| Objective To investigate the effect of different drugs on tracheal stenosis induced by transforming growth factor-?/rapamycin target protein(TGF-?/mTOR)signaling pathway.Methods In the animal experiment,30 rabbits were completely randomly divided into normal control(NC)group,normal saline(NS)group,penicillin(PNC)group,budesonide(BUD)group and erythromycin(EM)group.The normal control group was not treated,and the remaining groups were established with a tracheal stenosis model.The normal control group was not treated,and the other groups were established with tracheal stenosis model.On the 1st to10 th day after modeling,each group was administered with normal saline(0.75ml/kg,2 times/d),intramuscular injection of penicillin(40 000 U/kg,2 times/d),erythromycin.Gastric administration(12.5 mg/kg,2 times/d),inhalation of budesonide(0.05 mg/kg,2 times/d).Rabbits were sacrificed on the 11 th day after surgery,and tracheal specimens were collected to measure the degree of tracheal stenosis.Detection of interleukin-6(IL-6),transforming growth factor-?(TGF-?),Type I collagen(COL-1),Type ? collagen(COL-3),and Sirtuin 1(SIRT-1)by Real-time quantitative reverse transcription polymerase chain reaction(RT-qPCR)Relative expression level of related mRNA;immunohistochemical detection of mTOR,Phosphorylated protein kinase B(p-AKT),vascular endothelial growth factor(VEGF),SIRT-1 protein positive expression;Western blotting detection of Nuclear factor ?B(NF-?B),Phosphorylated nuclear factor ?B(p-NF-?B),protein kinase B(AKT),p-AKT,mTOR protein expression.In the cell experiment,lipopolysaccharide 100 ng/ml(LPS)was used to stimulate human embryonic lung fibroblasts,and four groups were set: normal control(NC)group,lipopolysaccharide 100 ng/ml(LPS)group,and erythromycin 10 mg/L(EM)group,budesonide 10 nM(BUD)group.Erythromycin and budesonide group were pre-intervention with related drugs,and intervention in lipopolysaccharide.The relative expression levels of IL-6,SIRT-1,TGF-? and mTOR mRNA were detected by real-time PCR.Results Animal experiment(1)Tracheal stenosis: There were significant differences in tracheal stenosis between normal control group,saline group,penicillin group,budesonide group and erythromycin group [(14.02±2.86)%,(64.14±3.21)%(49.11±2.96)%,(39.52±2.09)%,(32.60±4.27)%](all P<0.05).(2)Real-time quantitative reverse transcription polymerase chain reaction(RT-qPCR):except for IL-6 mRNA in the erythromycin group and the normal control group,IL-6 mRNA(1.00±0.00,9.02±1.50,4.25±0.87,2.53±0.17,1.31±0.56),TGF-? mRNA(1.00±0.00,6.92±0.84,3.83±0.44,2.13±0.25,1.40±0.15),COL-1 mRNA(1.00±0.00,11.83±2.49,7.19±0.59,3.93±0.45,2.87±0.48),COL-3(1.00±0.00,5.40±0.45,3.54±0.70,1.75±0.40,1.27±0.09),the relative expression differences were statistically significant(both P< 0.05).The relative expression levels of SIRT-1 mRNA(1.00±0.00,0.21±0.04,0.38±0.03,0.56±0.03,0.67±0.03)were statistically significant(P<0.05).(3)Immunohistochemistry: In addition to the SIRT-1 protein comparison,there was no significant difference between the erythromycin group and the budesonide group(P>0.05),and the other groups were mTOR protein(9.28±4.56,58.18±8.12,44.75±5.56,32.82±5.99,24.73±3.56),p-AKT protein(16.57±4.86,61.79±6.66,42.98±5.99,32.79±5.34,24.00±4.40),SIRT-1 protein(16.93±2.28,4.77±1.45,7.70±0.61,10.76±1.04,11.03±1.10),VEGF protein(10.39±8.38,124.29±27.50,86.02±13.07,62.72±10.90,38.78±10.08),the relative expression differences were statistically significant(both P < 0.05).(4)Western blot: In addition to mTOR protein comparison,there was no significant difference between the erythromycin group and the normal control group(P>0.05),and the other groups of protein p-NF-?B / NF-?B ratio(0.71±0.12,1.51±0.20,1.22±0.15,0.48±0.06,0.39±0.04),p-AKT/AKT ratio(0.50±0.10,2.13±0.24,1.87±0.19,1.17±0.18,0.89±0.10),mTOR protein(1.05±0.11,3.24±0.19,2.27±0.14,1.86±0.85,1.53±0.05)were statistically significant.(all P < 0.05).Cell experiment(1)RT-qPCR: The relative expression levels of IL-6mRNA(1.00±0.00,5.24±0.12,2.60±0.21,2.24±0.33),SIRT-1 mRNA(1.00±0.00,0.21±0.03,0.57±0.12,0.60±0.26),TGF-? mRNA(1.00±0.00?9.41±0.85?5.41±0.98?2.88±0.74),mTOR mRNA(1.00±0.00?120.49±11.3?28.27±4.74?8.04±0.51)were compared.The differences were statistically significant(P<0.05).Conclusion 1.The research reveals that Penicillin,erythromycin and budesonide probably alleviate inflammation by increasing SIRT-1,alleviate tracheal scar hyperplasia induced by TGF-beta/mTOR pathway and improve the degree of tracheal stenosis in rabbits.2.Erythromycin and budesonide probably reduce the inflammatory response of fibroblasts stimulated by lipopolysaccharide by increasing the expression of SIRT-1,thereby reducing fibroblast proliferation. |
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