| 1 ObjectiveUnder the guidance of the Traditional Chinese Medicine theory according to "spleen deficiency to deliver Bi " and "spleen deficiency to deliver stasis ","blood stasis syndrom" as the bre AKThrough point,study and explore the effect and the mechanism of traditional Chinese medicine on platelet microparticles(PMPs)in rheumatoid arthritis(RA).By copying the animal models of RA,to observe the expression of the dynamic changes of paw swelling ? arthritis index(AI)? ultrastructure of synoviocyte ?ultrastructure of platelets?PMPs?platelet parameters?inflammatory factor?growth factor?changes of PI3 K / AKT,JAK / STAT Pathway Related Genes and Protein Expression in Platelet Cells,evaluates influence of Chinese medicine Xinfeng Capsule(XFC)as part of "Jianpihuashi Tongluo herbs",and explores the mechanism of XFC on inhibiting PMPs Release.2 Methods2.1 Theoretical studyCollecting and organizing the relevant literature of the RA platelet microparticles,screening and summarizing internal and external newest information on the abnormal expression mechanism of the RA platelet microparticles,from the theoretical point of view,we analyzed the mechanism of cytokines ? PI3K/AKT and JAK/STAT signaling pathway regulating the release of PMPs and the relationship between them.This paperbased on the basic theories of TCM,we discussed the relationship between spleen deficiency and blood stasis,explored the pathogenesis of arthromyodynia Based on the relationship between "spleen" and "Bi",explained the mechanism of “Jianpihuashi Tongluo therapy” in the treatment of RA blood stasis syndrom.2.2 Experimental Study60 SD rats were randomly divided into six groups: normal control(NC)group,model control(MC)group,LY294002 group,AG490 group,XFC group,and Tripterygium glycosides piece(TPT)group,each group contains 10 rats,n=10.In addition to the NC group,right rear foot plantar skin of the rest rats were injected with Freund's complete adjuvant(CFA)0.1ml to duplicate proinflammatory modeling,at the fifteenth day,in order to strengthen the immune system,the rats were injected at the tail by using CFA 0.05 ml,and the AA model was induced.At nineteenth day after modeling,drug delivery dosage and dosing method are as follows: NC and MC group: physiological saline 1ml/100g/d;LY 294002 group: physiological saline 1ml/100g/d,the rats were injected intraperitoneally with LY29400(5mg/kg)for twice a week.AG490 group:physiological saline 1ml/100g/d;rats intraperitoneally infused with AG490(20 mg/kg)for twice a week.XFC group: XFC is fully dissolved in physiological saline,0.034g/1ml/100g/d,TPT group: TPT is also fully dissolved in physiological saline,0.57mg/1ml/100g/d.Rats in each group were given continuous medication for 30 d.During the test,body mass,paw swelling,arthritis index(AI)of rats were observated.The changes of PMPs expression were detected by flow cytometry;the changes of platelets were examined by using an auto hemocytometer;ultrastructure of platelets were observed through electron microscope;enzyme-linked immuno-sorbent assay was used to detect the expression of serum cytokines IL-6,IL-10,TNF-?in blood serum;thechanges of IL-10,TNF-?,VEGF and ES in synovial membrane were detected with immunohistochemical method.The expression of PI3K/AKT,JAK/STAT pathway gene and protein in platelet cells were detected by QT-PCR?Western blot.3 Results3.1 The theoretical research results3.1.1 Activation of PI3K/AKT pathway promotes PMPs releasae,and mediate immune inflammation and pannus formation.After the activation of PI3 K,phosphorylated AKT,and activation of AKT control its downstream target protein expression by phosphorylation,improve the expression of PMPs release,promote the expression of growth factor?inflammatory factor,and adhesion molecule,mediate RA synovial immune inflammation and pannus formation.3.1.2 JAK/STAT signaling pathway mediates platelet lesionsProinflammatory cytokine IL-6 can activate the JAK2/STAT3 pathway,IL-6 was associated with the related receptors on the cell membrane surface,then STAT can be transferred to the nucleus and combining with the corresponding DNA domain,to regulate target gene expression.SATA3 can regulate the differentiation and development of megakaryocytes,regulate the function of platelet cells,and mediate platelet diseases.3.1.3 Mi R-21 and PTEN can connect to the PI3K/AKT,JAK/STAT pathway,regulate