Expression Of TIPE2 In Patients With Osteoporosis And Its Preliminary Mechanisms In The Pathogenesis Of Osteoporosis

[Objective]Osteoporosis(OP)is a common senile disease,and its pathological features are characterized by decreased bone mass and destruction of bone micro-structure,resulting in decreased bone strength,increased fragility and easy fracture posibility.Patients with spinal and hip fractures caused by osteoporosis can cause serious complications such as pulmonary infection and thrombotic diseases due to prolonged bed rest,which ultimately leads to fatal harm.At present,osteoporosis has become one of the senile diseases worldwide.Epidemiological investigations show that the risk of fracture in osteoporosis patients is as high as 40%,and about half of white women have osteoporosis after menopause.China has now become the country with the most elderly population in the world.With the extension of life expectancy.the incidence of this disease is gradually increasing.Therefore,in-depth study of the mechanisms of osteoporosis,identification of its key pathogenic genes,search for new targets for the early diagnosis and treatment of osteoporosis and the development of new drugs need to be further explored.It is clear that chronic inflammation 1s closely related to the occurrence of osteoporosis,and activation of NF-κB is the most important stage.Studies have shown that the loss of p50 and p52 can cause osteoclast deficiency,which causes severe osteopetrosis.This study strongly suggests that osteoclast differentiation depends on RANKL-induced NF-κB activation.TIPE2(tumor necrosis factor-a induced protein 8-like 2,TNFAIP8L2)belongs to the TNFAIP8 family together with TNFAIP8L(TIPE),TNFAIP8L1(TIPE1)and TNFAIP8L3(TIPE3).It is an important new inflammation and immune related family discovered in 2008.The imbalance of expression of TIPE2 is closely related to inflammatory diseases such as systemic lupus erythematosus,hepatitis B and diabetic nephritis.During inflammatory process,RANKL-RANK activates NF-κB to cause excessive activation of osteoclasts,leading to imbalance of bone turnover,which leads to decreased bone mass and even osteoporosis;TIPE2 may affect NF-κB activation during LPS-induced inflammation,but whether or not it regulates NF-κB during osteoclast activation is still unknown.Based on the previous literature,this study will use a variety of molecular biology methods to explore how TIPE2 affects the activation of osteoclasts through negative regulation of NF-κB pathway,and thus participates in the process of osteoporosis.[Methods]1.Clinical specimens were analyzed the correlation between TIPE2 and inflammatory factors,osteoclast activation markers.The study populations(n=80)were collected from Zibo Central Hospital Affiliated to Shandong University.The age-and sex-matched healthy controls(n=90)were randomly recruited from Zibo Central Hospital Medical Center and excluded from having any bone disease and metabolic or other inflammatory related diseases.The study was approved by the local ethics committee of Zibo Central Hospital Affiliated to Shandong University.All subjects provided informed eonsent to join the study.PBMCs were isolated by density gradient centrifugation from the peripheral blood anticoagulated with sodium citrate.Cell pellets were lysed with TRIzol reagent and then total RNA was extracted according to the instructions of manufacturer,quantified by photometrical measurement.Serum was separated and prepared for the analysis.Commercially available sandwich enzyme-linked immunosorbent assay(ELISA)kits were used to measure TNF-α,IL-1,IL-6 levels according to the manufacturer’s instructions.Serum fasting PINP and β-CTX were measured by the chemiluminescence method.Ⅱ.The mechanisms of TIPE2 involved in osteoporosis.Mouse bone marrow-derived macrophages and RAW264.7 cell line were infected with TIPE2 siRNA lentiviral vector(TIPE2-si),and then stimulated with RANKL to detect osteoclast activation markers TRAP,Mmp9,Cathepsin K,NFATcl and NF-kB signaling pathway involved genes such as IL-6,Birc3,Tnf-α,Cox-2;Western blot method was to detect the p-p65 levels;detection of inflammatory factors(IL-1β,IL-6,TNF)in supernatant using ELISA method.