Alleviation of Airway Inflammation Induced by Chronic Obstructive Pulmonary Disease in Rats by Total Flavonoids of Gnaphalium affine Combination with Antibiotics
[Objective]This study was aimed to investigate the therapeutic effect of total flavonoids of Gnaphalium affine(G.affine)combined with antibiotics on chronic obstructive pulmonary disease induced by Klebsiella pneumoniae and lipopolysaccharides(LPS),and to explore the related anti-inflammatory mechanisms at the level of related proteins and genes to provide a new idea for the treatment of this disease.[Method]54 Wistar rats were divided into 6 groups:Control group,model group,aminophylline(AM)group,total flavonoids of G.affine(TFG)group,antibiotic(AB)group,and total flavonoids of G.affine+antibiotic(TFG+AB)group,with 9 rats in each group.Among them,chronic obstructive pulmonary disease(COPD)was established in all groups except for control group by tracheal instillation of Klebsiella pneumoniae and lipopolysaccharide(LPS).Rats in control group was treated with equal volume of normal saline instead of Klebsiella pneumoniae and LPS.After successful modeling of COPD for 12 weeks,the rats in treatment groups were treated with aminophylline(50 mg/kg BW)and total flavonoids of G.affine(50 mg/kg BW)by gavage,and ceftazidime(200 mg/kg BW)by intramuscular injection every day.The rats in TFG+antibiotic group were treated with total flavonoids of G.affine(50 mg/kg BW)and ceftazidime(200 mg/kg BW)every day.Control and model groups were not treated.After 7 days of continuous treatment,peak expiratory flow(PEF),peak inspiratory flow(PIF),and the ratio of forced expiratory volume in 0.3 s to forced vital capacity(FEV0.3/FVC)were measured by pulmonary function tester.HE staining and TUNEL staining were used to detect the pathological morphology of rat lung tissue and the level of apoptosis in rat lung tissue,respectively.Western blotting was used to detect the protein expression of Bax,Bcl-2,Cleaved Caspase-3,MMP-2,MMP-9 and TIMP-1 in lung tissues of rats.Real-time quantitative PCR was used to detect the expression of MMP-2,MMP-9 and TIMP-1 genes mRNA in lung tissues of rats.ELISA was used to detect the levels of IL-1β,IL-6 and TNF-a in serum and BALF of rats.Flow cytometry was used to detect the proportion of Th17 and Treg cells in lung tissues of rats.[Result]Compared with control group,PEF,PIF,FEV0.3/FVC,Bcl-2 and Treg cell ratio of rats in model group were significantly decreased(P＜0.05).While the positive rate of TUNEL,the expression of Bax and Cleaved Caspase-3 proteins,Th17 cell ratio,Th17/Treg ratio,the expression of MMP-2,MMP-9,TIMP-1,IL-1β,IL-6 and TNF-α were significantly increased(P＜0.05).Compared with model group,PEF,PIF,FEV0.3/FVC,Bcl-2 and Treg cell ratio in aminophylline,total flavonoids of G.affine,antibiotic and total flavonoids of G.affine+antibiotics groups were significantly increased(P＜0.05).While the positive rate of TUNEL,the expression of Bax,Cleaved Caspase-3,Th17 cell ratio,Th17/Treg ratio,expression of MMP-2,MMP-9,TIMP-1,IL-1β,IL-6 and TNF-α were significantly decreased(P＜0.05).Compared with total flavonoids of G.affine and antibiotic groups,all the proportion of PEF,PIF,FEV0.3/FVC,Bcl-2 and Treg cell ratio in total flavonoids of G.affine+antibiotics group were significantly increased(P＜0.05).While the positive rate of TUNEL,the expression of Bax and Cleaved Caspase-3,Th17 cell ratio,Th17/Treg ratio,the expression of MMP-2,MMP-9,TIMP-1,IL-1β,IL-6 and TNF-α were significantly decreased(P＜0.05).[Conclusion]Both total flavonoids of G.affine and antibiotics could reduce airway inflammatory injury in rats with chronic obstructive pulmonary disease,and the combination of them showed better effect.The mechanism was related to the inhibition of apoptosis,the inhibition of matrix metalloproteinases(MMPs)and inflammatory response,and also the reduction of Th17/Treg ratio by total flavonoids of G.affine and antibiotics.
total flavonoids of Gnaphalium affineantibioticschronic obstructive pulmonary diseaseairway inflammationapoptosismatrix metalloproteinases
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