| ObjectiveTo explore whether Yanghe Pingchuan Granules can regulate the immune imbalance of Th17/Treg cells by regulating PD1/PDL1 signal pathway,so as to improve the airway inflammation of asthma model rats.Methods1 In vivo experimentsAsthma rat model was established by ova sensitization and aerosol challenge and divided into normal group,model group,Yanghe Pingchuan Granules low,medium and high dose groups and positive drug group(dexamethasone),and the rats were challenged for 4 weeks.Observation and detection indicators:(1)The general state and behavioral changes of the rats were observed during the aerosol challenge.(2)HE staining of lung tissue pathological sections.(3)Count and classification of inflammatory cells in bronchoalveolar lavage fluid.(4)The expression levels of serum IL-10,TGF-β1 and IL-17 were detected by ELISA.(5)The ratio of Th17 and Treg cells in rat spleen was detected by flow cytometry.(6)RT-PCR detection of Th17 cell transcription factor RORγt m RNA expression and Treg cell transcription factor Foxp3m RNA expression in lung tissue.(7)Western-blot detection of Th17 cell transcription factor RORγt protein level and Treg cell transcription factor Foxp3 protein level in lung tissue.(8)Immunohistochemical detection of PD1 and PDL1 levels in rat lung tissue.2 In vitro experimentsThe immune imbalance model of Th17/Treg cells in vitro was established by adding TGF-β,IL-6 and IL-23 to CD4~+T cells.PDL1 si RNA was transfected to inhibit PD1/PDL1 pathway in vitro,and PDL1 overexpression vector was transfected to promote PD1/PDL1 pathway.The experiment was carried out in three stages:(1)Experiment 1:Divided into normal group,model group,model+Yanghe Pingchuan Granules optimal drug-containing serum group,model+positive drug group(dexamethasone);(2)Experiment 2:Screening the best small interference of PDL1After si RNA,the experiment was divided into normal group,model group,model+Yanghe Pingchuan Granules optimal drug-containing serum group,model+PDL1 small interference-NC group,model+PDL1 small interference group,model+PDL1 small interference+Yanghe Pingchuan Granules optimal drug-containing serum group.(3)Experiment 3:After constructing PDL1overexpression vector and transfecting cells,the experiment was divided into normal group,model group,model+Yanghe Pingchuan Granules optimal drug-containing serum group,model+PDL1 overexpression-NC group,model+PDL1 Overexpression group,model+PDL1 overexpression+Yanghe Pingchuan granules optimal drug-containing serum group.After corresponding intervention,flow cytometry was used to detect the proportion of Th17 and Treg cells in each group.Elisa detected the levels of Th17-related cytokines TGF-β1,IL-17 and Treg-related cytokines IL-10.RT-PCR and Western blot were used to detect the gene and protein expression levels of TGF-β1,RORγt,IL-17 related to Th17 cell differentiation and Foxp3,IL-10related to Treg cell differentiation,respectively.RT-PCR,Western blot detection of PD1-PDL1 pathway PD1 and PDL1 gene and protein expression levels.Results1 Results of in vivo experiments(1)Effects of Yanghe Pingchuan Granules on the behavior of asthmatic rats:The rats in the model group and each drug treatment group all developed asthma-like symptoms and decreased appetite after 1 week of challenge,like to lie down,poor spirit,loose stools,lodging hair,rough and tarnished,blue tongue,fear of cold and other phenomena,which were normal.There was no obvious abnormal performance in the group.After the intervention of Yanghe Pingchuan Granules and dexamethasone,the asthma-like symptoms and other symptoms and signs of the rats were improved to varying degrees compared with the model group.(2)Effects of Yanghe Pingchuan Granules on the pathology of lung tissue in asthmatic rats:Compared with the normal group,the lung tissue structure of the rats in the model group was severely damaged,the bronchial epithelium was shed,the lumen was narrow,the smooth muscle of the tube wall was thickened,the interior was filled with a large amount of mucus,and a large number of inflammatory cells were infiltrated around the tube wall.Compared with the model group,the inflammatory infiltration of the lung tissue in the Yanghe Pingchuan Granules low,medium and high dose groups and the dexamethasone group were improved to varying degrees,mainly manifested in the reduction of the bronchial lumen stenosis and smooth muscle thickening,and the degree of inflammatory cell infiltration.and reduced viscous secretions.(3)Effects of Yanghe Pingchuan Granules on inflammatory cells in bronchoalveolar lavage fluid of asthmatic rats:Compared with the normal group,the counts of various inflammatory cells in the bronchoalveolar lavage fluid of the model group were significantly increased(P<0.01);The difference in granulocyte and lymphocyte counts was statistically significant(P<0.05),and the difference in neutrophil count in bronchoalveolar lavage fluid between the high-dose Yanghe Pingchuan Granules group and the dexamethasone group was statistically significant(P<0.05).