| Objective: In this study,we observed the effects of total flavonoids of Zhuang medicine Jiulongteng and methotrexate on peripheral blood Th17/Treg cell ratio,inflammatory factor secretion,Th17/Treg protein expression level in rat synovial tissue,Th17/Treg protein expression level in rat colonic and mesenteric lymph node tissues and gene transcription level;and the effect of changes in rat synovial tissue from Zhuang medicine Gu Dao-Long Lu-Bone to explore the mechanism of action of total flavonoids of Zhuang medicine Jiulongteng on rheumatoid arthritis by inducing RA in SD rats with complete Freund’s adjuvant.Methods: Forty healthy male SD rats were selected and acclimatized and fed for one week,and 30 of them were selected to replicate the AIA model using the complete Freund’s adjuvant model,and the remaining 10 were the normal group.The rats with successful modeling were randomly divided into model group,methotrexate group(MTX group),and total flavonoids group of Jiulongteng,with 10 rats in each group.Each rat was given 1ml/100 g by gavage on one day.The daily group was given 2.1mg/kg of total flavonoid of Jiulongteng,and the methotrexate group was given0.9mg/kg of methotrexate solution by gavage every week,and saline was given on the remaining 6 days.The rest of the model group and normal group were given saline,and the body weight,swelling of both hind toes and AI score of each group were measured every 3 d after drug administration,and the specimens were collected one week after drug withdrawal after 28 days of administration.Whole blood serum was collected from rats for ELISA to measure serum IL-1β,IL-10,IL-17A;flow cytometry to detect the proportion of Th17 and Treg cells;colonic and mesenteric lymph nodes of rats were collected for RT-q PCR to detect the transcript levels of IL-10,IL-17 A,Foxp3and RORγt genes in each group.The protein expression of IL-10,IL-17 A,Foxp3 and RORγt in rat intestine and mesenteric lymph nodes was determined by Western-Blot assay;the protein expression of IL-10,IL-17 A,Foxp3 and RORγt in rat synovial tissues was detected by Western Blot;and rat ankle tissues were collected for histopathological HE staining analysis.The data were counted by SPASS 25.0 statistical software,and the graphs were soft-crafted by Graph Pad Prism 8.0.Results: 1.Rat body weight,toe volume,AI score results: during the administration period,the normal group rats grew well,and the model group rats grew slowly in body weight,compared with the normal group,from 12 days of administration to the end of the administration of the model group rats grew slowly in body weight(P < 0.05).Compared with the model group,the MTX group showed a significant increase in body weight from 12 days to the end of administration(P < 0.05).The BCF group showed a significant increase in body weight on 24 days of administration(P < 0.05),and there was no statistical significance between the MTX and BCF groups(P > 0.05).In terms of toe volume,the rate of increase in toe volume from 3 days to the end of dosing increased in the model group compared with the normal group(P < 0.05),and decreased in the BCF group compared with the model group from 21 days to the end of dosing(P < 0.05),and decreased in the BCF group from 18 to 24 days of dosing(P < 0.05).The AI score was significantly higher in the model group(P < 0.05),and the AI score decreased in the medicated group compared with the model group from 21 days to the end of the administration(P < 0.05).2.HE pathological staining results: The ankle joint of the normal group was smooth,no hyperplasia was observed,the distribution of chondrocytes was regular and clear,and no inflammatory infiltration was seen in the periarticular connective tissue.The synovial tissue did not invade the cartilage and no vascular opacification was seen.In the model group,the synovial surface was not smooth and obvious hyperplasia was seen,the joint cavity was narrowed,a large number of inflammatory cells infiltrated,the synovial membrane invaded the bone surface and the joint surface was heavily damaged.The inflammatory infiltration was reduced in the western medicine group,the chondrocytes were more evenly distributed and neatly arranged,the inflammatory exudation was not obvious,the synovial layer was reduced,and the surface was smoother,the inflammatory infiltration was reduced in the total flavonoids group,the chondrocytes were more evenly distributed and neatly arranged,the lymphocytes were reduced,the inflammatory exudation was not obvious,and the synovial invasion of the joint surface was reduced.3.serum ELISA test results: compared with the normal group,serum IL-1β and IL-17 A levels were increased and serum IL-10 levels were significantly reduced in the model