The Role Of Angiotensin ? And Its Receptor Signaling In Vascular Injury In Adjuvant Arthritis Rat

Rheumatoid arthritis(RA)is considered to be an unconventional factor that increases the incidence and mortality of cardiovascular disease(CVD).Clinical study has shown that endothelial function of RA patients was destroyed,the degree of carotid atherosclerosis and average thickness of carotid artery were increased compared with general population.Suggesting that changes of structure and function of vascular in RA patients may be an important factor for high incidence and mortality of CVD.It is important to explore the relationship between RA and CVD and its possible mechanism.Angiotensin?(Ang?)is the octapeptide compound produced by the hydrolysis of Ang? under the action of angiotensin converting enzyme(ACE),plays a physiological role mainly through two receptors on the cell membrane,namely AT1 R and AT2 R.Ang? plays an important role in contracting blood vessels,regulating blood pressure and maintaining electrolyte balance.Study has shown that Ang? was infused in adjuvant-induced arthritis(AA)rats,and the impact of systemic inflammation onAng?–induced hypertension,endothelial dysfunction,and vascular hypertrophy was evaluated,while the effect of elevated Ang? level on the severity of inflammation and the structure and function of AA and its mechanism is to be studied in depth.RA has been widely recognized to increase the risk of CVD,with mortality rates from 1.5 to 2-fold increase compared with the general population,so proper treatment of RA disease is important for reducing the incidence of CVD.Synovial inflammation is a major pathological feature of RA and joint synovial involved first.Proliferation of fibroblast-like synoviocyte(FLS)is one of the major pathologic features in RA.Study has shown that the level of ACE was increased in RA patients' serum,synovial fluid,which can convert AngI into Ang?,indirectly prompted that Ang? involved in the process of RA disease.Ang? combined with AT1 R plays pro-inflammatory and pro-liferative effects,coupled with AT2 R plays anti-inflammatory and anti-proliferative effects.The preliminary study of the research showed that the AT1 R antagonist(Losartan)and the AT2 R agonist(CGP42112)all ameliorated the inflammatory response in AA rats.Whether AT1 R blocker,AT2 R agonist play therapeutic role by inhibiting FLS proliferation and whether the expression of AT1 R is reduced and AT2 R expression is enhanced? It is rarely reported.Objective:To establish AHD model,then to observe the interaction between 2K1C-HT and AA diseases,and to explore the effect of high level Ang? on inflammation and vascular injury in AA rats and some mechanism,and to provide experimental evidence for the clinical understanding of the risk of CVD in RA patients.Methods:Rats were injected with 0.1 ml complete freund's adjuvant(CFA)in the left hind paw of the rat to establish the AA model.The two-kidney-one-clip hypertension(2K1C-HT)model was established by narrowing the left renal artery with 0.25 mm silver clamp.At the same time,the above two methods were applied to establish AHD model.After immunization for 11 days,the weight,paw swelling and inflammation scores of rats were recorded every three days;the blood pressure of the rats were measured weekly using the tail-cuff apparatus;the rats were sacrificed on day 35,and the left kidney,right kidney and heart were removed and weighed,and the joint,spleen and thoracic aorta were collected for pathological examination;Western blot and laser confocal microscopy were used to detect the expression and localization of AT1 R,AT2R in FLS isolated from AA,2K1C-HT,AHD rats;the vascular ring experiment was used to detect the endothelium-dependent vasodilatation function of the thoracic aorta;the changes of serum Ang? and TNF-? were detected by enzyme-linked immunosorbent assay(ELISA);the expression and localization of Ang? and its receptor were detected by immunohistochemistry;the expression of Ang? and recepters and downstream signal molecules(p-ERK1/2,p-NF-?B)in thoracic aorta were detected by Western blot;effects of Losartan,CGP42112 and PD123319 on the proliferation of FLS were observed by CCK-8;the effect of losartan,CGP42112 and PD123319 on the expression of AT1 R and AT2 R of FLS were detected by Western blot.Results:1.The AHD model could be successfully established by the combination of 2K1 C combined with CFA foot plant immunization methodsCompared with normal rats,weight of AA rats was significantly reduced,joint swelling,inflammation index scores were significantly increased;the left renal index of2K1C-HT rats was significantly decreased and the blood pressure was significantly