摘要
旨在以AMPK-SIRT3正反馈环路为切入点,探讨白果内酯改善白介素1β(IL-1β)诱导的ATDC5软骨细胞炎性损伤的作用机制。采用IL-1β(10 ng·mL^(-1))诱导ATDC5软骨细胞炎性损伤来构建体外骨关节炎模型,随机分为对照组,IL-1β组,IL-1β和白果内酯共同处理组,其中,共同处理组按照白果内酯的应用浓度又分为低、中和高(15、30和60μmol·L^(-1))3个不同剂量组。免疫印迹法(Western blot)和实时荧光定量PCR(qRT-PCR)方法检测各组软骨细胞中ADAMTS4、PGC-1a、Collagen Type II、MMP-3、NRF-1和Fis1的蛋白与mRNA表达情况。试剂盒检测各组ATP含量,并通过免疫荧光方法检测SIRT3表达水平。Western blot检测AMPK-SIRT3正反馈环路相关蛋白p-AMPK、AMPK和SIRT3表达水平。使用Compound C和3-TYP处理ATDC5软骨细胞,构建AMPK-SIRT3信号通路阻断模型,检测下游PGC-1a、NRF-1和Fis1蛋白变化。结果显示,白果内酯通过下调ADAMTS4和MMP-3表达(P<0.05),促进Type II collagen表达(P<0.05)来调节ECM代谢平衡,并促进ATP合成。在机制上,白果内酯干预软骨细胞后p-AMPK、SITR3、PGC-1a和NRF-1水平显著升高(P<0.05),而Fis1蛋白水平显著降低(P<0.05),并且使用Compound C和3-TYP预处理软骨细胞后,PGC-1a、NRF-1和Fis1蛋白水平被不同程度抑制。综上所述,白果内酯通过AMPK-SIRT3正反馈环路激活PGC-1a,调节线粒体生物发生改善ATDC5软骨细胞炎性损伤。
This study takes the AMPK-SIRT3 positive feedback loop as the starting point.The purpose is to explore the mechanism of improvement of bilobalide on inflammatory damage induced interleukin-1β(IL-1β)in ATDC5 chondrocytes.Using IL-1βto induce inflammatory damage of ATDC5 chondrocytes to construct an osteoarthritis model in vitro.Randomly divided into 5 groups(a control group,IL-1βgroup,IL-1βgroup cotreated with bilobalide)which cotreatment group is further divided into low,medium,and high dose groups based on the application concentration of bilobalide(15,30 and 60μmol·L^(-1)).Western blot and real-time fluorescence quantitative PCR(qRT-PCR)methods were used to detect the protein and mRNA expression of ADAMTS4,PGC-1a,Collagen Type II,MMP-3,NRF-1,and Fis1 in each group.A reagent kit was used to detect the ATP content in each group.The expression level of SIRT3 was detected by immunofluorescence.Western blot was used to detect the levels of AMPK-SIRT3 positive feedback loop related proteins(p-AMPK,AMPK,and SIRT3).Construct an AMPK-SIRT3 signaling pathway blockade model by treating ATDC5 chondrocytes with Compound C and 3-TYP.Mainly detecting downstream related protein changes(PGC-1a,NRF-1,and Fis1).The results showed that bilobalide down-regulated the expression of ADAMTS4 and MMP-3(P<0.05),promoted the expression of Type II collagen(P<0.05),regulated ECM metabolic balance,and promoted ATP synthesis.Mechanistically,after intervention with bilobalide,p-AMPK,SITR3,PGC-1a and NRF-1 levels(P<0.05)significantly increased while Fis1 levels(P<0.05)significantly decreased.After pre-treatment with Compound C and 3-TYP in ATDC5 chondrocytes,PGC-1a,NRF-1 and Fis1 protein levels were inhibited to varying degrees.In summary,bilobalide activates the expression of PGC-1a through the AMPK-SIRT3 positive feedback loop and regulates mitochondrial biogenesis to improve inflammatory damage in ATDC5 chondrocytes.
作者
李亚楠
马天文
马玉辉
魏成威
LI Ya nan;MA Tianwen;MA Yuhui;WEI Chengwei(Heilongjiang Key Laboratory of Animal Disease Pathogenesis and Comparative Medicine,College of Veterinary Medicine,Northeast Agricultural University,Harbin 150030,China;Animal Husbandry and Veterinary Development Center,Zhaosu County,Ili Kazakh Autonomous Prefecture,Zhaosu 835500,China)
出处
《畜牧兽医学报》
CAS
CSCD
北大核心
2024年第8期3714-3724,共11页
ACTA VETERINARIA ET ZOOTECHNICA SINICA
基金
中国博士后科学基金项目(2023TQ0053)
黑龙江省自然科学基金项目(LH2023C025,YQ2023C015)。
作者简介
李亚楠(1992-),女,黑龙江哈尔滨人,讲师,主要从事大动物普通病研究,E-mail:13796685756@163.com;马天文(1995-),男,满族,内蒙古呼伦贝尔人,博士,主要从事大动物普通病研究,E-mail:dnmatianwen@163.com;李亚楠和马天文为同等贡献作者通信作者:魏成威,主要从事动物疾病诊疗与研究,E-mail:neauweiwei@126.com。
