身痛逐瘀汤干预小鼠类风湿关节炎的效果及其作用机制OA

Objective:To investigate the therapeutic effects of Shentong Zhuyu Tang(身痛逐瘀汤,SZT)on rheumatoid arthritis(RA)in mice,and to explore its underlying mechanisms.Methods:①Animal experiment.Six tumor necrosis factor(TNF)-α-overexpressing negative transgenic mice were assigned into the normal control group,1exate(MTX)g8 TNF-α-overexpressing positive transgenic mice were randomized into model group,SZT group,and methotrroup,with 6 ones in each group.After that,interventions were performed on all mice once a day,5 consecutive days per week,for 8 consecutive weeks.The mice in the normal control group and model group were intervened by intra-gastric administration with 0.2 mL sterile saline each time;the ones in the SZT group with 0.2 mL SZT decoction;and those in the MTX group with0.2 mL MTX solution(containing15 μg MTX)on the first and fourth days per week,while 0.2 mL sterile saline on the other three days.After the end of the intervention,the body weight of the mice was measured.The ankle joint deformity of the mice was evaluated using the joint deformity score.The left ankle joints of the mice were scanned by micro-CT,and the volume of the talus was statistically ana-lyzed.Moreover,the tissue sections of the right ankle and knee joints were prepared and stained with Alcian blue-hematoxylin-eosin-Orange G to analyze the talar area,as well as the cartilage area and inflammatory area of these two joints.Additionally,the tissue sections of the two joints were stained with tartrate-resistant acid phosphatase(TRAP)to analyze the osteoclast area in these two sites.②Network pharmacology analysis.The effective active ingredients of each herb in SZT were retrieved and screened using the traditional Chinese medicine systems pharmacology database and analysis platform(TCMSP),and their corresponding potential targets were identified.The RA-related targets were obtained via retrieving the DisGeNET database.The overlapping targets between the corresponding potential targets of effective active ingredients of SZT and RA-related targets were identified using network pharmacology analysis software.Subsequently,these common targets were subjected to GO enrichment analysis(covering cellular components,biological processes,and immune-related functions)and KEGG signaling pathway enrichment analysis.Results:①Findings from animal experiment.No significant differences in body weight were observed between SZT group and model group or MTX group(P=0.957,P=1.000,P=0.942).Furthermore,compared with the model group,the mice in the SZT group exhibited a lower ankle joint deformity score(P=0.017),increased talar volume,talar area,as well as cartilage are-as in both ankle and knee joints(P=0.002,P=0.022,P=0.027,P=0.032),but decreased inflammatory and osteoclast areas in these two joints(inflammatory area:P=0.026,P=0.000;osteoclast area:P=0.000,P=0.002).Besides,compared with the model group,the talar volume increased,the inflammatory and osteoclast areas in both ankle and knee joints decreased in MTX group(talar volume:P=0.030;inflammatory area:P=0.022,P=0.000;osteoclast area:P=0.000,P=0.000),but the ankle joint deformity score,talus area,as well as cartilage areas in both joints were not significantly different between the two groups(P=0.055,P=0.751,P=0.123,P=0.571).In addition,no significant differences were found between SZT group and MTX group in any of the above parameters(P=0.945,P=0.555,P=0.163;P=0.873,P=1.000,P=0.829;P=0.347,P=1.000,P=0.948).②Findings from network pharmacology analysis.A total of 219 effective active ingredients were screened out from SZT,corresponding to 229 potential targets;1 841 RA-related targets were identi-fied,and 123 common targets were yielded via overlapping the 229 corresponding potential targets of effective active ingredients of SZT with the 1 841 RA-related targets.GO function enrichment analysis revealed that the cellular components mainly involved the presynaptic mem-brane,vesicle lumen,and membrane rafts;the biological processes mainly involved the positive regulation of B cell apoptosis,regulation of chronic inflammatory response to antigen stimulus,and positive regulation of vitamin D biosynthesis;and the immune-related functions main-ly involved T cell activation,regulation of cytokine secretion in immune response,positive regulation of leukocyte migration,leukocyte differ-entiation,and the Fc-epsilon receptor sialic acid pathway.KEGG signaling pathway enrichment analysis revealed that the common targets between the corresponding potential targets of effective active ingredients of SZT and the RA-related targets were primarily enriched in the phosphoinositide 3-kinase(PI3K)-protein kinase B(PKB)signaling pathway,TNF signaling pathway,forkhead box O(FOXO)transcription factor signaling pathway,mitogen-activated protein kinase(MAPK)signaling pathway,and Toll-like receptor(TLR)signaling pathway.Conclusion:SZT can suppress inflammatory response and bone erosion,protect bone and cartilage,and improve ankle joint deformity in RA mice,with efficacy comparable to MTX.It may exert anti-RA effects through multi-component,multi-target,and multi-pathway synergistic regulation of immune and inflammatory networks.