| 1 Objective The changes of VEGF,miR-126,coagulation indexes,platelet parameters,cytokines and related laboratory indexes in RA patients were observed through clinical studies,and the effects of XFC on them were observed.The changes of joint pathological morphology,vascular endothelial cell ultrastructure,synovial MVD,CD34,CD105,cytokines,miR-126,VEGF/PI3K/Akt signaling pathway and the effect of XFC on AA rats were observed,and the mechanism of XFC regulating synovial angiogenesis in RA was explored from the perspective of miR-126-regulated PI3K/AKT.2 Methods2.1 Clinical studiesSixty RA patients were selected from Rheumatology Department of Anhui Hospital of Traditional Chinese Medicine and randomly divided into XFC group(treatment group)and LEF group(control group),with 30 patients in each group,and 20 healthy subjects were selected from the physical examination center of the hospital as the healthy control group.Using automatic blood coagulation instrument detecting blood clotting index,using platelet parameters of detector,automatic blood cell analysis using biochemical analyzer test and biochemical indicators,miR-126 was evaluated by fluorescence quantitative PCR method,VEGF,using ELISA to detect the IL-6,TNF alpha,westergren method is used to detect the ESR,adopts questionnaire inventory records RA patients’ quality of life,TCM syndrome integral,observation of XFC the curative effect of patients with RA and the influence of the synovial membrane angiogenesis.2.2 Experimental studySixty rats were randomly divided into normal control group(NC group),model group(MC group),Xinfeng capsule high-dose group(H-XFC group),Xinfeng capsule mediumdose group(M-XFC group),Xinfeng capsule low-dose group(L-XFC group)and Tripterygum wilfordii polyglycosides tablet group(TPT group),with 10 rats in each group.In addition to the NC group,to each rat right foot plantar intradermal injection of 0.1 ml FCA inflammatory,13 days after initial inflammatory,injection of 0.05 ml FCA enhanced inflammation,again on the 19 th day,dose is as follows: the XFC to shell at the end of the study,adding saline suspension liquid(0.3 g/ml),XFC three groups respectively according to 0.5 ml / 100 g(L-XFC group),1 ml / 100 g(M-XFC group),2 ml / 100 g(H-XFC group)dose lavage,1 / d;TGT was ground into fine powder,and normal saline was added to make suspension(1mg/ml).TGT group was gavaged at 1ml/100 g dose,once a day.Normal group and model group were gavaged with normal saline,1ml/100 g,once a day.All groups were gavaged continuously for 30 days.Observed between groups of rats body quality,foot metatarsus swelling degree,AI,synovial pathological changes and ultrastructure of vascular endothelial cells,immune organized method is used to detect synovial MVD,CD34,CD105 expression,using real-time fluorescent quantitative PCR detection of miR-126,VEGF,HIF-1 alpha level,the synovial membrane of rats was evaluated by ELISA method,IL-1,HIF-1 alpha,IL-10 express,adopts WB detection PIK3R2,PI3 K,and AKT,p-AKT,e NOS protein expression level.3 The results3.1 Clinical study results3.1.1 Changes of coagulation indexes and platelet parameters in RA patients Compared with normal controls,D-D and FBG in peripheral blood of 60 RA patients were significantly increased(P<0.05),while TT,APTT and PT were not significantly changed(P>0.05).Peripheral blood PLT,PCT and MPV were significantly increased(P<0.05),but PDW had no significant change(P>0.05).3.1.2 Changes of cytokines and laboratory indicators in RA patientsCompared with normal controls,the levels of IL-6 and TNF-α in peripheral blood of60 RA patients were significantly increased(P<0.05).The levels of CRP,ESR,RF,CCP and Ig G in peripheral blood were significantly increased(P<0.05),but there were no significant changes in Ig A,Ig M,C3 and C4(P>0.05).3.1.3 Changes of serum VEGF and plasma miR-126 levels in RA patients Compared with normal controls,the levels of serum VEGF and plasma miR-126 in 60RA patients were significantly increased(P<0.05).3.1.4 Correlation analysis of serum VEGF and various indexes in RA patients VEGF in RA patients was positively correlated with blood coagulation indexes D-D,FBG,APTT and PT(P<0.05),but had no correlation with TT(P>0.05).It was