Irisin Mitigates Oxidative Stress, Chondrocyte Dysfunction and Osteoarthritis Development through Regulating Mitochondrial Integrity and Autophagy methods
Aim. Evidence-backed execution summary for Irisin Mitigates Oxidative Stress, Chondrocyte Dysfunction and Osteoarthritis Development through Regulating Mitochondrial Integrity and Autophagy methods from Irisin Mitigates Oxidative Stress, Chondrocyte Dysfunction and Osteoarthritis Development through Regulating Mitochondrial Integrity and Autophagy.
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mouse
Subject model for the experiment.
- Use
- confirm full cohort details in the source paper
2.3. Purification of Irisin Recombinant Protein
reagent used in the protocol.
- Use
- DNA coded Irisin (NM027402.4) was cloned and amplified using PCR protocols with primers (forward, 5′-ATTTCATATGAGCCCCTCAGCCCCT-3′; reverse, 5′-CCGCTCGAGCACTCCTTCAT GGTCAC-3′). The clone was ligated in between the NotI and BamHI restriction enzyme sites of pET28a vector (Novagen Inc., Merck KG...
2.6. Histomorphometry and Immunohistochemistry
reagent used in the protocol.
- Use
- Knee joints between tibiae and femurs were decalcified and embedded in paraffin. Articular cartilage morphology at proximal tibiae was stained using Safranin-O Staining Kits (3-H Biomedical, Uppsala, Sweden), according to the manufacturer's manuals. The severity of cartilage damage was quantified using the OAR...
2.7. Chondrocyte Cultures
reagent used in the protocol.
- Use
- After euthanasia, articular cartilage from the hips, femurs and tibiae of 7-day-old mice were dissected under a surgical microscope and incubated in 1 unit/mL collagenase (Sigma-Aldrich, St. Louis, MO, USA) to isolate chondrocytes, as previously described [ ]. Cells were incubated in DMEM with 10% fetal bovine serum...
2.8. Assessment of Chondrocytic Activity
reagent used in the protocol.
- Use
- For analysis of glycosaminoglycan production, 5 × 10 5 IL-1β, Irisin, IL-1β + Irisin and vehicle-treated chondrocytes in 20 µL medium were pipetted to form a drop on the culture plates (24-well plates) and incubated in a 5% O 2, 37 °C humidified chamber for 72 h. Micromass cultures were sta...
2.9. Reverse Transcription-Polymerase Chain Reaction (RT-PCR)
reagent used in the protocol.
- Use
- The articular cartilage of mouse knee joints and 10 6 chondrocytes were mixed with TRI Reagent™ Solution (Thermo Fisher Scientific Inc., Waltham, MA, USA) to extract total RNA, according to makers' manuals. Reverse transcription of 1 µg total RNA was performed using High-Capacity cDNA Reverse Transc...
2.10. Analysis of Autophagosome and Mitophagosome Formation
reagent used in the protocol.
- Use
- The autophagic vacuoles in cell cultures were probed using Autophagy Detection Kits (Abcam, Cambridge, UK), according to maker's manuals. In brief, IL-1β, Irisin, IL-1β + Irisin and vehicle-treated chondrocytes (10 3 cells/slide) were incubated on Falcon™ chambered cell culture slides (Thermo F...
2.11. Mitochondrial Dynamics Assay
reagent used in the protocol.
- Use
- Mitochondrial morphology in IL-1β, Irisin, IL-1β + Irisin and vehicle-treated cell cultures (10 3 cells/slide) was probed using MitoSOX Red (Thermo Fisher Scientific Inc., Waltham, MA, USA) and evaluated using a laser confocal microscope. Mitochondrial morphology was categorized using MicroP software, as p...
2.12. Mitochondrial Membrane Potential, ATP Production and Reactive Oxygen Radical (ROS) Level Assay
reagent used in the protocol.
- Use
- IL-1β, Irisin, IL-1β + Irisin, and vehicle-treated chondrocytes (2 × 10 5 cells) were probed using JC1-1 Mitochondrial Membrane Potential Assay Kits (Abcam, Cambridge, UK). In brief, cells were washed with 1 × Dilution Buffer and stained with 20 µM JC-1 at 37 °C in the dark for 15 min. T...
2.5. Gait Profile Analysis
The walking patterns and gait characteristics of injured legs were investigated using the Catwalk analysis system (Noldus Information Technology, Leesburg, VA, USA), as previously described [ ]. Footprint histograms were computed using CatWalk software 9.1 (Noldus Information Technology, Leesburg, VA, USA), accordin...
- Use
- The walking patterns and gait characteristics of injured legs were investigated using the Catwalk analysis system (Noldus Information Technology, Leesburg, VA, USA), as previously described [ ]. Footprint histograms were computed using CatWalk software 9.1 (Noldus Information Technology, Leesburg, VA, USA), accordin...