group,which was statistically significant.compared with the model group,serum IL-β levels were significantly reduced(P<0.01),IL-17 A levels were significantly reduced(P<0.01)and IL-10 was significantly increased in the MTX group(P< 0.01);serum IL-1β level was significantly reduced(P<0.05),IL-17 A level was significantly reduced(P<0.01)and IL-10 level was significantly increased(P<0.05)in the BCF group,which were statistically significant.4.Flow cytometry results: Compared with the normal group,Treg cells were significantly decreased(P<0.005),Th17 cells were increased(P<0.01),and Treg/Th17 was significantly increased(P<0.005)in the model group;compared with the model group,Treg was significantly increased(P<0.01)and Th17 was significantly decreased(P<0.05)in the MTX group.Treg/Th17 increased significantly(P<0.01),Treg increased significantly(P<0.05),Th17 increased significantly(P<0.05),Treg/Th17 increased significantly(P<0.05)in the BCF group.5.Western-Blot test results:Synovial membrane: compared with the normal group,the relative expression of IL-17 A and RORγt in the synovial membrane of rats in the model group increased significantly(P<0.01);the relative expression levels of IL-10 and Foxp3 in the model group decreased significantly(P<0.01).Compared with the model group,the relative expression of IL-17 A and RORγt in synovial membrane was significantly decreased(P<0.05)and the relative expression of IL-10 and Fox P3 protein was increased(P<0.05)in MTX group as well as BCF group.Colon:Compared with the normal group,the relative expression of IL-17 A was significantly increased(P<0.01),the relative expression of RORγt was significantly increased(P<0.05),and the relative expression of IL-10 and Foxp3 was significantly decreased(P<0.01)in the model group.Compared with the model group,the relative expression of IL-17 A and RORγt was significantly decreased in the MTX group as well as the BCF group(P<0.05),IL-10 and Foxp3 relative expression increased significantly(P<0.05);mesenteric lymph nodes: IL-17 A and RORγt relative expression increased significantly(P<0.01),Foxp3 and IL-17 A relative expression decreased significantly(P<0.01)in the model group compared with the normal group;IL-17 A and RORγt relative expression increased significantly(P<0.01)in the MTX group compared with the model group and the BCF group compared with the BCF group.-17 A,RORγt relative expression significantly decreased(P<0.05),IL-10,Foxp3 significantly increased(P<0.05);the differences were statistically significant.6.RT-q PCR test results: colon: compared with the normal group,the relative expression levels of IL-17 Am RNA increased significantly(P<0.005),RORγtm RNA expression increased significantly(P<0.01),IL-10 m RNA and Foxp3 m RNA expression decreased significantly(P<0.005)in the model group.Compared with the model group,the relative expression of IL-17 Am RNA and RORγtm RNA in the colon of MTX group was significantly decreased(P<0.01),and the relative expression of IL-10 m RNA and Foxp3 m RNA was increased(P<0.01);the relative expression of IL-17 Am RNA in the colon of BCF group was decreased(P<0.05),and the relative expression of RORγtm RNA was significantly decreased(P<0.01).expression was significantly decreased(P<0.01),IL-10 m RNA relative expression was significantly increased(P<0.01)and Foxp3 m RNA relative expression was increased(P<0.05)in the BCF group.Mesenteric lymph nodes: compared with the normal group,the relative expression of IL-17 m RNA and RORγtm RNA in the mesenteric lymph nodes of the model group were significantly increased(P<0.005),the relative expression of IL-10 m RNA was significantly decreased(P<0.01),and the relative expression of Foxp3 m RNA was significantly decreased(P<0.005),compared with the model group,the relative expression of IL-17 and RORγtm RNA in the MTX group was significantly decreased(P<0.01).The relative expression of IL-17 Am RNA and IL-10 m RNA decreased in the BCF group(P<0.05)and the relative expression of RORγtm RNA decreased in the MTX group(P<0.01)and the relative expression of IL-10 m RNA and Foxp3 m RNA increased in the MTX group(P<0.005).RORγtm RNA relative expression decreased(P<0.01),Foxp3 m RNA relative expression increased significantly(P<0.01).Conclusion(s): 1.bauhinia championii flavoneon regulate Th17/Treg balance in AIA rats,down-regulate the function of Th17,up-regulate Treg level,improve immune homeostasis,reduce IL-1β and IL-17 A levels,increase IL-10 level,regulate Th17 and Treg transcription,reduce joint inflammation and improve bone destruction in RA rats.2.Bauhinia championii flavoneon can synergistically regulate the Th17/Treg balance of Guido-Longlu-Bone to treat RA. |
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