The Effect And Mechanism Of RhoA/ROCK1 On Granulation Tissue Hyperplasia Of Benign Airway Stenosis

ObjectivesTo explore the role of Rho A/ROCK1 signaling pathway in the granulation tissue hyperplasia of benign airway stenosis as well as the effect and mechanism of ROCK inhibitor on the granulation tissue hyperplasia after tracheal injury.Methods1.5 granulation tissue specimens from patients with benign tracheal stenosis and 5normal tracheal mucosal ones from healthy volunteers were collected by biopsy forceps.Immunohistochemistry was performed to detect the expression and location of Rho A and ROCK1 proteins.2.Establishment of the animal model for granulation tissue hyperplasia followed by tracheal injury in rats.The SD rats bought from Beijing Vital River Laboratory Animal Technology were divided into 3 groups: Negative control group,Scraping group(injured by nylon brush),Tracheal anterior wall incision and suture group,Chemical injury group(injured by dilute hydrochloric acid),and Long-term tracheal intubation group.HE staining was applied to observe the pathological changes of tracheal tissues on D0,D7,and D14 after injury.Granulation tissue hyperplasia was analyzed after acute and chronic tracheal injury in rats.Meanwhile,normal tracheal tissue and tracheal granulation tissue samples from Scraping group rats were obtained.The expression and localization of Rho A and ROCK1 proteins were detected by immunohistochemistry,and the expression level of Rho A protein in both groups was detected by western blotting.3.The rats were divided into 3 groups: Negative control group,Tracheal stenosis model group,and Treatment group(treated by ROCK inhibitor fasudil).The tracheal stenosis/granulation tissue hyperplasia model was established by scraping rat tracheal mucosa using a nylon brush.The Treatment group was nebulized by fasudil when the rat trachea was scraped by nylon brush and the other two groups were nebulized with 0.9%saline.The survival status,weight change,respiratory rate were recorded.All the live rats were sacrificed on Day 7 to obtain the tracheal tissue.HE staining was applied to observe the hyperplasia of granulation tissue.The degree of tracheal stenosis was calculated.Immunohistochemistry and immunofluorescence staining were adopted to detect the expression of αSMA and COL1A1 proteins.4.The tissue block adherence method was used to obtain primary rat tracheal fibroblasts.The cells were identified by vimentin,collagen 1α1,αSMA and other markers by immunofluorescence.Passages 3~5 cells were used for the following in vitro experiment.10 μmol/L ROCK inhibitor Y-27632 were adopted to observed the effects of it on proliferation,migration,activation,extracellular matrix production,and contraction of the fibroblasts.cell proliferation was detected by CCK8 and cell migration ability was observed by Transwell.Cell contractile ability was determined by collagen gel contraction test and western blotting was used to detect the expression of COL1A1.Cells were treated with PBS,TGF-β1,TGFβ1+Y-27632 for 48 hours respectively,and then harvested to detect their expression of αSMA,Vimentin and F-actin as well as the subcellular localization of MRTF-A protein by immunofluorescence.Western blotting was used to detect the phosphorylation levels of LIMK(p-LIMK),Cofilin(p-Cofilin)and MLC2(p-MLC2)when the cells were stimulated for 2 h,and the expression of αSMA and COL1A1 after the cells were treated by MRTF-A inhibitor CCG-1423 for 48 hours.Results1.Immunohistochemistry showed that Rho A ROCK1 expression was increased in granulation tissue samples obtained by biopsy forceps under tracheoscope compared with normal mucosa samples.2.A rat model of granulation tissue hyperplasia for acute or chronic tracheal injury was established.The tracheal stenosis on Day 7 was(77.95±6.37)% for the nylon brush scraping group,(40.0± 4.75)% for the tracheotomy group,(36.1±7.85)% for the dilute hydrochloric acid injury group,and(26.9±1.97)% for the tracheal intubation group respectively.Among these models,the most severe stenosis,thickest mucosal layer,and the most obvious tracheal granulation hyperplasia was found in the nylon brush scraping group,which was applied for the following study.There was no obvious tracheal stenosis in the anterior tracheal wall incision and suture group with except for the mild mucosal thickening and fibrosis at the incision site.Granulation tissue hyperplasia could be distinguished in the dilute hydrochloric acid injury group,and the long-term tracheal