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. 2024 Jun;44(3):468-477.
doi: 10.19852/j.cnki.jtcm.20240402.001.

Acupotomy alleviates knee osteoarthritis in rabbit by regulating chondrocyte mitophagy Pink1-Parkin pathway

Zhu Wenting  1 , Guo Changqing  2 , D U Mei  2 , M A Yunxuan  2 , Cui Yongqi  2 , Chen Xilin  2 , Guo Changqing  3
Affiliations

Acupotomy alleviates knee osteoarthritis in rabbit by regulating chondrocyte mitophagy Pink1-Parkin pathway

Zhu Wenting et al. J Tradit Chin Med. 2024 Jun.

Abstract

Objective: To investigate the effect of acupotomy, on mitophagy and the Pink1-Parkin pathway in chondrocytes from rabbits with knee osteoarthritis (KOA).

Methods: A KOA model was established via the modified Videman method. Rabbits were randomly divided into a control group (CON), KOA group and KOA + acupotomy group (Acu). Rabbits in the acupotomy group were subjected to acupotomy for 4 weeks after model establishment. The behavior of the rabbits before and after intervention was recorded. Cartilage degeneration was evaluated by optical microscopy and fluorescence microscopy. The level of mitophagy was evaluated by transmission electron microscopy, immunofluorescence and enzyme-linked immunosorbent assay (ELISA). The expression of phosphatase and tensin homolog (PTEN)-induced kinase 1 (Pink1)-Parkin mitophagy pathway components was evaluated by immunofluorescence, Western blotting and real-time polymerase chain reaction.

Results: In rabbits with KOA, joint pain, mobility disorders and cartilage degeneration were observed, the Mankin score was increased, collagen type Ⅱ (Col-Ⅱ) expression was significantly decreased, mitophagy was inhibited, mitochondrial function was impaired, and factors associated with the Pink1-Parkin pathway were inhibited. Acupotomy regulated the expression of Pink1-Parkin pathway-related proteins, the mitophagy-related protein microtubule-associated protein-1 light chain-3, the translocase of the outer membrane, and the inner mitochondrial membrane 23; increased the colocalization of mitochondria and autophagosomes; promoted the removal of damaged mitochondria; restored mitochondrial adenosine-triphosphate (ATP) production; and alleviated cartilage degeneration in rabbits with KOA.

Conclusions: Acupotomy played a role in alleviating KOA in rabbits by activating mitophagy in chondrocytes via the regulation of proteins that are related to the Pink1-Parkin pathway.

Keywords: PTEN phosphohydrolase; acupuncture therapy; cartilage; mitophagy; osteoarthritis, knee.

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Figures

Figure 1
Figure 1. Cartilage degeneration under microscope
A: safranin O-fast green staining of the knee cartilage (× 200); A1: CON group; A2: KOA group; A3: Acu group; B: Col-Ⅱ immunofluorescence staining in knee cartilage (× 200); B1-B3: CON group; B4-B6: KOA group; B7-B9: Acu group; C: analysis of the Mankin score; D: analysis of the Col-Ⅱ fluorescence intensity. CON group: no modelling or no intervention; KOA group: 6 weeks of modeling followed by no intervention; Acu group: 6 weeks of modeling followed by acupotomy intervention for 4 weeks. Col-Ⅱ: type Ⅱ collagen; CON: control; KOA: knee osteoarthritis; Acu: acupotomy. The data were presented as the mean ± standard deviation (n = 6). Compared with the CON group, aP < 0.01; compared with the KOA group, bP < 0.01.
Figure 2
Figure 2. Ultramicrostructure of chondrocyte mitochondria
A, B, C: chondrocytes at low magnification (× 1200); D, E, F: chondrocytes at high magnification (× 8000); A, D: CON group; B, E: KOA group; C, F: Acu group; CON group: no modeling or no intervention; KOA group: 6 weeks of modeling followed by no intervention; Acu group: 6 weeks of modeling followed by acupotomy intervention for 4 weeks. The yellow arrow indicates mitochondria; the red arrow indicates mitochondrial autophagosomal vesicles.
Figure 3
Figure 3. Immunofluorescence staining of mitophagy and mitochondrial function
A: double immunofluorescence staining for TOM20 and LC3B (× 400); A1, A2, A3, A4: CON group; A5, A6, A7, A8: KOA group; A9, A10, A11, A12: Acu group; A1, A5, A9: DAPI staining; A2, A6, A10: TOM20 staining; A3, A7, A11: LC3B staining; A4, A8, A12: merge of TOM20 and LC3B staining; B: level of ATP; C: colocalization coefficient; D, E: analysis of TOM20 and LC3B fluorescence intensity. CON group: no modeling or no intervention; KOA group: 6 weeks of modeling followed by no intervention; Acu group: 6 weeks of modeling followed by acupotomy intervention for 4 weeks. ATP: adenosine triphosphate; PCC: Pearson's correlation coefficient; TOM20: translocase of the outer membrane 20; LC3B: microtubule-associated protein-1 light chain-3B; CON: control; KOA: knee osteoarthritis; Acu: acupotomy; DAPI: 4',6-diamidino-2-phenylindole. The data were presented as the mean ± standard deviation (n = 6). aP < 0.01 compared with the control group; bP < 0.05, cP < 0.01, compared with the KOA group.
Figure 4
Figure 4. Expression of factors associated with the Pink1-Parkin pathway
A: double immunofluorescence staining for Pink1 and Parkin (× 400); A-1A4: CON group; A5-A8: KOA group; A9-A12: Acu group; A1, A5, A9: DAPI staining; A2, A6, A10: Pink1 staining; A3, A7, A11: Parkin staining; A4, A8, A12: merge of Pink1 and Parkin staining; B: Pink1, Parkin, LC3Ⅱ/Ⅰ, TOM20 and TIM23 expression was detected by Western blot analyses. C: colocalization coefficient; D: mRNA expression of Pink1; E: mRNA expression of Parkin; F: Western blotting analysis of Pink1 expression; G: Western blotting analysis of Parkin expression; H: Western blotting analysis of LC3Ⅱ/Ⅰ expression; I: Western blotting analysis of TOM20 expression; J: Western blotting analysis of TIM23 expression. CON group: no modeling or no intervention; KOA group: 6 weeks of modeling followed by no intervention; Acu group: 6 weeks of modeling followed by acupotomy intervention for 4 weeks. PCC: Pearson's correlation coefficient; Pink1: PTEN-induced putative kinase 1; LC3Ⅱ/Ⅰ: microtubule-associated protein-1 light chain-3Ⅱ/Ⅰ; TOM20: translocase of the outer membrane 20; TIM23: translocase of the inner membrane 23; CON: control; KOA: knee osteoarthritis; Acu: acupotomy; DAPI: 4', 6-diamidino-2-phenylindole; PTEN: phosphatase and Tensin Homolog. The data were presented as the mean ± standard deviation (n = 6). aP < 0.01 and dP < 0.01 compared with the control group; bP < 0.01 and cP < 0.05, compared with the KOA group.
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