2.6. Histomorphometry and Immunohistochemistry
Knee joints between tibiae and femurs were decalcified and embedded in paraffin. Articular cartilage morphology at proximal tibiae was stained using Safranin-O Staining Kits (3-H Biomedical, Uppsala, Sweden), according to the manufacturer's manuals. The severity of cartilage damage was quantified using the OAR...
- Use
- Knee joints between tibiae and femurs were decalcified and embedded in paraffin. Articular cartilage morphology at proximal tibiae was stained using Safranin-O Staining Kits (3-H Biomedical, Uppsala, Sweden), according to the manufacturer's manuals. The severity of cartilage damage was quantified using the OAR...
2.7. Chondrocyte Cultures
After euthanasia, articular cartilage from the hips, femurs and tibiae of 7-day-old mice were dissected under a surgical microscope and incubated in 1 unit/mL collagenase (Sigma-Aldrich, St. Louis, MO, USA) to isolate chondrocytes, as previously described [ ]. Cells were incubated in DMEM with 10% fetal bovine serum...
- Use
- After euthanasia, articular cartilage from the hips, femurs and tibiae of 7-day-old mice were dissected under a surgical microscope and incubated in 1 unit/mL collagenase (Sigma-Aldrich, St. Louis, MO, USA) to isolate chondrocytes, as previously described [ ]. Cells were incubated in DMEM with 10% fetal bovine serum...
2.8. Assessment of Chondrocytic Activity
For analysis of glycosaminoglycan production, 5 × 10 5 IL-1β, Irisin, IL-1β + Irisin and vehicle-treated chondrocytes in 20 µL medium were pipetted to form a drop on the culture plates (24-well plates) and incubated in a 5% O 2, 37 °C humidified chamber for 72 h. Micromass cultures were sta...
- Use
- For analysis of glycosaminoglycan production, 5 × 10 5 IL-1β, Irisin, IL-1β + Irisin and vehicle-treated chondrocytes in 20 µL medium were pipetted to form a drop on the culture plates (24-well plates) and incubated in a 5% O 2, 37 °C humidified chamber for 72 h. Micromass cultures were sta...
2.9. Reverse Transcription-Polymerase Chain Reaction (RT-PCR)
The articular cartilage of mouse knee joints and 10 6 chondrocytes were mixed with TRI Reagent™ Solution (Thermo Fisher Scientific Inc., Waltham, MA, USA) to extract total RNA, according to makers' manuals. Reverse transcription of 1 µg total RNA was performed using High-Capacity cDNA Reverse Transc...
- Use
- The articular cartilage of mouse knee joints and 10 6 chondrocytes were mixed with TRI Reagent™ Solution (Thermo Fisher Scientific Inc., Waltham, MA, USA) to extract total RNA, according to makers' manuals. Reverse transcription of 1 µg total RNA was performed using High-Capacity cDNA Reverse Transc...
2.10. Analysis of Autophagosome and Mitophagosome Formation
The autophagic vacuoles in cell cultures were probed using Autophagy Detection Kits (Abcam, Cambridge, UK), according to maker's manuals. In brief, IL-1β, Irisin, IL-1β + Irisin and vehicle-treated chondrocytes (10 3 cells/slide) were incubated on Falcon™ chambered cell culture slides (Thermo F...
- Use
- The autophagic vacuoles in cell cultures were probed using Autophagy Detection Kits (Abcam, Cambridge, UK), according to maker's manuals. In brief, IL-1β, Irisin, IL-1β + Irisin and vehicle-treated chondrocytes (10 3 cells/slide) were incubated on Falcon™ chambered cell culture slides (Thermo F...
2.11. Mitochondrial Dynamics Assay
Mitochondrial morphology in IL-1β, Irisin, IL-1β + Irisin and vehicle-treated cell cultures (10 3 cells/slide) was probed using MitoSOX Red (Thermo Fisher Scientific Inc., Waltham, MA, USA) and evaluated using a laser confocal microscope. Mitochondrial morphology was categorized using MicroP software, as p...
- Use
- Mitochondrial morphology in IL-1β, Irisin, IL-1β + Irisin and vehicle-treated cell cultures (10 3 cells/slide) was probed using MitoSOX Red (Thermo Fisher Scientific Inc., Waltham, MA, USA) and evaluated using a laser confocal microscope. Mitochondrial morphology was categorized using MicroP software, as p...
2.12. Mitochondrial Membrane Potential, ATP Production and Reactive Oxygen Radical (ROS) Level Assay
IL-1β, Irisin, IL-1β + Irisin, and vehicle-treated chondrocytes (2 × 10 5 cells) were probed using JC1-1 Mitochondrial Membrane Potential Assay Kits (Abcam, Cambridge, UK). In brief, cells were washed with 1 × Dilution Buffer and stained with 20 µM JC-1 at 37 °C in the dark for 15 min. T...
