Discovery of the Active Compounds of the Ethyl Acetate Extract Site of Ardisia japonica (Thunb.) Blume for the Treatment of Acute Lung Injury.

The objective of this study was to identify and evaluate the pharmacodynamic constituents of Ardisiae Japonicae Herba (AJH) for the treatment of acute lung injury (ALI). To fully analyze the chemical contents of various extraction solvents (petroleum ether site (PE), ethyl acetate site (EA), n-butanol site (NB), and water site (WS)) of AJH, the UPLC-Orbitrap Fusion-MS technique was employed. Subsequently, the anti-inflammatory properties of the four extracted components of AJH were assessed using the lipopolysaccharide (LPS)-induced MH-S cellular inflammation model. The parts that exhibited anti-inflammatory activity were identified. Additionally, a technique was developed to measure the levels of specific chemical constituents in the anti-inflammatory components of AJH. The correlation between the "anti-inflammatory activity" and the constituents was analyzed, enabling the identification of a group of pharmacodynamic components with anti-inflammatory properties. ALI model rats were created using the tracheal drip LPS technique. The pharmacodynamic indices were evaluated for the anti-inflammatory active portions of AJH. The research revealed that the PE, EA, NB, and WS extracts of AJH included 215, 289, 128, and 69 unique chemical components, respectively. Additionally, 528 chemical components were discovered after removing duplicate values from the data. The EA exhibited significant anti-inflammatory activity in the cellular assay. A further analysis was conducted to determine the correlation between anti-inflammatory activity and components. Seventeen components, such as caryophyllene oxide, bergenin, and gallic acid, were identified as potential pharmacodynamic components with anti-inflammatory activity. The pharmacodynamic findings demonstrated that the intermediate and high doses of the EA extract from AJH exhibited a more pronounced effect in enhancing lung function, blood counts, and lung histology in a way that depended on the dosage. To summarize, when considering the findings from the previous study on the chemical properties of AJH, it was determined that the EA contained a group of 13 constituents that primarily contributed to its pharmacodynamic effects against ALI. The constituents include bergenin, quercetin, epigallocatechingallate, and others.

Comparative Pharmacokinetics Research of 13 Bioactive Components of Jieyu Pills in Control and Attention Deficit Hyperactivity Disorder Model Rats Based on UPLC-Orbitrap Fusion MS.

Jieyu Pills (JYPs), a Chinese medicine consisting of 10 herbal elements, have displayed promising clinical effectiveness and low by-effects in the treatment of depression. Prior investigations mostly focused on elucidating the mechanism and therapeutic efficacy of JYPs. In our earlier study, we provided an analysis of the chemical composition, serum pharmacochemistry, and concentrations of the main bioactive chemicals found in JYPs. However, our precise understanding of the pharmacokinetics and metabolism remained vague. This study involved a comprehensive and meticulous examination of the pharmacokinetics of 13 bioactive compounds in JYPs. Using UPLC-Orbitrap Fusion MS, we analyzed the metabolic characteristics and established the pharmacokinetic parameters in both control rats and model rats with attention deficit hyperactivity disorder (ADHD) following oral administration of the drug. Before analysis, plasma samples that were collected at different time intervals after the administration underwent methanol pre-treatment with Puerarin used as the internal standard (IS) solution. Subsequently, the sample was chromatographed on a C18 column employing gradient elution. The mobile phase consisted of methanol solution containing 0.1% formic acid in water. The electrospray ionization source (ESI) was utilized for ionization, whereas the scanning mode employed was selected ion monitoring (SIM). The UPLC-Orbitrap Fusion MS method was subjected to a comprehensive validation process to assess its performance. The method demonstrated excellent linearity (r ≥ 0.9944), precise measurements (RSD < 8.78%), accurate results (RE: -7.88% to 8.98%), and appropriate extraction recoveries (87.83-102.23%). Additionally, the method exhibited minimal matrix effects (87.58-101.08%) and satisfactory stability (RSD: 1.52-12.42%). These results demonstrated adherence to the criteria for evaluating and determining biological material. The 13 bioactive compounds exhibited unique pharmacokinetic patterns in vivo. In control rats, all bioactive compounds except Ferulic acid exhibited linear pharmacokinetics within the dose ranges. In the ADHD model, the absorption rate and amount of most of the components were both observed to have increased. Essentially, this work is an important reference for examining the metabolism of JYPs and providing guidelines for clinical therapy.

