| Rheumatoid arthritis is an autoimmune diseases,with the lesions of joints and synovium being the first and foremost damage.Patients' condition waving,lingering and difficulty to cure usually leads Joint deformities and disability to patients,and that affects patients' health and life seriously.Only Anti-inflammatory analgesic is not the basic target of curing RA,while the prevention and remission against patients' bone destruction is being more and more important.Recent study shows that traditional Chinese medicine is valid,safe and reliable in curing RA.The root of Thladiantha Dubia Bunge is the dry and mature roots of Thladiantha Dubia Bunge,a kind of Cucurbitaceae vines herbaceous plants for years.The root of Thladiantha Dubia is a kind of herbs used in the Manchu,Mongolian folk for long,used to cure Lumbocrural pain and other diseases,with outstanding treatment effect and without poison,side effect.The research team's previous studies have shown that Total saponins of Thladiantha Dubia Root is the effective parts of anti-inflammatory and analgesic effect,the effect is certain.While have studied the positive feedback factor IL-1?,IL-6 and TNF-? about excitation of Osteoclasts and OPG/RANKL/NF-?B by Adjuvant arthritis rat model.This study use CIA model rats,with Tripterygium Glycoside being Positive control drug,study the effect of cytokines IL-7,IL-15 and IL-34 on OC with different experimental methods in the level of gene and protein,and study the Protective effect of TSTR on CIA,trying to seek more cure points for RA,and providing experimental evidence for further study of Thladiantha Dubia Root.Objective:To observe the therapeutic effect of TSTR and the effects of OC positive feedback factors IL-7,IL-15 and IL-34 in blood and knee joint synovium onCIA in rats,and to explore the protective mechanism of TSTR on bone destruction of CIA in rats.Methods:1 Preparation of rat CIA modelEighty male Wistar rats were selected,and ten of them were randomly taken as normal control group.The remaining rats were multi point injected the mixed emulsion of bovine type II collagen and complete Freund's adjuvant in the back and tail root.After 7 days,it was carried out with the same operation.14 days after the first injection,those with arthritis index(AI value)more than 4 cent of the rats as successfully modeling.2 Grouping and administration of test animalsThe successful model of the rats were randomly divided into model group,TSTR 20,40,80 mg·kg-1 and TG(12 mg·kg-1)positive drug group.Rats in each group were intragastriced with corresponding drugs for 35 days,NC group and MC group were given equal volume of distilled water,1time/day.3 Index detection3.1 calculate AI value changes,observe the severity of arthritis.3.2 The serum levels of IL-7,IL-15 and IL-34 were evaluated by ELISA.3.3 The morphologic changes of synovium were observed with HE staining3.4 The expression of IL-7 m RNA,IL-15 m RNA and IL-34 m RNA in rat synovium were detected by RT-PCR.3.5 Western-Blot was used to detect the expression of IL-7,IL-15 and IL-34 in synovium.Results:1 Preparation of rat CIA modelA total of 70 rats were established.The model was composed of only 55 rats.The rate of rat model was about 78.6%.2.1 Effect of TSTR on AI in CIA ratsBefore administration,the AI value of MC group and drug group was significantly higher than that of NC group(P <0.01).There was no significant difference between drug groups and the MC group,the model was established successfully.After administration,Compared with the MC group,the AI values of TSTR group and TG group decreased significantly(P <0.01).2.2 Effect of TSTR on IL-7,IL-15 and IL-34 expression in serum of CIA rats2.2.1 Effect of TSTR on IL-7 expression in serum of CIA ratsThe expression of IL-7 in serum of MC group was significantly higher than that of NC group(P <0.01);The expression of IL-7 in TSTR 20,40mg·kg-1 dose group and TG group rats was significantly lower than that in MC group(P <0.05 or P <0.01);The expression of IL-7 in TSTR 20 mg·kg-1 dose group was lower than that in TSTR 40,80 mg·kg-1 dose group(P <0.01).2.2.2 Effect of TSTR on IL-15 expression in serum of CIA ratsThe expression of IL-15 in MC group was significantly higher than that in NC group(P <0.01);The expression of IL-15 in TSTR 20,40 mg·kg-1 dose group and TG rats was significantly lower than that in MC(P <0.05 or P<0.01);The expression of IL-15 in TSTR 20 mg·kg-1 dose group was lower than that in TSTR 40,80 mg·kg-1 dose group(P <0.01).2.2.3 Effect of TSTR on IL-34 expression in serum of CIA ratsThe