- Use
- IL-1β, Irisin, IL-1β + Irisin, and vehicle-treated chondrocytes (2 × 10 5 cells) were probed using JC1-1 Mitochondrial Membrane Potential Assay Kits (Abcam, Cambridge, UK). In brief, cells were washed with 1 × Dilution Buffer and stained with 20 µM JC-1 at 37 °C in the dark for 15 min. T...
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2.3. Purification of Irisin Recombinant Protein
DNA coded Irisin (NM027402.4) was cloned and amplified using PCR protocols with primers (forward, 5′-ATTTCATATGAGCCCCTCAGCCCCT-3′; reverse, 5′-CCGCTCGAGCACTCCTTCAT GGTCAC-3′). The clone was ligated in between the NotI and BamHI restriction enzyme sites of pET28a vector (Novagen Inc., Merck KGaA, Darmstadt, Germany). Plasmids were transferred into BL-21SI competent cells. Upon incubating the transformed cells in 3 mM NaCl for 4 h at 30 °C, cells were centrifuged at 6000 × g and 4 °C for 5 min which was followed by mixing with lysis buffer (pH 8.0) with 20 mM imidazole, 150 mM NaCl, 1 mM EDTA, 1 mM PMSF, 1 µg/mL aprotinin, 1 µg/mL leupeptin and 1 µg/mL pepstatin. The mixtures were homogenized and centrifuged at 9000 × g and 4 °C for 30 min. Irisin recombinant proteins in the supernatants were mixed with 1 mL Ni-NTA beads for...
2.4. Intra-Articular Injection of Irisin
20 mg/mL of Irisin recombinant protein were dissolved in normal saline and filtered through 0.22 µm filters. At 1 week postoperatively, a total of 10 µL Irisin mixtures were intra-articularly injected into DMM-injured knees using an insulin needle under the guidance of sonography (10-22 MHz, LOGIO TM, GE Healthcare, Princeton, NJ, USA), as previously described [ ]. At 8 weeks postoperatively, mice in the sham ( n = 5), DMM ( n = 5) and DMM + Irisin ( n = 5) groups were euthanatized, and knee joints were dissected.
2.8. Assessment of Chondrocytic Activity
For analysis of glycosaminoglycan production, 5 × 10 5 IL-1β, Irisin, IL-1β + Irisin and vehicle-treated chondrocytes in 20 µL medium were pipetted to form a drop on the culture plates (24-well plates) and incubated in a 5% O 2, 37 °C humidified chamber for 72 h. Micromass cultures were stained using Alcian Blue Stain Kits (ab150662; Abcam, Cambridge, UK). The stain in the micromass culture was dissolved in 50 µL of 6 M guanidine-HCl, and the absorbance of the mixture was detected using a spectrophotometer at a 620 nm wavelength and normalized with the total proteins of the micromass culture, as previously described [ ].
2.10. Analysis of Autophagosome and Mitophagosome Formation
The autophagic vacuoles in cell cultures were probed using Autophagy Detection Kits (Abcam, Cambridge, UK), according to maker's manuals. In brief, IL-1β, Irisin, IL-1β + Irisin and vehicle-treated chondrocytes (10 3 cells/slide) were incubated on Falcon™ chambered cell culture slides (Thermo Fisher Scientific Inc., Waltham, MA, USA) for 24 h. Upon removing medium, specimens were rinsed with PBS, fixed with 4% formaldehyde and washed with 1 × Assay buffer. The slides were incubated in Green Detection Reagent with fluorescent monodansylcadaverine in the dark for 30 min, and then washed with 1 × Assay buffer. The slides were covered with Prolong Gold Antifade Reagent (Cell Signaling Technology, Danvers, MA, USA). In some experiments, mitophagosome was probed using Mitophagy Detection Kits (Dojindo Laboratories, Kumamoto, Japan), according to maker's i...
2.12. Mitochondrial Membrane Potential, ATP Production and Reactive Oxygen Radical (ROS) Level Assay
IL-1β, Irisin, IL-1β + Irisin, and vehicle-treated chondrocytes (2 × 10 5 cells) were probed using JC1-1 Mitochondrial Membrane Potential Assay Kits (Abcam, Cambridge, UK). In brief, cells were washed with 1 × Dilution Buffer and stained with 20 µM JC-1 at 37 °C in the dark for 15 min. The red (FL2) and green (FL1) fluorescence reactions of each cell were analyzed using BD Accuri C6 flow cytometer (BD Biosciences, San Jose, CA, USA). Mitochondrial membrane potential was calculated from the ratio of FL2/FL1. In some experiments, ATP production of chondrocyte was investigated using ATP Assay Kits (ab83355, Abcam, Cambridge, UK), according to manufacturer's instructions. In brief, 5 × 10 5 chondrocytes were homogenized in ATP Assay Buffer. Aliquots of cell lysates were incubated with ATP Reaction Mix for 30 min. Absorbance of the mixtures was quant...
