| 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 cells 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 24h, 48h and 72h was detected by CCK8 assay. The expression of alkaline phosphatase (ALP) was 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. Results ①At 24h, 48h and 72h, 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 under the intervention of different drugs, and the effect of tonifying kidney and strengthening spleen group was the most obvious; ③ Alizarin red staining showed that the cells in each group formed different degrees of mineralized nodules, and the area of nodules in the invigorating kidney and strengthening spleen group was the largest and the bone formation effect was the best; ④The results of mitochondrial fluorescence co-localization showed that drug intervention could improve the level of mitochondrial autophagy, and the LC3 fluorescence intensity was the highest in the kidney-invigorating group; ⑤Mitochondrial autophagy protein detection results showed that compared with the model group, the expression of ULK1, p-ULK1 (Ser555), FUNDC1, p-FUNDC1 (Ser17) proteins in all treatment groups could be increased, and the ratio of LC3-Ⅱ/LC3-Ⅰ indicated that the level of mitochondrial autophagy in the kidney-strengthening spleen group was the highest. Conclusion Invigorating kidney and invigorating spleen therapy may promote osteogenic differentiation of BMSCs by regulating the mitochondrial autophagy pathway mediated by ULK1/FUNDC1. |