The Role And Mechanism Of VEGF Release Mediated By Acidosis Through ASIC1a In Synovial Fibroblast

Rheumatoid arthritis is a chronic systemic autoimmune syndrome that affects approximately 1%of the world’s population.The current treatment for rheumatoid arthritis is mainly symptomatic treatment,which cannot prevent the development of RA disease and the disability of patients.Our previous studies have shown that treatments for synovial vascular scabs can block the development of RA.Therefore,it is the key to rheumatoid arthritis treatment to actively find the causes of synovial vascular hyperplasia and lesions,clarify the formation mechanism of synovial vascular crests,and find effective intervention targets to provide effective interventions.Articular cavity acidification in rheumatoid arthritis is one of the main characteristics of the disease.This is due to ischemia and hypoxia in the synovial inflammation area of RA,which leads to increased oxygen consumption and increased glycolysis,which causes lactic acid accumulation.Therefore,RA patients The p H of the joint cavity often drops below 6.0.Previous studies have shown that the extracellular environment with low p H is related to the formation of vascular crests in synovial tissue of RA patients.But how is this acidic signal transmitted into the cell and how does it mediate the formation of vascular crests?Acid-sensitive ion channels（ASICs）are a class of cation channels activated by the acidification of the extracellular environment and belong to a family of epithelial sodium channels.Among them,ASIC1a can simultaneously mediate NA⁺and Ca²⁺inflow when stimulated by extracellular acidification.Transduces extracellular low p H signals into cells.Previous studies have found that ASIC1a can be closely involved in the development of RA.Then,can the extracellular acid environment in RA activate ASIC1a to transmit extracellular low-p H signals into cells and further mediate synovial blood vessels?What about puppet formation?Therefore,the research group explored whether the extracellular acid environment in RA can regulate the formation and mechanism of pannus by activating ASIC1a through the following experiments.Method:1. Observe the difference in the expression of acid-sensitive ion channel 1a in normal human synovium and rheumatoid arthritis patients.The expression of ASIC1a in normal human synovial tissue and rheumatoid arthritis patients’synovial tissue was observed by immunohistochemistry.The knee synovial tissue（n=3）of patients after amputation due to accidents and the synovial tissue of patients with synovectomy and joint replacement（n=42）were collected.Immunohistochemistry was used to investigate the difference in expression of ASIC1a in the two tissues.The adherent method was used to extract primary human synovial fibroblasts（NSF,n=1）and rheumatoid arthritis patients’primary synovial fibroblasts（RASF,n=3）.Flow cytometry was used to investigate the differences in the expression of membrane antigen CD55 and membrane protein ASIC1a on NSF and RASF cells.Total NSF and RASF proteins were extracted,and Western blotting was used to detect differences in the expression of ASICs and histones in the total protein.The NSF and RASF membrane proteins were extracted,and Western blotting was used to investigate the difference in expression of the membrane proteins ASIC1a.Immunofluorescence was used to detect the difference in expression of ASIC1a between unbroken membrane-treated NSF and RASF.2.To explore whether acid stimulation can promote the release of VEGF by synovial fibroblasts through ASIC1a.ELISA was used to explore the ability of RASF to synthesize and release VEGF under different treatments.The time gradient of acid stimulation（0h,45min,1.5h,3h.6h）was designed to detect the expression level of VEGF in RASF cells after stimulation at different times using Western blotting and immunofluorescence.ELISA method was used to detect the concentration of VEGF in the medium without acid stimulation or after 0h,3h,6h,12h and 24h.ASIC1a silencing cell line was constructed,RASF was pretreated with ASIC1a specific inhibitor Pc Tx-1,and the concentration of VEGF in the medium after acid stimulation and acid stimulation at 0h,12h and 24h was detected by ELISA method.The ASIC1a overexpression cell line was constructed,and the concentration of VEGF in the medium after acid-free stimulation and acid