Jiang Lingzhi, Wang Mingshan, Shen Ye
Department of Rehabilitation Medicine, Center for Rehabilitation Medicine, Zhejiang Provincial People's Hospital (Affiliated People's Hospital, Hangzhou Medical College), Rehabilitation and Sports Medicine Research Institute of Zhejiang Province, Hangzhou 310014, Zhejiang, China.
Department of Infectious Diseases, Center for General Practice Medicine, Zhejiang Provincial People's Hospital (Affiliated People's Hospital, Hangzhou Medical College), Hangzhou 310014, Zhejiang, China. Corresponding author: Wang Mingshan, Email:
Zhonghua Wei Zhong Bing Ji Jiu Yi Xue. 2025 Jun;37(6):576-582. doi: 10.3760/cma.j.cn121430-20240329-00298.
To observe the degree of myocardial cell injury and the changes in autophagy level in rats with myocardial ischemia/reperfusion (I/R) injury induced by cardiopulmonary bypass (CPB), and to explore the regulatory role of the long non-coding RNA-urothelial carcinoma antigen 1-microRNA-143-Notch1 axis (lncRNA-UCA1-miR-143-Notch1 axis) in myocardial I/R injury induced by CPB.
Healthy male Sprague-Dawley (SD) rats were randomly divided into the following groups using the random number method: Sham operation (Sham) group, myocardial I/R injury model group (model group), empty lentivirus group, lncRNA-UCA1 upregulation group, miR-143 downregulation group, and lncRNA-UCA1 upregulation+miR-143 upregulation group, with 9 rats in each group. The rat model of myocardial I/R injury induced by CPB was established by thoracotomy aortic ligation under cardiopulmonary bypass support; in the Sham group, only threading was performed without ligation, and other procedures were the same. Seventy-two hours before modeling, the lncRNA-UCA1 upregulated group was injected with 100 μL of myocardial tissue-specific adeno-associated virus (AAV) overexpression vector of lncRNA-UCA1 via tail vein, the miR-143 downregulated group was injected with 100 μL of AAV short hairpin RNA (shRNA) vector of miR-143 via tail vein, the lncRNA-UCA1 upregulation+miR-143 upregulation group was injected with 100 μL of myocardial tissue-AAV overexpression vector of lncRNA-UCA1 and 100 μL of AAV overexpression vector of miR-143 via tail vein, and the empty vector lentivirus group was injected with 100 μL of AAV empty vector (virus titers were 1×10 TU/mL); the Sham group and the model group were injected with equal amounts of normal saline. The animals were euthanized 24 hours after intervention and cardiac tissue specimens were collected. After hematoxylin eosin (HE) staining, the damage of myocardial cells and the changes of muscle fiber tissue were observed under a light microscope; after dual staining with uranyl acetate and lead citrate, the ultrastructural damage of heart tissue was observed under a transmission electron microscopy; the expression of lncRNA-UCA1, miR-143, and Notch1 mRNA in myocardial tissue was detected by real-time fluorescence quantitative reverse transcription-polymerase chain reaction (RT-PCR); the expression of microtubule 1 light chain 3-II/I (LC3-II/I) and Notch1 protein in myocardial tissue was detected by Western blotting.
Compared with the Sham group, the myocardial cells of rats in the model group were enlarged, the intercellular space increased, autophagosomes increased, the arrangement of myocardial fibers was disordered, mitochondrial proliferated and deformed. The expression levels of lncRNA-UCA1 and Notch1 mRNA, as well as the protein expression levels of LC3-II/I and Notch1 were significantly increased, while the expression level of miR-143 was significantly decreased. Compared with the model group, the degree of myocardial cell injury in the lncRNA-UCA1 upregulation group and miR-143 downregulation group was significantly alleviated, the expression levels of Notch1 mRNA, LC3-II/I, and Notch1 protein were significantly increased [Notch1 mRNA (2): 2.66±0.24, 2.03±0.23 vs. 1.45±0.13, LC3-II/I: 2.10±0.21, 1.92±0.19 vs. 1.39±0.14, Notch1 protein (Notch1/GAPDH): 1.72±0.16, 1.57±0.16 vs. 1.34±0.13, all P < 0.05], and the expression level of miR-143 was significantly decreased (2: 0.50±0.06, 0.52±0.06 vs.0.71±0.06, P < 0.05). The expression level of lncRNA-UCA1 in the lncRNA-UCA1 upregulated group was significantly higher than that in the model group (2: 2.47±0.22 vs. 1.43±0.14, P < 0.05), while there was no significant difference in the miR-143 downregulation group compared with the model group (2: 1.50±0.16 vs. 1.43±0.14, P > 0.05). There was no significant difference in the degree of myocardial cell injury in the empty load lentivirus group and the lncRNA-UCA1 upregulation+miR-143 upregulation group compared to the model group. There were no significant differences in the expression of miR-143, Notch1 mRNA, and the autophagy level in these two groups compared to the model group. The expression level of lncRNA-UCA1 in the lncRNA-UCA1 upregulation+miR-143 upregulation group was significantly higher than that in the model group (2: 2.47±0.20 vs. 1.43±0.14, P < 0.05).
