The Bcr-Abl Inhibitor DCC-2036 Inhibits Necroptosis And Ameliorates Osteoarthritis By Targeting RIPK1 And RIPK3 Kinases

Objectives:Osteoarthritis(OA)is a chronic progressive disease characterized by cartilage degeneration,the clinical symptoms of which are pain,stiffness and movement limitation.On the pathological level,OA mainly manifests as articular cartilage injury,synovial inflammation and subchondral bone changes.Because of its complex pathogenesis,difficult to cure,OA has become an increasingly urgent public health problem.At present,pain relief and anti-inflammatory therapy are the main treatment of OA in the early stage.With the progressive aggravation of the disease,many patients finally have to accept joint replacement or other surgical treatments.Therefore,it is urgent to further clarify the pathogenesis of OA and develop drugs to delay the progression of OA and even reverse the disease.Necroptosis belongs to programmed cell death forms,characterized by the activation of the serine/threonine kinase receptor-interacting protein kinase 1(RIPK1),receptor-interacting protein kinase 3(RIPK3)and mixed lineage kinase domain-like protein(MLKL).When necroptosis occurs,the cell membrane ruptures and releases a series of damage associated molecular patterns(DAMPs),thus make the inflammation in the surrounding cell matrix and the reaction aggravates further,thus causes the organ damage.Therefore,necroptosis is closely related to the occurrence and development of many inflammatory and injury diseases.Studies have shown that necroptosis is an important mode of chondrocyte death,expression of RIPK3 and phosphorylated mixed lineage kinase domain-like protein(p-MLKL)is high in the highly degenerated cartilage tissue of human OA knee.Inhibition of chondrocyte necroptosis can significantly reduce the degree of cartilage destruction,therefore,the regulation of necroptosis-related factors may be a new attempt to treat OA in the future.At present,many necroptosis inhibitors targeting on RIPK1,RIPK3 and MLKL have been developed.However,its poor efficacy,low stability and strong cytotoxicity have limited its further clinical trial study.Bcr-Abl tyrosine-kinase inhibitor has been widely used as a gene-targeted drug in the treatment of chronic myeloid leukemia and other diseases,and its drug stability and low cytotoxicity have been fully verified.Whereas the Glu-in/DLG-out conformation of RIPK1 is very similar to that of Abl,suggesting that Bcr-Abl inhibitors have potential anti-necroptotic effects.In this study,the recent common Bcr-Abl inhibitors were used to inhibit cell necroptosis in rat chondrocytes,Rebastinib(also called DCC-2036)was found to be more effective than other drugs in inhibiting necroptosis of rat chondrocytes.DCC-2036 can not only act on articular chondrocytes,but also inhibit the necroptosis of synovial fibroblasts(SF cells).In this study,chondrocytes and SFs of rats were selected to build necroptosis model by TSZ(TNF-α、Smac mimeticz、z-VAD-fmk)and other classical methods.It was found that DCC-2036 could inhibit the necroptosis obviously.In the animal experiment,the OA model group by the method of joint cavity injection,was used to explore the anti-necroptosis and anti-inflammatory effect of DCC-2036.Methods:1.Rat articular cartilage cell line(RCCs-1)and SF cells were cultured,and the necroptosis model was made by TSZ.The model was interfered by DCC-2036 at appropriate concentration,stained by Propidium Iodide(PI)and photographed by fluorescence,to study the protective effect of DCC-2036 on these two kinds of cell necroptosis.2.Rat chondrocytes were treated with different concentration gradients of DCC-2036,the toxic range and the optimal protective concentration of DCC-2036 were determined,and the 50% concentration of maximal effect(EC 50)was calculated.3.The expressions of pMLKL,MLKL,pRIPK1,RIPK1,pRIPK3 and RIPK3 were detected by Western blot.The CO-IP method confirmed that the mechanism of action is the formation of RIPK1-RIPK3-MLKL necrosome.4.The target of DCC-2036 in inhibiting necroptosis was identified by Drug affinity responsive target stability assay(DARTS),in vitro kinase assay and molecular docking analysis.5.Establishment and treatment of OA model in SD rats: 30 μL of DCC-2036 containing 200 μg MIA was injected into the articular cavity to establish OA model in rats,and DCC-2036 was administered intragastrically at a dose of 1 mg/kg body weight ratio.Body weight monitoring was used to evaluate the side effects of drugs,and serum TNF-α and IL-1β were detected by ELISA to evaluate the systemic inflammation.6.The evaluation of knee joint OA in each group: X-ray,HE and toluidine blue staining were performed after knee tissue section,then modified Mankin and OARSI score were performed.7.Western blot and qPCR were used to detect the expression of cartilage-associated proteins,and immunohistochemistry was used to detect the expression of cartilage-associated proteins.Results:1.DCC-2036 could significantly inhibit the TSZ-induced necroptosis of rat chondrocytes and SFs.In molecular mechanism,DCC-2036 inhibits necroptosis by targeting the activity of RIPK1 and RIPK3.2.Oral administration of DCC-2036 ameliorats the necroptosis of chondrocytes and ameliorates the inflammatory response of OA,and protects chondrocytes in MIA-injected rats.Conclusions:In this study,we found that the multi-targeted kinase inhibitor DCC-2036 is able to inhibit TSZ-induced chondrocytes and SFs necroptosis.In addition,we found that oral administration of DCC-2036 inhibits chondrocyte damage in OA model created by MIA injection.Mechanistic studies have shown that DCC-2036 can directly inhibit RIPK1 and RIPK3 activity to block necroptosis,thereby reducing the pro-inflammatory response to OA and protecting chondrocytes.Taken together,our study indicates that DCC-2036 is a novel necroptotic inhibitor targeting RIPK1 and RIPK3 activity with great clinical potential in the treatment of OA,which provides a new way for the research and treatment of OA.