| Angiotensin II is an octapeptide produced from the substrate angiotensinogen through sequential enzymatic cleavages by renin and angiotensin converting enzyme (ACE) or non ACE-dependent pathway by chymase. Besides AT1R, angiotensin II can also act through AT2R, which has a similar affinity to AT1R. Angiotensin II is well known to act as a cardiovascular mediator by binding to ATIR in the circulation and has a primary role in the control of body fluid volume homoeostasis and blood pressure. The clinical beneficial of ACE inhibitors (ACEIs) and/or ATIR blockers (ARBs) in hypertension and cardiovascular disease was achieved by limiting angiotensin II synthesis or blocking the interaction between angiotensin Ⅱ and ATIR. Researches have demonstrated that the independent presence of renin-angiotensin system distributes widely in monocytes/macrophages, dendritic cells, lymphocytes. Angiotensin II gets involved in monocytes chemotaxis and migration, dendritic cells differentiation and mature, lymphocytes activation and proliferation through binding to AT1R which expressed on inflammatory and immune cells in the autocrine and/or paracarine manner. Actually, the presence and up-regulation of angiotensin Ⅱ have previously been described in the development of rheumatoid arthritis (RA), and the pathological role of angiotensin II-AT1R in RA was confirmed by therapeutic effect of drugs that limit angiotensin Ⅱ production by ACEIs or blockade of angiotensin II-AT1R action by ARBs. Researches about the AT2R genetically altered animals showed that when AT2R-knockout mice were exposed to disease models (such as brain ischemia-reperfusion injury, atherosclerosis, arterial remodeling after wire injury), loss of the AT2R in these animals caused a deterioration of pathology compared to wild-type controls. In contrast, the beneficial effects under pathological conditions (such as hypertrophy, atherosclerosis) were observed in AT2R-transgenic animals, strongly suggesting a possible protective role in diseases development. Research of AT2R with pharmacological tools suggested that AT2R agonist may be suitable for the treatment of stroke, inflammation, cardial fibrosis, or myocardial infarction. Generally speaking, recent researches suggested that increased levels of free angiotensin II after AT1R blockade being diverted to and activating the AT2R. So, the benefical effects on inflammation and injury achieved with ARBs are contributed to not only by blockade of the AT1R, but also by increasing angiotensin II effects transduced through the AT2R. But AT2R still remains obscure in RA. In the present study, we firstly established the adjuvant-induced arthritis (AIA) model, and investigated the change and role of AT2R in the inflammatory and immune response with AIA rats, and further studied the effects of AT2R agonist with experimental arthritis and the mechanism, providing the experimental basis for the role and new target of AT2R in regulation of inflammatory and immune response with RA. Objectives:In the present study, we firstly established the AIA model, and investigated the change and role of AT2R in the AIA rats and RA patients in vivo, and further studied the therapeutic effects of intra-articular injection with AT2R agonist in experimental arthritis in vivo, and in vitro study we explored the role of AT2R in monocytes viability and chemotaxis, providing the experimental basis for the role of AT2R in the regulation of inflammatory and immune response with RA and the possibility of new therapeutic target of AT2R in RA.Methods:The AIA model was generated by immunizing the rats with a single intradermal injection of0.1ml complete Freund’s adjuvant into the right hind metatarsal rat footpad. AIA rats were treated with losartan (5,10and15mg/kg, once a day) and methotrexate (MTX;0.5mg/kg) orally. The treatment started on day14after immunization and continued until day28, lasting about15days. Arthritis was evaluated by the arthritis index and histological examination. Angiotensin II, tumor necrosis factor-a (TNF-a), and vascular endothelial growth factor (VEGF) levels were examined by ELISA. The expression of AT1R and AT2R in spleen and synovium was detected by Western blot, and further determined the Spearman correlation between the expression of AT1R/AT2R and polyarthritis index. The expression of AT1R and AT2R in the isolated monocytes was detected by Western blot.We collected peripheral blood mononuclear cells (PBMCs) from patients with active RA and healthy subjects, and further investigated the change of ATIR and AT2R expression using quantitative polymerase chain reaction (qPCR) and flow cytometry.To study the therapeutic effects of AT2R agonist CGP42112on rats with AIA, CGP42112(5,10and20μg/kg, once every3days, total three times) and triamcinolone acetonide (TA,1mg/kg, once every3days, total three times) were administered by intra-articular injection into the left hind (non-injected) paws from day14to day28. Arthritis was evaluated by the arthritis index, joints volume and histological examination, and CD11b positive cell infiltration in synovium. We detected the change of synoviocytes proliferation by CCK-8kit.In the in vitro