| ObjectiveReported data show that the cytotoxicity of bacterial lipopolysaccharides(LPS)from microbiota or infection is associated with many disorders in clinic,including embryonic resorption,intra-uterine fetal death(IUFD),Intra-uterine growth restriction(IUGR)and preterm Labor and so on.Most of the intrauterine growth retardation is accompanied by skeletal growth retardation.However,the mechanism by which LPS affects bone development is not clear.In this study,chick embryos were employed to explore the potential mechanism of LPS-induced embryonic skeletal growth retardation.MethodsThe chick embryos were incubated for 1.5 days and then treated with 200μl of LPS(10μg/ml through the air chamber every other day.The embryos were harvested at embryonic 8-day and 17-day respectively.The overall skeletal development was observed by Alcian Blue & Alizarin Red double staining.Morphological analysis were done by H&E staining of paraffin-embedded sections,to show the changes of differernt zones in phalanges growth plate.The expression of key genes regulating skeletogenesis were detected by biochemistry methods.In vitro cell transfection of si RNA/sh RNA was employed to evaluate the modulatory effects of Sox9 and Nrf2 in the LPS-induced skeletal retardation.ResultsIt was demonstrated that LPS exposure inhibited long bone length of the 8-day and 17-day chick embryos by Alcian Blue & Alizarin Red-staining respectively.Further analysis of the growth plates in phalanges showed that the proportion of proliferating zone(PZ)increased and hypertrophic zone(HZ)decreased following LPS exposure,while there was no significant change on chondrocytes’ proliferation in the growth plates.Combining results of immunofluorescent staining,Western blot and quantitive PCR revealed that Sox9 and Col2a1 were up-regulaged by LPS exposure at both m RNA and protein level.Furthermore,LPS exposure caused down-regulation of Runx2 and Col10a1 expression in 8-day chick embryos’ hindlimbs,also suppressed Vegfa expression in embryonic 17-day phalanges.Knocking-down Sox9 in ATDC5 cells by si RNA transfection led to the down-regulation of Col2a1,Runx2,Col10a1 expression,implying the vital role of Sox9 in the process of LPS-induced delay from proliferating chondrocytes to hypertrophic chondrocytes in the growth plate.Moreover,Nrf2 was highly expressed in presence of LPS,and the activities of SOD1,SOD2 and GLRX rose in both 17-day embryonic phalanges and ADTC5 cells,so was intracellular ROS.When Nrf2 expression was knocked-down in ATDC5 cells,the expressions of Sox9,Col2a1,Runx2,Col10a1 and Vegfa were going down as well.ConclusionsOur data suggested that LPS exposure during development could restrict the conversion of chondrocytes from proliferating to hypertrophy in the growth plate,in which LPS-induced elevation of Sox9 plays a crucial role to trigger the cascade of downstream genes by excessive ROS production and Nrf2 elevation. |
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