目的 探讨组织激肽释放酶(TK)对酸中毒损伤神经元的保护作用及对酸敏感离子通道1a(ASICIa)介导的氧化应激反应的影响.方法 取原代培养8～10d的新生SD大鼠皮质神经元,随机分为正常对照组,酸中毒组(pH =6.0的细胞外液处理4h),TK( 100 nmol/L)、缓激肽B2受体(B2R)激动剂(缓激肽,100 nmol/L)、ASIC1a阻断剂(狼蛛毒素,100 ng/ml)及B2R拮抗剂( HOE140)预处理组.TK、B2R激动剂、ASICla阻断剂预处理组在给予酸性液处理前,先给予相应药物预处理30 min；B2R拮抗剂预处理组,在TK干预前30 min,给予HOE140(500 nmol/L),30 min后给予酸性液处理.采用活细胞计数试剂盒( CCK-8)测定各组神经元的存活率.应用不同的荧光探针标记细胞内活性氧(ROS),一氧化氮(NO),线粒体膜电位(MMP)以及细胞内游离Ca2+,采用荧光酶标仪测定上述各组细胞内各荧光物质的相对强度,计算各物质的相对含量. 结果 ①正常对照组,TK、ASIC1a阻断剂及B2R激动剂预处理组的神经元存活率分别为(96.6±2.6)％、(79.7±5.9)％、(74.2±4.6)％、(77.7±5.0)％,与酸中毒组的(59.0±6.0)％比较,差异均有统计学意义(P＜0.01).B2R拮抗剂预处理组为(64.6±3.8)％,与TK预处理组比较差异有统计学意义(P＜0.01).②与正常对照组比较,酸中毒组细胞内ROS、NO及游离Ca2+水平显著增高(P＜0.01),MMP水平显著下降(P＜0.01)；与酸中毒组比较,TK、ASIC1a阻断剂及B2R拮抗剂预处理组细胞内ROS、NO及Ca2+水平显著下降,MMP水平显著增高(P＜0.01或P＜0.05)；而与TK预处理组比较,B2R拮抗剂预处理组细胞内ROS、NO含量,游离Ca2+水平明显增高,MMP水平明显下降,P＜0.01.结论 在酸性环境下,ASIC1a激活介导了神经元内氧化应激反应,诱导神经元损伤；TK通过B2R能够减轻ASIC1a介导的氧化应激性损伤.%Objective To investigate the protective effect of tissue kallikrein (TK) on acidosia induced neuronal injury and the effect of TK on acid-sensing ion channel la (ASIC1a) mediated oxidative stress reaction. Methods Primary cultured 8-10 day cortical neurons of newborn SO rate were randomly assigned to normal control, acidosis (cells incubated in pH 6. 0 extracellular fluid for 4 hours), TK( 100 nmol/L), B2R agonist(bradykinin 100 nmol/L), ASICla blocker (PcTX, 100 ng/mL), and B2R antagonist (HOE140) pretreatment groups. Before giving the acid liquid treatment, the corresponding drug pretreatmenJ was given for 30 min in the TK, B2R agonist and ASICla hlocker pretreatment groups. At 30 min before TK intervention, B2R antagonist HOE140 (500 nmol/L) was administered, and acid liquid treatment was conducted after 30 min. A Cell counting Kit-8 (CCK-8) was used to detect the survival rate of neurons in each group. Different fluorescent probes were used to label intracellular reactive oxygen species ( ROS), nitric oxide ( NO), mitochondrial membrane potential ( MMP), and intracellular free Ca2+. A fluorescence microplale reader was used to delect the relative intensity of the fluorescent substances in cells in the above groups. The relative contents of various substances were calculated. Results ①The survival rates of neurons in the normal control, TK, ASIC la blocker, and B2R agonist groups were 96.6 ± 2.6% , 79.7 ± 5.9%, 74.2 ± 4.6% , and 77.7 ± 5. 0% , respectively. Compared to the acidosis group(59.0±6.0%), there were significant differences (P<0.01). The survival rate of neurons in the B2R antagonist group was 64.6 ± 3.8%. Compared to the TK group, there were significant differences (P < 0.01). ② Compared to the normal control group, the intracellular ROS, NO and free Ca2* levels were significantly higher (P<0.01), and the MMP level decreased significantly in the acidosis group (P<0.01); compared to the acidosis group, the intracellular ROS, NO, and Ca2+ levels decreased significantly, and the MMP level increased significantly in the TK, ASIC1a antagonist, and B2R antagonist groups (P<0.01 or P<0.05); and compared to the TK group, the intracellular ROS, NO and free Ca2+ levels increased significantly, and the MMP level decreased significantly in the B2R antagonist group (P<0.01). Conclusion In an acid environment, ASIC1 a activation mediates the neuronal oxidalive stress reaction and induces neuronal damage. TK may attenuate ASIC1a mediated oxidative stress injury via B2R activation.
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