Protective Effect Of PNS Combine With Tripterygium To Cardio-vascular Research On Collagen-induced Arthritis Rat

Objective:Rheumatoid arthritis is a chronic, systemic, inflammatory autoimmunedisease. Recently study, RA increased incidence of cardiovascular disease. Ithas become the first cause of death with RA. So explore the pathogenesis andmeasures of prevention become one of the important issues. The aim of thestudy is that establish collagen-induced arthritis rat model, to study thepathogenesis of cardiovascular damage, to detect tumor necrosis factor(TNF-α), interleukin-6(IL-6), monocyte chemoattractant protein-1(MCP-I),plasminogen activator inhibitor (PAI-1), vascular endothelial growth factor(VEGF) levels in serum, to assay thoracic aorta and cardiac tissue MCP-1,VEGF expression by immunohistochemical, to study mechanism of action toprotect cardiovascular system and to provide an experimental basis for clinicaluse.Method:Choosing Wistar rats weight in150-180g, the number of male and femalewere equal. Measuring rats weight by electronic scale and the volume ofbilateral feet. they were randomly divided into control and experimentalgroups (CIA-made modules). CIA groups were injected with0.2ml collagenfrom chicken sternal cartilage type II in the tail, right hind limb and backtogether, then boosted at the14th day to make manufacture the model ofCIA．Normal group were injected saline in the same way. On21st day aftermodeling, to measure body weight and paw volume,arthritis index. And thenrandomly divided into model group, Tripterygium glycosides (TG), TG+group of PNS (PNS), leflunomide group (LEF), each group included eightsamples. Normal group and Model group were given equivalent volume ofsaline. On49nd day and63nd after modeling, to measure body weight, two hind paw volume. The next day after gavage end, drawn of blood byabdominal aortas under10%chloralic hydras to get serum. Applicating proteinchip technology to measure TNF-α, IL-6, MCP-1, PAI-1, VEGF levels inserum, immediately opened chest after blood, remove the thoracic aorta andcardiac tissue, separated fat and connective tissue surrounding, fixed,dehydrated, wrapped and HE dyed, determined expression of MCP-1andVEGF in vascular endothelium cell and cardiac tissue byimmunohistochemical.Data Statistics: All data is analysed by SPSS for windows13.0and the resultsare expressed asX±S. The comparison of mean among groups is evaluatedby one-way analysis of variance(ANOVA). Statistical significance was set at(P <0.05).Results：1Changes in body weight in ratsBefore modeling the weight among every groups was not statisticallysignificant difference (P>0.05). After modeled, the21st days, model group,LEF group, TG and TG+PNS group body weight was lower than the controlgroup (P<0.01). Among the model group, LEF group, TG and TG+PNS groupwere no statistical significance (P>0.05). After administered four weeks,model group, LEF group, TG and TG+PNS group body weight wassignificantly lower than control group (P<0.01). Model group, LEF group, TGand TG+PNS group were no statistical significance (P>0.05). But TG+PNSgroup weight-gain is higher than the model group (P<0.05). Afteradministration six weeks, model group, LEF group, TG and TG+PNS groupbody weight was significantly lower than the control group (P<0.01). Modelgroup, LEF group, TG group and TG+PNS group were no statisticalsignificance (P>0.05). But TG+PNS group weight-gain was significantlyhigher than model group (P<0.01) and higher than LEF group (P<0.05).2Changes in the rats paw swelling rate and inhibition rateAfter administration four weeks, LEF group, TG group and TG+PNSgroup modeled side (right) paw swelling was significantly lower than model group (P<0.01). Among LEF group, TG group, TG+PNS group were nosignificant difference (P>0.05), but TG+PNS group on modeled side pawinhibition rate is slightly better than the LEF and TG group (37.35%,30.16%,27.58%). LEF group, TG and TG+PNS group non-modeled side averagedegree of swelling was significantly lower than model group (P<0.01).Among LEF group, TG group, TG+PNS group were no statistical significance(P>0.05). After administration6weeks, LEF group, TG group and TG+PNSgroup the average swelling rate of modeling side (right) paw weresignificantly lower than model group (P<0.01). TG+PNS group swelling waslower than TG group (P<0.05). Comparing with LEF group, TG+PNS groupwas no statistically difference, but TG+PNS group inhibition rate was betterthan LEF group (52.89%VS46.65%). LEF group, TG group and TG+PNSgroup non-modeled side paw swelling were significantly lower than modelgroup (P<0.01). TG+PNS group swelling was lower than TG group (P<0.05).TG+PNS group compared with LEF group inhibition rate was slightly betterthan LEF group (51.60%VS44.10%).3Changes in the Arthritic Index (AI) in each groupOn21stday after modeling, TG group, LEF group, TG+PNS group,Compared with model group, they were no statistical differences (P＞0.05).After administration four and six weeks, Compared with model group, TGgroup, LEF group, TG+PNS group were statistical differences (P＜0.01). AIof TG+PNS group was the lowest at6th week after administration.4Comparison of serum TNF-α, IL-6, MCP-1, PAI-1, VEGF levelComparison of serum TNF-α level (pg/ml): Model group, LEF group, TGgroup were