Integrated bioinformatics analysis for the identification of idiopathic pulmonary fibrosis-related genes and potential therapeutic drugs.

OBJECTIVE: The pathogenesis of idiopathic pulmonary fibrosis (IPF) remains unclear. We sought to identify IPF-related genes that may participate in the pathogenesis and predict potential targeted traditional Chinese medicines (TCMs). METHODS: Using IPF gene-expression data, Wilcoxon rank-sum tests were performed to identify differentially expressed genes (DEGs). Protein-protein interaction (PPI) networks, hub genes, and competitive endogenous RNA (ceRNA) networks were constructed or identified by Cytoscape. Quantitative polymerase chain reaction (qPCR) experiments in TGF-ß1-induced human fetal lung (HFL) fibroblast cells and a pulmonary fibrosis mouse model verified gene reliability. The SymMap database predicted potential TCMs targeting IPF. The reliability of TCMs was verified in TGF-ß1-induced MRC-5 cells. MATERIALS: Multiple gene-expression profile data of normal lung and IPF tissues were downloaded from the Gene Expression Omnibus database. HFL fibroblast cells and MRC-5 cells were purchased from Wuhan Procell Life Science and Technology Co., Ltd. (Wuhan, China). C57BL/12 mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). RESULTS: In datasets GSE134692 and GSE15197, DEGs were identified using Wilcoxon rank-sum tests (both p < 0.05). Among them, 1885 DEGs were commonly identified, and 87% (1640 genes) had identical dysregulation directions (binomial test, p < 1.00E-16). A PPI network with 1623 nodes and 8159 edges was constructed, and 18 hub genes were identified using the Analyze Network plugin in Cytoscape. Of 18 genes, CAV1, PECAM1, BMP4, VEGFA, FYN, SPP1, and COL1A1 were further validated in the GeneCards database and independent dataset GSE24206. ceRNA networks of VEGFA, SPP1, and COL1A1 were constructed. The genes were verified by qPCR in samples of TGF-ß1-induced HFL fibroblast cells and pulmonary fibrosis mice. Finally, Sea Buckthorn and Gnaphalium Affine were predicted as potential TCMs for IPF. The TCMs were verified by qPCR in TGF-ß1-induced MRC-5 cells. CONCLUSION: This analysis strategy may be useful for elucidating novel mechanisms underlying IPF at the transcriptome level. The identified hub genes may play key roles in IPF pathogenesis and therapy.

Serum metabolomics using ultra-high performance liquid chromatography-Q-Exactive tandem mass spectrometry reveals the mechanism of action of exercise training on chronic obstructive pulmonary disease rats.

Exercise training is the cornerstone component of pulmonary rehabilitation, which results in symptom-reducing, psychosocial, and health economic benefits for chronic obstructive pulmonary disease (COPD) patients. However, the potential mechanisms of its action are poorly understood. This study conducted serum metabolomics using ultra-high performance liquid chromatography-Q-Exactive tandem mass spectrometry to determine the metabolic changes in COPD rats, and the effects of exercise training on improvement in COPD were further investigated. Twelve differential metabolites-which are primarily related to tryptophan metabolism, sphingolipid metabolism, glycerophospholipid metabolism, riboflavin metabolism, pantothenate and CoA biosynthesis, and lysine degradation-were identified in relation to COPD. After the intervention of exercise training, the levels of most metabolites were restored, and the changes in five metabolites were statistically significant, which suggested that exercise training provided effective protection against COPD and might play its role by rebalancing disordered metabolism pathways. This work enhanced our comprehension of the protective mechanism of exercise training on COPD.

