The Role And Mechanism Of SHH Signaling Pathway In Endotoxin Induced Acute Lung Injury And Metanephric Explant Culture In Mouse Model

Sonic Hedgehog(SHH)signaling play's a critical role in embryogenesis and adult tissue homeostasis.SHH ligand binds to the receptor Patched(PTCH).This results in the activation of a second transmembrane protein Smoothened(SMO).Once activated,SMO results in stabilization and nuclear accumulation of GLI family members GLI family zinc finger 1(GLI1),GLI family zinc finger 2(GLI2)and GLI family zinc finger 3(GLI3),defined as canonical Hh signaling.The sonic hedgehog(SHH)signaling pathway is an important signal transduction pathway for the formation of embryonic lung structure and its developmental processes.The activity of the pathway is greatly reduced after birth and in adult tissues,but is reactivated in some lung diseases,including pulmonary hypoplasia,interstitial lung disease,chronic obstructive pulmonary disease(COPD),asthma,and lung cancer.However,the role of the SHH signaling pathway in endotoxin induced ALI remains unclear.The SHH signaling pathway plays an important role during mammalian kidney development.Deletion of the SHH gene in humans has been linked to kidney malformations such as hydroureter.Abrrent SHH signaling is believed to be associated with the VACTERL syndrome,which involves renal anomalies.In the mouse,homozygous inactivation of SHH generates a series of defects including renal aplasia or dysplasia.The presence of renal hypoplasia/dysplasia of mice SHH deficient in the ureteric bud lineage demonstrates a crucial role for SHH signaling during mammalian renal development.However,the regulatory effect of how SHH protein modulates kidney development in mouse remains to be elucidated.In this study,we investigated the function of the SHH pathway in the response of ALI by using a LPS-induced mouse ALI model:and we also investigated the influence of SHH on embryonic kidney tissue culture to further clarify its role during the kidney development.Part I Sonic Hedgehog Signaling:Evidence for its Protective Role in Endotoxin Induced Acute Lung Injury in Mouse Model Objective:To investigate the protective role of the sonic hedgehog(SHH)signaling associated with a lipopolysaccharide(LPS)-induced acute lung injury(ALI)in a mouse model.Methods:Male BALB/c mice were randomly divided into four groups,control,LPS,LPS-cyclopamine group and cyclopamine group.ALI was induced by LPS ip injection(5 mg/kg).The sonic hedgehog inhibitor cyclopamine(50 mg/kg)was given to the LPS-cyclopamine group at 30 min after LPS injection as well as normal mice as control.Lung injury was observed histologically in hematoxylin and eosin(HE)stained tissue sections,semi-quantified by lung tissue injury score,and the lung tissue mass alteration was measured by wet to dry weight ratio(W/D).mRNA expression levels of TNF-a,SHH,Patched(PTC)and GLI1 in lung tissue were studied with real time quantitative PCR(RT-PCR),while the protein expression of SHH and GLI1 was determined by western blot analysis.Results:Lung tissue injury score,thickness of alveolar septa,W/D,and TNF-a mRNA expression levels were significantly higher in the ALI mice than the normal mice(P<0.05).The mRNA expressionlevels of SHH,PTC,and GLI1 in the ALI mice were significantly higher at 12h and 24h after LPS injection,but not at the 6h time point.Protein production of SHH and GLI1 at 6h,12h,and 24h in the lungs of ALI mice significantly increased,in a time-dependent manner,compared with that in normal mice.Cyclopamine alone has no effect on pathological changes in normal mice.Intervention with cyclopaminein ALI mice led to a reduction in mRNA levels of SHH,PTC,and GLI1 as well as SHH and GLI1 protein levels;meanwhile,the pathological injury scores of lung tissues,thickness of alveolar septa,W/D,and mRNA expression levels of TNF-a increased compared with mice receiving LPS only.Conclusion:The SHH signaling pathway was activated in response to LPS-induced ALI,and up-regulation of SHH expression could alleviate lung injury and be involved in the repair of injured lung tissue.Part II The SHH regulates FGF8 expression in metanephric explant culture from BALB/c mouse:possible mechanism related to renal morphogenesisAims:The morphogen sonic hedgehog(SHH)regulates cell differentiation and controls a hierarchy of genes during renal morphogenesis.To date,the effect of SHH on fibroblast growth factors(FGFs)in embryonic kidney development is still unknown.In this study,the embryonic kidney tissue culture from BALB/c mouse was used to elucidate the role of exogenous SHH on FGF8 and FGF10 expression in vitro.Main methods:Ureteric bud branches and epithelial metanephric derivatives were used to determine the renal morphogenesis with Dolichos Biflorus Agglutininin or Hematoxylin-eosin staining.The mRNA expression levels were quantitated using real-time reverse transcriptase PCR and the protein levels were examined using immunohistochemistry and western blot method.Key findings:The mRNA expression of Shh,Fgf8,and Fgf10 began at relatively low levels at E11.5 and increased with the development of metanephros.Exogenous SHH protein increased the number of ureteric bud branches and enhanced the formation of nephrons(p<0.05).In addition,SHH decreased FGF8 expression,while cyclopamine(a SHH-Smoothened receptor inhibitor)significantly increased FGF8 expression at both the mRNA and protein levels(p<0.05).Therefore,neither exogenous SHH protein nor cyclopamine modulates FGF10 changes in terms of the mRNA and protein levels(p>0.05).Significance:These results indicated that the modulation effects of SHH on metanephric explant culture from BALB/c mouse might involve FGF8 expression,which provided a possible explanation of renal morphogenesis.