补肾健脾法在BMSCs成骨分化中ULK1/FUNDC1介导线粒体自噬研究  

Study on ULK1/FUNDC1 mediated mitochondrial autophagy in osteogenic differentiation of BMSCs using kidney tonifying and spleen strengthening methods

作　　者：付夜平 钟妍苑 杨芳[1] 胡楠 孙鑫[1] 刘洋[1] 李俊儒 杨蓝鑫 FU Yeiping;ZHONG Yanyuan;YANG Fang;HU Nan;SUN Xin;LIU Yang;LI Junru;YANG Lanxin(Liaoning University of Traditional Chinese Medicine,Shenyang 110847,China;Huizhou Hospital of Traditional Chinese Medicine,Guangzhou University of Chinese Medicine,Huizhou 516001,China;Affiliated Hospital of Liaoning University of Traditional Chinese Medicine,Shenyang 110000,China)

机构地区：[1]辽宁中医药大学,辽宁沈阳110847 [2]广州中医药大学惠州市中医医院,广东惠州516001 [3]辽宁中医药大学附属医院,辽宁沈阳110000

出　　处：《中国骨质疏松杂志》2025年第2期157-162,共6页Chinese Journal of Osteoporosis

基　　金：国家自然科学基金(82104709);辽宁省应用基础研究计划(2022JH2/101300105);辽宁省博士后启动基金(2023-BSBA-225);辽宁省应用基础研究项目(2023JH2/101700241)。

摘　　要：目的探究补肾健脾法对大鼠骨髓间充质干细胞(BMSCs)成骨分化过程中ULK1/FUNDC1介导的线粒体自噬的影响。方法将72只SD大鼠分为六组:正常组、诱导组、补肾组、健脾组、补肾健脾组和补肾健脾+3-MA组。正常组和诱导组给予超纯水,其他组给予特定药物,补肾健脾组和补肾健脾+3-MA组给予相同药物。7 d后收集血液制备含药血清。BMSCs复苏后,转移到相应的培养瓶中,按照大鼠的分组进行培养,并使用含药血清的培养基培养15 d。通过CCK8实验检测细胞在24、48、72 h的增殖情况;使用ELISA法测定碱性磷酸酶(ALP)及活性氧(ROS)的表达;通过茜素红染色观察成骨细胞(OB)的矿化结节;免疫荧光染色检测线粒体远红外荧光探针(Mito-Tracker Deep Red FM)与线粒体自噬相关蛋白LC3的共定位情况,并通过Image J软件分析细胞的平均荧光强度;采用蛋白免疫印迹法(Western blot)检测ULK1、p-ULK1(Ser555)、FUNDC1、p-FUNDC1(Ser17)和LC3-Ⅱ/LC3-Ⅰ蛋白的表达。结果①在24、48、72 h,细胞增殖呈上升趋势,补肾健脾组的促进效果最为显著;②ALP表达在不同药物干预下增加、ROS降低,补肾健脾组的效果最为明显;③茜素红染色显示,各组细胞形成不同程度的矿化结节,补肾健脾组的结节面积最大,成骨效果最佳;④线粒体荧光共定位结果表明,药物干预可提高细胞线粒体自噬水平,补肾健脾组的LC3荧光强度最高;⑤线粒体自噬蛋白检测结果显示,与诱导组相比,各治疗组均能提升ULK1、p-ULK1(Ser555)、FUNDC1、p-FUNDC1(Ser17)蛋白的表达,而LC3-Ⅱ/LC3-Ⅰ的比值表明补肾健脾组的线粒体自噬水平最高。结论补肾健脾治法可能通过调节ULK1/FUNDC1介导的线粒体自噬途径,促进BMSCs的成骨分化。Objective To investigate the effect of invigorating kidney and invigorating spleen on mitochondrial autophagy mediated by ULK1/FUNDC1 during the osteogenic differentiation of rat bone marrow mesenchymal stem cells(BMSCs).Methods 72 SD rats were divided into six groups:control group,model group,tonifying kidney group,invigorating spleen group,invigorating kidney and invigorating spleen group and invigorating kidney and spleen+3-MA group.The control group and model group were given ultra-pure water,the other groups were given specific drugs,and the same drugs were given to the kidney-invigorating spleen group and kidney-invigorating spleen+3-MA group.After 7 days,the blood was collected to prepare drug-containing serum.After resuscitation,BMSCs were transferred to corresponding culture bottles,cultured according to the groups of rats,and cultured with medicated serum medium for 15 days.The proliferation of cells at 24 h,48 h and 72 h was detected by CCK8 assay.The expression of alkaline phosphatase(ALP)and reactive oxygen species(ROS)were determined by ELISA.The mineralization nodules of osteoblast(OB)were observed by alizarin red staining.The co-localization of mitochondrial far infrared fluorescence probe(Mito-Tracker Deep Red FM)and mitochondrial autophagy associated protein LC3 was detected by immunofluorescence staining,and the average fluorescence intensity of cells was analyzed by Image J software.Western blot was used to detect the expression of ULK1,p-ULK1(Ser555),FUNDC1,p-FUNDC1(Ser17)and LC3-Ⅱ/LC3-Ⅰ.Results①At 24 h,48 h and 72 h,cell proliferation showed an increasing trend,and the promoting effect of tonifying kidney and strengthening spleen group was the most significant;②The expression of ALP increased and ROS decreased under the intervention of different drugs,and the effect of tonifying kidney and strengthening spleen group was the most obvious;③Alizarin red staining showed that the cells in each group formed different degrees of mineralized nodules,and the area of nodules in the invigorat

关 键 词：BMSCS 线粒体自噬 ULK1/FUNDC1 脾肾相关 补肾健脾 

分 类 号：R68[医药卫生—骨科学]