(4)Effects of Yanghe Pingchuan Granules on Th17 and Treg cytokines in asthmatic rats:Compared with the normal group,the serum IL-10 content of the model group was significantly decreased(P<0.01);compared with the model group,the serum IL-10content of the Yanghe Pingchuan Granules group and the dexamethasone group was significantly increased(P<0.01);compared with the normal group,the serum levels of IL-17 and TGF-β1 in the model group were significantly increased(P<0.01);compared with the model group,the Yanghe Pingchuan granules group and the dexamethasone group The contents of IL-17 and TGF-β1 in serum were significantly decreased(P<0.01).(5)Effects of Yanghe Pingchuan Granules on the ratio of Th17 cells and Treg cells in spleen tissue of asthmatic rats:Compared with the normal group,the proportion of Th17cells in the spleen of the model group was significantly increased(P<0.01),and the proportion of Treg cells was significantly decreased(P<0.01).The proportion of Th17cells in the spleen of the dose group and dexamethasone group was significantly decreased(P<0.01),and the proportion of Treg cells was significantly increased(P<0.01).(6)Effects of Yanghe Pingchuan Granules on Th17 cells and Treg cell-specific transcription factor genes and proteins in the lung tissue of asthmatic rats:(1)Compared with the normal group,the m RNA expression of Th17 cell-specific transcription factor RORγt in the model group was significantly increased(P<0.01);The expression of sex transcription factor RORγt m RNA was significantly decreased(P<0.01).Compared with the normal group,the expression of Treg cell-specific transcription factor Foxp3m RNA in the model group was significantly decreased(P<0.01).Significantly increased(P<0.01).(2)Compared with the normal group,the expression of Th17cell-specific transcription factor RORγt protein in the model group was significantly increased(P<0.01);Protein expression was significantly decreased(P<0.01).Compared with the normal group,the expression of Treg cell-specific transcription factor Foxp3protein in the model group was significantly decreased(P<0.01);Significantly increased(P<0.01);(7)Effects of Yanghe Pingchuan Granules on PD1/PDL1 pathway proteins in lung tissue of asthmatic rats:Lung tissue immunohistochemistry showed that compared with the normal group,the expression of PD1 and PDL1 proteins in the model group increased;compared with the model group,the expressions of PD1 and PDL1 in the Yanghe Pingchuan granule group and the dexamethasone group decreased.2 Results of in vitro experiments(1)Experiment 1:(1)The effect of Yanghe Pingchuan granule-containing serum on Th17cells and Treg cytokines in vitro:Compared with the normal group,the Th17 cytokines IL-17 and TGF-β1 in the model group were significantly increased(P<0.01),Treg cytokine IL-10 decreased significantly(P<0.01).Compared with the model group,the Th17 cytokines IL-17 and TGF-β1 in the Yanghe Pingchuan granule group and the dexamethasone group were significantly decreased(P<0.01),and the Treg cytokine IL-10 was significantly increased(P<0.01).(2)The effect of Yanghe Pingchuan granule-containing serum on the proportion of Th17 cells and Treg cells in vitro:Compared with the normal group,the proportion of Th17 cells in the model group was significantly increased(P<0.01),and the proportion of Treg cells was significantly decreased(P<0.01).Compared with the model group,the proportion of Th17 cells in the Yanghe Pingchuan granule group and the dexamethasone group was significantly decreased(P<0.01),and the proportion of Treg cells was significantly increased(P<0.01).(3)Effect of Yanghe Pingchuan granule-containing serum on genes related to Th17 and Treg cell differentiation and PD1/PDL1 pathway-related genes in vitro:Compared with the normal group,the model group showed Th17 cell differentiation-related TGF-β1,IL-17,RORγt and PD1/PDL1 The relative expressions of pathway-related PD1 and PDL1 m RNA were significantly increased(P<0.01);compared with the model group,the relative expressions of TGF-β1,IL-17,RORγt,PD1,and PDL1 m RNA in the Yanghe Pingchuan granule group and the dexamethasone group were higher than those in the model group.Significantly decreased(P<0.01).Compared with the normal group,the relative expressions of IL-10 and Foxp3 m RNA related to Treg cell differentiation in the model group were significantly decreased(P<0.01).The relative expression levels were significantly increased(P<0.01).(4)Effect of Yanghe Pingchuan Granule-containing serum on Th17 and Treg cell differentiation-related and PD1/PDL1 pathway-related proteins in vitro:Compared with the normal group,the relative expressions of TGF-β1,IL-17,RORγt,PD1,and PDL1proteins in the model group Compared with the model group,the relative expressions of TGF-β1,IL-17,RORγt,PD1,PDL1 in Yanghe Pingchuan granules group and IL-17and RORγt in dexamethasone group were significantly decreased.(P<0.05).Compared with the normal group,the relative expressions of IL-10 and Foxp3 in the model group were significantly decreased(P<0.01);compared with the model group,the relative expression of IL-10 in the Yanghe Pingchuan Granules group was significantly increased(P<0.05).