the PMPs release togetherExpression of mi R-21 can be raised after activation of JAK/STAT.PTEN mi R-21 is one of the downstream target genes,there is a significant negative correlation between mi R-21 and PTEN.High expression of mi R-21 can inhibit the expression of PTEN,PTEN is the major negative regulatory protein of PI3K/AKT signaling pathway,downregulation of PTEN can activate PI3K/AKT and promote the release of PMPs.3.1.4 Blood stasis syndrome runs through the course of RAAt the beginning of the arthromyodynia,it may be due to six exogenous pathogens leading to abnormal circulation of Qi and blood.With the development of the disease,the confrontation of healthy tendency and perverse trend can easily lead to stagnation of Qi and blood stasis symptom.With the further course of disease,chronic illness into the collaterals,and blood stasis syndrome is increased.Thus the blood stasis runs through of course.3.1.5 "Spleen Deficiency" is the pathological basis of blood stasis syndrome in RA.Weakness of spleen and stomach,dysfunction of the spleen in transport,failure of transportation and transformation,lack of Qi and blood biochemical deficiency,Qi and blood deficiency,yin body fluid boring can lead to blood stasis and be caused by rheumatism;asthenia of splenic qi,failure of transportation and transformation,which fails to distribute fluid and leads to attack of fluid retention,spleen deficiency cannot control blood circulation,and lead to disorders of blood circulation,eventually become blood stasis to bi.3.1.6 Elevated platelet of RA is an important features of blood stasis syndromeThe abnormal expression of PMPs promotes the immune inflammatory reaction,thrombosis,arteriosclerosis,angiogenesis and other physiological and pathological processes related to blood stasis,which is an important material basis for the occurrence of blood stasis syndrome.3.1.7“Jianpihuashitang Tongluo Therapy”can obviously improve blood stasis syndrome of RAAccording to the TCM pathogenesis of RA blood stasis syndrome“asthenia of spleen and stomach,lack of sources,general weakness,endogenous phlegm-dampness” and “inclusion of asthenia and sthenia”characteristics.According to characteristics of etiology and pathogenesis,we use "strengthen body resistance and eliminating evil " clinical principle of treatment,applicate "Jianpihuashitang Tongluo" treatment.The use of XFC in the treatment of patients with blood stasis syndrome of RA can significantly inhibit the activation of platelet,adjust the parameters of platelet,and improve the blood stasis in patients with RA.3.2 The experimental study results3.2.1 Effects of XFC on PMPs,platelet parameters and ultrastructure in AA ratsCompared with NC group,the MC rats PMPs,platelet parameters PLT,PCT increased(P <0.05 or P<0.01).Under electron microscope,in MC group,platelet ultrastructure changes of platelet shape slightly swollen,less structured,pseudopodium,alpha particles in the cytoplasm and mitochondria was decreased,platelet structure was destroyed,part of the platelet surface visible platelet microparticle formation.Compared with MC group,the MC group had significantly decreased in platelet parameters PLT and PCT(P<0.05 or P<0.01).Under electron microscope,in MC group,platelets present oval shape,slightly a little pseudopodium,no obvious damage of cytoplasmic structure,alpha granule and in mitochondrial increased significantly in MC group.Compared with TPT group,PMPs and PLT decreased in XFC group;compared with XFC group,PLT increased in LY294002 group,PMPs and PLT increased in AG490 group(P <0.05 or P<0.01).Platelet pseudopodia of TPT group increased more than XFC group,alpha particles and mitochondriain in the cytoplasm were less than XFC group.In LY294002 and AG490 group,the shape of platelet is not regular,it is short of pseudopodia,and has alpha granules and mitochondria.3.2.2 Effects of XFC on AA rats' paw swelling,AI,body mass change in each groupCorrelation analysis showed that the PMPs in peripheral blood of AA rats was positively correlated with paw swelling and AI;and was negatively correlated with body mass(P <0.01 or P<0.05);Compared with the NC group,the MC group had significantly increased in paw swelling,AI,and decreased in body mass(P <0.05 or P<0.01).Compared with the MC group,paw swelling and AI were significantly decreased in the XFC group,body mass was increased(P <0.05 