| Objective:We investigated the intervention effects of the water extract of Sophora tonkinensis and its main active ingredients on type II collagen-induced arthritis(CIA)in mice,and explored its mechanism of anti-rheumatoid arthritis(RA)bone destruction in combination with transcriptome sequencing technology.The study will provide a reference for clinical use and a basis for the development of new drugs based on the Sophora tonkinensis.Methods:CIA models were prepared in 8-10 weeks old male DBA/1J mice and randomly grouped into five groups: normal group(Control),model group(Model),positive drug group(MTX),and Sophora tonkinensis low and high dose groups(0.1 g/kg and0.2 g/kg).Daily intragastric administration was started one week before the second immunization,and the drug was administered for 26 consecutive days,while the degree of joint swelling in CIA model mice was observed every 3 d,and the scores were recorded to calculate the incidence.The extent of histopathological changes and osteoclasts genesis in the knee joint were observed by HE staining and TRAP staining.The degree of bone destruction was evaluated using Micro-CT scan.Meanwhile,the effect of Sophora tonkinensis on the liver was initially evaluated by HE staining.Based on the model of RANKL-induced differentiation of BMMs cells to osteoclasts,the main active components of Sophora tonkinensis against osteoclast formation were screened by TRAP staining.The effect on osteoclast formation was evaluated using immunofluorescence staining of actin rings and toluidine blue staining of bone fragments.The expression of signature proteins of osteoclast differentiation was detected by RT-PCR technique,Westen blot assay,and immunofluorescence staining.On the basis of screening out the effective components of Sophora tonkinensis,a CIA model was established by using 8-10-week-old male DBA/1J which randomly divided into normal group(Control),model group(Model),positive drug group(MTX),low and high dose groups(5 mg/kg and 10 mg/kg)of Trifolirhizin.The therapeutic effect of the main active ingredient of Sophora tonkinensis(Trifolirhizin)on CIA mice was evaluated.After the above cellular experiments,the main active components of Sophora tonkinensis against osteoclast differentiation were clarified.The CIA mouse model was established and randomly grouped into normal group(Control),model group(Model),positive drug group(MTX),and low and high dose groups(5 mg/kg and 10mg/kg)of Trifolirhizin,to evaluate the therapeutic effects of the main active ingredient(Trifolirhizin)of Sophora tonkinensis on CIA mice.Transcriptomic sequencing analysis was performed on osteoclasts,differential genes were screened by volcano plot and Wayne plot,PPI protein interaction network analysis was constructed with the help of STRING database and Cytoscape software,potential targets of trichothecene on osteoclast differentiation process were screened,and the candidate targets were validated by RT-PCR and Western blot techniques in vivo and in vitro experiments to investigate the possible mechanism of action of Trifolirhizin against RA bone destruction.Results:1.The water extract of Sophora tonkinensis significantly inhibited bone destruction in CIA model miceThe results of Micro-CT scan showed that the low and high dose groups of the water extract of Sophora tonkinensis could alleviate the bone destruction in the knee joint of CIA mice;HE staining results suggested that compared with the Model group,the administration groups of the water extract of Sophora tonkinensis significantly inhibited synovial inflammatory infiltration,cartilage proliferation,vascular opacity formation and bone destruction(P<0.01).TRAP staining showed that the water extract of Sophora tonkinensis could significantly inhibit the level of bone erosion in CIA mice,and the low dose of Crocus sativus was more effective.In addition,the safety evaluation of the three aspects of body weight,organ coefficient and liver tissue staining of CIA mice showed that no significant changes were observed in the groups administered with Sophora tonkinensis.2.Trifolirhizin significantly inhibited osteoclastogenesis and bone destruction in CIA mice in vitroThe TRAP staining and quantification results showed that the degree of inhibition of osteoclasts by the five monomers of Sophora tonkinensis were:Trifolirhizin > maackiain > euchrenone > matrine > oxymatrine,based on which we selected Trifolirhizin for the subsequent experimental study.The results of MTS showed that Trifolirhizin no significant effect on the activity of BMMs below 80 μM.The results of TRAP staining and F-actin staining showed that trichothecene(10,20 and 40 μM)could dose-dependently inhibit the formation of osteoblasts.Trifolirhizin dose-dependently inhibited the bone resorption function of osteoclasts,and the expression levels of osteoclast differentiation-related factors TRAP,CTSK and MMP-9 proteins and genes were significantly reduced,with statistically significant differences(P<0.01 or P<0.05).Compared with the CIA model group,each administration group of Trifolirhizin significantly reduced the arthritis score and incidence of CIA mice,improved the degree of bone destruction in knee joint tissues,reduced the number of osteoclasts in knee joints of CIA mice,and also significantly inhibited the expression of TRAP,NFATc1,MMP-9 proteins and genes,with statistically significant differences(P<0.01 or P<0.05).3.Transcriptomics-based exploration of the mechanism of anti-RA bone destruction effect of TrifolirhizinLibrary sequencing was performed using osteoclast samples to analyze the differential genes of Trifolirhizin inhibition of osteoclast formation.The results showed that there were 2520 differential genes after RANKL induction,including1470 up-regulated genes and 1050 down-regulated genes compared with Control.Compared with the RANKL induction group,there were 606 differential genes after Trif action,of which 59 genes were up-regulated and 547 genes were down-regulated.By constructing PPI protein interaction network analysis through STRING database and Cytoscape software,the top 20 important targets of Trifolirhizin on osteoblast differentiation were screened,and RT-PCR experiments were verified and combined with literature analysis,FPR2 protein was screened as a key target of Trifolirhizin Triptolide significantly up-regulated FPR2 m RNA and protein levels,significantly inhibited TRAF6 protein expression and decreased p38 and ERK phosphorylation levels in osteoclasts and CIA mouse models.3.Exploring the mechanism of anti-RA bone destruction of Trifolirhizin based on transcriptomicsThe mice osteoclast samples were used for library construction and sequencing,and the differential genes of Trifolirhizin inhibiting osteoclast formation were analyzed.There were 2520 differential genes in the RANKL vs Control comparison group,including 1470 up-regulated genes and 1050 down-regulated genes.There were 606 differential genes in RANKL vs Trif,of which 59 genes were up-regulated and 547 genes were down-regulated.The STRING database and Cytoscape software were used to construct the PPI protein interaction network analysis,and the key target of Trifolirhizin in the process of osteoclast differentiation was screened out.Real time-PCR was used to verify and screen the important target FPR2.Subsequent in vitro and in vivo experiments showed that pterostilbene could significantly upregulate FPR2 m RNA and protein levels in osteoclast models,significantly inhibit TRAF6 protein expression,and down-regulate p38 and ERK phosphorylation levels.The vitro experiments also confirmed this phenomenon.Conclusion:1.The water extract of Sophora tonkinensis(0.1 g/kg,0.2 g/kg)significantly alleviated the progression of CIA mice,especially significantly inhibited bone destruction.2.Trifolirhizin significantly inhibited osteoclast differentiation and bone destruction in CIA mice in vitro,suggesting that it may be the main active component of the antiRA activity of Sophora tonkinensis.3.Trifolirhizin inhibits osteoclast differentiation by inhibiting FPR2/TRAF6/NFATc1 pathway activation,which may be the main pathway of Trifolirhizin against bone destruction in RA. |
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