ABSTRACT
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.
Subject(s)
Asthma , Proteomics , Mice , Animals , Ovalbumin , Asthma/drug therapy , Asthma/metabolism , Lung/pathology , Inflammation/metabolism , Bronchoalveolar Lavage Fluid , Cytokines , Disease Models, Animal , Mice, Inbred BALB CABSTRACT
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.
Subject(s)
Acupuncture Therapy , Anti-Asthmatic Agents , Asthma , Ferroptosis , Pneumonia , Animals , Mice , Anti-Asthmatic Agents/therapeutic use , Anti-Asthmatic Agents/pharmacology , Asthma/therapy , Asthma/drug therapy , Coenzyme A Ligases/metabolism , Coenzyme A Ligases/pharmacology , Disease Models, Animal , Glutathione Disulfide/adverse effects , Inflammation , Interleukin-4/pharmacology , Ovalbumin/therapeutic use , Pneumonia/drug therapy , Arachidonate 15-Lipoxygenase/metabolismABSTRACT
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.
Subject(s)
Asthma , NF-kappa B , Animals , Bronchoalveolar Lavage Fluid , Cytokines/metabolism , Disease Models, Animal , Female , Glycogen Synthase Kinase 3 beta/metabolism , Inflammation/pathology , Interleukin-1 Receptor-Like 1 Protein/metabolism , Interleukin-33/metabolism , Lung/metabolism , Mice , Mice, Inbred BALB C , NF-kappa B/metabolism , Ovalbumin , TOR Serine-Threonine Kinases/metabolismABSTRACT
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.
Subject(s)
Asthma , Cytokines , Animals , Bronchoalveolar Lavage Fluid , Chemokines , Disease Models, Animal , Inflammation , Iridoid Glycosides , Iridoids , Lung , Mice , Mice, Inbred BALB C , Ovalbumin , PyransABSTRACT
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.
Subject(s)
Asthma , Interleukin-33 , Animals , Asthma/drug therapy , Bronchoalveolar Lavage Fluid , Disease Models, Animal , Immunoglobulin E , Immunoglobulin G , Inflammation/drug therapy , Interleukin-33/metabolism , Interleukin-4/metabolism , Interleukin-5/metabolism , Interleukin-9/metabolism , Interleukin-9/therapeutic use , Lung/pathology , Metaplasia/metabolism , Metaplasia/pathology , Mice , Mice, Inbred BALB C , Mucus , Ovalbumin , PhenotypeABSTRACT
Allergic asthma is associated with T helper (Th) 2 cell-biased immune responses and characterized by the airway hyperresponsiveness (AHR). Studies have shown that the acupoint catgut-embedding therapy (ACE) has a therapeutic effect on allergic asthma. However, the relevant mechanism is poorly understood. In present study, female BALB/c mice were sensitized and challenged with ovalbumin (OVA) to establish a model of allergic asthma. AHR was evaluated by using airway resistance (R L ) and lung dynamic compliance (Cdyn). Airway inflammation and mucus hypersecretion were observed by HE and PAS staining. Inflammatory cells were counted, and related cytokines in bronchoalveolar lavage fluid (BALF) were detected by enzyme-linked immunosorbent assay (ELISA). Pulmonary group 2 innate lymphoid cell (ILC2s) proportions were analyzed by flow cytometry. The expression of nuclear factor κB (NF-κB) and cyclooxygenase-2 (COX-2) was detected by immunostaining. Our results showed that OVA induction resulted in a significant increase in R L , accompanied by a significant decrease in Cdyn. The levels of interleukin- (IL-) 4, IL-13, OVA-specific IgE in BALF, and the percentage of ILC2 in the lungs were markedly increased accompanied by a significant decreased in interferon-γ (IFN-γ). Furthermore, the expressions of p-NF-κB p65 and COX-2 in airways were significantly upregulated. After ACE treatment, the indicators above were significantly reversed. In conclusion, ACE treatment inhibited the secretion of Th2 cytokines and the proliferation of ILC2s in the lungs, thereby dampening the inflammatory activity in allergic asthma. The underlying mechanism might be related to the inhibition of NF-κB/COX-2 pathway.
