【作者】 赵峰；

【导师】 钱忠来；

【作者基本信息】 苏州大学 ， 外科学（骨科）， 2022， 博士

【摘要】 第一部分:木兰花碱抑制“M1”极化巨噬细胞诱导的髓核细胞损伤对改善椎间盘退变的效果分析目的:探讨木兰花碱(MAG)对M1极化巨噬细胞介导的髓核(NP)细胞凋亡的影响,为椎间盘退变(IDD)的治疗提供参考依据。方法:运用脂多糖(LPS)对THP-1细胞诱导“M1”极化巨噬细胞进行处理,在不同浓度MAG作用下,ELISA法经过接种、培养、离心、分离等步骤检测THP-1细胞中NLRP3炎症小体、NF-κB、MyD88、HMGB1以及促炎细胞因子(IL-18、TNF-α、IL-6、IL-1β)的表达。选择取自THP-1细胞的条件培养基(CM)对人NP细胞进行处理,对NP细胞凋亡和活力情况进行观察;运用Western blot(WB)经过蛋白提取、电泳、转膜、封闭、抗体孵育、检测等步骤对NP细胞中凋亡相关蛋白(Caspase9、Caspase3、Bax)、细胞外基质(ECM)成分(aggr ecan和collagen Ⅱ)以及分解代谢酶(ADAMTS-5、ADAMTS-4、MMP-13、MM P-3)的表达进行检测。采用细胞免疫荧光法经过接种、固定、一抗孵育、洗涤、染色等步骤检测THP-1细胞中的p-NF-κB和NLRP3的表达。结果:(1)LPS促进THP-1细胞中促炎细胞因子和HMGB1的表达、NLRP3炎症小体的表达,促进MyD88-NF-κB通路的活,并且MAG能够抑制THP-1细胞的“M1”极化;(2)“M1”极化THP-1细胞降低NP细胞活力,导致ECM成分减少、分解代谢酶上调、相关蛋白凋亡以及细胞凋亡,而MAG可减轻“M1”极化THP-1细胞对NP细胞的损伤;(3)MAG可以抑制NP细胞中的HMGB1表达,灭活NLRP3炎症小体和抑制MyD88/NF-κB通路。结论:MAG通过灭活NLRP3炎症小体和HMGB1-MyD88-NF-κB通路来减轻“M1”极化巨噬细胞介导的NP细胞损伤,可以为IDD治疗提供一定的参考依据。第二部分:木兰花碱抑制炎症反应诱导的髓核细胞损伤的体内研究目的:验证木兰花碱体内是否可抑制炎症反应介导的椎间盘退变。方法:本实验部分将3月龄SD大鼠分为3个组,每组10只。具体分组如下:空白组,不做任何的侵入性操作,即大鼠无针刺操作;PBS组:大鼠尾椎间盘穿刺(18G针)后,盘内注射5 μLPBS;MAG组:大鼠椎间盘穿刺(18G针)后,盘内注射5 μL MAG,在术后4周和8周采用MRI、组织病理评估、免疫组化、酶联免疫吸附试验(ELISA),Western blot蛋白免疫印迹法(WB),实时定量PCR(Quantitative Real-Time PCR),细胞免疫荧光对不同组别椎间盘退变情况进行分析。结果:随着穿刺时间的延长,PBS组MRI T2像显示髓核结构为低信号,椎间隙变窄;MAG组MRI T2像虽也可见髓核结构显示为低信号,椎间隙变窄但是比PBS组轻。组织学染色可见PBS组髓核面积显著缩小,纤维环排列紊乱,与髓核组织界限不清,而MAG组在8周时髓核面积相对缩小,纤维环排列较为紊乱,但是髓核区界限分明。MAG组NP组织和细胞HMGB1/MyD88/NF-κB通路和NLRP3炎症小体以及IL-18、TNF-α、IL-6以及IL-1β炎症因子表达明显低于穿刺组。结论:本实验通过影像学分析、免疫组化、酶联免疫吸附试验(ELISA),Western blot 蛋白免疫印迹法(WB),实时定量 PCR(Quantitative Real-Time PCR),细胞免疫荧光观察证实了在椎间盘退变大鼠体内木兰花碱通过抑制HMGB1/MyD88/NF-κB通路和NLRP3炎症小体减弱炎症反应介导的髓核细胞损伤导致的椎间盘退变。与体外实验结论一致。更多还原

