Effects Of Acupuncture On Endoplasmic Reticulum Stress-autophagy On Asthma Inflammation And T Cell Subsets Based On PI3K/AKT Pathway

Objective:The high incidence of bronchial asthma and its characteristic of recurrent chronic disease cause great social and economic pressure.Studies have shown that endoplasmic reticulum stress and autophagy are involved in the pathophysiological process of multiple subtypes of bronchial asthma,directly regulating the occurrence of airway inflammation and affecting steroid tolerance.PI3K/AKT pathway is a major signaling pathway that regulates autophagy.PI3 K activates Akt through phosphorylation and increases m TORC activity,thereby inhibiting the initiation of autophagy mediated by ER stress.In this study,the effects of acupuncture on airway inflammation in OVA-sensitized asthmatic mice were investigated by intervening PI3K/Akt pathway,and the effects of endoplasmic reticulum stress and autophagy process were observed.Methods:1.BALB/c mice model of bronchial asthma was established by OVA-AAI combined sensitization.Acupuncture of Dazhui,Feishu and Zusanli and PI3K/AKT pathway inhibitor LY294002 were used for intervention treatment.2.The pathological changes of lung tissues were detected by HE staining,Masson staining and Sirius red staining.The inflammatory factors in serum and bronchoalveolar lavage fluid(BALF)were detected by ELISA to evaluate the inflammatory levels of mice.3.Lung tissue electron microscopy,immunofluorescence staining,PCR and Western blot were used to verify the differences in the expression of autophagy in each group.4.The changes of ER stress levels and the expression of key molecules in PI3K/AKT pathway were detected by PCR,immunohistochemistry and Western blot.Results:1.Histological pathology tests show that compared with the control group,model group of epithelial cells in the lung tissue disorder,inflammatory cells,secretion is more transparent and eosinophil infiltration,wall thickening,obvious increase of collagen deposition,gas pipe wall thickening of muscle fibers and bronchial asthma airway inflammation and remodeling changes,acupuncture group and inhibitor symptoms significantly reduced;The changes of lung tissue and airway in combination group were between acupuncture group and model group.ELISA results showed that serum levels of Ig-E were significantly increased(P< 0.01),pro-inflammatory factors TGF-?,IL-4,IL-17 A were increased,anti-inflammatory factors IFN-? was decreased(P< 0.01),and the changes in each group were consistent with pathological changes.These results indicate that acupuncture therapy can reduce the degree of allergic inflammation and airway remodeling in asthmatic airway,and it is related to PI3K/AKT pathway.2.Compared with the blank group,the levels of ER stress and autophagy in the model group were significantly increased,and the levels of PI3 K,AKT and m TOR,the representative proteins of PI3K/AKT pathway,were significantly increased(P< 0.01),while the levels of ER stress and autophagy in the intervention groups were significantly decreased.The representative protein of PI3K/AKT pathway was significantly decreased(P< 0.01),indicating that ER stress and autophagy were positively correlated,and acupuncture could reduce ER stress and autophagy degree,which were related to PI3K/AKT pathway,and inhibition of PI3K/AKT pathway could inhibit ER stress and autophagy process.3.At the gene level,compared with the blank group,the levels of ER stress and autophagy in the model group were significantly increased,and the representative genes of PI3K/AKT pathway were significantly increased(P< 0.01).However,significantly different from the protein results,there were no significant changes in ER stress and autophagy genes in the acupuncture group and the combination group(P> 0.05).It is suggested that acupuncture therapy may regulate endoplasmic reticulum stress and autophagy in asthma by intervening post-transcriptional translation rather than changing gene expression level.Conclusion:Acupuncture can alleviate airway inflammation and remodeling by mediating endoplasmic reticulum stress-autophagy process in mice with bronchial asthma,and it is related to the regulation of PI3K/AKT pathway.Objective:Airway inflammation and immune regulation are the focus of the treatment of bronchial asthma.Bronchial asthma is closely related to the Ig-E-related immune response mainly mediated by