摘要目的:探讨黄芪甲苷（AS-Ⅳ）对高位脊髓损伤（SCI）大鼠急性期心肌损伤的影响。方法:选取24只8~10周龄、体重250~300 g的健康雄性SD大鼠，按随机数字表法分为四组：假手术组、高位SCI组（SCI组）、高位SCI+AS-Ⅳ组（SCI+AS-Ⅳ组）和高位SCI+AS-Ⅳ+沉默信息调节因子1（SIRT1）抑制剂EX527组（SCI+AS-Ⅳ+EX527组），每组6只。采用改良Allen法建立SCI模型，假手术组仅暴露脊髓。SCI+AS-Ⅳ组在伤后即刻腹腔注射40 mg/kg AS-Ⅳ；SCI+AS-Ⅳ+EX527组于伤前1 h腹腔注射5 mg/kg EX527，并在伤后即刻腹腔注射40 mg/kg AS-Ⅳ；假手术组和SCI组腹腔注射等量生理盐水。伤后苏醒即刻观察并记录各组大鼠的后肢运动功能，并采用BBB法评估。伤后24 h，透射电镜下观察心肌细胞超微结构；ELISA法检测血清肌钙蛋白I（cTnI）、心肌组织炎症因子白细胞介素（IL）-18和IL-1β水平；二氢乙啶（DHE）法检测心肌组织活性氧（ROS）水平；生化法检测超氧化物歧化酶（SOD）活性和丙二醛（MDA）水平；RT-PCR和Western blot检测核苷酸结合寡聚化结构域样受体3（NLRP3）、含半胱氨酸的天冬氨酸蛋白水解酶-1（caspase-1）、消皮素D（GSDMD）、SIRT1和过氧化物酶体增殖物激活受体γ共激活因子-1α（PGC-1α）mRNA及蛋白表达水平；caspase-1和TUNEL免疫荧光双染色法检测心肌细胞焦亡率。结果:伤后苏醒即刻，假手术组后肢活动正常，BBB评分均为21（21，21）分；其余各组双后肢呈弛缓性瘫痪，自主排泄消失，BBB评分均为0（0，0）分。伤后24 h透射电镜显示，假手术组心肌细胞超微结构未见明显异常，SCI组细胞出现不同程度改变。伤后24 h ELISA法检测结果显示，SCI组血清cTnI水平为（1 435.3±148.1）pg/ml，高于假手术组的（619.6±95.4）pg/ml（ P<0.01）；SCI+AS-Ⅳ组cTnI水平为（1 154.0±80.0）pg/ml，低于SCI组（ P<0.01）；SCI+AS-Ⅳ+EX527组cTnI水平为（1 321.8±50.2）pg/ml，高于SCI+AS-Ⅳ组（ P<0.05）。SCI组心肌组织IL-18和IL-1β水平分别为（493.0±145.0）pg/ml、（936.7±93.2）pg/ml，均高于假手术组的（131.1±62.5）pg/ml和（281.7±83.6）pg/ml（ P<0.01）；SCI+AS-Ⅳ组心肌组织IL-18和IL-1β水平分别为（182.4±45.6）pg/ml、（573.4±99.5）pg/ml，均低于SCI组（ P<0.01）；SCI+AS-Ⅳ+EX527组心肌组织IL-18和IL-1β水平分别为（337.4±72.0）pg/ml、（742.6±82.7）pg/ml，均高于SCI+AS-Ⅳ组（ P<0.05），仍低于SCI组（ P<0.01）。伤后24 h DHE法和生化法检测结果显示，SCI组心肌组织ROS和MDA水平分别为（65±6）%、（1.97±0.27）nmol/mg，均高于假手术组的（19±10）%和（1.03±0.16）nmol/mg（ P<0.01）；SCI+AS-Ⅳ组心肌组织ROS和MDA水平分别为（37±10）%、（1.39±0.11）nmol/mg，均低于SCI组（ P<0.01）；SCI+AS-Ⅳ+EX527组心肌组织ROS和MDA水平分别为（52±7）%、（1.70±0.14）nmol/mg，均高于SCI+AS-Ⅳ组（ P<0.05）。SCI组心肌组织SOD水平为（658.48±77.56）U/mg，低于假手术组的（1 059.55±71.91）U/mg（ P<0.01）；SCI+AS-Ⅳ组心肌组织SOD水平为（901.74±32.30）U/mg，高于SCI组（ P<0.01）；SCI+AS-Ⅳ+EX527组心肌组织SOD水平为（799.86±26.70）U/mg，低于SCI+AS-Ⅳ组（ P<0.05）。伤后24 h RT-PCR显示，SCI组心肌组织NLRP3、caspase-1和GSDMD mRNA表达水平分别为2.07±0.25、2.46±0.28、1.82±0.12，均高于假手术组的1.10±0.13、0.95±0.17和1.03±0.08（ P<0.01）；SCI+AS-Ⅳ组心肌组织NLRP3、caspase-1和GSDMD mRNA表达水平分别为1.47±0.24、1.51±0.16、1.42±0.13，均低于SCI组（ P<0.01）；SCI+AS-Ⅳ+EX527组心肌组织NLRP3、caspase-1和GSDMD mRNA表达水平分别为1.93±0.28、1.97±0.31、1.65±0.16，均高于SCI+AS-Ⅳ组（ P<0.05）。SCI组心肌组织SIRT1和PGC-1α mRNA表达水平分别为0.41±0.09、0.56±0.07，均低于假手术组的1.20±0.14和1.29±0.20（ P<0.01）；SCI+AS-Ⅳ组心肌组织SIRT1和PGC-1α mRNA表达水平分别为0.78±0.08、1.01±0.19，均高于SCI组（ P<0.01）；SCI+AS-Ⅳ+EX527组心肌组织SIRT1和PGC-1α mRNA表达水平分别为0.53±0.12和0.72±0.22，均低于SCI+AS-Ⅳ组（ P<0.05）。伤后24 h Western blot显示，SCI组心肌组织NLRP3、caspase-1和GSDMD蛋白表达水平分别为1.00±0.20、0.60±0.19、0.77±0.15，均高于假手术组的0.27±0.09、0.18±0.10和0.28±0.08（ P<0.01）；SCI+AS-Ⅳ组心肌组织NLRP3、caspase-1和GSDMD蛋白表达水平分别为0.59±0.10、0.25±0.11、0.33±0.11，均低于SCI组（ P<0.01）；SCI+AS-Ⅳ+ EX527组心肌组织NLRP3、caspase-1和GSDMD蛋白表达水平分别为0.85±0.15、0.54±0.12、0.55±0.13，均高于SCI+AS-Ⅳ组（ P<0.05）。SCI组心肌组织SIRT1和PGC-1α蛋白表达水平分别为0.44±0.16、0.28±0.10，均低于假手术组的0.93±0.22和0.75±0.16（ P<0.01）；SCI+AS-Ⅳ组心肌组织SIRT1和PGC-1α蛋白表达水平分别为0.78±0.19和0.55±0.12，均高于SCI组（ P<0.01）；SCI+AS-Ⅳ+ EX527组心肌组织SIRT1和PGC-1α蛋白表达水平分别为0.46±0.16、0.35±0.07，均低于SCI+AS-Ⅳ组（ P<0.05）。伤后24 h caspase-1和TUNEL免疫荧光双染色显示，SCI组心肌细胞焦亡率为（34.5±6.7）%，高于假手术组的（5.3±2.9）%（ P<0.01）；SCI+AS-Ⅳ组心肌细胞焦亡率为（13.4±3.0）%，低于SCI组（ P<0.01）；SCI+AS-Ⅳ+EX527组心肌细胞焦亡率为（22.5±5.9）%，高于SCI+AS-Ⅳ组（ P<0.01），仍低于SCI组（ P<0.01）。 结论:AS-Ⅳ能显著减轻高位SCI大鼠急性期心肌损伤，其机制可能涉及激活心肌中的SIRT1/PGC-1α信号通路，对线粒体产生保护作用，提高抗氧化应激能力，并有效抑制NLRP3炎症小体介导的细胞焦亡途径。

