基于mTOR信号通路探讨鹿茸多肽对大鼠退变椎间盘髓核细胞自噬的影响  

Effects of Pilose Antler Polypeptide(PAP)on Autophagy of Nucleus Pulposus Cells in Degenerative Intervertebral Disc in Rats Based on mTOR Signaling Pathway

作　　者：姚啸生[1] 马南廷 戚晓楠 崔海舰 杨鸫祥[1] YAO Xiaosheng;MA Nanting;QI Xiaonan;CUI Haijian;YANG Dongxiang(The Affiliated Hospital of Liaoning University of Traditonal Chinese Medicine,Shenyang 110032,Liaoning,China;Liaoning University of Traditonal Chinese Medicine,Shenyang 110847,Liaoning,China)

机构地区：[1]辽宁中医药大学附属医院,辽宁沈阳110032 [2]辽宁中医药大学,辽宁沈阳110847

出　　处：《中华中医药学刊》2023年第7期21-25,共5页Chinese Archives of Traditional Chinese Medicine

基　　金：国家自然科学基金面上项目(82174398);辽宁省教育厅科学技术研究项目(20170540600)。

摘　　要：目的 探讨鹿茸多肽(pilose antler polypeptides, PAP)对大鼠退变椎间盘髓核细胞自噬的影响。方法 购入1月龄的60只健康SD大鼠,随机分为正常组(normal)、对照组(control)、鹿茸多肽组(PAP)、雷帕霉素组(Rapamycin, Rap)、鹿茸多肽+雷帕霉素组(PAP+Rap)5组,每组12只。后4组采用双前肢去势直立法复制大鼠腰椎间盘退变模型。造模6周后,正常组普通饲养不做任何处置,对照组用0.9%氯化钠注射液灌胃,鹿茸多肽组用4.5 mg/mL浓度的鹿茸多肽灌胃,雷帕霉素组腹腔注射0.2 mg/mL浓度的雷帕霉素,鹿茸多肽+雷帕霉素组腹腔注射0.2 mg/mL浓度的雷帕霉素联合4.5 mg/mL浓度的鹿茸多肽灌胃,各组均给药8周。给药结束后,5组中,半数样本取L5-L6椎间盘及上下相邻椎体,用10%的甲醛溶液固定后使用苏木精-伊红染色和阿利新蓝染色进行切片染色,置于显微镜下观察;另半数样本取完整L5-L6椎间盘组织,经液氮罐速冻后移入-80℃冰箱冻存,Western blot检验Akt-mTOR信号通路相关蛋白p-mTOR、p-Akt和自噬相关蛋白LC3-Ⅰ、LC3-Ⅱ、LC3-Ⅱ/LC3-Ⅰ的表达。结果 Alcian blue染色及苏木精-伊红染色下,与对照组比较,PAP组、Rap组、PAP+Rap组均可显著延缓椎间盘退变。蛋白质印迹法检测p-Akt、p-mTOR的表达水平排列为:PAP+Rap>PAP>control>Rap(P<0.05);LC3-Ⅱ的表达水平排列为:Rap>PAP+Rap>control>PAP(P<0.05);LC3-Ⅱ/Ⅰ的比例排列为:Rap>PAP+Rap>control>PAP(P<0.05)。结论 鹿茸多肽具有抑制大鼠椎间盘髓核细胞自噬的作用,进而延缓大鼠椎间盘退变,这可能与激活mTOR信号通路有关。Objective To explore the effects of pilose antler polypeptide(PAP)on autophagy of nucleus pulposus cells in degenerative intervertebral disc in rats.Methods Sixty one-month-old healthy SD rats were randomly divided into five groups:normal group,control group,PAP group,rapamycin(Rap)group and PAP+Rap group.There were 12 rats in each group and the latter 4 groups were subject to the vertical model of double forelimb castrated rats.After 6 weeks of modeling,the normal group was fed without any treatment,while the control group was gavaged with normal saline.The PAP group was given PAP(4.5 mg/mL)by gavage.The Rap group was injected intraperitoneally with Rap at a concentration of 0.2 mg/mL and the PAP+Rap group was given Rap(0.2 mg/mL)by intraperitoneal injection and PAP(4.5 mg/mL)by gavage.The administration lasted for 8 weeks.After administration,L_(5)-L_(6) intervertebral discs and upper and lower vertebral bodies of half of the rats in the 5 groups were taken out,fixed with 10% formaldehyde solution,and then impregnated with hematoxylin-eosin incidence and Alcian blue boron,which were further observed under the microscope.The intact L_(5)-L_(6) intervertebral disc tissues of the rest half of the rats were taken out and were frozen in a liquid nitrogen tank and then transferred to-80℃freezer for storage.The expressions of Akt-mTOR signaling pathway related proteins(p-mTOR and p-Akt)and autophagy-related proteins(LC3-Ⅰ,LC3-Ⅱand LC3-Ⅱ/LC3-Ⅰ)were examined with Western-blot.Results Under Alcian blue staining and hematoxylin-eosin staining,compared with the control group,PAP group,Rap group and PAP+Rap group can significantly delay the degeneration of the intervertebral disc.The expressions of p-Akt and p-mTOR detected by Western blot were arranged as:PAP+Rap>PAP>control>Rap(P<0.05).The expression of LC3-II was arranged as follows:Rap>PAP+Rap>control>PAP(P<0.05).The ratio of LC3-Ⅱ/I was arranged as follows:Rap>PAP+Rap>control>PAP(P<0.05).Conclusion Rather than promoting autophagy in rat intervertebral disc

关 键 词：鹿茸多肽 髓核细胞 自噬 椎间盘退变 

分 类 号：R285.5[医药卫生—中药学]