Uncovering mechanisms of Baojin Chenfei formula treatment for silicosis by inhibiting inflammation and fibrosis based on serum pharmacochemistry and network analysis.

BACKGROUND: Baojin Chenfei formula (BCF), a Chinese herbal formula, has significant effects on improving the clinical symptoms of patients with silicosis. However, its active compounds and the underlying mechanisms have not yet fully been elucidated. PURPOSE: This study aimed to explore the underlying mechanisms of BCF in treating silicosis. METHODS: The rat model of silicosis was developed via a single intratracheal instillation of SiO2 suspension to examine the therapeutic impacts of BCF on silicosis. Subsequently, the active compounds, targets, and mechanisms of BCF were analyzed based on serum pharmacochemistry and network analysis. Finally, the underlying mechanisms of representative compounds of BCF were validated in vitro experiments. RESULTS: BCF significantly alleviated SiO2-induced silicosis in rats, evidenced by improved lung function, decreased pathological injury, and reduced inflammatory response and fibrosis. 19 active compounds were identified from the rat serum samples after BCF gavage. Subsequently, 299 targets for these 19 compounds in BCF and 257 genes related to silicosis were collected. 26 overlapping targets, including AKT1, TNF, IL6, MAPK3, EGFR, and others, were obtained from the intersection of the 299 BCF-related targets and 257 silicosis-associated genes. These overlapping targets mainly corresponded to glycyrrhetic acid and paeoniflorin and were mainly associated with positive regulation of smooth muscle cell proliferation, positive regulation of MAP kinase activity, and inflammatory response. In vitro experiments also demonstrated that the representative compounds of BCF (glycyrrhetic acid and paeoniflorin) could suppress inflammatory response by the MAPK pathway, and also inhibited fibroblast activation by the EGFR-PI3K-AKT pathway. CONCLUSION: Active compounds of BCF, such as glycyrrhetic acid and paeoniflorin, could suppress inflammatory response by the MAPK pathway and suppress fibroblast activation by the EGFR-PI3K-AKT pathway. These might be the mechanisms of BCF in treating silicosis.

Comparative study of cigarette smoke, Klebsiella pneumoniae, and their combination on airway epithelial barrier function in mice.

BACKGROUND: The airway epithelium acts as a physical barrier to protect pulmonary airways against pathogenic microorganisms and toxic substances, such as cigarette smoke (CS), bacteria, and viruses. The disruption of the structural integrity and dysfunction of the airway epithelium is related to the occurrence and progression of chronic obstructive pulmonary disease. PURPOSE: The aim of this study is to compare the effects of CS, Klebsiella pneumoniae (KP), and their combination on airway epithelial barrier function. METHODS: The mice were exposed to CS, KP, and their combination from 1 to 8 weeks. After the cessation of CS and KP at Week 8, we observed the recovery of epithelial barrier function in mice for an additional 16 weeks. To compare the epithelial barrier function among different groups over time, the mice were sacrificed at Weeks 4, 8, 16, and 24 and then the lungs were harvested to detect the pulmonary pathology, inflammatory cytokines, and tight junction proteins. To determine the underlying mechanisms, the BEAS-2B cells were treated with an epidermal growth factor receptor (EGFR) inhibitor (AG1478). RESULTS: The results of this study suggested that the decreased lung function, increased bronchial wall thickness (BWT), elevated inflammatory factors, and reduced tight junction protein levels were observed at Week 8 in CS-induced mice and these changes persisted until Week 16. In the KP group, increased BWT and elevated inflammatory factors were observed only at Week 8, whereas in the CS + KP group, decreased lung function, lung tissue injury, inflammatory cell infiltration, and epithelial barrier impairment were observed at Week 4 and persisted until Week 24. To further determine the mechanisms of CS, bacteria, and their combination on epithelial barrier injury, we investigated the changes of EGFR and its downstream protein in the lung tissues of mice and BEAS-2B cells. Our research indicated that CS, KP, or their combination could activate EGFR, which can phosphorylate and activate ERK1/2, and this effect was more pronounced in the CS + KP group. Furthermore, the EGFR inhibitor AG1478 suppressed the phosphorylation of ERK1/2 and subsequently upregulated the expression of ZO-1 and occludin. In general, these results indicated that the combination of CS and KP caused more severe and enduring damage to epithelial barrier function than CS or KP alone, which might be associated with EGFR/ERK1/2 signaling. CONCLUSION: Epithelial barrier injury occurred earlier, was more severe, and had a longer duration when induced by the combination of CS and KP compared with the exposure to CS or KP alone, which might be associated with EGFR/ERK signaling.