positively correlated with platelet parameters PLT,PCT and MPV(P<0.05),but had no correlation with PDW(P>0.05).Was positively correlated with cytokines IL-6 and TNF-α(P<0.05);It was positively correlated with CRP,ESR,CCP and Ig G(P<0.05),but had no correlation with RF,Ig A,Ig M,C3 and C4(P>0.05).It was positively correlated with miR-126(P<0.05).3.1.5 Correlation analysis of serum VEGF with TCM syndromes and quality of life in RA patientsIn RA patients,VEGF was positively correlated with joint pain,joint swelling and joint tenderness(P<0.05),but not with pain aggravation at night and morning stiffness(P>0.05).It was positively correlated with loss of appetite,tiredness and weakness,abdominal distension after eating and loose stools(P<0.05),but not with lack of qi,lazy speech and joint weight(P>0.05).It was positively correlated with joint pain,lip color and skin nail error(P<0.05),negatively correlated with tongue texture(P<0.05),and had no correlation with pulse condition and subcutaneous ecchymosis(P<0.05).In RA patients,VEGF was positively correlated with AQT,physiological function,social function and healthy self-awareness(P<0.05),but not with mental function(P>0.05).3.1.6 Clinical efficacy of XFC in RAAfter treatment,the significant efficiency was 63.3% in XFC group and 43.3% in LEF group.The effective rate was 26.7% in XFC group and 40% in LEF group.The total effective rate was 90% in the XFC group and 83.3% in the LEF group.In conclusion,the significant efficiency of XFC group was higher than that of LEF group(P<0.05),and the total effective rate was similar(P>0.05).3.1.7 Influence of XFC on coagulation indexes and platelet parameters in RA patients Compared with before treatment,the coagulation indexes D-D,FBG and platelet parameters PLT were significantly decreased in the two groups after treatment(P<0.05),while the coagulation indexes APTT,PT,TT and platelet parameters PCT,MPV and PDW had no significant changes(P>0.05).Compared with the LEF group,the decrease of coagulation index D-d and platelet parameter PLT was more significant in the XFC group(P<0.05).3.1.8 Effects of XFC on cytokines and laboratory indicators in RA patients Compared with before treatment,cytokines IL-6,TNF-α and laboratory indexes CRP,ESR,RF,CCP and Ig G were significantly decreased in both groups after treatment(P<0.05).Compared with the LEF group after treatment,the levels of cytokines IL-6,TNF-αand laboratory indicators of CRP,ESR and CCP were significantly lower in XFC group after treatment(P<0.05).3.1.9 Effects of XFC on miR-126 and VEGF in RA patientsCompared with before treatment,miR-126 and VEGF levels in both groups were decreased after treatment(P<0.05).Compared with the LEF group,the levels of miR-126 and VEGF decreased more significantly in the XFC group after treatment(P<0.05).3.1.10 Influence of XFC on TCM syndrome score and quality of life score in RA patientsCompared with before treatment,the indexes of main joint symptoms,spleen deficiency syndrome and blood stasis syndrome were significantly improved in both groups after treatment(P<0.05);Compared with the LEF group,the improvement of joint pain,joint tenderness,anorexia,abdominal distension,loose stools,joint tingling,lip color and skin defect in the XFC group was more obvious after treatment(P<0.05).Compared with before treatment,the quality of life indexes in both groups were significantly improved after treatment(P<0.05);Compared with the LEF group after treatment,the XFC group improved the quality of life more significantly(P<0.05).3.2 Experimental research results3.2.1 Body weight,toe swelling degree and AI changes of AA rats and the effects of XFC on themBefore modeling,there were no significant differences in body weight,plantar swelling and AI among all groups(P>0.05).After building,in addition to the NC group of rats,the rest of the group of rats right rear toes gradually appear red and swollen,tight skin,burning,inflammatory swelling,2-3 days after 6-7 day part inflammatory toe local occurrence canker,8-10 days ease swelling degree,local skin temperature basic returned to normal,13-14 days before and after the toes swelling again,part of the contralateral toes swelling of rats.Before administration,toe