The Effect Of Notch1 Pathway In IL-1β Induced Inflammation In Temporomandibular Chondrocytes

BackgroudTMD is a series of conditions that cause pain and dysfunction in the muscles and the joints between the lower jaw and the base of the skull.The prevalence of TMD varied from 20%to 40%depending on different epidemiological studies,diagnostic criteria,and the means of assessment.TMD is a complex and multi-factorial disease,where physical,social and psychological factors are usually associated with its etiology.Although remarkable progress has been made,the precise pathogenesis of TMD still remains uncertain,which leads to the difficulty of diagnosis and therapy.TMJ OA is an important type of TMD,with degenerative morphological changes in TMJ such as articular cartilage loss,destruction and articular cartilage abnormalities.The prevalence of TMJ OA is 25%.It is closely related to cytokines such as IL-1β,TNF-α,IL-6 and the Notch signaling.IL-1 is a potent pro inflammatory cytokine and functions as the gatekeeper for inflammation.It is produced by a variety of cells and acts on virtually every organ system of the body.IL-1 consists of two distinct proteins(IL-1α and IL-1β)that bind to the type 1 IL-1 receptor(IL-1R1)to activate a downstream pro inflammatory pathway.It has the critical position in the process of inflammation and chondrocyte apoptosis in TMD.IL-1β may stimulate the secretion of TNF-a,IL-6,ICAM-1 and other inflammatory cytokines and inhibit the synthesis of collagen,TIMPS and others to interfere with the metabolism of articular cartilage,which lead to chondrocyte degeneration,and promote the occurrence of chondrocytes and intracellular catabolism.It can obviously affect the synthesis of articular cartilage cells,and cause joint inflammation and destruction.It may be an important inflammatory factor for the damage of TMJ.The Notch signaling pathway consists of Notch receptors,Notch ligands,intracellular effector molecules,other effectors and Notch regulatory molecules.It plays a critical role in the growth,differentiation,and survival of various cell types in diverse tissues.It also plays an important part in maintaining articular chondrocyte phenotype and cartilage matrix metabolic balance.Dysregulation of this signaling pathway can lead to OA.There are few Notch molecules in healthy adult articular cartilage.But a large number of Notch signaling molecules appear in the damaged cartilage.However,the role of Notch1 signaling in IL-1β induced inflammation and destruction of temporomandibular articular chondrocytes has not been addressed.In this study,the in vitro model of TMJ chondrocytes were established.Chondrocytes were isolated from Spraguee Dawley rats and stimulated by IL-1β at lOng/ml.Notchl expression was inhibited by siRNA.The expression of Notchl protein,NICD protein,ICAM-1 protein,iNOS protein,MMP-1,MMP-9,TIMP-1,TNF-a,IL-6,ICAM-1 mRNA,Notchl mRNA,iNOS mRNA and phosphoric NF-κB p65 and NF-κB p65 protein in the cytoplasm and cytoplasm were detected by ELISA,RT-qPCR and WB.The research investigated the role of Notch1 siRNA in IL-1βinduced inflammation and destruction of temporomandibular articular chondrocytes,providing new research ideas for the clinical treatment of TMD.Material and MethodsIn this study,the in vitro model of TMJ chondrocytes were established.Chondrocytes were isolated from Spraguee Dawley rats and cultured in DMEM.They were serum-starved overnight before treatment,and from passage 1 to 3 were used in all experiments.The experiments were divided into 5 parts according to the different interference factors.experiment oneChondrocytes were divided into control group,the PBS group and IL-1β group.The chondrocytes in IL-1β group were stimulated with IL-1β.The expression of Notch1 mRNA,Notchlprotein and NICD protein was measured by RT-qPCR and WB.experiment twoChondrocytes were divided into control group,the Notch1 siRNA group and Notch1 Si-NC group.The chondrocytes in Notch1 siRNA group were stimulated with Notch1 siRNA.The chondrocytes in Notch]Si-NC group were stimulated with Notchl Si-NC.The expression of Notchl mRNA,Notch1protein was measured by RT-qPCR and WB.experiment threeChondrocytes were divided into control group,IL-1β group,the Notchl siRNA+IL-1β group and Notch1 Si-NC+ IL-1β group.The level of TNF-αand IL-6 secretion was measured by ELISA.The expression of ICAM-1 mRNA,iNOS mRNA,ICAM-1 protein,iNOS protein was measured by RT-qPCR and WB.experiment fourChondrocytes were divided into control group,IL-1β group and the Notchl siRNA+ IL-1β group.The expression of MMP-1,MMP-9,TIMP-1 was measured by WB.experiment fiveChondrocytes were divided into control group,IL-1β group and the Notch1 siRNA+ IL-1β group.The expression of phosphoric NF-κB p65 and NF-κB p65 protein in the cytoplasm and cytoplasm was measured by WB.Results1.IL-1β increased the expression of Notch1 and NICD.2.Notchl siRNA reduced the mRNA and protein expression of Notchl.3.Notchl siRNA reduced IL-1β-induced inflammatory response by reducing TNF-a and IL-6 secretion levels and reducing ICAM-1 and iNOS mRNA and protein expression.4.Notch1 siRNA reduced MMPs expression and increased TIMP-1 expression.5.Notch1 siRNA suppressed IL-1β induced NF-κB activation in chondrocytes.ConclusionThese data suggested that IL-1β could induce inflammation response in rat temporomandibular chondrocytes by activating the Notchl and NF-κB singnal pathway.Notch1 siRNA could inhibit the expression of mRNA and protein of Notch1 in rat temporomandibular chondrocytes.It prevented IL-1β induced inflammation response partly by inhibiting the nuclear translocation and phosphorylation of NF-κB p65 in temporomandibular chondrocytes which inhibited the activation of NF-κB pathway.Therefore,it could be concluded that Notch1 siRNA could reduce the inflammation and damage response of IL-1β induced chondrocytes in rat TMJ.It might be a theoretical basis for the treatment of TMD.