硫化氢可通过下调内质网应激改善糖尿病大鼠的心肌纤维化

发布时间：2018-05-08 09:55

本文选题：硫化氢 + 内质网应激　；参考：《南华大学》2015年硕士论文

【摘要】：研究背景及目的有研究表明,内质网(Endoplasmic reticulum,ER)应激可以通过多种途径诱导细胞凋亡,从而参与糖尿病(Diabetes mellitus,DM)心肌纤维化,因此,ER应激可能与糖尿病心肌病(Diabetic cardiomyopathy,DC)发生发展过程有关,最终影响心脏功能。然而,目前没有特殊的药物来预防和治疗DC。硫化氢(Hydrogen sulfide,H2S)是第3种气体信号分子,有多种生理和生物学作用,其中包括抗ER应激。之前有研究证明,H2S可以抑制DM大鼠心肌纤维化。但是,H2S抑制心肌纤维化的机制尚不明确。本研究旨在建立DM模型,探讨ER应激是否参与DM心肌纤维化,而H2S是否可通过下调ER应激,减少心肌细胞凋亡,从而改善DM大鼠心脏重构。方法成年雄性SD(Sprague-Dawley,SD)大鼠,随机分为5组:对照组(Control);DM模型组(STZ);低浓度保护组(C1);高浓度保护组(C2);H2S对照组(H2S)。然后对STZ组、C1组和C2组大鼠腹腔注射(Intraperitoneal injection,ip)链脲佐菌素(Streptozotocin,STZ)(40 mg/kg)建立DM模型。STZ注射后的72 h,尾静脉采血测空腹血糖,高于16.7 mmol/L,认为DM模型成功建立。对C1组(30μmol/kg)、C2组(100μmol/kg)和H2S组(100μmol/kg)使用相应浓度硫氢化钠(Sodium hydrosulfide,Na HS)溶液腹腔注射作为H2S供体。实验8周后取大鼠心脏组织,用免疫组化染色法检测I、III型胶原纤维;马森(Masson)染色检测心肌胶原纤维;碱水解法检测羟脯氨酸表达水平;用苏木精伊红(Hematoxylin-eosin,HE)染色观察心肌病理变化;Western blotting法检测各组心肌组织ER应激相关蛋白糖调节蛋白78(Glucose regulated protein 78,GRP78)、C/EBP同源蛋白(C/EBP homologous protein,CHOP)、Caspase-12,H2S生成酶胱硫醚-γ-裂解酶(Cystathionine-gamma-lyase,CSE)、巯基丙酮酸转硫酶(Mercaptopyruvate sulfurtransferase,MPST)以及凋亡相关蛋白Cleaved Caspase-3的表达;透射电镜观察ER及心肌纤维形态。结果1.成功建立DM大鼠模型。STZ注射72 h后检测血糖,结果显示血糖明显升高,且每只大鼠血糖均高于16.7 mmol/L。2.H2S对DM大鼠BW、HW、HW/BW的影响。实验结束后DM大鼠BW和HW明显轻于对照组,HW/BW值明显高于对照组,提示DM大鼠心脏可能出现肥大,而H2S对DM大鼠BW,HW及HW/BW并无影响。3.H2S可改善DM大鼠心肌纤维化。免疫组化结果显示:DM大鼠心脏组织中I型和III型胶原纤维表达较对照组明显上升,且胶原纤维排列紊乱;而H2S干预的DM大鼠心脏组织中I型和III型胶原纤维表达明显下降。Masson染色结果显示:DM大鼠心脏组织中心肌胶原纤维沉积较对照组明显增加,而H2S明显减少DM大鼠心肌胶原纤维沉积面积;碱水解法结果显示:脯氨酸在DM大鼠心脏组织中的表达明显高于对照组。而H2S减少了DM大鼠心脏组织中羟脯氨酸的表达。HE染色结果显示:DM大鼠心脏胶原排列紊乱,心肌纤维间隙增宽,而H2S可以改善DM大鼠心肌病理变化。透射电镜结果显示:DM大鼠心肌纤维出现水肿、排列紊乱、灶性溶解坏死;而H2S可以减轻心肌纤维水肿,减少心肌纤维坏死。4.H2S可上调DM大鼠H2S生成酶的表达。Western blotting法检测结果显示:DM大鼠心脏组织中H2S生成酶CSE及MPST的表达明显下降,而H2S可上调DM大鼠CSE及MPST的表达。5.H2S可下调STZ诱导的DM大鼠心肌ER应激。Western blotting法检测结果显示:与对照组相比,DM大鼠心脏组织中GRP78,CHOP和Caspase-12的表达明显升高,而H2S明显抑制了DM大鼠心脏组织中GRP78,CHOP和Caspase-12的表达。透射电镜结果显示:DM大鼠心肌ER出现不同程度的水肿;而H2S可以改善ER水肿。6.H2S可减轻STZ诱导的DM大鼠心肌细胞凋亡。Western blotting法检测结果显示:DM大鼠心脏组织中Cleaved Caspase-3的表达较对照组明显升高,而H2S明显抑制了DM大鼠心脏组织中Cleaved Caspase-3的表达。结论1.DM大鼠出现心肌纤维化,其机制可能与内源性H2S生成减少,ER应激上调相关。2.H2S可抑制DM大鼠心脏纤维化,机制可能与下调ER应激水平相关。
[Abstract]:Research background and purpose have shown that Endoplasmic reticulum (ER) stress can induce apoptosis through a variety of pathways and thus participate in diabetic (Diabetes mellitus, DM) myocardial fibrosis. Therefore, ER stress may be related to the development of diabetic cardiomyopathy (Diabetic cardiomyopathy, DC), and ultimately affect cardiac function. However, there are no special drugs to prevent and treat DC. sulfide (Hydrogen sulfide, H2S) are third kinds of gas signal molecules, which have many physiological and biological effects, including anti ER stress. Previous studies have shown that H2S can inhibit myocardial fibrosis in DM rats. However, the mechanism of H2S inhibition of myocardial fibrosis is not clear. This study is not clear. The aim of this study is to establish a DM model to explore whether ER stress is involved in DM myocardial fibrosis, and whether H2S can improve cardiac remodeling by reducing ER stress and reducing cardiomyocyte apoptosis. Methods adult male SD (Sprague-Dawley, SD) rats were randomly divided into 5 groups: control group (Control), DM model group (STZ), low concentration protection group and high concentration protection group (C2); H2S control group (H2S). Then the rats in group STZ, C1 and C2 were injected intraperitoneally (Intraperitoneal injection, IP) streptozotocin (Streptozotocin, STZ) (Streptozotocin, STZ) (40 mg/kg) to establish the model after injection, and the caudal vein was collected to measure the fasting blood glucose, which was higher than 16.7. Group 2S (100 mu mol/kg) was injected with the corresponding concentration of sodium hydrosulfide (Sodium