Objective: To explore the mechanism of CD38-mediated cardiac damage under hypoxic-ischemic (H/I) conditions. Methods: Twenty CD38(-/-) male mice (8-week-old) and 20 wild-type (WT) male C57BL/6J mice (8-week-old) were randomly selected to construct the model of approximately 25% of the total body surface area (TBSA) burn injury. The cardiomyocytes (CMs) were separated from neonatal mice (1day) to construct the H/I injury model. Ad-CD38 adenovirus was transfected into CD38(-)/- primary CMs to callback CD38 expression. Animal experiments were grouped into WT-control group, CD38(-/-)-control group, WT-burn group, and CD38(-/-)-burn group (10 mice in each group). Primary CMs were divided into 6 groups: WT-normoxia group, CD38(-/-)-normoxia group, CD38(-/-)+Ad-CD38-normoxia group, WT-H/I group, CD38(-/-)-H/I group, CD38(-/-)+Ad-CD38-H/I group. The release of lactic dehydrogenase (LDH) from CMs and the cell viability were measured to estimate the level of myocardial injury. Ultrastructure of cardiomyocytes was examined by electron microscope. CD38 protein level and mitochondrial apoptosis-related proteins were detected by Western blot. Flow cytometry was used to detect mitochondrial reactive oxygen species (MitoSOX) of CMs under H/I condition. Cardiac function of mice was detected by ultrasonic apparatus. Results: (1) Animal experiments: The expression level of cardiac CD38 in WT-burn group was significantly higher than that in sham group (P<0.001). The heart function of CD38(-/-)-burn group was obviously better than WT-burn group [ejection fraction (EF)%: (84.70±2.31)% vs (76.10±2.96)%, shortening fraction (FS)%: (48.90±5.00)% vs (38.10±2.80)%] (both P<0.001). (2) Cell experiments: The expression level of cardiac CD38 in WT CMs under H/I condition was significantly higher than that in WT CMs under normoxia condition (P<0.05). The level of LDH, apoptotic cell and MitoSOX in CD38(-/-)-H/I group were fewer than WT-H/I group and CD38(-/-)+Ad-CD38(-)H/I group [(11.2±3.0)% vs (18.2±3.4)% and (17.6±4.0)%, (13.0±2.8)% vs (23.1±4.9)% and (23.3±6.0)%, (162±11)% vs (228±18)% and (220±18)%] (all P<0.001). The levels of cleaved-caspase3, Cytochrome-C in CD38(-/-)-H/I group were significantly lower than those in WT-H/I group and CD38(-/-)+Ad-CD38-H/I group (P<0.001). The cell viability in CD38(-/-)-H/I group was higher than that in WT-H/I group and CD38(-/-)+Ad-CD38-H/I group (0.355±0.043 vs 0.280±0.051 and 0.291±0.024) (all P<0.05). Electron microscopy results showed that structure of mitochondria in CD38(-/-)-H/I group was better than in WT-H/I group and CD38(-/-)+Ad-CD38-H/I group. Conclusion: Overexpression of CD38 contributes to cardiac damage by stimulating mitochondrial apoptotic pathway.
目的： 探讨CD38分子介导烧伤小鼠缺血缺氧性心肌损伤的机制。 方法： 随机选取8周龄CD38(-/-)雄性小鼠和野生型（WT）雄性C57BL/6J小鼠各20只构建烧伤模型，动物实验分为WT对照组、CD38(-/-)对照组、WT烧伤组、CD38(-/-)烧伤组各10只，烧伤24 h后取材进行实验。利用出生1 d的小鼠乳鼠培养原代心肌细胞构建细胞缺血缺氧模型。细胞实验分为WT常氧组、CD38(-/-)常氧组、CD38(-/-)+Ad-CD38常氧组、WT缺血缺氧组、CD38(-/-)缺血缺氧组、CD38(-/-)+Ad-CD38缺血缺氧组。通过检测乳酸脱氢酶（LDH）释放及细胞活性明确心肌细胞缺血缺氧损伤程度；电镜检测心肌细胞超微结构；免疫印迹检测心肌细胞中CD38蛋白及线粒体凋亡相关蛋白水平；流式细胞技术检测心肌细胞线粒体活性氧（MitoSOX）水平；凋亡试剂盒检测心肌细胞凋亡情况；小动物超声仪检测小鼠心功能。 结果： （1）动物实验：WT烧伤组CD38表达水平显著高于WT对照组（P<0.001），CD38(-/-)烧伤组心功能明显优于WT烧伤组[射血分数：（84.70±2.31）%比（76.10±2.96）%；短轴缩短率：（48.90±5.00）%比（38.10±2.80）%]（均P<0.001）。（2）细胞实验：WT缺血缺氧组CD38表达水平高于WT常氧组（P<0.05）；CD38(-/-)缺血缺氧组LDH释放水平、心肌细胞凋亡率、MitoSOX水平均显著低于WT缺血缺氧组和CD38(-/-)+Ad-CD38缺血缺氧组[（11.2±3.0）%比（18.2±3.4）%和（17.6±4.0）%、（13.0±2.8）%比（23.1±4.9）%和（23.3±6.0）%、（162±11）%比（228±18）%和（220±18）%]（均P<0.001），活化的半胱氨酸天冬氨酸蛋白酶（caspase）3、细胞色素C也有相似结果（均P<0.001）；细胞活性均显著高于上述两组（0.355±0.043比0.280±0.051和0.291±0.024）（均P<0.05）。电镜结果显示CD38(-/-)缺血缺氧组心肌细胞内线粒体结构优于WT缺血缺氧组及CD38(-/-)+Ad-CD38缺血缺氧组。 结论： 心肌细胞内高表达的CD38分子促进线粒体凋亡因子释放，诱导心肌细胞凋亡，介导烧伤小鼠缺血缺氧性心肌损伤。.
Keywords: Antigens, CD38; Burns; Mitochondrial apoptosis; Myocardial ischemia.