A Pharmacokinetic Study of Sixteen Major Bioactive Components of Jinshui-Huanxian Granules in Pulmonary Fibrosis Model and Control Rats Using Orbitrap Fusion Mass Spectrometry.

Jinshui-Huanxian granules (JHGs), a Chinese herbal compound prescription, have shown a therapeutic effect in reducing lung tissue damage, improving the degree of pulmonary fibrosis, replenishing lungs and kidneys, relieving cough and asthma, reducing phlegm, and activating blood circulation. However, these active compounds' pharmacokinetics and metabolic processes were unclear. This study aimed to compare the pharmacokinetics, reveal the metabolic dynamic changes, and obtain the basic pharmacokinetic parameters of 16 main bioactive compounds after intragastric administration of JHGs in control and pulmonary fibrosis (PF) model rats by using Orbitrap Fusion MS. After administration of JHGs, the rat plasma was collected at different times. Pretreating the plasma sample with methanol and internal standard (IS) solution carbamazepine (CBZ), and it was then applied to a C18 column by setting gradient elution with a mobile phase consisting of methanol 0.1% formic acid aqueous solution. Detection was performed on an electrospray ionization source (ESI), and the scanning mode was SIM. Pharmacokinetic parameters were analyzed according to the different analytes' concentrations in plasma. The matrix effect was within the range of 79.01-110.90%, the extraction recovery rate was 80.37-102.72%, the intra-day and inter-day precision relative standard deviation (RSD) was less than 7.76%, and the stability was good, which met the requirements of biological sample testing. The method was validated (r ≥ 0.9955) and applied to compare the pharmacokinetic profiles of the control group and PF model group after intragastric administration of the JHGs. The 16 analytes exhibited different pharmacokinetic behaviors in vivo. In the pathological state of the PF model, most of the components were more favorable for metabolism and absorption, and it was more meaningful to study the pharmacokinetics. Above all, this study provided an essential reference for exploring the mechanism of action of JHGs and guided clinical medication as well.

Poly I:C Exacerbates Airway Inflammation and Remodeling in Cigarette Smoke-Exposed Mice.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) is a chronic respiratory disorder characterized by chronic inflammation and airway remodeling. Cigarette smoke (CS) and respiratory viruses are major causes of COPD development and exacerbation, but the mechanisms of these compounding factors on inflammation and pathological changes in airway structure still need further investigation. PURPOSE: This work aimed to investigate the effects and mechanisms of Poly I:C on pathological changes in CS-induced COPD mice, such as airway inflammation and remodeling. METHODS: From 1 to 8 weeks, the mice were exposed to CS, Poly I:C, or a combination of both. To compare the pathological changes among different groups over time, the mice were sacrificed at week 4, 8, 16, and 24, then the lungs were harvested to measure pulmonary pathology, inflammatory cytokines, and airway remodeling. RESULTS: Our data revealed that the fundamental characteristics of COPD, such as pulmonary pathological damage, the release of inflammatory mediators, and the remodeling of airway walls, were observed at week 8 in CS-exposed mice and these pathological changes persisted to week 16. Compared with the CS group, the pathological changes, including decreased lung function, inflammatory cell infiltration, alveolar destruction, and airway wall thickening, were weaker in the Poly I:C group. These pathological changes were observed at week 8 and persisted to week 16 in Poly I:C-induced mice. Furthermore, Poly I:C exacerbated lung tissue damage in CS-induced COPD mice. The decreased lung function, airway inflammation and remodeling were observed in the combined group at week 4, and these pathological changes persisted to week 24. Our research indicated that Poly I:C enhanced the expression of p-P38, p-JNK and p-NF-κB in CS-exposed mice. CONCLUSION: Poly I:C could promote airway inflammation and remodeling in CS-induced COPD mice probably by NF-κB and MAPK signaling.

