The Mechanism Of TREM-1 In IL-1? Induced Osteoarthritis

BackgroundOsteoarthritis(OA)is the most common degenerative joint disease and a major cause of pain and disability in adult individuals.Osteoarthritis is becoming an increasingly crucial risk factor of disability of the elderly.With the acceleration of aging of the population in our country,osteoarthritis has become a growing burden of the whole country.The etiology of OA includes joint injury,obesity,aging,and heredity.However,the detailed molecular mechanisms of OA initiation and progression remain poorly understood and,currently,there are no interventions available to restore degraded cartilage or decelerate disease progression.The triggering receptor expressed on myeloid cells-1(TREM-1),a novel cell surface receptor,is a member of the immunoglobulin superfamily that potently amplifies inflammatory responses via promoting secretion of pro-inflammatory mediators.A previous study reported that TREM-1 expression was increased in inflamed synovial membrane compared with normal/reactive synovial membrane obtained from the same patient with OA.However,the specific role of TREM-1 in OA remains unclear.Objective1.To detect the expression of TREM-1 in normal and osteoarthritis human cartilage tissues.2.To make a preliminary research on the effects of TREM-1 knockdown on the expression of TNF-?,MMP-13 and Type II collagen in IL-1?-stimulated chondrocytes.3.To make a preliminary research on the effects of TREM-1 knockdown on the proliferation and apoptosis in IL-1?-stimulated chondrocytes.4.To explore the effect of TREM-1 knockdown on expression of NF-?B pathway related genes,reveal the mechanisms of TREM-1 modulate the expression of TNF-?,MMP-13 and Type II collagen,and the proliferation and apoptosis in osteoarthritis chondrocytes.5.To explore the effect of TREM-1 knockdown on expression of MMP-13,Type II collagen and NF-?B pathway related genes in mice osteoarthritis modelin order to supply experimental evidence for the application of TREM-1 as a target for molecular therapy.Methods1.Sequential enzymatic digestion was used to harvest human knee articular chondrocytes.Chondrocytes were identified through toluidine blue staining and immunocytochemical staining of Type II collagen.2.The TREM-1 expression in human OA articular cartilage specimens and the normal articular cartilage tissues was analyzed by qRT-PCR and Western blot assay.3.Short hairpin RNA(shRNA)targeting TREM-1(shTREM-1)was synthesizedand subcloned into p LL3.7 vector,lentivirus was harvested 48 h after co-transfection of p LL3.7-shRNA with psPAX2 and pMD2.G into cells using Lipofectamine 2000 reagent.The expression of TREM-1 in chondrocytes transduced with shTREM-1 or NC was detected using qRT-PCR and Western blot assay.4.qRT-PCR,ELISA and Western blot were used to detect the effects of TREM-1 on the expression of TNF-?,MMP-13 and Type II collagen in IL-1?-stimulated chondrocytes transduced with shTREM-1.5.MTT was used to verify the effects of TREM-1 on proliferation of IL-1?-stimulated chondrocytes transduced with shTREM-1.6.The influence of TREM-1 on the apoptosis of IL-1?-stimulated chondrocytes transduced with shTREM-1 was detected by flow cytometry.7.The expression levels of NF-?B pathway related genes I?B? and p65 were detected by Western blot assay.8.Localization of cytoplasmic and nuclear NF-?B p65 in the indicated cells,determined with immunofluorescence staining.9.Mice articular chondrocytes were transfected with lentivirus-mediated TREM-1 shRNAs or with no-load lentivirus as the control.The suppression efficiency was measured using qRT-PCR and western blotting.OA was induced in male mice model by performing anterior cruciate ligament transection(ACLT)and partial medial meniscectomy(OA group).Half of the animals received an intra-articular injection of lentivirus-mediated shTREM-1(OA+shTREM-1 group).The sham operation group as the control group(NC group).Results1.The mRNA and protein expression levels of TREM-1 in human OA articular cartilage specimens were significantly elevated compared withthe normal articular cartilage tissues(P< 0.05).2.The chondrocytes were successfully harvested after digestion with trypsin and Type II collagen.Immunocytochemical staining of Type II collagen and Toluidine blue staining showed positive results and the purity of chondrocytes met the standard of this experiment.shTREM-1 and negative control was successfully transfected into articular chondrocytes with a high efficiency.The mRNA and protein expression of TREM-1 was significantly decreased in chondrocytes transduced with shTREM-1 compared with negative control group(P< 0.05).3.Knockdown of TREM-1 expression significantly alleviated IL-1?-induced downregulation of Type II collagen and upregulation of MMP-13 and TNF-?(P< 0.05);Data from the MTT assay indicated that cell viability is suppressed with IL-1? treatment,and conversely enhanced upon TREM-1 silencing;The percentage of apoptosis was increased in IL-1?-induced chondrocytes and reduced after transfection with shTREM-14.IL-1? induced activation of the NF-?B signaling pathway through increasing p65 and reducing I?B? expression,IL-1? also reduced cell viability and induced cell apoptosis in chondrocytes,which were inhibited by both PDTC,(an NF-?B blocker)and TREM-1 knockdown.Immunofluorescence analysis revealed that IL-1? induces rapid nuclear translocation of NF-?B,accompanied by a decrease in cytoplasmic NF-?B.Treatment with PDTC and TREM-1 knockdown alleviated these effects of IL-1?.5.The expression of TREM-1 was obviously higher in OA articular cartilage than the normal articular cartilage,but a significant decrease in TREM-1 expression in OA articular cartilage after treatment with shTREM-1.The significantly increase in MMP-13 and p65 expression,and the decrease in Type II collagen and I?B? expression of OA articular cartilage were alleviated by treatment with TREM-1 knockdown.Conclusion1.TREM-1 was highly expressed in human OA articular cartilage specimens.And the expression of TREM-1 was may be closely related to the occurrence and development of osteoarthritis.2.The chondrocytes could be successfully harvested after digestion with trypsin and Type II collagen with a high purity and vitality.shTREM-1 was successfully transfected into articular chondrocytes with a high efficiency.The expression of TREM-1 was significantly decreased in chondrocytes transduced with shTREM-1 compared with negative control group.3.TREM-1 knockdown could inhibit the expression of TNF-? and MMP-13,promote the expression of Type II collagen in human articular chondrocytes in vitro,which could make TREM-1 a potential target of molecular therapy of osteoarthritis.TREM-1 knockdown could promotethe proliferation and inhibit the apoptosis of human articular chondrocytes in vitro,which suggested that TREM-1 might play crucial role in the pathogenesis of osteoarthritis.4.IL-1? induced activation of the NF-?B signaling pathway through increasing p65 and reducing I?B? expression,and IL-1? reduced cell viability and induced cell apoptosis in chondrocytes,which was inhibited by bothPDTC,(an NF-?B blocker)and TREM-1 knockdown.These results suggest that knockdown of TREM-1 delays OA development via inhibiting the NF-?B pathway.5.The significantly increase in MMP-13 and p65 expression,and the decrease in Type II collagen and I?B? expression of OA articular cartilage were alleviated by treatment with TREM-1 knockdown.These results again suggest that knockdown of TREM-1 delays OA development via inhibiting the NF-?B pathway.