中国骨质疏松杂志

补肾健脾法对OP大鼠肌骨中ULK1/FUNDC1介导的线粒体自噬的影响

The effect of reinforcing kidney and invigorating spleen method on ULK1/FUNDC1 mediated mitochondrial autophagy in osteoporosis rats

DOI：10.3969/j.issn.1006-7108.2024.07.003

中文关键词: 骨肉不相亲 ULK1/FUNDC1通路线粒体自噬骨质疏松补肾健脾法

英文关键词:flesh and blood do not match with each other ULK1/FUNDC1 channel mitochondrial autophagy osteoporosis reinforcing kidney and invigorating spleen method

基金项目:国家自然科学基金（81973719，82104709）；沈阳市中青年科技创新人才计划（RC200160）；辽宁省应用基础研究计划（2022JH2/101300105）；辽宁中医药大学博士后启动基金（21601A2110）

作者	单位
付夜平杨芳* 孙鑫刘洋李俊儒王致远杨蓝鑫刘彤张李博	辽宁中医药大学，辽宁沈阳 110847

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中文摘要:

目的探究补肾健脾法对骨质疏松（osteoporosis，OP）大鼠肌骨中ULK1/FUNDC1介导的线粒体自噬的影响。方法 72只2月龄雄性SD大鼠随机分为正常组、模型组、补肾组、健脾组、补肾健脾组、西药组（福善美），每组12只，模型组及各个用药组采用尾部悬吊法进行造模，造模时间为5周。正常组与模型组予以超纯水灌胃，其余各组予以相应药物进行灌胃。造模结束后行腹主动脉采血，酶联免疫吸附法（ELISA）测定血清碱性磷酸酶（ALP）、抗酒石酸酸性磷酸酶（TRACP）含量；计算机微断层扫描（Micro-CT）检测大鼠左侧股骨远端形态学变化；双能X线行股骨骨密度（bone mineral density，BMD）检测；马松（Masson）染色观察腓肠肌病理变化；蛋白免疫印迹法（Western blot）检测股骨、腓肠肌中线粒体蛋白ULK1、FUNDC1、LC3-Ⅱ/LC3-Ⅰ蛋白表达。结果与正常组相较，模型组ALP（P＜0.01）降低、TRACP（P＜0.01）升高，骨小梁面积、BMD（P＜0.01）均下降，提示造模成功。Masson染色提示在中医不同治法干预下腓肠肌组织纤维化程度得到缓解。Western blot结果显示，在中医不同治法干预下与模型组股骨相比ULK1、LC3-Ⅱ/LC3-Ⅰ、FUNDC1表达降低（P＜0.05），线粒体自噬水平降低；与模型组腓肠肌相比ULK1、FUNDC1、LC3-Ⅱ/LC3-Ⅰ蛋白表达升高（P＜0.05），线粒体自噬水平升高。结论补肾健脾中医治法对于OP模型大鼠的治疗效果可能是通过ULK1/FUNDC1通路调控线粒体自噬发挥作用，综合来看补肾健脾法效果较优。

英文摘要:

Objective To explore the effect of reinforcing kidney and invigorating spleen method on ULK1/FUNDC1 mediated mitochondrial autophagy in osteoporosis rats. Methods Seventy-two 2-month-old male SD rats were randomly divided into normal group, model group, kidney-tonifying group, kidney-tonifying group, spleen-strengthening group, and Western medicine group (Fosamaxa), with 12 rats in each group. Rats in the model group and each medication group were modeled with tail suspension method. The modeling time lasted for 5 weeks. Rats in the normal group and model group received ultra-pure water. Rats in the other groups received corresponding drugs. After the modeling, blood samples were collected from the aorta. The serum alkaline phosphatase (ALP) and tartrate-resistant acid phosphatase (TRACP) were determined with enzyme-related immunosorbent assay (ELISA). The morphological changes of the left femur were detected with micro-CT. Bone mineral density (BMD) of the left femur was detected with dual-energy X-ray. The pathological changes of the left gastrocnemius were observed using Masson staining. The expressions of mitochondrial proteins ULK1, FUNDC1, and LC3-Ⅱ/LC3-Ⅰ extracted from the left femur and gastrocnemius were detected using Western blotting. Results Compared to those in the normal group, ALP decreased (P<0.01), TRACP increased (P<0.01), trabecular area and BMD decreased in model group (P<0.01), indicating successful modeling. Masson staining results suggested that the degree of gastrocnemius fibrosis was relieved after intervention with different treatment methods of traditional Chinese medicine. Western blotting results showed that the expressions of ULK1, LC3-Ⅱ/LC3-Ⅰ, and FUNDC1 decreased (P<0.05) and the level of mitochondrial autophagy decreased compared to those in the model femur group after different treatment methods of traditional Chinese medicine. Compared to those in the model group, the protein expressions of ULK1, FUNDC1, and LC3-Ⅱ/LC3-Ⅰ increased (P<0.05), and the level of mitochondrial autophagy increased. Conclusion The therapeutic effect of TCM treatment of reinforcing kidney and invigorating spleen on OP model rats may be due to the regulation of mitochondrial autophagy through ULK1/FUNDC1 pathway, and the effect of reinforcing kidney and invigorating spleen method is better.

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