or P<0.01).Compared with XFC group,paw swelling was significantly increased in the LY294002 group and AG490 group(P<0.05),there was no significant difference between TPT group and XFC group;compared with XFC group,AI was significantly increased in the LY294002 group and TPT group,no significant difference in the AG490 group;compared with XFC group,the body mass of rats decreased significantly in other groups after treatment(P<0.05).3.2.3 The morphological changes of AA rats and the impact of XFC on itNC group of toe synovial tissue under light microscope,no obvious inflammatory cell infiltration,no obvious synovial hyperplasia and pannus formation,joint surface is smooth and complete.Under electron microscope,the synovial cells in the NC group there were more mitochondria,shape normalization,endoplasmic reticulum,regular shape,uniform distribution of chromosome.MC rats toe joint synovial tissue under light microscope we will see a large number of inflammatory cell infiltration,typical pannus formation erosion of articular surface,articular cartilage was damaged.The number of mitochondria in the cytoplasm of MC group under electron icroscope,less endoplasmic reticulum fracture,chromosome aggregation.Under light microscope,toe joint synovitis cells of XFC group become lesser,articular cartilage integrity.Under the electron microscope,the the cytoplasm of synovial cells in XFC group were rich in mitochondria and endoplasmic reticulum,and the chromosomes were uniform,most of the cristae arranged complete.Under the light microscope,TPT group has a lot of inflammatory cell infiltration,a small amount of atypical pannus formation,largely intact articular surface;In LY294002 group and AG490 group,the synovial membrane of the rats showed inflammatory cells infiltrated,a small number of vascular proliferation,synovial tissue embedded in the joint cavity.Under electron microscope,shape of mitochondria in the cytoplasm of TPT group were slightly deformed,some Chromosome aggregation.In LY294002 group,the mitochondria were deformed,the number was slightly reduced,the endoplasmic reticulum was not rich,the cristae was shorter.In the AG490 group,the number of mitochondria in the cytoplasm was decreased,the endoplasmic reticulum was slightly reduced,the chromosomes were scattered in the cytoplasm,and the cristae were not regular arranged.3.2.4 The Changes of serum cytokines in AA rats and the impact of XFC on itCorrelation study showed that the expression of PMPs in peripheral blood of AA rats was positively correlated with serum cytokines IL-6,TNF-?,Th1/Th2,and negatively correlated with IL-10(P <0.05 or P <0.01);Compared with NC group,the expression of IL-6 and TNF-?in rats' serumof MC group were increased,IL-10 was decreased,Th1/Th2 was increased(P <0.05 or P <0.01).Compared with MC group,the expression of IL-6 and TNF-? in rats' serum of XFC group were decreased,the expression of IL-10 was increased,and the expression of Th1/Th2 was decreased significantly(P <0.05 or P<0.01).Compared with TPT group,the expression of IL-6 in rats' serum of XFC group were decreased,IL-10 was increased,Th1/Th2 was decreased.Compared with XFC group,the expression of IL-6 and TNF-? in rats' serum of LY294002 group TNF-? were increased,IL-10 was decreased,the expression of IL-6 in AG490 group was increased,IL-10 was decreased(P<0.05 or P < 0.01).3.2.5 Changes of inflammatory factor in synovitis of AA rats and the impact of XFC on itThe correlation analysis showed that expression of PMPs in peripheral blood of AA rats were positively correlated with TNF-? and Th1/Th2 in toe joint of synovial tissue,were negatively correlated with IL-10(P <0.05 or P<0.01);Compared with NC group,the expression of TNF-?in synovial tissue of MC group was increased,Th1/Th2 was increased and IL-10 was decreased(P <0.05 or P <0.01);Compared with MC group,the expression of TNF-? was decreased in synovial tissue of XFC group,the expression of Th1/Th2 was decreased and IL-10 was increased(P <0.05 or P <0.01);Compared with TPT group,the expression of IL-10 in serum of XFC group was increased and Th1/Th2 was decreased.Compared with XFC group,the expression of IL-10 in LY294002 group was decreased and Th1/Th2 was increased.The expression of IL-10 in AG490 group was decreased and Th1/Th2 was increased(P <0.05 or P <0.01).3.2.6.Changes of growth factor and MVD in synovial tissue of