姜黄素调控脂多糖诱导成骨细胞线粒体功能改变及凋亡研究

Effect of curcumin and lipopolysaccharide on mitochondrial function and cell apoptosis in osteoblasts

摘要:

目的：研究脂多糖（lipopolysaccharide，LPS）和姜黄素（curcumin，Cur）对成骨细胞线粒体功能活性改变及凋亡的影响。方法：体外培养小鼠原代成骨细胞，并将细胞分为4组：无任何处理的对照组（N组）；10 μmol/L姜黄素预处理2 h，使用正常培养基培养24 h组（Cur组）；用1 mg/L LPS处理24 h组（LPS组）；用10 μmol/L姜黄素预处理2 h，然后再用1 mg/L LPS处理成骨细胞24 h组（Cur +LPS组）。利用线粒体荧光探针测量线粒体活性氧的产生，检测线粒体氧化呼吸链酶活性、线粒体膜电位、细胞内ATP的合成及细胞凋亡情况。结果：在经过1 mg/L LPS刺激后，小鼠成骨细胞线粒体内活性氧（reactive oxygen species，ROS）生成为对照组的2.59倍；与对照组相比，LPS组氧化呼吸链酶Ⅰ活性降低21%，氧化呼吸链酶Ⅲ活性降低26%，氧化呼吸链酶Ⅳ活性降低28%；线粒体细胞膜电位降低32%，ATP合成量为（0.79±0.06） μmol/mg蛋白，细胞凋亡数目增加3.2倍；而Cur+LPS组较LPS组相比，小鼠成骨细胞内ROS水平降低（1.48±0.21 vs. 2.59±0.32，P=0.003），氧化呼吸链酶Ⅰ活性升高（0.98±0.02 vs. 0.79±0.03，P=0.000），氧化呼吸链酶Ⅲ活性升高（0.93±0.03 vs. 0.74±0.03，P=0.004），氧化呼吸链酶Ⅳ活性升高（0.92±0.05 vs. 0.72±0.02，P=0.009），线粒体膜电位增加（0.91±0.04 vs. 0.68±0.04，P=0.002），ATP合成量增加（1.53±0.05 vs. 0.79±0.06，P=0.000），细胞凋亡数目减少（1.67±0.42 vs. 5.33±0.80，P=0.002）。结论：姜黄素预处理组的成骨细胞线粒体功能得以明显改善，并且细胞凋亡数目明显减少，为姜黄素可能被进一步应用于临床治疗慢性牙周炎提供一定的实验依据。

Abstract:

Objective：To investigate the effect of lipopolysaccharide（LPS） and curcumin（Cur） on the changes in mitochondrial func－tion and activity and cell apoptosis in osteoblasts. Methods：Primary mouse osteoblasts were cultured in vitro and then divided into control group（N group without any treatment），Cur group（pretreated with 10 μmol/L Cur for 2 hours and then cultured with normal medium for 24 hours），LPS group（treated with 1 mg/L LPS for 24 hours），and Cur+LPS group（pretreated with 10 μmol/L Cur for 2 hours and then treated with 1 mg/L LPS for 24 hours）. A mitochondrial fluorescent probe was used to detect reactive oxygen species （ROS），and the activity of mitochondrial respiratory chain complex，mitochondrial membrane potential，synthesis of ATP in cells，and cell apoptosis were also measured. Results：After the stimulation with 1 mg/L LPS，ROS in the mitochondria was 2.59 times that in the control group. Compared with the control group，the LPS group had a 21% reduction in the activity of respiratory chain complexⅠ， a 26% reduction in the activity of respiratory chain complex Ⅲ，and a 28% reduction in the activity of respiratory chain complex Ⅳ， as well as a 32% reduction in mitochondrial membrane potential，an amount of ATP synthesis of 0.79±0.06 μmol/mg protein，and a 3.2-fold increase in the number of apoptotic cells. Com－pared with the LPS group，the Cur+LPS group had a significant reduction in ROS（1.48±0.21 vs. 2.59±0.32，P=0.003），signifi－cant increases in the activities of respiratory chain complexⅠ（0.98±0.02 vs. 0.79±0.03，P=0.000），Ⅲ（0.93±0.03 vs. 0.74±0.03，P=0.004），and Ⅳ（0.92±0.05 vs. 0.72±0.02，P=0.009），a sig－nificant increase in mitochondrial membrane potential（0.91±0.04 vs 0.68±0.04，P=0.002），a significant increase in ATP synthesis（1.53±0.05 vs. 0.79±0.06，P=0.000），and a significant reduction in the number of apoptotic cells（1.67±0.42 vs. 5.33±0.80，P=0.002）. Conclusion：Osteoblasts pretreated with Cur had a significant improvement in mitochondrial function and a significant reduction in the number of apoptotic cells，which provide an experimental basis for the application of Cur in clinical treatment of periodontitis.