Effects Of Qingjin Huatan Granule On Beclin-1? LC3 And Inflammatory Factors In AECOPD Rats

Objective:This topic using molecular biology techniques from the perspective of airway epithelial cell autophagy levels to explore the influence of qing jin phlegm decoction to AECOPD rats,observe the qing jin phlegm decoction intervene airway epithelial cells after autophagy after the formation of the autophagy related protein Beclin 1,LC3 expressed in airway epithelial cells in rats in AECOPD inflammation with the change of situation,in order to make clear its targets.Thus,the relationship between autophagy,cytokines and inflammatory mediators in airway epithelial cells of AECOPD rats was elaborated.Objective basis was provided for the prevention and treatment mechanism of AECOPD by qingjinhuatan decoction at the cellular and molecular level.Methods: A total of 120 healthy male SPF SD rats,weighing 180 ~ 220 g,were randomly selected,of which 60 were used to make drug-containing serum,and 50 were used to make models by using smoke poisoning +LPS(lipopolysaccharide)combined with the classical preparation method,and 10 were used as blank control.The drug serum was divided into 6 groups according to the random number method(the high dose group of qingjinhuatan decoction was 30 g/kg·d;15 g/kg·d in the medium dose group,7.5 g/kg·d in the low dose group,0.0175 g/kg·d in the roxithromycin positive control group),and 20 in each model group and blank control group.After successful modeling,50 AECOPD rats and 10 blank controls were cultured with primary airway epithelial cells.The prepared serum containing qingjinhuatan decoction was divided into groups to intervene the primary cultured airway epithelial cells in accordance with the medium of DMEM-F12 and drug serum 10:1 ratio.HE staining was used to observe the changes of lung tissue.Meanwhile,the expressions of beclin-1 and LC3 m RNA in airway epithelium were detected by RT-PCR.Beclin-1,LC3-II/I proteins in airway epithelial cells were detected by Western Blot,and the contents of IL-6 and IL-8 inflammatory cytokines were detected by ELISA.Results:1.Pathological section results of lung tissueAfter the establishment of the model,HE staining of pathological sections of rat lung tissue showed that inflammatory cells infiltrated around the bronchial tube,thickened tracheal wall,occluded and narrowed lumen,and obvious destruction of alveolar structure,alveolar expansion and fusion into pulmonary vesicles,and exudate could be seen in the alveolar cavity.2.Immunofluorescence identification of cellsThe keratin labeled by green secondary antibody showed positive green fluorescence.DAPI staining nuclei showed blue fluorescence.The positive rate of epithelial cells in the merge green and orange staining images was above 99%.3.Beclin-1 and LC3 m RNA expression levels of rat airway epithelial cells after serum intervention in each groupBeclin-1 and LC3 m RNA levels in rat airway epithelial cells after serum intervention were significantly higher in each group than in the normal group(P<0.01).Compared with the model control group,Beclin-1 and LC3 expressions were significantly decreased in the western medicine group and the qingjinhuatan decoction high,medium and low dose groups after serum intervention(P<0.01),and there was no significant difference between the western medicine group and the qingjinhuatan decoction high dose group.4.Changes in autophagy related Beclin-1,LC3-II /I and protein expression levels after serum intervention in rat airway epithelial cells in each groupBeclin-1,LC3-I and LC3-II protein expressions in rat airway epithelial cells after serum intervention were significantly higher in each group than in the normal group(P<0.01).Compared with the model control group,beclin-1 and lc3 expressions were significantly decreased in the western medicine group and the qingjinhuatan decoction high,medium and low dose groups after serum intervention(P<0.01),and the decrease was more obvious in the western medicine group and the qingjinhuatan decoction high dose group,with no statistical significance between the two groups.5.Changes of inflammatory cytokines IL-6 and IL-8 in rat airway epithelial cells after serum intervention in each groupELISA used the standard concentration as the ordinate and the OD value as the abscissa to draw the linear regression curve of the standard,and calculated the concentration of each sample according to the curve equation.Compared with the model group,the levels of IL-6 and IL-8 in rats in the western medicine group and the qingjinhuatan decoction groups with high,medium and low doses were significantly decreased after the serum intervention.Conclusions : Qingjin huatan decoction can reduce the inflammation of airway epithelial cells in AECOPD rats and play a protective role in airway epithelium.The mechanism of action may be to inhibit the increase of inflammatory cytokines IL-6 and IL-8 through the regulation of autophagy in airway epithelial cells,so as to reduce the inflammatory response and exert the anti-inflammatory effect to inhibit the acute attack and aggravation of COPD.