Effective-component compatibility of Bufei Yishen formula III ameliorated COPD by improving airway epithelial cell senescence by promoting mitophagy via the NRF2/PINK1 pathway.

BACKGROUND: Effective-component compatibility of Bufei Yishen formula III (ECC-BYF III) demonstrates positive effects on stable chronic obstructive pulmonary disease (COPD). PURPOSE: To investigate the mechanisms of ECC-BYF III on COPD rats from the aspect of airway epithelial cell senescence. METHODS: COPD model rats (Sprague-Dawley rat) were treated with ECC-BYF III for 8 weeks, and the efficacy was evaluated. Cigarette smoke extract (CSE)-induced senescence model of airway epithelial cells was treated with ECC-BYF III, and related enzymes and proteins involved in oxidative stress and mitophagy were detected. RESULTS: ECC-BYF III markedly rescued pulmonary function and histopathological changes, which might be associated with the amelioration of lung senescence, including the reduction of malondialdehyde (MDA) and tumor necrosis factor-α (TNF-α), interleukin (IL)-6 and matrix metalloproteinase (MMP)-9 levels, increase of the level in total superoxide dismutase (T-SOD), and decease in the p21 level in the airways. Furthermore, ECC-BYF III suppressed p16 and p21 expressions and senescence-associated ß-galactosidase (SA-ß-Gal) in CSE-induced airway epithelial cells. Moreover, ECC-BYF III upregulated mitophagy-related proteins, including the co-localizations of TOM20 and LC3B, PINK1 and PARK2, and improved mitochondrial function by upregulating mitochondrial mitofusin (MFN)2 and reducing dynamin-related protein 1 (DRP1) expression. ECC-BYF III enhanced the activities of T-SOD and GSH-PX by up-regulating NRF2, thus inhibiting oxidative stress. After intervention with NRF2 inhibitor, the regulation effects of ECC-BYF III on oxidative stress, mitophagy and senescence in airway epithelial cells were significantly suppressed. CONCLUSIONS: ECC-BYF III exerts beneficial effects on COPD rats by ameliorating airway epithelial cell senescence, which is mediated by inhibiting oxidative stress and subsequently enhancing mitophagy through the activation of NRF2 signaling.

Effective-component compatibility of Bufei Yishen formula protects COPD rats against PM2.5-induced oxidative stress via miR-155/FOXO3a pathway.

