Objective To investigate whether miR-15b-5p can alleviate airway inflammation in asthma by negatively regulating ubiquitin specific peptidase 9X (USP9X) to down-regulate the expression of phosphatidylinositol 4, 5-diphosphate 3-kinase catalytic subunit α/AKT serine/threonine kinase 1 (PIK3CA/AKT1) pathway. Methods USP9X was predicted to be a direct target of miR-15b-5p by using an online database (miRWalk), and the luciferase reporter gene assay was performed to verify it. Co-immunoprecipitation (CO-IP) was used to verify the direct binding between USP9X and PIK3CA and the role of USP9X and its small molecule inhibitor WP1130 in the deubiquitination of PIK3CA. C57 mice were randomly divided into Control group, OVA group, OVA combined with NC group and miR-15b-5p agomir group, with 10 mice in each group. BEAS-2B cells were induced with interleukin 13 (IL-13) and treated with miR-15b-5p mimic. HE, Masson, PAS, immunohistochemistry, immunofluorescence staining, flow cytometry, Western blot and quantitative real-time PCR(qRT-PCR) were performed. Results It was found that the administration of miR-15b-5p agomir and mimic could reduce peribronchial inflammatory cells and improve airway inflammation, and miR-15b-5p could target negative regulation of USP9X. USP9X could directly bind to PIK3CA and regulate PIK3CA level in a proteasome-dependent manner, and USP9X could deubiquitinate K29-linked PIK3CA protein. Down-regulation of USP9X could increase PIK3CA ubiquitination level. WP1130, a small molecule inhibitor of USP9X, has the same effect as knockdown of USP9X, both of which could increase the ubiquitination level of PIK3CA and reduce the protein level of PIK3CA. Conclusion The miR-15b-5p/USP9X/PIK3CA/AKT1 signaling pathway may provide potential therapeutic targets for asthma.
Animals ; MicroRNAs/metabolism* ; Asthma/pathology* ; Class I Phosphatidylinositol 3-Kinases/genetics* ; Ubiquitin Thiolesterase/metabolism* ; Proto-Oncogene Proteins c-akt/genetics* ; Mice ; Signal Transduction ; Mice, Inbred C57BL ; Humans ; Inflammation/genetics* ; Cell Line ; Female ; Male

Objective To explore the mechanism of Glaucocalyxin A(GLA)in inhibiting ovalbumin(OVA)-induced airway remodeling in asthmatic mice through the topoisomerase Ⅱ α(TOP2A)/cyclin-dependent kinase 1(CDK1)signaling pathway.Methods Forty Balb/c mice were randomly divided into 5 groups:the control group,the model group,the low-dose GLA group,the high-dose GLA group and the Dexamethasone group,with 8 mice in each group.The effect of GLA on airway remodeling was examined by immunohistochemical staining,ELISA and other methods,and bioinformatics methods were used to predict new targets of GLA.The action targets of TOP2A were screened using the STRING database,and the interaction relationship between the two was verified by co-immunoprecipitation.In vitro,GLA and siRNA were used to interfere with interleukin-4(IL-4)-stimulated human airway epithelial cells BEAS-2B.The expressions of TOP2A,epidermal growth factor receptor(EGFR),Integrin β1,focal adhesion kinase(FAK),β-catenin,CDK1 and DRP1 were detected by Western Blot.Results GLA intervention could significantly reduce OVA-induced asthma airway remodeling,airway smooth muscle thickening,collagen deposition around the airway,the number of eosinophils in alveolar lavage fluid,the expression of pro-inflammatory cytokines such as IL-4,and the level of serum IgE.The new target of GLA screened out was TOP2A,which was highly expressed in the lung tissue of the asthma airway remodeling model.GLA intervention could down-regulate its expression.In vitro,intervention with GLA and si-TOP2A could significantly down-regulate the expressions of IL-4-induced TOP2A,EGFR,Integrin β1,FAK and β-catenin.Further studies have found that TOP2A had an interaction relationship with CDK1.si-TOP2A could downregulate the expression of CDK1,and knockdown of CDK1 could significantly down-regulate the expression of phosphorylated DRP1.Conclusion GLA may alleviate asthma airway remodeling by targeting the TOP2A/CDK1 signaling pathway,providing experimental evidence for the clinical diagnosis and treatment of asthma airway remodeling in asthma.

