| The development of modern surgery has brought new ideas and techniques for the treatment of fracture.However,the incidence of nonunion is still high.Therefore,the mechanism and influencing factors of fracture healing remains to be further studied.Osteoblasts(OBs)play an important role in bone fracture healing,the proliferation and differentiation of OBs is closely related to microenvironment in fracture sites,Therefore,it is important to study how OBs behave in the complex fracture microenvironment.Autophagy plays a pivotal role in maintaining cellular homeostasis and defending the cell against adverse microenvironments.Studies have shown that ischemia,oxidative stress and hypoxia can lead to cell autophagy in vitro.However,an acidic microenvironment also induced in the fracture regions,Which remains unclear whether acidic microenvironment induces autophagy in osteoblasts.Therefore,the study of autophagy and related mechanisms of osteoblasts in acidic microenvironment may provide new ideas for improved osteogenesis and the prevention and treatment of bone nonunion.Purpose:By simulating the acid microenvironment of the fracture sites,aim to investigate the influence of acidic media with low pH value on cell proliferation and apoptosis of osteoblasts.Also we detect the autophagy flu in osteoblasts,hoping to provide more evidence for improving the survival rate of fracture sites osteoblasts and accelerate the development of bone tissue engineering.Methods:1.Experiment in vivo27 KM rats(6week,male)were selected and divided into three groups A,B,C and bone fracture mode was established on the right legs of the rats,while the left legs was designed as the control group.Rats in group A,B and C were killed in 6h,24h,36h respectively.These samples were fixed,decalcified and embedded.Immunofluorescence staining was used to evaluate the expression of LC3 and p62.2.Experiment in vitroWe divided the cells into three groups according to the different pH:pH 6.4,6.8 and 7.4.Osteoblasts were cultured in 96-well plates,after the cells were exposed to different pH(6.4,6.8,7.4)for different time(12h,24h,48h).Cell viability was detected by MTT assay.After the cells were exposed to different pH for 24h,apoptosis was determined by flow cytometry.LC3 expressed in cells was detected by immunofluorescence staining after incubated for 6h.TEM was carried out to confirm the number of cytolysosome and the changes of morphological in the osteoblastic cells after cultured in acidic media for 6h.Western blot was used to analyze of autophagy marker protein LC3 and p62 to monitor acidic conditions of osteoblast autophagy flow.In order to study the relationship between apoptosis and autophagy,MC3T3-E1 cells were exposed to autophagy inhibitor CQ,which the apoptosis was detected as above.SPSS 16.0 was used to analyze the datas,one-way ANOVA was applied to evaluate the difference between groups.Results:1.Immunofluorescence staining in vivo:Immunofluorescence staining was used to detect the expression of LC3 and p62.When compared with the control group,samples in experimental group show a stronger fluorescence of LC3(p<0.05),while p62 was weaker(p<0.05).It indicated that autophagy was induced in the fracture sites.2.MTT assay results:Both pH values of 6.4 and 6.8 induced a significant decrease in cell viability compared with that of pH 7.4(p<0.05),and a significant difference in viability between pH value of 6.4 and 6.8(p<0.05).Furthermore,a pH-and time dependent decrease in cell viability was observed.pH inhibits cell viability in osteoblastic cells line.3.Flow cytometry results:After exposed in media for 24h,compared with cells cultured at pH 7.4,those at pH 6.4 and 6.8 exhibited statistically significantly increased levels of apoptosis and pH 6.4 is the most(p<0.05).pH induced cell apoptosis in osteoblastic cell line.4.TEM results:The morphology of the cells was changed after exposure to low pH media 6h,with more autophagosomes observed in the pH6.4 conditions.The typical double-membrane structures surrounded with cytoplasm and mitochondria were found.5.Western blot analysis:It showed that after 6h of cell culture,an inverse correlation was observed between the increasing expression of LC3 in OBs and decreasing pH of the media.Under prolonged exposure to acidic conditions,the expression of LC3 in OBs decreased(p<0.05)?Furthermore,inhibition of autophagy using chloroquine(CQ)markedly increased the expression of LC3-II(p<0.05).Our results suggested that reduction of environmental pH increased the expression of LC3 and reduced the levels of P62.6.Immunofluorescence staining analysis:In addition,this study has shown the cellular location of LC3 is restricted to in the cytoplasm of osteoblasts under acid environment,and with the increase of pH,fluorescent expression decreased(p<0.05).Acid environment induce the autophagy.7.Apoptosis of the cells in acid media after CQ was added:The rate of cellular apoptosis increased when cells were treated with CQ in parallel with exposure to acidic conditions(p<0.05).Cells inhibit cell apoptosis by enhancing autophagy.Conclusions:1.Acid environment is harmful to the cell growth.It can inhibit the cell vitality,accelerate cell apoptosis and induce the autophagy of cells in vitro.2.Autophagy was induced in the fracture sites in vivo.3.Cells inhibit cell apoptosis by enhancing autophagy,to improve the survival rate of OBs in the acid environment. |
PDF Full Text Request