【Abstract】 Part Ⅰ:Analysis of the effect of Magnoflorine on improving intervertebral disc degeneration by inhibiting nucleus pulposus cell injury induced by "M1" polarized macrophagesObjective:To investigate the effect of Magnoflorine(MAG)on M1-polarized macrophage-mediated apoptosis of nucleus pulposa(NP)cells,and to provide reference for the treatment of intervertebral disc degeneration(IDD).Methods:Thp-1 cell induced "M1" polarized macrophages were treated with lipopolysaccharide(LPS Inoculation,cultivation,centrifugation,separation).Under the increased concentration of MAG,the expressions of NLRP3 inflammasome,NF-κB,MyD88,HMGB1 and pro-inflammatory cytokines(IL-18,TNF-α,IL-6,IL-1β)in THP1 cells were detected.At the same time,human NP cells were treated with conditional medium(CM)from THP-1 cells,and the apoptosis and viability of NP cells were observed.Western blot(WB)(Protein sample preparation、Electrophoresis、Transfer、Blocking、Antibody incubation、Detection of proteins)was used to detect apoptosis-related proteins(Caspase9,Caspase3,Bax)and extracellular matrix(ECM)components(Aggrecan and collagen)in NP cells II)and the expression of catabolic enzymes(ADAMTS-5,ADAMTS-4,MMP-13,MMP-3).The expression of p-NF-κB and NLRP3 in THP-1 cells was detected by immunofluorescence assay(Inoculation,fixation,primary antibody incubation,washing,and staining).Results:(1)LPS could promote the expression of pro-inflammatory cytokines and HMGB1,the expression of NLRP3 inflammatory body and the activation of myd88-nfκB in thp-1 cells,and MAG could inhibit the "M1" schedule of thp-1 cells.(2)CM treatment of "M1" polarized THP-1 cells reduced the viability of NP cells,leading to the reduction of ECM components,up-regulation of catabolic enzymes,apoptosis of related proteins and apoptosis of cells,and MAG treatment could reduce the injury of NP cells.(3)MAG can inhibit HMGB1 expression in NP cells,inactivate NLRP3 inflammasome and MyD88/NF-κB pathway.Conclusions:MAG alleviates NP cell damage mediated by M1 polarized macrophages by inactivating NLRP3 inflammaome and HMGB1MyD88-NF-κB pathway,which can provide a reference for IDD treatment.Part Ⅱ:Magnoflorine alleviates intervertebral disc degeneration by inhibiting nucleus pulposus cell damage induced by inflammation in vivoObjective:To verify whether magnoflorine attenuates inflammatory response-mediated disc degeneration in vivo.Methods:Three-month-old SD rats were divided into three groups of 10 rats each.The specific groups are as follows:blank group,without any invasive operation,that is,rat needle-free operation;In the PBS group,5 μL PBS was injected into the disc after the tail disc puncture(18G needle).MAG group:After intervertebral disc puncture(18G needle),5 μL MAG was injected into the disc of rats.MRI,histopathological evaluation,immunohistochemistry,enzyme-linked immunosorbent assay(ELISA),Western blot(WB)were performed at 4 and 8 weeks after operation.Quantitative Real-Time PCR and cell immunofluorescence were used to analyze intervertebral disc degeneration in different groups.Results:With the extension of puncture time,MRI T2 images in PBS group showed that the nucleus pulposus structure was low signal and the intervertebral space was narrowed.The MRI T2 images of the MAG group also showed hyposignal nucleus pulposus structure and narrowing of the intervertebral space,but they were lighter than those of the PBS group.Histological staining showed that the area of nucleus pulposus in PBS group was significantly reduced,the annulus fibrosus was disordered,and the boundary between nucleus pulposus and nucleus pulposus was unclear,while the area of nucleus pulposus in MAG group was relatively reduced at 8 weeks,the annulus fibrosus was disordered,but the boundary between nucleus pulposus was clear.The expressions of HMGB1/MyD88/NF-κB pathway,NLRP3 inflammasome,IL-18,TNF-α,IL-6 and IL-1β in NP cells in MAG group were significantly lower than those in puncture group.Conclusion:In this study,imaging analysis,immunohistochemistry,enzyme-linked immunosorbent assay(ELISA),Western blot,Quantitative Real-Time PCR were performed.Cellular immunofluorescence observation confirmed that magnolithine attenuates intervertebral disc degeneration induced by nucleus pulposus cell injury mediated by inflammation by inhibiting HMGB1/MyD88/NF-κB pathway and NLRP3 inflammasome in rats with intervertebral disc degeneration.更多还原