Autophagy is involved in the pathological process of myocardial I/R injury induced by CPB. The lncRNA-UCA1-microRNA-143-Notch1 axis may regulate the autophagy level to participate in the I/R injury process.
观察体外循环（CPB）诱导的心肌缺血/再灌注（I/R）损伤大鼠的心肌细胞损伤程度及自噬水平变化，探讨长链非编码RNA-尿路上皮癌抗原1-微小RNA-143-Notch1轴（lncRNA-UCA1-miR-143-Notch1轴）在CPB诱导的心肌I/R损伤中的调控作用。
采用随机数字法将健康雄性Sprague-Dawley（SD）大鼠随机分为以下几组：假手术（Sham）组、心肌I/R损伤模型组（模型组）、空载体慢病毒组、lncRNA-UCA1上调组、miR-143下调组、lncRNA-UCA1上调+miR-143上调组，每组9只大鼠。在CPB支持下通过开胸主动脉结扎建立CPB诱导的心肌I/R损伤大鼠模型；Sham组仅穿线不结扎，其他操作相同。建模前72小时，lncRNA-UCA1上调组经尾静脉注射100 μL lncRNA-UCA1的心肌组织特异性腺相关病毒（AAV）过表达载体，miR-143下调组经尾静脉注射100 μL miR-143的AAV短发夹RNA（shRNA）载体，lncRNA-UCA1上调+miR-143上调组经尾静脉注射100 μL lncRNA-UCA1的心肌组织-AAV过表达载体和100 μL miR-143的AAV过表达载体，空载体慢病毒组经尾静脉注射100 μL AAV空载体（病毒滴度为1×10 TU/mL）；Sham组和模型组注射等量生理盐水。干预24小时后处死动物，采集心脏组织标本。苏木精-伊红（HE）染色后，在光学显微镜下观察心肌细胞损伤及肌纤维组织变化；醋酸铀和柠檬酸铅双重染色后，在透射电子显微镜下观察心脏组织超微结构损伤；采用实时荧光定量逆转录-聚合酶链反应（RT-PCR）检测心肌组织中lncRNA-UCA1、miR-143和Notch1 mRNA的表达；采用蛋白质免疫印迹法检测心肌组织中微管相关蛋白1轻链3-II/I（LC3-II/I）和Notch1蛋白的表达。
与Sham组相比，模型组大鼠心肌细胞增大，细胞间隙增宽，自噬体增多，心肌纤维排列紊乱，线粒体增生变形。lncRNA-UCA1和Notch1 mRNA表达水平以及LC3-II/I和Notch1蛋白表达水平显著升高，而miR-143表达水平显著降低。与模型组相比，lncRNA-UCA1上调组和miR-143下调组心肌细胞损伤程度显著减轻，Notch1 mRNA、LC3-II/I和Notch1蛋白表达水平显著升高[Notch1 mRNA（2）：2.66±0.24，2.03±0.23 vs. 1.45±0.13，LC3-II/I：2.10±0.21，1.92±0.19 vs. 1.39±0.14，Notch1蛋白（Notch1/GAPDH）：1.72±0.16，1.57±0.16 vs. 1.34±0.13，均P < 0.05]，miR-143表达水平显著降低（2：0.50±0.06，0.52±0.06 vs.0.71±0.06，P < 0.05）。lncRNA-UCA1上调组lncRNA-UCA1表达水平显著高于模型组（2：2.47±0.22 vs. 1.43±0.14，P < 0.05），而miR-143下调组与模型组相比差异无统计学意义（2：1.50±0.16 vs. 1.43±0.14，P > 0.05）。空载体慢病毒组和lncRNA-UCA1上调+miR-143上调组心肌细胞损伤程度与模型组相比差异无统计学意义。与模型组相比，这两组miR-143、Notch1 mRNA表达及自噬水平差异无统计学意义。lncRNA-UCA1上调+miR-143上调组lncRNA-UCA1表达水平显著高于模型组（2：2.47±0.20 vs. 1.43±0.14，P < 0.05）。
自噬参与CPB诱导的心肌I/R损伤的病理过程。lncRNA-UCA1-微小RNA-143-Notch1轴可能通过调节自噬水平参与I/R损伤过程。
