Semaphorin 3A mitigates lipopolysaccharide-induced chondrocyte inflammation, apoptosis and extracellular matrix degradation by binding to Neuropilin-1

Figures

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  Figure 1.. SEMA3A expression decreased in ATDC5 cells under LPS stimulation. (a) Cell viability was observed via CCK-8 assay following stimulation with LPS (5 ug/mL) for 6, 12 and 24 h. (b-c) SEMA3A and NRP-1 expression was detected by RT-qPCR and western blot following stimulation with LPS (5 ug/mL) for 6, 12 and 24 h. *P < 0.05, **P < 0.01, ***P < 0.001 vs. control

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  Figure 2.. SEMA3A overexpression improved cell viability and alleviated inflammatory injury of ATDC5 cells stimulated with LPS (5 ug/mL) for 12 h. (a-b) SEMA3A expression was detected by RT-qPCR and western blot after transfection of pcDNA 3.1(+)/SEMA3A into LPS-stimulated ATDC5 cells. (c) Cell viability was detected by the use of CCK-8. (d-g) The release of pro-inflammatory cytokines was assayed by RT-qPCR. (h) The expression of inflammatory mediator p-NF-κB p65, NF-κB p65 and COX2 was determined by western blot analysis. ***P < 0.001 vs. control; #P < 0.05, ##P < 0.01, ###P < 0.001 vs. LPS+Ov-NC

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  Figure 3.. SEMA3A overexpression alleviated LPS-induced ATDC5 cell apoptosis. (a-b) The apoptosis of LPS-stimulated ATDC5 cells was observed though TUNEL assay after overexpression of SEMA3A. (c) The protein expression of pro-apoptotic Bax and cleaved caspase3 and that of anti-apoptotic Bcl-2 was detected by western blotting. ***P < 0.001 vs. control; ###P < 0.001 vs. LPS+Ov-NC

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  Figure 4.. SEMA3A overexpression attenuated LPS-induced ATDC5 cell cartilage matrix degradation. (a-b) ECM-degrading MMP3, MMP9, MMP13 and ADAMTS-4 as well as collagen II, the main component of ECM, were assayed by RT-qPCR and western blot. ***P < 0.001 vs. control; ##P < 0.01, ###P < 0.001 vs. LPS+Ov-NC

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  Figure 5.. SEMA3A and NRP-1 bound to each other in ATDC5 cells. (a) Predicted binding of SEMA3A to NRP-1 on GeneMANIA database. (b-c) NRP-1 expression was detected by RT-qPCR and western blot assay in LPS-stimulated ATDC5 cells in the absence or presence of SEMA3A overexpression. ***P < 0.001 vs. control; ###P < 0.001 vs. LPS+Ov-NC. (d) The binding relationship between SEMA3A and NRP-1 was verified by co-IP assay

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  Figure 6.. NRP-1 interference reversed SEMA3A-ameliorated ATDC5 viability injury and inflammation induced by LPS. (a-b) NRP-1 interference level was detected by RT-qPCR and western blot after transfection of siRNAs targeting NRP-1 (siRNA-NRP-1-1/2). ***P < 0.001 vs. siRNA-NC. (c) The viability of LPS-stimulated ATDC5 cells following SEMA3A overexpression with or without NRP-1 knockdown was detected by CCK-8 assay. (d-g) The expression of pro-inflammatory cytokines was detected by RT-qPCR and western blot. (h) The expression of inflammatory mediator p-NF-κB p65, NF-κB p65 and COX2 was determined by western blot analysis. ***P < 0.001 vs. control; ###P < 0.001 vs. LPS; ΔP<0.05, ΔΔP<0.01, ΔΔΔP<0.001 vs. LPS+Ov-SEMA3A+siRNA-NC

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  Figure 7.. NRP-1 interference reversed SEMA3A-ameliorated ATDC5 apoptosis induced by LPS. (a-b) The apoptosis of LPS-stimulated ATDC5 cells following SEMA3A overexpression with or without NRP-1 knockdown was observed via TUNEL assay. (c) Apoptosis-related protein expression was determined by western blot analysis. ***P < 0.001 vs. control; ###P < 0.001 vs. LPS; ΔP<0.05, ΔΔP<0.01, ΔΔΔP<0.001 vs. LPS+Ov-SEMA3A+siRNA-NC

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  Figure 8.. NRP-1 interference reversed SEMA3A-ameliorated ATDC5 cartilage matrix degradation induced by LPS. (a-b) The expression of MMP3, MMP9, MMP13, ADAMTS-4 and collagen II was detected by RT-qPCR and western blot to reflect the state of extracellular matrix degradation. ***P < 0.001 vs. control; ###P < 0.001 vs. LPS; ΔP<0.05, ΔΔP<0.01, ΔΔΔP<0.001 vs. LPS+Ov-SEMA3A+siRNA-NC