益气活血方调节自噬促进破裂型腰椎间盘突出症重吸收  

Yiqi Huoxue Formula Promotes Reabsorption of Ruptured Lumbar Disc Herniation Via Regulating Autophagy

作　　者：白雪 孙孝先 刘锦涛[3] 刘孟敏 郭杨[1] 林顺 马勇[1,2] 姜宏[3] BAI Xue;SUN Xiao-xian;LIU Jin-tao;LIU Meng-min;GUO Yang;LIN Shun;MA Yong;JIANG Hong(Laboratory of New Techniques of Restoration&Reconstruction of Orthopedics and Traumatology,Nanjing University of Chinese Medicine,Nanjing,210023;School of Integrated Chinese and Western Medicine,Nanjing University of Chinese Medicine,Nanjing,210023;Department of Orthopaedics,Suzhou Hospital of Traditional Chinese Medicine,Nanjing University of Chinese Medicine,Jiangsu,215008)

机构地区：[1]南京中医药大学骨伤修复与重建新技术实验室,南京210023 [2]南京中医药大学中西医结合学院,南京210023 [3]南京中医药大学附属苏州市中医院骨伤科,南京215008

出　　处：《中国中西医结合杂志》2024年第10期1242-1251,共10页Chinese Journal of Integrated Traditional and Western Medicine

基　　金：国家自然科学基金面上项目(No.82074467,No.82374220);国家中医药管理局高水平中医药重点学科建设项目(No.国中医药人教函[2023]85号);江苏省研究生科研与实践创新计划(No.KYCX23-2202)。

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-1αmRNA表达。结果干预4、8周后,与假手术组比较,模型组大鼠L_(4)-L_(5)、L_(5)-L_(6)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-1αmRNA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-1αmRNA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�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-1αin intervertebral disc tissues.Results After 4 and 8 weeks of treatment,compared with sham operation group,the signal of L_(4)-L_(5) and L_(5)-L_(6) 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-αand 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

关 键 词：益气活血方 腰椎间盘突出症 重吸收 低氧诱导因子-1α/Bcl-2-腺病毒E1B相互作用蛋白3/苄氯素1通路 自噬 中药复方 中西医结合 

分 类 号：R285.5[医药卫生—中药学]