3.2. Irisin Attenuated Articular Cartilage Loss and Irregular Gait of DMM-Injured Knees
Irisin is a soluble peptide cleaved from the extracellular domain of FNDC5, regulating the apoptotic program or autophagic reaction in various cell types [, ]. The analyses of decreased FNDC5 signaling in osteoarthritic chondrocytes in human OA prompted us to isolate Irisin recombinant proteins and verify whether the gained Irisin function changed knee OA development. We constructed expression vector coding for Irisin and isolated the recombinant protein corresponding to 15 kDa. Immunoblotting confirmed the Irisin immunoactivity of the recombinant protein ( a). We adopted the destabilization of medial meniscus (DMM) in the knee joints of mice as a well-established in vivo model of OA development [ ]. The recombinant protein was intra-articularly injected into DMM-injured knee joints 1 week postoperatively ( b). DMM-injured joints showed articular cartilage degradation, synovium hyper...
3.4. Irisin Alleviated ECM Production of Inflamed Chondrocytes
The investigations of Irisin-mediated repression of articular cartilage destruction in the development of OA prompted us to characterize organelle machinery by which Irisin promoted chondrocyte survival of the injured cartilage. Chondrocytes were incubated in 5 ng/mL IL-1β as an in vitro model simulating inflamed chondrocytes in the OA microenvironment [ ]. IL-1β significantly decreased Alcian blue-stained glycosaminoglycan production of chondrocyte micromass cultures 72 h after incubation ( a). Irisin dose-dependently promoted ECM accumulation above the baseline, as well as attenuating ECM underproduction in the inflamed chondrocytes ( b). The treatment with 10 ng/mL Irisin had the greatest promotion and was chosen for succeeding experiments. IL-1β significantly decreased FNDC5 expression ( c) and cell growth after 24 h of incubation ( d). Chondrocytic markers collagen...
Measurement outputs
What raw and processed outputs should exist?
DNA coded Irisin (NM027402.4) was cloned and amplified using PCR protocols with primers (forward, 5′-ATTTCATATGAGCCCCTCAGCCCCT-3′; reverse, 5′-CCGCTCGAGCACTCCT...
- Raw artifact
- Per-run gait capture with paw placement, timing, and stride features for each animal
- Processed artifact
- Cleaned gait metrics table and recovery trend summary across timepoints
- Reported as
- Group comparisons of gait indices, stride metrics, or recovery curves
The walking patterns and gait characteristics of injured legs were investigated using the Catwalk analysis system (Noldus Information Technology, Leesburg, VA, USA), as previous...
- Raw artifact
- Per-run gait capture with paw placement, timing, and stride features for each animal
- Processed artifact
- Cleaned gait metrics table and recovery trend summary across timepoints
- Reported as
- Group comparisons of gait indices, stride metrics, or recovery curves
Knee joints between tibiae and femurs were decalcified and embedded in paraffin. Articular cartilage morphology at proximal tibiae was stained using Safranin-O Staining Kits (3-...
- Raw artifact
- Per-run gait capture with paw placement, timing, and stride features for each animal
- Processed artifact
- Cleaned gait metrics table and recovery trend summary across timepoints
- Reported as
- Group comparisons of gait indices, stride metrics, or recovery curves
For analysis of glycosaminoglycan production, 5 × 10 5 IL-1β, Irisin, IL-1β + Irisin and vehicle-treated chondrocytes in 20 µL medium were pipetted to form a...
- Raw artifact
- Per-run gait capture with paw placement, timing, and stride features for each animal
- Processed artifact
- Cleaned gait metrics table and recovery trend summary across timepoints
- Reported as
- Group comparisons of gait indices, stride metrics, or recovery curves
Analysis plan
How should the outputs become interpretable results?
Acquisition
Capture run-level gait data for each animal and preserve the timepoint or treatment labeling.
inferred from protocolPreprocessing / cleaning
Differences between the investigations of OA and non-OA in human specimens were analyzed using Wilcoxon test.
from paperScoring or quantification
Quantify the primary readouts for this experiment: DNA coded Irisin (NM027402.4) was cloned and amplified using PCR protocols with primers (forward, 5′-ATTTCATATGAGCCCCTCAGCCCCT-3′; reverse, 5′-CCGCTCGAGCACTCCT...; The walking patterns and gait characteristics of injured legs were investigated using the Catwalk analysis system (Noldus Information Technology, Leesburg, VA, USA), as previous...; Knee joints between tibiae and femurs were decalcified and embedded in paraffin. Articular cartilage morphology at proximal tibiae was stained using Safranin-O Staining Kits (3-...; For analysis of glycosaminoglycan production, 5 × 10 5 IL-1β, Irisin, IL-1β + Irisin and vehicle-treated chondrocytes in 20 µL medium were pipetted to form a....
from paperStatistical comparison
Differences between the investigations of OA and non-OA in human specimens were analyzed using Wilcoxon test. Differences of analyses among 3 groups in the experimental OA model...; Irisin is a soluble peptide cleaved from the extracellular domain of FNDC5, regulating the apoptotic program or autophagic reaction in various cell types [, ]. The analyses of...
