Expression And Significance Of Autophagy In Rabbits' Tracheal Stenosis

Objective To investigate the expression and activation of autophagy in rabbits' tracheal stenosis model.Then to explore the significance of autophagy in the pathogenesis of in rabbits' tracheal stenosis.Methods 18 male rabbits were equally apportioned into 3 groups blank control group(C),saline group(NS)and erythromycin group(EM)in accordance with the random number table.After rabbits' tracheal stenosis model established according to the literature,no treatment was done with C group.NS group was atomized with saline(0.54 mg/kg,2 times/day),EM group was fed on erythromycin(7.5 mg/kg,2 times/day)about 7 days before operation and 9days after the operation.On the tenth day,rabbits were killed,and their trachea were collected.The pathological changes of tracheal mucosal epithelium(EPI)and lamina propria(LP)was observed via HE staining.Tracheal specimens were processed for determining alterations in the thicknesses of tracheal EPI and LP via HE staining.The proportion of collagen fiber area of EPI and LP was assessed by Masson staining.The tracheal m RNA(assessed by RT-PCR)expressions of the following fibrotic-related factors were determined:transforming growth factor-?1(TGF-?1),collagen type ?(COL1A1),collagen type ?(COL3A1),autophagy associated gene-3(ATG3)and autophagy associated gene-5(ATG5).The protein levels of TGF-?1,COL1A1 and COL3A1 were determined through immunohistochemistry and integrated optical densities.The protein of microtubule-associated protein 1 light chain 3(LC3B),ATG3 and ATG5 were assessed by Western blot.Results(1)Measurement the thickness of EPI and LP: In rabbits' tracheal stenosis model,the thickness of EPI and LP in NS group was maximum(1.49±0.28 mm),which was significantly higher than C group(0.24±0.39 mm)(P < 0.05).After erythromycin intervention,the thickness of EPI and LP in EM group(0.47±0.08mm)was statistically significant lower than NS group(P <0.05).(2)Masson staining: The proportion of collagen fiber area of tracheal EPI and LP of C group was(6.79±0.67)%,NS group was(40.55±5.40)%,EM group was(27.48±0.43)%.The differences between the groups was statistically significant(all,P<0.05).(3)RT-PCR: The m RNA expression of TGF-?1,COL1A1,COL3A1,ATG3 and ATG5 was closely related to the EPI and LP thickness of trachea.The m RNA expression of TGF-?1,COL1A1 and COL3A1 in NS group was significantly higher than C group,however ATG3 m RNA and ATG5 m RNA was significantly lower than C group,the differences between the groups was statistically significant(all,P<0.05).After erythromycin intervention,the m RNA expression of TGF-?1,COL1A1 and COL3A1 was significantly lower than NS group,but ATG3 and ATG5 was significantly higher than NS group(all,P<0.05).(4)Immunohistochemistry: The proteins TGF-?1,COL1A1 andCOL3A1 were found in the EPI and LP,but primarily in the LP,and specifically in the cytoplasm of the mucosal epithelial cells and mesenchymal cells.The protein levels of TGF-?1,COL1A1 and COL3A1 in NS group were significantly higher than C group(all,P<0.05).The proteins in EM group was significantly lower than NS group(all,P<0.05).(5)Western Blot: The protein levels of LC3B-?/?,ATG3 and ATG5 in NS group were significantly lower than C group(all,P<0.05).After erythromycin intervention,all the protein levels were significantly higher than the NS group(all,P<0.05).Conclusions(1)In tracheal stenosis model,the fibrotic-related factors that TGF-?1,COL1A1 and COL3A1 may associated in the occurrence and development of tracheal stenosis.(2)The expression of autophagy was decreased in rabbits' tracheal stenosis model.Erythromycin could increase the expression of autophagy and at the same time improve the degree of fibrosis of the tracheal mucosa.Erythromycin may modify the process of tracheal fibrosis through up-regulating the expression level of autophagy.