Icariin attenuates asthmatic airway inflammation via modulating alveolar macrophage activation based on network pharmacology and in vivo experiments.

BACKGROUND: Icariin (ICA) inhibits inflammatory response in various diseases, but the mechanism underlying ICA treating airway inflammation in asthma needs further understood. We aimed to predict and validate the potential targets of ICA against asthma-associated airway inflammation using network pharmacology and experiments. METHODS: The ovalbumin-induced asthma-associated airway inflammation mice model was established. The effects of ICA were evaluated by behavioral, airway hyperresponsiveness, lung pathological changes, inflammatory cell and cytokines counts. Next, the corresponding targets of ICA were mined via the SEA, CTD, HERB, PharmMapper, Symmap database and the literature. Pubmed-Gene and GeneCards databases were used to screen asthma and airway inflammation-related targets. The overlapping targets were used to build an interaction network, analyze gene ontology and enrich pathways. Subsequently, flow cytometry, quantitative real-time PCR and western blotting were employed for validation. RESULTS: ICA alleviated the airway inflammation of asthma; 402 targets of ICA, 5136 targets of asthma and 4531 targets of airway inflammation were screened; 216 overlapping targets were matched and predicted ICA possesses the potential to modulate asthmatic airway inflammation by macrophage activation/polarization. Additionally, ICA decreased M1 but elevated M2. Potential targets that were disrupted by asthma inflammation were restored by ICA treatment. CONCLUSIONS: ICA alleviates airway inflammation in asthma by inhibiting the M1 polarization of alveolar macrophages, which is related to metabolic reprogramming. Jun, Jak2, Syk, Tnf, Aldh2, Aldh9a1, Nos1, Nos2 and Nos3 represent potential targets of therapeutic intervention. The present study enhances understanding of the anti-airway inflammation effects of ICA, especially in asthma.

TMT-based quantitative proteomics revealed protective efficacy of Icariside II against airway inflammation and remodeling via inhibiting LAMP2, CTSD and CTSS expression in OVA-induced chronic asthma mice.

BACKGROUND: Asthma is a chronic inflammatory disorder in airways with typical pathologic features of airflow limitation, airway inflammation and remodeling. Icariside II (IS), derived from herbal medicine Herba Epimedii, exerts an anti-inflammatory property. However, underlying mechanisms with specifically targeted molecular expression by IS in asthma have not been fully understood, and whether IS could inhibit remodeling and EMT still remains unclear. PURPOSE: The study aimed to clarify therapeutic efficacy of IS for attenuating airway inflammation and remodeling in asthma, and illustrate IS-regulated specific pathway and target proteins through TMT-based quantitative proteomics. STUDY DESIGN AND METHODS: Murine model of chronic asthma was constructed with ovalbumin (OVA) sensitization and then challenge for 8 weeks. Pulmonary function, leukocyte count in bronchoalveolar lavage fluid (BALF), lung histopathology, inflammatory and fibrotic cytokines, and markers of epithelial-mesenchymal transition (EMT) were evaluated. TMT-based quantitative proteomics were performed on lung tissues to explore IS-regulated proteins. RESULTS: IS contributed to alleviative airway hyperresponsiveness (AHR) evidenced by declined RL and increased Cdyn. After IS treatment, we observed a remarked down-regulation of leukocyte count, inflammatory cytokines in BALF, and peribronchial inflammation infiltration. Goblet cell hyperplasia, mucus secretion and peribronchial collagen deposition were attenuated, with the level of TGF-ß and MMP-9 in BALF declined. Furthermore, IS induced a rise of Occludin and E-cadherin and a decline of N-cadherin and α-SMA in lung tissues. These results proved the protective property of IS against airway inflammation, remodeling and EMT. To further investigate underlying mechanisms of IS in asthma treatment, TMT-based quantitative proteomics were performed and 102 overlapped DEPs regulated by IS were identified. KEGG enrichment exhibited these DEPs were enriched in lysosome, phagosome and autophagy, in which LAMP2, CTSD and CTSS were common DEPs. WB, q-PCR and IHC results proofed expressional alteration of these proteins. Besides, IS could decrease Beclin-1 and LC3B expression with increasing p62 expression thus inhibiting autophagy. CONCLUSIONS: The study demonstrated IS could ameliorate AHR, airway inflammation, remodeling and EMT in OVA-induced chronic asthma mice. Our research was the first to reveal that inhibition of LAMP2, CTSD and CTSS expression in autophagy contributed to the therapeutic efficacy of IS to asthma.

