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目的探讨糖尿病小鼠主动脉平滑肌活性氧簇(ROS)/核因子-κB(NF-κB)信号通路对肌肉环指蛋白(Muscle RING finger,MuRF)1介导大电导钙激活钾通道β1亚基(BK-β1)降解的调控机制。方法腹腔注射链脲霉素制备1型糖尿病小鼠模型,将雄性C57/BL6J小鼠分为6组(每组15只):对照组,对照+N-乙酰半胱氨酸(NAC,ROS清除剂)组,对照+吡咯烷二硫代氨基甲酸盐(PDTC,NF-κB抑制剂)组,糖尿病组,糖尿病+NAC组,糖尿病+PDTC组。对照+NAC组和糖尿病+NAC组给予NAC 100 mg/(kg·d)腹腔注射治疗,对照+PDTC组和糖尿病+PDTC组给予PDTC 50 mg/(kg·d)腹腔注射治疗,对照组和糖尿病组给予同等体积的生理盐水腹腔注射。药物治疗12周后,Western blot检测胸主动脉BK-β1、MuRF1、Rel A/p65的表达水平;动脉血管舒张反应性实验检测胸主动脉血管环对BK通道的激动剂NS-1619的反应性;酶消化法急性分离小鼠胸主动脉平滑肌细胞,全细胞膜片钳实验技术记录小鼠胸主动脉平滑肌细胞BK通道的电流。结果与对照组、对照+NAC组和对照+PDTC组相比,糖尿病组的BK-β1的表达显著降低(P<0.05),MuRF1和Rel A/p65的表达显著增加(P<0.05),胸主动脉血管环对NS-1619的舒张反应性降低(P<0.05),当刺激电压>50 mV时,糖尿病组胸主动脉平滑肌细胞BK通道电流显著降低(P<0.05);与糖尿病组相比,糖尿病+NAC组和糖尿病+PDTC组的BK-β1的表达显著增加(P<0.05),MuRF1和Rel A/p65的表达显著降低(P<0.05),胸主动脉血管环对NS-1619的舒张反应性显著改善(P<0.05),当刺激电压>50 mV时,糖尿病+NAC组和糖尿病+PDTC组胸主动脉平滑肌细胞BK通道电流显著增加(P<0.05)。结论 NAC和PDTC可抑制糖尿病小鼠胸主动脉平滑肌MuRF1介导的BK-β1降解,ROS/NF-κB信号通路可能是参与调控MuRF1介导的BK-β1降解。
Objective To investigate the effect of muscle reactive oxygen species (ROS) / nuclear factor-κB (NF-κB) signaling pathway on muscarinic RING finger (MuRF) 1-mediated large-conductance calcium-activated potassium channel β1 subunit (BK-β1) degradation mechanism of regulation. Methods The model of type 1 diabetic mice was established by intraperitoneal injection of streptozotocin. The male C57 / BL6J mice were divided into 6 groups (n = 15): control group, NAC, ROS scavenging (PDTC, NF-κB inhibitor) group, diabetic group, diabetic + NAC group, diabetic + PDTC group. The rats in control group and NAC group were given intraperitoneal injection of NAC 100 mg / (kg · d), PDTC 50 mg / (kg · d) in control + PDTC group and diabetes + PDTC group, and the control group and diabetes mellitus Group given the same volume of saline intraperitoneal injection. Twelve weeks after the drug treatment, the expression of BK-β1, MuRF1 and Rel A / p65 in the thoracic aorta was detected by Western blot. The reactivity of the thoracic aortic ring to the BK channel agonist NS-1619 Acute pulmonary artery smooth muscle cells were isolated by enzymatic digestion and the whole cell patch clamp technique was used to record the current of BK channels in mouse thoracic aortic smooth muscle cells. Results Compared with the control group, the control + NAC group and the control + PDTC group, the expression of BK-β1 in diabetic group was significantly decreased (P <0.05), and the expression of MuRF1 and Rel A / p65 was significantly increased The vasorelaxation of NS-1619 was decreased in aortic rings (P <0.05). When stimulation voltage was> 50 mV, the currents of BK channels in thoracic aorta smooth muscle cells were significantly decreased (P <0.05). Compared with diabetic group (P <0.05), and the expression of MuRF1 and Rel A / p65 in diabetic + NAC group and diabetic + PDTC group were significantly decreased (P <0.05). The expression of B7- (P <0.05). When the stimulation voltage was> 50 mV, the currents of BK channels in thoracic aorta smooth muscle cells in diabetic + NAC group and diabetic + PDTC group were significantly increased (P <0.05). Conclusion NAC and PDTC can inhibit MuRF1-mediated degradation of BK-β1 in diabetic rat aortic smooth muscle. ROS / NF-κB signaling pathway may be involved in the regulation of MuRF1-mediated BK-β1 degradation.