expression of IL-34 in serum of MC group was significantly higher than that of NC group(P <0.01);The expression of IL-34 in TSTR dose group and TG group was significantly lower than that in MC group(P <0.01);The expression of IL-34 in TSTR 20 mg·kg-1 dose group was lower than that of TSTR 40,80 mg·kg-1 dose group(P <0.01).2.3 Effects of TSTR on pathological changes of synovial membrane in CIA ratsObserved under the optical microscope,the arrangement of synovial tissue in the NC group was more loose than the model group.There was no congestion,edema and other inflammatory phenomena,synovial cell2 Index detectionproliferation and pannus formation were also invisible.Compared with the NC group,the arrangement of synovial tissue cells was dense and messy in the MC group,the number of synovial tissue cells was more,congestion and a large number of inflammatory cells,lymphocytes can be observed.Compared with the MC group,the TSTR 20 mg·kg-1 dose group and TG group had a significant decrease in synovial tissue congestion and inflammatory cells,and the pathological changes of synovial tissue were not obvious in the TSTR 40,80 mg·kg-1 dose group.2.4 Effect of TSTR on expression of IL-7 m RNA,IL-15 m RNA and IL-34 m RNA in synovial membrane of CIA rats2.4.1 The effect of TSTR on IL-7 m RNA expression in synovial membrane of CIA ratsThe expression of ?-actin m RNA was homogeneous in each group,the expression of IL-7 m RNA in MC group was significantly higher than that in NC group(P <0.01);IL-7 m RNA in TSTR 20,40 mg·kg-1 dose group and TG group was significantly lower than that in MC group(P<0.01);The expression of IL-7 m RNA in TSTR 20 mg·kg-1 dose group was lower than that in TSTR40,80 mg·kg-1 and TG group(P <0.05 or P <0.01).2.4.2 The effect of TSTR on the expression of IL-15 m RNA in synovial membrane of CIA ratsThe expression of ?-actin m RNA was homogeneous in each group,The expression of IL-15 m RNA in MC group was significantly higher than that in NC group(P <0.01);The expression of IL-15 m RNA in TSTR dose group and TG group was significantly lower than that in MC group(P <0.01);The expression of IL-15 m RNA in TSTR 20 mg·kg-1 dose group was lower than that in TSTR 40,80 mg·kg-1 dose group,but higher than that in TG group(P<0.05 or P <0.01).2.4.3 The effect of TSTR on IL-34 m RNA expression in synovial membrane of CIA ratsThe expression of ?-actin m RNA was homogeneous in each group,The expression of IL-34 m RNA in MC group was significantly higher than that inNC group(P <0.01);The expression of IL-34 m RNA in TSTR dose group and TG group was significantly lower than that in MC group(P <0.01);The expression of IL-34 m RNA in TSTR 20 mg·kg-1 dose group was significantly lower than that in TSTR 40,80 dose mg·kg-1(P <0.01).2.5 Effect of TSTR on the expression of IL-7,IL-15 and IL-34 protein in synovial membrane of CIA rats2.5.1 The expression of IL-7 protein in the synovium of CIA ratsThe expression of ?-actin protein was homogeneous in each group,The expression of IL-7 protein in MC group was significantly higher than that in NC group(P <0.01);The expression of IL-7 protein in TSTR 20,40 mg·kg-1dose group and TG rats was significantly lower than that in MC group(P<0.01);The expression of IL-7 protein in TSTR 20 mg·kg-1 dose group was lower than that in TSTR 40,80 mg·kg-1 and TG group(P <0.01).2.5.2 The expression of IL-15 protein in the synovium of CIA ratsThe expression of ?-actin protein was homogeneous in each group,The expression of IL-15 protein in MC group was significantly higher than that in NC group(P <0.01);The expression of IL-15 protein in TSTR dose group and TG group was significantly lower than that in MC group(P <0.01);The expression of IL-15 protein in TSTR 20 mg·kg-1 dose group was lower than that in TSTR 40,80 mg·kg-1 dose group(P <0.01).2.5.3 The expression of IL-34 protein in the synovium of CIA ratsThe expression of ?-actin protein was homogeneous in each group,The expression of IL-34 protein in MC group was significantly higher than that in NC group(P <0.01);The expression of IL-34 protein in TSTR dose group and TG group was significantly lower than that in MC group(P <0.05 or P <0.01).The expression of IL-34 protein in TSTR 20 mg·kg-1 dose group was lower than that in TSTR 40,80 mg·kg-1 dose group but higher than TG group(P<0.01).ConclusionThe therapeutic effect of TSTR on articular bone destruction in CIA rats may be related to the inhibition of the expression of IL-7,IL-15 and IL-34 inblood and synovial tissue. |
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