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

Abstract

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

CLC Number:

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.

Figures/Tables 7

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References 36

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

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