Albumin-based formononetin nanomedicines for lung injury and fibrosis therapy via blocking macrophage pyroptosis.

Pulmonary fibrosis that occurs following lung injury is a progressive and fatal disease since continual damage to lung tissue triggers the dysregulated inflammation response and accompanying abnormal healing process. Pyroptosis of alveolar macrophages has been found to play an essential role in the deterioration of lung injury and fibrosis. However, the lack of inhibitors against this inflammatory cell death in macrophages and the dense stroma pose major barriers to lung injury and fibrosis treatment. Herein, we developed an albumin-based nanoformulation to realize active delivery of formononetin (FMN) to improve the treatment of lung injury and fibrosis. The obtained nanoparticle, FMN@BSA NPs, could efficiently accumulate at the impaired lesion benefiting from the leaky vasculatures and the affinity between albumin and the overexpressed SPARC protein. Through blocking the NLRP3 inflammasome-involved pyroptosis process of macrophages, FMN@BSA NPs remarkably improved lung function and prolonged animal survival in the bleomycin (BLM)-induced lung injury and fibrosis model without noticeable side effects. Meanwhile, we proved FMN as a pyroptosis inhibitor and the corresponding lipid metabolism-related mechanisms through multi-omics analysis. This study first employed an albumin-based nanoparticle to deliver the pyroptosis inhibitor to the impaired lung tissue actively, providing a promising strategy for lung injury and fibrosis treatment.

Network pharmacology and experiments in vivo and in vitro reveal that the Jia-Wei-Bu-Shen-Yi-Qi formula (JWBSYQF) and its active ingredient baicalein ameliorate BLM-induced lung fibrosis in mice via PI3K/Akt signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Jia-Wei-Bu-Shen-Yi-Qi formula (JWBSYQF), a classical traditional Chinese herbal formula consisting of five herbs, is used clinically in China to treat inflammatory lung diseases, including asthma, chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary fibrosis (IPF). Its mechanism for treating asthma and COPD has been reported, however, how it works against IPF remains unclear. RESEARCH PURPOSE: Our study aims to observe the therapeutic effect of JWBSYQF on pulmonary fibrosis and further identify the potential active ingredients and molecular pathways. RESEARCH METHODS: In this study, we used a bleomycin-induced mouse model to investigate the therapeutic effect of JWBSYQF on pulmonary fibrosis. To further explore the potential effective ingredients and molecular pathways, we used the network pharmacology approach to construct a drug-ingredient-target network of JWBSYQF. Then, the common target set was established for JWBSYQF, fibroblast, and lung fibrosis. Analyses of the KEGG pathway, GO enrichment, and network topology were performed to identify key biological processes and molecular pathways for the common targets. Finally, a TGF-ß-induced NIH/3T3 proliferation and activation model was used to validate the possible active ingredients and signaling pathways. RESEARCH RESULTS: JWBSYQF reversed BLM-induced balf leukocyte levels, pulmonary inflammatory lesions and fibrotic collagen deposition in mice and reduced the levels of a-SMA, Col1a1 and TGF-ß. A total of 86 active ingredients were identified, 12 of which were considered as potential effective ingredients, while only baicalein effectively improved TGF-ß-induced proliferation and activation of NIH/3T3. KEGG results showed that PI3K/Akt signaling pathway may be the potential action mechanism, and Western Blot demonstrated that both JWBSYQF and baicalein downregulated the protein levels of p-PI3K and p-Akt. The molecular docking results suggest that baicalein may have a direct effect on the catalytic and regulatory subunits of P13K, which is stronger than direct binding to Aktl. CONCLUSIONS: Our study revealed that baicalein may be the material basis for JWBSYQF in the treatment of pulmonary fibrosis, and the PI3K/Akt signaling pathway may be a common pathway of action for JWBSYQF and baicalein.

Transcriptomics reveals apigenin alleviates airway inflammation and epithelial cell apoptosis in allergic asthma via MAPK pathway.

