Therapeutic Effect And Mechanism Of AH On Collagen-Induced Arthritis Mice

ObjectiveIn this study,mouse models of collagen-induced arthritis were treated in combination with Hydroxychloroquine Sulfate(HS)and Artemisinin(ART)to treat rheumatoid arthritis(RA),and the artemisinin-hydroxychloroquine(AH)was observed for the treatment of collagen-induced arthritis(CIA)mice and to explore whether the combination of drugs could reduce the dose of hydroxychloroquine and the occurrence of its adverse reactions,and try to find its possible mechanism.It may provide experimental evidence for clinical applications.Methods1.DBA/1 mice were randomly divided into groups of Normal,Model,Methotrexate,AH low dose,AH medium dose,AH high dose,ART and HS.Establishment of mouse CIA:Emulsified the bovine type Ⅱ collagen solution and complete Freund’ s adjuvant thoroughly then injected 0.1 mL to the base of the tail on day 1.A booster injection of 0.1 mL bovine type Ⅱ collagen solution and incomplete Freund’ s adjuvant thoroughly emulsified was applied on day 21.2.Administration began on the day 42 after immunization,with a dose of 20 mL·kg-1 daily until the day 84,and the methotrexate group was administered once a week.The normal group and model group were treated with 0.5%sodium carboxymethyl cellulose(CMC-Na)solution.3.Mice were observed daily and weighed.every 3 days.The thickness of hind limbs and metatarsal was measured by a Vernier caliper on day 0 and after the onset of inflammation every 3 days.The arthritis index of four limbs was scored by a four-level scoring method.4.After the experiment,the blood,thymus,spleen and the right posterior hind foot ankle of the mice were collected.Serum was isolated by centrifugation.Thymus index and spleen index were calculated.Histopathological changes of ankle joint were observed by HE staining.The IL-6,IL-17 A/F and TGF-β1 cytokines in the serum were detected by ELISA.The RORγt and Foxp3 mRNA in the spleen was detected by qPCR.Results1.Therapeutic effect of AH on CIA miceCompared with the normal group,the model group was generally in poor condition,weight loss(P=0.010),foot swelling(P=0.000),arthritis index(P=0.001),spleen index(P=0.000)and ankle joint pathological score(P=0.000)were significantly increased.There was no difference in thymus index(P>0.05).Compared with the model group,there was no difference in weight,spleen index and thymus index between the AH groups(P>0.05).There was no significant difference in the degree of foot swelling,arthritis index and ankle joint pathological scores between the AH groups(P>0.05),but from the mean results,it can be seen that AH medium and high dose group has a tendency to reduce the swelling degree of the foot,arthritis index and ankle joint pathological scores;2.Effect of AH on serum cytokines in CIA miceCompared with the normal group,IL-6 in the serum of the model group was significantly increased(P=0.024),IL-17A/F and TGF-β1 was elevated but both not statistically significant(P>0.05).Compared with the model group,the concentrations of IL-6 and IL-17A/F decreased and the concentration of TGF-β1 increased in each group.There was no significant difference in the decrease of IL-6,IL-17A/F and the increase of TGF-β1 in each dose group of AH.From the mean results,the AH groups had a decreasing effect on IL-6,and the AH medium and high dose groups had a tendency to decrease IL-17A/F cytokines,and the AH groups had a rise tendency in TGF-β1.Compared with the normal group,the model group was generally in poor condition,weight loss(P<0.05 or P<0.01),foot swelling was significantly increased(P<0.01 or P<0.001),and the arthritis index was significantly increased.(P<0.01 or P<0.001),spleen index was significantly increased(P<0.001)and ankle joint pathological score was significantly increased(P<0.001).There was no significant difference in thymus index(P>0.05).IL-6,IL-17A/F increased significantly(P<0.01,P<0.05),and TGF-β1 increased slightly but not statistically(P>0.05).The expression of Foxp3 and ROR γt mRNA in the spleen of the model group were increased but no significance(P>0.05).3.Effect of AH on the expression of Foxp3 and ROR γ t mRNA in spleen of CIA miceCompared with the normal group,the expression of Foxp3 and ROR y t mRNA in the spleen of the model group were increased,but there was no significance(P>0.05).Compared with the model group,the expression of Foxp3 mRNA was significantly different in the AH medium dose group(P=0.000)and the high AH dose group(P=0.006).There was no statistically significance(P>0.05)of the ROR γ t mRNA expression in the AH groups,but the expression of ROR γ t mRNA showed a decreasing trend from the mean results.Conclusion1.Therapeutic effect of AH on CIA miceIn CIA model mice,the paws were obviously swollen after the onset of arthritis,and the CIA models were successfully induced.The AH administration group had no significant effect on the change of.weight of CIA mice.AH had a certain decrease trend on the foot swelling degree and arthritis index of CIA mice,which may indicate that AH may have anti-inflammatory effect on alleviating CIA.The three doses of AH may had a certain tendency to reduce the spleen index and thymus index,and there may be a dose-dependent manner,suggesting that the three doses of AH may inhibit the immune function of spleen and thymus.CIA mice had obvious lesions in the ankle joint.The AH groups had a tendency to reduce the pathological score,suggesting that the AH groups may alleviate the joint lesions of mouse CIA to some degrees.2.Effect of AH on serum cytokines in CIA miceAH may be effective in inhibiting the production of inflammatory cytokines and promoting the production of anti-inflammatory factors,and has certain anti-inflammatory effects,which may be one of the therapeutic mechanisms of AH to alleviate inflammation of RA.3.Effect of AH on the expression of Foxp3 and ROR γ t mRNA in spleen of CIA miceThe medium and high dose groups of AH signif icantly promoted the expression of Foxp3 mRNA,and the AH groups may have a decreased expression of RORγt mRNA.AH may regulate the balance of Foxp3/RORγ t,which may regulate the balance of Treg/Th17 cells,which may be one of the mechanisms of AH to alleviate joint inflammation and improve joint pathology.