白果内酯调控AMPK-SIRT3正反馈环路介导的线粒体生物发生改善ATDC5软骨细胞炎性损伤

doi:10.11843/j.issn.0366-6964.2024.08.043

畜牧兽医学报 ›› 2024, Vol. 55 ›› Issue (8): 3714-3724.doi: 10.11843/j.issn.0366-6964.2024.08.043

白果内酯调控AMPK-SIRT3正反馈环路介导的线粒体生物发生改善ATDC5软骨细胞炎性损伤

李亚楠¹(), 马天文¹(), 马玉辉², 魏成威¹^,*()

1. 东北农业大学动物医学学院，黑龙江省动物疾病致病机制与比较医学重点实验室，哈尔滨 150030
2. 新疆伊犁哈萨克自治州昭苏县畜牧兽医发展中心，昭苏 835500

收稿日期:2023-10-26 出版日期:2024-08-23 发布日期:2024-08-28
通讯作者: 魏成威 E-mail:13796685756@163.com;dnmatianwen@163.com;neauweiwei@126.com
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
第一联系人：
李亚楠和马天文为同等贡献作者
基金资助:
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);黑龙江省自然科学基金项目(YQ2023C015)

Bilobalide Regulates Mitochondrial Biogenesis Mediated by AMPK-SIRT3 Positive Feedback Loop and Improves Inflammatory Damage of ATDC5 Chondrocytes

Ya’nan LI¹(), Tianwen MA¹(), Yuhui MA², Chengwei WEI¹^,*()

1. Heilongjiang Key Laboratory of Animal Disease Pathogenesis and Comparative Medicine, College of Veterinary Medicine, Northeast Agricultural University, Harbin 150030, China
2. Animal Husbandry and Veterinary Development Center, Zhaosu County, Ili Kazakh Autonomous Prefecture, Zhaosu 835500, China

Received:2023-10-26 Online:2024-08-23 Published:2024-08-28
Contact: Chengwei WEI E-mail:13796685756@163.com;dnmatianwen@163.com;neauweiwei@126.com

摘要/Abstract

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60 μmol·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。

关键词: 软骨细胞, 炎性损伤, 白果内酯, AMPK-SIRT3正反馈环路, 线粒体生物发生

Abstract:

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-1β to induce inflammatory damage of ATDC5 chondrocytes to construct an osteoarthritis model in vitro. Randomly divided into 5 groups (a control group, IL-1β group, IL-1β group 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 60 μmol·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.

Key words: chondrocytes, inflammatory damage, bilobalide, AMPK-SIRT3 positive feedback loop, mitochondrial biogenesis

中图分类号:

李亚楠, 马天文, 马玉辉, 魏成威. 白果内酯调控AMPK-SIRT3正反馈环路介导的线粒体生物发生改善ATDC5软骨细胞炎性损伤[J]. 畜牧兽医学报, 2024, 55(8): 3714-3724.

Ya’nan LI, Tianwen MA, Yuhui MA, Chengwei WEI. Bilobalide Regulates Mitochondrial Biogenesis Mediated by AMPK-SIRT3 Positive Feedback Loop and Improves Inflammatory Damage of ATDC5 Chondrocytes[J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(8): 3714-3724.

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基因Gene	上游引物(5′→3′) Forward prime	下游引物(5′→3′) Reverse primer
Type Ⅱ Collagen	TCCAGATGACTTTCCTCCGTCTA	AGGCGATGCTGTTCTTACA
MMP-3	TTTGGCCGTCTCTTCCATCC	GCATCGATCTTCTGGACGGT
ADAMTS4	AGGAGGCGCCCTTAACTCTG	CTACTCAGCGAAGCGAAGCG
PGC-1a	CGCAGCCCTATTCATTGT	GCATCCTTTGGGGTCTTT
NRF-1	AGCACGGAGTGACCCAAAC	AGGATGTCCGAGTCATCATAAGA
Fis1	TACTGGACCATGGAGACTGTG	GAACCAGGCACCAGGCATATT
β-actin	GTGACGTTGACATCCGTAAAGA	GCCGGACTCATCGTACTCC

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白果内酯调控AMPK-SIRT3正反馈环路介导的线粒体生物发生改善ATDC5软骨细胞炎性损伤

Bilobalide Regulates Mitochondrial Biogenesis Mediated by AMPK-SIRT3 Positive Feedback Loop and Improves Inflammatory Damage of ATDC5 Chondrocytes

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