Persistent chronic inflammation of the lungs and airway remodeling are important pathological features that cannot be ignored in patients with chronic asthma. Apigenin (API) is a natural small molecule compound with good anti-inflammatory and antioxidant activity that has been widely reported in recent years, but its role in chronic asthma is not well defined. Our study began with oral gavage intervention using API (10, 20 mg/kg) or dexamethasone (DEX, 2 mg/kg) in a BALB/c mouse model of ovalbumin (OVA) sensitization. Different doses of API intervention effectively reduced airway resistance in the administered group. Additionally, inflammation was downregulated, mucus secretion was reduced, and airway remodeling was inhibited in the API intervention group compared with the model group. Asthma-related inflammatory cytokines, such as IgE, IL-4, IL-5, IL-13, and IL-17, were downregulated in alveolar lavage fluid. Moreover, the apoptosis level of the administered group was found to be lower than that of the model group in the Tunel staining experiment. By analyzing transcriptome sequencing results, we found that API may exert anti-inflammatory and anti-apoptotic effects by inhibiting the MAPK pathway. Our subsequent results supported this conclusion, showing that the phosphorylation levels of ERKs, JNKs, and p38 MAPKs were inhibited in the administered group relative to the model group. Downstream expression of the apoptosis-related protein B-cell lymphoma-2 (Bcl-2) was upregulated, and the expression of Bcl-2-associated × protein (Bax) and cleaved caspase-3 was downregulated. To further investigate the specific mechanism by which API acted, we established an in vitro model with house dust mite (HDM) stimulation, using API (10, 20 µM) for administration intervention. The results showed that API was able to improve cell viability, inhibit ROS production, and reverse HDM-induced decreases in mitochondrial membrane potential (MMP) and apoptosis in airway epithelial cells via the MAPK pathway.

Apigenin ameliorates non-eosinophilic inflammation, dysregulated immune homeostasis and mitochondria-mediated airway epithelial cell apoptosis in chronic obese asthma via the ROS-ASK1-MAPK pathway.

BACKGROUND: Obese asthma is one of the important asthma phenotypes that have received wide attention in recent years. Excessive oxidative stress and different inflammatory endotypes may be important reasons for the complex symptoms, frequent aggravation, and resistance to traditional treatments of obese asthma. Apigenin (API), is a flavonoid natural small molecule compound with good anti-inflammatory and antioxidant activity in various diseases and proved to have the potential efficacy to combat obese asthma. METHODS: In vivo, this study fed C57BL/6 J mice with high-fat diets(HFD)for 12 weeks and then stimulated them with OVA for 6 weeks to establish a model of chronic obese asthma, while different doses of oral API or dexamethasone were used for therapeutic interventions. In vitro, this study used HDM to stimulate human bronchial cells (HBEs) to establish the model and intervened with API or Selonsertib (SEL). RESULTS: This study clarified that OVAinduced a type of mixed granulocytic asthma with elevated neutrophils and eosinophils in obese male mice fed with long-term HFD, which also exhibited mixed TH17/TH1/TH2 inflammation. Apigenin effectively suppressed this complex inflammation and acted as a regulator of immune homeostasis. Meanwhile, apigenin reduced AHR, inflammatory cell infiltration, airway epithelial cell apoptosis, airway collagen deposition, and lung oxidative stress via the ROS-ASK1-MAPK pathway in an obese asthma mouse model. In vitro, this study found that apigenin altered the binding status of TRAF6 to ASK1, inhibited ASK1 phosphorylation, and protected against ubiquitin-dependent degradation of ASK1, suggesting that ROS-activated ASK1 may be an important target for apigenin to exert anti-inflammatory and anti-apoptotic effects. To further verify the intervention mechanism, this study clarified that apigenin improved cell viability and mitochondrial function and inhibited apoptosis by interfering with the ROS-ASK1-MAPK pathway. CONCLUSIONS: This study demonstrates for the first time the therapeutic effect of apigenin in chronic obese asthma and further clarifies its potential therapeutic targets. In addition, this study clarifies the specificity of chronic obese asthma and provides new options for its treatment.

Regulation of ferroptosis and ACSL4-15LO1 pathway contributed to the anti-asthma effect of acupuncture.

