JAK2/STAT5 Signaling In The Lipopolysaccharide (LPS)-induced Inhibition Of Osteoblast Differentiation

BackgroundThe normal function coupling of osteoblast cells and osteoclast cell is an important factor in fracture healing.Local infection of fracture can cause dysfunction of osteoblast and osteoclast at the ends of fracture,interfere the normal function coupling of osteoblast and osteoclast,and form independent dead bone from ends of fracture.The dead bone form a vicious cycle of uncontrollable infection and we named it refractory nonunion.Infection of fracture is usually mixed infection,and Gram-negative bacteria are involved.Lipopolysaccharide is one of the main components of the cell wall of Gram-negative bacteria.However,it is not clear whether lipopolysaccharide interferes with the osteoblasts to leads maladjustment of normal function coupling maladjustment.JAK-STAT signaling pathway plays an important role in the occurrence,maturation,differentiation and apoptosis of osteoblasts.Based on the previous literatures and the experimental results,we demonstrate that LPS can activate JAK2/STAT5 pathway,and interfere the differentiation of osteoblast;Blocking the JAK2/STAT5 signaling pathway can reverse the function of osteoblast differentiation.Objective1.In vitro,we study the effects of lipopolysaccharide at varying concentrations on osteoblast activity and osteogenesis function index,RUNX2,BMP-2,PINP,TLR-2,TLR-4,STAT5b,phosphorylation STAT5b and JAK2 protein expression,and provide the molecular biological basis for lipopolysaccharide acting on osteoblast.2.Constructing siRNA-JAK2 by transient transfection technology in MC3T3-El cell,to detect the effects of different concentrations of LPS on osteoblast activity,osteogenesis function index RUNX2,BMP-2 and PINP and TLR-2,TLR-4 expression.3.In vivo,by using animal model,we randomly assigned control group,fracture+LPS group,fracture of +LPS+WP1066 group to observe the changes of fracture healing at 2 weeks and 4 weeks.We detect the bone mineral density,trabecular separation index,by using safranin fast green staining,HE staining and Immunohistochemical staining of OCN,to measure the fracture degree,cartilage bone changes of osteoblast number.It indicates if JAK2-STAT5 signaling pathway play a role in affection of lipopolysaccharide on osteoblast growth and differentiation and lay the foundation for the treatment of refractory nonunion.Materials and Methods:Part 11.Cell cultureMC3T3-E1 cells were cultured in a-minimum essential medium.(a-MEM;containing 50 ?g/ml of ascorbic acid and 10 mM of ?-glycerophosphat)(Life tech,USA).2.Mineralized nodules staining.ALP activity detection Assay.3.Detection of ALP,BMP-2,RunX-2,and PINP expression assay4.MTT cell proliferation assay5.Western Blot Analysis:To test the expressions of TLR-2?TLR-4?STAT5b protein in osteoblast interfered by lipopolysaccharide.Part21.Cell culture2.Small Interference RNA(siRNA)Assay3.Western Blot Analysis:The Jak2 expression in MC3T3-E1 cell were tested after transfection by using westen blot.4.Mineralized nodules staining:The mineralized points in siRNA-Jak2 MC3T3-E1 cell were counted by using von Kossa staining.5.Quantitive Real time-PCR:The expression of TLR2.TLR4 were tested by using quantitive Real time-PCR.6.ELISA assay:The protein expressions of ALP,RUNX,BMP-2 and PINP were detected by enzyme linked immunosorbent assay(ELISA)after transfection.Part 31.Mouse model and surgical methods.The middle and upper tibial fracture in C57b1/6 mice was treated with intramedullary nail fixation.2.The fracture healing were tested by X-ray examination and bone mineral density(BMD)and bone trabecular separation(Tb.Sp)were tested by Micro-CT.3.The osteogenesis of chondrocytes were observed by using safranin O fast green staining.4.The number of osteoblasts were detected by using HE staining,and OCN immunohistochemical staining.Statistical AnalysisThe IBM SPSS Statistics 21 was used for statistical analysis.Data are depicted as the mean± standard deviation that is calculated from three independent experiments.For the data which basically conform to normal distribution or partial normal distribution,the independent group t test(Independent-Samples T Test)and one-way ANOVA were used to compare the mean values between 2 groups or more than 2 groups.For data which do not accord with normal distribution,two groups of independent samples and nonparametric tests were used to compare the mean values between 2 groups or 2 groups.The test methods were Mann-Whitney U test and Kruskal-Wallis H test respectively.P<0.05 indicates a statistical difference.Conclusion1.With the rising of lipopolysaccharide concentration,MC3T3 E1 osteoblast mineralization-point droped,the level of the mineralized points and the concentration of lipopolysaccharides were dose dependent.2.Lipopolysaccharides interfed MC3T3 E1 osteoblast,activity of alkaline phosphatase levels decreased.Lipopolysaccharides interfeed MC3T3 El cell,osteoblast cell activity decreased,osteogenesis differentiation related proteins RUNX2,BMP-2 and PINP content gradually decreased,the three levels and LPS are dose dependent.3.With lipopolysaccharide interference MC3T3-E1 cell,steoblast toll-like receptors after 2 and 4 mRNA expression level increased gradually,the expression level depend to the lipopolysaccharide.With difference concentrations of lipopolysaccharide interference MC3T3 E1 cell,except STAT5b,TLR-2,TLR-4,phosphorylation STAT5b and the expression of JAK2'protein expression levels increased,and there was dose dependence.4.JAK2-specific siRNA(siRNA-JAK2)transfection MC3T3 El osteoblast,JAK2 mRNA and protein expression levels drop.5.Lipopolysaccharide induce JAK2-specific siRNA(siRNA-JAK2)transfection of MC3T3 E1 cell,osteoblast ossification-mineralized points transfection group were more than of transfection cell mineralization points.Lipopolysaccharides interference JAK2-specific siRNA(siRNA-JAK2)transfection osteoblast MC3T3 E1-after osteogenetic differentiation related proteins RUNX2,BMP-2 and PINP increased.6.2 weeks after surgery,the chondrocyte ossification in the control group and the treatment group were higher than that in the inflammatory group,and the fracture healing in the control group and the treatment group was faster than that in the inflammation group.The tibial fracture was healed and the healing process of the inflammatory group was slower compare with the control group and the treatment group.7.2 weeks after surgery the bone mineral density in the control group and the treatment group were higher compare with it in the inflammatory group.The bone trabecular separation in control group and the treatment group were lower than those in the inflammatory group.8.2 weeks,4weeks after surgery,the number of osteoblasts in the control group and the treatment group was more than that in the inflammatory group,and the osteogenesis was active.