Studies On The Effects And Mechanism Of IL-13 In Adjuvant-induced Arthritis Rats

Objective:Rheumatoid arthritis (RA) is a kind of chronic and systemic immune inflammatory disease with complex etiology and non-specific diagnostic indicators. It involvs multiple joints and extra-articular organs and seriously affects the patient’s physical and mental health. At present, there have not been specific effective and no side effective drugs to cure AA. Therefore, to explore the effective and no side effects or smaller side effects of natural active ingredients has become a hot research spot in RA drugs. Interleukin-13 (IL-13) is a natural anti-inflammatory cytokines, with a wide range of biological effects. In view of the coexistence of immunostimulatory and immunosuppressive biological activity of IL-13, it has become an important target of a variety of allergic and autoimmune diseases in clinical research and treatment; while IL-13 also has a strong anti-inflammatory effect, it can inhibit the progress of a series of TNF-a induced diseases. Therefore, IL-13 is expected to become a kind of natural active ingredients for the treatment of RA. In this study, rheumatoid arthritis A adjuvant-induced arthritis (AA) animal model in rats was prepared, and we investigate the effectsand mechanism of IL-13 on AA rats.Methods:1. Adopting complete Freund adjuvant to establish arthritis of rats model, then rat weighted by using analytical balance measurement, using digit volume measuring apparatus was developed for the determination of rats toes volume, using the scoring method (0-4 points) to evaluate index of experimental arthritis of rats. Eventually, AA rats are evaluated through weight gain rate, toes swelling degree, arthritis index and spleen index;2. Through weight gain rate, toes swelling degree, arthritis index and spleen index to test the effect of IL-13 on AA rats, and adopting ELISA method to measure the concentration of IL-1β、TNF-α and IL-6 in AA rats;3. Establish in vitro inflammatory cells model by employing LPS to induce RAW264.7 cell, and to study the mechanism of IL-13 on AA arthritis.Application of MTT method to determined the group cell proliferation curve of LPS model cells with IL-13, ELISA method to measure the concentration of IL-1β、TNF-α and IL-6 in cell culture supernatant, Western blotting method to detecte the protein phosphorylation related with MAPK and NF-κB signaling pathway.Results:1. Preparation and evaluation of the adjuvant-induced arthritis animal model in rats:Compared with control group rats, the percent of weight gain of model group rats dropped sharply starting from the 14th day, and with the extension of time, the difference increased gradually. That was 54.98 percent of model groug rats at 30th day, while the control group rats have been to 94.87 percent; At 14th day after immunization, the model group rats appeared obvious arthritis symptoms, includingnon-injected limb ankle and voix pedis became obviously red and edematous and its swelling ration was up to 65.73%(P< 0.01).At about 22th day, the swelling ration reached the peak, by this time, the model group rats were difficult to get about, This experiment using CFA AA rat model was established successfully, can be used for further study, and bigger gap about bone; Finally, it also proved evident spleen hyperplasia of the model group rats. In conclusion, this experiment established successfully AA rat model using CFA method, and it could be used for further study.2.The effects of IL-13 in adjuvant-induced arthritis rats:Compared with the AA model group, low, medium and high doses of IL-13 could increase the rate of weight gain in rats; middle dosage (1.5μg/kg bw)、high dosage (3.0μg/kg bw) of IL-13 could significantly reduce the arthritis index (P<0.05):at 30th d of inflammatory, the arthritis index of AA model rats was 3.25, while the arthritis index of medium and high doses of IL-13 rats was reduced to 1.31 and 1.21; Different concentrations of IL-13 could reduce the toes swelling rate with the extension of the time:at 30th d of inflammatory, the toes swelling rate of AA model rats was 65.85%, while the toes swelling rate of medium and high doses of IL-13 rats was 56.75% and 54.26%; Each dose of IL-13 groups could reduce the spleen index in different degree, medium and high dose groups could decrease it significantly compared with model group (P<0.05, P< 0.01); The result of ELISA displayed, middle and high dosage of IL-13 couldsignificantly lower the concentration of IL-1β (74.35 pg/mL、63.75 pg/mL)、 TNF-α (223.76 pg/mL 190.45 pg/mL) and IL-6 (106.85 pg/mL,92.86 pg/mL) of AA ratS。Therefore, this study suggests that IL-13 can reduce some arthritis symptoms and recover part of the immune function of AA rats, which may be caused by reducing the concentration of serum inflammatory factor IL-1β、TNF-α and IL-6.3. The mechanism of IL-13 in adjuvant-induced arthritis rats:LPS could increase the concentration of IL-1β, TNF-α and IL-6 and avtivate MAPK and NF-κB signaling pathway in RAW264.7 cells. Under the presence of different concentrations of IL-13, it found that final concentration of 2μM,10 μM and 50μM of IL-13 could reduce the concentration of IL-1β, TNF-α and IL-6 in the experimental cells supernatant; inal concentration of 10μM and 50 μM of IL-13 could obviously inhibit the degree of phosphorylation of P38 and ERK; under final concentration of 2μM,10 μM and 50 μM of IL-13,the degree of phosphorylation of IκB、P65 could obviously lower. In conclusion, IL-13 can obviously inhibit the activation of MAPK and NF-κB signaling pathway, that may be how IL-13 inhibit AA pathological process.Conclusion:This experiment established AA rats model which has the typical symptoms of rheumatoid arthritis with the CFA, and proved IL-13 could improve the AA rats model, reduce the concentration of serum inflammatory factors, control arthritis disease progress; Cells in vitro experiments showed that IL-13 could obviously reduce the content of inflammatory cytokines, inhibit the activation of MAPK and NF-κB signaling pathway, that suggest IL-13 has strong anti-inflammatory effect, which may be the mechanism of IL-13 on the effect of inhibition of AA pathological process.Significance:1. Study the effect of IL-13 on AA rats;2. Study the mechanism of anti-arthritis about IL-13;3. Provide new view for the study and treatment of rheumatoid arthritis.