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蒋佳豪,彭兴宁,吴官保,谭婉俊,冯帅华.基于Klotho、PI3K、Akt蛋白表达水平探讨补肾活血汤对绝经后骨质疏松大鼠骨骼、血管及肾脏功能的影响[J].湖南中医药大学学报英文版,2024,44(1):9-15.[Click to copy
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| 基于Klotho、PI3K、Akt蛋白表达水平探讨补肾活血汤对绝经后骨质疏松大鼠骨骼、血管及肾脏功能的影响 |
| 蒋佳豪,彭兴宁,吴官保,谭婉俊,冯帅华 |
| (昆山市第一人民医院, 江苏 苏州 215300;湖南省中医药研究院附属医院, 湖南 长沙 410006;湖南中医药大学, 湖南 长沙 410208) |
| 摘要: |
| 目的 通过观察补肾活血汤对去势大鼠股骨近端、主动脉外膜及肾小管中可罗索（Klotho）蛋白，大鼠股骨近端磷脂酰肌醇3-激酶（phosphatidylinositol 3-kinase，PI3K）蛋白及蛋白激酶B（protein kinase B，Akt）表达水平的影响，探讨补肾活血汤治疗绝经后骨质疏松症（postmenopausal osteoporosis，PMOP）的作用机制。方法 选取6月龄SD雌性大鼠31只，第1次随机分为空白组8只、假手术组5只、手术组18只，手术造模8周后随机处死空白组和手术组大鼠各3只，评估造模成功后，手术组按随机数字表法第2次分组：模型组5只、补肾活血汤组5只、戊酸雌二醇组5只。然后分组给药：空白组、假手术组及模型组予以蒸馏水灌胃，补肾活血汤组予补肾活血汤3.32 mg/kg灌胃，戊酸雌二醇组予戊酸雌二醇0.1 mg/kg灌胃，连续给药8周。观察大鼠的一般情况；分光光度法测定血清钙含量；偶联反应法测定血清磷含量；骨密度扫描仪测量股骨近端的骨密度；电镜观察股骨近端的骨细胞；HE染色观察大鼠股骨近端的病理变化；ELISA法检测大鼠股骨近端、主动脉外膜及肾小管中Klotho蛋白的水平；Western blot检测大鼠股骨近端中Klotho、PI3K及Akt蛋白的相对表达水平。结果 与模型组比较，补肾活血汤组和戊酸雌二醇组大鼠的平均体质量较重、活力较好，饮食及毛发情况未见明显差异。与模型组比较，补肾活血汤组和戊酸雌二醇组大鼠股骨近端细胞、骨小管形态数量均有不同程度恢复，股骨近端骨组织骨质疏松病变均得到不同程度的修复。与空白组、假手术组比较，模型组、补肾活血汤组血清钙含量降低（P<0.01，P<0.05）；与模型组比较，戊酸雌二醇组血清钙含量增加（P<0.05）。与空白组、假手术组比较，模型组骨密度明显降低（P<0.01）；股骨近端、主动脉外膜及肾小管中Klotho蛋白含量明显减少（P<0.01）；骨组织中Klotho蛋白表达水平显著降低（P<0.01），PI3K、Akt蛋白表达水平显著增加（P<0.01）。与模型组比较，补肾活血汤组、戊酸雌二醇组股骨近端、主动脉外膜及肾小管中Klotho蛋白含量明显增加（P<0.01）；骨组织中Klotho蛋白表达水平显著增加（P<0.01），PI3K、Akt蛋白表达水平降低（P<0.05，P<0.01）。与补肾活血汤组比较，戊酸雌二醇组股骨近端及肾小管中Klotho蛋白含量增加（P<0.05，P<0.01），骨组织中PI3K、Akt蛋白表达水平降低（P<0.05）。各组大鼠血清磷含量比较，差异无统计学意义（P>0.05）。结论 补肾活血汤可通过提高股骨近端、主动脉外膜及肾小管中Klotho蛋白的表达，抑制股骨近端中PI3K蛋白、Akt蛋白的表达，一定程度上缓解去势大鼠的骨质疏松样病变，其作用机制可能与抑制骨细胞凋亡、改善肾脏功能及循环系统功能相关。 |
| 关键词: 补肾活血汤 绝经后骨质疏松症 可罗索蛋白 磷脂酰肌醇3-激酶 蛋白激酶B |
| DOI：10.3969/j.issn.1674-070X.2024.01.002 |
| Received:June 20, 2023 |
| 基金项目:湖南中医药大学中医学双一流课题（2021ZYX07）；湖南省科技创新计划项目（2021SK51008）；湖南省中医药科研计划项目（B2023100）。 |
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| Effects of Bushen Huoxue Decoction on skeletal, vascular, and renal functions in postmenopausal osteoporosis rats based on protein expressions of Klotho, PI3K, and Akt |
| JIANG Jiahao,PENG Xingning,WU Guanbao,TAN Wanjun,FENG Shuaihua |
| (The First People's Hospital of Kunshan, Suzhou, Jiangsu 215300, China;The Hospital of Hunan Academy of Chinese Medicine, Changsha, Hunan 410006, China;Hunan University of Chinese Medicine, Changsha, Hunan 410208, China) |
| Abstract: |
| Objective To observe the effects of Bushen Huoxue Decoction (BSHXD) on the protein expression level of Klotho in the proximal femur, adventitia of aorta, and renal tubules as well as those of phosphatidylinositol 3-kinase (PI3K) and protein kinase B (Akt) in the proximal femur of ovariectomized rats, so as to explore the mechanism of action of BSHXD in treating postmenopausal osteoporosis (PMOP). Methods Thirty-one 6-month-old female SD rats were randomly divided into blank group (n=8), sham-operated group (n=5), and surgery group (n=18). After eight weeks of surgical modeling, three rats from blank group and another three from surgery group were randomly selected and sacrificed. After successful modeling, the remaining rats in the surgery group were subdivided into model group (n=5), BSHXD group (n=5), and estradiol valerate group (n=5) according to the random