| Rheumatoid arthritis（RA）is a common autoimmune disease.As RA patients are in a state of chronic inflammation for a long time,their synovial membrane and cartilage and other tissues are infiltrated in the inflammatory environment for a long time,which eventually leads to serious joint destruction.Angiotensin II（Ang II）,as an important hormone in the body,has vasoconstriction and other functions.Ang II participates in local activation of immune system through Ang II type 1 receptor（Agtr1）signals in immune cells,mesangial cells and vascular smooth muscle cells,and promotes vasoconstriction,inflammatory response and tissue damage.Ang II can also induce vasodilation and anti-inflammation through the signal of Ang II type 2 receptor（Agtr2）,which are opposite to Agtr1-mediated physiological effects.Angiotensin and its receptors have recently been found to play an important role not only in the cardiovascular system,but also in autoimmune diseases.Angiotensin receptor inhibitors not only alleviate synovitis of RA and its experimental models（collagen-induced arthritis and adjuvant induced arthritis）,but also play a protective role in improving cardiovascular damage caused by inflammatory environment in RA patients and reducing cardiovascular risk.However,the existing studies mainly focus on the regulatory role of Ang II-Agtr1 in inflammatory immune response,while there are relatively few studies on Ang II-Agtr2.In RA,monocytes/macrophages are recruited and activated,and are an important source of pro-inflammatory cytokines,which cause inflammatory response in the acute stage of RA and damage to articular cartilage,and are an important factor in the occurrence and development of RA.The presence of large numbers of monocytes/macrophages in RA joints was positively correlated with the severity of arthritis.Many inflammatory factors and chemokines that cause joint inflammation are secreted by monocytes/macrophages that are recruited into the joint and reside in the tissue.Monocytes/macrophages can also help recruit extra white blood cells to the inflamed joint and play an important role in the occurrence and development of RA.Over the years,our research group has focused on the urgent needs of RA diagnosis and treatment,and conducted in-depth studies on the pathological mechanism and therapeutic drugs of RA.Previous studies of our group have found that Agtr2 is involved in the therapeutic effect of losartan on experimental arthritis,and Agtr2 agonist CGP42112 alleviates adjuvant induced synovitis in rats with arthritis,suggesting that Agtr1/Agtr2 imbalance may play an important role in the pathological mechanism of articular synovitis.Therefore,the research group explored the effect of Agtr2 on macrophage typing in RA and its related mechanism by activating Agtr2.The experimental results confirmed that the activation of Agtr2 can reverse the M1-type polarization of macrophages caused by the imbalance of Agtr1/Agtr2 and thus alleviate the progression of RA disease.The possible mechanism is that the activation of Agtr2can promote the transfer of overexpressed GRK2 on the cell membrane to the cytoplasm,and combine with the abnormal increase of p-ERK to form a complex,thus inhibiting the activation of p-ERK on NF-κB signaling pathway.Monocytes/macrophages collected from the synovial membrane of arthritis are the key to triggering joint inflammation,but it is obviously not enough to summarize the therapeutic effect of Agtr2 in arthritis only from the perspective of macrophage typing.Therefore,this study use Agtr2 gene knockout mice to investigate whether Agtr2 to macrophages in mononuclear cells in the process of differentiation has by changing the monocyte/macrophage differentiation,in turn,affect the incidence of RA development,further defined Agtr2 protective role in RA,for the future research and development to provide more experimental basis Agtr2 agonist treatment of RA.Purpose:1.The effects of Agtr2 on the course of collagenous antibody induced arthritis in mice and the activation of monocytes/macrophages in synovial membrane of arthritis were elaborated.2.It was demonstrated that Agtr2 can be used as a regulatory target to inhibit abnormal activation of monocytes/macrophages and then participate in the treatment of RA.3.Reveals the potential value of Agtr2 agonist as a new drug for the treatment of RA,and provides experimental basis for this.Method:1.The CRISPR/Cas9 technology was jointly designed and implemented with Jiangsu Gem Pharmatech Co.,Ltd.,and the Agtr2 gene knockout(Agtr2-/-)mice（F0 generation）were obtained by Polymerase chain reaction（PCR）genotype identification.Agtr2-/-mice of the F1 generation were produced by sexual maturation of Agtr2-/-mice of the F0generation and wild type mice of the same litter.The reliability of PCR identification results was verified by Western blot on protein level.2.The CAIA model of Agtr2 gene engineered mice was established by using collagen antibody.On day 7,Agtr2 agonist CGP42112（20μg/kg/2d）was injected intraperitoneally and Agtr2 agonist C21（0.3mg/kg/d）was injected intraperitoneally.From day 0 of modeling,arthritis score,paw thickness,paw swelling number and other overall indicators of mice were observed.The changes of blood flow signal and joint effusion in knee joint of mice were observed by small animal ultrasound.The osteopathies of knee joint in mice were observed by X-ray of small animals.The mice were sacrificed on the 15th day for related tests:H&E