Figures & data
Figure 1. CAB39 was down-regulated in a mouse OA model.
Chondrocytes (ATDC5) were treated with varying concentrations of IL-1β (5–100 ng/mL) for 24 hours. A-B: The CAB39 expression was validated by qRT-PCR and WB. ATDC5 cells were treated with IL-1β (10 ng/mL) for 6, 12, 24, and 48 hours, respectively. C-D: qRT-PCR and WB gauged the CAB39 profile. M1 phenotype was induced in RAW264.7 cells by LPS (100 ng/mL) + IFNγ (20 ng/mL) treatment for 24 hours. Meanwhile, RAW264.7 cells were treated with IL-4 (20 ng/mL) + IL-13 (20 ng/mL) to induce the M2 phenotype. E-F: qRT-PCR and WB were conducted to check the CAB39 expression in RAW264.7 cells and M1- and M2-polarized macrophages. The mice were subjected to right knee DMM surgery to construct OA models and were executed four weeks after surgery to collect the right knee joint. G: The CAB39 profile in mouse joint tissues was ascertained by qRT-PCR. H: Immunohistochemical detection of CAB39-positive cell percentage in mouse joint tissues. Scale bar = 50 μm. ns P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001 (vs. con group), **P < 0.01, ***P < 0.001 (vs.RAW 264.7 group). N = 3 ***P < 0.001 (vs.sham group). N = 5
Figure 2. Overexpression of CAB39 facilitated M1 to M2 polarization of macrophages and attenuated IL-1β-mediated chondrocyte injury.
Vectors and CAB39 overexpression plasmids were transferred into RAW264.7 cells. A: qRT-PCR tested the CAB39 profile. RAW264.7 cells were processed with LPS (100 ng/mL) + IFNγ (20 ng/mL) and IL-4 (20 ng/mL) + IL-13 (20 ng/m) to induce the M1 phenotype and M2 phenotype, respectively. B-D: WB was employed to evaluate the expression of CD86, iNOS, CD206, and Arg1. Vectors and CAB39 overexpression plasmids were transfected into ATDC5 cells. E: The CAB39 profile in ATDC5 cells was checked by qRT-PCR. ATDC5 cells were exposed to IL-1β (10 ng/mL). F: Cell viability was evaluated by CCK-8. G. Caspase3 activity detection kit checked cell apoptosis. H-I. The profiles of Bax, Bcl-2, Caspase-3, MMP3, MMP13, and Aggrecan were verified by WB. ***P < 0.001 (vs.vector group), *P < 0.05, **P < 0.01, ***P < 0.001 (vs.con group), &&P < 0.01, &&&P < 0.001 (vs.M1+ vector group), &&&P < 0.001 (vs.M2+ vector group), &&P < 0.01, &&&P < 0.001 (vs.IL-1β+vector). N = 3
Figure 3. Knockdown of CAB39 curbed the M2 phenotype of macrophages and enhanced IL-1β-mediated chondrocyte injury.
Si-NC and Si-CAB39 were transfected into RAW264.7 cells and A: qRT-PCR evaluated the CAB39 expression. The above cells were exposed to LPS (100 ng/mL) + IFNγ (20 ng/mL) and IL-4 (20 ng/mL) + IL-13 (20 ng/mL) to induce the M1 and M2 phenotype, respectively. B-D: The expression of CD86, iNOS, CD206, and Arg1 was measured by WB. Si-NC and Si-CAB39 were transfected into ATDC5 cells. E: qRT-PCR testified the CAB39 profile in ATDC5 cells. ATDC5 cells were treated with IL-1β (10 ng/mL). F: Cell viability was evaluated by CCK-8. G. Caspase3 activity detection kit checked cell apoptosis. H-I: WB was implemented for gauging the profiles of Bax, Bcl-2, Caspase-3, MMP3, MMP13, and Aggrecan. **P < 0.01, ***P < 0.001 (vs. Si-NC group), *P < 0.05, **P < 0.01, ***P < 0.001 (vs. con group), &&P < 0.01, &&&P < 0.001(vs. M1+ Si-NC group), &&P < 0.01, &&&P < 0.001 (vs. M2+ Si-NCgroup), &P < 0.05, &&P < 0.01, &&&P < 0.001 (vs. IL-1β+Si-NC group). N = 3
Figure 4. Overexpressing CAB39 alleviated chondrocyte injury induced by macrophages.