swelling degree and AI in MC group and all treatment groups were significantly higher than those in NC group(P<0.05),body weight was significantly lower than that in NC group(P<0.05),and there was no significant difference between MC group and all treatment groups(P>0.05).After 30 days of administration,compared with NC group,toe swelling degree and AI in MC group were increased(P<0.05),while body weight was significantly decreased(P<0.05).Compared with MC group,the degree of plantar swelling and AI in treatment groups were significantly decreased(P<0.05),and body weight was significantly increased(P<0.05).Compared with the three XFC high-dose,medium-dose and low-dose groups,the plantar swelling degree and AI of M-XFC group were significantly decreased(P <0.05),and the body weight of M-XFC group was significantly higher than that of H-XFC group,but there was no significant difference between M-XFC and L-XFC group(P > 0.05).Compared with M-XFC group,AI in TPT group was higher(P <0.05),but there were no significant differences in body weight and plantar swelling degree(P>0.05).3.2.2 Pathomorphological changes of joints in AA rats and the effects of XFC on them Under light microscope,the synovial tissue structure of NC group rats was complete,without hyperplasia,infiltration of inflammatory cells,and monolayer cells.Compared with the NC group,the synovium of the joint in the MC group was hyperplasia and the number of blood vessels was increased.The synovium presented villous protrusion into the joint cavity,with a large number of inflammatory cell infiltration,and the synovium lining cells were thickened and multilayered.After drug intervention,M-XFC group had the best recovery of synovial structure,synovial stratification and hyperplasia were significantly improved,inflammatory cells were reduced,and the articular surface was more orderly.Compared with M-XFC group,the synovial membrane and its surrounding tissues in H-XFC and L-XFC groups showed infiltration of inflammatory cells,more proliferative blood vessels,thickened synovial lining cells,obvious stratification,and less regular articular surface.The synovial cells in TPT group were thickened and stratified,and the articular surface was less regular.3.2.3 Ultrastructural changes of vascular endothelial cells in AA rats and the effect of XFC on themUnder electron microscope,the mitochondria of the NC group were basically without deformation and swelling,with abundant rough endoplasmic reticulum,clear nuclear membrane boundary,uniform chromatin distribution and regular ridge arrangement.In MC group,endothelial cells were deformed,mitochondrial vacuolization was more,rough endoplasmic reticulum was reduced and destroyed,nuclear membrane boundary was not clear,ridges were irregular and part disappeared.After drug intervention,the endothelial cells in M-XFC group showed a small number of deformed mitochondria,more rough endoplasmic reticulum,clearer nuclear membrane boundary,more uniform chromatin,and complete arrangement of most cristae.In H-XFC group,mitochondria and rough endoplasmic reticulum were decreased,and most cristae disappeared.In L-XFC group,mitochondria of endothelial cells were slightly swollen and deformed,cytoplasmic edema was observed,rough endoplasmic reticulum was slightly reduced and expanded obviously,and cristae were arranged in disorder.In TPT group,mitochondria of endothelial cells were slightly swollen,cristae were partially destroyed,rough endoplasmic reticulum was visible and moderately dilated,and cytoplasm was slightly edema.3.2.4 Changes of synovial microvessel density,CD34 and CD105 in AA rats and the effects of XFC on themAfter 30 days of administration,compared with NC group,MVD,CD34 and CD105in synovium of MC group were significantly increased(P<0.05).Compared with MC group,MVD,CD34 and CD105 in treatment groups were significantly decreased(P<0.05).Compared with XFC high-dose,medium-dose and low-dose groups,the MVD,CD34 and CD105 in M-XFC group were significantly decreased(P<0.05).Compared with M-XFC group,there were no significant differences in MVD,CD34 and CD105 in TPT group(P>0.05).3.2.5 Changes of cytokines in AA rats and the effects of XFC on themAfter 30 days of