Drug Screening And Mechanism Of A Novel Tylophoirne Analog NK-007in Collagen Induced Arthritis

Rheumatoid arthritis (RA) is a chronic inflammatory polyarthritis that can affectany synovial-lined diarthrodial joint, although it has a predilection for the wrist andsmall joints of the hand. The typical natural history of RA is one of progressivearticular damage leading to joint deformities and disability. Recently, there has beenconsiderable progress in elucidating the mechanisms that mediate synovitis andarticular damage. Indeed, recognition that tumor necrosis factor alpha (TNF-α)plays an important role in joint inflammation and damage has led to the introductionof therapies that target TNF-α and that have had a major impact on the care ofpatients with RA.Our previous study reported that a novel Tylophorine analog DCB-3503showedsignificant effect on the development and progression of CIA through inhibiting theinnate immune responses. Based on this result, we synthesized more derivatives ofthis compound and setup assays to evaluating their effect on LPS-triggered TNF-αproduction. NK-007, as one of these compounds, exhibited a strong inhibition ofTNF-α and showed better sensitivity and water solubility.To test the anti-inflammatory properties of NK-007, we used different cells andexperiments in our studies. Lipopolysaccharides (LPS)-triggered tumor necrosisfactor-α (TNF-α) production by murine splenocytes and macrophage cell line (Raw264.7) were determined by ELISA, intracellular cytokine staining as well as WesternBlot. NK-007significantly suppressed LPS-induced TNF-α production by bothmurine splenocytes and macrophage cell line (Raw264.7) in vitro, and the inhibitoryeffect was dose-time-dependent.Therefore, CIA model with or without LPS-boosted was adopted, NK-007orvehicle was administered at different time points post immunization. The mice weremonitored for the clinical severity, the joint tissues were used for histologicalexamination, cytokine detection and immunohistochemical staining. Spleens anddraining lymph nodes obtained from CIA mice on day35postimmunization were analyzed. Administration of NK-007completely blocked CIA development andprogression. Furthermore, treatment with NK-007at the onset of arthritissignificantly inhibited the progress of joint inflammation. Administration ofNK-007also suppressed production of TNF-α, IL-6and IL-17A in joint, and reducedIL-17positive cells in CD4+and γδ T cells in draining lymph nodes.To elucidate the mechanism of NK-007, we analysis the cell treated by NK-007.NK-007showed on significant effect on the transcription of TNF-α mRNA but actingon the stability of TNF-α. NK-007significantly reduced the level of p-p38andMK2, which in turn enhanced the expression of TTP and mediated the instability ofTNF-α mRNA.Furthermore, Th17differentiation was studied using flow cytometry. We furtherdemonstrated that NK-007inhibited IL-6production of BMDC, and reduced Th17differentiation indirectly, although it also had direct effect on Th17differentiation.In addition, it significantly inhibited IL-6and IL-17A level in human co-cultureassay.Based on its effects on the development, progression and therapeutic effect onCIA, NK-007has a great potential to be a therapeutic agent for human RA.