自噬在气管狭窄家兔模型中的表达及意义

Expression and significance of autophagy in rabbit model of tracheal stenosis

目的初步探讨自噬在气管狭窄家兔模型中的表达及意义。方法18只家兔根据随机数字表法均分成空白对照组、生理盐水组、红霉素组3组。建立家兔气管狭窄模型后，术前7d至术后10d期间，空白对照组不予任何处理，生理盐水组予以生理盐水0．54mg／kg雾化，红霉素组予以红霉素7．5mg／kg喂食，均为2移’／d。术后第11天处死家兔，收集气管标本。利用Masson染色检测气管黏膜层及固有层中胶原纤维面积比例；运用实时荧光定量PCR检测气管黏膜中自噬相关基因3（ATG3）、自噬相关基因5（ATG5）的mRNA相对表达量；运用Western印迹法检测微管相关蛋白1轻链B3（MAP1-LC3B，简称LC3B）、ATG3、ATG5蛋白的表达量。结果空白对照组、生理盐水组、红霉素组气管黏膜层及固有层中胶原纤维面积所占比例分别为（6．79±0．67）％、（40．55±5．40）％、（27．48±0．43）％，组间差异均有统计学意义（均P〈0．05）。生理盐水组ATG3mRNA和ATG5 mRNA的相对表达量均显著低于空白对照组（均P〈0．01），红霉素组均显著高于生理盐水组（均P〈0．01）。生理盐水组LC3B-Ⅱ／Ⅰ蛋白比值、ATG3和ATG5蛋白表达水平均显著低于空白对照组（均P〈0．01），红霉素组蛋白表达水平均显著高于生理盐水组（均P〈0．01）。结论气管狭窄家兔自噬表达水平降低，小剂量红霉素可提高其表达，同时减轻气管黏膜纤维化程度；自噬可能通过上调其表达，从而改善气管纤维化。

Objective To investigate the expression and significance of autophagy in rabbit model of tracheal stenosis. Methods A total of 18 rabbits were equally divided into 3 groups （blank control group, saline group, erythromycin group） in accordance with the random number table. After rabbit model of tracheal stenosis was established, no treatment was done with blank control group. Saline group was atomized with saline （0. 54 mg/kg, 2 times/day ）, and erythromycin group was fed on erythromycin （7.5 mg/kg, 2 times/day） for 7 days before and 10 days after the operation. On the eleventh day, rabbits were executed, and their trachea were collected. The proportion of collagen fiber area of tracheal lamina propria （LP） and epithelium （EP） was assessed by Masson staining. The mRNA of autophagy associated gene-3 （ATG3） and autophagy associated gene-5 （ATG5） of tracheal mucosa were assessed by Real-Time Polymerase Chain Reaction （ RT-PCR）. The protein of microtubule-associated protein 1 light chain 63 （ LC3B）, ATG3 and ATG5 were assessed by Western blot. Results The proportion of collagen fiber area of tracheal LP and EP of blank control group was （6.79 ± 0. 67 ） % , saline group was （40. 55 ± 5.40） % , erythromycin group was （27.48 ± 0.43 ）%. The differences between any two groups was all statistically significant （ all P 〈 0. 01 ）. The relative value of ATG3 mRNA and ATG5 mRNA in saline group were significantly lower than blank control group （ all P 〈 0.01 ） . Those value in erythromycin group were significantly higher than the saline group （ all P 〈 0. 01 ）. The protein levels of LC3B- II/I , ATG3 and ATG5 in saline group were significantly lower than blank control group （ all P 〈 0. 01 ）. After low dose of erythromycin intervention, all the protein levels were significantly higher than the saline group （ all P 〈 0. 01 ）. Conclusions The expression of autophagy is decreased in rabbit model of trachea stenosis. Low dose of erythromycin could increase the expression of autophagy and at the same time alleviate the degree of fibrosis of the tracheal mucosa. Autophagy may alleviate tracheal fibrosis through up-regulating its expression level and play a protective role.