study, we detected the effects of CGP42112(10-6M) and losartan (10-6M) on AT1R/AT2R expression of the isolated monocytes obtained from AIA rats, the effects of CGP42112(10-10to10-5M) on viability of monocytes using CCK-8kit, and the effects of CGP42112(10-8to10-5M) and losartan (10"6M) on monocytes chemotaxis.Results:1. AT2R expression increased in AIA rats and RA patients, and up-regulation of AT2R expression correlated positively with reduction of the polyarthritisCompared to normal groups, polyarthritis index of rat from model group was increased significantly. The characteristic histopathology of arthritis was observed in AIA rats, specifically, marked proliferation of synoviocytes and infiltration of dense mononuclear cells, extensive deep cartilage degradation and pannus formation. Compared to normal groups, ELISA results showed angiotensin Ⅱ, TNF-a, and VEGF levels increased significantly in serum and synovium from AIA rats. Western blot results showed ATIR and AT2R expression were both significantly increased in the spleen, synovium, and monocyte of model group. Compared to the healthy subjects, the levels of ATIR and AT2R mRNA in PBMCs from RA patients were both significantly increased by qPCR. Flow cytometry results showed that expression of ATIR and AT2R was significantly increased in PBMCs from RA patients.Treatment with losartan (10and15mg/kg) and MTX (0.5mg/kg) significantly decreased the polyarthritis index on day22and day26. The administration of losartan (10and15mg/kg) and MTX (0.5mg/kg) significantly alleviated the abnormalities in the joints of AIA rats, including marked proliferation of synoviocytes and infiltration of dense mononuclear cells, extensive deep cartilage degradation and pannus formation. Treatment with losartan (5,10, and15mg/kg) decreased significantly levels of angiotensin II, TNF-a, VEGF in the serum and synovium. Western blot result showed down-regulation of AT1R expression and up-regulation of AT2R expression within the spleen and synovium of AIA rats after treatment with losartan. Compared to model group, treatment with losartan (15mg/kg) decreased significantly the AT1R expression and increased significantly the AT2R expression in monocytes from AIA rats. The correlation analysis showed that the down-regulation of AT1R expression and the up-regulation of AT2R expression in the spleen and synovium of AIA rats correlated positively with a reduction of the polyarthritis index.2. AT2R stimulation ameliorated the inflammatory and immune response in AIA rat during intra-articular injection with CGP42112Our study showed AT2R expression increased in AIA rats and RA patients, and up-regulation of AT2R expression correlated positively with reduction of the polyarthritis. To clarify the role of AT2R in the inflammatory and immune response in AIA rat, we investigated the therapeutic effects of intra-articular injection with AT2R agonist CGP42112on rats with AIA. Compared to normal groups, arthritis index and joints volume of rat from model group was increased significantly. Intra-articular injection of CGP42112(10,20μg/kg) and TA (1mg/kg) significantly decreased the arthritis index and joints volume of the left hind paw on day22and day26. The rats from model group had secondary synovitis what was characterised by marked synoviocytes proliferation, dense mononuclear cells infiltration, pannus formation, and cartilage erosion. Intra-articular injection with CGP42112(10,20μg/kg) and TA (1mg/kg) alleviated significantly the histological abnormalities, and CD11b positive cell infiltration in synovium. Additionally, treatment with CGP42112(10,20μg/kg) and TA (1mg/kg) inhibited significantly proliferation of synoviocytes from AIA rats. 3. AT2R stimulation inhibited the monocytes viability in vitroTo explore the mechanism of AT2R stimulation ameliorated the inflammatory and immune response in AIA rats, the isolated monocytes were obtained from AIA rats, treatment with CGP42112(10"6M) or losartan (10"6M) significantly increased AT2R expression, and also decreased AT1R expression. Treatment with CGP42112(10-7to10-5M) inhibited monocyte viability, IC50is5.21×10-6M. Treatment with CGP42112(10"7to10-5M) and losartan (10"6M) suppressed significantly the chemotaxis of IL-1β-stimulated AIA monocytes.Conclusion:1. AT2R expression increased in spleen, synovium, monocyte from AIA rats and PBMCs from RA patients, and up-regulation of AT2R expression correlated positively with reduction of the polyarthritis, strongly suggesting a possible role of AT2R in the inhibition of inflammatory and immune response with RA.2. Intra-articular injection of AT2R agonist CGP42112significantly alleviated secondary arthritis, inhibited proliferation of synoviocytes from AIA rats, strongly indicating AT2R stimulation alleviates AIA inflammatory and immune response in vivo, and also a possibility of new therapeutic target of AT2R in RA.3. Treatment with CGP42112increased AT2R expression, and AT2R stimulation inhibited the viability of monocytes in vitro, strongly suggesting AT2R stimulation alleviates AIA rats secondary arthritis via inhibiting monocytes-mediated inflammatory and immune response. |
PDF Full Text Request