hydrosulfide, Na HS) as H2S donor in the abdominal cavity. After 8 weeks, the rat heart tissue was collected, I and III collagen fibers were detected by immunohistochemical staining. The myocardial collagen fibers were detected by Mason (Masson) staining, and the level of hydroxyproline expression was detected by alkali hydrolysis. The pathological changes of myocardium were observed by red (Hematoxylin-eosin, HE) staining, and Western blotting method was used to detect the ER stress related protein 78 (Glucose regulated protein 78, GRP78), C/EBP homologous protein (C/EBP homologous). The expression of thiol pyruvate (Mercaptopyruvate sulfurtransferase, MPST) and apoptosis related protein Cleaved Caspase-3; transmission electron microscopy (TEM) to observe the morphology of ER and myocardial fiber. Results 1. successfully established the DM rat model after.STZ injection of 72 h to detect blood glucose, the results showed that blood glucose was obviously elevated, and the blood sugar of each rat was higher than 16.7 mmol/L.2.H2. The effect of S on BW, HW and HW/BW in DM rats. After the experiment, BW and HW in DM rats were significantly lighter than those in the control group. The HW/BW value was significantly higher than that of the control group. The expression of primary fiber was significantly higher than that of the control group, and the arrangement of collagen fibers was disorderly, while the expression of I and III collagen fibers in the heart tissue of DM rats with H2S intervention was significantly decreased by.Masson staining results showed that the deposition of collagen fibers in the cardiac muscle collagen fibers in the cardiac tissue of DM rats increased significantly, while H2S significantly reduced the collagen fibrous deposition in DM rats. The results of alkaline hydrolysis showed that the expression of proline in the cardiac tissue of DM rats was significantly higher than that in the control group. H2S decreased the expression of hydroxyproline in the cardiac tissue of DM rats by.HE staining, and the results showed that the cardiac collagen arrangement in DM rats was disordered and the gap of myocardial fibers widened, and H2S could improve the pathological changes of myocardium in DM rats. Transmission electron microscope junction The results showed that the myocardial fibrosis in DM rats appeared edema, disorder and focal dissolving necrosis, while H2S could reduce myocardial fibrosis and decrease myocardial fibrous necrosis,.4.H2S could up regulate the expression of H2S producing enzyme in DM rats. The results of.Western blotting assay showed that the expression of H2S generating enzyme CSE and MPST in DM rat heart tissue decreased obviously, while H2S could be superior. The expression of CSE and MPST in DM rats could down regulate the blotting method of ER stress.Western blotting method in DM rats induced by STZ. Compared with the control group, the GRP78, CHOP and expressions in the cardiac tissue of DM rats were significantly higher than those of the control group. The results showed that the myocardial ER of DM rats appeared to varying degrees of edema, while H2S could improve the ER edema.6.H2S to reduce the.Western blotting assay of STZ induced cardiomyocyte apoptosis in DM rats. The expression of Cleaved Caspase-3 in the cardiac tissue of DM rats was significantly higher than that in the control group. Aspase-3 expression. Conclusion 1.DM rats have myocardial fibrosis, the mechanism may be reduced by endogenous H2S formation, ER stress up-regulated.2.H2S can inhibit cardiac fibrosis in DM rats, the mechanism may be related to the downregulation of ER stress level.

【学位授予单位】：南华大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R587.2

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