The Role And Mechanism Of Roundabout In Synovium Of Knee Osteoarthritis

Background:Osteoarthritis(OA)is a common cause of joint pain in middle-aged and elderly people,and one of the clinicopathological changes of OA is synovitis.The Roundabout(Robo)protein family,which includes Robol,Robo2,Robo3 and Robo4,is major cell membrane surface receptors in the Slit-Robo signaling pathway,and plays important roles in cell proliferation,inflammatory response and angiogenesis.OA Synovitis is characterized by tissue hyperplasia,inflammatory cell infiltration and vascular hyperplasia,which involves a variety of signaling pathways,but there are few reports about the Slit-Robo in synovium.Objectives:To explore the expression of Robo 1-4 proteins in human Synovium(SM)and Synovial Fibroblasts(SF),and to investigate the expression and mechanism of Robol-4 in SF by adding inflammatory factors such as TNF-α,IL-1β and IL-6,to stimulate SF under the inflammatory environment in vitro.Methods:Choose the synovium of knee OA patients requiring joint replacement as the OA group,and choose the synovium of knee ligament or cartilage injury and no synovium lesions requiring arthroscopic surgery treatment as the Normal Control group(NC group).The synovium of the two groups was studied histologically and cytologically by Hematoxylin Eosin(HE)staining,Western Blot(WB),Immunohistochemistry(IHC),cell culture and identification,to explore the expression of Robol-4 and related protein.Results:(1)The intraoperative and arthroscopic observation of the synovium showed that SM in the OA group was significantly proliferative and swollen than NC group.The HE staining of SM in the OA group showed a large number of tissue proliferation,inflammatory cell infiltration and angiogenesis than NC group.(2)WB and IHC were used to detect the expression of Robol-4 proteins in SM.The results showed that the expression of Robol and Robo4 in SM of OA group was increased,compared with NC group,while the expression of Robo2 and Robo3 in SM was very low,both in NC group and OA group.WB and IHC techniques were also used to detect the expression of Vascular Endothelial Growth Factor(VEGF)and Uncoordinated 5-homolog B(UNC5B)in SM.The results showed that the expression of VEGF in SM group was increased compared with NC group,and there was no significant difference in UNC5B expression.These results suggest that there may be Robol/Robo4-VEGF-dependent angiogenesis in SM of OA group.(3)Primary SF cells were isolated and cultured from synovium,using cell culture technology,and the cultured primary cells were identified by Short Tandem Repeat(STR).The results showed that the purity of SF satisfied the requirements of the next experiment.(4)WB test was used to detect the expression of Robol-4 in SF.The results showed that the expression of Robol-4 was very low in SF,and there was no significant difference between the OA group and the NC group,indicating that the increased Robol and Robo4 in the SM of the OA group may not be expressed by SF.(5)CCK-8 showed that proliferative activity of SF in OA group was lower than NC group.After TNF-α and IL-1β stimulation,the proliferation activity of SF in OA group was significantly higher than that in NC group.After Slit2 stimulation,there was no significant change in the SF proliferation activity between the OA and NC group.These results suggest that NF-κB signaling pathway may be associated with increased proliferation activity in OA synovial fibroblasts.(6)WB was used to detect the OASF after TNF-α IL-1β and IL-6 stimulation,the results showed that the expression of Robo1-4 did not change.The results of CCK-8 assay showed that Slit2 had no effect on the proliferation of TNF-α or IL-1β dependent in SF.These results suggest that there may be no direct regulation between Slit-Robo and NF-κB in SF.(7)The transcriptome sequencing analysis of SM in the OA group and the NC group was performed.A total of 3952 differential genes were screened out,among which 2135 genes up and 1817 genes down.And cluster analysis was performed on the differential genes.Conclusions:In summary,synovium and blood vessels were significantly hyperplasia in OA.The expression of Robol and Robo4 was increased in OA synovium,which suggests Robol/4-VEGF might mediate angiogenesis in OA synovium.The expression of Robol-4 in OA synovial fibroblasts was not increased or decreased,which suggests that the expression of Robol and Robo4 in OA synovium may not expresse in synovial fibroblasts.The increased of NF-κB dependent proliferation activity in OA synovial fibroblasts might one of the reasons for the hyperplasia of OA synovium.In synovial fibroblasts,there may be no direct regulation between Slit-Robo and NF-κB.Slit-Robo in synovial fibroblasts may not be involved in the development of OA synovium.