Subject(s)
Asthma , NF-kappa B , Acupuncture Points , Animals , Asthma/drug therapy , Asthma/therapy , Bronchoalveolar Lavage Fluid , Catgut , Cyclooxygenase 2/metabolism , Cytokines/metabolism , Disease Models, Animal , Female , Immunity, Innate , Lung/metabolism , Lymphocytes/metabolism , Mice , Mice, Inbred BALB C , NF-kappa B/metabolism , OvalbuminABSTRACT
BACKGROUND: Jia-Wei Bu-Shen-Yi-Qi formula (JWBSYQF), a Chinese herbal formula, is a commonly used prescription for treating asthma patients. However, the targeted proteins associated with JWBSYQF treatment remain unknown. PURPOSE: Present study aims to evaluate the therapeutic efficacy of JWBSYQF and identify the targeted proteins in addition to functional pathways. STUDY DESIGN: The ovalbumin (OVA)-induced murine asthma model was established to explore the therapeutic effect of JWBSYQF treatment. Proteomic profiling and quantifications were performed using data-independent acquisition (DIA) methods. Differentially expressed proteins (DEPs) were validated via western blot (WB) and immunohistochemistry (IHC). METHODS: A murine asthma model was made by OVA sensitization and challenge, and JWBSYQF (2.25, 4.50, 9,00 g/kg body weight) or dexamethasone (1 mg/ kg body weight) were administered orally. Airway hyperresponsiveness (AHR) to methacholine (Mch), inflammatory cell counts and classification in bronchoalveolar lavage fluid (BALF), lung histopathology, and cytokine levels were measured. Furthermore, DIA proteomic analyses were performed to explore the DEPs targeted by JWBSYQF and were further validated by WB and IHC. RESULTS: Our results exhibited that JWBSYQF attenuated AHR which was mirrored by decreased airway resistance and increased lung compliance. In addition, JWBSYQF-treated mice showed reduced inflammatory score, mucus hypersecretion, as well as reduced the number of BALF leukocytes along with decreased content of BALF Th2 inflammatory cytokines (IL-4, IL-5, IL-13) and serum IgE. Proteomics analysis identified 704 DEPs between the asthmatic mice and control group (MOD vs CON), and 120 DEPs between the JWBSYQF-treatment and the asthmatic mice (JWB-M vs MOD). A total of 33 overlapped DEPs were identified among the three groups. Pathway enrichment analysis showed that DEPs were significantly enriched in IL-17 signaling pathway, in which DEPs, Lcn2, TGF-ß1, Gngt2, and Ppp2r5e were common DEPs between three experimental groups. WB and IHC results further validated expressional levels and tendency of these proteins. Our results also showed that JWBSYQF affects mitogen activated protein kinase (MAPK) and nuclear factor-κB (NF-κB) signaling pathways, that are activated by IL-17 signaling. CONCLUSION: The present study suggested that JWBSYQF could attenuate AHR and airway inflammation in OVA-induced asthmatic mice. In addition, proteomics analysis revealed that suppression of IL-17 signaling pathways contributes to the therapeutic effects of JWBSYQF.
Subject(s)
Asthma , Drugs, Chinese Herbal/pharmacology , Interleukin-17 , Proteomics , Signal Transduction , Animals , Asthma/drug therapy , Bronchoalveolar Lavage Fluid , Cytokines , Disease Models, Animal , Lung , Mice , Mice, Inbred BALB C , OvalbuminABSTRACT
Cycloastragenol (CAG), a secondary metabolite from the roots of Astragalus zahlbruckneri, has been reported to exert antiinflammatory effects in heart, skin and liver diseases. However, its role in asthma remains unclear. The present study aimed to investigate the effect of CAG on airway inflammation in an ovalbumin (OVA)induced mouse asthma model. The current study evaluated the lung function and levels of inflammation and autophagy via measurement of airway hyperresponsiveness (AHR), lung histology examination, inflammatory cytokine measurement and western blotting, amongst other techniques. The results demonstrated that CAG attenuated OVAinduced AHR in vivo. In addition, the total number of leukocytes and eosinophils, as well as the secretion of inflammatory cytokines, including interleukin (IL)5, IL13 and immunoglobulin E were diminished in bronchoalveolar lavage fluid of the OVAinduced murine asthma model. Histological analysis revealed that CAG suppressed inflammatory cell infiltration and goblet cell secretion. Notably, based on molecular docking simulation, CAG was demonstrated to bind to the active site of autophagyrelated gene 4microtubuleassociated proteins light chain 3 complex, which explains the reduced autophagic flux in asthma caused by CAG. The expression levels of proteins associated with autophagy pathways were inhibited following treatment with CAG. Taken together, the results of the present study suggest that CAG exerts an antiinflammatory effect in asthma, and its role may be associated with the inhibition of autophagy in lung cells.
