3-甲基腺嘌呤对风湿性心脏病瓣膜纤维化的影响.pdf

MM微创医学2 0 2 3年第18 卷第4期Journal of Minimally Invasive Medicine,2023,18(4)435.3-甲基腺嘌呤对风湿性心脏病瓣膜纤维化的影响一朱晓莉谢晓勇*黄柳柳罗程黎玉贵温大帅贺洲（广西医科大学第一附属医院心胸外科，广西南宁市530021)【摘要】目的探讨3-甲基腺嘌呤(3-MA)对风湿性心脏病(RHD)瓣膜纤维化相关指标的影响。方法取30 只Lewis雌性大鼠,利用电脑随机数将大鼠分为正常组、模型组和实验组，每组10 只。模型组和实验组通过后足垫、腹部皮下注射抗原I、抗原I 造模，大鼠多处关节出现红肿,B超检查显示二尖瓣有不同程度反流，表明RHD模型建立成功。建模成功后,第8 9周,实验组大鼠于腹部皮下注射3-MA2.5mg/kg，1次/周，模型组大鼠腹部皮下注射等量生理盐水；第1024周，模型组、实验组大鼠腹部皮下注射0.5mL抗原I，1次/周，正常组大鼠在相同时间以同样方式注射等量生理盐水。采用免疫印迹（Westernblot）法检测3组大鼠二尖瓣组织中PI3K、Akt、-平滑肌肌动蛋白（-SMA）、LC3I及I型胶原蛋白的相对表达量；采用ELISA法检测肿瘤坏死因子-（T NF-）、白介素-6（IL-6）、白介素-8（IL-8）水平。结果正常组、实验组、模型组大鼠的二尖瓣组织中PI3K、A k t、LC3 I的相对表达量依次升高，组间差异均有统计学意义（均P0.05）；模型组-SMA、I 型胶原蛋白的相对表达量均高于正常组、实验组,差异均有统计学意义（均P0.05）。正常组、实验组、模型组大鼠的二尖瓣组织中TNF-、I L-6、I L-8 水平依次升高,组间差异均有统计学意义（均P0.05）。结论3-MA能够有效抑制RHD大鼠二尖瓣组织自噬，减轻心脏瓣膜纤维化；调控PI3K/Akt信号通路可能是3-MA起抑制纤维化的作用机制之一;自噬作用的调控可能与TNF-、IL-6 及IL-8水平具有一定的相关性。【关键词】风湿性心脏病;3-甲基腺嘌呤;PI3K/Akt;瓣膜纤维化【中图分类号】R541.2【文献标识码】A【文章编号】16 7 3-6 57 5（2 0 2 3)0 4-0 435-0 5D0I:10.11864/j.issn.1673.2023.04.05Effect of 3-methyladenine on valve fibrosis in rheumatic heart diseaseZHU Xiaoli,XIE Xiaoyong,HUANG Liuliu,LUO Cheng,LI Yugui,WEN Dashuai,HE Zhou(Department of Cardiothoracic Surgery,the First Affliated Hospital of Guangxi Medical University,Nanning530021,Guangxi,China)Abstract)Objective To investigate the effect of 3-methyladenine(3-MA)on related indexesof valve fibrosis in rheumatic heart disease(RHD).Methods Thirty female Lewis rats were dividedinto normal group,model group and experimental group by computer random number method,with10 rats in each group.The model group and experimental group were modeled by subcutaneous injectionof antigen I and antigen I into the hind foot pad and abdomen.Multiple joints of the rats showedredness and swelling,and two-dimensional ultrasonography showed different degrees of mitral valveregurgitation,indicating that the model was successfully established.After successful modeling,rats inthe experimental group were injected subcutaneously with 3-MA 2.5 mg/kg once a week in the abdomenat the 8th to 9th week,while rats in the model group were injected subcutaneously with the same amountof normal saline in the abdomen.From 10th to 24th week,rats in the model group and experimentalgroup were injected with 0.5 mL antigen II subcutaneously in the abdomen once a week,while rats inthe normal group were injected with the same amount of normal saline at the same time and in the same基金项目：广西自然科学基金（编号：2 0 19GXNSFAA185055）*通信作者436way.The relative expressions of