| Objective:Establishing a model of airway inflammation injury in rats with bronchitic asthma and inducing human airway epithelial cells（16HBE）inflammation injury model with lipopolysaccharide（LPS）in vitro,the protective effect of pingpaning on airway inflammation in bronchial asthma and its molecular mechanism were studied,which provided experimental basis and new ideas for the prevention and treatment of asthma and its complications.MethodsPart one:The role of autophagy in relieving airway inflammation in rats with bronchial asthmaThere were 105 grade SPF SD rats,The high,middle and low dose groups were given 14.578,7.289,3.645 g·kg-1 respectively,the dexamethasone group was given0.405 mg·kg-1,the Guilongkechuanning group was given 0.405 g·kg-1,and the normal group and the model group were given the same amount of normal saline respectively.The general conditions of rats in each group were observed,including body mass,water intake and food intake.Detection of bronchoalveolar lavage fluid（BALF）.Part two:The role of autophagy in relieving the 16HBE inflammatory model1 To establish an in vitro model of LPS induced 16HBE inflammatory injuryCell lines of 16HBE were cultured in vitro.According to the LPS concentration,they were divided into normal medium without cells,normal medium with cells,200ng/ml LPS group,400ng/ml LPS group,800ng/ml LPS group,1000ng/ml LPS group,1200ng/ml LPS group,1600ng/ml LPS group,and 2000ng/ml LPS group.Cell viability was determined by CCK8.2 Study on the protective mechanism of pingpanning on lps-induced inflammatory injury model of human airway epithelial cellsThe cells were divided into complete culture medium without cells and normal culture medium cells.5%PCN+LPS group;10%PCN+LPS group;15%PCN+LPS group;20%PCN group containing pharmaceutical serum+LPS group.Cell morphology was observed by optical inverted mic ROScope.Cell viability was determined by CCK8.Then,the second part of the experiment was conducted,which was divided into five groups:normal control group,LPS group（LPS1200ng/ml concentration）,3-MA group（LPS with concentration of 1200ng/ml after concentration of 2.5mm pre-protection）,RAPA group（LPS with concentration of1200ng/ml after concentration of 0.1um pre-protection）,and PCN group（LPS with concentration of 1200ng/ml after concentration of 10%PCN pre-protection）.The ROS release was detected by flow cytometry,the ultrastructure of cells in each group was observed by TEM,the expression of Beclin-1,Bcl-2,and ATG5 in cells was detected by IF,the expression of Beclin-1,Bcl-2,LC3 and ATG5 in cells was detected by WB,and the levels of inflammatory cytokines IL-6,IL-8,and TNF-in cells were detected by ELISA.3 Antiasthmatic ning alleviates the damage of the 16HBE asthma inflammatory model through the ROS/HMGB1/Beclin-1 pathwayThe 16HBE cells were divided into normal control group,LPS group（1200ng/ml concentration LPS）,Glycyrrhizin group（1200ng/ml concentration LPS added after pre-protection of HMGB1 inhibitor with a concentration of 0.15um）and PCN group（1200ng/ml concentration LPS added after pre-protection with a concentration of 10%PCN）.The cell processing and detection methods were as follows:ROS release was detected by flow cytometry,ultrastructure of cells in each group was observed by TEM,protein expression of HMGB1 and Beclin-1 Bcl-2 in cells was detected by IF method,and protein expression of HMGB1,Beclin-1,Bcl-2,LC3 and ATG5 in cells was detected by WB method.ResultsPart one:The role of autophagy in relieving airway inflammation in rats with bronchial asthma1 Changes in the general condition of asthmatic model rats and the effect of PCNOn day 0 of modeling,there was no difference in body mass of rats in each group（P>0.05）.After 21 days of modeling,compared with the normal group,the body mass of rats in each group decreased significantly（P<0.01）.On the 35th day of modeling,the body mass of rats in each modeling group still showed a decreasing trend compared with that in the control group（P<0.01）.By the end of the experiment,the body mass of rats in each group was significantly lower than that in the normal group（P<0.01）.Compared with the model group,the body mass of rats in each treatment group increased（P<0.01）.There was no statistical difference in the food intake of rats in each group （P>0.05）.After 21 days of modeling,there was no significant difference in food intake in each group（P>0.05）.On the 35th day of modeling,the body mass of rats in each modeling group was decreased compared with that in the control group（P<0.01）.On the 49d at the end of the experiment,compared with the normal group,the food intake of rats in each group was reduced（P<0.01）.Compared with the model group,the food intake of rats in each treatment group was higher than that in the model group （P<0.01）.On day 0,there was no difference in water intake in each group（P>0.05）.After21 days of modeling,there was no significant difference in water intake in each group（P>0.05）.On the 35th day of modeling,the water intake of rats in each modeling group was less than that in the control group（P<0.01）.At the end of the experiment（49d）,compared with the normal group,the water intake of rats in each group decreased（P<0.01）.Compared with the model group,the water intake of rats in each treatment