APPL1减轻LPS诱导的心肌细胞损伤的实验研究

目的:探讨APPL1对脂多糖(LPS)诱导的心肌细胞损伤的影响。方法:用LPS处理心肌细胞,RT-qPCR和Western blot检测细胞中APPL1的表达变化。用过表达APPL1重组慢病毒载体转染心肌细胞,经LPS处理后,RT-qPCR和Western blot检测过表达效果。MTT测定心肌细胞活力,流式细胞术测定心肌细胞凋亡变化,Western blot测定心肌细胞中活化的caspase-3蛋白水平,用硫代巴比妥酸法检测心肌细胞中丙二醛(MDA)含量,用2,4-二硝基苯肼显色法检测心肌细胞培养液中乳酸脱氢酶(LDH)水平,用黄嘌呤氧化酶法检测心肌细胞中超氧化物歧化酶(SOD)活性,5,5’-二硫代双(2-硝基苯甲酸)比色法测定心肌细胞中谷胱甘肽过氧化物酶(GSH-Px)活性,DCFH-DA法检测心肌细胞中活性氧(ROS)水平。结果:与对照心肌细胞比较,LPS处理后心肌细胞中APPL1的mRNA和蛋白水平均显著降低(P<0.05)。与单纯LPS处理的心肌细胞比较,过表达APPL1重组慢病毒载体可以显著提高LPS条件下心肌细胞中APPL1 mRNA和蛋白水平(P<0.05)。LPS处理后的心肌细胞活力降低,细胞凋亡率和细胞中活化的caspase-3蛋白水平升高,细胞中MDA水平升高,培养液中LDH水平也升高,细胞SOD活性、GSH-Px活性降低,ROS水平升高,(P<0.05)。过表达APPL1后的心肌细胞经LPS诱导后,细胞活力升高,细胞凋亡率及细胞中活化的Caspase-3蛋白水平降低,MDA水平降低,培养液中LDH水平降低,细胞中SOD活性、GSH-Px活性升高,ROS水平降低,(P<0.05)。结论:过表达APPL1能够降低LPS诱导的心肌细胞氧化损伤,减少细胞凋亡,具有减轻LPS诱导的心肌细胞损伤的作用。

APPL1 reduces injury of H9c2 cardiomyocytes induced by LPS

AIM: To study the effect of APPL1(adaptor protein, phosphotyrosine interacting with PH domain and leucine zipper 1) on H9 c2 cardiomyocyte injury induced by lipopolysaccharides(LPS). METHODS: The H9 c2 cells were treated with LPS. RT-qPCR and Western blot were used to detect the expression of APPL1 in the H9 c2 cells. The recombinant APPL1 lentiviral vector was used to transfect into the H9 c2 cells. After LPS treatment, the over-expression efficiency was detected by RT-qPCR and Western blot. The viability was measured by MTT assay. The apoptosis was analyzed by flow cytometry. The protein level of activated caspase-3 in the H9 c2 cells was determined by Western blot. The content of malonaldehyde(MDA) in the H9 c2 cells and the level of lactate dehydrogenase(LDH) in the culture medium were detected. The activity of superoxide dismutase(SOD) and glutathione peroxidase(GSH-Px), and the level of reative oxygen species(ROS) in the H9 c2 cells were also examined. RESULTS: The expression of APPL1 at mRNA and protein levels in LPS-treated H9 c2 cells was decreased significantly(P<0.05). Over-expression of APPL1 by transfection of recombinant lentiviral vector significantly increased the level of APPL1 at mRNA and protein levels in the H9 c2 cells with LPS treatment(P<0.05). LPS treatment reduced the viability, but increased the apoptotic rate of the H9 c2 cells, the protein level of activated caspase-3, the content of MDA and the level of LDH in the culture medium. The activity of SOD and GSH-Px was reduced, while the level of ROS was increased as compared with control group(P<0.05). Over-expression of APPL1 elevated the viability of H9 c2 cells treated with LPS, and the apoptotic rate and the protein level of activated caspase-3 were decreased. The content of MDA and the level of LDH in the culture medium were reduced, the activity of SOD and GSH-Px was elevated, and the level of ROS was reduced as compared with the H9 c2 cells treated with LPS alone(P<0.05). CONCLUSION: Over-expression of APPL1 reduces oxidative damage and apoptosis of the H9 c2 cells induced by LPS.