Hedgehog Signaling Pathway Inhibitor Vismodegib Induces Hypoglossia In Mice And Its Related Mechanisms

Background:Tongue is an important organ of chewing,swallowing and articulation.Hypoglossia has seriously affected people’s lives.It is generally believed that the development of the tongue is influenced by genetic and environmental factors.The development of tongue is the result of the interaction between mesenchymal cells from nerve spines and myogenic cells from occipital nodules.Hedgehog signaling pathways are involved in regulating the development,differentiation and formation of the tongue muscle.Objective:In this study,Vismodegib(GDC-0449),a small molecule inhibitor,was used to specifically inhibit the Hedgehog signaling pathway in the early stage of mouse tongue muscle development to establish a stable model of hypoglossia,observe and study the deformity model and further explore its possible molecular regulation mechanism.Methods:1.SPF-grade ICR pregnant mice were taken,and the experimental group received150 mg/kg Vismodegib orally at E10.5,while the control group received the same amount of solvent through gavage,and the fetal mice were disemboweled at E15.5.The incidence of malformation of tongue was observed and statistically analyzed under the stereomicroscope.2.RT-q PCR was used to dynamically detect the expression of Ptch1 and Gli1 in the first branchial arch,and analyze the expression of Hedgehog signaling pathway after single dose administration of Vismodegib.3.HE staining was performed on E11.5-E15.5 fetal mice to observe the morphological characteristics of the development of hypoglossia.4.The experimental group of E11.5-E15.5 and control group fetal mice were subjected to immunofluorescence staining of phosphorylated histone H3 as a proliferation indicator and TUNEL fluorescence staining as a apoptosis indicator,to analyze the proliferation and apoptosis of tongue muscle cells.5.Immunofluorescence staining was performed on E11.5-E15.5 experimental group and fetal mice of the control group to detect Myod expression characteristics of myogenic regulatory factor family members,and immunofluorescence staining was performed on E13.5 experimental group and control group to detect Myf5 expression distribution characteristics of myogenic regulatory factor family members.Results:1.At E10.5,150mg/kg Vismodegib in the experimental group after single dose gavage had a higher incidence of tongue deformity(90.5%)and a lower mortality(4.7%).2.RT-q PCR dynamic analysis showed that the expression levels of Gli1 and Ptch1 genes were significantly down-regulated after single dose gavage of 150mg/kg Vismodegib(P<0.01).3.HE staining showed E11.5 experimental group and the control group no significant morphological differences,E12.5 the development of tongue was obviously inhibited in the experimental group,E13.5-E15.5 experimental tongue muscle decreased significantly compared with control group,At high magnification of E15.5,the muscle fibers of the experimental group and the control group showed a significant difference in muscle fibers walking,In the control group,the vertical muscle fibers of tongue and striated muscle fibers interspersed with each other,In the experimental group,the muscle fibers were differentiated abnormally and disorderly,with less striated muscle,but there was no significant difference in the development of genioglossus.4.Immunofluorescence results showed that the expression of Myod showed no significant difference between the experimental group and the control group at day E11.5,but the expression of PHH3 in the experimental group was lower than that in the control group.On E12.5 days,the expression of Myod and PHH3 in the control group was higher than that in the experimental group.On E13.5 days,the expression of Myf5 in the control group was higher than that in the experimental group,but there was no significant difference in the expression of PHH3 and Myod between the experimental group and the control group;The expression of PHH3 and Myod in E15.5 striated muscle in the control group was higher than that in the experimental group.TNNEL fluorescence of E11.5-E15.5 showed increased apoptosis in the experimental group.Conclusion:Hedgehog signaling inhibitor Vismodegib was administered at E10.5 to effectively inhibit Hedgehog signaling and establish a stable mouse model of malformation of tongue.The formation of hypoglossia is related to the decreased proliferation ability and the enhanced apoptotic ability of neural crest derived mesenchymal cells after Hedgehog signal inhibition,and the down-regulated expression of myogenic regulatory factors.