| Background Osteoarthritis(OA)is a common joint degenerative disease that can lead to joint pain and functional loss.It is the primary cause of disability in elderly patients.OA affects about 7% population worldwide which results in a huge burden on social and health economies.The pathogenesis of OA is complicated,including cellular metabolic disorder,cartilage wear,and inflammatory response of joint.All of the pathological changes contribute to the degeneration of articular cartilage.Nowadays,there is no effective treatment that can stop the course of OA.Therefore,exploring the pathophysiological changes of OA and find out new therapeutic targets to delay or even stop the progression of OA is an important research goal.Semaphorin 3A(Sema3A)is an axon guidance molecular.It can collapse the growth cone cytoskeleton and change the direction of axon growth.Recently,Sema3 A has been found to play a role in various pathophysiological processes such as regulating angiogenesis,interfering with tumor invasion,and maintaining bone homeostasis.Sema3 A can bind to the Neuropilin-1(NRP-1)protein presented on the cell surface and transmits downstream signals through the intracellular domain of Plexin-A1,which forms a receptor complex with NRP-1.This signal was found to inhibit cell-matrix contact by interfering with the activation of integrin proteins.Meanwhile,integrin protein plays an important role in the development of OA by mediating various cellular functions such as mechanotransduction and matrix metalloproteinase expression.Previous studies have shown that the expression of Sema3 A in chondrocytes can competitively bind to NRP-1 protein with vascular endothelial growth factor(VEGF),thereby inhibiting the migration and inflammatory expression of chondrocytes,confirming the role of Sema3 A as a receptor antagonist.However,so far,no studies have elucidated the effects of Sema3 A regulating Integrin protein on chondrocyte function.Aim To investigate the expression of Sema3 A and its receptor in OA cartilage tissue,to study the regulation of Integrin-α5β1 by Sema3 A and its effect on chondrocyte function.And to find out the effect of Sema3 A gene knockout on the course of knee OA in mice.Materials and Methods 1.Pathology and transcriptomic analysis revealing the expression of Sema3 A and its receptor in OA and normal knee cartilage The tissue of femoral condyle cartilage was collected from patients who underwent total knee arthroplasty due to primary knee OA and above-knee amputation due to trauma.The wear status of the cartilage was assessed by histochemical staining to identify normal,mild,and severe wear groups.Immunohistochemical staining(IHC)was used to evaluate the expression of Sema3 A,NRP-1,and Plexin-A1 proteins in cartilage,and to establish the relationship between cartilage wear severity and the expression of the proteins.The zonal specific expression of the proteins was analyzed by calculating the positive rate of IHC staining of the different zones.Finally,the relationship between the m RNA expression of proteins and the layers of cartilage tissue was further determined by RT-q PCR.2.The effect of Sema3 A on integrin-α5β1 in chondrocyte was investigated in vitro by cell function assay and immunofluorescence staining Femoral condyle cartilage from patients who underwent total knee arthroplasty due to primary knee OA and knee cartilage from mice aged 5 days were collected for primary chondrocyte culture.Identification of the chondrocyte was performed with microscope observation,Alcian blue staining,and immunocytochemistry(ICC)staining of type II collagen.100ng/ml Sema3 A was used to intervene chondrocytes,and a blank control was set.The adhesion and migration experiment were completed under the fibronectin(FN)coated condition to determine the effect of Sema3 A on the adhesion and migration functions of chondrocytes.The effect of Sema3 A on the survival of chondrocytes was analyzed by flow cytometry using Annexin V and propidium iodide staining.The effect of Sema3 A on Integrin-α5β1 on chondrocyte surface was determined by ICC staining using antibodies that recognized the activated state of integrin-α5β1.We used the SNAKA51 antibody to activated Integrin-α5β1 and determine whether activation of Integrin-α5β1 could antagonize the effects of Sema3 A on chondrocyte adhesion and migration.3.Sema3 A inhibited the expression of matrix metalloproteinases induced by fibronectin fragment(FN-f)was analyzed by ELISA and Western-blot 120 k Da FN-f was chosen for the intervention.We set control group,FN-f group,FN-f+Sema3A group,and FN-F +Sema3A+ SNAKA51 group.The supernatant of the chondrocytes was collected and the expression of MMP-13 was analyzed by enzymelinked immunosorbent assay(ELISA).The phosphorylation of ERK1/2 protein in the control group,FN-f group,and FN-f+Sema3A group was analyzed by Western-blot assay.4.In vivo knee OA model was established to investigate the expression of Sema3 A and its receptor and exploring the effect of Sema3 A gene knockout on OA cartilage tissue 10 wild-type(WT)C57 mice were selected and the OA model was established by Anterior Cruciate Ligament Transection(ACLT)plus Destabilization of Medial Meniscus(DMM)operation on the right knee as the OA group,and sham operation on the left knee as the control group.The knee joint samples were collected after 4 weeks.Cartilage wear in