| ObjectiveObserve the effect of Qingre Huoxue Decoction on synovial angiogenesis of rats with collagen-induced arthritis(CIA)and the effect on angiogenesis of zebrafish intersegment vessels(ISV)by exploring the mechanism of action related to miR-125b-PIGF/Fltl pathway,to provide scientific evidence and treatment prospects of Qingre Huoxue Decoction on treatment of active rheumatoid arthritis in clinical practice.MethodsThe healthy female SD rats were randomly divided into 7 groups after weighing,named as Normal group,CIA model group,Qingre Huoxue Decoction 15g/kg,20g/kg,25g/kg in treatment group,the inhibitor ZD6474 group(50mg/kg)and the MTX group(10mg/kg).Inject type II collagen emulsifier in the root of rat tail subcutaneously,then needed a booster shot on day 7,to establish the CIA model.Groups of rats with CIA were treat with Qingre Huoxue Decoction?MTX and ZD6474 orally accrording to the group from the first immune day,and the normal group and CIA model group were given the same volume of saline,which continuously administrated 21 days.During the process,pathological observe:The activities and behavior changes,changes of body weight,joint swelling and were observed and recorded;observe inflammation?angiogenesis and bone erosion by joint pathology?synovium pathology and immunohistochemistry.Test the body indicator:The serum levels of interleukin-1?(IL-1?),interleukin-6(IL-6),vascular endothelial growth factor(VEGF),tumor necrosis factor-a(TNF-a),basic fibroblast growth factor(bFGF)and transforming growth factor-?(TGF-?)were test by ELISA.The protein levels of VEGFR2,MMP9 and bFGF of synovial tissue were tested by Western Blot.The mRNA levels of placental growth factor(PIGF)and its receptors Fltl,Flkl of synovial tissue were tested Real-time PCR.Establishment of zebrafish angiogenesis model,intervene which by the different concentration Qingre Huoxue Decoction.then the effect of Qingre Huoxue Decoction on the angiogenesis of zebrafish was observed by fluorescence microscopy.The area of intersegmental vessels(ISV)was calculated and the difference of neovascularization between the two groups was compared.The effects of Qingre Huoxue Decoction on the organs and tissues of zebrafish were observed by toxicity test.Results1.Effects of Qingre Huoxue Decoction on symptoms,joint swelling and weight on rats with CIACompared with the normal group,the swelling of the model group was significantly increased(P<0.01);Compared with the CIA model group,the swelling tendency of the rats in the ZD6474 group and the MTX group was significantly inhibited(P<0.01);the low dose of Qingre Huoxue Decoction group also had a significant effect on the swelling of the ankle joint in rats(P<0.05).Compared with the normal group,the weight gain of the model group was slower than that of the model group,from the 1st to 10th day after immunization,the rat*s weight in each group was increased,and the growth rate was increased tolerably.From the 10th day to the 18th day of the immunization,the weight of the rats in the normal group decreased continuously,and the weight of the rats in other groups aslo decreased.On the 18th day,the weight of rats in the normal group were 262.20±8.23g(n = 10):the rat*s weight of the model group was 239.00±12.51g(n = 10);there was significant difference between the two groups(P<0.05).2.Effects of Qingre Huoxue Decoction on the levels of serum IL-1??IL-6?TNF??VEGF?bFGF?TGF in rats with CIAThe expression of IL-6,TNFaand VEGF in the model group was significantly higher than that in the normal group(P<0.01),and the expression of IL-1?,bFGF and TGF was significantly increased(P<0.01);compared with the model group,the middle dose group of Qingre Huoxue Decoction could reduce the expression of the above indexes in different degrees.The ZD6474 group and the MTX group could reduce the expression of related indexes.3.Effects of Qingre Huoxue Decoction on protein levels of VEGFR2?MMP-9?bFGF in synovial tissueCompared with the normal group,the expression