瑞芬太尼对椎间盘退变模型大鼠椎间盘的影响及机制

Effect of remifentanil on the intervertebral disc of rats with intervertebral disc degeneration and its mechanism

摘要:

目的 观察瑞芬太尼对椎间盘退变（intervertebral disc degeneration，IDD）模型大鼠椎间盘的保护作用，并探讨其作用机制。方法 将40只健康SD大鼠随机分为假手术组、模型组和瑞芬太尼低、中、高剂量组，每组8只；采用针刺纤维环法构建IDD模型；瑞芬太尼低、中、高剂量组于造模后以0.2、0.6、1.2 μg/（kg·min）经大鼠尾静脉泵注瑞芬太尼30 min，模型组和假手术组给予等量生理盐水泵注。采用苏木精-伊红（hematoxylin-eosin，HE）染色观察椎间盘组织病理变化；酶联免疫吸附法（enzyme linked immunosorbent assay，ELISA）检测血清肿瘤坏死因子-α（tumor necrosis factor α，TNF-α）、白细胞介素-6（interleukin-6，IL-6）、白细胞介素-1β（interleukin-1β，IL-1β）水平；Western blot检测椎间盘组织过氧化物酶体增殖物激活受体γ共激活因子-1α（peroxisome proliferator-activated receptor-1α，PGC-1α）、线粒体转录因子（mitochondrial transcription factor A，TFAM）、半胱氨酸天冬氨酸蛋白酶1（cysteine aspartic protease-1，Caspase-1）、消皮素D（gasdermin D，GSDMD）、NOD样受体热蛋白结构域3（NOD-like receptor family pyrin domain containing 3，NLRP3）、缺氧诱导因子-1α（hypoxia-inducible factor 1α，HIF-1α）、血管内皮生长因子（vascular endothelial growth factor，VEGF）蛋白表达。结果 与假手术组比较，模型组大鼠髓核缩小，纤维环排列紊乱，髓核细胞数量减少；血清TNF-α、IL-6、IL-1β水平升高（P<0.05）；椎间盘组织PGC-1ɑ、TFAM蛋白表达水平降低，Caspase-1、GSDMD、NLRP3、HIF-1α、VEGF蛋白表达水平增加（P<0.05）。与模型组比较，瑞芬太尼中、高剂量组纤维环排列紊乱明显改善，髓核细胞数量明显增多；血清TNF-α、IL-6、IL-1β水平降低（P<0.05）；椎间盘组织PGC-1α、TFAM蛋白表达水平增加，Caspase-1、GSDMD、NLRP3、HIF-1α、VEGF蛋白表达水平降低（P<0.05）。结论 瑞芬太尼可改善IDD模型大鼠椎间盘退变，其机制与抑制椎间盘炎症反应、细胞焦亡，调控HIF-1α/VEGF信号通路有关。

Abstract:

Objective To observe the protective effect of remifentanil on intervertebral disc of rats with intervertebral disc degeneration （IDD），and to explore its mechanism of action.Methods A total of 40 healthy SD rats were randomly divided into sham-operation group，model group，low-dose，middle-dose and high-dose remifentanil groups，8 cases in each group. IDD models were constructed by fibrous annulus puncturing. After modeling，low-dose，medium-dose and high-dose remifentanil groups were given intravenous injection of remifentanil through tail vein at rates of 0.2，0.6 and 1.2 μg/（kg·min） for 30 minutes，while model group and sham operation group were given the same volume of normal saline. The pathological changes of intervertebral disc tissues were observed by hematoxylin-eosin（HE） staining. The levels of serum tumor necrosis factor α（TNF-α），interleukin 6（IL-10） and interleukin 1β（IL-1β） were detected by enzyme linked immunosorbent assay（ELISA）. The expressions of peroxisome proliferator-activated receptor-1α（PGC-1α），mitochondrial transcription factor A（TFAM），cysteine aspartic protease-1（Caspase-1），gasdermin D（GSDMD），NOD-like receptor family pyrin domain containing 3（NLRP3），hypoxia-inducible factor 1α（HIF-1α） and vascular endothelial growth factor（VEGF） in intervertebral disc tissues were detected by Western blot.Results Compared with sham operation group，nucleus pulposus was shrunk，arrangement of fibrous annulus was disordered and the number of nucleus pulposus cells was decreased in model group. The levels of serum TNF-α，IL-6 and IL-1β were increased（P<0.05）. The expression levels of PGC-1α and TFAM in intervertebral disc tissues were decreased，and expression levels of Caspase-1，GSDMD，NLRP3，HIF-1α and VEGF were increased（P<0.05）. Compared with model group，disorders of fibrous annulus were significantly improved in medium-dose and high-dose remifentanil groups，and number of nucleus pulposus cells was significantly increased. The levels of serum TNF-α，IL-6 and IL-1β were decreased（P<0.05）. The expression levels of PGC-1α and TFAM in intervertebral disc tissues were increased，and expression levels of Caspase-1，GSDMD，NLRP3，HIF-1α and VEGF were decreased（P<0.05）.Conclusion Remifentanil can improve IDD in rats，and its mechanism is related to inhibiting inflammatory response and pyroptosis of intervertebral disc，and regulating HIF-1α/VEGF signaling pathways.