| ObjectiveBronchial asthma(referred to as asthma)is one of the most common chronic inflammatory diseases of the airway.According to the epidemiological data,there are currently about 300 million asthma patients worldwide,with a morbidity rate of 1.24%.The number of asthma patients in China is about 30 million,and most of them have uncontrolled symptoms and recurrent attacks,which seriously affects their quality of life.At present,the first-line drugs for the treatment of asthma are glucocorticoids.Although these drugs can alleviate symptoms effectively,about 40%of asthma patients do not respond to corticosteroid treatment.In addition,it usually takes a relatively long period for glucocorticoids to treat asthma and the long-term use of these drugs has a variety of side effects.In recent years,it has been found that Th17/Treg cell imbalance plays an important role in the pathogenesis of asthma,which provides new promise for the treatment of asthma.Researches on the development of new drugs to improve immunomodulatory function is warranted.In this study,ovalbumin(OVA),aluminum hydroxide(AL(OH)3)intraperitoneal injection and OVA nebulization were used to establish an allergic asthma airway inflammation model in BALB/c mice.The rLZ-8 was used to treat asthma mice,compared with normal mice,model group mice and dexamethasone intervention group mice,to clarify the effect of rLZ-8 on the airway inflammation and immune mechanism of asthmatic mice.In addition,the rLZ-8 intervention was used to culture Spleen T cells of mice in vitro for the observation of the effect of rLZ-8 on the proliferation and differentiation of T cells.This study aimed to explore the anti-inflammatory effect of rLZ-8 on asthma mice and its immune regulation mechanism,therefore providing theoretical basis for potential treatment options for asthma.Methods1.The effect and immune mechanism of rLZ-8 on asthma miceThe BALB/C mice were divided into 4 groups by random number method:control group,model group,dexamethasone group(DEX),rLZ-8 group,8 mice in each group.The asthma model was established by OVA induction,and treated with physiological saline,DEX,rLZ-8 and other drugs for 2 weeks.The levels of cytokines IL-17A,IL-10,IFN-γ and IL-4 in the bronchoalveolar lavage fluid(BALF)of each group of mice were detected by ELISA.The pathological changes of lung tissue were observed by HE staining.Immunohistochemical method was used to detect the level of eosinophil cationic protein(ECP)and dendritic cell(DC)surface molecules(CD11c and CD86)in lung tissue,and flow cytometry was used to detect CD4+ IFN-in spleen cells of asthmatic mice.Proportion of IFNγ+ Th1 cells,CD4+ IL-4+ Th2 cells,CD4+ IL-17A+ Th17 cells,CD4+ Foxp3+ Treg cells,CD11c+ CD80+ and CD11c+ CD86+ mature dendritic cells.Real-time PCR was used to detect the expression levels of transcription factors T-bet,IFN-γ,ROR y t,and Foxp3 mRNA in lung tissue.Western Blotting was used to detect the expression of STAT3 pathway in lung tissue.2.Immune mechanism of rLZ-8 on asthma airway inflammation in vitroThe spleen CD3+ T cells,CD4+ CD25+ T cells and CD4+ CD25-T cells of BALB/C mice were purified by flow cytometry for experiments in vitro.Apoptosis was detected by annexin/pi method to clarify the cytotoxicity of rLZ-8 and the safe concentration of that could be used in the experiment.After CD3+ T cells were activated and intervened with DEX and rLZ-8,the levels of cytokines IL-1β,IL-10 and TGF-β1 in the culture supernatant were detected by ELISA.CD3+ T cells were stimulated with CFSE Differentiation was induced,the level of CD4+ IL-17A+ was determined by flow cytometry after DEX and rLZ-8 intervention.The expression level of transcription factor ROR γ t was detected by Real-time PCR to explore the differentiation of CD3+ T cells.Flow sorting CD4+ CD25+ Treg cells and labeling the nucleus with CFSE.After DEX and LZ-8 intervention,flow cytometry was used to measure the proliferation of Treg cells and induce the differentiation of purified CD4+ CD25-T cells by flow cytometry.After DEX or rLZ-8 treatment,flow cytometry was used to determine the proportion of CD4+Foxp3+ Treg cells to explore the differentiation of Treg,and T cell protein was detected by Western Blotting to investigate the expression of STAT3 protein in each group of cells.Results1.The effect and immune mechanism of rLZ-8 on asthma miceLung tissue HE staining and immunohistochemistry results showed that the lung pathology of OVA-induced asthma model group showed obvious characteristics of asthma airway inflammation.HE staining showed that rLZ-8 and DEX reduced the airway inflammatory infiltration in mice,immunohistochemistry It showed that rLZ-8 and DEX reduced lung mature DC and eosinophils(P<0.01).Assay of BALF in asthmatic mice found that rLZ-8 reduced the level of IgE(P<0.01),and reduced the level of Th17 cytokine IL-17A(P<0.01)and increased the level of Treg cytokine IL-10 Levels(P<0.01),but rLZ-8 did not change the levels of Th1 cytokine IFN-γ and Th2 cytokine IL-4(P>0.05).Real-time PCR of lung tissue of asthmatic mice showed that rLZ-8 reduced the level of Th17 cell transcription factor RORγt and increased the level of Treg cell transcription factor Foxp3(P<0.01),but rLZ-8 had a significant effect on Thl cell transcription factor T-The level of bet and the level of Th2-related transcription factor GATA3 had no effect(P>0.05)In the spleen of mice,rLZ-8 significantly reduced the proportion of CD11c+CD86+ and CD11c+ CD80+ mature DC in the spleen of the mouse,while down-regulating the level of CD4+ IL-17A+ Th17 in the spleen of the mouse and upregulating the CD4+ Foxp3+ Treg Ratio(P<0.01),but rLZ-8 had no significant change in the ratio of CD4+ IL-4+ Th2 cells and CD4+ IFN-γ+ Th1 cells(P>0.05).Western Blotting detection of lung tissue suggested that rLZ-8 inhibited the expression of STAT3 signaling pathway in lung tissue(P<0.01).2.Immune mechanism of rLZ-8 on asthma airway inflammation in vitrorLZ-8 inhibited the production of Th17-related cytokine IL-1β in CD3+ T cell culture fluid(P<0.01),and promoted the production of Treg cytokines IL-10 and TGF-β1(P<0.01).In the in vitro differentiation and proliferation study of CD3+ T cells,it was found that rLZ-8 reduced the level of Th17 cell transcription factor RORγt mRNA,inhibited the activation and proliferation of CD4+ IL-17A+(P<0.01),and the differentiation of naive T cells into TH17 cells was inhibited..CD4+CD25+ Treg cell proliferation and CD4+CD25-T cell differentiation study in vitro found that rLZ-8 did not show the effect of promoting CD4+CD25+ T cell proliferation(P>0.05),but induced the differentiation of CD4+CD25+ T cells into CD4+Foxp3+ Treg(P<0.01),while DEX is shown to promote the proliferation of CD4+CD25+ T cells(P<0.01),but does not induce Treg differentiation(P>0.05).Western Blotting detection of T cells cultured in vitro indicated that rLZ-8 inhibited the activation of STAT3 signaling pathway in cultured T cells in vitro(P<0.01).ConclusionThe results of this study revealed the immunomodulatory effect of rLZ-8 in relieving airway inflammation in asthmatic mice.The mechanism might be related to the inhibition of the STAT3 signaling pathway and the differentiation of Th17 cells and the production of related cytokines.Meanwhile,rLZ-8 induced CD4+ Foxp3+ Treg differentiates and promoted the production of its cytokines,regulated Th17/Treg cell balance,and inhibited DC maturation,thereby playing an immunomodulatory role in the improvement of airway inflammation in asthma mice.Therefore,rLZ-8,which is an extract of Ganoderma lucidum,could be a potential medicine for asthma. |
