The Preliminary Study On The Role And Rrgulation Of Fibrous Tissue Hyperplasia And Inflammation On Tracheal Stenosis Induced By Tracheal Injury In Rabbits

In recent years,with the improvement of the diagnostic level,more patients with tracheal stenosis have been found.Among them,tracheal stenosis after injury(such as tracheal intubation and tracheotomy)is particularly common in clinical practice.Tracheal stenosis induced by injury affects the patients' physical health and quality of life in a direct way,threatens the patients' life safety severly,and increases heavy burden on family and society.Therefore,it is of great clinical significance to study the role and regulation of stenosis induced by tracheal injury.Tracheal stenosis after pretreatment should focus on a series of pathophysiological changes such as fibrosis and inflammatory response of tracheal tissue,and take effective measures to reduce or prevent the proliferation of fibroblasts and the synthesis of collagen fibers.Many studies have shown that there is inflammation and fibrosis in the tracheal trauma model of rabbits.Histone deacetylase 2(HDAC2)plays an important role in inhibiting inflammation and organ fibrosis.Nintedanib is a multiple tyrosine kinase inhibitor.As a new-type anti-fibrotic drug,it can not only reduce the inflammatory response,but also directly inhibit lung fibrosis.At present,there is no related research on the use of nintedanib in tracheal stenosis at home and abroad.Whether nintedanib can regulate the expression of HDAC2,inhibit tracheal fibrosis and inflammatory response,and prevent and treat tracheal stenosis induced by injury remains to be studied.This study explores the role and regulation of fibrosis on tracheal stenosis induced by injury in rabbits.The expressions of HDAC2 and multiple inflammatory factors in rabbit tracheal stenosis granulation tissue and human embryo lung fibroblasts were observed at the tissue and cell level,and related research on drug intervention was conducted.The research of this topic is divided into the following three parts:Part I Study on the Changes of Stenotic Fibrosis and Inflammation in Tracheal Stenosis Induced by Injury in Rabbits and the RegulationOBJECTIVE: To investigate the changes of fibrosis and inflammation indexes in rabbits' tracheal induced by injury,and to study the effects of nintedanib and budesonide on tracheal stenosis.Methods: Thirty New Zealand rabbits were divided into 5 groups randomly,6 in each group.Among them,group A is the normal control(NC)group,group B is the model group,group C is the normal saline(Model + NS)group,group D is the Nintedanib(Model+Nintedanib)group,and group E is Budesonide(Model + Budesonide)group.The normal control group was left untreated,and the other four groups were established with tracheal stenosis models induced by injury.Except for group A,the remaining four groups were treated with corresponding drugs on the 1st to 10 th days after modeling.Serum and alveolar lavage fluid were collected on the 11 th day after operation,and the concentrations of TGF-?1,VEGF,and IL-8 in the serum and alveolar lavage fluid of each group were measured by ELISA method.Tracheal specimens were collected,tracheal stenosis of each group was measured,and HE staining was performed to observe the damage and repairation of tracheal tissue in each group.Results: 1.The levels of TGF-?1,VEGF,and IL-8 in serum and alveolar lavage fluid of group B-E were higher than those in group A,and the difference was statistically significant(P<0.05).We found no significant difference in comparison between group B and C,and the levels in group D and E were lower than those in group B(P<0.05).Comparison between intervention groups,the levels in group D was lower than those in group E(P<0.05).2.Tracheal stenosis: Compared with group A,trachea in group B to E showed stenosis caused by granulation tissue hyperplasia in varying degrees.The tracheal stenosis of group AE was [(20.52±3.23)%,(62.79±5.55)%,(61.48±3.77)%,(31.62±2.44)%,(35.05±2.00)%)],group B stenosis was the highest while the lowest is group A.There was no statistically significant difference between the group B and C.The stenosis in group D and E was lower than that in group B,and the difference was statistically significant(P<0.05).Comparison between intervention groups,the stenosis in group D was lower than that in group E.3.HE staining group B-E showed different degrees of fibroblast proliferation,collagen fiber proliferation,and inflammatory cell infiltration,among which group B and C were the most obvious.Fibroblast proliferation and inflammatory cell infiltration were more significant in group D and E than in group B.The group was significantly reduced.Conclusions: After tracheal injury in rabbits,tracheal stenosis shows obvious fibrosis and inflammatory changes.Both Nintedanib and budesonide can reduce tracheal stenosis induced by injury,which may be related to the reduction of fibrosis and inflammation caused by tracheal injury.The response is related,of which Nintedanib is more effective.Part II Study on The Expression and Regulation of HDAC2 in Tracheal Stenosis Granulation Tissue Induced by Injury in RabbitsOBJECTIVE: To investigate the expression of HDAC2 in tracheal stenosis granulation tissue induced by injury in rabbits,and to study the interventional effects of nintedanib and budesonide on HDAC2 expression.Methods: Thirty