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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(ASIC1a)介导的氧化应激反应的影响。方法取原代培养8～10 d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4 h),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、ASIC1a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 effects of tissue kallikrein (TK) on neurons injured by acidosis and its effect on oxidative stress induced by acid-sensitive ion channel 1a (ASIC1a). Methods Primary cortical neurons of neonatal SD rats were cultured for 8-10 days. They were randomly divided into normal control group, acidosis group (pH = 6.0 for 4 h), TK (100 nmol / L) (B2R) agonist (bradykinin, 100 nmol / L), ASIC1a blocker (tiboxanide 100 ng / ml) and B2R antagonist (HOE140). TK, B2R agonist and ASIC1a pretreatment group were pretreated with appropriate drugs for 30 min prior to acidic solution pretreatment. In B2R antagonist pretreatment group, HOE140 (500 nmol / L ), After 30 min give acidic solution. The viability of neurons in each group was determined by using live cell counting kit (CCK-8). Different fluorescent probes were used to label intracellular reactive oxygen species (ROS), nitric oxide (NO), mitochondrial membrane potential (MMP) and intracellular free Ca2 +. Fluorescence microplate reader was used to measure the relative fluorescence intensity Strength, calculate the relative content of each substance. Results ① The survival rates of neurons in the normal control group, TK, ASIC1a inhibitor and B2R agonist pretreatment group were (96.6 ± 2.6)%, (79.7 ± 5.9)%, (74.2 ± 4.6)%, (77.7 ± 5.0%), compared with (59.0 ± 6.0)% in acidosis group, the differences were statistically significant (P <0.01). B2R antagonist pretreatment group was (64.6 ± 3.8)%, compared with TK pretreatment group, the difference was statistically significant (P <0.01). ②Compared with the normal control group, ROS, NO and free Ca2 + levels were significantly increased (P <0.01) and MMP levels were significantly decreased in the acidosis group (P <0.01). Compared with the acidosis group, TK, ASIC1a blockers and B2R antagonist pretreatment group intracellular ROS, NO and Ca2 levels decreased significantly, MMP levels were significantly increased (P <0.01 or P <0.05), while compared with the TK pretreatment group, B2R antagonist pretreatment group intracellular ROS, NO Content, free Ca2 + level was significantly increased, MMP levels decreased significantly, P <0.01. Conclusion In acidic environment, ASIC1a activation mediates oxidative stress in neurons and induces neuronal damage. TK can reduce ASIC1a-mediated oxidative stress-induced injury through B2R.