staining was used to observe the pathological changes of the joints of the mice;Changes in serum levels of rheumatoid factor and C-reactive protein were detected by biochemical assay.The expression of monocytes/macrophages and related surface molecules in mouse synovial tissues were detected by flow cytometry.3.Mouse bone marrow cells were taken and stimulated by LPS（100ng/ml）for 48h,and treated with CGP42112(10-6M)and C21(10-6M)at the same time.The supernatant of the cells was collected and the nitric oxide content was detected by Griese kit,and the expression changes of MHCII and CD11b were detected by immunofluorescence assay.M-CSF was used to stimulate the differentiation of mouse bone marrow monocytes into macrophages.M1-type polarization of macrophages was stimulated by LPS（100ng/ml,24h）and CGP42112(10-6M)and C21(10-6M)were treated simultaneously.The expression of F4/80 and MHCII was detected by immunofluorescence assay after the supernatant of cells was collected.Result:1.Using CRISPR/Cas9 technology,PCR gene identification and Western blot verification,Agtr2-/-gene engineering mice were successfully constructed.2.Both wild-type mice and Agtr2-/-mice showed obvious signs of arthritis after induction of CAIA arthritis model,but Agtr2-/-mice showed more obvious signs of arthritis than wild-type mice.Compared with CAIA wild-type mice,CAIA Agtr2-/-mice showed more significant weight loss,paw thickness,paw swelling number,spleen index,whole body score and arthritis index.CAIA wild-type mice were treated with Agtr2agonists CGP42112 and C21,which significantly improved arthritis in wild-type mice.After CAAIA induced mouse arthritis model,both wild-type mice and Agtr2-/-mice showed obvious signs of increased blood flow signal and joint effusion,but the Agtr2-/-mice showed more obvious signs of increased blood flow signal and joint effusion than wild-type mice.After CAIA wild-type mice were treated with Agtr2 agonists CGP42112and C21,joint blood flow signals and joint effusion were significantly alleviated in wild-type mice.After CAIA induced arthritis in mice,both wild-type mice and Agtr2-/-mice showed obvious synovial lesions,but the synovial lesions in Agtr2-/-mice were more significant than those in wild-type mice.Compared with CAIA wild-type mice,CAIA Agtr2-/-mice showed more obvious inflammatory cell infiltration,and synovial tissue thickening,joint space narrowing,synovial structure disorder,joint surface deformation and other pathological changes caused by synovial cell proliferation were more significant.After treatment with CGP42112 and C21 in CAIA wild-type mice,inflammatory cell infiltration was reduced,synovial tissue hyperplasia was relieved,joint space was increased,articular surface was more intact,and pathological manifestations of arthritis were significantly improved.After CAIA induced arthritis in mice,the number of monocytes/macrophages in synovial tissue of wild-type mice and Agtr2-/-mice was significantly increased,but the number of monocytes/macrophages in synovial tissue of CAIA Agtr2-/-mice was more obvious than that of CAIA wild-type mice.They also showed a pro-inflammatory mononuclear/macrophage phenotype,with high expression of LY6C and MHCII and low expression of MERTK.CAIA wild-type mice treated with CGP42112 and C21 activated Agtr2,reduced the number of monocytes/macrophages in synovial tissue and decreased the expression of inflammatory markers.Immunofluorescence results showed that the number of monocytes/macrophages in synovial joints of CAIA Agtr2-/-mice was significantly higher than that of wild-type mice,and the expression of i NOS was also increased.However,the number of monocytes/macrophages in synovial joints of CAIA Agtr2-/-mice was decreased and the expression of i NOS was decreased after the administration of CGP42112 and C21 excited Agtr2.3.In the in vitro experiment,the expression of MHCII and the production of nitric oxide in bone marrow monocytes of Agtr2-/-mice were increased when LPS was used to stimulate the bone marrow monocytes of mice compared with wild-type mice.CGP42112 and C21 activated the Agtr2 in bone marrow monocytes of wild-type mice,the level of nitric oxide was significantly reduced,and the expression of MHCII was also significantly down-regulated.Compared with wild-type mice,the expression of MHCII and the production of nitric oxide in the M1-type macrophages of Agtr2-/-mice were relatively higher.The expression of MHCII and the production of nitric oxide in CAIA wild-type mice were significantly decreased after CGP42112 and C21 were activated Agtr2.Conclusion:1.The absence of Agtr2 intensifies the pathological manifestations of CAAIA arthritis model in mice.Excitation of Agtr2 with CGP42112 and C21 alleviates the severity of arthritis in CAAIA mice.2.The lack of Agtr2 promoted the CAIA arthritis synovial tissue monocyte/macrophage in mice gathered,and present a proinflammatory cellular phenotypes,high expression of Ly6C and MHCII,lower expression Mer TK,promoted the progress of joint inflammation,using CGP42112 and C21 excited Agtr2 decreased in synovial tissue monocyte/macrophage accumulation,alleviate the severity CAIA arthritis in mice.3.The absence of Agtr2 can promote the LPS-induced M1-type polarization of macrophages and promote the progression of inflammation,while the activation of Agtr2 may inhibit the M1-type polarization of macrophages and alleviate inflammatory conditions. |
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