黄芪影响缺氧微环境中骨髓间充质干细胞增殖活性的PI3K-AKT信号通路分析

doi:10.11843/j.issn.0366-6964.2024.01.032

摘要/Abstract

摘要： 基于PI3K/AKT信号通路研究黄芪对缺氧环境中骨髓间充质干细胞（bone marrow mesenchymal stem cells， BMSCs）成骨分化的细胞活性的影响。分别以黄芪冻干粉溶液低、中、高剂量（100、200和300 μg·mL^-1）干预低氧浓度（10%）环境中成骨分化培养的BMSCs，通过高内涵实时成像系统观察BMSCs动态增殖情况及分化代数；进行无标记示踪模拟细胞运动轨迹，分析各组细胞运动速度、位移距离和路程的情况；通过激光共聚焦显微镜观察细胞线粒体膜电位，通过免疫荧光和RT-PCR检测PI3K/AKT信号通路相关蛋白和基因表达水平的变化。结果显示：与对照组相比，10%低氧浓度条件下BMSCs增殖减慢、分化代数减少，运动速度减慢，位移距离和路程减少，线粒体膜电位活性降低，p-PI3K、p-AKT蛋白表达降低，PI3K和AKT基因表达水平降低，差异有统计学意义（P<0.01）；与低氧组相比，黄芪冻干粉溶液干预后，能显著维持缺氧环境中BMSCs增殖和分化活性，运动活力升高，线粒体膜电位活性提高，p-JAK2、p-STAT3蛋白和PI3K、AKT基因表达升高，差异有统计学意义（P<0.05或0.01）。黄芪可能通过激活PI3K/AKT信号通路维持缺氧条件下BMSCs的增殖活性。

关键词: 缺氧, 骨髓间充质干细胞, 黄芪, 增殖活性, PI3K/AKT信号通路

Abstract: This study aimed to explore the effect of Astragalus membranaceus on the cell activity of osteogenic differentiation of BMSCs in hypoxic environments based on the PI3K/AKT signaling pathway. BMSCs cultured in osteogenic differentiation at low oxygen concentration (10%) were intervened with astragalus lyophilized powder solution at low, medium and high doses (100, 200 and 300 μg·mL^-1), respectively, and the dynamic proliferation and differentiation generations of BMSCs were observed by high-intrinsic real-time imaging system; label-free tracing was performed to simulate cell movement trajectories and analyze the cell movement speed, displacement and movement distance of each group. The cellular mitochondrial membrane potential was observed by laser confocal microscopy, and the changes of PI3K/AKT signaling pathway-related proteins and gene expression levels were detected by immunofluorescence and RT-PCR. Results were as follows: Compared with the control group, 10% hypoxia concentration inhibited the proliferation of BMSCs, reduced their differentiation generations, slowed down the motility, displacement and movement distance of BMSCs; at the same time, the mitochondrial membrane potential activity decreased, p-PI3K and p-AKT protein expression decreased, PI3K and AKT gene expression level decreased, the difference was statistically significant (P<0.01). Compared with the hypoxia group, astragalus lyophilized powder solution intervention significantly maintained the proliferation and differentiation activity of BMSCs in hypoxic environment, increased motor viability, increased mitochondrial membrane potential activity, and increased expression of p-JAK2, p-STAT3 proteins and PI3K and AKT genes, with statistically significant differences (P<0.05 or 0.01). Astragalus maybe maintain the proliferative activity of BMSCs under hypoxic conditions by activating PI3K/AKT signaling pathway.

Key words: hypoxia, bone marrow mesenchymal stem cells, astragalus, proliferative activity, PI3K/AKT signaling pathway

中图分类号:

S853.7

田启会, 张亮, 龙亚丽. 黄芪影响缺氧微环境中骨髓间充质干细胞增殖活性的PI3K-AKT信号通路分析[J]. 畜牧兽医学报, 2024, 55(1): 346-354.

TIAN Qihui, ZHANG Liang, LONG Yali. Study on the Effect of Astragalus on Proliferation of Bone Marrow Mesenchymal Stem Cells in Anoxic Microenvironment based on PI3K-AKT Signal Pathway[J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(1): 346-354.

