The Effects Of GRHL2 On The Epithelial-mesenchymal Transition In Pulmonary Fibrosis Rats And Its Mechanisms

ObjectiveThis study aimed to observe the differential expression of GRHL2 in normal rats and pulmonary fibrosis rats by treatment of valproic acid. By studying the expression of E-cadherin, vimentin and β-catenin, we explored the mechanism which VPA suppressed pulmonary fibrosis and effect of Wnt-β-catenin signaling on this process.MethodsThe forty-five female SD rats were randomly divided into three groups, 15 rats in each group, which were normal saline group(NS), blcomycin-induced pulmonary fibrosis group(BLM) and valproic acid-treated group(VPA) respectively. NS group rats were injected with sterile physiological saline, dose was 0.3ml. BLM group and VPA group were given bleomycin by intratracheal injection respectively, dose was5mg/kg. VPA group accepted sodium valproate by intraperitoneal injection, dose was200 mg/kg/d. The rats in BLM were treated intraperitoneally with sterile physiological saline by 0.5 ml/d.On day 7, 14 and 28 after treament, 15 rats in each group were killed randomly. HE and Masson staining were used for observing collagen in lung tissue of each group. Ashcroft score, hydroxyproline(HYP) content were detected for dynamic changes in pulmonary fibrosis in each group at three times.Immunohistochemistry of GRHL2 were used to observe differential expression in three groups. The immunohistochemistry of E-cadherin and vimentin were detected in each group for judging the degree of epithelial mesenchymal transition. The immunohistochemistry of β-catenin was detected to analyse the difference of Wnt-β-catenin signaling in each group.Results(1) HE staining showed the structure of lung tissue in NS group was normal. In BLM group on day 7, 14 and 28, normal alveolar structure was destroyed, markedalveolar exudation of inflammatory cells and fibers appeared, alveolar septum significantly widened. In VPA group on day 7, 14 and 28, alveolar structure was still surviving, fiber exudation was less than BLM group, along with mildly inflammatory exudation.(2) Masson staining in NS group showed no collagen fibers. In BLM group,alveolar septal edema was significant, normal alveolar structure was occupied by a large number of inflammatory cells. On day 28, collagen became markedly more than the one on day 7(P<0.05). In VPA group, alveolar structure was still surviving,collagen was significantly less than BLM group(P<0.05).(3) In BLM group on day 7,14 and 28, HYP contents and Ashcroft score of lung tissue significantly increased compared with NS group(P<0.05). In VPA group, HYP contents and Ashcroft score were between NS and BLM group, were significantly different compared between NS group(P<0.05). In BLM, HYP contents and Ashcroft score were significantly differential among day 7, 14 and 28(P<0.05).(4) The immunohistochemistry of GRHL2 and E-cadherin in NS group showed a large number of positive regions in lung tissue. In BLM group on day 7, 14 and 28, the severer pulmonary fibrosis was,the less positive area was observed. The difference was statistically significant compared with NS group(P<0.05). In BLM, the immunohistochemistry of GRHL2 and E-cadherin were significantly differential among day 7, 14 and 28(P<0.05). In VPA group, the expression trend was similar with BLM group. Since pulmonary fibrosis was milder than BLM group, positive area in VPA group was obviously more than BLM(P<0.05).(5) The immunohistochemistry of vimentin and β-catenin in NS group was almost no expression in NS group. In BLM group on day 7, 14 and 28,alveolar structure was destroyed, the severer pulmonary fibrosis was, the more positive area was observed. The difference was statistically significant compared with NS(P<0.05). In BLM group, the immunohistochemistry of vimentin and β-catenin were significantly differential among day 7, 14 and 28(P<0.05). In VPA group, the expression trend of vimentin and β-catenin was consistent with BLM group, butpositive regions were obviously less than that in BLM group(P<0.05).Conclusion(1) The treatment of VPA in the pulmonary fibrosis rats can significantly alleviate the alveolar inflammation, prevent inflammatory cells, collagenous fibers exudation, reduce lung tissue fibrosis.(2) By regulating the expression of GRHL2,VPA may suppress pulmonary fibrosis by inhibiting EMT process.(3) The Wnt-β-catenin signaling may play an important role in the process of alleviating pulmonary fibrosis in rats by VPA.