from paperReporting output
Report representative outputs alongside summary comparisons for DNA coded Irisin (NM027402.4) was cloned and amplified using PCR protocols with primers (forward, 5′-ATTTCATATGAGCCCCTCAGCCCCT-3′; reverse, 5′-CCGCTCGAGCACTCCT..., The walking patterns and gait characteristics of injured legs were investigated using the Catwalk analysis system (Noldus Information Technology, Leesburg, VA, USA), as previous..., Knee joints between tibiae and femurs were decalcified and embedded in paraffin. Articular cartilage morphology at proximal tibiae was stained using Safranin-O Staining Kits (3-..., For analysis of glycosaminoglycan production, 5 × 10 5 IL-1β, Irisin, IL-1β + Irisin and vehicle-treated chondrocytes in 20 µL medium were pipetted to form a....
inferred from protocolStructured statistical methods
Differences between the investigations of OA and non-OA in human specimens were analyzed using Wilcoxon test. Differences of analyses among 3 groups in the experimental OA model...; Irisin is a soluble peptide cleaved from the extracellular domain of FNDC5, regulating the apoptotic program or autophagic reaction in various cell types [, ]. The analyses of...
source structuredSource and audit
What supports the facts on this page?
Evidence quotes (7)
DNA coded Irisin (NM027402.4) was cloned and amplified using PCR protocols with primers (forward, 5′-ATTTCATATGAGCCCCTCAGCCCCT-3′; reverse, 5′-CCGCTCGAGCACTCCTTCAT GGTCAC-3′). The clone was ligated in between the NotI and BamHI restriction enzyme sites of pET28a vector (Novagen Inc., Merck KGaA, Darmstadt, Germany). Plasmids were transferred into BL-21SI competent cells. Upon incubating the transformed cells in 3 mM NaCl for 4 h at 30 °C, cells were centrifuged at 6000 × g and 4 °C for 5 min which was followed by mixing with lysis buffer (pH 8.0) with 20 mM imidazole, 150 mM NaCl, 1 mM EDTA, 1 mM PMSF, 1 µg/mL aprotinin, 1 µg/mL leupeptin and 1 µg/mL pepstatin. The mixtures were homogenized and centrifuged at 9000 × g and 4 °C for 30 min. Irisin recombinant proteins in the supernatants were mixed with 1 mL Ni-NTA beads for 20 min and eluted through nickel-gel column affinity chromatography (GE Pharmacia, Princeton, NJ, USA) using an elution buffer (pH 7.0) with 20 mM phosphate buffer, 250 mM imidazole and 150 mM NaCl. Irisin recombinant proteins were freeze-dried and stored at -80 °C.
20 mg/mL of Irisin recombinant protein were dissolved in normal saline and filtered through 0.22 µm filters. At 1 week postoperatively, a total of 10 µL Irisin mixtures were intra-articularly injected into DMM-injured knees using an insulin needle under the guidance of sonography (10-22 MHz, LOGIO TM, GE Healthcare, Princeton, NJ, USA), as previously described [ ]. At 8 weeks postoperatively, mice in the sham ( n = 5), DMM ( n = 5) and DMM + Irisin ( n = 5) groups were euthanatized, and knee joints were dissected.
For analysis of glycosaminoglycan production, 5 × 10 5 IL-1β, Irisin, IL-1β + Irisin and vehicle-treated chondrocytes in 20 µL medium were pipetted to form a drop on the culture plates (24-well plates) and incubated in a 5% O 2, 37 °C humidified chamber for 72 h. Micromass cultures were stained using Alcian Blue Stain Kits (ab150662; Abcam, Cambridge, UK). The stain in the micromass culture was dissolved in 50 µL of 6 M guanidine-HCl, and the absorbance of the mixture was detected using a spectrophotometer at a 620 nm wavelength and normalized with the total proteins of the micromass culture, as previously described [ ].
The autophagic vacuoles in cell cultures were probed using Autophagy Detection Kits (Abcam, Cambridge, UK), according to maker's manuals. In brief, IL-1β, Irisin, IL-1β + Irisin and vehicle-treated chondrocytes (10 3 cells/slide) were incubated on Falcon™ chambered cell culture slides (Thermo Fisher Scientific Inc., Waltham, MA, USA) for 24 h. Upon removing medium, specimens were rinsed with PBS, fixed with 4% formaldehyde and washed with 1 × Assay buffer. The slides were incubated in Green Detection Reagent with fluorescent monodansylcadaverine in the dark for 30 min, and then washed with 1 × Assay buffer. The slides were covered with Prolong Gold Antifade Reagent (Cell Signaling Technology, Danvers, MA, USA). In some experiments, mitophagosome was probed using Mitophagy Detection Kits (Dojindo Laboratories, Kumamoto, Japan), according to maker's instruction. In brief, cells were washed with PBS and incubated in 100 nM Mitophagy Dye Solution for 30 min, followed by incubating cell cultures in medium with IL-1β or Irisin for 24 h. Upon removing the culture medium and washing with PBS, specimens were incubated in 1 mM Lyso Dye Solution for...