Acupuncture has been frequently used in China for the treatment asthma for thousands of years. Ferroptosis was recently revealed to be involved in several pathological conditions including asthma. However, the detailed links between ferroptosis and airway inflammation in asthma, as well as the detailed regulation of acupuncture on these disorders remains unclear. Our results demonstrated that the non-haem Fe2+ level increased markedly in the lung tissue of mouse asthma model, and positively correlated with RL and IL-4 level in BALF. Furthermore, lipid peroxidation markers MDA and GSSG increased remarkably in OVA-induced experimental asthma mice. Up-regulation of lipid peroxidation associated proteins ACSL4 and15-LO1 was also observed in OVA-induced experimental asthma mice. To demonstrate the role of ferroptosis in asthma and the effect of acupuncture on these disorders, ferroptosis-induction agent erastin and ferroptosis-inhibition agent fer-1 were used, and our data demonstrated that erastin could augment lung inflammation and lipid peroxidation in OVA induced asthma model. Fer-1 was able to relieve AHR, lung inflammation, non-haem Fe2+ level, lipid peroxidation and ferroptosis related pathway ACSL4-15LO1 in OVA-induced experimental asthma mice. Acupuncture treatment alleviated RL, lung inflammation as well as type 2 cytokines IL-4 and IL-13 levels induced by OVA inhalation. What's more, acupuncture significantly reduced the MDA and GSSG levels, the non-haem Fe2+ level and ACSL4-15-LO1 proteins expression. Acupuncture also relieved erastin-induced exacerbation in lung inflammation and lipid peroxidation in ferroptosis. Acupuncture treatment could relieve ferroptosis related exacerbation in airway inflammation. Our study provided insights into the underlying mechanisms for the protective effects of acupuncture and highlighted a therapeutic potential of acupuncture treatment in the attenuation of lipid peroxidation and ferroptosis in asthma.

TMT-Based Quantitative Proteomic Analysis Reveals Downregulation of ITGAL and Syk by the Effects of Cycloastragenol in OVA-Induced Asthmatic Mice.

Background: Cycloastragenol (CAG) has been reported to alleviate airway inflammation in ovalbumin- (OVA-) induced asthmatic mice. However, its specific mechanisms remain unclear. Objective: This study is aimed at investigating the effects of CAG on asthma, comparing its efficacy with dexamethasone (DEX), and elucidating the mechanism of CAG's regulation. Methods: The asthma mouse model was induced by OVA. CAG at the optimal dose of 125 mg/kg was given every day from day 0 for 20-day prevention or from day 14 for a 7-day treatment. We observed the preventive and therapeutic effects of CAG in asthmatic mice by evaluating the airway inflammation, AHR, and mucus secretion. Lung proteins were used for TMT-based quantitative proteomic analysis to enunciate its regulatory mechanisms. Results: The early administration of 125 mg/kg CAG before asthma happened prevented asthmatic mice from AHR, airway inflammation, and mucus hypersecretion, returning to nearly the original baseline. Alternatively, the administration of CAG during asthma also had the same therapeutic effects as DEX. The proteomic analysis revealed that the therapeutical effects of CAG were associated with 248 differentially expressed proteins and 3 enriched KEGG pathways. We then focused on 3 differentially expressed proteins (ITGAL, Syk, and Vav1) and demonstrated that CAG treatment downregulated ITGAL, Syk, and Vav1 by quantitative real-time PCR, western blot analysis, and immunohistochemical staining. Conclusion: These findings suggest that CAG exerts preventive and protective effects on asthma by inhibiting ITGAL, Syk, and the downstream target Vav1.

Louki Zupa decoction attenuates the airway inflammation in acute asthma mice induced by ovalbumin through IL-33/ST2-NF-κB/GSK3ß/mTOR signalling pathway.