AA rats and the impact of XFCThe correlation analysis showed that PMPs was positively correlated with the integral optical density of VEGF and MVD,which was a negativelycorrelated with the integral optical density of ES(P <0.05 or P <0.01)?Compared with NC group,expression of VEGF and MVD were significantly increased in rats' toe joint of synovial tissue,ES was decreased(P <0.05 or P <0.01).Compared with MC group,the expression of VEGF and MVD in synovial tissue of XFC group was significantly decreased,and the expression of ES was increased(P <0.05 or P <0.01);Compared with TPT group,the expression of ES in XFC group was higher than that,compared with XFC group,the VEGF level of LY294002 group was increased,ES was decreased,and the expression of ES in AG490 group was decreased.(P <0.05 or P <0.01).3.2.7 Expression of JAK2/STAT3 pathway in platelet cells of AA cells and the impact of XFC on itCorrelation analysis showed that PMPs in peripheral blood of AA rats was positively correlated with JAK2,STAT3,mi R-21 m RNA,and PMPs was positively correlated with P-JAK2,STAT3 and P-STAT3 protein(P <0.05 or P <0.01);Compared with NC group,the expression of JAK2 m RNA,STAT3 m RNA,mi R-21 m RNA,JAK2,P-JAK2,STAT3,P-STAT3 protein in MC group were increased(P <0.05 or P <0.01)Compared with MC group,expression of JAK2 m RNA,STAT3 m RNA,mi R-21,JAK2,P-JAK2,STAT3,P-STAT3 in XFC group decreased(P <0.05 or P <0.01).Compared with TPT group,the expression of mi R-21 m RNA,JAK2,P-JAK2,P-STAT3 in XFC group were decreased(P <0.05 or P <0.01).Compared with XFC group,the expression of JAK2 m RNA,mi R-21 m RNA,JAK2,P-JAK2 were increased in LY294002 group,the expression of STAT3 m RNA and STAT3 were increased in AG490 group(P <0.05 or P<0.01).3.2.8 Expression of PI3K/AKT pathway in platelet cells of AA cells and the impact of XFC on itCorrelation analysis showed that PMPs was positively correlated with PI3 K and AKT in AA rats,and negatively correlated with PTEN m RNA,PMPs was negatively correlated with PTEN protein,and positively correlated with PI3 K and P-AKT protein;Compared with NC group,the expression of PTEN m RNA in platelet cells was decreased in MC group,the expression of PI3 K and AKT was increased,the expression of PTEN protein was decreased,the expression of PI3 K,AKT and P-AKT protein were increased.Compared with MC group,the expression of PTEN m RNA in XFC group was increased,PI3 K,AKT m RNA was decreased,PTEN protein was increased,PI3 K,AKT,P-AKT protein were decreased.Compared with XFC group,the expression of PTENm RNA,PTEN protein in LY294002 group was significantly increased,PI3 K protein was significantly increased,PI3 K protein,AKT,AKT protein expression in AG490 group were increased(P <0.05 or P <0.01).3.2.9 Expression of PI3K/AKT pathway in platelet cells of AA cells and the impact of XFC on itCorrelation analysis showed that there was a significant positive correlation between the expression of PMPs and BCL-2 m RNA,BCL-2protein in AA rats,and significantly negative correlation with BAD m RNA and BCL-2 protein(P <0.05 or P <0.01).Compared with NC group,the expression of BCL-2 m RNA and BCL-2protein was significantly increased in MC group,the expression of BAD m RNA and BAD protein in MC group was significantly increased(P <0.05 or P <0.01).Compared with MC group,the expression of BCL-2 m RNA and BCL-2protein in XFC group were decreased,the expression of BAD m RNA and BAD protein were increased in XFC group(P <0.05 or P<0.01).Compared with TPT group,the expression of BAD m RNA and BAD in platelet cells of XFC group were increased;compared with XFC group,the expression of BAD m RNA and BADprotein in platelet cells of AG490 group were increased;the expression of BAD m RNA and BAD protein in platelet cells of rats in AG490 group was decreased(P <0.05).4 Conclusions4.1 Changes of PMPs in AA rats4.1.1 Characteristics of PMPs change in AA ratsThere is abnormal expression of PMPs in AA rats,which showed increasing release of PMPs,and it was positively correlated with proinflammatory cytokines and vascular growth factor,but it was negatively correlated with inflammation inhibitor,inhibition of vascular endothelial growth factor.These results suggest that PMPs mediated inflammation and angiogenesis in AA rats.4.1.2 PI3K/AKT and JAK/STAT signaling pathways mediate the release of PMPsIn the activation of inflammatory cytokines IL-6,it