Research On The Immune Regulatory Mechanism Of Apitherapy In Rats With Type Ⅱ Collagen-Induced Arthritis

Part 1.Effects of Apitherapy on Th1/Th2 Balance in Rats with Type Ⅱ Collageninduced ArthritisObjectiveTo observe the effects of apitherapy on Th1/Th2 balance and expression of related specific transcription factors and cytokines in rats with type II collagen-induced arthritis(CIA).Investigate the mechanism of apitherapy in rheumatoid arthritis treatment.MethodsFifty female SD rats were divided into five groups:normal,model,apitherapy,methotrexate and etanercept groups.Except for the normal group,rats were induced by immunizing with type II collagen to establish CIA model.Four groups of CIA rats were treated by saline,apitherapy,methotrexate(2.5mg/kg/w)or etanercept(6mg/kg/w)subcutaneous injection at "Zusanli" acupoint per week for 4 weeks respectively.Recorded the changes of arthritis index and observed the histological changes of knee joint by staining with H&E.The expression of T-bet and GATA-3 mRNA of spleen were detected by Real-time PCR.The concentration of serum cytokine IFN-y,IL-10 and IL4 were detected by ELISA.ResultsOn the 28th day after booster immunization,the toe joints,feet and ankles of rats appeared swelling,and the CIA model was successfully established.After 4 weeks treatment,compared with model group,apitherapy alleviated the arthritis index and the damage of synovium and cartilage of knee joints.The results of Real-time PCR showed that T-bet mRNA expression and T-bet/GATA-3 ratio were declined in apitherapy group(P<0.05).The ELISA results showed that the concentration of IFN-y was decreased and the concentration of IL-10 and IL-4 were increased in apitherapy group(P<0.05).ConclusionApitherapy alleviates the arthritis index and ameliorates the knee joints damage of CIA model.The mechanism possibly related to restoration of the balance between Th1/Th2 by regulating the expression of T-bet mRNA and T-bet/GATA-3 ratio and the concentration of IFN-y,IL-10 and IL-4.Part 2.Effects of Apitherapy on Th17/Treg Balance in Rats with Type Ⅱ Collageninduced ArthritisObjectiveTo observe the effects of apitherapy on Th17/Treg balance and expression of related specific transcription factors and cytokines in rats with type Ⅱ collagen-induced arthritis.Investigate the mechanism of apitherapy in rheumatoid arthritis treatment.MethodsFifty female SD rats were divided into five groups:normal,model,apitherapy,methotrexate and combination of apitherapy and methotrexate groups.Except for the normal group,rats were induced by immunizing with type Ⅱ collagen to establish CIA model.Four groups of CIA rats were treated by saline(i.p.),apitherapy(once every three days,at "Zusanli" acupoint),methotrexate(0.75mg/kg/w,i.p.)or combination of apitherapy and methotrexate for 4 weeks respectively.Recorded the changes of arthritis index for evaluating the severity of arthritis.Observed the histological changes of ankle and knee joint by staining with H&E and scored the histological severity.The proportion of Th17 and Treg cells in peripheral blood were examined by flow cytometry.The expression of RORyt and Foxp3 mRNA of spleen were detected by Real-time PCR.The concentration of serum cytokine IL-17 and TGF-β were detected by ELISA.ResultsOn the 21st day after booster immunization,the CIA model was successfully established.Compared with model group,apitherapy declined the arthritis index and pathological score(P<0.05),as well as ameliorated the synovial hyperplasia,inflammations,pannus formation and cartilage destruction of ankle and knee joints.The results of flow cytometry showed that apitherapy treatment reduced the proportion of Th17 cells and increased the proportion of Treg cells(P<0.05).The results of Realtime PCR showed that RORyt mRNA expression was declined and Foxp3 mRNA expression was increased in rats of apitherapy group(P<0.05).The ELISA results showed that the concentration of IL-17 was down-regulated and the concentration of TGF-β was up-regulated in apitherapy group(P<0.05).ConclusionApitherapy alleviates the arthritis index and pathological score,and ameliorates the ankle and knee joints damage of CIA model.The mechanism possibly related to restoration of the balance between Th17/Treg by regulating the proportion of Th17,Treg cells and the expression of RORyt,Foxp3 mRNA and the concentration of IL-17 and TGF-β.