CONTEXT: Asthma is a common respiratory system disease. Louki Zupa decoction (LKZP), a traditional Chinese medicine, presents a promising efficacy against lung diseases. OBJECTIVE: To investigate the pathogenic mechanism of asthma and reveal the intervention mechanism of LKZP. MATERIALS AND METHODS: Forty-eight female Balb/c mice were randomly divided into 6 groups: normal control group (NC), ovalbumin (OVA)/saline asthma model group, OVA/LL group, OVA/LM group, OVA/LH group and OVA/DEX group (n = 8 per group). The asthmatic mice were modelled through intraperitoneal injecting and neutralizing OVA. LKZP decoction was administrated by gavage at the challenge stage for seven consecutive days (2.1, 4.2 and 8.4 g/kg/day). We investigated the change in lung function, airway inflammation, mucus secretion and TH-1/TH-2-related cytokines. We further verify the activated status of the IL-33/ST2/NF-κB/GSK3ß/mTOR signalling pathway. RESULTS: LKZP was proved to improve asthmatic symptoms, as evidenced by the down-regulated airway resistance by 36%, 58% and 53% (p < 0.01, p < 0.001 vs. OVA/saline group), up-regulated lung compliance by 102%, 114% and 111%, decreased airway inflammation and mucus secretion by 33%, 40% and 33% (p < 0.001 vs. OVA/saline group). Moreover, the content of cytokines in BALF related to airway allergy (such as IgE) and T helper 1/T helper 2 cells (like IL-2, IL-4, IL-5, IL-13, TNF-α and IFN-γ), were also markedly reduced by 13-65% on LKZP intervention groups compared with model group. Mechanistic research revealed that the IL-33/ST2-NF-κB/GSK3ß/mTOR signalling pathway was activated in the OVA/saline group and LKZP significantly down-regulated this pathway. DISCUSSION AND CONCLUSION: LKZP improves lung function, airway inflammation, mucus secretion and correct immune imbalance by intervening with the IL-33/ST2-NF-κB/GSK3ß/mTOR signalling pathway, presenting a promising therapeutic choice for asthma.

A novel iridoid glycoside leonuride (ajugol) attenuates airway inflammation and remodeling through inhibiting type-2 high cytokine/chemokine activity in OVA-induced asthmatic mice.

BACKGROUND: Asthma is a chronic airway disorder with a hallmark feature of airflow obstruction that associated with the remodeling and inflammation in the airway wall. Effective therapy for controlling both remodeling and inflammation is still urgently needed. Leonuride is the main pharmacological component identified from Bu-Shen-Yi-Qi-Tang (BSYQT) which has been traditionally used in treatment of lung diseases. However, no pharmacological effects of leonuride in asthma were reported. PURPOSE: Here we aimed to investigated whether leonuride provided a therapeutic efficacy in reversing asthma airway remodeling and inflammation and uncover the underlying mechanisms. STUDY DESIGN AND METHODS: Mouse models of chronic asthma were developed with ovalbumin (OVA) exposure for 8 weeks. Respiratory mechanics, lung histopathology and asthma-related cytokines were examined. Lung tissues were analyzed using RNA sequencing to reveal the transcriptional profiling changes. RESULTS: After oral administration with leonuride (15 mg/kg or 30 mg/kg), mice exhibited a lower airway hyperresponsiveness in comparison to asthmatic mice. Leonuride suppressed airway inflammation evidenced by the significant reductions in accumulation of inflammatory cells around bronchi and vessels, leukocyte population counts and the abundance of type 2 inflammatory mediators (OVA specific IgE, IL-4, IL-5 and IL-13) in bronchoalveolar lavage fluid (BALF). On the other hand, leonuride slowed down the process of active remodeling as demonstrated by weaker goblet cell metaplasia and subepithelial fibrosis in lung histopathology and lower transforming growth factor (TGF)-ß1 levels in serum and BALF in comparison to mice treated with OVA only. Furthermore, we uncovered transcriptional profiling alternations in lung tissue of mice after OVA exposure and leonuride treatment. Gene sets belonging to type-2 cytokine/chemokine activity stood out in leonuride target transcripts. Those upregulated (Bmp10, Ccl12, Ccl22, Ccl8, Ccl9, Cxcl15, Il13, Il33, Tnfrsf9, Il31ra, Il5ra, Il13ra2 and Ccl24) or downregulated (Acvr1c and Il18) genes in asthmatic mice, were all reversely regulated by leonuride treatment. CONCLUSIONS: Our results revealed the therapeutic efficacy of leonuride in experimental chronic asthma for the first time, and implied that its anti-inflammatory and antifibrotic properties might be mediated by regulation of type-2 high cytokine/chemokines responses.

