Effect of Autophagy on Cardiac Myocytes Injury in Uremic Cardiomyopathy Mice

doi:10.3969/j.issn.1007-9572.2016.36.017

Abstract

Abstract: Objective　To explore the effect and mechanism of autophagy on cardiac myocytes injury in uremic cardiomyopathy mice.Methods　From August to December 2015，fifty male C57BL/6-PAX6 mice in a SPF grade were selected in our study.The mice were randomly divided into control group（6）,sham operation group（8）,test groups〔5/6 nephrectomy（NC） group （6）,5/6NC+saline group（10）,5/6NC+wortmannin group（10） and 5/6NC+Beclin-1 group（10）〕.The control group did not receive any intervention in the study.The sham operation group only received surgery and anesthesia procedures simultaneous with the test groups,but the kidneys were not removed.5/6 mice nephrectomy model were established in the test groups.Six weeks after model establishment,5/6 NC group did not receive intervention.5/6NC+saline group was given 0.9% sodium chloride solution 1 ml intraperitoneal injection,1 time every other day.5/6NC+wortmannin group was given wortmannin 1 mg/kg intraperitoneal injection,1 time/day.5/6NC+Beclin-1 group was given adenovirus carrying Beclin-1 1 ×109 pfu intramuscular injection,1 time/once.12 weeks after model establishment,mice heart function was measured by ultrasonic cardiograph,and renal function was measured by ELISA.In addition,we analyzed the expression of autophagy and its associated protein in mice myocardium using Western blotting and immunohistochemical staining after sacrificed.Results　12 weeks after model establishment,compared with control group and sham operation group,LVEDD significantly increased,LVFS significantly increased and serum creatinine and urea nitrogen level significantly increased in the 5/6NC group and 5/6NC+saline group（P<0.05）.Compared with 5/6 NC+saline group,LVEDD significantly decreased,LVFS significantly increased,serum creatinine and urea nitrogen level significantly decreased in the 5/6NC+wartmannin group（P<0.05）.Compared with control group and 5/6 NC+saline group,LVEDD significantly increased,LVFS significantly decreased,serum creatinine and urea nitrogen level significantly increased in the 5/6 NC+Beclin-1 group （P<0.05）.Immunohistochemical staining showed that the expression of Atg5,Cathepsin-D and Rab 7 in the myocardial cells of 5/6NC group were significantly higher than that in control group（P< 0.05）.There were significant differences in the rate of Atg5,Cathepsin-D and Rab7 positive cells between the control group and 5/6NC group（P<0.05）.The expressions of autophagy-related protein LC3,Beclin-1 and AKT in the 5/6 NC+saline group and 5/6NC+Beclin-1 group were significantly higher than those in the control group （P<0.05）.The expressions of LC3,Beclin-1 and AKT in 5/6 NC+wortmannin penicillin group were higher than those in control group,but lower than those in 5/6NC+saline group,5/6NC+Beclin-1 group （P<0.05）.The expression of LC3,Beclin-1 and AKT in the 5/6 NC+Beclin-1 group were higher than those in 5/6 NC+saline group and 5/6 NC+wartmannin group（P<0.05）.Conclusion　Autophagy was closely related to cardiomyocytes injury in uremic mice.Enhancement or inhibition of autophagy could aggravate or relieve cardiac and renal function in uremic mice.Beclin-1 gene and PI3K/AKT signal proteins played an important role in regulation cardiomyocytes autophagy.

Key words: Cardiomyopathies, Uremia, Autophagy, Autophagy associated protein, PI3K/AKT signal protein

摘要： 目的　探讨自噬与尿毒症小鼠心肌细胞损伤的关系及其发生的可能机制。方法　2015年8—12月选取SPF级C57BL/6-PAX6雄性小鼠50只，采用随机数字表法将小鼠分成对照组（6只）、假手术组（8只）、实验组〔5/6NC组（6只）、5/6NC+生理盐水组（10只）、5/6NC+渥曼青霉素组（10只）以及5/6NC+Beclin-1组（10只）〕。对照组研究过程中不做任何干预。假手术组仅与实验组同期进行两次手术，但不切除肾脏。实验组制备5/6肾切除尿毒症小鼠模型。造模6周时，5/6NC组不进行干预，5/6NC+生理盐水组、5/6NC+渥曼青霉素组、5/6NC+Beclin-1组分别给以0.9%氯化钠溶液1 ml 腹腔内注射，隔日1次；渥曼青霉素1 mg·kg-1·次-1，腹腔内注射，1次/d；腺病毒携带的Beclin-1 1×109 pfu腿部肌肉注射，1次/只。造模12周时采用超声心动图检测各组小鼠心功能，ELISA法检测肌酐、尿素氮，采用免疫组化及Western blotting法检测处死后小鼠的心肌细胞自噬相关蛋白表达情况。结果　造模12周时，与对照组及假手术组比较，5/6NC组、5/6NC+生理盐水组左心室舒张末期内径（LVEDD）增厚、左心室短轴缩短率（LVFS）降低，肌酐及尿素氮上升（P<0.05）；与5/6NC+生理盐水组比较，与5/6NC+渥曼青霉素组LVEDD降低，LVFS升高，肌酐、尿素氮降低（P<0.05）；与对照组、5/6NC+生理盐水组比较，5/6NC+Beclin-1组LVEDD增加，LVFS降低，肌酐及尿素氮升高（P<0.05）。免疫组化染色显示5/6NC组心肌细胞Atg5、Cathepsin-D、Rab 7表达较对照组增强；对照组与5/6NC组Atg5、Cathepsin-D、Rab 7阳性细胞率比较，差异均有统计学意义（P<0.05）。5/6NC+生理盐水组、5/6NC+Beclin-1组心肌组织细胞中自噬相关蛋白LC3、Beclin-1及AKT表达均较对照组升高（P<0.05），5/6NC+渥曼青霉素组LC3、Beclin-1及AKT表达虽较对照组升高，但低于5/6NC+生理盐水组、5/6NC+Beclin-1组（P<0.05），5/6NC+Beclin-1组LC3、Beclin-1、AKT表达较5/6NC+生理盐水和5/6NC+渥曼青霉素组升高（P<0.05）。结论　尿毒症小鼠心肌细胞的损伤与自噬密切相关，增强或抑制自噬可以加重或缓解尿毒症小鼠心肾功能，Beclin-1基因及磷脂酰肌醇3-激酶（PI3K）/丝氨酸/苏氨酸蛋白激酶（AKT）信号蛋白在调节尿毒症心肌细胞自噬中发挥重要作用。

关键词: 心肌疾病, 尿毒症, 自噬, 自噬相关蛋白, PI3K/AKT信号蛋白

GONG Jian-guang, JIN Juan, ZHAO Li, LIN Bo, LIANG Shi-kai, HE Qiang, LI Yi-wen. Effect of Autophagy on Cardiac Myocytes Injury in Uremic Cardiomyopathy Mice[J]. Chinese General Practice, 2016, 19(36): 4489-4494.

龚建光, 金娟, 赵黎, 林波, 梁世凯, 何强, 李一文. 自噬在尿毒症小鼠心肌细胞损伤中的作用研究[J]. 中国全科医学, 2016, 19(36): 4489-4494.

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