abstractsObjective:To investigate the effects of astragaloside IV (AS-IV) on acute myocardial injury in rats with high-level spinal cord injury (SCI).Methods:Twenty-four healthy male SD rats, aged 8-10 weeks with a body weight of 250-300 g, were randomly divided into 4 groups using a random number table method: sham operation group, high-level SCI group (SCI group), high-level SCI+AS-IV group (SCI+AS-IV group) and high-level SCI+AS-IV+silent information regulator 1 (SIRT1) inhibitor EX527 group (SCI+AS-IV+EX527 group), with 6 rats in each group. The SCI model was established using the modified Allen method and the sham operation group underwent the spinal cord exposure only. In the SCI+AS-IV group, 40 mg/kg of AS-IV was injected intraperitoneally immediately after injury. SCI+AS-IV+EX527 group received an intraperitoneal injection of 5 mg/kg EX527 at one hour before injury and another injection of 40 mg/kg AS-IV in the same way immediately after injury. The sham operation group and the SCI group received an equal volume of saline via intraperitoneal injection. Immediately after awakening from injury, the hind limb motor function of the rats in each group was observed, recorded and then evaluated using the BBB method. At 24 hours after injury, the ultrastructure of the cardiomyocytes was examined under a transmission electron microscope; the levels of serum cardiac troponin I (cTnI), myocardial tissue inflammatory factors interleukin (IL)-18 and IL-1β were quantified by the ELISA method; the level of reactive oxygen species (ROS) of the myocardial tissue was assessed utilizing the dihydroethidium (DHE) assay; biochemical analyses were employed to determine the superoxide dismutase (SOD) activity and malondialdehyde (MDA) concentrations; mRNA and protein expression levels of nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3), cysteinyl aspartate specific proteinase-1 (caspase-1), gasdermin D (GSDMD), SIRT1 and peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) were examined using RT-PCR and Western blot; cardiomyocyte pyroptosis rate was evaluated by caspase-1 and TUNEL double-labeled fluorescence staining.Results:Immediately after awakening from injury, the sham operation group exhibited normal hind limb activity, with BBB scores of 21(21, 21)points, while the remaining groups displayed flaccid paralysis in both hind limbs, accompanied by the cessation of