Effects Of Hedgehog Signaling Pathway On The Goblet Cell Metaplasia In Asthma

Background and objectives:Bronchial asthma (asthma) is one of the most common respiratory diseases. The chronic inflammatory of airway involved a variety of cells and cell components. Hedgehog (HH) signaling pathway not only plays key roles on embryonic development, but also on postnatal development and maintenance of tissue/organ integrity and function. In recent years, it has been found that repair of respiratory system after acute injury is similar to the development of fetal lung and HH signaling pathway is activated obviously in the process of repair. Goblet cells are situated in the epithelium of the conducting airways and they form a physical barrier complemented by the mucociliary escalator to provide the first line of defense. Goblet cell metaplasia and mucus overproduction are important features in asthmatic airway remodeling. However, the relationship between the HH signaling pathway and goblet cell metaplasia has not been reported. Thus, this study was set to investigate the effect of HH signaling pathway on goblet cell metaplasia in asthma. Methods:In vivo, we investigated the effects of HH signaling pathway on goblet cell metaplasia in mice model of asthma. First, we used the ICR female mice to establish the asthmatic model with ovalbumin (OVA) and treated them with cyclopamine (lmg/ml,3mg/ml,6mg/ml) which is a smoothed (Smo) protein inhibitor. To exclude the proinflammatory effect of cyclopamine, we used intratracheal administration of adenovirus-expressing ere to Smof/f mice to obtain airway epithelium Smo knockout mice. Western Blot and immunohistochemistry were used to confirm the efficiency of Smo knockout. Also, the Smo knockout mice were sensitized and challenged with OVA. Then, the total and differential leukocytes were analysised in bronchoalveolar lavage fluid (BALF) to evaluate the alveolar inflammation. Lung tissues were stained with hematoxylin and eosin (HE), Periodic Acid-Schiff (PAS) and immunofluorescence of Muc5ac to determinate the inflammation and mucin in lung. Immunohistochemistry (IHC) of patchedl (Ptch1) and Glil in lung tissues was performed as well.In vitro, we want to investigate the mechanism of the HH signaling pathway in the goblet cell metaplasia. We used purmorphamine, an agonist of Smo, to induce human bronchial epithelial cells (HBE). Then, we measured the levels of Muc5ac, Sterile alpha motif-pointed domain-containing Ets transcription factor (Spedf) and Forkhead box A2(FoxA2) as well as other key signal molecules in HH signaling pathways by quantification real-time polymerase chain reaction (qRT-PCR).Results:(1) Effect of HH inhibitor cyclopamine on goblet cell metaplasia in asthma: Compared with model group, Ptchl and Glil levels were lower in cyclopamine-treated group. The total number of white blood cells and eosinophils in BALF were decreased in cyclopamine-treated group than these in the model group. HE staining showed alleviative change of lung inflammation in cyclopamine-treated group as well except for, the lmg/ml cyclopamine-treated group. It was interesting that the level of Muc5ac in3cyclopamine-treated groups was decreased compared with model group although the lmg/ml cyclopamine treatment had in spite no effect on inflammation.(2) Effect of Smo knockout on the goblet cell metaplasia in asthma:Compared with control virus group, Smo deficiency of the airway epithelium did not relieve OVA-stimulated white blood cells increase and severity allergic lung inflammation. Immunofluorescence analysis showed that the Muc5ac expression was inceased significantly in epithelium of asthmatic model. It was notable that Muc5ac was decreased obviously after Smo knockout from epithelium, which was consistent with those HH signaling proteins.(3) Mechanism of the HH signaling pathway in the goblet cell metaplasia:Smo agonist can effectively increase Ptchl and Gli1gene expression and significantly induced Muc5ac expression by promoting Spdef expression and inhibiting FoxA2expression in HBE cells.Conclusions:HH signaling pathway is abnormally activated in asthma. It is involved in the secretion of mucus and it may contribute to goblet cell metaplasia. The mechanisms of HH signaling pathway induced mucus secretion and goblet cell metaplasia may be associated with Spdef activeing and FoxA2inhibiting. These suggest that HH signaling pathway may promote the process of goblet cell metaplasia. It is likely to be a potential target for the treatment of asthma.