基于TCTP/AKT/P53信号通路探讨中药血必净注射液治疗脓毒症诱导大鼠心肌损伤作用机制研究

Investigation of the mechanism of Xuebijing Injection in treating sepsis-induced myocardial injury in rats via the TCTP/AKT/P53 signaling pathway

关键词:  脓毒症心肌病  血必净注射液  TCTP/AKT/P53信号通路

Key words:  sepsis-induced cardiomyopathy  Xuebijing Injection  TCTP/AKT/P53 signaling pathway

摘要:

[目的] 探讨中药血必净注射液（XBJ）通过翻译控制肿瘤蛋白（TCTP）/蛋白激酶B（AKT）/肿瘤蛋白53（P53）信号通路改善脓毒症心肌病（SICM）的作用机制研究。[方法] 将18只SD大鼠随机分配至假手术组（Sham）、盲肠结扎穿孔术组（CLP）和血必净治疗组（XBJ），每组6只。给予连续3 d的相应处理后，采用苏木素-伊红染色（HE）观察心肌组织的病理变化。使用超声技术评估大鼠的左心室射血分数（LVEF）及左心室缩短分数（LVFS）。通过全自动生化分析仪测定血清中的肌酸激酶（CK）和乳酸脱氢酶（LDH）浓度。通过酶联免疫吸附法（ELISA）检测血清中的肿瘤坏死因子α（TNF-α）、白细胞介素-6（IL-6）和白细胞介素-1β（IL-1β）的表达水平。蛋白免疫印迹法（Western blot）用于检测心肌组织中B细胞淋巴瘤-2（Bcl-2）、Bcl-2相关X蛋白（Bax）、剪切型含半胱氨酸的天冬氨酸蛋白水解酶3（Cleaved Caspase3）、TCTP、p-AKT、P53蛋白表达。采用免疫组化观察Bcl-2、Bax、Cleaved Caspase3的蛋白表达。最后，采用免疫荧光法对心脏组织中的TCTP、p-AKT及P53的表达情况进行观察。[结果] XBJ治疗后可显著降低CLP组大鼠的CK、LDH、TNF-α、IL1-β及IL-6的表达水平，提高其LVEF和LVFS，差异具有统计学意义（P<0.05）。超声心动图、HE染色的结果表明XBJ治疗可改善SICM大鼠心肌损伤。Western blot结果表明，与CLP组相比，XBJ治疗后降低了Bax、Cleaved Caspase3、TCTP和p-AKT的表达水平，并上调了Bcl-2和P53蛋白的表达水平，差异具有统计学意义（P<0.05）。免疫组化结果显示，XBJ治疗后可上调Bcl-2的表达，而下调Bax、Cleaved Caspase3的表达。同时免疫荧光结果也显示与CLP组相比，XBJ组心肌组织中TCTP、p-AKT水平降低，而P53水平升高。[结论] XBJ连续治疗可能通过调节TCTP/AKT/P53信号通路抑制心肌细胞凋亡和炎症反应，改善SICM。

Abstract:

[Objective] To investigate the mechanism by which Xuebijing Injection（XBJ） improves sepsis-induced cardiomyopathy（SICM） through the translationally controlled tumor protein（TCTP）/protein kinase B（AKT）/tumor protein 53（P53） signaling pathway. [Methods] Eighteen SD rats were randomly assigned to the sham group，cecum ligation and puncture（CLP） group，and XBJ group，with 6 rats in each group. After 3 days of continuous treatment，histopathological changes in myocardial tissue were observed using hematoxylin and eosin（HE） staining. left ventricular ejection fraction（LVEF） and left ventricular fractional shortening（LVFS） were assessed by ultrasound. Serum levels of creatine kinase（CK） and lactate dehydrogenase（LDH） were measured using an automated biochemical analyzer. Enzyme-linked immunosorbent assay（ELISA） was performed to measure the expression levels of tumor necrosis factor-α（TNF-α），lnterleukin-6（IL-6），and lnterleukin-1β（IL-1β） in serum. Western blotting was used to detect the protein expression levels of TCTP，p-AKT，P53，B-cell lymphoma 2（Bcl-2），Bcl-2-associated X protein（Bax），and Cleaved Caspase3 in myocardial tissue. Immunohistochemistry was employed to observe the expression of Bax，Bcl-2，and Cleaved Caspase3 proteins. Finally，immunofluorescence was used to examine the expression levels of TCTP，p-AKT，and P53 in cardiac tissue. [Results] XBJ treatment significantly reduced the serum levels of CK，LDH，TNF-α，IL-1β，and IL-6，while improving LVEF and LVFS in CLP rats，with statistically significant differences（P<0.05）. Ultrasound，HE staining，and electron microscopy results indicated that XBJ treatment alleviated myocardial injury in SICM rats. Western blot analysis showed that XBJ treatment decreased the expression levels of Bax，Cleaved Caspase3，TCTP，and p-AKT，while increasing the expression of Bcl-2 and P53 compared to the CLP group，with statistically significant differences（P<0.05）. Immunohistochemical analysis confirmed that XBJ treatment upregulated Bcl-2 and downregulated Bax and Cleaved Caspase3. Moreover，immunofluorescence results revealed that XBJ treatment decreased the expression of TCTP and p-AKT，while increasing P53 expression in myocardial tissue compared to the CLP group. [Conclusion] XBJ may improve SICM by regulating the TCTP/AKT/P53 signaling pathway，inhibiting myocardial cell apoptosis and inflammatory responses.