spontaneous excretion, with BBB scores of 0(0, 0)points. At 24 hours after injury, transmission electron microscopy did not reveal any significant abnormalities in the ultrastructure of the myocardiomyocytes in the sham operation group, while changes of varying degrees were observed in the SCI group. The ELISA results indicated that at 24 hours after injury, the serum cTnI level in the SCI group was (1 435.3±148.1)pg/ml, higher than (619.6±95.4)pg/ml in the sham operation group ( P<0.01); the cTnI level was (1 154.0±80.0)pg/ml in the SCI+AS-IV group, lower than that in the SCI group ( P<0.01); the cTnI level was (1 321.8±50.2)pg/ml in the SCI+AS-IV+EX527 group, higher than that in the SCI+AS-IV group ( P<0.05). The levels of IL-18 and IL-1β in the myocardial tissue in the SCI group were (493.0±145.0)pg/ml and (936.7±93.2)pg/ml, higher than (131.1±62.5)pg/ml and (281.7±83.6)pg/ml in the sham operation group ( P<0.01); the levels of IL-18 and IL-1β in the SCI+AS-IV group were (182.4±45.6)pg/ml and (573.4±99.5)pg/ml, lower than those in the SCI group ( P<0.01); the levels of IL-18 and IL-1β in the SCI+AS-IV+EX527 group were (337.4±72.0)pg/ml and (742.6±82.7)pg/ml, higher than those in the SCI+AS-IV group ( P<0.05), yet lower than those in the SCI group ( P<0.01). At 24 hours after injury, DHE and biochemical assays showed that the levels of ROS and MDA in the myocardial tissue in the SCI group were (65±6)% and (1.97±0.27)nmol/mg, higher than (19±10)% and (1.03±0.16)nmol/mg in the sham operation group ( P<0.01); the ROS and MDA levels in the SCI+AS-IV group were (37±10)% and (1.39±0.11)nmol/mg, lower than those in the SCI group ( P<0.01); the ROS and MDA levels in the SCI+AS-IV+EX527 group were (52±7)% and (1.70±0.14)nmol/mg, higher than those in the SCI+AS-IV group ( P<0.05). The SOD level in the myocardial tissue of the SCI group was (658.48±77.56)U/mg, lower than (1 059.55±71.91)U/mg in the sham operation group ( P<0.01); the SOD level in the SCI+AS-IV group was (901.74±32.30)U/mg, higher than that in the SCI group ( P<0.01); the SOD level in the myocardial tissue in the SCI+AS-IV+EX527 group was (799.86±26.70)U/mg, lower than that in the SCI+AS-IV group ( P<0.05). At 24 hours after injury, RT-PCR showed that the mRNA expression levels of NLRP3, caspase-1 and GSDMD in the myocardial tissue of the SCI group were 2.07±0.25, 2.46±0.28 and 1.82±0.12 respectively, which were higher than 1.10±0.13, 0.95±0.17 and 1.03±0.08 in the sham operation group ( P<0.01); the mRNA expression levels of NLRP3, caspase-1 and GSDMD in the SCI+AS-IV group were 1.47±0.24, 1.51±0.16 and 1.42±0.13 respectively, which were lower than those in the SCI group ( P<0.01); the mRNA expression levels of NLRP3, caspase-1 and GSDMD in the SCI+AS-IV+EX527 group were 1.93±0.28, 1.97±0.31 and 