[Results]1.Analysis of the correlation between TIPE2 and inflammatory factors and osteoclast activation markers1.TIPE2 expression level in patients with osteoporosis was significantly lower than that of healthy controls both in mRNA and protein levels.Interestingly,the TIPE2 mRNA expression was inversely correlated with serum IL-1,IL-6 and TNF-α.Those results indicated that TIPE2 may participate in osteoporosis,and suppress the secretion of inflammatory cytokines in the pathogenesis of osteoporosis.2.We analyzed the relationship between TIPE2 and p-CTX or PINP.The results showed that TIPE2 inversely correlated with β-CTX.However,there was not reflected in an association between PINP and TIPE2.The inverse association between bone resorption assessed using β-CTX and TIPE2 suggested that TIPE2 might exert its function via suppression of osteoclastogenesis.3.The results showed that TIPE2 had a significant positive correlation withFemoral neck(FN)and Lumbar spine(L1-L4)in patients with osteoporosis,suggesting that TIPE2 serves as a protective function involved in osteoporosis.4.We observed its predictive function for bone fracture using Simple Logistic Regression method.TIPE2 expression was significantly lower in fracture group than that of control group.More interestingly,TIPE2 exhibited significant odds ratios to predict bone fracture in patients with osteoporosis.The ROC analysis was performed and AUC value was calculated for the predictive function of TIPE2.5.The clinical investigation has preliminarily demonstrated that TIPE2 serves as a protective factor in the pathogenesis of osteoporosis.In order to further investigate its underlying function in osteoporosis,the monocytes from three patients with osteoporosis were separated and cultured in DMEM,and then stimulated with LPS after transfected with or without TIPE2.The result showed that there had either decreased proinflammatory cytokines in culture supernatant or blunted activity of the NF-kB pathway after TIPE2 transfection,indicating that TIPE2 can weaken the sensitivity of monocytes to LPS.Ⅱ.To study the mechanisms of TIPE2 involved in osteoporosis.1.Mouse bone marrow-derived macrophages and RAW264.7 cell lines were infected with TIPE2 siRNA lentiviral plasmid(TIPE2-si),and then stimulated with RANKL,TRAP staining showed that there was a significant increase regarding osteoclast activation after downregulting TIPE2 expression.2.Mouse bone marrow-derived macrophages and RAW264.7 cells were interfered with TIPE2-S1,and then stimulated with RANKL,osteoclast activation markers such as Trap,Mmp9,Cathepsin K(Ctsk),Nfatcl and NF-κB pathway activation indicators 11-6,Birc3,Tnf-a and Cox-2 were significantly up-regulated.3.ELISA method was used to detect the NF-kB regulatory genes such as TNF-α,IL-1β,IL-6 in the culture supernatant,and the results showed that the inflammation factors were elevated after TIPE2-si.4.The result showed that elevated activity of the NF-κB pathway after TIPE2-si.[Conclusion]I.1.Decreased TIPE2 expression level in PBMCs from patients with osteoporosis and 1s negatively correlated with proinflammatory cytokines,fasting serum β-CTX,but not with PINP.TIPE2 may suppress the secretion of inflammatory cytokines and the osteoclastogenesis in the pathogenesis of osteoporosis.2.TIPE2 expression level is positively correlated with BMD,which can predict the risk of bone fracture3.TIPE2 protects monocytes from being stimulated with LPS in patients with osteoporosisⅡ.1.TRAP staining showed that there was a significant increase regarding osteoclast activation after downregulting TIPE2 expression.2.Osteoclast activation markers such as Trap,Mmp9,Cathepsin K(Ctsk),Nfatcl and NF-κB pathway activation indicators 11-6,Birc3,Tnf-α and Cox-2 were significantly up-regulated when interfered with TIPE2-si.3.NF-kB regulatory genes such as TNF-α,IL-1β,IL-6 were elevated after TIPE2-si.4.The activity of the NF-κB pathway was elevated after TIPE2-si.