IL-1β, Irisin, IL-1β + Irisin, and vehicle-treated chondrocytes (2 × 10 5 cells) were probed using JC1-1 Mitochondrial Membrane Potential Assay Kits (Abcam, Cambridge, UK). In brief, cells were washed with 1 × Dilution Buffer and stained with 20 µM JC-1 at 37 °C in the dark for 15 min. The red (FL2) and green (FL1) fluorescence reactions of each cell were analyzed using BD Accuri C6 flow cytometer (BD Biosciences, San Jose, CA, USA). Mitochondrial membrane potential was calculated from the ratio of FL2/FL1. In some experiments, ATP production of chondrocyte was investigated using ATP Assay Kits (ab83355, Abcam, Cambridge, UK), according to manufacturer's instructions. In brief, 5 × 10 5 chondrocytes were homogenized in ATP Assay Buffer. Aliquots of cell lysates were incubated with ATP Reaction Mix for 30 min. Absorbance of the mixtures was quantified using a spectrophotometer at a 570 nm wavelength. ATP levels were calculated from a standard curve plotted by the serial dilution of authentic ATP. The ROS levels in cells were probed using 5-6-chloromethyl-2′,7-dichlorodihydrofluorescein diacetate acetyl ester (CM-H 2 DCFAD; Invitrogen,...
Irisin is a soluble peptide cleaved from the extracellular domain of FNDC5, regulating the apoptotic program or autophagic reaction in various cell types [, ]. The analyses of decreased FNDC5 signaling in osteoarthritic chondrocytes in human OA prompted us to isolate Irisin recombinant proteins and verify whether the gained Irisin function changed knee OA development. We constructed expression vector coding for Irisin and isolated the recombinant protein corresponding to 15 kDa. Immunoblotting confirmed the Irisin immunoactivity of the recombinant protein ( a). We adopted the destabilization of medial meniscus (DMM) in the knee joints of mice as a well-established in vivo model of OA development [ ]. The recombinant protein was intra-articularly injected into DMM-injured knee joints 1 week postoperatively ( b). DMM-injured joints showed articular cartilage degradation, synovium hypertrophy and subchondral bone exposure histopathology, as compared to the sham group 8 weeks postoperatively. The Irisin-treated joints displayed less cartilage injury and synovitis than the DMM group ( c). Likewise, the severity of articular cartilage damage and synovitis was significantly increased...
The investigations of Irisin-mediated repression of articular cartilage destruction in the development of OA prompted us to characterize organelle machinery by which Irisin promoted chondrocyte survival of the injured cartilage. Chondrocytes were incubated in 5 ng/mL IL-1β as an in vitro model simulating inflamed chondrocytes in the OA microenvironment [ ]. IL-1β significantly decreased Alcian blue-stained glycosaminoglycan production of chondrocyte micromass cultures 72 h after incubation ( a). Irisin dose-dependently promoted ECM accumulation above the baseline, as well as attenuating ECM underproduction in the inflamed chondrocytes ( b). The treatment with 10 ng/mL Irisin had the greatest promotion and was chosen for succeeding experiments. IL-1β significantly decreased FNDC5 expression ( c) and cell growth after 24 h of incubation ( d). Chondrocytic markers collagen II, aggrecan and Sox9 were significantly downregulated in the inflamed chondrocytes ( e), whereas synovitis-promoting factors matrix metalloproteinase 9 (MMP9) and vascular endothelium growth factor (VEGF) ( f) were increased. Irisin significantly reversed the IL-1β-mediated loss of FNDC5 sign...
Machine-readable layer
[
{
"@context": "https://schema.org",
"@type": "HowTo",
"name": "Irisin Mitigates Oxidative Stress, Chondrocyte Dysfunction and Osteoarthritis Development through Regulating Mitochondrial Integrity and Autophagy methods",
"description": "Evidence-backed execution summary for Irisin Mitigates Oxidative Stress, Chondrocyte Dysfunction and Osteoarthritis Development through Regulating Mitochondrial Integrity and Autophagy methods from Irisin Mitigates Oxidative Stress, Chondrocyte Dysfunction and Osteoarthritis Development through Regulating Mitochondrial Integrity and Autophagy.",
"totalTime": "PT4850M",
"step": [
{
"@type": "HowToStep",
"position": 1,
"name": "2.3. Purification of Irisin Recombinant Protein",
"text": "DNA coded Irisin (NM027402.4) was cloned and amplified using PCR protocols with primers (forward, 5′-ATTTCATATGAGCCCCTCAGCCCCT-3′; reverse, 5′-CCGCTCGAGCACTCCTTCAT GGTCAC-3′). The clone was ligated in between the NotI and BamHI restriction enzyme sites of pET28a vector (Novagen Inc., Merck KGaA, Darmstadt, Germany). Plasmids were transferred into BL-21SI competent cells. Upon incubating the transformed cells in 3 mM NaCl for 4 h at 30 °C, cells were centrifuged at 6000 × g and 4 °C for 5 min which was followed by mixing with lysis buffer (pH 8.0) with 20 mM imidazole, 150 mM NaCl, 1 mM EDTA, 1 mM PMSF, 1 µg/mL aprotinin, 1 µg/mL leupeptin and 1 µg/mL pepstatin. The mixtures were homogenized and centrifuged at 9000 × g and 4 °C for 30 min. Irisin recombinant proteins in the supernatants were mixed with 1 mL Ni-NTA beads for..."
