Journal of Traditional Chinese Medicine ›› 2024, Vol. 44 ›› Issue (3): 468-477.DOI: 10.19852/j.cnki.jtcm.20240402.001
• Original articles • Previous Articles Next Articles
Acupotomy alleviates knee osteoarthritis in rabbit by regulating chondrocyte mitophagy via Pink1-Parkin pathway
ZHU Wenting1, GUO Changqing2(
), DU Mei2, MA Yunxuan2, CUI Yongqi2, CHEN Xilin2, GUO Changqing3
- 1 the Third Affiliated Hospital of Beijing University of Chinese Medicine, Beijing 100029, China
2 School of Acupuncture-Moxibustion and Tuina, Beijing University of Chinese Medicine, Beijing 100029, China
3 Department of Medical Technology, Shijiazhuang Medical College, Hebei 050599, China
-
Received:2023-08-11Accepted:2023-12-19Online:2024-06-15Published:2024-04-02 -
Contact:Mrs. GUO Changqing, Shijiazhuang Medical College, Hebei 050599, China.925499172@qq.com Telephone: +86-311-82517626 -
Supported by:Key Project of Beijing University of Chinese Medicine: to Investigate the Effect of Acupotomy Mechanics on Oxidative Injury of Rabbit Chondrocytes with Knee Osteoarthritis Based on Mitophagy Mediated by Pink1/Parkin Signaling Pathway(2020-JYB-ZDGG-059);National Natural Science Foundation of China: to Investigate the Mechanism of Acupotomy "Regulating Tendon and Treating Bone" in the Treatment of Knee Osteoarthritis based on the Vascularization of Hypoxic Cartilage Mediated by Hypoxia-Inducible Factor 1alpha/ Vascular Endothelial Growth Factor Pathway(82074523)
Cite this article
ZHU Wenting, GUO Changqing, DU Mei, MA Yunxuan, CUI Yongqi, CHEN Xilin, GUO Changqing. Acupotomy alleviates knee osteoarthritis in rabbit by regulating chondrocyte mitophagy via Pink1-Parkin pathway[J]. Journal of Traditional Chinese Medicine, 2024, 44(3): 468-477.
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Table 1 Behavioral assessment of rabbits from the 3 groups (scores, $\bar{x} \pm s$)
| Item | Group | n | Before intervention | After intervention |
|---|---|---|---|---|
| PROM | CON | 6 | 141.2±2.1 | 140.7±3.5 |
| KOA | 6 | 31.2±6.5a | 53.2±10.0a | |
| Acu | 6 | 40.3±4.6a | 97.3±12.5ab | |
| Lequesne MG | CON | 6 | 0.0±0.0 | 0.0±0.0 |
| KOA | 6 | 7.3±0.5 | 6.5±1.0 | |
| Acu | 6 | 7.3±0.8 | 3.3±0.8b |
Table 1 Behavioral assessment of rabbits from the 3 groups (scores, $\bar{x} \pm s$)
| Item | Group | n | Before intervention | After intervention |
|---|---|---|---|---|
| PROM | CON | 6 | 141.2±2.1 | 140.7±3.5 |
| KOA | 6 | 31.2±6.5a | 53.2±10.0a | |
| Acu | 6 | 40.3±4.6a | 97.3±12.5ab | |
| Lequesne MG | CON | 6 | 0.0±0.0 | 0.0±0.0 |
| KOA | 6 | 7.3±0.5 | 6.5±1.0 | |
| Acu | 6 | 7.3±0.8 | 3.3±0.8b |
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 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 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 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 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 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 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.
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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