| Objective:Temporomandibular joint osteoarthritis(TMJOA)is a maxillofacial disease which affects the oral and maxillofacial function.Condyle cartilage defect can hardly self-heal and its molecular mechanism of degeneration is unknown.Mechanical stimulation directly affects the cartilage,inflammation is widely involved in the degeneration of condylar cartilage.Cells release a large amount of inflammatory cytokines such as Interleukin-1beta(IL-1?)and Interleukin-18(IL-18)during cell pyroptosis and anticipate chronic inflammation.However,there are few studies on pyroptosis in chondrocytes.The present goal of this study is to explore the expression of NLRP3/Caspase-1 canonical pathway in chondrocytes induced by abnormal stress and provide clues for the targeted therapy of TMJOA.Methods:Part 1:(1)In clinical study,The synovial fluid of patients with temporomandibular disorders in the First Affiliated Hospital of Xinjiang Medical University was collected and divided into TMJOA group with condylar bone destruction and temporomandibular joint degeneration group without condylar bone destruction according to cone beam computed tomography(CBCT)or magnetic resonance imaging(MRI)detection.The clinical data were recorded.(2)The expression of inflammatory mediators of IL-1?,IL-18,Caspase-1and NLRP3 were detected by ELISA.Part 2:(2)At the histological level,TMJOA model was established by Unilateral anterior crossbite(UAC)stimulation in SD rats for 4 weeks,8 weeks and 12 weeks.The morphology and structure of cartilage were observed by stereomicroscope,BV/TV(trabecular bone area fraction),BS/BV(bone surface area and bone volume ratio)and Tb.Th(Trabecular thickness),Tb.N(Number of trabeculae)were detected by Micro-CT and analyzed with Mimics 20.0 software to evaluate the remodeling of subchondral bone.The characteristics of cartilage lesions were evaluated by histopathological examination and scoring system.(2)Immunohistochemical technique was used to detect the expression and distribution of IL-1?and IL-18,NLRP3,ASC,Cysteinyl aspartate specific proteinase(Caspase-1),gasdermin D(GSDMD)in normal group and UAC-induced group.Real-time polymerase chain reaction(RT-PCR)and Western blot(WB)were used to detect the expression of key genes and proteins of pyroptosis in the two groups.Part 3:(1)At the molecular level,chondrocytes of SD rats were isolated and cultured,and chondrocytes were treated with Fluid Flow Shear Stress(FFSS)of 0 dyn/cm~2,4 dyn/cm~2,12 dyn/cm~2,16 dyn/cm~2for 1 hour respectively and FFSS of 12 dyn/cm~2 for 1hour,2 hours and 4 hours respectively.The expression of IL-18,IL-1?in the supernatant of chondrocyte culture medium was analyzed by ELISA technique.(2)The expression of cell death in each group was compared by AO-EB staining.(3)The expression of IL-1?and IL-18,NLRP3,ASC,Caspase-1,GSDMD in each group was detected by Western blot technique.Results:Part 1:(1)TMJOA patients with condylar bone destruction and TMJID patients without condylar bone destruction tend to exist in young and middle-aged women and had symptoms of pain and dysfunction of temporomandibular joint,the number of functional disorders in TMJOA group was relatively large.(2)The expression of IL-1?and IL-18,Caspase-1,NLRP3 related inflammatory mediators in synovial fluid of TMJOA and TMJID group was compared.It was proved that the concentration of IL-1?and IL-18,Caspase-1,NLRP3 in TMJOA patients with condylar bone destruction was higher than that in TMJID(P<0.05).Part 2:(1)TMJOA could be induced by UAC stimulation,cartilage deformation could be seen under stereomicroscope.With the prolongation of UAC,the lesion was more serious than that of normal control group at the same time.Micro-CT showed that BV/TV decreased,subchondral bone loss was obvious,and BS/BV increased at 4 weeks after UAC induction with a short time bone remolding,there was no significant difference between control group and UAC induction for 8 weeks and 12 weeks.Tb.Th decreased at 12 weeks after UAC induction,Tb.N decreased at 8 weeks and 12 weeks after UAC induction,(P<0.05).The histopathological score increased in UAC induction group.(2)Immunohistochemistry showed that IL-1?and IL-18,NLRP3,ASC,Caspase-1,GSDMD were highly expressed in the cartilage layer of UAC group.RT-PCR and Western blot also showed that the expression of pyroptosis related genes and proteins in UAC group was higher than that in normal control group at the same time(P<0.05).Part 3:(1)ELISA showed that the expression of IL-1?and IL-18 in the culture medium supernatant of 4dyn/cm~2,12 dyn/cm~2and 16 dyn/cm~2FFSS intervention groups was higher than that of 0dyn/cm~2 FFSS control group(P<0 05).Under the stimulation of 12 dyn/cm~2 FFSS,the expression of pyroptosis molecules of chondrocyte increased with the prolongation of FFSS intervention time(P<0.05).(2)AO-EB staining showed that the number of cell pyroptosis increased with the increase of FFSS load size and stimulation time.(3)Western blot showed that the expression of IL-1?and IL-18,NLRP3,ASC,Caspase-1,GSDMD of chondrocyte was up-regulated with the increase of FFSS load and the prolongation of action time(P<0.05)Conclusion:(1)Inflammatory mediators related to pyroptosis were higher expressed in synovial fluid of OA.(2)UAC can induce TMJOA lesion,pyroptosis related proteins are expressed in the condylar cartilage layer,the expression level was increased with the severity of OA lesions.(3)The pyroptosis of chondrocytes was aggravated with the increase of the loads size and time of FFSS. |
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