},
{
"@type": "HowToStep",
"position": 2,
"name": "2.4. Intra-Articular Injection of Irisin",
"text": "20 mg/mL of Irisin recombinant protein were dissolved in normal saline and filtered through 0.22 µm filters. At 1 week postoperatively, a total of 10 µL Irisin mixtures were intra-articularly injected into DMM-injured knees using an insulin needle under the guidance of sonography (10-22 MHz, LOGIO TM, GE Healthcare, Princeton, NJ, USA), as previously described [ ]. At 8 weeks postoperatively, mice in the sham ( n = 5), DMM ( n = 5) and DMM + Irisin ( n = 5) groups were euthanatized, and knee joints were dissected."
},
{
"@type": "HowToStep",
"position": 3,
"name": "2.8. Assessment of Chondrocytic Activity",
"text": "For analysis of glycosaminoglycan production, 5 × 10 5 IL-1β, Irisin, IL-1β + Irisin and vehicle-treated chondrocytes in 20 µL medium were pipetted to form a drop on the culture plates (24-well plates) and incubated in a 5% O 2, 37 °C humidified chamber for 72 h. Micromass cultures were stained using Alcian Blue Stain Kits (ab150662; Abcam, Cambridge, UK). The stain in the micromass culture was dissolved in 50 µL of 6 M guanidine-HCl, and the absorbance of the mixture was detected using a spectrophotometer at a 620 nm wavelength and normalized with the total proteins of the micromass culture, as previously described [ ]."
},
{
"@type": "HowToStep",
"position": 4,
"name": "2.10. Analysis of Autophagosome and Mitophagosome Formation",
"text": "The autophagic vacuoles in cell cultures were probed using Autophagy Detection Kits (Abcam, Cambridge, UK), according to maker's manuals. In brief, IL-1β, Irisin, IL-1β + Irisin and vehicle-treated chondrocytes (10 3 cells/slide) were incubated on Falcon™ chambered cell culture slides (Thermo Fisher Scientific Inc., Waltham, MA, USA) for 24 h. Upon removing medium, specimens were rinsed with PBS, fixed with 4% formaldehyde and washed with 1 × Assay buffer. The slides were incubated in Green Detection Reagent with fluorescent monodansylcadaverine in the dark for 30 min, and then washed with 1 × Assay buffer. The slides were covered with Prolong Gold Antifade Reagent (Cell Signaling Technology, Danvers, MA, USA). In some experiments, mitophagosome was probed using Mitophagy Detection Kits (Dojindo Laboratories, Kumamoto, Japan), according to maker's i..."
},
{
"@type": "HowToStep",
"position": 5,
"name": "2.12. Mitochondrial Membrane Potential, ATP Production and Reactive Oxygen Radical (ROS) Level Assay",
"text": "IL-1β, Irisin, IL-1β + Irisin, and vehicle-treated chondrocytes (2 × 10 5 cells) were probed using JC1-1 Mitochondrial Membrane Potential Assay Kits (Abcam, Cambridge, UK). In brief, cells were washed with 1 × Dilution Buffer and stained with 20 µM JC-1 at 37 °C in the dark for 15 min. The red (FL2) and green (FL1) fluorescence reactions of each cell were analyzed using BD Accuri C6 flow cytometer (BD Biosciences, San Jose, CA, USA). Mitochondrial membrane potential was calculated from the ratio of FL2/FL1. In some experiments, ATP production of chondrocyte was investigated using ATP Assay Kits (ab83355, Abcam, Cambridge, UK), according to manufacturer's instructions. In brief, 5 × 10 5 chondrocytes were homogenized in ATP Assay Buffer. Aliquots of cell lysates were incubated with ATP Reaction Mix for 30 min. Absorbance of the mixtures was quant..."