Subject(s)
Anti-Asthmatic Agents/pharmacology , Asthma/etiology , Autophagy/drug effects , Drugs, Chinese Herbal/pharmacology , Sapogenins/pharmacology , Animals , Asthma/drug therapy , Asthma/metabolism , Autophagy-Related Proteins/antagonists & inhibitors , Autophagy-Related Proteins/chemistry , Autophagy-Related Proteins/metabolism , Biomarkers , Biopsy , Bronchial Hyperreactivity/drug therapy , Bronchial Hyperreactivity/etiology , Bronchial Hyperreactivity/metabolism , Bronchoalveolar Lavage Fluid , Cytokines/metabolism , Disease Management , Disease Models, Animal , Disease Susceptibility , Female , Immunoglobulin E/blood , Immunoglobulin E/immunology , Immunohistochemistry , Inflammation Mediators/metabolism , Mice , Microtubule-Associated Proteins/antagonists & inhibitors , Microtubule-Associated Proteins/chemistry , Microtubule-Associated Proteins/metabolism , Sapogenins/chemistry , Structure-Activity RelationshipABSTRACT
Airway remodeling is one of the typical pathological characteristics of asthma, while the structural changes of the airways in asthma are complex, which impedes the development of novel asthma targeted therapy. Our previous study had shown that Bu-Shen-Yi-Qi formula (BSYQF) could ameliorate airway remodeling in chronic asthmatic mice by modulating airway inflammation and oxidative stress in the lung. In this study, we analysed the lung transcriptome of control mice and asthmatic mouse model with/without BSYQF treatment. Using RNA-sequencing (RNA-seq) analysis, we found that 264/1746 (15.1%) of transcripts showing abnormal expression in asthmatic mice were reverted back to completely or partially normal levels by BSYQF treatment. Additionally, based on previous results, we identified 21 differential expression genes (DEGs) with fold changes (FC) > (±) 2.0 related to inflammatory, oxidative stress, mitochondria, PI3K/AKT, and MAPK signal pathways which may play important roles in the mechanism of the anti-remodeling effect of BSYQF treatment. Through inputting 21 DEGs into the IPA database to construct a gene network, we inferred Adipoq, SPP1, and TNC which were located at critical nodes in the network may be key regulators of BSYQF's anti-remodeling effect. In addition, the quantitative real-time polymerase chain reaction (qRT-PCR) result for the selected four DEGs matched those of the RNA-seq analysis. Our results provide a preliminary clue to the molecular mechanism of the anti-remodeling effect of BSYQF in asthma.
ABSTRACT
BACKGROUND: Group 2 innate lymphoid cells (ILC2s) are known to serve important functions in the pathogenesis of allergic airway inflammation. Studies have shown that acupuncture has an anti-inflammatory effect in the airways. However, how acupuncture treatment affects innate immunity, especially with regard to the function of ILC2s in ovalbumin (OVA)-induced allergic airway inflammation, is poorly understood. METHODS: BALB/c mice were injected and subsequently challenged with OVA ± treated with manual acupuncture. At the end of the experimental course, lung function was assessed by measurement of airway resistance (RL) and lung dynamic compliance (Cdyn). Cytokine levels were detected by enzyme-linked immunosorbent assay (ELISA). ILC2 proportions in the lung were analyzed by flow cytometry. RESULTS: The results showed that airway inflammation and mucus secretion were significantly suppressed by acupuncture treatment. RL decreased while Cdyn increased after acupuncture treatment. There was an apparent decrease in the bronchoalveolar lavage fluid (BALF) concentrations of interleukin (IL)-5, IL-13, IL-9, IL-25 and IL-33 and an increase in soluble IL-33 receptor (sST2) levels compared with untreated asthmatic mice. Acupuncture also reduced the lin-CD45+KLRG1+ST2+ cell proportion in the lung. CONCLUSION: In conclusion, this study has demonstrated that acupuncture treatment alleviates allergic airway inflammation and inhibits pulmonary ILC2 influx and IL-5, IL-9 and IL-13 production. The inhibition of ILC2s by acupuncture may be associated with the IL-33/ST2-signaling pathway and IL-25 levels, thereby offering protection from the respiratory inflammation associated with asthma.
