丙二醛对大鼠肝线粒体呼吸功能及相关脱氢酶活性影响

Malonaldehyde inhibits respiratory function and enzyme activities in isolated rat liver mitochondria

目的:探索脂质氧化终产物丙二醛(MDA)在体外对线粒体呼吸链复合物及线粒体内关键酶活性的影响。方法:采用不同浓度MDA体外干预大鼠肝线粒体,氧电极法检测线粒体呼吸控制率(RCR)、磷氧比(P/O),测定呼吸链复合物及α酮戊二酸脱氢酶、丙酮酸脱氢酶、苹果酸脱氢酶活性。结果:线粒体经MDA作用后,以苹果酸/谷氨酸或琥珀酸作底物,线粒体两条呼吸途径对MDA呈现不同的耐受力,前者在MDA100μmol/L时RCR显著降低(P<0.05),400μmol/L时P/O降低(P<0.05);后者MDA浓度达到400和800μmol/L时,线粒体P/O及RCR值显著降低(P<0.05)。丙酮酸脱氢酶及α酮戊二酸脱氢酶分别在50μmol/L和100μmol/L时活性下降(P<0.05),而MDA浓度达到1mmol/L时,苹果酸脱氢酶活性降低(P<0.05)。呼吸链复合物Ⅰ、Ⅱ分别在MDA400和800μmol/L时最大反应速度(Vm)降低(P<0.05),但MDA(0～3.2mmol/L)对呼吸链复合物Ⅲ、Ⅳ的Vm及米氏常数(Km)没有影响。结论:MDA对线粒体呼吸链及α酮戊二酸脱氢酶、丙酮酸脱氢酶、苹果酸脱氢酶存在不同程度的损伤作用,不同酶对MDA损伤的敏感程度有所差异,本研究中相关酶受MDA损伤的次序可能是:丙酮酸脱氢酶、α酮戊二酸脱氢酶、呼吸链复合物Ⅰ、呼吸链复合物Ⅱ、苹果酸脱氢酶、呼吸链复合物Ⅲ、Ⅳ。

Objective：To study the in vitro effects of malonaldehyde （MDA） on rat liver mitochondrial complex and key dehydrogenases in mitochondria. Methods：Rat liver mitochondria were treated with different concentrations of MDA in vitro and mitochondrial respiration （ADP/O ratio, respiration control ratio, RCR） was measured by oxygen electrode. The kinetics of complex Ⅰ,Ⅱ,Ⅲ, Ⅳ and the activities of related dehydrogenases （α-ketoglutaric dehydrogenase, pyruvate dehydrogenase, malate dehydrogenase） in mitochondria were determined by spectrophotometry. Results： Mitochondrial NADH linked RCR and ADP/O ratio declined when MDA at 100μmol/L （0.2μmol/mg protein） and 400μmol/L, respectively（P〈0.05）; succinate linked mitochondrial RCR and ADP/O declined when MDA at 800 and 400μmol/L, respectively（P〈0.05）. Activities of pyruvate dehydrogenase, α-ketoglutaric dehydrogenase and malate dehydrogenase significantly decreased when MDA at 50μmol/L, 100 /μmol/L and 1 mmol/L, respectively（P%0.05）. Vm of the complex Ⅰand Ⅱ was depressed by MDA at 400 and 800μmol/ L, respectively（P%0.05）. However, MDA did not inhibit Km of complex Ⅰ,Ⅲ,Ⅳand Vm of complex Ⅲ ,Ⅳ in present study（0-3.2 mmol/L MDA）. Conclusion： MDA can cause mitochondrial dysfunction by inhibiting mitochondrial respiration and enzyme activity, and the sensitivity of the enzymes to MDA is in the order of PDH〉KGDH〉 complex Ⅰ , Ⅱ 〉MDH〉complex Ⅲ, Ⅳ.