of MMP-9 in the synovium of CIA rat model group was significantly higher than that of the normal group(P<0.01).Compared with the CIA model group,the ZD6474 group,the MTX group and the traditional Chinese medicine group could decrease the expression of MMP-9(P<0.05).Compared with the model group,the expression of bFGF in the synovial tissue of CIA rats model group was significantly higher than that in the model group(P<0.05),and the expression of bFGF was significantly inhibited by ZD6474 group,MTX group and traditional Chinese medicine(P<0.05).Compared with the normal group,the expression of VEGFR2 in the synovial tissue of CIA rat model group was significantly higher than that of the control group(P<0.05);compared with the model group,the ZD6474 group,MTX group and the traditional Chinese medicine group VEGFR2 expression level significantly decrease(P<0.01),and the MTX group,low Qingre Huoxue Decoction group can also reduce the expression of VEGFR2(P<0.05).4.Effects of Qingre Huoxue Decoction on mRNA levels of PIGF?FIt1l?Flkl and miR-125b in synovial tissueThe expression of PIGF,F1k1F1tlmRNA in the model group was significantly increased after 21 days of administration(P<0.05),while the and miR-125b was decreased;compared with the CIA model group,the expression of PIGF mRNA could be decreased in the middle Qingre Huoxue Decoction group and the ZD6474 group(P<0.05),Flkl mRNA decreased in the middle Qingre Huoxue Decoction group and the MTX group(P<0.05),FltlmRNA decreased in the middle Qingre Huoxue Decoction group?the ZD6474 group and the MTX group(P<0.05)and the expression of miR-125b could be increased in the middle Qingre Huoxue Decoction group and the ZD6474 group(P<0.05).5.Effects of Qingre Huoxue Decoction on intersegment vessels Angiogenesis of ZebrafishCompared with the blank control group,the ISV of the zebrafish in the positive control group showed incomplete and vascular defect under the fluorescence microscope;the degree of zebrafish vascular defect was similar to that of the positive drug group at the time of 100?g/ml of traditional Chinese medicine;with the increase of concentration of Qingre Huoxue Decoction the degree of vascular defect in zebrafish was positively correlated with the concentration of Qingre Huoxue Decoction.6.The toxic effects of Qingre Huoxue Decoction on zebrafish1/10MNLD Qingre Huoxue Decoction only affected the zebrafish egg yolk absorption delay;1/3MNLD of which can cause zebrafish intestinal enlargement;MNLD and LD50 Qingre Huoxue Decoction could cause zebrafish intestinal degeneration,and the latter was more obvious.The Different concentrations of Qingre Huoxue Decoction had different effect on the heart rate of zebrafish.It could inhibit the heart rate of zebrafish When the concentration of Qingre Huoxue Decoction was 60p,g/ml;the zebrafish heart rate increased,when the concentration of Qingre Huoxue was higher than 200?g/ml,the heart rate of zebrafish increased obviously.Compared with the blank group,10%of the zebrafish intestine became smaller at 6?g/ml;30%of the zebrafish intestinal folds became smaller at 200?g/ml;at 60?g/ml,20%of the zebrafish intestinal degenerated;at 1200 ?g/ml,90%of the zebrafish tail fins became smaller.Conclusion1.The effect of Qingre Huoxue Decoction could reduce the symptoms of joint swelling and decrease the degree of joint swelling in CIA rats,and also reduce the levels of proinflammatory cytokines IL-1?,IL-6,TNF-?,TGF-?and angiogenesis related factors bFGF,VEGF levels,down-regulated the expression of PIGF,Fltl,Flkl mRNA and inreased miR-125b in synovial tissue,thereby relieved the clinical symptoms of CIA rats,reducing synovial inflammation,inhibiting the formation of neovascularization and delaying the progress of arthritis.2.Different concentrations of Qingre Huoxue Decoction could inhibition of zebrafish angiogenesis,to different degree;toxicity experiment showed that Qingre Huoxue Decoction is relatively safe. |
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