CXCL12/CXCR4 Axis Regulates Aggrecanase Activation And Cartilage Degradation In A Post-Traumatic Osteoarthritis Rat Model

Objective1.We first analyzed the level of CXCL12/CXCR4 and ADAMTS-5 in subjects with OA compared to non-arthritic control subjects..2.Then,we cultured rat primary chondrocytes with untreated control,siNC+ CXCL12a,CXCL12a,or siRNA CXCR4 + CXCL12a and confirmed RUNX2,SOX9,ACAN,and ADAMTS-4/5 mRNA and protein levels in vitro.Finally,we explored the role of NF-κB,MAPK,and canonical Wnt/β-catenin signaling in CXCL12/CXCR4-mediated AD AMTS activation.3.At last,we induced OA in rats through surgical destabilization of the medial meniscus(DMM),andMethods1.We analyzed the expression of the CXCL12 and CXCR4 proteins in the synovial fluid from the knees of rats,along with the cartilage of OA rats and non-arthritic controls.The expressions of CXCL12 and CXCR4 in the synovial tissue and cartilage of OA rats and healthy controls were also studied by immunofluorescence.Furthermore,we analyzed levels of ADAMTS-4/5 in a murine model of OA.2.Primary rat chondrocytes pretreated with IL-1(24 h,10 ng/mL)were cultured with CXCL12a,siNC +CXCL12a,or siRNA CXCR4 + CXCL12a in vitro,and assessed for the expression of ACAN,RUNX2,ADAMTS-4/5,and SOX9 mRNA and protein levels at 24 and 72 h.Relatively marks of NF-κB,MAPK,and Wnt/β-catenin cellular pathways were evaluated by western blotting3.For OA induction,28 8-week-old male Spague-Dawley rats(200 g ± 10 g)were divided randomly into three groups.DMM/AMD3100-treated rats(n = 9)underwent destabilization of the medial meniscus(DMM)on the right knee.Rats in this group were treated with AMD3100(3 mg/day)via a constant infusion osmotic mini-pump.DMM/PBS-treated rats(n = 9)underwent the same microsurgery of the right knee as described for the first group,followed by treatment with phosphate buffered saline(PBS)via constant infusion osmotic mini-pump.The sham control group(n = 10)underwent sham surgery on the right knee and received empty pumps.All animals were euthanized eight weeks after surgery.The degree of OA damage in each of these three groups was quantified using the Mankin score.Cartilage sections from sham,AMD3100-treated,and PBS-treated groups were processed for immunohistochemicaldetection of the marker proteins ADAMTS-5,aggrecan neoepitope,RUNX2 and SOX9.Results1.High expression of CXCL12/CXCR4 and ADAMTS-5 in OA rats.2.CXCR4 knockdown inhibits IL-induced expression of ACAN,RUNX2,and ADAMTS-4/5,and enhances the expression of SOX9.CXCL12a-mediated aggrecanase activation of chondrocytes is dependent on the NF-κB,MAPK,and Wnt/(3-Catenin pathways.3.Inhibition of the CXCL12/CXCR4 axis results in reduced cartilage destruction and alleviates OA severity.The blocking CXCL12/CXCR4 axis protects osteoarthritic cartilage from proteoglycan loss accompanied by reduced ADAMTS-5 and aggrecan neoepitope staining.ConclusionTaken together,our results demonstrate that nhibition of SDF-1α/CXCR4 signaling axis was able to inhibit aggrecanase expression and lessen cartilage degeneration in post-traumatic osteoarthritis rats.