Effect And Mechanism Of Siweixizangmaoru Decoction On The Treatment Of Rheumatoid Arthritis Rats(Ⅰ)

Siweixizangmaoru decoction（SXD）,with Rhamnella gilgitica Mansf.et Melch.as the main medicine and Gentiana macrophylla Pall.,Berberis kansuensis Schneid and Terminalia chebula Retz.as the auxiliary medicines,is a commonly traditional prescription for treatment of rheumatoid arthritis in Tibet.In the previous study,we obtained the best compatibility of SXD and confirmed its anti-inflammatory and anti-rheumatic effects,while there is still no study report on its potential mechanism of action.Therefore,this study is designed to further explore and elucidate the underlying action mechanism of SXD on the premise of confirming its anti-rheumatoid effect.SXD was used as experimental materials,and based on the single factor experiment,the ultrasonic-assisted enzyme extraction of total flavonoids from SXD was optimized by response surface method.Meanwhile,the anti-inflammatory activity of total flavonoids in SXD extracted by ultrasonic-assisted enzymatic extraction was comparatively studied with those extracted by ultrasonic extraction,enzymatic extraction and reflux extraction.In vivo,typeⅡcollagen-induced arthritis rats（CIA）model was established by injecting collagen into tail,and the body weight,arthritis index,ankle joint and paw swelling were examined every 7 days during the administration-period.Organs（liver,kidney,spleen and thymus）indexes were measured after the death of rats,and serum samples were taken to detect the levels of oxidative stress,inflammatory factors and matrix metalloproteinases.HE staining and saffron O-fast green staining were used to observe the pathological changes（synovial and cartilage tissue）,and the protein expression of inflammatory signaling pathways in synovial tissue were detected by immunohistochemistry.Meanwhile,lipopolysaccharides（LPS）-induced RAW264.7 cells were used to investigate the anti-inflammatory action of SXD.The relevent indexes of oxidative stress were measured with kit and immunofluorescence,and ELISA kit was used to detect the inflammation,and the protein expression of related inflammatory signaling pathways were detected by western blotting and immunofluorescence.In vivo,the results showed that SXD effectively improve the body weight,arthritis index,ankle joint and paw swelling,organ index and the serum level of oxidative stress,inflammatory factors and matrix metalloproteinases in CIA rats.The results of HE and saffron O-fast green staining indicated that SXD could obviously improve the infiltration of inflammatory cells and the cartilage erosion in CIA rats.The immunohistochemistry results showed that SXD significantly inhibit the protein phosphorylations of NF-κB,MAPK and JAK2/STAT3 signaling pathways in synovial tissue.In vitro,SXD effectively improved the imbalance of oxidative stress stimulated by LPS,elavated the activities of SOD and GSH-Px,reduced the production of ROS and MDA,decreased the levels of TNF-α,IL-6,IL-1βand IL-17.Additionally,the results of immunofluorescence and western blotting showed that SXD suppressed the protein expression of i NOS/COX-2 signaling pathway and inhibited the protein phosphorylation of NF-κB,MAPK（P38,JNK,ERK）and JAK2/STAT3 signaling pathways.Additionally,the results showed that the optimal extraction parameters were cellulase concentration of 0.40 mg·m L^-1,enzymolysis p H of 5.0,enzymolysis time of2.2 h,enzymolysis temperature of 50℃,ultrasonic temperature of 50℃,solid-liquid ratio of 1:60(g·m L^-1),and ultrasonic time of 30 min.Under the above conditions,the extraction amounts of total flavonoids from SXD was 19.11 mg·g^-1.Compared with ultrasonic extraction,enzymatic extraction and reflux extraction,the content of total flavonoids extracted by ultrasonic-assisted enzymatic extraction is higher,which shows a better effect of reducing NO production.Summarily,ultrasonic-assisted enzymatic extraction method effectively elevated the total flavonoids amounts in SXD.In vivo,SXD significantly improved the joint lesions in CIA rats,and in vitro,SXD significantly improved the LPS-induced cell inflammation,and its underlying mechanism may be related to the inhibition with the protein expression of signaling pathways such as i NOS/COX-2,NF-κB,MAPK and JAK2/STAT3.This study confirmed the therapeutic effect of SXD on rheumatoid arthritis and further explored and studied its potential mechanism of action.