Irisin Mitigates Oxidative Stress, Chondrocyte Dysfunction and Osteoarthritis Development through Regulating Mitochondrial Integrity and Autophagy methods
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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...
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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
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"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..."
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"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 [ ]."
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"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..."
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"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..."
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"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..."
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