Study On The Effect Of Fengshi Qutong Capsule On Synovial Angiogenesis In Treating Rheumatoid Arthritis

1 ObjectiveIn order to explore the effect and mechanism of Fengshi Qutong Capsule on Rheumatoid arthritis(RA)primarily on AKT and MAPK signaling path ways,we used the type II collagen induced arthritis(CIA)rats and a variety of in vivo and in vitro models about RA synovial angiogenesis.2 Method(1)SD rats,according to body quality,were randomly divided into Control group,Model group,Fengshi Qutong Capsule low-dose group(FSQTC 0.25 g/kg),Fengshi Qutong Capsule dose group(FSQTC 0.5 g/kg),Fengshi Qutong Capsule high-dose group(FSQTC 1 g/kg),methotrexate(MTX 0.2 mg/kg)as positive control group,10 samples in each group.The rats were established CIA models except the control group.To evaluate the effect of FSQTC on RA,the degree of swelling joints,incidence clinical score and so on were used;(2)The therapeutic effects and characteristics of FSQTC on CIA rats were observed by imaging and histopathology(HE),combining with immunohistochemistry(IHC-P)and immunofluorescence(IF)double staining,and the number,length and area of vessels of rat thoracic aorta induced by VEGF(20ng/mL)cured by FSQTC(0.02?0.1?0.5ng/mL)was observed;(3)To explore the influence and mechanism of angiogenesis of FSQTC(0.02?0.1?0.5 ng/mL)on vascular endothelial cell function,Human Umbilical Vein Endothelial Cells(HUVEC)induced by VEGF(20ng/mL)simulate the process of angiogenesis in vitro RA through proliferation,migration,adhesion,invasion and tube formation experiments;(4)As inflammatory proliferation of RA synovial cells is closely related to angiogenesis,human rheumatoid arthritis fibroblasts(MH7A)induced by TNF?(20ng/mL)were used to observe the indirect effect on the angiogenesis process of FSQTC(0.02,0.1,0.5 ng/mL)on the function of fibroblast synovial cells though the proliferation,migration,adhesion and invasion.(5)Enzyme-linked ImmunoSorbent Assay(ELISA),immunohistochemistry(IHC-P),Western Blot and/or real time PCR were used to detect the serum,synovial tissue angiogenic regulators,interleukin 1?(IL-1?)and tumor necrosis factor ?(TNF?),vascular endothelial growth factor(VEGF),transforming growth factor ?(TGF-?),platelet-derived growth factor(PDGF),of CIA rats.TNF alpha,vascular endothelial growth factor(VEGF),platelet derived factor(PDGF),matrix metalloproteinase 3(MMP-3),nuclear factor kappa B receptor activating factor ligand(RANKL),platelet endothelial cell adhesion molecule-1(CD31),Alpha smooth muscle actin,(?SMA)and VEGF receptor 2(VEGFR2)and downstream protein kinase B(AKT)and mitogen-activated protein kinase(MAPK)signaling pathway.3 Results 3.1 The therapeutic effect of Fengshi Qutong Capsule of CIA ratsFengshi Qutong capsule(0.25,0.5,1 g/kg)dose-dependently improved the symptoms of inflammation,redness,swelling and deformity of joints of CIA rats,and reduced clinical scores and morbidity,and inhibited histopathological changes such as synovitis,pannus,cartilage and bone destruction.The effect of the high-dose Fengshi Qutong capsule group was similar to that of methotrexate as the positive control group.3.2 The effect of Fengshi Qutong Capsule on synovial angiogenesis of CIA ratsFengshi Qutong Capsule(0.25,0.5,1 g/kg)inhibited synovial angiogenesis of the CIA rats: reduced the density of CIA rats joint synovial vascular,declined the positive expression of CD31,CD31+/?SMA-immature vessels and total positive vessels IOD values,but had no obvious effect on the positive expression of CD31+/? SMA+ mature vessels.And the medium and high dose of FSTQC show significantly effect,the methotrexate was similar to high dose.3.3 The effect of Fengshi Qutong Capsule on angiogenesis in rat thoracic aorta.Fengshi Qutong Capsule(0.02,0.1,0.5ng/mL)significantly reduced the number,length and area of vessels induced by VEGF in rat aorta.3.4 The effect of Fengshi Qutong Capsule on HUVEC's proliferation,migration,invasion,adhesion and tube formationFengshi Qutong Capsule(0.02,0.1,0.5ng/mL)dose-dependently inhibited HUVEC proliferation activity after 48 h,and significantly inhibited HUVEC induced by VEGF(20 ng/mL)about proliferation,migration,adhesion,invasion and tube formation within 24 h.3.5 The effect of Fengshi Qutong Capsule on the proliferation,migration,invasion and adhesion of MH7AFengshi Qutong Capsule(0.02,0.1,0.5ng/mL)was administered in vitro to TNF?-induced(20 ng/mL)MH7A for 24 h,and it had no significant effect on proliferation activity.For 48 h,the proliferation activity of TNF?-induced MH7 A was reduced,and there was a concentration-dependent phenomenon.Within 24 h,the migration,adhesion and invasion of TNF?-induced MH7 A were significantly reduced.3.6 The effect of Fengshi Qutong Capsule on angiogenesis regulatory factor of CIA rats and cells in vitroFengshi Qutong Capsule(0.02,0.1,0.5ng/mL)significantly reduced VEGF(20 ng/mL)in the inflammatory joints and serum of CIA rats,and reduced IL-1?,TNF?,VEGF,TGF-?,PDGF and VEGFR2 in the serum of CIA rats.The effect of the high-dose was similar to that of the methotrexate as positive control;and Fengshi Qutong Capsule decreased the IL-1? and VEGF in supernatant of MH7 A induced by TNF-? significantly in vitro.3.7 The effect of Fengshi Qutong Capsule on VEGFR2 or p-VEGFR2 protein and gene expression of CIA rats and in vitroFengshi Qutong Capsule(0.25,0.5,1 g/kg)significantly reduced VEGFR2 in the serum of CIA rats.The effect of Fengshi Qutong Capsule(0.02,0.1,0.5 ng/mL)on the phosphorylation,protein and gene expression of VEGFR2 of CIA rats and HUVEC in vitro.3.8 The effect of Fengshi Qutong Capsule on AKT or MARK signaling pathways of CIA rats and in vitroFengshi Qutong Capsule significantly reduced the expression of p-AKT,p-p38,p-ERK and p-JNK proteins in the inflammatory joints of CIA rats,HUVEC and MH7 A.4.Conclusion:According to the above results,Fengshi Qutong Capsule can effectively prevent and cure experimental RA,and have obviously inhibitory effect on synovial angiogenesis(pannus formation)of RA.The relevant mechanism may be related to the abnormal activation of angiogenesis regulatory factor and the downstream signaling pathways of p-AKT,p-JNK,p-p38 and p-ERK1/2,so as to inhibit the migration,adhesion,invasion and/or tube formation of Human Umbilical Vein Endothelial Cells and human rheumatoid arthritis fibroblasts.The study provides experimental reference for further exploration of Fengshi Qutong Capsule on the theory of “Tong Luo Kai Bi” and the characteristics of worm-snake-medicine.