Iristectorigenin A exerts novel protective properties against airway inflammation and mucus hypersecretion in OVA-induced asthmatic mice: Iristectorigenin A ameliorates asthma phenotype.

BACKGROUND: Despite the substantial amount of efforts made to reduce morbidity and improve respiratory management, asthma control remained a major challenge for severe patients. Plant isoflavones, one of the most estrogenic compounds, are considered a potential alternative therapy for asthma. Iristectorigenin A, a naturally occurring isoflavone, is extracted from a variety of medical plants and its biological activity has not been reported previously. PURPOSE: In present study, we aim to reveal the potential therapeutic role of Iristectorigenin A against acute asthmatic mice. STUDY DESIGN: We established ovalbumin (OVA) induced asthmatic murine model and orally administrated Iristectorigenin A at concentration of 5 and 10 mg/kg and dexamethasone as a positive control substance. METHODS: Asthmatic murine model was established with OVA sensitization and challenge. Lung function was assessed with FinePoint Ventilation system recording lung resistance (RI) and lung compliance (Cydn). White cells were sorted and counted in BALF. Histopathological assessment was conducted by H&E, PAS, and Masson's trichrome staining on paraffin embedded lung tissues. BALF content of IL-4, IL-5, IL-33, IL-13, INF-γ, IL-9 and serum IgE, IgG1 were measured using ELISA kit. Expression levels of mRNAs associated with inflammatory cytokines and goblet cell metaplasia were evaluated via quantitative RT-PCR. Protein expression levels of FOXA3, MUC5AC, SPDEF were estimated by immunohistochemistry on lung tissue, while NOTCH1 and NOTCH2 expressions were evaluated by western blotting analysis. RESULTS: Iristectorigenin A resulted in improved airway hyperresponsiveness (AHR) mirrored by decreased RI and increased Cydn. With Iristectorigenin A, we also observed reduced number of BALF leukocytes, improved inflammatory cell infiltration in lung tissue, decreased content of BALF IL-4, IL-5, IL-33, but not IL-13, INF-γ, IL-9, and their mRNA levels, along with decreased levels of OVA-specific IgE, IgG1 in asthmatic mice. Additionally, Iristectorigenin A exhibited significant therapeutic potential on attenuating mucus production reflected by mitigated FOXA3 and MUC5AC immunostaining on the airway epithelium, as well as decreased mRNAs associated with goblet cell metaplasia. At last, a decrease in elevated expression level of NOTCH2, but not NOTCH1, in asthmatic mice lung tissue was observed by western blotting analysis. CONCLUSION: Our study provides strong evidence that Iristectorigenin A can be potential therapeutic agent ameliorating airway inflammation and mucus hypersecretion in allergic asthma. This is a first research reported the potential of Iristectorigenin A as an alternative therapeutic agent.

N6-Methyladenosine Methylomic Landscape of Lung Tissues in Murine Acute Allergic Asthma.

Allergic asthma is well known as a common respiratory disorder comprising an allergic inflammatory nature and excessive immune characteristic. N6-methyladenosine (m6A) methylation is an RNA epigenetic modification that post-transcriptionally regulates gene expression and function by affecting the RNA fate. Currently, m6A methylation is gaining attention as a mechanism of immunoregulation. However, whether m6A methylation engages the pathological process of asthma remains uncertain. Here, we present the m6A methylomic landscape in the lung tissues of ovalbumin-induced acute asthma mice using MeRIP-seq and RNA-seq. We identified 353 hypermethylated m6A peaks within 329 messenger RNAs (mRNAs) and 150 hypomethylated m6A peaks within 143 mRNAs in the lung tissues of asthmatic mice. These differentially methylated mRNAs were found to be involved in several immune function-relevant signaling pathways. In addition, we predicted 25 RNA-binding proteins that recognize the differentially methylated peak sites by exploring public databases, and the roles of these proteins are mostly related to mRNA biogenesis and metabolism. To further investigate the expression levels of the differentially methylated genes, we performed combined analysis of the m6A methylome and transcriptome data and identified 127 hypermethylated mRNAs (107 high and 20 low expression) and 43 hypomethylated mRNAs with differential expressions (9 high and 34 low expression). Of these, there are a list of mRNAs involved in immune function and regulation. The present results highlight the essential role of m6A methylation in the pathogenesis of asthma.