(2)Experiment 2:(1)Effect of Yanghe Pingchuan Granule-containing serum on inhibiting of PD1/PDL1 pathway Th17 cells and Treg cytokines in vitro:Compared with the model group,the Th17 cytokines IL-17 and TGF-β1 in the small interference group were significantly decreased(P<0.01),Treg cytokine IL-10 was significantly increased(P<0.01);compared with the small interference group,the Treg cytokine IL-10of the small interference+Yanghe Pingchuan granule was significantly increased(P<0.01).(2)The effect of Yanghe Pingchuan granule-containing serum on the proportion of Th17 cells and Treg cells that inhibit PD1/PDL1 pathway in vitro:Compared with the model group,the proportion of Th17 cells in the small interference group was significantly decreased(P<0.01),and the proportion of Treg cells was significantly increased(P<0.01);compared with the small interference group,the proportion of Th17 cells in the small interference+Yanghe Pingchuan granules was significantly decreased(P<0.01),and the proportion of Treg cells was significantly increased(P<0.01).(3)Effect of Yanghe Pingchuan Granule-containing serum on inhibiting of PD1/PDL1 pathway Th17 and Treg cell differentiation-related genes and PD1/PDL1 pathway-related genes in vitro:Compared with the model group,the small interference group showed Th17 cell differentiation-related IL-17,TGF-β1,RORγt,and pathway-related PD1 and PDL1 m RNA ratios decreased significantly(P<0.01),while Treg cell differentiation-related IL-10 and Foxp3 m RNA ratios significantly increased(P<0.01).Compared with the small interference group,the small interference+Yanghe Pingchuan granules The m RNA levels of IL-17,TGF-β1,RORγt,PD1 and PDL1decreased significantly(P<0.01),while the m RNA levels of IL-10 and Foxp3 increased significantly(P<0.01).(4)Effect of Yanghe Pingchuan Granule-containing serum on inhibiting PD1/PDL1 pathway Th17 cells in vitro,Treg cell differentiation-related and PD1/PDL1 pathway-related proteins:Compared with the model group,the proportion of TGF-β1 protein related to Th17 cell differentiation in the small interference group was significantly higher.Compared with the small interference group,the RORγt protein of small interference+Yanghe Pingchuan granules decreased significantly(P<0.05).(3)Experiment 3:(1)Effect of Yanghe Pingchuan Granule-containing serum on Th17and Treg cytokines in the PD1/PDL1 pathway promoted in vitro:Compared with the model group,the Th17 cytokines IL-17 and TGF-β1 in the overexpression group were significantly increased(P<0.01),Treg cytokine IL-10 decreased significantly(P<0.01);compared with overexpression group,Th17 cytokine IL-17 and TGF-β1 decreased significantly in overexpression+Yanghe Pingchuan Granules(P<0.05),Treg cytokine IL-10 increased significantly(P<0.01).(2)The effect of Yanghe Pingchuan granule-containing serum on the proportion of Th17 cells and Treg cells promoting PD1/PDL1pathway in vitro:Compared with the model group,the proportion of Th17 cells in the overexpression group was significantly increased(P<0.01),and the proportion of Treg cells was significantly decreased(P<0.01);compared with the overexpression group,the proportion of Th17 in overexpression+Yanghe Pingchuan granules was significantly decreased(P<0.01),and the proportion of Treg cells was significantly increased(P<0.01).(3)The effect of Yanghe Pingchuan granule-containing serum on the genes related to the differentiation of Th17 cells and Treg cells promoting the PD1/PDL1pathway in vitro,and the genes related to the PD1/PDL1 pathway:Compared with the model group,the Th17 cell differentiation-related IL-17,TGF-βin the overexpression group-β1,RORγt and pathway-related PD1,PDL1 m RNA ratios increased significantly(P<0.01),while Treg cell differentiation-related IL-10 and Foxp3 m RNA ratios significantly decreased(P<0.01);The m RNA levels of IL-17,TGF-β1,RORγt,PD1,and PDL1 in Yanghe Pingchuan granules were significantly decreased(P<0.01),and the m RNAs of IL-10 and Foxp3 were significantly increased(P<0.01).(4)Effect of Yanghe Pingchuan granule-containing serum on promotion of PD1/PDL1 pathway Th17 and Treg cell differentiation-related and PD1/PDL1pathway-related proteins in vitro:Compared with the model group,Th17 cell differentiation-related IL-17,TGF-β1,RORγt and pathway-related PDL1 proteins were significantly increased(P<0.05);compared with the overexpression group,the IL-17,TGF-β1,RORγt,and PDL1 proteins of the overexpression+Yanghe Pingchuan granules were significantly decreased(P<0.05).Conclusion(1)Yanghe Pingchuan Granules can effectively improve the airway inflammation of asthma model rats and regulate the immune imbalance of Th17/Treg cells in vivo.(2)Yanghe Pingchuan Granules can inhibit PD1/PDL1 signal pathway,reduce Th17cells and related factors,increase Treg cells and related factors,and regulate the immune imbalance of Th17/Treg cells,so as to treat asthma. |
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