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目的研究牙龈卟啉单胞菌脂多糖(Pg-LPS)对人牙龈成纤维细胞(HGF)自噬功能的影响。方法以10μg/mL Pg-LPS刺激HGF 12 h或24 h,采用雷帕霉素作为阳性对照,Western blot法检测微管相关蛋白1轻链3B(LC3B)的表达,间接免疫荧光技术检测自噬体的分布;同时使用MitoSOX Red荧光探针标记线粒体活性氧(mtROS)、间接免疫荧光法检测自噬体,观察Pg-LPS处理后HGF mtROS与线粒体自噬水平;分别以叔丁基-4-羟基茴香醚(BHA)、N-乙酰半胱氨酸(NAC)以及辅酶Q10(CoQ10)阻断ROS,Western blot法检测Pg-LPS刺激后LC3B的表达量。结果 Pg-LPS处理后LC3BⅡ/LC3BⅠ的比值与自噬体数量均显著升高,并且24 h处理组高于12 h处理组,同时mtROS生成量与线粒体自噬均明显增加。此外CoQ10可有效降低Pg-LPS诱导的HGF自噬。结论 Pg-LPS在HGF中通过触发mtROS活化自噬,并且自噬参与受损线粒体的降解以维持细胞内环境稳态。
Objective To study the effect of Pg-LPS on autophagy in human gingival fibroblasts (HGF). Methods HGF was stimulated with 10 μg / mL Pg-LPS for 12 h or 24 h, rapamycin was used as a positive control, the expression of microtubule-associated protein 1 light chain 3B (LC3B) was detected by Western blot and autophagy was detected by indirect immunofluorescence The mitochondrial reactive oxygen species (mtROS) were labeled with MitoSOX Red fluorescent probe, and the autophagosomes were detected by indirect immunofluorescence. The mtROS and mitochondrial autophagy of HGF after Pg-LPS treatment were observed. ROS were detected by hydroxyanisole (BHA), N-acetylcysteine (NAC) and coQ10 (CoQ10), and the expression of LC3B after Pg-LPS stimulation was detected by Western blot. Results After treatment with Pg-LPS, the ratio of LC3BⅡ / LC3BⅠ and the number of autophagosomes were significantly increased, and the levels of mtROS production and mitochondrial autophagy in 24 h treatment group were higher than those in 12 h treatment group. In addition CoQ10 can effectively reduce Pg-LPS induced HGF autophagy. Conclusion Pg-LPS activates autophagy by activating mtROS in HGF, and autophagy participates in the degradation of damaged mitochondria to maintain the homeostasis of the cell.