摘要
目的 探索柠檬苦素(Limonin)对高糖(HG)诱导的髓核(NP)细胞损伤的保护机制。方法 将人髓核细胞分为空白对照组(0μmol·L^(-1),Ctl组)、HG组(200 nmol·L^(-1))、Limonin(50μmol·L^(-1))+HG组(200 mmol·L^(-1))。使用CCK-8检测不同浓度(50、100、200、300 mmol·L^(-1))HG处理24 h后细胞存活率,检测不同浓度(12.5、25、50、100、200μmol·L^(-1))Limonin处理24、48 h后细胞存活率;Western blot检测凋亡相关指标Bcl-2、Bax表达水平,氧化应激相关指标Nrf2表达水平,以及AKT、p-AKT蛋白表达水平;Nrf2检测核转位水平;TUNEL法检测细胞凋亡水平。结果 CCK-8结果显示,不同浓度(50、100、200、300 mmol·L^(-1))HG对NP细胞活力有不同的影响,与Ctl组相比,在HG浓度200 mmol·L^(-1)时,NP细胞活力下降(P<0.05);在300 mmol·L^(-1)时出现大量死亡的情况(P<0.01),所以选用200 mmol·L^(-1)HG进行后续实验。与Ctl组相比,不同浓度(12.5、25、50、100、200μmol·L^(-1))Limonin与NP细胞共培养24 h时,200μmol·L^(-1)时细胞活力下降(P<0.05);在48 h时,当Limonin浓度在100μmol·L^(-1)时NP细胞活力下降(P<0.05),200μmol·L^(-1)时细胞活力下降(P<0.05),因此选用50μmol·L^(-1)浓度进行后续实验。Western blot结果显示,与Ctl组相比,HG处理后的髓核细胞出现损伤,主要表现为凋亡相关蛋白Bax/Bcl-2表达上调(P<0.01),氧化应激相关蛋白Nrf2表达下调(P<0.05),信号通路相关蛋白p-AKT/AKT表达下调(P<0.05),经Limonin处理后Bax/Bcl-2表达下调(P<0.01);Nrf2表达上调(P<0.05);p-AKT/AKT表达上调(P<0.05)。Nrf2染色结果显示,Ctl组NP细胞中Nrf2在细胞质内保持相对高水平阳性表达,HG诱导损伤后,NP细胞质内Nrf2阳性表达水平较低,细胞总数减少(P<0.05)。Limonin干预后能够明显增强HG诱导损伤后Nrf2的核转位情况(P<0.01)。TUNEL染色结果显示,与Ctl组相比,HG组阳性细胞数量增加(P<0.01);与HG组相比,Limonin+HG组在经过Limonin处理后,HG诱导NP细胞凋亡的作用减�
Objective Exploring the protective mechanism of Limonin against high glucose(HG)induced damage to nucleus pulposus(NP)cells.Methods Divide human NP cells into control group(0μmol·L^(-1),Ctl group),HG group(200 nmol·L^(-1)),HG(200 mmol·L^(-1))+Limonin(50μmol·L^(-1))group(Limonin+HG group).CCK-8 was used to detect cell survival rates after 24 hours of treatment with different concentrations of HG(50,100,200,300 mmol·L^(-1)),and different concentrations of Limonin(12.5,25,50,100,200μmol·L^(-1))after 24 and 48 hours of treatment;Western blot was used to detect the expression levels of apoptosis related indicators Bcl-2 and Bax,oxidative stress related indicators Nrf2 expression levels,as well as AKT and p-AKT protein expression levels;Nrf2 was used to detection of nuclear translocation levels;TUNEL was used to detect the level of cell apoptosis.Results CCK-8 assay showed that HG had different effects on the viability of NP cells at different concentrations(50,100,200,300 mmol·L^(-1)).Compared with the Ctl group,NP cell viability decreased at a HG concentration of 200 mmol·L^(-1)(P<0.05),and a large number of deaths occurred at 300 mmol·L^(-1)(P<0.01).Therefore,200 mmol·L^(-1) HG were selected for subsequent experiments.Compared with the Ctl group,different concentrations(12.5,25,50,100,200μmol·L^(-1))of Limonin showed different cell viability when co-cultured with NP cells for 24 hours,at 200μmol·L^(-1) the activity significantly decreased(P<0.05);At 48 hours,when the concentration of Limonin were 100μmol·L^(-1) the activity of NP cells showed the decreased trend(P<0.05),When the concentration were 200μmol·L^(-1),the vitality significantly decreased(P<0.05),so 50μmol·L^(-1) were selected for further experiments.Western blot results showed that compared with the Ctl group,with nucleus pulposus cells HG treated were damaged,mainly manifested by upregulation of apoptosis related protein Bax/Bcl-2 expression(P<0.01),downregulation of oxidative stress related protein Nrf2 expression(P<0.05),downregu
作者
吕权
高金金
杨玉林
孙甫
LYU Quan;GAO Jinjin;YANG Yulin;SUN Fu(First Clinical Medicine College of Ningxia Medical University,Yinchuan 750004,China;Xi’an Medical University,Xi’an 710077,China;The First Affiliated Hospital of Xi’an Medical University,Xi’an 710077,China)
出处
《宁夏医科大学学报》
2024年第4期325-330,337,共7页
Journal of Ningxia Medical University
基金
西安医学院第一附属医院院内配套经费项目(XYFYPT-2020-05)。
关键词
髓核细胞
凋亡
氧化应激
信号通路
nucleus pulposus cells
apoptosis
oxidative stress
signaling pathways