group increased compared with that in the model group（P<0.01）.2 Changes in lung function and the effect of PCN in asthmatic ratsCompared with the normal group,model FEV0.3,FVC and FEV0.3/FVC all had differences（P<0.01）.Compared with the model group,there were differences in FEV,FVC and FEV/FVC in each treatment group（P<0.01）.3 Changes of inflammatory cell number in BALF and the effect of PCN in asthmatic ratsCompared with the normal group,the number of neutrophils,eosinophils,lymphocytes,macrophages and leukocytes in BALF in the model group was statistically different（P<0.01）.Compared with the model group,the number of neutrophils,eosinophils,lymphocytes,macrophages and white blood cells in BALF of the gellong kechuatanin group and dexamethasone group were statistically different（P<0.01）.4 The change of lung pathological morphology and the effect of PCN in asthmatic ratsThe lung tissues of rats in each group were stained with HE and PAS,and the observation under light mic ROScope showed that:the normal group showed no obvious infiltration of inflammatory cells around the bronchi,clear lung structure,normal pulmonary septum,intact bronchial wall and alveolar structure,no exudation in the trachea,and no obvious pathological changes.The pathological changes in the lung tissues of rats in the model group were obvious.and the lumen was narrowed compared with the normal group.The submucosal mucus of the trachea threatens to proliferate and dilate.PAS staining in bronchioles revealed goblet cell proliferation and significantly increased airway mucus secretion.The pathological changes of the rats in the drug group were improved to different degrees.The degree of proliferation of peribronchiolar goblet cells and the amount of mucus secretion were significantly reduced.The infiltration of bronchial inflammatory cells was reduced in the low-dose group and the medium-dose group.There were differences between the model group and the normal group （P<0.01）.Compared with the model group,there was no difference between HE inflammation score and PAS score in the pinglow group（P>0.05）,and the rest of the treatment groups decreased（P<0.01）.5 Changes of ROS,serum MDA and SOD in lung tissues and effects of PCN in asthmatic ratsThe ROS,MDA and SOD values of the model group were different from those of the normal group（P<0.01）.Compared with the model group,ROS,MDA and SOD values in each treatment group were different（P<0.01）.6 Ultrastructural changes of lung tissue and the effect of PCN in asthmatic ratsWith transmission electron mic ROScope each level autophagy in rat lung tissue cells,observed between groups are visible autophagy vesicles,model group rats have more autophagy vesicles,and normal cells,and other treatment group observed electron mic ROScopy figure autophagy bubble is much less than the model group,in addition,heavy osmium lamellar corpuscle emptying obviously,disordered arrangement of mitochondrial crest and edema,and the treatment group of lamellar corpuscle emptying II type cells and mitochondria change is lighter.7 The expression levels of HMGB1,Beclin-1,ATG5 and PCN were detected by immunofluorescence method in the lung tissue of asthmatic ratsThe immunofluorescence density of Beclin-1,HMGB1 and ATG5 increased in the model group compared with the normal group（P<0.01）.Compared with the model group,there was no difference in the HMGB1 immunofluorescence density in the low-dose antiasthmatic group,and there were differences in the other groups compared with the model group（P<0.01）.8 The expression levels of HMGB1,Beclin-1,Bcl-2,ATG5,LC3 and PCN in lung tissues of asthmatic rats were detected by WB methodThe expression of Beclin-1,HMGB1,ATG5 and Bcl-2 proteins and the LC3II/LC3I ratio in the model group were statistically different from that in the normal group（P<0.01 or P<0.05）.Compared with the model group,Beclin-1,HMGB1 and ATG5 protein expression were down-regulated,Bcl-2 protein expression was up-regulated and LC3II/LC3I ratio was down-regulated（P<0.01 or P<0.05）.Compared with the kelong kechuanning group,the expressions of Beclin-1,HMGB1,ATG5,and Bcl-2 proteins and the LC3II/LC3I ratio in the low-dose antipanning group were statistically different（P<0.01）,and the expressions of Beclin-1 and HMGB1 proteins in the medium-dose antipanning group were up-regulated（P<0.05）.9 The expression levels of HMGB1,Beclin-1,Bcl-2,ATG5m RNA and PCN were detected by WB method in the lung tissues of asthmatic ratsCompared with the normal group,HMGB1m RNA,Beclin-1mrna,ATG5m RNA and Bcl-2m RNA in the model group were all different（P<0.01 or P<0.05）.Compared with the model group,there was no statistical difference in Beclin-1 mrna in the low and medium dose groups,and there was a statistical difference in the other drug intervention groups compared with the model group（P<0.01 or P<0.05）.10 Changes in IFN-γ,IL-6,IL-1β,IL-8,IL-1β,and IFN-γlevels and the effects of PCN on lung tissue in asthmatic ratsThere were statistically significant differences between the model group and the normal group（P<0.01 or P<0.05）.Compared with the model group,all the drug intervention groups were different（P<0.01）.Part two:The effect of pingpanning on autophagy of 16HBE inflammatory model cells1 Establishment of an in vitro model