Decreased RIPK1 Expression In Chondrocytes Alleviates Osteoarthritis Via The TRIF/MyD88-RIPK1-TRAF2 Negative Feedback Loop

Background Osteoarthritis(OA)is a common,age-related,degenerative disease characterized by the loss of articular cartilage integrity in the joint area,with varying degrees of articular osteophyte formation,subchondral bone remodeling,and synovitis.Receptor interacting serine/threonine kinase 1(RIPK1),a protein that regulates cell death and inflammation,is ubiquitously expressed in the limbs,lungs,liver,intestines.RIPK1 regulates apoptosis and necroptosis via kinase-dependent and-independent functions,which are essential for cell fate and inflammation.Our team has recently found that the phosphorylation level of RIPK1 was lower in articular cartilage of osteoarthritis mice than in the normal control group.Because whether the expression level of RIPK1 and its phosphorylation level have an effect on osteoarthritis is still unclear,we tried to study the role and mechanism of RIPK1 in osteoarthritis.Methods1.Adult male C57BL/6 mice(age = 12 weeks)were used to induce the OA model via DMM surgery on the right knee.After wound healing,intra-articular injection of 10 μL Ad-sh RIPK1 or Ad-negative adenoviruses was administered to the mice for 8 weeks.Knee joint tissue sections were stained with crocus green,HE,and immunohistochemical staining to observe histomorphological changes and expression levels of inflammation-related marker proteins.Mouse primary chondrocytes were extracted from the bilateral knee joint cartilage of newborn C57BL/6 mice.Primary chondrocytes were treated with IL-1β(5ng/ml)in the presence of Ad-sh RIPK1 or adenovirus overexpressing RIPK1 or vehicle for 48 h.Inflammation-related proteins(MMPs),TRAF2,MyD88,and TRIF expression levels were detected by WB.Elisa kits were used to quantify TNF-α secretion levels in cell culture media.2.Primary chondrocytes were treated with IL-1β(5ng/ml)in the presence of si-NC、si-MYD8 or si-TRIF RNA.MMPs and p-RIPK1 were detected by WB.Next,we used Pam3CSK4,an agonist of TLR2 that exclusively engages MyD88,and poly(I:C),an agonist of TLR3 that exclusively engages TRIF,to assess the role of MyD88 and TRIF in inflammation.We also overexpressed TRAF2 in chondrocytes to determine whether it modulates OA via TRIF/MyD88-RIPK1-TRAF2 negative feedback loop.3.Immunohistochemical staining and TUNEL staining were performed on articular cartilage in DMM and sham operation groups to determine whether apoptosis and necrotic apoptosis occurred in osteoarthritis models.We examined the phosphorylation of MLKL,a marker of necroptosis,in vivo and in vitro.Chondrocytes were incubated with IL-1β in the presence or absence of the pan-caspase inhibitor Z-VAD.Results1.HE and Safranin O/Fast Green staining showed that Ad-sh RIPK1 significantly ameliorated cartilage destruction,based on the OARSI scores.The severity of synovitis showed the same trend as the OARSI score.Immunohistochemical results that the expression levels of inflammatory proteins such as MMP1,MMP3,and MMP13 were also significantly reduced.The results of in vitro experiments are consistent with the results in vivo.Meanwhile,the knockdown of RIPK1 decreased the expression of TRAF2、MyD88 and TRIF.2.Knockdown of MyD88 suppressed the IL-1β–induced production of MMPs,and knockdown of TRIF suppressed that of MMP1 and MMP13.Knockdown of RIPK1 decreased the Pam3CSK4-and poly(I:C)-induced production of MMPs.The IL-1β–induced phosphorylation of RIPK1 was significantly reduced by TRAF2 overexpression.Furthermore,the increase in the MyD88,TRIF,and MMP levels was similar to that caused by overexpression of TRAF2.3.There was no significant difference in MLKL phosphorylation between the OA and control groups.RIPK1 knockdown reduced the IL-1β–induced increased expression of cleaved caspase-3 and cleaved PARP and the number of TUNEL-positive cells in vivo and in vitro.The upregulation of MMPs induced by IL-1β was abrogated by Z-VAD.Conclusion1.The knockdown of RIPK1 can significantly inhibit osteoarthritis in vivo and in vitro.2.RIPK1 regulates osteoarthritis through MyD88 / TRIF-RIPK1-TRAF2 negative feedback signal pathway.3.RIPK1 kinase-mediated osteoarthritis is dependent on apoptosis but not MLKL-dependent necroptosis.