| ObjectiveTo investigate the effects of Qingre Huoxue Decoction on disease activity and JAK2/STAT3 pathway,and to explain the possible mechanism of Qingre Huoxue Decoction in the treatment of active RA.MethodsThese male SD rats were divided into 5 groups randomly(envelope method):group of control,model,Chinese traditional medicine(Qingre Huoxue Decoction),methotrexate(MTX),TCM and Western medicine(Qingre Huoxue Decoction+MTX).On day 1 and day 8 of the experiment,CIA modeling was performed on SD rats in 4 groups except the control group using emulsifier made of immunogenic bovine type Ⅱ collagen mixed with Freund incomplete adjuvant.On day 1,0.2mL/rat of modeling agent was subcutaneously injected into left tail root of rats for the first modeling,and 0.1mL/rat of modeling agent was subcutaneously injected into right tail root of rats for the second modeling 1 week later to enhance immunity,so as to build CIA rat model.The drug group was given intragastric administration from the first day of the experiment at the same time as modeling,1mL/100g rats,the cycle was 28 days,control group and model group were given 0.9%NaCL solution in equal amount.Observation of disease activity in rats:Behavioral status,body weight,toe volume and Arthritis Index(AI)of rats in 5 groups were observed and recorded throughout the experiment.HE staining was used to detect the pathology of synovial and ankle joint of rats in the 5 groups,and observe inflammation,cell morphology and other conditions.CRP,RF and APF in serum of rats in 5 groups were detected by ELISA.The mRNA expression were detected by QRT-PCR.The expression of proteins were detected by Western Blot.Results1 Effect of Qingre Huoxue Decoction on disease activity in CIA ratsWeight:During the 28-day experiment,the body weight of all 5 groups increased over time.The weight gain of the model group was slower than that of the control group.After comparing with model group,Qingre Huoxue Decoction can reduce the influence of modeling on the weight of CIA rats,and make them gain more weight,which is beneficial to alleviate the disease development of RA(P<0.05).Toe volume:With the advance of time after immunization,the swelling degree of joints of the four groups of CIA rats increased in different degrees.On day 14 after the second immunization,or day 21 of the experiment,CIA rats showed the most obvious joint swelling,reaching the maximum toe volume,and CIA rats showed dragging feet and difficulty in walking.It can be seen that after enhanced immunization,After comparing with control group,the swelling degree of model group was significantly increased(P<0.01),and the volume of toes increased rapidly.Compared with model group,joint swelling of CIA rats in TCM group,MTX group and TCM and Western medicine group increased slowly with gentle slope(P<0.05).The results showed that Qingre Huoxue Decoction could reduce joint swelling in CIA rats,and thus play a role in treating RA and delaying the progression of RA.AI score:On the 6th day after the second immunization,joints of the four groups of CIA rats began to show redness and swelling,and AI scores began to increase.Compared with the control group,AI scores of the model group increased rapidly.The AI score reached its peak on the 17th day of the experiment cycle.Compared with model group,Qingre Huoxue Decoction could reduce arthritis index and promote the recovery of RA in CIA rats(P<0.05).Pathological findings of synovial tissue:The synovial cells were orderly and normal in shape without hyperplasia in the control group.The synovial cells were disordered,the structure was abnormal,and the inflammatory cells were infiltrated more in the model group.After Comparing with model group,there were fewer abnormal synovial cells and infiltrating inflammatory cells in the three drug treatment groups.Pathological findings of the ankle joint:Pathological sections showed that the surface of ankle cartilage is smooth in the control group,with normal joint space.There was no proliferation of synovial tissue and no inflammatory cells.The cartilage surface of the ankle joint was rough,synovial tissue was hyperplasia,there were a lot of inflammatory cells and inflammatory exudate in the joint space in the model group.Compared with the model group,the TCM group,MTX group and TCM and Western medicine group had better articular cartilage surface smoothness,mild synovial tissue hyperplasia and less inflammatory cell infiltration.CRP,RF and APF expression levels:Model group compared with the control group,the contents of CRP,RF and APF in serum of CIA rats were increased significantly(P<0.01).After comparing with the model group,the contents of CRP arid RF in serum of CIA rats in TCM group,MTX group and TCM and Western medicine group were decreased(P<0.05).The content of APF in TCM and Western medicine group was also lower(P<0.05).2 Effect of Qingrehuoxue Decoction on JAK2/STAT3 pathway in CIA ratsThe expression levels of JAK2 and STAT3 mRNA in ankle joint:These two mRNA in CIA rats in model group were significantly increased compared with normal rats in control group(P<0.01).After Comparing with model rats,the expression of JAK2 mRNA in CIA rats in TCM group,MTX group and TCM and Western medicine group was decreased to varying degrees after drug gavage(P<0.05,P<0.05,P<0.01).Similarly,After Comparing with model rats,STAT3 mRNA expression in the three drug groups also decreased to varying degrees after gavage treatment(P<0.05,P<0.01,P<0.05).The mRNA expression levels of JAK2 and STAT3 in peripheral blood:Expression of 2 genes in model group were significantly higher than control group(P<0.01).Compared with the model group,the JAK2 mRNA expression of CIA rats in the TCM group,MTX group and TCM and Western medicine group was decreased to varying degrees after drug gavage,and the difference was statistically significant in the TCM and Western medicine group(P<0.05).After Comparing with model group,STAT3 mRNA expression in the three drug groups was declined to different degrees after drug gavage(P<0.05).The expression levels of JAK2 and STAT3 protein in ankle joint:The results showed that compared with the control group,the JAK2 protein in CIA rats in model group were significantly raised(P<0.01).Compared with model group,JAK2 protein expression in three drug groups was significantly decreased(P<0.05).After Comparing with control group,STAT3 protein in model group was significantly increased(P<0.05).After Comparing with model group,STAT3 protein expression in the three drug groups decreased to varying degrees,and the difference was statistically significant in the TCM and Western medicine group(P<0.05).The expression levels of P-JAK2 and P-STAT3 protein in ankle joint:After Comparing with control group,the expression of P-JAK2 and P-STAT3 protein in the model group were significantly raised(P<0.01).After Comparing with model group,the P-JAK2 protein in the TCM group,MTX group and TCM and Western medicine group was significantly declined(P<0.05,P<0.01,P<0.01).Compared with model group,the P-STAT3 protein in three drug groups was also declined(P<0.05).Conclusions1 The treatment of Qingre Huoxue Decoction can promote weight gain in CIA rats,reduce joint redness and swelling,reduce AI score,maintain the normal morphology of synovial cells,reduce vascular proliferation and inflammatory cell infiltration,maintain the structural integrity of ankle joint,reduce synovial proliferation,delay the destruction of ankle cartilage,and reduce the contents of CRP,RF and APF in serum,so as to inhibit the disease activity of CIA rats.2 The treatment of Qingre Huoxue Decoction can reduce the content of JAK2 and STAT3 mRNA in local ankle joint and systemic peripheral blood of CIA rats at the gene level,reduce the level of JAK2 and STAT3 protein in ankle joint of CIA rats at the protein level,and reduce the level of activated p-jak2 and p-STAT3 protein at the protein level.3 The mechanism of Qingre Huoxue Decoction inhibiting the disease activity of CIA rats may be by regulating JAK2/STAT3 pathway. |
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