Therapeutic Effects And Mechanism Of Baicalin On Rheumatoid Arthritis Model Mice

Although the exploration of etiology,pathogenesis,diagnosis and treatment of rheumatoid arthritis(RA)has gone through a long process,there are still many doubts about etiology and pathogenesis,diagnosis and treatment.RA is associated with decreased quality of life and life expectancy.The incidence rate of RA is 0.5%-1%,which is more common in women.The disease causes progressive destruction of articular cartilage and bone,high rate of disability and malformation,and is accompanied by cardiopulmonary vascular metabolism and psychological abnormalities.It is the main manifestation of symmetrical peripheral joint involvement in primary patients.With the progression of the disease,they lose the ability to work because the joint dysfunction occurs.Later in the disease,it leads to death that cardiopulmonary vascular and metabolic complications caused by chronic inflammation.In the pathogenesis of RA,the genetic structure of RA has been better understood by traditional and genome-wide approaches.More than 100 loci are associated with risk and progression of the disease,most of which involve products of gene regulation and immune effects.Autoimmune tolerance is altered by factors such as lung infection,vitamine D(VitD)deficiency,obesity and disorders of gastrointestinal flora.Autoantibodies are produced under the abnormal immune response,which are an important cause of RA.There is no cure for RA.Rheumatologists around the world have faced a difficult problem for a long time.First-line drugs for RA include NSAIDs and glucocorticoids.They only relieve swelling and pain in the joints and have no effect on the progression of the disease.Second-line medicines are disease modifying antirheumatic drugs.However,these drugs generally have the disadvantages of slow onset,high side effects,and individual differences after medication.Although targeted biologics TNF-a and IL-6 show significant efficacy,30%-40%of patients still have poor response to treatment.These drugs also have the inherent side effects.Therefore,the application of biologics in some RA patients may be limited.Scutellaria baicalensis is a traditional Chinese medicine.In ancient times,it was regarded as the golden medicine and applied to the treatment of many diseases.In recent years,studies on the pharmacological mechanism of baicalin(Ba)have found that it can reduce the joint injury of osteoarthritis rats by inhibiting oxidative stress and the production of inflammatory cytokines induced by IL-1?.Baicalin alleviated the inflammatory response in adjuvant-induced mice arthritis by inhibiting IL-17.Currently,there is a lack of in-depth discussion about the effect of baicalin on the mechanism of RA.As a potential drug to RA,we have conducted further studies.In this study,We established arthritis mouse models by collagen antibody and lipopolysaccharide(LPS)to investigate the effects of baicalin on cytokines(TNF-a,IL-1?,IL-6),inducible nitric oxide synthase(iNOS),cyclooxygenase(COX)-2,matrix metalloproteinase(MMP)-2/9,apoptosis of mononuclear cells in the fluid and the extent of cartilage degradation.The effect on Janus kinase 1(JAK1)/signal transducer and activator of transcription 3(STAT3),mitogen-activated protein kinases(MAPKs)signaling pathway was further discussed to confirm the therapeutic effects of baicalin on RA and its related mechanismRA model mice establishment:The mixture of type II collagen monoclonal antibody(5mg/kg)was intraperitoneally injected every day for 10 consecutive days,and 100?g lipopolysaccharide was intraperitoneally injected at the same time on day 1 and day 4,to establish the RA models.Experimental design and grouping(10 for each group):(1)Control group:normal mice were intraperitoneally injected with 0.9%NaCl(30mg/kg)every day for 5 days.(2)RA+NaCl group:from the first day after the establishment of the models,the mice were intraperitoneally injected with 0.9%NaCl(30mg/kg)every day for 5 days.(3)RA+Ba group:from the first day after the establishment of the models,the mice were intraperitoneally injected with baicain(30mg/kg)every day for 5 days.After 5 days of treatment,blood samples were taken from the retroorbital veins of mice.Under anesthesia,the synovial membrane,synovial fluid and cartilage specimens of the mice were taken.The observed indicators are as follows:1.The general conditions of the mice in each group were observed,and pressure pain thresholds were measured using an electronic pressure sensor at before and after modeling as well as after 5 days of treatment.2.During 10 days of mouse modeling and 5 days of treatment,the macroscopic scoring system was used to evaluate mice joint inflammation in each group.3.Levels of TNF-?,IL-1? and IL-6 in peripheral blood of mice were analyzed by ELISA under different experimental conditions.4.Western Blot was used to detect the expression of iNOS,COX-2,MMP-2/9,JAK1/STAT3,pJAKl/pSTAT3,p38,JNK,ERK1/2 and pp38,pJNK,pERK1/2 in the synovium and synovial fluid of mice.5.Nuclear morphological changes of