Dysbacteriosis-induced LPS Elevation Disturbs The Development Of Muscle Progenitor Cells By Interfering With Retinoic Acid Signaling

ObjectiveUnder normal physiological conditions,intestinal flora and human are mutually beneficial and symbiotic.Currently,many literatures have reported that intestinal flora disorders may affect human health.Intestinal flora disorders can increase the exposure of intestinal bacterial products,especially the exposure of LPS,which can cross the barrier of placenta and thus affect the growth and development of fetus.Although the LPS-induced negative influence on human health and even the developing fetus has been studied as described,we still know very little about if or how the embryonic somitogenesis and myogenic cell differentiation,the primordium of prenatal myogenesis,are affected by the exposure of maternal LPS.Combined with the advantages of the animal models of chicken embryo and mouse embryo,this study explored the effect of the increased LPS caused by the imbalance of intestinal flora on embryonic somitogenesis and myogenic cell differentiation,and its potential mechanism.MethodsFirstly,we use mouse embryos to construct a model of intestinal flora imbalance.We have determined the level of LPS in the plasma of female mice,assessed their embryonic growth and development,and then directly apply LPS to chicken embryos.H&E staining and immunofluorescence are uesd to observe the effects of LPS on chicken embryonic somitogenesis,myogenic cell differentiation and even muscle.Next,RNA sequencing,Elisa,QPCR,in situ hybridization and other methods were used to detect the changes of retinoic acid metabolism pathway,oxidative stress,and NF-B signaling pathway,so as to further explore the potential mechanism of the effects mentioned above.ResultsFirstly,our experimental results demonstrate that the imbalance of the maternal intestinal flora can expose the fetus to higher levels of LPS,which can inhibit the growth and development of the embryo,leading to embryonic somite development and muscle-derived cell differentiation disorders.Through RNA sequencing and later confirmatory experiments,we found that the metabolic pathway of retinoic acid plays a crucial role in this series of changes caused by LPS.At the same time,we found that LPS can activate oxidative stress and the NF-κB signaling pathway,so we used the antioxidant NAC and the inhibitor of the NF-κB signaling pathway Bay11-7082 to inhibit the relevant pathways and then detect retinoic acid metabolism and myogenesis.The results showed that the oxidative stress rather than the NF-κB signaling pathway plays a vital role in regulating retinoic acid metabolism and myogenesis.And then after AGN193109 inhibiting the effect of retinoic acid,we found that the protective effect of NAC on embryonic myogenesis disappeared,which further indicated that oxidative stress caused embryonic somitogenesis and myogenic cell differentiation disorder by affecting the metabolic pathway of retinoic acid.Finally,fecal microbial transplantation was used to improve the microbial community in the intestinal tract of mice with dysbacterial flora,further explaining that a series of negative reactions caused by intestinal bacterial flora disturbance were mainly due to LPS-induced retinoic acid metabolism disorders.ConclusionsIn summary,the results of this study suggest that the increase of LPS caused by the imbalance of intestinal flora can reduce the concentration of retinoic acid in the embryo by affecting the level of oxidative stress,and inhibit the development of embryonic somitogenesis and myogenic cell differentiation.