Objective:To explore the effects of imperatorin(IMP)on airway remodeling in the bronchial asthma mice,and to elucidate the possible mechanisms.Methods:Forty SFP male BALB/c mice were randomly divided into control group,model group,low dose of IMP group(IMP-L group),high dose of IMP group(IMP-H group)and dexamethasone group,with 8 mice in each group.Except for contol group,the mice in the other groups were injected with an ovalbumin(OVA)suspension intraperitoneally to induce the asthma models.After one week,the daily asthma symptoms of the mice were observed and scored.After 8 weeks,the enhanced pause(Penh)values of the mice in various groups were detected to evaluate the airway reactivities.The percentages of eosinophils in the bronchoalveolar lavage fluid(BALF)of the mice in various groups were detected by flow cytometry.The levels of serum IgE,interleukin interferon-gamma(IL)-13,IL-5,IL-4 and interferon-gamma(IFN-γ)in BALF of the mice in various groups were measured by enzyme-linked immunosorbent assay(ELISA)method.HE,PAS and Masson staining were applied to observe the pathomorphology,the number of goblet cells and collagen deposition of the lung tissue of the mice in various groups.Immunohisto chemistry method was applied to detect the expressions of α-smooth muscle actin(α-SMA)and mouse mammary tumor virus(MMTV)wingless type MMTV intergration site family member 5A(Wnt5A)proteins in lung tissue of the mice in various groups.The expression levels of Wnt5A,cellular myelocytomatosis oncogene(c-Myc),β-catenin and α-SMA in lung tissue of the mice in various groups were detected by Western blotting method.The expression levels of α-SMA protein in lung tissue of the mice in various groups were detected by immunofluorescence method.Results:Compared with control group,the score of asthma symptoms of the mice in model group was increased(P<0.01);the Penh value was significantly increased(P<0.01);the serum IgE levels and the levels of IL-13,IL-5,IL-4 in BALF,as well as the percentage of eosinophils(EOS)in BALF were significantly increased(P<0.05 or P<0.01),and the level of IFN-γ was reduced(P<0.05);the expression levels of α-SMA and Wnt5A proteins in lung tissue were markedly increased(P<0.01);the expression levels of proteins associated with the Wnt/β-catenin signaling pathway in the lung tissue were significantly increased(P<0.01);the immofluorescence method results showed the expression level of α-SMA protein in lung tissue was significantly increased(P<0.01).Compared with model group,the scores of asthma symphtoms of the mice in IMP-L group,IMP-H group,and dexamethasone group were decereased(P<0.01),and the Penh values of the mice in IMP-H group were decreased(P<0.05);the serum IgE levels and the levels of IL-13,IL-5,IL-4 in BALF,as well as the percentages of EOS in BALF of the mice in IMP-L group,IMP-H group,and dexamethasone group were decreased(P<0.05 or P<0.01),and the levels of IFN-γ were increased(P<0.05);the expression levels α-SMA and Wnt5A proteins in lung tissue were decreased(P<0.05 or P<0.01);the expression levels of proteins related to the Wnt/β-catenin signaling pathway in the lung tissue were decreased(P<0.05 or P<0.01);the immunofluorescence method results showed that expression levels of the α-SMA protein in the lung tissue were reduced(P<0.05 or P<0.01).Conclusion:IMP has an improving effect on airway remodeling in the asthmatic mice and can inhibit the expression levels of Wnt/β-catenin pathway-related proteins.

Objective To explore the role of Erianin in atopic dermatitis(AD)and its regulatory mechanism involving the high-mobility group box 1(HMGB1)/receptor for advanced glycation end products(RAGE)-Ras homolog gene family member A(RhoA)/Rho-associated protein kinase 1(ROCK1)signaling pathway.Methods An AD model was induced in BALB/c mice using 1-chloro-2,4-dinitrobenzene(DNCB).Skin thickness and spleen and lymph node weight were measured and pathological changes in the back skin and ears were detected using methylamine blue and hematoxylin and eosin staining.Inflammatory factors were detected by enzyme-linked immunosorbent assay.An in vitro model of AD was established in HaCaT cells stimulated by tumor necrosis factor(TNF)-α.Cellular reactive oxygen species(ROS)were detected by flow cytometry and mitochondrial ROS(mtROS)were detected by immunofluorescence assay.Cell apoptosis was detected by terminal deoxynucleotidyl transferase dUTP nick-end labeling.HMGB1,RAGE,RhoA,and ROCK1 proteins were detected by Western blot.Results Erianin inhibited the increase in skin thickness,reduced the spleen and lymph node weights,improved the infiltration of inflammatory cells and the degranulation of mast cells,and reduced the levels of inflammatory factors(P＜0.05).Erianin also reduced the production of cellular ROS and mtROS induced by TNF-α in vitro(P＜0.01),and decreased the protein expression of HMGB1,RAGE,RhoA,and ROCK1(P＜0.01).Treatment of HMGB1-stimulated HaCaT cells with a RAGE-specific blocker(TFA)had no effect on HMGB1 expression,while expression levels of RAGE,RhoA,and ROCK1 were decreased(P＜0.01).Cells treated with the Rho kinase inhibitor Y-27632+r-HMGB1 group showed similar result to the TFA+r-HMGB1 group,except for RAGE.Conclusions Erianin relieves AD by regulating the HMGB1/RAGE-RhoA/ROCK signaling pathway.