monocytes were detected by immunofluorescence assay and apoptosis was analyzed by flow cytometry.6.The degradation of articular cartilage in mice was detected by immunohistochemistry.The research results of this subject are as follows:1.General condition of mice:The mice in control group were in good spirits,responsive well,no swelling on the paws,normal skin temperature,activities,drinking and eating.The mice in RA+Ba group showed relatively little activities,swellen paws.Skin temperatures were not high.The mental states were OK,but the reactions were a bit poor.Eating and drinking water were basically normal.The mice in RA+NaCl group showed activities reduction significantly and swollen paws noticeably.Skin temperatures were high.The mice were in a low spirit,poor response and irritable.Drinking and eating were less.2.Pain threshold measurement:After 5 days of baicalin treatment,the pain threshold of RA+Ba group was significantly higher than that of the RA+NaCl,with significant difference(P<0.05).However,compared with the control group,the pain threshold was still at a low level,with a significant difference(P<0.01).This indicated that although baicalin had certain effects on pain relief,it could not restore the normal level of pain perception behavior in mice.3.Arthritis score:during the 10 days of modeling,the arthritis score of mice in the model group gradually increased,and significantly increased on the 8th day,the disease began to attack,and the arthritis score reached a peak on the 10th day.Compared with the RA+NaCl group,the arthritis score of mice in the RA+Ba group significantly decreased on the 4th day and the 5th day of treatment,and the difference was statistically significant(P<0.05).4.After 5 days of baicalin treatment,the expression levels of TNF-?,IL-1?,and IL-6 in the RA+Ba group were significantly down-regulated compared with that in the RA+NaCl group(P<0.05).However,compared with the control group,TNF-?,IL-1?,and IL-6 remained at higher levels,with significant differences(P<0.01).5.After 5 days of baicalin treatment,compared with the RA+NaCl group,the expression levels of iNOS and COX-2 in the RA+Ba group were significantly down-regulated with statistically significant differences(P<0.05).However,compared with the control group,the expression of iNOS and COX-2 in the RA+Ba group remained at a higher level with statistically significant differences(P<0.01).Compared with the RA+NaCl group,the expression levels of MMP-2 and MMP-9 in the RA+Ba group were significantly down-regulated,with statistically significant differences(P<0.05).6.Morphology and apoptosis analysis of mononuclear cells:5 days after baicalin treatment,the nucleus of synovial mononuclear cells in the RA+Ba group showed more aggregation.The apoptosis rate was significantly increased in RA+Ba group by flow cytometry.7.Five days after baicalin treatment,Although local inflammatory cell infiltration,proteoglycan consumption and cartilage erosion was observed in the tissues of mice in the RA+Ba group,the damage was less than that in the RA+NaCl group.8.Five days after baicalin treatment,the expression levels of pJAK1 and pSTAT3 in the RA+Ba group were significantly down-regulated compared with that in the RA+NaCl group,and the difference was statistically significant(P<0.05).However,compared with the control group,they still remained at a high level with significant differences(P<0.01).Compared with the RA+NaCl group,the expression level of pp38 in RA+Ba group showed no significant change.Compared with the control group,the expression level of pp38 in RA+Ba group was up-regulated with significant difference(P<0.01).Compared with the RA+NaCl group,the expression level of pJNK in the RA+Ba group showed no significant change.Compared with the control group,the expression level of pJNK in the RA+Ba group was significantly up-regulated,with statistically significant(P<0.01).Compared with the RA+NaCl group,there was no significant change in the expression level of pERKl/2 in RA+Ba group.Compared with the control group,the expression level of pERK1/2 in RA+Ba group was significantly up-regulated,and the difference was statistically significant(P<0.01).The research results of this project show that:1.The mice model of rheumatoid arthritis can be successfully established by intraperitoneal injection of collagen type II monoclonal antibody and lipopolysaccharide for laboratory study.2.Baicalin can effectively reduce the levels of TNF-?,IL-1? and IL-6 in peripheral blood of RA model mice,and has the effect of alleviating the disease state of RA.3.Baicalin inhibited the expression of iNOS and COX-2 in synovium and synovial fluid of RA mice and promoted the apoptosis of monocytes.4.Baicalin can reduce the expression of MMP-2 and MMP-9 proteins in synovium and synovial fluid of RA mice and reduce the degradation of cartilage.5.Baicalin inhibited the activation of the pJAKl/pSTAT3 signaling pathway in the synovial membrane and synovial fluid of RA mice,but had no significant effect on the pMAPKs(pERKl/2,pJNK and pp3 8)signaling pathway.