| ObjectiveTo observe and compare the infiltration of B cells and plasma cells in the synovial tissue of rats with collagen-induced arthritis(CIA)by Qingre Huoxue Recipe,tripterygium glycosides and methotrexate(MTX).And observe the effects of interleukin(IL)-21,B lymphocyte chemotactic factor(CXCL13),and anti-cyclic citrullinated peptide antibody(CCP)expression function.To explore the mechanism of the effects of three drugs on rheumatoid arthritis on the related functions of synovial tissue CD19+B cells.It also to find the new experimental evidence of the efficacy of traditional Chinese medicine Qingre Huoxue Recipe in the treatment of RA.MethodsSixty healthy SD rats(male and female rats in equivalent number and respectively feeding)were divided into 5 groups according to the components of the random weight group.12 were left untreated for the normal control group,and the rest were injected with bovine type Ⅱ collagen and complete Freund’s adjuvant to forming a CIA arthritis model,and the first and second exemptions were separated by one week.After modeling,12 rats of the 1 group were kept as the model control group,and the other 3 groups were fixed to the Tripterygium glycosides,Qingre Huoxue Recipe,and methotrexate(MTX)by intragastric administration on the 10th day after the first immunization.Tripterygium wilfordii glycosides were prepared into a suspension of 0.625mg/ml concentration.Qingrehuoxue decoction was concentrated to a concentration of 2g/ml,and methotrexate was at a concentration of 1mg/mL,strictly at a dose of 0.1ml/kg,feeding into tomach.Qingre Huoxue Recipe and Tripterygium wilfordii glycosides once a day,MTX twice a week.During the medication intervention,the body weight and hind limb swelling were recorded twice a week.After 32 days of treatment,the rats in each group were anesthetized with chloral hydrate.The blood was collected from the abdominal aorta and centrifuged.The serum IL-21,CXCL13 and CCP were quantified by ELISA.The synovial tissue of the lower extremity knee joint was obtained.Each group of 3(2 male,1 female)rats’ synovial tissue was fixed in neutral paraformaldehyde solution,and the B cells in the synovial membrane were detected by immunohistochemistry.The specific membrane protein molecule CD 19,plasma cell membrane specific protein molecule CD138 was expressed,and the average optical density(MOD)value of the specific labeling molecule was statistically analyzed using ImageProPlus6.0 software.The synovial tissue of the remaining rats was detected by ELISA.Real-time PCR was used to detect the expression of IL-21,CXCL13 and activation-induced cytosine deaminase(AID)mRNA in synovial tissue of each group.and detectiing the AID activation in synovial tissue through Western Blot.The paws below the knee joint of the lower limbs were taken,and using pathological HE staining of the lower extremity ankle joint.observing and comparing the differences of synovial hyperplasia and bone destruction between the ankle joints of each groupResultsEach treatment group had a slowing effect on the degree of swelling of the rat foot.The Qingre Huoxue recipe group had the lowest degree of swelling,better than Tripterygium wilfordii and the Methotrexate group.In the normal control group,the-joint structure was intact,the articular surface and joint space were smooth and intact,and no synovial tissue hyperplasia was observed.Synovial tissue hyperplasia was observed obviously,and a large number of B lymphocytes and plasma cells infiltrated of the model rats group,This group also showed signs of fusion of synovial with the edge of the bone,and the bone showed irregular erosion and ablation,and the bilateral joint space was obviously narrow or disappeared.But only little B lymphocyte marker molecules around the blood vessels were observed,and there was almost no plasma cells infiltration in the synovial tissue of the normal group.Compared with the model group,the synovial hyperplasia of the rats in the Qingre Huoxue Recipe group was improved,and the curative effect was better than that of tripterygium glycosides and methotrexate.Qingre Huoxue Recipe reduced the degree of infiltration of B cells and plasma cells in synovial membrane,while methotrexate and tripterygium glycosides only showed independent inhibition on B cells and plasma cells.Compared with the normal control group,the serum levels of IL-21,CXCL13 and CCP were significantly increased in the CIA model group(P<0.001).The serum levels of IL-21,CXCL13 and CCP were significantly reduced in the rats treated with Qingre Huoxue Recipe than the model group(P<0.001).Both Tripterygium wilfordii and MTX reduced serum IL-21 concentration,but had no significant effect on serum CXCL13(P>0.05).However,tripterygium glycosides can reduce the serum CCP antibody content,and there is statistical difference compared with the model group,while methotrexate can not(P=0.008,<0.01).The overall level of serum synovial plasma IL-21 and CXCL13 was significantly higher than that of serum,but CCP did not.The concentrations of IL-21 and CCP in the synovial tissue of the model group were significantly higher than those in the normal group(P<0.001).The increase of CXCL13 concentration was statistically different from the normal group(P<0.05).Tripterygium glycosides,Qingre Huoxue Recipe and MTX can significantly reduce the expression of IL-21 in synovial tissue(P<0.001).However,the concentration of CXCL 13 in the synovial tissue of Tripterygium wilfordii and MTX did not show a decrease,and the mean value of the sample increased in inverse proportion to the model group.Although Qingre Huoxue Recipe can reduce the concentration of CXCL 13 in synovial tissue,it showed no statistical difference compared with the model group.(P>0.05),may be related to too small sample size(n=6).All three drugs were able to reduce the level of CCP antibody in synovial tissue.The effect of Qingre Huoxue Recipe was the most significant(P<0.01),and the effect of tripterygium glycosides was similar to that of methotrexate(P<0.05).The mRNA expression levels of IL-21 and CXCL 13 of the model group were higher than those of the normal controled group(P<0.01).Tripterygium glycosides,Qingrehuoxue and MTX showed similar expression of IL-21 mRNA in synovial tissue.Inhibition(P<0.05).Only Qingre Huoxue Recipe showed inhibition of CXCL 13 mRNA’s experssion(P<0.05).This study found that activation-induced cytidine deaminase(AID)increased after CIA modeling,and was statistically different from the normal group(P<0.05).All three drugs showed a decrease in AID enzyme,but there was no statistical difference(P>0.05).The expression of AID enzyme in synovial tissue was detected by Western Blot.The three drugs had different degrees of AID enzyme in the synovial membrane.Compared with MTX and Tripterygium wilfordii glycosides,Qingre Huoxue Recipe has a stronger inhibitory effect on AID enzyme,and similar results with PCR experiments.Conclusion1.Qingre Huoxue Recipe,Tripterygium wilfordii glycosides and MTX can alleviate the joint swelling degree of CIA rats.Qingre Huoxue Recipe and Tripterygium wilfordii glycosides can alleviate bone destruction in CIA rats.2.Qingre Huoxue Recipe can significantly inhibit the infiltration of B cells and plasma cells in synovial tissue of CIA rats.The inhibitory effect of tripterygium glycosides on plasma cell infiltration was more obvious than that of MTX.MTX showed inhibition of synovial tissue B cells,but had little effect on plasma cell invasion.3.Qingre Huoxue Recipe can significantly inhibit IL-21,CXCL 13 and CCP antibodies in serum synovial tissue of CIA model rats.Tripterygium glycosides and MTX showed a certain inhibitory effect on serum and synovial tissue IL-21,but did not show significant effects on CXCL13.4.Qingre Huoxue Decoction may inhibit the infiltration of B cells and plasma cells of synovial tissue of CIA rats by inhibiting the expression of IL-21 and CXCL13,thereby inhibiting synovial hyperplasia and joint bone destruction. |
PDF Full Text Request