Nowadays,the incidence of allergic diseases is increasing worldwide,which seriously affects the quality of human life.Recent studies have shown that sex hormones are closely related to the occurrence of allergic diseases.Sex hormones can directly affect the function and development of immune cells,as well as the susceptibility to autoimmune cell responses,resulting in different prevalence and clinical manifestations of allergic diseases in men and women.This article reviews the different roles and potential mechanisms of sex hormones in the occurrence and development of common allergic diseases.In atopic dermatitis,the amount of dehy-droepiandrosterone sulfate into dehydroepiandrosterone sulfate is higher in females than in males.Therefore,females are more suscep-tible to the influence of dehydroepiandrosterone,which inhibits the proliferation of Th2,resulting in a series of clinical symptoms.In allergic asthma,estrogen can aggravate type 2 airway inflammation and androgens can reduce type 2 airway inflammation.Studies have found that there are estrogen and progesterone receptors in the nasal mucosa.When the estrogen concentration increases,the two re-ceptors are also up-regulated,resulting in clinical manifestations such as increased nasal secretions and nasal mucosal swelling.

Objective To explore the role of Erianin in atopic dermatitis(AD)and its regulatory mechanism involving the high-mobility group box 1(HMGB1)/receptor for advanced glycation end products(RAGE)-Ras homolog gene family member A(RhoA)/Rho-associated protein kinase 1(ROCK1)signaling pathway.Methods An AD model was induced in BALB/c mice using 1-chloro-2,4-dinitrobenzene(DNCB).Skin thickness and spleen and lymph node weight were measured and pathological changes in the back skin and ears were detected using methylamine blue and hematoxylin and eosin staining.Inflammatory factors were detected by enzyme-linked immunosorbent assay.An in vitro model of AD was established in HaCaT cells stimulated by tumor necrosis factor(TNF)-α.Cellular reactive oxygen species(ROS)were detected by flow cytometry and mitochondrial ROS(mtROS)were detected by immunofluorescence assay.Cell apoptosis was detected by terminal deoxynucleotidyl transferase dUTP nick-end labeling.HMGB1,RAGE,RhoA,and ROCK1 proteins were detected by Western blot.Results Erianin inhibited the increase in skin thickness,reduced the spleen and lymph node weights,improved the infiltration of inflammatory cells and the degranulation of mast cells,and reduced the levels of inflammatory factors(P＜0.05).Erianin also reduced the production of cellular ROS and mtROS induced by TNF-α in vitro(P＜0.01),and decreased the protein expression of HMGB1,RAGE,RhoA,and ROCK1(P＜0.01).Treatment of HMGB1-stimulated HaCaT cells with a RAGE-specific blocker(TFA)had no effect on HMGB1 expression,while expression levels of RAGE,RhoA,and ROCK1 were decreased(P＜0.01).Cells treated with the Rho kinase inhibitor Y-27632+r-HMGB1 group showed similar result to the TFA+r-HMGB1 group,except for RAGE.Conclusions Erianin relieves AD by regulating the HMGB1/RAGE-RhoA/ROCK signaling pathway.

