| Bronchial asthma is a chronic airway inflammatory disease characterized by airway inflammation,airway remodeling and airway hyper-responsiveness.Genetic susceptibility and atmospheric pollution determine the occurrence and development of asthma.In recent years,some progress has been made in research on the pathogenesis of asthma.The treatment of asthma is constantly improving,but the incidence of bronchial asthma continues to increase.According to incomplete statistics,the number of people with bronchial asthma worldwide is close to 300 million.The latest study found that autophagy is involved in the pathogenesis of bronchial asthma,and the level of autophagy determines the severity of asthma.mTOR is an important factor regulating cell growth and proliferation.At the same time,it is also a key regulator of autophagy initiation.mTOR phosphorylation can inhibit autophagy.Ketamine can significantly improve the clinical symptoms of asthma patients,especially for refractory asthma.In this study,the mouse macrophage cell line RAW264.7 was used as a research object to observe the effect of ketamine on the expression of mTOR induced by S.aureus in RAW264.7 cells at the cellular level.Allergic airway inflammation mice were established by sensitizing and challenging mice with OVA.Then,intraperitoneal injection of mice with ketamine to observe its effect on airway inflammation and mTOR phosphorylation in lung tissue.At the same time,we used the mTOR inhibitor rapamycin to pretreat wild-type mice to further explore the mechanism of ketamine inhibition of allergic airway inflammation.Objective1.Investigate the effect of ketamine on the mTOR signaling pathway of macrophages infected with S.aureus.2.To investigate the effects of intraperitoneal injection of ketamine on airway inflammation and mTOR signaling pathway in mice with allergic airway inflammation.Methods1.To observe the expression of mTOR and autophagy-related proteins in different time of S.aureus-infected macrophagesMouse mononuclear macrophage RAW264.7 cell line was seeded in a 12-well cell culture plate at a density of 2.5 x 105 per well.Stimulated with Staphylococcus aureus for 15 min,30 min,45 min,60 min,120 min.Detection of mTOR and autophagy protein expression by immunoblotting.2.Effect of ketamine on the expression of mTOR and autophagy-related proteins in the process of macrophage infection by Staphylococcus aureusDifferent concentrations of ketamine(10uM,100uM,1000uM)were applied to RAW264.7 cells for lh to detect the effect of cell viability.Then,the macrophages were infected with Staphylococcus aureus for 1 hours and measure the expression of mTOR and autophagy proteins by Western Blot.3.To evaluate the effect of intraperitoneal injection of ketamine on airway inflammation and expression of autophagy-related proteins in allergic airway inflammation miceC57BL/6 female mice were randomly divided into 5 groups:control group(Con group),Allergic airway inflammation(OVA group),ketamine 25 mg/kg group(OVA+25 mg/kg group),ketamine 50 mg/kg group(OVA+50 mg/Kg group),ketamine 100mg/kg group(OVA+100mg/kg group).Mouse lung tissue and bronchoalveolar lavage fluid(BALF)were collected.HE staining was used to observe airway inflammation and PAS staining to observe the secretion of goblet cells and mucus.The expression of P-mTOR in lung tissue of mice was observed by immunohistochemistry.The expression of autophagosome in lung tissue of mice was observed by electron microscopy.The number of inflammatory cells in alveolar lavage fluid was counted by Wright's staining.The concentrations of IL-13,IL-6,TNF-? and IL-10 in the alveolar lavage fluid of mice were detected by ELISA kit.The expression of mTOR and downstream autophagy-related proteins in lung tissue were detected by immunoblotting.4.To observe the effect of intraperitoneal injection of rapamycin on airway inflammation and autophagy-related protein expression in ketamine-pretreated allergic airway inflammation micePretreatment of C57BL/6 female mice by intraperitoneal injection of rapamycin 1 mg/kg 30 min before ketamine injection.Mouse lung tissue and bronchoalveolar lavage fluid(BALF)were collected.HE staining was used to observe airway inflammation and PAS staining to observe the secretion of