stimulation at 0h,12h,and 24h was detected by ELISA.3.To explore whether the regulation of VEGF by ASIC1a is involved in the formation of pannus in arthritis.An adjuvant arthritis rat（AA rat）model was constructed,and ASIC1a specific inhibitor Pc Tx-1 was injected into the articular cavity after successful modeling.Pathological changes of the articular cavity of AA rats before and after administration were observed.The HE staining method was used to observe the pathological changes of normal human synovial tissue and rheumatoid arthritis patients.Immunohistochemical method was used to investigate the changes of VEGF and microvessel density marked by CD34 in the synovial tissue of normal people and rheumatoid arthritis patients.Construct AA rat model,divide AA rats into control group,model group,Pc Tx-1 0.5μg/kg group,Pc Tx-1 1μg/kg group,Pc Tx-1 2μg/kg group and triamcinolone acetonide（TA）group.The HE staining method was used to observe the pathological changes of AA rat joint cavity before and after modeling.The changes of microvessel density in the joint cavity of AA rats were observed by immunohistochemistry.Pc Tx-1 and TA were administered every two days for 26 days,and HE staining technique was used to observe the pathological changes of the joint cavity of AA rats after the administration.Immunohistochemical method was used to observe the changes of ASIC1a,VEGF and microvessel density marked by CD34 in the joint cavity of AA rats after the administration.Result:1. The expression of ASIC1a is significantly increased in the synovial tissue of patients with rheumatoid joints.Among ASICs family proteins,ASIC1a and ASIC3 were significantly increased in RASF.The expression of ASIC1a membrane protein is increased in rheumatoid arthritis.The above results indicate that the expression level of ASIC1a in rheumatoid arthritis and the transfer to the cell membrane are enhanced,indicating that ASIC1a may be involved in the development of rheumatoid arthritis disease.2. Acid stimulation can promote synovial fibroblasts to release VEGF through ASIC1a.After stimulation with acid for different periods of time,the expression of VEGF in synovial fibroblasts and the concentration of VEGF in the medium increased,and the expression level of VEGF in the cells reached the highest level at 3h,and then slightly decreased,while the expression of VEGF in the medium The level increase is time-dependent.The immunofluorescence results are consistent with Western blotting results,which indicates that the expression of VEGF in synovial fibroblasts may have undergone a process from synthesis to release.Silencing or blocking ASIC1a can significantly reduce the release of VEGF by acid-stimulated cells.After overexpression of ASIC1a,synovial fibroblasts increased the release of VEGF under acid stimulation.3. The regulation of VEGF by ASIC1a may be involved in the development of pannus in arthritis.In the pathological examination of human synovial tissue,it was found that compared with normal people,the synovial tissue of rheumatoid arthritis patients has obvious hyperplasia,inflammatory infiltration and pannus.Immunohistochemical results showed that the expression level of VEGF and the density of microvessels marked by CD34 in the synovial tissue of patients with rheumatoid arthritis were significantly increased.The pathological examination of AA rats revealed that the joint cavity of AA rats showed obvious inflammation and cartilage damage,and the microvessel density increased.After treatment with ASIC1a specific inhibitor Pc Tx-1,AA rat joint inflammation was significantly reduced,cartilage damage was reduced,and different doses of Pc Tx-1reduced the expression of ASIC1a in the joint cavity.Immunohistochemical results showed that the expression level of VEGF and microvessel density in the joint cavity of AA rats decreased with the concentration-dependent decrease of Pc Tx-1.Immunofluorescence results showed that compared with normal group rats,AA rats synovial microvessels showed obvious angiogenesis,the blood vessel wall changed from a single layer to multiple layers,and different doses of Pc Tx-1 can reduce the joint cavity of AA rats.Lesions of microvessels.Conclusion:1.In rheumatoid arthritis,acid can promote RASF release of VEGF through ASIC1a.2.In rheumatoid arthritis,the regulation of VEGF by ASIC1a may be involved in the development of synovial pannus.