【摘要】 目的 观察益气活血方对破裂型腰椎间盘突出症模型大鼠的干预作用，探讨其促进破裂型腰椎间盘突出症重吸收的作用及机制。方法 将75只雄性SD大鼠按照随机数字表法分为假手术组，模型组，益气活血方高、中、低剂量组（15.48、7.74、3.87 g/kg），每组15只。除假手术组外，其余各组大鼠建立破裂型腰椎间盘突出症模型，各给药组大鼠灌胃益气活血方，假手术组和模型组给予等量生理盐水，每天给药1次，连续8周。磁共振成像（MRI）评估大鼠椎间盘突出严重情况；HE及Masson染色观察椎间盘组织形态学变化；ELISA检测血清中肿瘤坏死因子α(TNF-α)、血管内皮生长因子（VEGF）水平；免疫组织化学法检测椎间盘组织中微管相关蛋白轻链3B(LC3B)、苄氯素1(Beclin-1)阳性细胞百分比；蛋白质印迹法（Western Blot）检测椎间盘组织中VEGF、LC3B、p62、核因子κB(NF-κB) p65、低氧诱导因子-1α(HIF-1α)、Bcl-2-腺病毒E1B相互作用蛋白3(BNIP3)、Beclin-1蛋白表达；实时荧光定量聚合酶链式反应（RT-PCR）检测椎间盘组织中TNF-α、NF-κB p65、HIF-1α mRNA表达。结果 干预4、8周后，与假手术组比较，模型组大鼠L4-L5、L5-L6髓核信号降低，与纤维环界限模糊，Pfirmann分级评分显著增加（P<0.05）;TNF-α、VEGF含量升高（P<0.05），椎间盘组织中Beclin-1、LC3B阳性细胞及蛋白表达增加（P<0.05）,NF-κB p65、HIF-1α、BNIP3、Beclin-1蛋白表达水平显著降低（P<0.05）,TNF-α、NF-κB p65及HIF-1α mRNA表达增加（P<0.05）。与模型组比较，益气活血方高、中、低组髓核细胞形状较完整，纤维环轻度紊乱；椎间盘组织中LC3B、Beclin-1阳性细胞比率增加（P<0.01）；血清中VEGF、TNF-α表达增加（P<0.05）；椎间盘组织中VEGF、LC3B、NF-κB p65、HIF-1α、BNIP3、Beclin-1蛋白表达增高（P<0.05）,P62蛋白表达降低（P<0.05）;NF-κB p65、TNF-α及HIF-1α mRNA表达升高（P<0.05）。结论 益气活血方能促进破裂型腰椎间盘突出症重吸收，具体机制可能与其促进椎间盘新生血管VEGF表达，聚集炎性因子TNF-α激活NF-κB信号通路，通过NF-κB信号通路实现HIF-1α高表达，进一步正向调控HIF-1α/BNIP3/Beclin-1通路，增强椎间盘的自噬水平有关。更多还原

【Abstract】 Objective To observe the therapeutic effect of Yiqi Huoxue Formula（YQHXF） on ruptured lumbar disc herniation model rats,and to explore the effect and mechanism of YQHXF on promoting the reabsorption of ruptured lumbar disc herniation. Methods Totally 75 male SD rats were randomly divided into the sham operation group,the model group,and the high,medium and low YQHXF group(15.48,7.74,3.87 g·kg^-1),with 15 rats in each group. Except for the sham operation group,the ruptured lumbar disc herniation model was established in the other groups.The rats in each administration group were given YQHXF by intragastric administration,the sham operation group and model group were intragastrically administered equal volume normal saline for 8 consecutive weeks. Magnetic resonance imaging（MRI） was used to assess the severity of disc herniation in rats. The histomorphologic changes of intervertebral disc were observed by HE and Masson staining. The serum levels of tumor necrosis factor-α（TNF-α） and vascular endothelial growth factor（VEGF） were detected by enzyme-linked immunosorbent assay（ELISA）. The percentage of microtubuleassociated protein light chain 3B（LC3B） and Beclin-1 positive cells in intervertebral disc tissues were detected by immunohistochemistry. Western Blot was used to detect VEGF, LC3B, p62, NF-κB p65 and hypoxia-inducible factor-1α（HIF-1α）, Bcl-2-adenovirus E1B interacting protein 3（BNIP3）, and Beclin-1 protein in intervertebral disc tissue. Real-time PCR was used to detect the mRNA expression of TNF-α, NF-κB p65 and HIF-1α in intervertebral disc tissues. Results After 4 and 8 weeks of treatment,compared with sham operation group,the signal of L4-L5 and L5-L6 nucleus pulposus in model group decreased,the boundary between L4-L5 and L5-L6 was blurred, and the Pfirmann grading score significantly increased（P<0.05）. The contents of TNF-α and VEGF increased（P<0.05）,Beclin-1 and LC3B positive cells and their protein expressions increased（P<0.05）, and the expression levels of NF-κB p65,HIF-1α,BNIP3 and Beclin-1 protein significantly decreased（P<0.05）.The mRNA expressions of TNF-α,NF-κB p65 and HIF-1α increased（P<0.05）. Compared with the model group,the shape of nucleus pulposus cells in high,medium and low YQHXF groups were more complete and the annulus fibrosus were slightly disturbed. The proportion of LC3B and Beclin-1 positive cells in intervertebral disc tissues increased（P<0.05）. The expressions of VEGF and TNF-α in serum increased（P<0.01）. The protein expressions of VEGF,LC3B,NF-κB p65,HIF-1α,BNIP3 and Beclin-1 in intervertebral disc tissues increased（P<0.05）,while the protein expressions of p62 decreased（P<0.05）. The mRNA expressions of NF-κB p65,TNF-α and HIF-1α increased（P<0.05）. Conclusions YQHXF can promote the reabsorption of ruptured lumbar intervertebral disc herniation. The specific mechanism may be related to the promotion of the expression of VEGF in intervertebral disc neovascularization,and the aggregation inflammatory factor TNF-α by activating the NF-κB signaling pathway,through which HIF-1α is highly expressed, the futher positive regulation of HIF-1α/BNIP3/Beclin-1 signaling pathway and the enhancement autophagy in intervertebral discs.更多还原