goblet cells and mucus.The expression of P-mTOR in lung tissue of mice was observed by immunohistochemistry.The expression of autophagosome in lung tissue of mice was observed by electron microscopy.The number of inflammatory cells in alveolar lavage fluid was counted by Wright's staining.The concentrations of IL-13,IL-6,TNF-a and IL-10 in the alveolar lavage fluid of mice were detected by ELISA kit.The expression of mTOR and downstream autophagy-related proteins in lung tissue were detected by immunoblotting.Results1.S.aureus infection macrophages activate autophagy and inhibit the expression of mTOR phosphorylationThe expression of P-mTOR was decreased in RAW264.7 cells stimulated by Staphylococcus aureus.At the same time,the expression of autophagy proteins Beclin-1 and LC3-? was significantly increased after S.aureus stimulated RAW264.7 cells.Moreover,the level of autophagy in macrophages was most significant in 60 min of S.aureus stimulation.At the same time,the secretion of pro-inflammatorv factors TNF-? and IL-6 in the culture supernatant was significantly increased after S.aureus stimulated macrophages for 60 min.2.Ketamine activates mTOR phosphorylation and down-regulation of autophagy in macrophages infected with S.aureusDifferent concentrations of ketamine have no effect on the cell viability of RAW264.7 macrophage cell line.Ketamine ean activate the expression of mTOR phosphorylation in S.aureus-infected macrophages,and the expression levels of autophagy proteins Beclin-1 and LC3-? stimulated by S.aureus are significantly reduced.This inhibition was most pronounced in the ketamine concentration of 1000uM.3.Ketamine significantly inhibits airway inflammation and activates mTOR phosphorylation in allergic airway inflammation miceCompared with the control group,C57BL/6 female mice under the OVA challenge,the number of inflammatory cells around the bronchiel increased,the tracheal wall thickened,goblet cell hyperplasia,mucus secretion increased,the number of inflammatory cells in alveolar lavage fluid increased,the levels of pro-inflammatory factors including IL-13,IL-6,and TNF-a are significantly increased,and the level of anti-inflammatory factor IL-10 is decreased.Compared with the OVA group,ketamine 50mg/kg significantly reduced the infiltration of inflammatory cells around the bronchi of allergic mice.Goblet cell hyperplasia and mucus secretion are significantly reduced,and inflammatory cells and inflammatory factors in alveolar lavage fluid are significantly decreased.However,Ketamine 25 mg/kg and 100 mg/kg had no protective effect on airway inflammation.Compared with the control group,the expression of P-mTOR in the lung tissue of ova group mice were decreased,and the expression levels of the autophagy proteins of Beclin-1 and LC3-? increased significantly.Ketamine 50mg/kg group can activate the expression of phosphorylated mTOR in mouse lung tissue,and the expression levels of Beclin-1 and LC3-? autophagy protein decrease.Ketamine 25 mg/kg and 100 mg/kg had no effect on the expression of autophagy in lung tissue of mice.Rapamycin can reverse the protective effect of ketamine 50mg/kg on airway inflammation in allergic mice4.Rapamycin can reverse the protective effect of ketamine 50mg/kg on airway inflammation in allergic airway inflammation miceKetamine 50mg/kg can significantly reduce the infiltration of inflammatory cells in the lung tissue of allergic mice,the proliferation of goblet cells and the content of inflammatory factors in alveolar lavage fluid are also reduced.After rapamycin pretreatment,the protective effect of ketamine was reversed.At the same time,the expression of mTOR phosphorylation in lung tissue of mice in the rapamycin group were decreased,and the expression levels of Beclin-1 and LC3-? autophagy proteins were increased significantly.Conclusions(1)Ketamine activates mTOR phosphorylation and down-regulated autophagy expression in RAW264.7 cells stimulated by Staphylococcus aureus.(2)Ketamine 50 mg/kg reduces allergic airway inflammation in mice by activating mTOR phosphorylation and down-regulating autophagy.(3)Rapamycin can reverse the protective effects of ketamine,and its mechanism may be related to inhibition of mTOR phosphorylation. |
PDF Full Text Request