Subject(s)
Acupuncture Therapy , Asthma/therapy , Lymphocytes/immunology , Animals , Asthma/etiology , Asthma/genetics , Asthma/immunology , Bronchoalveolar Lavage Fluid/immunology , Female , Humans , Immunity, Innate , Interleukin-1 Receptor-Like 1 Protein/genetics , Interleukin-1 Receptor-Like 1 Protein/immunology , Interleukins/immunology , Lung/immunology , Mice , Mice, Inbred BALB C , Ovalbumin/adverse effectsABSTRACT
Asthma is a chronic airway inflammatory disease and acupuncture is frequently used in patients suffering from asthma in clinic. However, the regulatory mechanism of acupuncture treatment in asthma is not fully elucidated. We sought to investigate the effectiveness of acupuncture on asthma and the associated regulatory mechanism. An ovalbumin (OVA)-induced mouse asthma model was established and the effect of acupuncture on airway hyperresponsiveness (AHR), mucus hypersecretion and inflammation was assessed. Tandem mass tag (TMT)-based quantitative proteomics analysis of lung tissue and bioinformatics analysis were performed. Our results revealed that the OVA-induced mouse asthma model was successfully established with the significantly elevated AHR to methacholine (Mch), and acupuncture was effective in attenuation of AHR to Mch, peribronchial and perivascular inflammation and mucus production. The inflammatory cells around the airways, mucous secretion as well as levels of IgE, CCL5, CCL11, IL-17A in bronchoalveolar lavage fluid (BALF) and IL-4, IL-5 and IL-13 levels in serum were siginificantly inhibited by acupuncture. TMT-based quantitative proteomics analysis found that a total of 6078 quantifiable proteins were identiï¬ed, and 564 (334 up-regulated and 230 down regulated) differentially expressed proteins (DEPs) were identiï¬ed in OVA-induced asthma model group (A) versus normal control group (NC). Acupuncture treatment resulted in 667 DEPs (416 up-regulated and 251 down regulated) compared with A group, and 86 overlapping DEPs were identiï¬ed in NC, A and AA groups. Among the 86 overlapping DEPs, we identified 41 DEPs regulated by acupuncture. Based on the above data, we performed a systematic bioinformatics analysis of the 41 DEPs, and results showed that these 41 DEPs were predominantly related to 4 KEGG pathways including SNARE interactions in vesicular transport, ferroptosis, endocrine and other factor-regulated calcium reabsorption, and protein digestion and absorption. DEPs of SLC3A2 and ATP1A3 expression levels were verified by immumohistochemical staining. Mice in OVA-induced asthma model group had elevated SLC3A2 and ATP1A3 expression and acupuncture had the ability to downregulate SLC3A2 and ATP1A3 protein expression. Furthermore, acupuncture reduced the MDA level and increased the GSH and SOD levels in the lung tissue. Taken together, our data suggested that acupuncture was effective in treating asthma by attenuation of AHR, mucus secretion and airway inflammation, and the mechanism was associated with regulation of ferroptosis, SLC3A2 and ATP1A3 protein expression as well as oxidative stress. Results from our experiments revealed the anti-inflammatory effect of acupuncture in OVA-induced mouse asthma model, leading to a more effective approach to be chosen by patients in clinic.
