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吴门枳壳甘草汤调控HIF-1α/VEGF/Ang信号轴抑制退变椎间盘病理性血管新生的机制研究
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| 篇名： | 吴门枳壳甘草汤调控HIF-1α/VEGF/Ang信号轴抑制退变椎间盘病理性血管新生的机制研究 |
| TITLE： | Mechanism of Wumen Zhiqiao gancao decoction inhibiting pathological angiogenesis in degenerative intervertebral discs by regulating HIF-1α/VEGF/Ang signal axis |
| 摘要： | 目的 探讨枳壳甘草汤对退变椎间盘病理性血管新生的影响及机制。方法动物实验设置假手术组（生理盐水）、模型组（生理盐水）、缺氧诱导因子1α（HIF-1α）抑制剂（YC-1）组[2mg/（kg·d），尾静脉注射]和枳壳甘草汤低、中、高剂量组[3.06、6.12、12.24g/（kg·d），灌胃]，每组8只。除假手术组外，其余各组均构建椎间盘退变大鼠模型。造模成功后，每天给药1次，连续3周。末次给药后，观察大鼠椎间盘组织病理改变及血管新生情况，检测大鼠椎间盘组织中炎症因子[白细胞介素1β（IL-1β）、IL-6、肿瘤坏死因子α（TNF-α）]水平以及血管生成相关蛋白[HIF-1α、血管内皮生长因子（VEGF）、VEGF受体2（VEGFR2）、血管紧张素1（Ang1）、Ang2]表达水平。细胞实验中，提取并培养大鼠原代髓核细胞，以50ng/mLTNF-α诱导细胞退变。设置空白对照组（10%空白对照血清）、TNF-α组（10%空白对照血清）、YC-1组（10%空白对照血清+0.2mmol/LYC-1）和5%、10%、15%含药血清组（5%、10%、15%含药血清），干预24h后，将髓核细胞与人脐静脉内皮细胞（HUVEC）共培养。检测髓核细胞中Ⅱ型胶原蛋白（CollagenⅡ）、基质金属蛋白酶3（MMP-3）表达，检测HUVEC增殖、迁移、成管能力，检测HUVEC中HIF-1α/VEGF/Ang信号轴及血管生成相关蛋白（在动物实验基础上新增MMP-2、MMP-9）表达水平。结果动物实验结果显示，与模型组比较，各药物组大鼠椎间盘组织中CD31阳性表达减少（P＜0.05），炎症因子及血管生成相关蛋白表达均显著下调（P＜0.05），椎间盘病理改变减轻。细胞实验结果显示，与TNF-α组比较，各药物组髓核细胞中CollagenⅡ表达均显著上调、MMP-3表达均显著下调（P＜0.05），HUVEC增殖、迁移及成管能力显著减弱（P＜0.05），HUVEC中HIF-1α、VEGF、Ang2mRNA及蛋白和血管生成相关蛋白（除5%含药血清组的Ang2mRNA及HIF-1α、VEGFR2、Ang2蛋白）表达均显著下调（P＜0.05）。结论枳壳甘草汤可能通过抑制HIF-1α/VEGF/Ang信号轴，减弱血管内皮细胞的血管生成能力，改善椎间盘病理性血管新生，延缓椎间盘退变。 |
| ABSTRACT： | OBJECTIVE To explore the effect and mechanism of Zhiqiao gancao decoction （ZQGCD） on pathological angiogenesis of degenerative intervertebral disc. METHODS The rats were randomly divided into sham operation group （normal saline）， model group （normal saline）， hypoxia inducible factor-1α （HIF-1α） inhibitor （YC-1） group [2 mg/（kg·d）， tail vein injection]， and ZQGCD low-dose， medium-dose and high-dose groups [3.06， 6.12， 12.24 g/（kg·d）]， with 8 rats in each group. Except for sham operation group， lumbar disc degeneration model of rat was constructed in all other groups. After modeling， they were given relevant medicine once a day， for consecutive 3 weeks. After the last medication， pathological changes and angiogenesis of the intervertebral disc tissue in rats were observed； the levels of inflammatory factors [interleukin-1β （IL-1β）， IL-6， tumor necrosis factor-α （TNF-α）] and the expressions of angiogenesis-related proteins [HIF-1α， vascular endothelial growth factor （VEGF）， VEGF receptor 2 （VEGFR2）， angiotensin 1（Ang 1）， Ang 2] in the com intervertebral disc tissue in rats were all determined. In cell experiment， the primary nucleus pulposus cells were isolated and cultured from rats， and cellular degeneration was induced using 50 ng/mL TNF-α. The cells were divided into blank control group （10% blank control serum）， TNF-α group （10% blank control serum）， YC-1 group （10% blank control serum+0.2 mmol/L YC-1）， and 5%， 10%， 15% drug-containing serum group （5%， 10%， 15% drug-containing serum）. After 24 hours of intervention， the nucleus pulposus cells were co-cultured with HUVEC. The expressions of Collagen Ⅱ， matrix metalloproteinase-3 （MMP-3） in nucleus pulposus cells were detected. HUVEC proliferation， migration and tube forming ability were detected， and the expression levels of the HIF-1α/VEGF/Ang signal axis and angiogenesis- related proteins （add MMP-2， MMP-9） in HUVEC were detected. RESULTS Animal experiments had shown that compared with model group， the positive expression of CD31 in the intervertebral disc tissues of rats in each drug group was down-regulated （P＜ 0.05）， the levels of inflammatory factors and angiogenesis-related proteins were decreased significantly （P＜0.05）， and the pathological changes in the intervertebral disc were alleviated. Cell experiments had shown that compared with TNF-α group， the expression of Collagen Ⅱ in nucleus pulposus cells of all drug groups was significantly up-regulated （P＜0.05）， and the expression of MMP-3 was significantly down-regulated （P＜0.05）； the proliferation， migration and tubulogenesis of HUVEC were significantly weakened （P＜0.05）. The mRNA and protein expressions of HIF-1α， VEGF， Ang 2 as well as the expression of angiogenesis-related proteins （except for the expression of Ang 2 mRNA and HIF-1α， VEGFR2， Ang 2 protein in 5% drug- containing serum group） were significantly down-regulated （P＜0.05）. CONCLUSIONS ZQGCD may inhibit the HIF-1α/VEGF/ Ang signal axis to weaken the angiogenic ability of vascular endothelial cells， improve pathological angiogenesis in the intervertebral disc， and delay the degeneration of the intervertebral disc. |
| 期刊： | 2025年第36卷第07期 |
| 作者： | 黄泽灵；朱在师；李宇卫；徐波；陈俊名；张宝飞；陆斌杰；蔡学峰；陈华 |
| AUTHORS： | HUANG Zeling， ZHU Zaishi，LI Yuwei，XU Bo，CHEN Junming，ZHANG Baofei，LU Binjie，CAI Xuefeng，CHEN Hua |
| 关键字： | 枳壳甘草汤；椎间盘退变；病理性血管新生；HIF-1α/VEGF/Ang信号轴；髓核细胞退变；炎症因子 |
| KEYWORDS： | Zhiqiao gancao decoction； intervertebral disc degeneration； pathological angiogenesis； HIF-1α/VEGF/Ang signal |
| 阅读数： | 4 次 |
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