increased;weight of AHD rats was significantly reduced,joint swelling,inflammation index scores were significantly increased;joint pathology showed a large number of inflammatory cell infiltration,synovial hyperplasia,cartilage destruction;levels ofAng?,TNF-? in serum were increased.Meanwhile,blood pressure of AHD rats was increased at the 2 week,and reach a stable value at the 5 week,left kidney index of2K1C-HT group was significantly reduced,the heart index was increased,pathological examination of the thoracic aorta showed that the thickness medial(MT),the cross-sectional area(CSA)of the wall were increased significantly,lumen diameter(LD)was significantly reduced,cardiomyo-pathological examination showed arrhythmia of myocardium,rupture.It is suggested that AHD model has the characteristics of AA and2K1C-HT models,which indicates that AHD model has been successfully established.2.The incidence of AA was increased,and the joint and spleen pathology of AA rats were aggravated by 2K1C-HT and part mechanismsCompared with AA rats,the inflammation of AHD rats appeared early,and the incidence is high,paw swelling,arthritis index,joint and spleen pathology scores were significantly increased,level of Ang? in serum was significantly increased.Suggesting that 2K1C-HT increased the degree of inflammation of AA.Further study found that AT1 R expression in FLS isolated from AA rats was significantly increased,but AT2 R expression has no significant changes,AT1 R and AT2 R expression were significantly increased and ratio of AT1 R and AT2 R was significantly higher in FLS obtained from AHD rats,indicating that high level of Ang? induced by 2K1 C plays a major role in promoting inflammation through AT1 R.Meanwhile,we further observed the effect of Ang? receptor target drug on proliferation of local effector cell-synoviocyte of AA.The results showed that AT1 R blocker Losartan(10-6 mol/L)and AT2 R agonist CGP42112(10-6 mol/L)could inhibit the proliferation of AA-FLS,while AT2 R antagonist PD123319(10-6 mol/L)had no effect.Losartan combined with PD123319 had no significant effect on the inhibition of proliferation,while Losartan combined with CGP42112 could inhibit the ability of proliferation of FLS but no significant difference compared with used alone.Losartan,CGP42112 and PD123319 reduced theexpression of AT1 R,and the expression of AT1 R was significantly lower in Losartan combined with CGP42112 than in CGP42112 alone.CGP42112 significantly increased the expression of AT2 R,PD123319 significantly inhibited the expression of AT2 R,the expression of AT2 R was increased treated with Losartan combined with CGP42112 than the use of Losartan alone,and CGP42112 significantly increased AT2 R expression.3.The thoracic aortic and myocardial injury of AA rats were aggravated by2K1C-HTCompared with 2K1C-HT rats,the blood pressure and cardiac index of AHD rats were significantly higher.The MT,CSA of the thoracic aorta were significantly increased,LD was significantly reduced.Cardiomyo-pathological examination showed arrhythmia of myocardium,rupture.Cross-sectional area and axis length of myocardial cells were significantly increased.Cardiac pathology showed arrhythmia and rupture.The CSA and axis length of myocardial cell were significantly increased.Endothelium-dependent vasodilatation function of thoracic aorta was significantly reduced in AHD rats.It is suggested that AA chronic inflammation increased the vascular and myocardial injury of 2K1C-HT rats.4.Ang? involved in vascular injury of AHD rats via AT1 R / ERK1/2 signaling pathwayThe level of Ang? in serum in AHD rats was significantly higher,IHC results showed that the expression of Ang?,AT1 R,AT2R,GRK2 in the thoracic aortas isolated from AA,2K1C-HT,AHD rats were increased,and mainly expressed in the vascular wall medial expression.The expression of Ang? and downstream signaling molecules(p-ERK1/2 and p-NF-?B)were significantly increased in the thoracic aortas isolated from AHD rats compared with 2K1C-HT rats,and AT1R/AT2 R ratio was significantly higher.Indicating that Ang? mainly coupled with AT1 R and activatedERK1/2 / NF-?B signaling pathway may leading to vascular injury.Conclusion:1.The AHD model could be successfully established by the combination of 2K1 C combined with CFA foot plant immunization methods;2.The incidence of AA was increased,and the joint and spleen pathology of AA rats were aggravated by 2K1C-HT and part mechanisms;3.Ang? and its receptors played important roles in vascular injury in 2K1C-HT rat,and Ang?-induced vascular injury was exacerbated by AA inflammation;4.AT1 R / ERK1/2 signaling pathway activated by Ang? may be an important mechanism in exacerbated vascular injury in AA rats.