参考文献 19

[1]	FAN M K, SHI H Y, YAO H L, et al. Glutamate regulates gliosis of BMSCs to promote ENS regeneration through α-KG and H3K9/H3K27 demethylation[J]. Stem Cell Res Ther, 2022, 13(1):255.
[2]	YOU M R, AI Z S, ZENG J H, et al. Bone mesenchymal stem cells (BMSCs)-derived exosomal microRNA-21-5p regulates Kruppel-like factor 3 (KLF3) to promote osteoblast proliferation in vitro[J]. Bioengineered, 2022, 13(5):11933-11944.
[3]	赵雯, 邹彤, 吕阳欧, 等. 犬BMSCs复合3D打印支架制备组织工程半月板及体外成软骨诱导时间对其分化的影响[J]. 畜牧兽医学报, 2019, 50(7):1486-1492.ZHAO W, ZOU T, LV Y O, et al. Preparation of tissue engineering meniscus by canine BMSCs composite 3D printed scaffold and effect of chondrogenic induction time on its differentiation in vitro [J]. Acta Veterinaria et Zootechnica Sinica, 2019, 50(7):1486-1492. (in Chinese)
[4]	龙亚丽, 田启会. 骨碎补总黄酮对缺氧环境中犬骨髓间充质干细胞成骨分化潜能的影响[J]. 畜牧兽医学报, 2022, 53(4):1280-1288.LONG Y L, TIAN Q H. Effects of total flavonoids of Drynariae Rhizoma on osteogenic differentiation potential of canine bone marrow mesenchymal stem cells in hypoxic environment[J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(4):1280-1288. (in Chinese)
[5]	YAN C Q, WANG Z R, LIU W L, et al. Resveratrol ameliorates high altitude hypoxia-induced osteoporosis by suppressing the ROS/HIF signaling pathway[J]. Molecules, 2022, 27(17):5538.
[6]	伍志伟, 刘丹, 薛娜, 等. 黄芪多糖对低氧环境中BMSCs成骨分化的影响[J]. 中国细胞生物学学报, 2017, 39(6):732-738.WU Z W, LIU D, XUE N, et al. Effects of astragalus polysaccharides on osteogenic differentiation of BMSCs cultured in low oxygen environment[J]. Chinese Journal of Cell Biology, 2017, 39(6):732-738. (in Chinese)
[7]	石特, 王杨凯. 黄芪对间充质干细胞增殖及分化的影响[J]. 解剖学杂志, 2019, 42(2):194-196.SHI T, WANG Y K. Effects of astragalus membranaceus on proliferation and differentiation of mesenchymal stem cells[J]. Chinese Journal of anatomy, 2019, 42(2):194-196. (in Chinese)
[8]	赵芳, 王洪新, 鲁美丽, 等. 黄芪甲苷对慢性间歇性缺氧诱导血管内皮功能障碍的保护作用[J]. 中成药, 2022, 44(10):3145-3150.ZHAO F, WANG H X, LU M L, et al. Protective effects of astragaloside Ⅳ on vascular endothelial dysfunction in mice with chronic intermittent hypoxia[J]. Chinese Traditional Patent Medicine, 2022, 44(10):3145-3150. (in Chinese)
[9]	谢耀锟, 曹金城, 周锦魁. 黄芪对体外培养的新生大鼠大脑皮层神经细胞缺氧性凋亡的保护机理研究[J]. 中国实用医药, 2021, 16(15):210-212.XIE Y K, CAO J C, ZHOU J K. The protective mechanism of Huangqi on hypoxic apoptosis of neonatal rat cortical neurons cultured in vitro[J]. China Practical Medical, 2021, 16(15):210-212. (in Chinese)
[10]	邓海艳, 鲁美丽, 王洪新. 黄芪多糖对低氧诱导肺动脉高压小鼠肺血管重构的保护作用[J]. 陆军军医大学学报, 2022, 44(11):1112-1118.DENG H Y, LU M L, WANG H X, et al. Astragalus polysaccharide protects mice against pulmonary vascular remodeling after hypoxia-induced pulmonary hypertension[J]. Journal of Army Medical University, 2022, 44(11):1112-1118. (in Chinese)
[11]	ROSSI A, PIZZO P, FILADI R. Calcium, mitochondria and cell metabolism:a functional triangle in bioenergetics[J]. Biochim Biophys Acta Mol Cell Res, 2019, 1866(7):1068-1078.
[12]	DÖHLA J, KUULUVAINEN E, GEBERT N, et al. Metabolic determination of cell fate through selective inheritance of mitochondria[J]. Nat Cell Biol, 2022, 24(2):148-154.
[13]	YAO C H, WANG R C, WANG Y H, et al. Mitochondrial fusion supports increased oxidative phosphorylation during cell proliferation[J]. Elife, 2019, 8:e41351.
[14]	侯茜, 张秋菊, 刘永琦, 等. 低氧环境下黄芪注射液对人骨髓间充质干细胞体外增殖的影响[J]. 中成药, 2015, 37(4):873-875.HOU Q, ZHANG Q J, LIU Y Q, et al. The effect of Huangqi injection on the proliferation of human bone marrow mesenchymal stem cells in vitro under hypoxic environment[J]. Chinese Traditional Patent Medicine, 2015, 37(4):873-875. (in Chinese)
[15]	SHI Y Q, LIU J, ZHANG R Y, et al. Targeting endothelial ENO1 (Alpha-Enolase) -PI3K-Akt-mTOR axis alleviates hypoxic pulmonary hypertension[J]. Hypertension, 2023, 80(5):1035-1047.
[16]	YU Z P, YU H Q, LI J, et al. Troxerutin attenuates oxygen-glucose deprivation and reoxygenation-induced oxidative stress and inflammation by enhancing the PI3K/AKT/HIF-1α signaling pathway in H9C2 cardiomyocytes[J]. Mol Med Rep, 2020, 22(2):1351-1361.
[17]	HAO J P, SHI H, ZHANG J, et al. Role of GSPE in improving early cerebral vascular damage by inhibition of Profilin-1 expression in a ouabain-induced hypertension model[J]. Eur Rev Med Pharmacol Sci, 2018, 22(20):6999-7012.
[18]	王君妍, 叶洁瑜, 梁恩瑜, 等. 血小板生成素通过PI3K/AKT通路防止CoCl2诱导内皮细胞凋亡[J]. 中国实验血液学杂志, 2018, 26(2):528-534.WANG J Y, YE J Y, LIANG E Y, et al. Thrombopoietin prevents CoCl2-inducing apoptosis of HUVEC through the PI3K/AKT pathway[J]. Journal of Experimental Hematology, 2018, 26(2):528-534. (in Chinese)
[19]	张贤益, 李文娟, 钟亮, 等. 白扁豆多糖对神经细胞缺氧性凋亡的保护机制[J]. 食品科学, 2018, 39(3):222-228.ZHANG X Y, LI W J, ZHONG L, et al. Protective mechanism of polysaccharides from dolichos bean seeds (Dolichos lablab L.) on hypoxia-induced neuronal apoptosis[J]. Food Science, 2018, 39(3):222-228. (in Chinese)