},
{
"@type": "HowToStep",
"position": 6,
"name": "3.2. Irisin Attenuated Articular Cartilage Loss and Irregular Gait of DMM-Injured Knees",
"text": "Irisin is a soluble peptide cleaved from the extracellular domain of FNDC5, regulating the apoptotic program or autophagic reaction in various cell types [, ]. The analyses of decreased FNDC5 signaling in osteoarthritic chondrocytes in human OA prompted us to isolate Irisin recombinant proteins and verify whether the gained Irisin function changed knee OA development. We constructed expression vector coding for Irisin and isolated the recombinant protein corresponding to 15 kDa. Immunoblotting confirmed the Irisin immunoactivity of the recombinant protein ( a). We adopted the destabilization of medial meniscus (DMM) in the knee joints of mice as a well-established in vivo model of OA development [ ]. The recombinant protein was intra-articularly injected into DMM-injured knee joints 1 week postoperatively ( b). DMM-injured joints showed articular cartilage degradation, synovium hyper..."
},
{
"@type": "HowToStep",
"position": 7,
"name": "3.4. Irisin Alleviated ECM Production of Inflamed Chondrocytes",
"text": "The investigations of Irisin-mediated repression of articular cartilage destruction in the development of OA prompted us to characterize organelle machinery by which Irisin promoted chondrocyte survival of the injured cartilage. Chondrocytes were incubated in 5 ng/mL IL-1β as an in vitro model simulating inflamed chondrocytes in the OA microenvironment [ ]. IL-1β significantly decreased Alcian blue-stained glycosaminoglycan production of chondrocyte micromass cultures 72 h after incubation ( a). Irisin dose-dependently promoted ECM accumulation above the baseline, as well as attenuating ECM underproduction in the inflamed chondrocytes ( b). The treatment with 10 ng/mL Irisin had the greatest promotion and was chosen for succeeding experiments. IL-1β significantly decreased FNDC5 expression ( c) and cell growth after 24 h of incubation ( d). Chondrocytic markers collagen..."
}
],
"tool": [
{
"@type": "HowToTool",
"name": "2.5. Gait Profile Analysis"
},
{
"@type": "HowToTool",
"name": "2.6. Histomorphometry and Immunohistochemistry"
},
{
"@type": "HowToTool",
"name": "2.7. Chondrocyte Cultures"
},
{
"@type": "HowToTool",
"name": "2.8. Assessment of Chondrocytic Activity"
},
{
"@type": "HowToTool",
"name": "2.9. Reverse Transcription-Polymerase Chain Reaction (RT-PCR)"
},
{
"@type": "HowToTool",
"name": "2.10. Analysis of Autophagosome and Mitophagosome Formation"
},
{
"@type": "HowToTool",
"name": "2.11. Mitochondrial Dynamics Assay"
},
{
"@type": "HowToTool",
"name": "2.12. Mitochondrial Membrane Potential, ATP Production and Reactive Oxygen Radical (ROS) Level Assay"
}
],
"supply": [
{
"@type": "HowToSupply",
"name": "2.3. Purification of Irisin Recombinant Protein"
},
{
"@type": "HowToSupply",
"name": "2.6. Histomorphometry and Immunohistochemistry"
},
{
"@type": "HowToSupply",
"name": "2.7. Chondrocyte Cultures"
},
{
"@type": "HowToSupply",
"name": "2.8. Assessment of Chondrocytic Activity"
},
{
"@type": "HowToSupply",
"name": "2.9. Reverse Transcription-Polymerase Chain Reaction (RT-PCR)"
},
{
"@type": "HowToSupply",
"name": "2.10. Analysis of Autophagosome and Mitophagosome Formation"
},
{
"@type": "HowToSupply",
"name": "2.11. Mitochondrial Dynamics Assay"
},
{
"@type": "HowToSupply",
"name": "2.12. Mitochondrial Membrane Potential, ATP Production and Reactive Oxygen Radical (ROS) Level Assay"
}
],
"isBasedOn": {
"@type": "ScholarlyArticle",
"headline": "Irisin Mitigates Oxidative Stress, Chondrocyte Dysfunction and Osteoarthritis Development through Regulating Mitochondrial Integrity and Autophagy",
"datePublished": "2020",
"author": [
{
"@type": "Person",
"name": "Feng-Sheng Wang"
},
{
"@type": "Person",
"name": "Chung-Wen Kuo"
},
{
"@type": "Person",
"name": "Jih-Yang Ko"
},
{
"@type": "Person",
"name": "Yu-Shan Chen"
},
{
"@type": "Person",
"name": "Shao-Yu Wang"
},
{
"@type": "Person",
"name": "Huei-Jing Ke"
},
{
"@type": "Person",
"name": "Pei-Chen Kuo"
},
{
"@type": "Person",
"name": "Chin-Huei Lee"
},
{
"@type": "Person",
"name": "Jian-Ching Wu"
},
{
"@type": "Person",
"name": "Wen-Bin Lu"
},
{
"@type": "Person",
"name": "Ming-Hong Tai"
},
{
"@type": "Person",
"name": "Holger Jahr"
},
{
"@type": "Person",
"name": "Wei-Shiung Lian"
}
],
"identifier": "10.3390/antiox9090810"
}
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