New Zealand rabbits were divided into 5 groups randomly,6 in each group.Among them,group A is the normal control(NC)group,group B is the model group,group C is the normal saline(Model + NS)group,group D is the Nintedanib(Model + Nintedanib)group,and group E is Budesonide(Model + Budesonide)group.The normal control group was left untreated,and the other four groups were established with tracheal stenosis models after tracheal injury.Except for group A,the remaining four groups were treated with corresponding drugs on the 1st to 10 th days after modeling.On the 11 th day after operation,the trachea tissues of rabbits were collected,and the relative expression levels of HDAC2,IL-8 and VEGF m RNA in the trachea tissue of each group were detected by real-time quantitative PCR.The HDAC2 protein in the trachea tissue of each group was detected by Western blot.The expression was detected by immunofluorescence.The expression of HDAC2 in each group was detected by immunofluorescence.The expression of Collagen ? and Collagen ? was detected by immunohistochemistry.Results: 1.q RT-PCR test: there was no statistically significant difference between the two groups of indicators in group B and C(P>0.05).Compared with group A,the expression of HDAC2 m RNA decreased and the expression of IL-8 and VEGF m RNA increased in groups B and C,the difference was statistically significant(P <0.05).Compared with group B,the expression of HDAC2 m RNA in group D and E increased significantly,and the expression of IL-8 and VEGF m RNA decreased significantly,the difference was statistically significant(P<0.05).2.Western blot detection: HDAC2 protein expression in tracheal tissue of group B and C was lower than that of group A,and the difference was statistically significant(P<0.05).Compared with group B,the expression of HDAC2 protein in groups D and E increased,and the difference was statistically significant(P<0.05).The expression of HDAC2 protein in groups D was higher than the group E,and the difference was statistically significant(P<0.05).3.Immunofluorescence: The immunofluorescence expression of HDAC2 in group B-E was lower than that in group A.There was no statistically significant difference between the group B and C(P>0.05).The HDAC2 immunofluorescence expression in group D and E were stronger than that in group B,and the difference was statistically significant(P<0.05).4.Immunohistochemistry: The expression levels of Collagen ? and Collagen ? proteins in group B-E were higher than those in group A.There was no statistically significant difference between the group B and C(P>0.05).Compared with group B,the protein expressions of collagen ? and collagen ? in group D and E were significantly lower than those in group B(P<0.05).Conclusions: 1.The expression of HDAC2 is down-regulated in tracheal stenosis granulation tissue induced by injury in rabbits.2.Nintedanib and budesonide may relieve the inflammation and fibrosis response of tracheal stenosis by increasing the level of HDAC2,and reduce the formation of Collagen? and Collagen?.Part III Preliminary Study of Nintedanib on Reducing Inflammation of Human Embryonic Lung FibroblastsOBJECTIVE: To investigate the effects of nintedanib on inflammation indexes of human embryo lung fibroblasts stimulated by lipopolysaccharide and possible intervention mechanism.Methods: Human embryo lung fibroblasts(WI-38)were cultured in vitro anddivided into normal control(NC)group,lipopolysaccharide(LPS)group,andnintedanib(NB)group.The lipopolysaccharide group was given 100 ng / mllipopolysaccharide to interfere with fibroblasts,and the nidanib(EM)group wasfirst given 10 mg / L nintedanib to interfere with the cells for 2 hours,and thenwith lipopolysaccharide for 24 hours.ELISA method was used to detect theexpression of IL-8 and VEGF in the cell supernatant of each group.Real-timequantitative PCR was used to detect the relative expression of HDAC2,IL-8,andVEGF m RNA in cells.Results: 1.Compared with the NC group,the concentrations of IL-8 and VEGF in the supernatant of the LPS group and the NB group increased,and the differences were statistically significant(P<0.05).Compared with the LPS group,the concentrations of IL-8 and VEGF in the supernatant of the NB group were reduced,and the difference was statistically significant(P<0.05).2.Real-time quantitative PCR detection: HDAC2 mRNA(1.00 ± 0.00,0.56 ± 0.06,0.85 ± 0.08),IL-8 m RNA(1.00 ± 0.00,4.99 ± 0.95,2.72 ± 1.11),VEGF m RNA(1.00 ± 0.00,2.81 ± 0.59,1.85 ± 0.27)The relative expression of each group was statistically significant(all P<0.05).Compared with the NC group,the relative expression of HDAC2 m RNA in the LPS group and the NB group decreased,and the relative expression of IL-8 m RNA and VEGF m RNA increased;compared with the LPS group,the relative expression of HDAC2 m RNA in the NB group increased,and IL-8 m RNA,The relative expression of VEGF m RNA decreased(all P<0.05).Conclusions: 1.The expression of HDAC2 in human embryo lung fibroblasts decreased under the stimulation of lipopolysaccharide,which reduced the inhibitory effect on inflammatory factors.2.Nintedanib may up-regulate the expression of HDAC2 in lipopolysaccharide-stimulated human embryonic lung fibroblasts,down-regulate the expression of IL-8 and VEGF,and reduce inflammation and fibrosis.3.Nintedanib is expected to become a potential therapeutic drug for tracheal stenosis induced by injury.