| Background:Rheumatoid arthritis (RA) is a chronic autoimmune disease which is characterized by the presence of inflammatory synovitis, the infiltration of inflammatory cells, the pannus formation, and the progressive destruction of cartilage and bone. Triptolide (TP), isolated from the root of Tripterygiumwilfordii Hook F (TW, a traditional Chinese medicine), is a diterpenetriepoxide biologically active natural product. Previous studies have indicated that TP had various biological functions including immunosuppression, anti-inflammation and anti-cancer. But the progress of disease is complicated, and more mechanisms of TP on RA should be fully elucidated.Objective:The aim of this study is to investigate the mechanisms of TP acting on RA by combining bioinformatics analysis with experiment validation.Methods:1. The human target proteins of Triptolide and the human target genes related with RA were searched in PubChem platform and National Center for Biotechnology Information (NCBI) Gene database, respectively. Then, using the IPA biological network analysis platform, interaction network of TP target protein and RA disease were constructed and function of network and related biological pathways were analyzed, which aimed to predict the advantageous biological pathways and the key molecular target of TP acting on RA.2. Experiment validation in vitro and in vivo. (1) Experiment in vitro:The inflammatory models of cells were induced by LPS and the anti-inflammatory effects of TP through TREM-1/DAP12 signal pathway were observed. Then, to further determine whether the inhibition of LPS-induced inflammatory cytokines production by TP is dependent on TREM-1 signal pathway, we conducted the TREM-1 knockdown experiment. (2) Experiment in vivo:The anti-inflammatory action of TP on RA through TREM-1/DAP12 signal pathway was observed by collagen-induced arthritis (CIA).Results:1. Eight hundred and thirty two genes related with RA were found from Gene database in NCBI. Then, eight human target proteins of TP were found from PubChem database.2. The top fifteen signaling pathways related with RA were focused on cellular immune response, cytokine signaling, humoral immune response and intercellular and second messenger signaling. Based on the classification of signaling pathways in IPA, we found most signaling related with TP targets were focused on cytokine and cellular immune. Further comparative analysis showed that TREM-1 signaling was detected to be the top one shared signaling pathway and involved in cytokine and cellular immune signaling and TP may take part in the regulation of TREM-1 signaling pathway.3. The results in vitro indicated that TREM-1 mRNA and protein expression was significantly decreased in TP-treated U937 cells compared with the LPS model group. Consistent with these results, TREM-1 expression on U937 cells surface was also significantly inhibited by TP. Furthermore, expression of DAP 12 and phosphorylation of JAK2 and STAT3 were suppressed by TP. In addition, LPS potently induced the production of TNF-a, IL-1β and IL-6 in U937 cells, and TP could remarkably inhibit the production of these cytokines.4. Our results showed that the expression of TREM-1 protein was significantly decreased in PMA-induced U937 cells transfected with TREM-siRNA, as compared to untransfected cells or cells transfected with control siRNA. Therefore, TREM-1 was remarkably slienced by the corresponding siRNA. TP and TREM-1 siRNA could remarkably inhibit the production of TNF-a, IL-1 β and IL-6 compared with LPS group. And TP still could decrease these cytokines expression after TREM-1 knockdown, but there were no significant differences between cells transfected with TREM-1 siRNA and cells transfected with TREM-1 siRNA in the presence of TP.5. The results in vivo showed that treatment with TP could alleviate the severity of arthritis. From day 9 after the treatment, the arthritic scores were decreased in the rats treated with TP at both dosages. We then analyzed the histopathological changes of the ankle joints in rats from each group. As shown in results, the infiltration of inflammatory cells and synovial hyperplasia as well as cartilage and bone destruction in CIA rats were clear. Whereas, TP treatment could alleviate those histopathological changes, which showed a potential therapeutic effect of TP on CIA rats.6. We observed that TP markedly decreased the mRNA and protein levels of TREM-1 and DAP 12(P<0.01, P<0.05). Phosphorylation of JAK2 and STAT3 were also significantly suppressed by TP treatment. Our data also indicated that TP could decrease the expression of TNF-a, IL-1β and IL-6 in serum of CIA rats (P<0.05). Furthermore, the mRNA levels of TNF-a, IL-1β and IL-6 in ankle joints of CIA rats treated with TP were also significantly decreased (P<0.01, P<0.05).Conclusion:1. We find out a new strategy, by which we can search TP’s new target and the possible mechanisms. Meanwhile, the consensus between the results analyzed by bioinformatics method and previous studies suggested bioinformatics analysis method was reliable. Our finding also suggested that bioinformatics analysis method was not only suitable for finding TP’s target and the possible mechanisms but also might be appropriate for that of other drugs.2. TP could modulate TREM-1 signal pathway to inhibit inflammatory response. Our results also indicated that TREM-1 might be a potential target in therapy of RA. |
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