| Objective: Rheumatoid arthritis (RA) is a chronic, systemic autoimmune disease that mainly affects the joints. Using with biologic agent and immunodepressive drug in these years, the life of RA patients has been prolonged,but the morbidity and mortality of cardiovascular disease are increasing. Thus, investigating the mechanism in it and the precautionary measure become one of the cardinal problem in RA clinical treatment. The aim of the study is to investigate the effect of Tripterygium Glycosides(TG) combine with TanshinoneⅡA(TanⅡA) in collagen-induced arthritis(CIA) mice to the level of tumor necrosis factor-α(TNF-α),von Willebrand Factor(vWF) in serum, the endothelial nitric oxide synthase,(eNOs),endothelin-1(ET-1)in arteriae aorta ,relaxant response depend on endothelium of isolated aortas and contractile response depend on accumulate concentration of endothe- lin-1(ET-1) , to investigate the cardiovascular dysfunction protective mechanism of traditional Chinese medicine active component and afford experimental evidence for clinical work.Methods: Choose Wistar rats weight from 120g to 140g, the number of male and female is equal. Injecting collagen solution(CS) into the subcutis of their back,tail and right rear paws after sterilizing regularly and eight of rats are injected equivalent volume of saline as control group. arthritis index(AI) were measured every week. On the 21th day after injecting, rats that AI>5 and presenting secondary response are divided randomly into Model group, MTX group, TG group, TG+TanⅡA group and TanⅡA group, each group includes 8 samples. After measuring the paw thickness with vernier caliper and weights of rats in each group, the treatment rats are given drug for successive 27 days. On the second day after treatment over, after measuring the paw thickness and the weights, rats are sacrificed by withdraw of blood from abdominal aortas under 4% chloraldurat anesthesia to get serum. TNF-α, vWF was assayed by ELISA, After drawing blood, thorax was cut open and removed thoracic aortas quickly. Then, it was cleared of adherent fatty and connective tissue, and was scissored rings of 3-4mm length. Determining relaxant response depend on endothelium and contractile response depend on accumulate concentration of 3,10,20,40nmol/L ET-1 by technique of aortas in organ chambers. The remanent part of aortas was fixed, dehydrated, wrapped and dyed to Hematoxylin and eosin stain(HE stain) and detect the level of ET-1,eNOs in vascular endothelium by immunohistochemical.Data Statistics: All data are analysed by SPSS for windows 13.0 and the results are expressed as X±S. The comparison of mean among groups are evaluated by one-way analysis of variance(ANOVA)Results:1 The effect of TG&TanⅡA to general condition of CIA rats Every group exclude control group appeared RF nodus in back and tail, the AI>5, the swelling of two side paws are significantly after injecting 21th. After model founded, the weight of model group, MTX group, TG group, TG&TanⅡA group and TanⅡA group is lower than control group significantly(P<0.01), but there is no significant difference among the CIA five groups(P>0.05).At the end of the experiment, the weight of model group, MTX group TG group and TanⅡA group is still lower than control group significantly(P<0.01), TG&TanⅡA group lower than control group(P<0.05), model group is lower than MTX group and TG group significantly(P<0.01), but there is no significant difference between model group and TanⅡA group(P>0.05),and no significant difference between MTX group and TG group(P>0.05).2 The depressive effect of TG&TanⅡA to paw swelling of CIA ratsAt the end of experiment, the average swelling degree(%)(CS-injected paws) of MTX group and TG&TanⅡA group is lower than model group and TanⅡA group significantly(P<0.01),TG group is lower than model group and TanⅡA group(P<0.05), there is no significant difference between MTX group,TG group and TG&TanⅡA group (P>0.05),but depressive effect of TG&TanⅡA group were the best compare with MTX group and TG group(46.44% VS 40.46%,33.90%). There is no significant difference between model group and TanⅡA group(P>0.05). The average swelling degrees(%) ( non-FCA-injected paws) of MTX group,TG group and TG&TanⅡA group is lower than model group and