MnSOD和SIRT3蛋白在佐剂性关节炎大鼠肝组织中表达和意义

发布时间：2018-05-12 09:33

本文选题：佐剂性关节炎 + 锰超氧化物歧化酶　；参考：《安徽医科大学》2016年硕士论文

【摘要】：背景：氧化应激性反应是导致类风湿关节炎(rheumatoid arthritis, RA)的一个重要因素,长期治疗、生活方式的改变、关节疼痛和功能受限等使RA患者处于应激状态,机体出现脂质过氧化反应,氧自由基(oxygen free radical, ROS)的生成增加,而清除ROS的能力下降。佐剂性关节炎(adjuvant arthritis, AA)大鼠模型由于和RA在形态学上相似,且造模简单,是目前较为常用的RA动物模型,常用于RA药物筛选模型。真核细胞线粒体中含有锰超氧化物歧化酶(manganese superoxide dismutase, MnSOD),属于金属抗氧化酶,能够特异性的将超氧阴离子自由基(superoxide anion free radical)(0.-)2转化为H202和O2。MnSOD主要存在于线粒体,而线粒体是细胞内氧化代谢和产生能量、自由基的重要场所,故其在抗氧化损伤中作用显著。去乙酰化酶(sirtuins, SIRTs)家族中的SIRT3主要位于线粒体,是具有去乙酰化酶活性的SIRTs,具有调节线粒体的能量代谢和抗氧化应激的作用。目的：通过建立弗氏佐剂性关节炎(AA)大鼠模型,观察分析持续性的炎症刺激有无引起肝脏生化及形态学上的改变,这种改变与氧化应激性反应是否具有相关性,并且探讨肝脏锰超氧化物歧化酶(MnSOD)、去乙酰化酶3(Sirtuin3, SIRT3)的改变与AA大鼠肝脏损伤的关系。方法：弗氏完全佐剂(Freund's complete adjuvant, FCA)足趾皮下注射诱导SD大鼠从,造模后d 12、19、26,分批处死大鼠,计算肝指数,检测血清谷草转氨酶(aspartate transaminase, AST)、谷丙转氨酶(glutamic-pyruvic transaminase,ALT)、肝匀浆检测丙二醛(methane dicarboxylic aldehyde, MDA)、谷胱甘肽过氧化物酶(glutathion peroxidase, GSH-Px)、超氧化物歧化酶(superoxide dismutase, SOD)的变化,肝组织免疫组化和Western blot检测MnSOD、SIRT3蛋白表达变化。结果：与正常组大鼠比较,弗氏完全佐剂诱导的SD大鼠关节炎模型在造模后的3d即出现急性炎症表现,足爪、关节发红肿胀,继而踝关节背屈/趾屈活动度减小,活动受限,继发性炎症进一步发展,说明从大鼠模型建立成功。从大鼠造模d12后,肝脏指数升高,血清转氨酶升高,肝匀浆中MDA升高,GSH-Px、SOD活性下降(P0.05或P0.01),肝组织中MnSOD、 SIRT3蛋白表达下降。从大鼠关节和肝脏HE染色可见：滑膜组织增生水肿,血管翳形成,滑膜组织内小血管增生,扩张充血,大量炎性细胞浸润。严重者出现软骨的破坏和骨的侵蚀。炎症后期关节间隙变窄,从而形成不可逆的关节改变；肝脏结构损伤逐渐严重,表现为炎细胞浸润,肝血窦充血,肝细胞有点片状坏死。结论：AA模型组大鼠存在肝损伤,肝匀浆中氧化应激指标升高,其机制与肝脏中MnSOD和SIRT3表达有一定的关联。
[Abstract]:Background: oxidative stress response is an important factor leading to rheumatoid arthritis (RA). Long-term treatment, changes in lifestyle, joint pain and limited function lead to stress and lipid peroxidation in RA patients. The formation of oxygen free radical, ROS) increased, while the ability of scavenging ROS decreased. Adjuvant arthritis (AA) rat model is a commonly used animal model for RA drug screening because it is similar to RA in morphology and simple in making model. The mitochondria of eukaryotic cells contain manganese superoxide dismutase, MnSODX, which belong to metal antioxidant enzymes, and can specifically transform superoxide anion free radical)(0.-)2 into H202 and O2.MnSOD mainly in mitochondria. Mitochondria are important sites for intracellular oxidative metabolism, energy production and free radical production, so mitochondria play a significant role in antioxidant damage. The SIRT3 of deacetylase sirtuins (SIRTs) family is mainly located in mitochondria and is a kind of SIRTs with deacetylase activity which can regulate mitochondrial energy metabolism and antioxidant stress. Aim: to establish a rat model of Freund's adjuvant arthritis (Freund's adjuvant arthritis) and to investigate whether the persistent inflammatory stimulation causes biochemical and morphological changes in the liver and whether the changes are related to the oxidative stress response. The relationship between liver manganese superoxide dismutase (MnSOD), deacetylase (3), Sirtuin 3 (SIRT3) and liver injury in AA rats was studied. Methods: Sprague-Dawley rats were induced by subcutaneous injection of Freundus complete adjuvant, FCA), a Freundus adjuvant. The rats were killed in batches and liver index was calculated. Serum glutamic-pyruvic transaminase (alt), aspartate transaminase (AST), glutamic-pyruvic transaminase (alt), malondialdehyde (MDA) methane dicarboxylic aldehyde, malondialdehyde (MDA), glutathione peroxidase (Glutathione peroxidase), glutathione peroxidase (GSH-PxD), superoxide dismutase (SOD), and the expression of MnSOD- SIRT3 protein in liver tissue were detected. Results: compared with the normal group, the SD rat model of arthritis induced by Freund's complete adjuvant showed acute inflammation, redness and swelling of the paw and joint on the 3rd day after the establishment of the model, and then the motion of the ankle joint was decreased and the movement was limited. Secondary inflammation was further developed, indicating success in the establishment of rat models. The liver index increased, serum aminotransferase increased, the activity of MDA increased in liver homogenate, the activity of GSH-PxX SOD decreased by P0.05 or P0.01A, and the expression of MnSOD and SIRT3 protein in liver tissue decreased. From the HE staining of the joints and livers of rats, the synovial tissue was proliferative and edema, pannus was formed, the small vessels in the synovial tissue proliferated, dilated and congested, and a large number of inflammatory cells infiltrated. Severe cases appear cartilage damage and bone erosion. In the late stage of inflammation, the joint space became narrower, which resulted in irreversible joint changes. The damage of liver structure was gradually serious, showing inflammatory cell infiltration, hepatic sinusoidal congestion, and hepatocyte necrosis. Conclusion there is liver injury and oxidative stress index in liver homogenate of rats in the weight AA group. The mechanism is related to the expression of MnSOD and SIRT3 in the liver.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R593.22

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