T lymphocytes,and Th2-inflammation has been proven to be related to some patients with severe asthma,mainly manifested by the significantly increased Ig E level and serum inflammation level.Combined with the regulatory effect of acupuncture on airway inflammation and endoplasmic reticulum stress-autophagy verified by animal experiments,this study selected the bronchial epithelial cell line BEAS-2B cells,used inhibitors and agonists for positive and negative intervention,and extracted the conditional transgenic Atg5-/-mouse primary CD4+T lymphocytes for intervention culture.To investigate the role of endoplasmic reticulum stress-autophagy mediated by PI3K/ Akt pathway in inflammatory environment and its effect on T cell subsets..Methods:1.The bronchial epithelial cell line BEAS-2B cells were cultured,and LPS was used to create the inflammatory model.The cell proliferation was detected by CCK8 to determine the concentration and time of the intervention,and the inflammatory cytokines in the cell supernatant were detected.2.Primary CD4+T lymphocytes of Atg5-/-mice were extracted for sorting and intervention,and cell proliferation was detected by CFSE staining to determine the intervention time;3.Endoplasmic reticulum stress levels and PI3K/AKT pathway typical molecular expression levels of BEAS-2B cells in each group were detected by PCR and Western blot.4.The balance changes of primary CD4+T lymphocyte subsets Th1/Th2 and Treg/Th17 in each group were detected by flow cytometry.Results:1.The results of CCK8 indicated that the intervention concentration was 1?g/ m L LPS solution and 0.25?g/ m L TM solution,and the BEAS-2B cells were used for 24 h after intervention.The results of CFSE staining indicated that the intervention time of lymphocytes was 72 h.2.Compared with Con group,the pro-inflammatory factors TGF-?,IL-4 and IL-17 A were increased in LPS group and TM group,while the anti-inflammatory factor IFN-? was decreased,with statistical difference(P< 0.01);Compared with TM group,pro-inflammatory factors TGF-?,IL-4,IL-17 A and anti-inflammatory factors IFN-? were decreased in LY group and GS group,while pro-inflammatory factors TGF-?,IL-4,IL-17 A and anti-inflammatory factors IFN-? were increased in RA group,with statistical difference(P< 0.01).3.At the protein level,compared with the Con group,the expression levels of autophagy protein LC3 B and Beclin-1 were significantly increased in the LPS group,while the expression levels of p62 were significantly decreased,with statistical differences(P<0.01).Compared with the LPS group,the protein expression levels of LC3 B and Beclin-1were significantly increased in the TM group,while the protein expression levels of p62 were significantly decreased(P< 0.01);the protein expression levels of LC3 B and Beclin-1were significantly decreased in the TU group,while the protein expression levels of p62 were significantly increased(P< 0.01).The protein expression levels of Beclin-1,LC3 B,p-PI3K/PI3 K,p-AKT/AKT,p-m TOR/m TOR in LPS group and TM group were significantly increased,with statistical difference(P< 0.01);the protein expression level of p62 was significantly decreased,with statistical difference(P < 0.01).Compared with the TM group,the p-m TOR/m TOR protein expression level in the RA group was significantly decreased,with statistical difference(P< 0.01);the p-PI3K/PI3 K protein expression level in the LY group was significantly decreased,with statistical difference(P< 0.01);the pAKT/AKT protein expression level in the GS group was significantly decreased.There was statistical difference(P < 0.01).At the gene level,the gene expression levels of LC3 b,Beclin-1,p62,PI3 K,Akt and m TOR were basically consistent with the protein expression levels.The results showed that inhibition of PI3K/AKT pathway could inhibit the activation of autophagy induced by ER stress.4.After intervention,the ratios of Th1/Th2 and Treg/Th17 in the primary T lymphocytes of transgenic mice were significantly decreased in the TM group,with statistically significant differences(P< 0.01).The ratios of Treg cells decreased,Th17 cells increased,Th1 cells decreased and Th2 cells increased.Compared with TM group,the subgroup imbalance of Atg5-/-group and TL group was reversed,suggesting that inhibition of PI3K/AKT pathway can inhibit endoplasmic reticulum stress and autophagy process,and can correct the balance of T lymphocyte differentiation.