Objective To explore the mechanism of Glaucocalyxin A(GLA)in inhibiting ovalbumin(OVA)-induced airway remodeling in asthmatic mice through the topoisomerase Ⅱ α(TOP2A)/cyclin-dependent kinase 1(CDK1)signaling pathway.Methods Forty Balb/c mice were randomly divided into 5 groups:the control group,the model group,the low-dose GLA group,the high-dose GLA group and the Dexamethasone group,with 8 mice in each group.The effect of GLA on airway remodeling was examined by immunohistochemical staining,ELISA and other methods,and bioinformatics methods were used to predict new targets of GLA.The action targets of TOP2A were screened using the STRING database,and the interaction relationship between the two was verified by co-immunoprecipitation.In vitro,GLA and siRNA were used to interfere with interleukin-4(IL-4)-stimulated human airway epithelial cells BEAS-2B.The expressions of TOP2A,epidermal growth factor receptor(EGFR),Integrin β1,focal adhesion kinase(FAK),β-catenin,CDK1 and DRP1 were detected by Western Blot.Results GLA intervention could significantly reduce OVA-induced asthma airway remodeling,airway smooth muscle thickening,collagen deposition around the airway,the number of eosinophils in alveolar lavage fluid,the expression of pro-inflammatory cytokines such as IL-4,and the level of serum IgE.The new target of GLA screened out was TOP2A,which was highly expressed in the lung tissue of the asthma airway remodeling model.GLA intervention could down-regulate its expression.In vitro,intervention with GLA and si-TOP2A could significantly down-regulate the expressions of IL-4-induced TOP2A,EGFR,Integrin β1,FAK and β-catenin.Further studies have found that TOP2A had an interaction relationship with CDK1.si-TOP2A could downregulate the expression of CDK1,and knockdown of CDK1 could significantly down-regulate the expression of phosphorylated DRP1.Conclusion GLA may alleviate asthma airway remodeling by targeting the TOP2A/CDK1 signaling pathway,providing experimental evidence for the clinical diagnosis and treatment of asthma airway remodeling in asthma.

Nowadays,the incidence of allergic diseases is increasing worldwide,which seriously affects the quality of human life.Recent studies have shown that sex hormones are closely related to the occurrence of allergic diseases.Sex hormones can directly affect the function and development of immune cells,as well as the susceptibility to autoimmune cell responses,resulting in different prevalence and clinical manifestations of allergic diseases in men and women.This article reviews the different roles and potential mechanisms of sex hormones in the occurrence and development of common allergic diseases.In atopic dermatitis,the amount of dehy-droepiandrosterone sulfate into dehydroepiandrosterone sulfate is higher in females than in males.Therefore,females are more suscep-tible to the influence of dehydroepiandrosterone,which inhibits the proliferation of Th2,resulting in a series of clinical symptoms.In allergic asthma,estrogen can aggravate type 2 airway inflammation and androgens can reduce type 2 airway inflammation.Studies have found that there are estrogen and progesterone receptors in the nasal mucosa.When the estrogen concentration increases,the two re-ceptors are also up-regulated,resulting in clinical manifestations such as increased nasal secretions and nasal mucosal swelling.

UNC-51-like kinase 1(ULK1)is an important factor involved in regulating the initiation of autophagy.ULK1 regu-lates inflammatory cytokines through autophagy and mitochondrial oxidative stress,and is involved in the pathological processes of var-ious diseases.ULK1 and its complexes are regulated by rapamycin(mTOR)and AMP-activated protein kinase(AMPK)to initiate au-tophagy,thereby exerting differential effects on a variety of inflammatory diseases.In inflammatory diseases,mitochondrial oxidative stress can induce ULK1 into the nucleus to accelerate apoptosis.Therefore,ULK1 plays different important roles in inflammatory dis-eases.For example,ULK1 initiates airway epithelial mitochondrial autophagy in asthma,participates in mitochondrial oxidative stress in acute liver failure,affects related inflammatory factors in atherosclerosis,and modulates beneficial effects of autophagy in diabetes.This article reviews the biological function of ULK1,its impact on inflammatory diseases and the research progress of targeted drugs.

We investigated whether FTY-720 might have an effect on bleomycin-induced pulmonary fibrosis through inhibiting TGF-β1 pathway, and up-regulating autophagy. The pulmonary fibrosis was induced by bleomycin. FTY-720 (1 mg/kg) drug was intraperitoneally injected into mice. Histological changes and inflammatory factors were observed, and EMT and autophagy protein markers were studied by immunohistochemistry and immunofluorescence. The effects of bleomycin on MLE-12 cells were detected by MTT assay and flow cytometry, and the related molecular mechanisms were studied by Western Blot. FTY-720 considerably attenuated bleomycin-induced disorganization of alveolar tissue, extracellular collagen deposition, and α-SMA and E-cadherin levels in mice. The levels of IL-1β, TNF-α, and IL-6 cytokines were attenuated in bronchoalveolar lavage fluid, as well as protein content and leukocyte count. COL1A1 and MMP9 protein expressions in lung tissue were significantly reduced. Additionally, FTY-720 treatment effectively inhibited the expressions of key proteins in TGF-β1/TAK1/P38MAPK pathway and regulated autophagy proteins. Similar results were additionally found in cellular assays with mouse alveolar epithelial cells. Our study provides proof for a new mechanism for FTY-720 to suppress pulmonary fibrosis. FTY-720 is also a target for treating pulmonary fibrosis.