intubation group was characterized by obvious thickened epithelial layer and squamous epithelial metaplasia.The expression of Rho A ROCK1 was also increased in rat tracheal granulation tissue.The results of western blot showed increased Rho A protein expression.3.The stenosis of the scraped model group(nebulized with 0.9% saline,NS)was(78.21±7.083)%,while that of the Fasudil group was(45.74±6.264)%.The survival curve analysis showed that the median survival time of the NS group was 4 days.The survival rate on Day 7 for the NS group was 10%,while that of the Fasudil group was 60%.Log-rank test of the survival curve p=0.0003,the difference was statistically significant.The results showed that atomized inhalation of fasudil could reduce granulation tissue hyperplasia induced by tracheal injury,the degree of tracheal stenosis,and improved the survival rate of rats after tracheal injury.It was shown by immunofluorescence that there was only some chondrocytes were positive for vimentin,and some small blood vessels were positive for αSMA in the control group.By contrast,there was a large number ofαSMA-and vimentin-positive mesenchymal cells demonstrating differentiation from fibroblasts into myofibroblasts in the NS group.While Fasudil treatment could inhibit this differentiation induced by scraping.Western blot results showed that Fasudil could reduce the expression of αSMA and COL1A1 in the granulation tissue induced by tracheal injury.4.Y-27632 inhibits the proliferation,migration,contraction and activation of primary rat tracheal fibroblasts,inhibits F-actin formation and the transportation of MRTF-A into the nucleus.1)Identification of fibroblast: The tissue block adherence method could obtain primary rat tracheal fibroblasts successfully,as was identified to be positive for vimentin,a marker for fibroblasts,by immunofluorescence and contain abundant rough endoplasmic reticulum and Golgi under the electron microscope.2)Change of contraction ability: TGF-β1 stimulation promoted fibroblasts to shrink,while Y-27632 treatment inhibited the increase of contraction ability induced by TGF-β1,as shown in the collagen gel contraction experiment.3)Activation of fibroblasts: When treated by 5ng/ml TGF-β1 and/or 10μmol/L Y-27632 for 48 h,the expression of αSMA was detected by immunofluorescence.The results demonstrated that Y-27632 could inhibit the up-regulation of αSMA induced by TGF-β1.The average fluorescence intensity of αSMA was higher in TGF-β1 group than that in the PBS group vs(P= 0.0053),and lower than that in the Y-27632 treatment group(P= 0.0281).The differences were statistically significant.Western blotting was used to detect the expression level of COL1A1 protein and the result showed that the difference was significant in TGF-β1 group vs.Y-27632 treatment group(P=0.0185).Y-27632 treatment significantly decreased the expression level of αSMA protein as compared with that in TGF-β1 group(P =0.0003)4)Exploration of the related mechanisms: Y-27632 could induce the upregulation of LIMK1 and MRTF-A proteins as well as the phosphorylation levels of Cofilin and MLC2 caused by TGF-β1 stimulation as detected by western blotting.TGF-β1 stimulation could increase F-actin,upregulate the expression of MRTF-A protein and promote its nuclear localization as detected by immunofluorescence.While Y-27632 treatment made F-actin depolymerize and inhibited the translocation of MRTF-A into nuclear stimulated by TGF-β1.When MRTF-A was intervened by its inhibitor CCG-1423,the upregulation ofαSMA and COL1A1 caused by TGF-β1 was blocked.The relative expression of COL1A1 protein in the CCG-1423 pretreatment group decreased significantly as compared with that in the TGF-β1 group(P=0.0212).Meanwhile,the expression of αSMA protein in the CCG-1423 pretreatment decreased significantly as compared with that in TGF-β1 group(P= 0.0021).Conclusion1.The expression of Rho A/ROCK1 proteins upregulated significantly in granulation tissue secondary to tracheal injury,and the Rho A/ROCK1 signaling was activated in granulation tissue.2.Nebulized inhalation of ROCK inhibitor fasudil could inhibit granulation tissue hyperplasia and tracheal stenosis caused by tracheal injury in rats,and alleviate the activation of fibroblasts and the production of extracellular matrix in the tracheal granulation tissue.3.ROCK inhibitor Y-27632 could inhibit the proliferation,migration and contraction of fibroblasts.It inhibited the activation of fibroblasts by down-regulating the LIMK/Cofilin/MRTF-A signaling pathway.