Effect Of Don On Expressions Of PAX1in Mouse Osteoblast

ObjectiveUSing the vitro Mouse osteoblastic cells model,we discuss the influence ofdeoxynivalenol on the proliferation, apoptosis, and mineralization of the osteoblast andthe Pax1, Bcl-2and Bax gene expression. Thus to reveale the molecular mechanism ofDON influencing osteoblast growth and differentiation and to make the theoretical basisfor the prevention of DON-induced skeletal deformities.Methodswe cultured the mouse osteoblasts in vitro, adding different concentrations of DON(0、500、1000、1500ng/ml) to the logarithmic growth phase of osteoblasts about48hours.Thiazolyl tetrazolium (MTT) assay to detect DON osteoblast proliferation; flowcytometry to detect osteoblast apoptosis; Trizol cracking cell extracts osteoblast total RNAand RT-PCR detect mRNA expression of Pax1gene; RIPA cell lysates detect the proteinlevel of Pax1gene.Results1The impact of DON on the proliferation of osteoblasts:Different concentrations of DON were added to the logarithmic growth phase ofosteoblasts, observing the role of DON on the proliferation of osteoblasts. MTT assayshowed that: the absorbance value was gradually decreased with increasing DONconcentration, treatment time. When DON concentrations were100ng/ml,200ng/ml,theabsorbance value did not change significantly. AS DON concentration of500ng/ml,1000ng/ml,1500ng/ml, significant cytotoxic effects were presented, the absorbance valuedecreased significantly with the blank control group were statistically significant (P <0.05).AT the group of the same concentration of DON, the longer the incubation time, the lowerthe absorbance value. Histogram can be seen from the inhibition of cell proliferation: the number of cells gradually decreased with the increase in DON concentration and treatmenttime，indicating that the DON can inhibit the proliferation of osteoblasts.2. DON affects osteoblast apoptosis:Adding different concentrations of DON to the logarithmic growth phase ofosteoblasts, the flow cytometry results showed that: the proliferation index decreased withincreasing DON concentration. Compared with the control group, there are significantdifferences.The AO-EB fluorescence staining results showed that the nucleus of the control groupwere all dyed green fluorescent with normal morphology. Afetr DON500、1000ng/ml48h，Most of the cell nucleus staining enhanced fluorescence, but the nuclei began to shrinkageor beaded, early apoptotic nuclei, but also part of the nuclear chromatin was orangefluorescence, and nuclei fragmentation occurs, the performance of late apoptotic nuclei.1500ng/mlDON for48h, the number of cells significantly reduced cell performance of lateapoptosis. Western blot showed that: the500,1000,1500ng/ml of DON,Bcl-2proteinexpression was decreased and Bax protein expression increase, the ratio of Bcl-2/Baxdecreased. The above experiments showed that the DON has significantly induce apoptosisof bone cells.3. DON affects PAX1gene expression:RT-PCR gel electrophoresis: DON significantly increased mRNA expression of Pax1gene in osteoblasts. And with the increase of the DON concentrations Pax1geneexpression gradually increased.500ng/ml,1000ng/ml,1500ng/ml groups compared withthe control group was statistically significant (P <0.01).Western blot showed that: DON significantly increased the protein levels of Pax1gene in osteoblasts. And with the increase in DON concentration, Pax1gene expressionlevels gradually increased. Pax1bands of gray value is gradually enhanced.500ng/ml,1000ng/ml,1500ng/ml groups compared with the control group was statistically significant(P <0.05).Conclusion1. DON has a direct toxic effect on osteoblasts and can inhibit the proliferation ofosteoblasts and osteoblast mineralization function to reduce.2. Adding DON to the osteoblasts, the Pax1and Bax expression increased,while Bcl-2gene expression decreased, thus DON affects the proliferation and apoptosis of osteoblasts.