each group was evaluated by histochemical staining and OARSI score.The expressions of Sema3 A,NRP-1,and Plexin-A1 proteins in the cartilage of each group were analyzed by IHC staining.Cartilage-specific Sema3 A gene knockout mice were screened by PCR.5 knockout mice and 5 WT mice were selected.After the ACLT+DMM or sham operation,they were divided into OA and control groups as mentioned above.The cartilage of the knockout mice and WT mice was evaluated with the same pathological methods,and the expression of MMP-13 protein in the cartilage of each group was analyzed by IHC staining.Result 1.Expression of Sema3 A and its receptor in OA and normal cartilage of knee joint The normal knee cartilage surface was smooth,the cell morphology was arranged regularly.Mild wear OA cartilage surface continuity was broken,the cells were slightly hypertrophied with disordered arrangement.In the severe wear OA cartilage,fissures were found on the superficial zone and chondrocytes appeared to be hypertrophic.Besides,chondrocyte clusters showed up in the severe wear cartilage.IHC staining revealed that the expressions of Sema3 A,NRP-1,and Plexin-A1 in OA cartilage were significantly increased,and were positively correlated with the severity of cartilage wear.The three proteins preferred to be expressed in the superficial zone of cartilage and were clearly stained in the chondrocyte clusters.After RT-q PCR analysis of m RNA extracted from different zones of cartilage,it was found that Sema3 A,NRP-1,and Plexin-A1 were significantly expressed in the superficial zone.2.The effect of Sema3 A on Integrin-α5β1 in chondrocyte The isolated primary cells were identified as chondrocytes.The adhesion and migration towards FN were significantly inhibited by Sema3 A.Flow cytometry analysis confirmed that 100ng/ml Sema3 A had no significant cytotoxic effect on chondrocytes after 24 h intervention,and it was also proved that the decrease in the number of adhesion and migration cells observed after Sema3 A intervention was not caused by cell apoptosis or death.ICC staining showed that the intervention of Sema3 A significantly reduced the activation state of Integrin-α5β1 in chondrocytes.Activation of Integrin-α5β1 by SNAKA51 antibody significantly antagonized the decrease in the number of adherent and migratory cells induced by Sema3 A.The downstream protein of the Sema3 A signal was identified as Integrin-α5β1.3.Sema3 A inhibits the expression of matrix metalloproteinases induced by fibronectin fragment(FN-f)120k Da FN-f could significantly promote the expression of MMP-13 in chondrocytes.After the addition of Sema3 A,the expression of MMP-13 was significantly inhibited.Further,activation of Integrin-α5β1 by SNAKA51 reverse the inhibition of MMP-13 expression caused by Sema3 A,suggesting that Sema3 A downregulates the expression of MMP-13 by inhibiting the binding of Integrin-α5β1 to 120 k Da FN-f.Meanwhile,through Western-blot assay,120 k Da FN-f intervention could induce the high expression of phosphorylated ERK1/2 protein in chondrocytes,while the total ERK1/2 protein remained the same,and the addition of Sema3 A could inhibit this process.4.Expression of Sema3 A and its receptor in knee OA cartilage of WT mice and the effect of Sema3 A gene knockout on OA cartilage tissue In the WT mice OA group,the continuity of knee cartilage surface was lost,the cartilage tissue was obviously worn,and the cartilage layer collapsed.While in the control group,the knee cartilage surface was complete,the cells were arranged regularly,and there was no obvious cell hypertrophy.The expressions of Sema3 A,NRP-1,and Plexin-A1 proteins were increased in the knee cartilage of the OA group,but not significantly stained in the control group.The OA cartilages of the knockout mice were more damaged than that of the WT mice confirmed by the safranin O staining.Meanwhile,the expression of MMP-13 in the OA cartilage of the knockout mice was significantly higher than that of WT mice.Conclusion In this study,we found that Sema3 A,NRP-1,and Plexin-A1 were highly expressed in articular cartilage of OA patients compared with normal.The expressions of the three proteins were all correlated with the severity of cartilage wear and were obviously stained in the superficial zone.In vitro,we found that Sema3 A inhibited the activation of integrin-α5β1 and interfered with the adhesion and migration ability of chondrocytes.We found that the 120 k Da FN-f promoted the phosphorylation of ERK1/2 protein in chondrocytes,resulted in significant expression of MMP-13.In the meantime,Sema3 A inhibited this process by blocking the binding of Integrin-α5β1 to FN-f.Through building a mouse knee OA model,we found that Sema3 A,NRP-1,and Plexin-A1 proteins were highly expressed in the knee cartilage of OA mice.Besides, using cartilage tissue-specific Sema3 A gene knockout mice,we found that lack of Sema3 A protein leads to worse destruction of OA knee cartilage,which accelerated the progression of OA and increased the expression of MMP-13 in knee cartilage.In conclusion,we have proved that Sema3 A protein can play a protective role in the progression of OA by inhibiting the activation of Integrin-α5β1 and preventing FN-finduced chondrocyte hypercatabolism,which provided a theoretical basis for finding new therapeutic targets for OA. |
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