TanⅡA group significantly(P<0.01) ,the average swelling degrees(%) of non-FCA-injected paws of TG&TanⅡA group is batter than MTX group and TG group(46.33% VS 42.30%,41.39%).3 The effect of TG&TanⅡA to relaxation depend on endothelium of CIA rats(%)The acetylcholine(Ach) induced endothelium dependent relaxation are decreased in aortas from CIA rats. Model group, MTX group and TG group are lower than control group significantly(P<0.01).But there is no significant difference between TG&TanⅡA group and TanⅡA group compare with control group (P>0.05).4 The antagonistic effect of TG&TanⅡA to ET-1 of CIA ratsWe noted that 3nmol/L ET-1 could relax vascular ring after an shrot time contraction in control group and TG&TanⅡA group. The average relax rate is 67.26±12.59% and 26.75±15.44% respectively, and other group haven't this phenomenon. The concentration of 10,20nmol/L ET-1 consecutive contraction and achieve peak, Model group, MTX group and TG group has significant difference to control group (P<0.01). There is no significant difference between control group, TG&TanⅡA group and TanⅡA group(P>0.05)but TG&TanⅡA group better than TanⅡA group. MTX group and TG group are lower than Model group in concentration of 20nmol/L ET-1(P<0.05). Concentration of 40nmol/L ET-1 has no obviously contraction compare with 10,20nmol/L ET-1.5 The effect of TG&TanⅡA to TNF-αand vWF in serum of CIA rats5.1The effect of TG&TanⅡA to TNF-αof CIA rats(pg/ml)Model group and TanⅡA group are higher than control group significantly(P<0.01),TanⅡA group is lower than model group(P<0.01).There is no significant difference between control group, MTX group, TG group and TG&TanⅡA group (P>0.05). TG&TanⅡA group had lowest level compare with MTX group and TG group(27.29±6.36), TG group higter than MTX group(36.50±14.88 VS 33.43±13.45).5.2The effect of TG&TanⅡA to vWF of CIA rats(mU/ml)Model group have the highest vWF level, and have significant difference compare with other group(P<0.01,0.05).There is no significant difference between control group, MTX group, TG group , TG&TanⅡA group and TanⅡA group(P<0.01,0.05),but TG&TanⅡA group and TanⅡA group are lower than MTX group, TG group.6 HE stain and ET-1,eNOs express in vascular endothelium cell of CIA rats In HE stain, vascular endothelium cell under the high power lens, the vascular endothelium of model group are unconsecutivly, lipidoses and foam cell formation on it, this phenomenon is fit for atherosclerosis second stage. A little of mononuclear cell were found in MTX group, TG group and TanⅡA group. Control group, TG&TanⅡA group no pathological change.Comparing the express of ET-1 in vascular endothelium cell, model group is higher than control group significantly (P<0.01), MTX group, TG group and TanⅡA group are higher than control group and TG&TanⅡA group(P<0.05), and there is no significant difference between TG&TanⅡA group and control group(P>0.05).Comparing the express of eNOs in vascular endothelium cell, model group and MTX group, TG group and TanⅡA group is lower than control group significantly(P<0.01), model group is lower than MTX group ,TG group and TanⅡA group significantly(P<0.05), and there is no significant difference between TG&TanⅡA group and control group(P>0.05).Conclusions:1 CIA rats have many factors engendering cardiovascular dysfunction. CIA rats TNF-αand vWF in serum are all increased significantly, relaxation dependent on endothelium is decreased significantly, endothelium sensitivity to ET-1 increased and vascular endothelium are unconsecutivly, and have foam cell formation on it 2 TG&TanⅡA can resist inflammation positively. TG combine with TanⅡA can remedy CIA significantly, decrease the paw swelling and the level of inflammatory cell factors such as TNF-α. TG&TanⅡA have better effect of resist inflammation than MTX and TG3 TanⅡA can protect vascular endothelium dysfunction of CIA rats. TG&TanⅡA increase relaxation dependent on endothelium, decrease the sensitivity to ET-1.4 The Protective effects and mechanism of TG&TanⅡA to cardiovascular dysfunction are concerned with decreased level of cell factors TNF-α,vWF in serum, antistatic the biological effect of ET-1, up-regulation eNOS and down-regulation ET-1in arteriae aorta. |
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