number table method. Then, blank, sham-operated, and model groups were given distilled water, BSHXD group was given BSHXD 3.32 mg/kg, and estradiol valerate group was given estradiol valerate 0.1 mg/kg. All groups were treated by gavage continuously for eight weeks. The general condition of rats was observed; the serum calcium content was measured by spectrophotometry; the serum phosphorus content was determined by coupled reaction method; bone mineral density of the proximal femur was measured by bone density scanner, and the bone cells were examined by electron microscopy. In addition, the pathological changes in the proximal femur were observed by HE staining; Klotho protein level in the proximal femur, aortic adventitia, and renal tubules was measured by ELISA; the relative expression levels of Klotho, PI3K, and Akt proteins in the proximal femur were checked by Western blot. Results Compared with model group, the rats in BSHXD and estradiol valerate groups showed higher average body weight and better vitality, and there was no significant difference in the condition of diet and hair; the number and form of bone cells and bone tubules in the proximal femur were restored to varying degrees, and the osteoporotic lesions were also repaired to different extent. Compared with blank and sham-operated groups, the serum calcium content of rats in model and BSHXD groups was lower (P<0.01, P<0.05); compared with model group, the serum calcium content of rats in estradiol valerate group was higher (P<0.05). Compared with blank and sham-operated groups, rats in model group showed a significant decrease in the bone mineral density (P<0.01), as well as in the Klotho protein content in the proximal femur, aortic adventitia, and renal tubules (P<0.01); the Klotho protein expression in the bone tissue was significantly reduced (P<0.01), while the protein expressions of PI3K and Akt were significantly elevated (P<0.01). Compared with model group, the Klotho protein content in the proximal femur, aortic adventitia, and renal tubules of rats in BSHXD and estradiol valerate groups increased (P<0.01); the Klotho protein expression in the bone tissue was significantly higher (P<0.01), while the protein expressions of PI3K and Akt were significantly lower (P<0.05, P<0.01). Compared with BSHXD group, the Klotho protein content in the proximal femur and renal tubules of rats in estradiol valerate group increased (P<0.05), while the protein expressions of PI3K and Akt in the bone tissue decreased (P<0.05). Moreover, there was no statistical difference in serum phosphorus content among the groups (P>0.05). Conclusion BSHXD can alleviate the osteoporosis-like lesions of ovariectomized rats to a certain extent by elevating the Klotho protein expression in the proximal femur, aortic adventitia, and renal tubules and inhibiting the protein expressions of PI3K and Akt in the proximal femur. And its mechanism of action may be related to inhibiting osteocyte apoptosis and improving renal function and circulatory system function. |
| Key words: Bushen Huoxue Decoction postmenopausal osteoporosis Klotho protein phosphatidylinositol 3-kinase protein kinase B |
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