(jìn)展[J];中國臨床藥理學(xué)與治療學(xué);2013年07期

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本文編號：2219374