1.65±0.16 respectively, which were higher than those in the SCI+AS-IV group, yet lower than those in the SCI group ( P<0.05). The mRNA expression levels of SIRT1 and PGC-1α in the myocardial tissue in the SCI group were 0.41±0.09 and 0.56±0.07, lower than 1.20±0.14 and 1.29±0.20 in the sham operation group ( P<0.01); the mRNA expression levels of SIRT1 and PGC-1α in the myocardial tissue in the SCI+AS-IV group were 0.78±0.08 and 1.01±0.19, higher than those of the SCI group ( P<0.01); the mRNA expression levels of SIRT1 and PGC-1α in the myocardial tissue of the SCI+AS-IV+EX527 group were 0.53±0.12 and 0.72±0.22, lower than those of the SCI+AS-IV group ( P<0.05). At 24 hours after injury, the western blot analysis showed that the protein expression levels of NLRP3, caspase-1 and GSDMD in the myocardial tissue in the SCI group were 1.00±0.20, 0.60±0.19 and 0.77±0.15 respectively, which were higher than 0.27±0.09, 0.18±0.10 and 0.28±0.08 in the sham operation group ( P<0.01); the protein expression levels of NLRP3, caspase-1 and GSDMD in the SCI+AS-IV group were 0.59±0.10, 0.25±0.11 and 0.33±0.11 respectively, lower than those in the SCI group ( P<0.01); the protein expression levels of NLRP3, caspase-1 and GSDMD in the myocardial tissue in the SCI+AS-IV+EX527 group were 0.85±0.15, 0.54±0.12 and 0.55±0.13 respectively, higher than those in the SCI+AS-IV group ( P<0.05). The protein expression levels of SIRT1 and PGC-1α in the myocardial tissue in the SCI group were 0.44±0.16 and 0.28±0.10, lower than 0.93±0.22 and 0.75±0.16 in the sham operation group ( P<0.01); the protein expression levels of SIRT1 and PGC-1α in the myocardial tissue in the SCI+AS-IV group were 0.78±0.19 and 0.55±0.12, higher than those in the SCI group ( P<0.01); the protein expression levels of SIRT1 and PGC-1α in the myocardial tissue in the SCI+AS-IV+EX527 group were 0.46±0.16 and 0.35±0.07, lower than those in the SCI+AS-IV group ( P<0.05). At 24 hours after injury, caspase-1 and TUNEL double-labeled fluorescence staining showed that the cardiomyocyte pyroptosis rate in the SCI group was (34.5±6.7)%, higher than (5.3±2.9)% in the sham operation group ( P<0.01); the cardiomyocyte pyroptosis rate in the SCI+AS-IV group was (13.4±3.0)%, lower than that in the SCI group ( P<0.01); the cardiomyocyte pyroptosis rate in the SCI+AS-IV+EX527 group was (22.5±5.9)%, higher than that in the SCI+AS-IV group ( P<0.01), yet lower than that in the SCI group ( P<0.01). Conclusions:AS-IV can significantly reduce acute myocardial injury in rats with high-level SCI. Its mechanism may involve activating the myocardial SIRT1/PGC-1α signaling pathway, protecting the mitochondria, enhancing the ability to resist oxidative stress, and effectively inhibiting the NLRP3 inflammasome-mediated pyroptosis pathway.