Subject(s)
Acupuncture Therapy/methods , Asthma/therapy , Fusion Regulatory Protein 1, Heavy Chain/metabolism , Inflammation/therapy , Sodium-Potassium-Exchanging ATPase/metabolism , Animals , Asthma/metabolism , Bronchoalveolar Lavage Fluid , Cytokines/metabolism , Disease Models, Animal , Female , Inflammation/metabolism , Lung/metabolism , Methacholine Chloride/metabolism , Mice , Mice, Inbred BALB C , Mucus/metabolism , Ovalbumin/adverse effects , Proteomics , Respiratory Hypersensitivity/therapyABSTRACT
ETHNOPHARMACOLOGICAL RELEVANCE: Loki Zupa (LKZP) decoction is one of the herbal prescriptions in traditional Uyghur medicine, which is commonly used for treating airway abnormality. However, underlying pathological mechanism and pathways involved has not been well studied. OBJECTIVES: In this paper, we aim to further confirmed the anti-inflammatory and anti-fibrotic role of LKZP decoction in airway, and uncover the passible mechanism involved via comprehensive quantitative proteomic DIA-MS analysis. MATERIALS AND METHODS: Mice asthmatic model was established with sensitizing and challenging with OVA. Lung function, pathological status, and inflammatory cytokines were assessed. Total of nine lung tissues were analyzed using proteomic DIA-MS analysis and 18 lung tissues were subjected to PRM validation. RESULTS: Total of 704 differentially expressed proteins (DEPs) (363 up regulated, 341 down regulated) were quantified in comparison of asthmatic and healthy mice, while 152 DEPs (91 up regulated, 61 down regulated) were quantified in LKZP decoction treated compared to asthmatic mice. Total of 21 proteins were overlapped between three groups. ECM-receptor interaction was significantly enriched and commonly shared between downregulated DEPs in asthma and upregulated DEPs in LKZP decoction treated mice. Total of 20 proteins were subjected to parallel reaction monitoring (PRM) analysis and 16 of which were quantified. At last, two proteins, RMB 10 and COL6A6, were validated with significant difference (P < 0.001) in protein abundance. CONCLUSIONS: Our results suggest that attenuated airway inflammation and fibrosis caused by LKZP decoction may associated with ECM-receptor interaction and RMB 10 and COL6A6 may be targeted by LKZP decoction in OVA-induced asthmatic mice.
Subject(s)
Anti-Asthmatic Agents/pharmacology , Anti-Inflammatory Agents/pharmacology , Asthma/drug therapy , Drugs, Chinese Herbal/pharmacology , Animals , Anti-Asthmatic Agents/isolation & purification , Anti-Inflammatory Agents/isolation & purification , Cytokines/metabolism , Disease Models, Animal , Female , Inflammation/drug therapy , Inflammation/pathology , Medicine, Chinese Traditional/methods , Mice , Mice, Inbred BALB C , Ovalbumin , Proteomics , Receptors, Cell Surface/metabolismABSTRACT
Ghrelin, through action on its receptor, GH secretagogue receptor type 1a (GHS-R1a), exerts a variety of metabolic functions including stimulation of appetite and weight gain and suppression of insulin secretion. In the present study, we examined the effects of novel small-molecule GHS-R1a antagonists on insulin secretion, glucose tolerance, and weight loss. Ghrelin dose-dependently suppressed insulin secretion from dispersed rat islets. This effect was fully blocked by a GHS-R1a antagonist. Consistent with this observation, a single oral dose of a GHS-R1a antagonist improved glucose homeostasis in an ip glucose tolerance test in rat. Improvement in glucose tolerance was attributed to increased insulin secretion. Daily oral administration of a GHS-R1a antagonist to diet-induced obese mice led to reduced food intake and weight loss (up to 15%) due to selective loss of fat mass. Pair-feeding experiments indicated that weight loss was largely a consequence of reduced food intake. The impact of a GHS-R1a antagonist on gastric emptying was also examined. Although the GHS-R1a antagonist modestly delayed gastric emptying at the highest dose tested (10 mg/kg), delayed gastric emptying does not appear to be a requirement for weight loss because lower doses produced weight loss without an effect on gastric emptying. Consistent with the hypothesis that ghrelin regulates feeding centrally, the anorexigenic effects of potent GHS-R1a antagonists in mice appeared to correspond with their brain exposure. These observations demonstrate that GHS-R1a antagonists have the potential to improve the diabetic condition by promoting glucose-dependent insulin secretion and promoting weight loss.