higher than control group (P<0.01). PNS+TG group was higherthan control group (P<0.05). Model group was significantly higher than LEFgroup&TG group&PNS+TG group (P<0.01). PNS+TG group was lower thanLEF group&TG group (P<0.05).Comparison of serum IL-6level (pg/ml): Model group, LEF group, TGgroup were higher than control group, the difference was statisticalsignificance (P<0.01). PNS+TG group was higher than control group (P<0.05). Model group was significantly higher than LEF group&TG group&PNS+TGgroup (P<0.01). PNS+TG group was lower than LEF group&TG group(P<0.05).Comparison of serum MCP-1level (pg/ml): Model group, LEF group,TG group were higher than control group (P<0.01). PNS+TG group washigher than control group (P<0.05). Model group was higher than LEFgroup&TG group&PNS+TG group (P<0.01). PNS+TG group was lower thanLEF group&TG group, the difference was statistical significance (P<0.05).Comparison of serum PAI-1level (pg/ml): Model group, LEF group, TGgroup were higher than control group, the difference was statisticalsignificance (P<0.01). PNS+TG group was higher than control group (P<0.05).Model group was significantly higher than LEF group&TG group&PNS+TGgroup (P<0.01). PNS+TG group was lower than LEF group&TG group(P<0.05).Comparison of serum VEGF level (pg/ml): Model group, LEF group, TGgroup were higher than control group, the difference was statisticalsignificance (P<0.01). PNS+TG group was higher than control group (P<0.05).Model group was significantly higher than LEF group&TG group&PNS+TGgroup (P<0.01). PNS+TG group was lower than LEF group&TG group(P<0.05).5The pathological change and VEGF, MCP-1express in thoracic aorta andmyocardium5.1The pathological change of thoracic aorta and myocardiumThe pathological change of thoracic aorta: the vascular endothelium ofmodel group were unconsecutivly, lipidoses and foam cell formation on it. Itwas fit for atherosclerosis Ⅲ type．A little of mononuclear cell were found inLEF group&TG group. It was fit for atherosclerosisⅠtype. PNS+TG groupand Control group have no pathological change．The pathological change ofmyocardium: model group extensive myocardial inflammatory lesions,myocardial interstitial edema, scattered lymphocytes surrounded myocardialcells and there are limitations myocardial cells degeneration. The lesions of LEF group and TG group were reduced. Part of myocardium werehypertrophy and interstitial edema, Local inflammatory cells wereaccumulated, and part of myocardium were visible disarray. PNS+TG groupwere seen only sporadically inflammatory cells. Myocardium structure of thenormal control group showed no abnormal changes.5.2MCP-1, VEGF expression in thoracic aorta and myocardiumThe expression of VEGF in thoracic aorta (gray value): Model group,LEF group, TG group were higher than control group, the difference wasstatistical significance (P<0.01). PNS+TG group was higher than controlgroup (P<0.05). Model group was higher than LEF group&TG group&PNS+TG group (P<0.01). PNS+TG group was lower than LEF group&TGgroup, the difference was statistical significance (P<0.05).The expression of MCP-1in thoracic aorta (gray value): Model group,LEF group, TG group were higher than control group, the difference wasstatistical significance (P<0.01). PNS+TG group was higher than controlgroup (P<0.05). Model group was higher than LEF group&TG group&PNS+TG group (P<0.01), PNS+TG group was lower than LEF group&TGgroup, the difference was statistical significance (P<0.05).The expression of VEGF in myocardium (gray value): Model group, LEFgroup, TG group were higher than control group, the difference was statisticalsignificance (P<0.01). PNS+TG group was higher than control group (P<0.05).Model group was higher than LEF group&TG group&PNS+TG group(P<0.01), PNS+TG group was lower than LEF group&TG group, thedifference was statistical significance (P<0.05).The expression of MCP-1in myocardium (gray value): Model group,LEF group, TG group were higher than control group, the difference wasstatistical significance (P<0.01). PNS+TG group was higher than controlgroup (P<0.05). Model group was higher than LEF group&TG group&PNS+TG group (P<0.01), PNS+TG group was lower than LEF group&TGgroup, the difference was statistical significance (P<0.05). Conclusions：1The level of MCP-1, PAI-1, VEGF in serum of Collagen-inducedarthritis rat were significantly elevated, lipid deposition in thoracic aorta andinfiltration of inflammatory cells in myocardium were showed in the lightmicroscope, the expression of MCP-1, VEGF in thoracic aorta andmyocardium increased, indicating CIA rats had relevant factors ofcardiovascular damage.2PNS+TG significantly reduced paw swelling, arthritic index and thelevels of TNF-α, IL-6in serum. The anti-inflammatory effect of PNS+TG wasbetter than TG or leflunomide in CIA rats. Compatibility of traditionalChinese medicine active ingredients has a synergistic effect on anti-inflammatory.3The aspect of prevention of CIA rats cardiovascular damage,compatibility of PNS+TG has significant synergy. The possible mechanism ofthe cardiovascular: reduce CIA rats serum PAL-1, MCP-1, VEGF levels;reduce lipid deposition in thoracic aorta and infiltration of inflammatory cellsin myocardium; reduced the expression of MCP-1, VEGF in the thoracic aortaand myocardium.