of lps-induced inflammation of human bronchial airway epithelial cellsIn this study,LPS were used to screen the model concentration in the classic in vitro model of asthma airway inflammation.With the increase of LPS dose and the extension of LPS action time,the cell survival rate was 45.9±6.0%after 1200ng/ml PS action for 12H,which was close to IC50.Therefore,this concentration of LPS action on 16HBE cells for 12H was selected to model airway inflammation.2 Effect of PCN on ROS changes after in vitro interventionCompared with the normal group,the ROS water level in the model group increased by（P<0.01）or（P<0.05）.Compared with the model group,ROS levels in all treatment groups decreased by（P<0.01）.3 Effects of PCN on ultrastructural changes of cells after intervention in vitroIn the normal group,the cell structure and morphology were basically normal,and the nucleus chromatin was abundant and evenly distributed.Endoplasmic reticulum,mitochondria and kurtosis were intact.Autophagy vesicles,the signature structure of autophagy,were observed in the cells modeled by LPS.In the RAPA group,autophagy vesicles increased,and a small number of mitochondria showed a mitolysis fracture.After the intervention of 3-MA and PCN groups,the morphology and ultrastructure of the cells were improved,and the cell morphology was good.Autophagic vesicles and complete mitochondria were observed.4 The effect of PCN on the expression of Beclin-1,Bcl-2,LC3 and ATG5 in cells detected by WB after in vitro interventionCompared with normal group,model group,ATG5 protein expression,LC3II Beclin-1/LC3I ratio increased（P<0.01 or P<0.05）,decreased Bcl-2 protein expression（P<0.01）,compared with model group,3-MA group and RAPA group and PCN Beclin 1,ATG5 protein expression,LC3II/LC3I ratio decrease（P<0.01 or P<0.05）,higher Bcl-2 protein expression（P<0.01）.5 The effect of PCN on the expression of Beclin-1,Bcl-2 and ATG5 in cells after in vitro intervention by immunofluorescence assayCompared with the normal group,the expression of ATG5 and Beclin-1 in the model group increased（P<0.01）,and the expression of Bcl-2 decreased （P<0.01）.Compared with the model group,the ATG5 protein expression was decreased,the Bcl-2 protein expression was increased,and the Beclin-1 protein expression was decreased in the RAPA group,the 3-MA group and the PCN group（P<0.01）.6 Effects of PCN on changes in levels of inflammatory cytokines IL-6,IL-8 and TNF-αafter intervention in vitroCompared with the normal group,the levels of IL-6,IL-8 and TNF-in the model group were all increased（P<0.01 or P<0.05）.Compared with the model group,IL-6,IL-8 and TNF-levels in the 3-MA group and the PCN group and IL-6 levels in the RAPA group all decreased（P<0.01 or P<0.05）,while IL-8 and TNF-levels in the RAPA group showed no difference compared with the model group（P>0.05）.CPart three:The effect of antiasthmatic on autophagy of 16HBE inflammatory model cells through ROS/HMGB1/Beclin-1 pathway1 Effect of PCN on ROS changes after in vitro interventionCompared with the normal group,ROS levels in the model group increased（P<0.01）,while ROS levels in both the GLY group and the PCN group decreased（P<0.01）.2 Effects of PCN on ultrastructural changes of cells after intervention in vitroAs shown in the figure,the basic structure and morphology of the cells in the normal control group were intact,and the nucleus chromatin was abundant and evenly distributed.Endoplasmic reticulum and mitochondria were seen,and the kurtosis was relatively complete.Autophagy vesicles,the hallmark structure of autophagy,were observed in the cells modeled by LPS.After intervention,the morphology and ultrastructure of the cells in the GLY and PCN groups were improved,and the basic morphology of the cells was better.3 The effect of PCN on the expression of Beclin-1,Bcl-2,LC3 and ATG5 in cells detected by WB after in vitro interventionCompared with the normal group,the protein expression of HMGB1,BECLIN and ATG5 in the model group increased（P<0.01 or P<0.05）,the protein expression of Bcl-2 decreased（P<0.01）,and the LC3II/LC3I ratio increased（P<0.01）.In the model group,HMGB1,BECLIN,ATG5 protein expression was decreased（P<0.01 or P<0.05）,Bcl-2 protein expression was increased（P<0.01）,and LC3II/LC3I ratio was decreased（P<0.01 or P<0.05）.4 The effect of PCN on the expression of Beclin-1,Bcl-2 and ATG5 in cells after in vitro intervention by immunofluorescence assayHMGB1 than with the normal group,model group,increasing Beclin 1 protein expression and Bcl-2 protein expression reduced（P<0.01）,compared with model group,GLY,PCN HMGB1,Beclin 1 protein expression and Bcl-2 protein expression increased（P<0.01 or P<0.05）.ConclusionPingchuan Ning can relieve airway inflammation and improve lung function in rats.The mechanism may be related to reducing oxidative stress levels,reducing excessive accumulation of ROS,inhibiting the combination of HMGB1 and Beclin-1,and achieving inhibition of autophagy and thus relieving inflammatory damage.Pingchuan Ning has a protective effect on 16 HBE of inflammatory injury induced by LPS,which may be related to the inhibition of autophagy and the reduction of inflammatory response by inhibiting overexpression of ROS/HMGB1/Beclin-1. |
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