PI3K,Akt,alpha-smooth muscle actin（-SMA,)LC3 I and typeI collagen in the mitral valve tissues of the three groups were detected by Western blot.The levels oftumor necrosis factor-（T NF-),interleukin-6（IL-6)a n d i n t e r l e u k i n-8 （IL-8)w e r e d e t e c t e d b yELISA.Results The relative expressions of PI3K,Akt and LC3 I in mitral valve tissue of normalgroup,experimental group and model group increased in turn,and the differences among the groupswere statistically significant(all P0.05).Compared with the normal group and the experimentalgroup,the relative expressions of-SMA,type I collagen were higher in the model group,withstatistically significant dfferences(all P0.05).The levels of TNF-,IL-6 and IL-8 in mitral valve tisue of normal group,experimental group and model group increased in turn,and the differences among the groups werestatistically significant(all P0.05).Conclusion 3-MA can effectively inhibit autophagy of mitralvalve tissue in RHD rats and reduce heart valve fibrosis.The regulation of PI3K/Akt signaling pathwaymay be one of the mechanisms of 3-MA inhibiting fibrosis.The regulation of autophagy may be relatedto the levels of TNF-,IL-6 and IL-8.Key words】Rh e u m a t i c h e a r t d i s e a s e;3-m e t h y l a d e n i n e;PI3K/A k t;Va l v e f i b r o s i s风湿性心脏病（rheumatic heart disease，RH D）是我国瓣膜性心脏病的主要构成部分，其中55.1%为风湿性瓣膜病变,2 1.3%为退行性瓣膜病变 。A 组乙型溶血性链球菌（group A streptococcus pyogens，GAS)侵犯心脏,可导致风湿性心瓣膜炎、心肌炎和心包炎,其中以风湿性心瓣膜炎最为常见。RHD的症状大多由瓣膜病变引起,但RHD的发病机制尚未明确,且缺乏有效的防治措施,需进一步研究其可能的发病机制，为临床预防和治疗提供更完善的理论依据。自噬是最热门的研究领域之一,是一种细胞内代谢降解的过程，包括自噬体吞噬细胞质、溶酶体降解、溶酶体回收等2 。本团队的前期研究3 发现,风湿性瓣膜病患者主动脉瓣膜的自噬水平明显高于正常瓣膜组织；而体外细胞实验41结果发现，LY294002自噬抑制剂在RHD的发病机制中发挥重要作用,可促进瓣膜间质细胞向肌成纤维细胞分化，同时对肌成纤维细胞的增殖有抑制作用。本研究通过动物在体实验，探讨自噬抑制剂3-甲基腺嘌呤（3-methyladenine，3-MA)对RHD大鼠瓣膜增生、纤维化的作用及其可能的机制,以期为临床预防和治疗RHD提供理论依据。1材料与方法1.1实验动物30 只8 周龄无特定病原体（specificpathogen free，SPF）级Lewis 雌性大鼠，体重16 0 180g，购于北京维通利华实验动物技术有限公司SCXK（京）2 0 2 1-0 0 0 6，大鼠购入后饲养于广西医Journal of Minimally Invasive Medicine,Aug.2023,Vol.18,No.4科大学实验动物中心SPF级实验动物房SYXK（桂）2020-0004,温度2 0 2 5,相对湿度40%7 0%。1.2主要试剂完全弗氏佐剂（complete Freundsadjuvant，CFA）为Sigma公司产品（生产批号：SLCF1289），3-M A 为美国Focus公司产品（生产批号：MA0318-Jul-10H），BCA 试剂购于武汉赛维尔生物科技有限公司（生产批号：G2026），抗-平滑肌肌动蛋白(alpha-smooth muscle actin，-SMA）抗体购于艾博抗（上海）贸易有限公司（生产批号：GR3447889-2），I型胶原蛋白抗体购于上海联迈生物工程有限公司（生产批号：LM-7158R），G A PD H 抗体（生产批号：AC220730007）、PI3K 抗体（生产批号：AC220830004）、Akt抗体（生产批号：AC220430017）、LC3抗体（生产批号：AC220330001）均购于武汉赛维尔生物科技有限公司,辣根酶标记山羊抗兔二抗购于北京博奥森生物科技有限公司（生产批号：AH11286402）。ELISA试剂盒（生产批号：SEKM-0325）、G A S菌株（生产