Vectors and CAB39 overexpression plasmids were transfected into RAW264.7 cells, which were intervened with LPS (100 ng/mL) + IFNγ (20 ng/mL) for 24 hours. The CM of macrophages was obtained and co-cultured with ATDC5 cells for 24 hours. A: The CCK-8 assay was applied for the ATDC5 cell viability test. B. Cell apoptosis was checked by Caspase3 activity detection kit. C-D: WB was implemented to verify the levels of Bax, Bcl-2, Caspase-3, MMP3, MMP13, and Aggrecan. *** P < 0.001 (vs.blank group), &&P < 0.01, &&&P < 0.001 (vs. M1 vector -CM group). N = 3
Figure 5. Overexpressing CAB39 motivated the AMPK/Sirt-1 pathway.
Vectors, CAB39 overexpression plasmids, Si-NC, and Si-CAB39 were transfected into RAW264.7 and ATDC5. RAW264.7 cells were processed with LPS (100 ng/mL) + IFNγ (20 ng/mL), while ATDC5 cells were exposed to IL-1β (10 ng/mL) for 24 hours. A-D: The AMPK/Sirt-1 pathway profile was ascertained by WB. **P < 0.01, ***P < 0.001 (vs.con group), &P < 0.05, &&P < 0.01, &&&P < 0.001 (vs.M1+ vector group), &&P < 0.01 (vs.M1+ Si-NC), &&P < 0.01 (vs.IL-1β+vector), &P < 0.05, &&P < 0.01 (vs. L-1β+Si-NC group). N = 3
Figure 6. Inhibition of the AMPK/Sirt-1 pathway attenuated CAB39’s promoting effect on the M2 phenotype of macrophages and its protective effect on chondrocytes.
Vectors and CAB39 overexpression plasmids were transfected into RAW264.7 cells. RAW264.7 cells were treated with LPS (100 ng/mL) + IFNγ (20 ng/mL) for 24 hours, followed by BML-275 (5 μM) treatment. A: The profiles of CD86, iNOS, CD206, and Arg1 were testified by WB. Vectors and CAB39 overexpression plasmids were transfected into ATDC5 cells, which were then processed with IL-1β (10 ng/mL) for 24 hours, followed by BML-275 treatment. B: Cell viability was assessed by CCK-8. C: Apoptosis was gauged by Caspase3 activity detection kit. D-H: WB was carried out to monitor the expression of Bax, Bcl-2, Caspase-3, MMP3, MMP13, Aggrecan and the AMPK/Sirt-1 pathway. **P < 0.01, ***P < 0.001 (vs.M1group), **P < 0.01, ***P < 0.001 (vs.IL-1β group), &&P < 0.01 (vs.M1+ CAB39 group), &&P < 0.01, &&&P < 0.001 (vs.IL-1β+CAB39 group). N = 3
Figure 7. Overexpressing CAB39 eased cartilage damage and inflammation in DMM mice.
DMM was performed to induce the mouse knee OA model, and lentiviruses coated with vectors and CAB39 overexpression plasmids were administered to the right knee cavity of mice. The mice’s blood and right knee joints were collected. A: ELISA was employed to test the expression of IL-6, IL-10 and IL-1β in mouse serum. B-D: WB estimated the expression of macrophage phenotypic markers (CD86, iNOS, CD206, and Arg1) and apoptosis-related proteins (Bax, Bcl-2, and Caspase-3). E-I: Immunohistochemical detection of Caspase-3 and the CAB39/AMPK/Sirt-1 pathway expression in medial interventricular cartilage of the knee joint. Scale bar = 50 μm. J-K: The AMPK/Sirt-1 pathway expression was assessed by WB. ***P < 0.001 (vs.sham group), &&P < 0.01, &&&P < 0.001 (vs.OA+LV-NC group). N = 5
Data availability statement
The data sets used and analyzed during the current study are available from the corresponding author on reasonable request.