不同药物对基于TGF-β/mTOR信号通路所致兔气管狭窄的影响机制

Effects of different drugs on bronchial stenosis by TGF-β/mTOR signaling pathway in rabbit model

目的探讨不同药物对基于转化生长因子-β/雷帕霉素靶蛋白(TGF-β/mTOR)信号通路所致气管狭窄的影响机制.方法将30只家兔完全随机分为正常对照组、生理盐水组、青霉素组、布地奈德组、红霉素组5组,正常对照组不做处理,其余各组均建立气管狭窄模型.建模后第1~10天,建模各组分别予以生理盐水灌胃(0.75 ml/kg,2次/d)、肌肉注射青霉素(40 000 U/kg,2次/d)、红霉素灌胃(12.5 mg/kg,2次/d)、雾化吸入布地奈德(0.05 mg/kg,2次/d)处理.术后第11天处死家兔,收集气管标本,测气管狭窄度.运用实时定量逆转录聚合酶链反应(RT-qPCR)检测白细胞介素(IL)-6、TGF-β、Ⅰ型胶原蛋白(COL-1)、Ⅲ型胶原蛋白(COL-3)、沉默信息调节因子2相关酶1(SIRT-1)mRNA的相对表达量;免疫组化检测mTOR、磷酸化蛋白激酶B(p-AKT)、血管内皮生长因子(VEGF)、SIRT-1蛋白阳性表达情况;运用Western印迹法检测核因子(NF)-κB及其磷酸化(p-NF-κB)、AKT,p-AKT、mTOR蛋白表达情况.结果正常对照组、生理盐水组、青霉素组、布地奈德组、红霉素组气管狭窄度差异均有统计学意义[(14.02±2.86)%、(64.14±3.21)%、(49.11±2.96)%、(39.52±2.09)%、(32.60±4.27)%](均P<0.05).除红霉素组与正常对照组IL-6 mRNA外,其余各组IL-6 mRNA(1.00±0.00、9.02±1.50、4.25±0.87、2.53±0.17、1.31±0.56)、TGF-βmRNA(1.00±0.00、6.92±0.84、3.83±0.44、2.13±0.25、1.40±0.15)相对表达量差异均有统计学意义(均P<0.05).除红霉素组与布地奈德组SIRT-1蛋白外,其余各组SIRT-1mRNA(1.000±0.000、0.209±0.042、0.375±0.034、0.555±0.028、0.667±0.032)及SIRT-1蛋白(16.93±2.28、4.77±1.45、7.70±0.61、10.76±1.04、11.03±1.10)的相对表达量差异均有统计学意义(均P<0.05).免疫组化各组mTOR蛋白(9.28±4.56、58.18±8.12、44.75±5.56、32.82±5.99、24.73±3.56)、p-AKT蛋白(16.57±4.86、61.79±6.66、42.98±5.99、32.79±5.34、24.00±4.40)相对表达量差异均有统计学意义(均P<0.05).Western印迹检测各组NF-κB、p-NF-κB、AKT、p-AKT、mTOR蛋白表达与免疫组化相关蛋白趋势一致,生理盐水组NF-κB、AKT、mTOR的蛋白相对表达量均高于其他各组,红霉素组低于布地奈德组、且均低于青霉素组;除红霉素组与正常对照组mTOR蛋白外,其余各组差异均有统计学意义(均P<0.05).结论青霉素、红霉素、布地奈德可通过升高SIRT-1减轻炎症反应,减轻基于TGF-β/mTOR通路所致的兔气管瘢痕增生,改善气管狭窄程度.

Objective To investigate the effect of different drugs on tracheal stenosis caused by transforming growth factor-β/rapamycin target protein (TGF-β/mTOR) signaling pathway.Methods Thirty rabbits were randomly divided into normal control group,normal saline group,penicillin group,budesonide group and erythromycin group.The normal control group was not treated,and tracheal stenosis models were established in the other groups.From the 1st to 10th day after modeling,each group was respectively administered with normal saline (0.75 ml/kg,2 times/d),intramuscular injection of penicillin (40 000 U/kg,2 times/d),gastric administration of erythromycin (12.5 mg/kg,2 times/d),inhalation of budesonide (0.05 mg/kg,2 times/d).Rabbits were sacrificed on the 1 lth day after surgery,and tracheal specimens were collected to measure the degree of tracheal stenosis.Relative mRNA expression level of interleukin-6 (IL-6),transforming growth factor-β (TGF-β),Type Ⅰ collagen (COL-1),Type Ⅲ collagen (COL-3),and Sirtuin 1 (SIRT-1) were detected by Real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR);protein expression of mTOR,phosphorylated protein kinase B (p-AKT),vascular endothelial growth factor (VEGF),SIRT-1 were detected by immunohistochemical analysis;protein expression of nuclear factor κB (NF-κB),phosphorylated nuclear factor κB (p-NF-κB),protein kinase B (AKT),p-AKT,mTOR were detected by Western blotting.Results The degree of stenosis of normal control group was (14.02±2.86)%,saline group was (64.14±3.21)%,penicillin group was (49.11 ±2.96)%,budesonide group was (39.52±2.09)%,erythromycin group was (32.60±4.27)%.The differences between any two groups were statistically significant (all P<0.05).Except between erythromycin group and normal control group,the differences in relative expression of IL-6 mRNA between any two groups (1.00±0.00,9.02± 1.50,4.25±0.87,2.53±0.17,1.31 ±0.56)was statistically significant (all P<0.05),and the differences in relative expression of TGF-β mRNA among all groups (1.00±0.00,6.92±0.84,3.83±0.44,2.13±0.25,1.40±0.15) were statistically significant (all P<0.05).The relative expression of SIRT-1 mRNA among all the groups (1.000±0.000,0.209±0.042,0.375±0.034,0.555 ± 0.028,0.667 ± 0.032) was statistically significant different (all P<0.05);except between erythromycin group and budesonide group,the protein levels of SIRT-1 among all other groups (16.93±2.28,4.77 ± 1.45,7.70±0.61,10.76 ± 1.04,11.03 ± 1.10) were statistically significant different (all P<0.05).The protein levels of mTOR (9.28±4.56,58.18±8.12,44.75±5.56,32.82±5.99,24.73±3.56) and p-AKT (16.57±4.86,61.79±6.66,42.98±5.99,32.79±5.34,24.00±4.40) determined through immunohistochemistry of all groups were statistically significant different (all P<0.05).The protein levels of NF-κB,p-NF-κB,AKT,p-AKT and mTOR determined through Western blotting had the same trend as that of determined through immunohistochemistry.The protein expression of NF-κB,AKT and mTOR in saline group were significantly higher than other groups;those protein expression of erythromycin group was lower than budesonide group and penicillin group.Except between the erythromycin group and the normal control group,the protein expression of mTOR in other groups was statistically significant different (all P<0.05).Conclusion Penicillin,erythromycin and budesonide can alleviate inflammation by increasing SIRT-1,alleviate tracheal scar hyperplasia induced by TGF-beta/mTOR pathway,and reduce the degree of tracheal stenosis in rabbits.