Abstract
Background Emerging studies have suggested an essential role of fibroblast metabolic reprogramming in the pathogenesis of arthrofibrosis. The metabolic modulator metformin appears to be a good candidate for fibrotic disorder treatment. We sought to determine the beneficial effect of metformin on arthrofibrosis and the underlying mechanism of action of this drug.
Methods Shoulder capsule samples were collected from patients with/without frozen shoulder to perform gene and protein expression analysis. Arthrofibrotic animal model was established to examine the anti-fibrotic effect of metformin. In vitro cell culture experiments were conducted to determine the mechanism by which metformin inhibits fibroblast activation.
Results Clinical sample analysis demonstrated that glycolysis was upregulated in human fibrotic joint capsules. In an arthrofibrotic animal model, metformin improved the joint range of motion, reduced capsular fibrosis and thickening, mitigated inflammatory reactions, and downregulated both fibrotic and glycolytic markers. The results of gene and protein expression analysis showed that metformin effectively inhibited TGF-β induced fibroblast activation. The metabolomics study confirmed a metformin-induced metabolic shift from oxidative phosphorylation (OXPHOS) to aerobic glycolysis in fibroblasts. Importantly, the inhibitory effect of metformin on fibroblast activation was mediated by metabolic reprogramming of fibroblasts. Interestingly, fibroblast-derived lactate induced M2-like macrophage polarization and diminished the inflammatory reaction.
Conclusions Metformin is a potential treatment alternative for arthrofibrosis, and antifibrotic effect of the drug involves metabolic reprogramming toward aerobic glycolysis in fibroblasts and lactate-induced M2-like macrophage polarization.
Competing Interest Statement
The authors have no relevant financial or non-financial interests to disclose.
Footnotes
Funding This work was supported by Chongqing Natural Science Foundation (Grant No. cstc2019jcyj-msxmX0831), the Science and Technology Research Program of Chongqing Municipal Education Commission (Grant No. KJQN202000430) and Chongqing Medical Scientific Research Project (Grant No. 2021MSXM032). This project was also supported by Innovation Project from Chongqing Municipal Education Commission (#CYB21169) to Z.Z.
Abbreviations
- AMPK
- AMP-activated protein kinase
- ANOVA
- Analysis of variance
- α-SMA
- α-Smooth muscle actin
- Arg-1
- Arginase-1
- CCK-8
- Cell Counting Kit-8
- DAPI
- 4’,6-diamidino-2-phenylindole
- ECM
- Extracellular matrix
- EDTA
- Ethylenediaminetetraacetic acid
- FBS
- Fetal bovine serum
- 18FDG-PET
- 18F-fluoro-deoxyglucose positron-emission tomography
- FN1
- Fibronectin
- GLUT1
- Glucose transporter 1
- HE
- Hematoxylin-eosin
- HK2
- Hexokinase-2
- IFN-γ
- Interferon-γ
- IHC
- Immunohistochemistry
- IL
- Interleukin
- iNOS
- Inducible nitric oxide synthase
- LC-MS
- Liquid Chromatograph-Mass Spectrometer
- LDHA
- Lactate dehydrogenase
- MΦ
- Macrophage
- NSAIDs
- Non-Steroidal Anti-Inflammatory Drugs
- OARSI
- Osteoarthritis Research Society International
- OD
- Optical density
- OXPHOS
- Oxidative phosphorylation
- PBS
- Phosphate buffered saline
- PFKFB3
- 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3
- PFKP
- Phosphofructokinase
- PLGA
- Poly (lactic-co-glycolic acid)
- PKM
- Pyruvate kinase M
- qRT-PCR
- Reverse transcription-quantitative polymerase chain reaction
- ROM
- Range of motion
- SEM
- Standard error of mean
- TCA
- Tricarboxylic acid
- TGF-β
- Transforming growth factor-β
- TNF-α
- Tumor necrosis factor-α