补肾健脾活血方调控Apaf-1/CHOP-Caspase-9/Bcl-2通路抑制成骨细胞氧化应激
The nourishing kidney, invigorating spleen, and promoting blood flow recipe inhibits oxidative stress damage in osteoblasts by regulating the Caspase-9/Bcl-2 signaling pathway through Apaf-1/CHOP
  
DOI:10.3969/j.issn.1006-7108.2024.05.005
中文关键词:  补肾健脾活血方  骨质疏松症  氧化应激  线粒体  成骨细胞
英文关键词:the nourishing kidney, invigorating spleen, and promoting blood flow recipe  osteoporosis  oxidative stress  mitochondria  osteoblasts
基金项目:国家自然科学基金面上项目(82374482);广东省自然科学基金项目(2022A1515011404);广东省中医药局科研基金项目(20211318);广州市科技计划市校(院)联合资助项目(202102010124);广州中医药大学“双一流”与高水平大学学科协同创新团队重点项目(2021XK21)
作者单位
李颖1, 4 林燕平2, 4 黄佳纯2, 4 杜书军1, 4 李燕南1, 4 郭海威2, 3, 4* 1 暨南大学祈福医院广东 广州 511495 2 广州中医药大学第三附属医院广东 广州 510378 3 广东省中医骨伤研究院广东 广州 510378 4 广州中医药大学岭南医学研究中心广东 广州 510405 
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中文摘要:
      目的 探讨补肾健脾活血方对减轻大鼠成骨细胞氧化应激损伤和调控线粒体介导的细胞凋亡的机制。方法 提取大鼠原代成骨细胞,通过CCK8实验检测补肾健脾活血方干预下对成骨细胞增殖活性的影响、碱性磷酸酶(alkaline phosphatase, ALP)染色检测成骨细胞ALP活性、茜素红染色检测成骨细胞的矿化情况、实时定量PCR检测Apaf-1和CHOP基因的mRNA表达、蛋白质印迹检测Caspase-9和Bcl-2蛋白表达量。结果 CKK8实验和碱性磷酸酶染色表明氧化应激损伤的大鼠成骨细胞增殖活性显著降低,成骨分化受抑制,同时Apaf-1和CHOP的mRNA表达以及Caspase-9的蛋白表达明显增加(P≤0.01),Bcl-2蛋白表达则显著减少(P≤0.01);经补肾健脾活血方干预后,成骨细胞增殖活性得到明显的增强、成骨分化增加,Apaf-1和CHOP的mRNA及Caspase-9的蛋白表达均显著减少(P<0.05),而Bcl-2的蛋白表达则显著升高(P≤0.000 1)。结论 补肾健脾活血方可以促进成骨细胞增殖和成骨分化,显著降低Apaf-1和CHOP的mRNA及Caspase-9的蛋白表达,升高Bcl-2的蛋白表达量。补肾健脾活血方在减轻成骨细胞的氧化应激损伤和调控线粒体介导的细胞凋亡中起着重要作用。
英文摘要:
      Objective To explore the mechanism of the nourishing kidney, invigorating spleen, and promoting blood flow recipe in reducing oxidative stress damage and in regulating mitochondria-mediated apoptosis in rat osteoblasts. Methods Rat primary osteoblasts were extracted. The CCK8 experiment was used to detect the effect of the nourishing kidney, invigorating spleen, and promoting blood flow recipe on osteoblast proliferation activity under the intervention of the nourishing kidney, invigorating spleen, and promoting blood flow recipe. Alkaline phosphatase (ALP) staining was used to detect the ALP activity of osteoblasts. Alizarin red staining was used to detect the mineralization of osteoblasts. Real-time quantitative PCR was used to detect the mRNA expressions of Apaf-1 and CHOP genes. Western blotting was used to detect the protein expressions of Caspase-9 and Bcl-2. Results The results of CKK8 experiment and alkaline phosphatase staining showed that the proliferation activity of rat osteoblasts damaged by oxidative stress was significantly reduced and the osteogenic differentiation was inhibited. At the same time, mRNA expressions of Apaf-1 and CHOP and protein expression of Caspase-9 increased significantly (P≤0.01). Bcl-2 protein expression decreased significantly (P≤0.01). After intervention with the nourishing kidney, invigorating spleen, and promoting blood flow recipe, osteoblast proliferation activity was significantly enhanced, osteogenic differentiation increased, mRNA expressions of Apaf-1 and CHOP as well as the protein expression of Caspase-9 were noticeably reduced (P<0.05), and the protein expression of Bcl-2 increased significantly (P≤0.000 1). Conclusion The nourishing kidney, invigorating spleen, and promoting blood flow recipe promotes osteoblast proliferation and osteogenic differentiation, significantly reduces the mRNA expression of Apaf-1 and CHOP and the protein expression of Caspase-9, and increases the protein expression of Bcl-2. The nourishing kidney, invigorating spleen, and promoting blood flow recipe plays an important role in alleviating oxidative stress damage in osteoblasts and in regulating mitochondria-mediated apoptosis.
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