administration,compared with NC group,cytokines IL-1 and TNF-αin MC group were significantly increased,while IL-10 was significantly decreased(P<0.05).Compared with MC group,IL-1 and TNF-α in treatment groups were significantly decreased,while IL-10 was significantly increased(P<0.05).Compared with XFC highdose,medium-dose and low-dose groups,IL-1 and TNF-α in M-XFC group were significantly decreased,and IL-10 was significantly increased(P<0.05).Compared with M-XFC group,TNF-α in TPT group was significantly increased(P<0.05),but IL-10 and IL-1 had no significant differences(P>0.05).3.2.6 Changes of miR-126 and HIF-1α in synovial membrane of AA rats and the effects of XFC on themAfter 30 days of administration,compared with NC group,the levels of HIF-1α and miR-126 in synovial membrane of MC group were significantly increased(P<0.05).Compared with MC group,the levels of HIF-1α and miR-126 in synovium in treatment groups were significantly decreased(P<0.05).Compared with XFC high-dose,medium dose and low-dose groups,the levels of HIF-1α and miR-126 in synovial membrane of M-XFC group were significantly decreased(P<0.05).Compared with M-XFC group,the level of HIF-1α in synovium of TPT group was significantly increased(P <0.05),but there was no significant difference in miR-126 level(P>0.05).3.2.7 Changes of VEGF/PI3K/ Akt signaling pathway in synovium of AA rats and the effect of XFC on itAfter 30 days of administration,compared with NC group,the synovial VEGF level,protein PI3 K,AKT,p-AKT and e NOS expressions in MC group were significantly increased(P<0.05),and protein PIK3R2 expression was significantly decreased(P<0.05).Compared with MC group,the synovial VEGF level,the expression of protein PI3 K,AKT,p-AKT and e NOS in treatment groups were significantly decreased(P<0.05),and the expression of protein PIK3R2 was significantly increased(P<0.05).Compared with each treatment group,the level of synovial VEGF in M-XFC group was significantly decreased(P<0.05);The expressions of PI3 K,Akt,p-Akt and e NOS in M-XFC histones were significantly lower than those in L-XFC group(P<0.05),and had no significant difference with those in H-XFC group and TPT group(P>0.05).The expression of PIK3R2 in M-XFC group was significantly higher than that in L-XFC group(P<0.05),and there was no significant difference between M-XFC group and H-XFC group and TPT group(P>0.05).4 conclusion4.1 Clinical efficacy of XFC in RA patients and its influence on angiogenesis XFC can significantly control the inflammatory response and disease activity,regulate the vascular environment,improve the joint symptoms and the overall quality of life in RA patients.XFC can significantly reduce the levels of plasma miR-126 and serum VEGF,regulate the level of vascular growth factor and inhibit angiogenesis in RA patients.4.2 Effects of XFC on joint symptoms and synovial angiogenesis in AA rats XFC can improve the structure of vascular endothelial cells and synovial membrane,and improve the pathological morphology of synovial membrane in AA rats.Adjust the fine;To improve the joint morphology and function and regulate the overall growth state of rats;XFC significantly down-regulated the expression of miR-126 in synovium of AA rats,promoted the secretion of PIK3R2,decreased the level of VEGF,and inhibited the activity of PI3K/AKT signaling pathway.4.3 Mechanism of action of XFC on synovial angiogenesisXFC can balance the cytokine network and reduce the stimulation of inflammation to synovial membrane.It can improve the structure of vascular endothelial cells and synovial membrane,relieve the functional damage of vascular endothelial cells and reduce the morphological and pathological changes of synovial membrane.Adjust vascular environment,reduce synovial lesions;Regulate HIF-1α secretion,reduce stress response,inhibit synovial microvascular stress proliferation;Regulating the expression of VEGF/PI3K/AKT pathway regulated by miR-126,by down-regulating the expression of miR-126,promoting the expression of PIK3R2,inhibiting VEGF secretion,reducing the secretion of PI3 K and Akt,down-regulating the activity of PI3K/AKT signaling pathway,and reducing angiogenesis. |
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