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目的研究钙蛋白酶抑制剂ALLN对缺血-再灌注(I-R)大鼠心肌肌浆网钙泵(SERCA)功能的影响。方法 24只雄性SD大鼠随机分为正常灌注组(对照组)、I-R组、ALLN+I-R组和二甲亚砜+I-R组(DMSO+I-R组),分别检测再灌注15min时冠状动脉流出量(CF)和冠脉流出液中乳酸脱氢酶(LDH)漏出量;荧光分光光度计测定荧光强度来反映钙蛋白酶活性;分别采用改良p-NPP法和Millipore过滤技术测定SERCA的活性和ATP依赖的SERCA45Ca2+摄取率,免疫印迹法检测心肌细胞SERCA蛋白表达水平。结果与I-R组相比,ALLN+I-R组CF、SERCA蛋白表达水平、SERCA的活性和45Ca2+摄取率显著增高(P<0.01),而LDH漏出量和钙蛋白酶活性显著降低(P<0.01)。结论 ALLN对离体大鼠心脏I-R损伤有保护作用,与其抑制钙蛋白酶活性,一定程度减轻I-R损伤对SERCA功能及蛋白表达水平的影响有关。
Objective To investigate the effect of calpain inhibitor ALLN on cardiac sarcoplasmic reticulum calcium pump (SERCA) function in rats with ischemia-reperfusion (I-R). Methods Twenty-four male SD rats were randomly divided into normal perfusion group (control group), IR group, ALLN + IR group and dimethyl sulfoxide + IR group (DMSO + IR group) (CF) and coronary fluid efflux, lactate dehydrogenase (LDH) leakage in coronary effluent, fluorescence intensity was measured by fluorescence spectrophotometer to reflect calpain activity, SERCA activity and ATP dependence were determined by modified p-NPP and Millipore filtration respectively SERCA45Ca2 + uptake rate, Western blotting was used to detect the expression of SERCA protein in cardiomyocytes. Results Compared with I-R group, the levels of CF, SERCA, SERCA and 45Ca2 uptake were significantly increased in ALLN + I-R group (P <0.01), while the LDH leakage and calpain activity were significantly decreased (P <0.01). Conclusion ALLN has a protective effect on I-R injury in isolated rat hearts, which is related to the inhibition of calpain activity and alleviating the effect of I-R injury on SERCA function and protein expression to a certain extent.