can increase the downstream target gene,induce mi R-21 to increase,and then inhibit the expression of PTEN through JAK/STAT.PTEN negatively regulates the activation of PI3K/AKT,which leads to the increase of the downstream target protein BCL-2,the decrease of BAD,the lack of platelet apoptosis and the release of PMPs.4.1.3 High expression of RA platelet microparticles showed that the pathogenesis of traditional Chinese medicine is characterized by spleen deficiency and blood stasis.The pathogenesis in traditional Chinese medicine of RA patients with blood stasis syndrome showed that is characterized by "asthenia of spleen and stomach,lack of sources,general weakness,endogenous phlegm-dampness" and " inclusion of asthenia and sthenia".The increasing expression of RA platelet microparticles is an important manifestation of blood stasis syndrome.4.2 Efficacy of XFC on AA rats4.2.1 XFC can improve the quality of the AA ratsXFC prescription containing astragalus and coix seed,which can have the function of invigorating Qi and strengthening spleen,can improve the function of gastrointestinal tract,increase the feeding and drinking water volume of rats,and increase the body weight.4.2.2 XFC can reduce the dynamic changes of paw swelling and AI in AA rats,and inhibit the pathological changes of synovial membrane.XFC can inhibit the immune inflammatory reaction obviously,toe joint can inhibit the immune inflammatory reaction induced by Freund's complete adjuvant,reduce the inflammatory response of toe and whole body joint,so as to improve the effect of joint symptoms.By reducing inflammatory cell infiltration in the synovial tissue,XFC can inhibit synovial hyperplasia and pannus formation,reduce bone destruction,inhibiting the ultrastructure of synovial tissue damage,improve joint symptoms.4.3 Mechanism of inhibition of platelet microparticles release by XFC4.3.1 It can inhibit synovial lesions,reduce inflammation,inhibit the expression of PMPs.XFC can down regulate the TNF-alpha and VEGF of synovial tissue,up regulate expression of IL-10 and ES,it can regulate the balance of Th1/Th2 in synovial tissue,reduce microvessel density,inhibit angiogenesis,inhibit the inflammatory reaction of synovial tissue,reduce the dynamicchanges of paw swelling in AA rat,improve joint function.4.3.2 It can adjust the balance of cytokine in AA rats,inhibition the expression of PMPs.XFC can increase the expression of inflammatory factors,regulate the balance of Th1/Th2,reduce the expression of inflammatory factors,reduce the systemic inflammatory response,reduce the arthritis index,improve the platelet parameters,increase body mass and improve the overall symptoms.4.3.3 It can improve platelet parameters and ultrastructure,inhibit the expression of PMPs.XFC can reduce platelet count,improve ultrastructural damage,inhibit platelet production,release PMPs.4.3.4 It can inhibit IL-6/JAK2/STAT3 signaling pathway,inhibit the expression of PMPs.XFC can regulate the balance of Th1/Th2 in peripheral blood of AA rats,and it was drifted to Th2.XFC can up regulate IL-10,reduce the expression of IL-6,down regulate the expression of JAK2,STAT3 and P-JAK2,P-STAT3,and then down regulate the release of PMPs.4.3.5 It can regulate expression of mi R-21 and PTEN,inhibit the expression of PMPs.XFC can down regulate the expression of mi R-21 by inhibiting the JAK2/STAT3 signaling pathway,up regulate the expression of PTEN,increase the expression of PTEN protein,inhibit the activation of PI3K/AKT signaling pathway,and inhibit the expression of PMPs through the cascade amplification effect.4.3.6 It can regulate PI3K/AKT signaling pathway and inhibits the expression of PMPs.XFC can reduce the expression of PMPs by inhibiting the activation of PTEN/PI3K/AKT signaling pathway.4.3.7 It can up regulate the expression of BAD,down regulate expressionof BCL-2 and inhibit expression of PMPs.XFC can down regulate the expression of BAD,down regulate the expression of anti apoptotic index BCL-2,improve platelet parameters,inhibit platelet activation,and down regulate the expression of PMPs.XFC can up regulate the expression of promoting apoptosis index BAD,down regulate the expression of anti apoptosis index BCL-2,in order to improve platelet parameters,inhibit platelet activation,and down regulate the expression of PMPs. |
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