Ambient particulate matter <2.5 µm (PM2.5) has been identified as a critical risk factor in chronic obstructive pulmonary disease (COPD) exacerbation, but therapies for this condition are limited. Effective-component compatibility of Bufei Yishen formula (ECC-BYF) exhibits beneficial efficacy on COPD rats. However, its effect on PM2.5-aggravated COPD rats are considered to be uncertain. In this study, we used an established PM2.5-aggravated COPD rat model in vivo to evaluate the protective effect of ECC-BYF, and focused on its antioxidative role in PM2.5-stimulated bronchial epithelial cells via regulating microRNA (miR)-155/ forkhead box class O3a (FOXO3a) pathway. As expected, PM2.5-aggravated COPD rats showed a reduction of lung function, persistent lung inflammation, and remodeling of lung tissue. In comparison, ECC-BYF administration significantly enhanced lung function, alleviated alveolar destruction, inflammatory cell infiltration, mucus hypersecretion, and collagen deposition, along with diminishing inflammatory cytokine production and oxidative stress. Furthermore, ECC-BYF pretreatment markedly decreased the fluorescence intensity of reactive oxygen species (ROS) in PM2.5-induced human bronchial epithelial (Beas-2B) cells and primary mouse tracheal epithelial cells (MTECs), as well as reversing the imbalance between oxidants and antioxidants in Beas-2B. Meanwhile, ECC-BYF elevated FOXO3a while inhibiting miR-155 expression dose -dependently. In vitro transfection of miR-155 mimic into Beas-2B significantly decreased FOXO3a protein expression, accompanied by the reduced superoxide dismutase 2 (SOD2) and catalase (CAT) expressions, thus eliminating the protective effect of ECC-BYF on PM2.5-evoked oxidative stress. Nonethless, FOXO3a overexpression could partially restore the antioxidative effect of ECC-BYF. In conclusion, ECC-BYF can protect pre-existing COPD against PM2.5 contamination by exerting a profound antioxidative influence via regulating miR-155/FOXO3a signaling.

Exposure to Air Pollution Exacerbates Inflammation in Rats with Preexisting COPD.

Particulate matter with an aerodynamic diameter equal or less than 2.5 micrometers (PM2.5) is associated with the development of chronic obstructive pulmonary disease (COPD). The mechanisms by which PM2.5 accelerates disease progression in COPD are poorly understood. In this study, we aimed to investigate the effect of PM2.5 on lung injury in rats with hallmark features of COPD. Cardinal features of human COPD were induced in a rat model by repeated cigarette smoke inhalation and bacterial infection for 8 weeks. Then, from week 9 to week 16, some of these rats with COPD were subjected to real-time concentrated atmospheric PM2.5. Lung function, pathology, inflammatory cytokines, oxidative stress, and mucus and collagen production were measured. As expected, the COPD rats had developed emphysema, inflammation, and deterioration in lung function. PM2.5 exposure resulted in greater lung function decline and histopathological changes, as reflected by increased Mucin (MUC) 5ac, MUC5b, Collagen I, Collagen III, and the profibrotic cytokine α-smooth muscle-actin (SMA), transforming growth factor- (TGF-) ß1 in lung tissues. PM2.5 also aggravated inflammation, increasing neutrophils and eosinophils in bronchoalveolar lavage fluid (BALF) and cytokines including Interleukin- (IL-) 1ß, granulocyte-macrophage colony-stimulating factor (GM-CSF), and IL-4. The likely mechanism is through oxidative stress as antioxidants levels were decreased, whereas oxidants were increased, indicating a detrimental shift in the oxidant-antioxidant balance. Altogether, these results suggest that PM2.5 exposure could promote the development of COPD by impairing lung function and exacerbating pulmonary injury, and the potential mechanisms are related to inflammatory response and oxidative stress.

miRNA-206 regulates human pulmonary microvascular endothelial cell apoptosis via targeting in chronic obstructive pulmonary disease.

Chronic obstructive pulmonary disease (COPD) is a leading cause of death due to tis high morbidity and mortality. microRNAs have emerged as new biomarkers for the prognosis and diagnosis of patients with COPD. In this study, we aimed to investigate the expression of microRNA-206 (miR-206) in lung tissues from COPD patients and to explore the regulatory role of miR-206 in the human pulmonary microvascular endothelial cells (HPMECs). Our results showed that cigarette smoke extract (CSE) promoted cell apoptosis, increased caspase-3 activity, and upregulated the expression of miR-206 in HPMECs, which was significantly reversed by the miR-206 knockdown. Transfection with miR-206 mimics led to cell apoptosis and was closely related to changes in the protein expression levels of caspase-3, caspase-9, and Bcl-2 in HPMECs. Further bioinformatics prediction analysis revealed that the 3'-untranslated region (3'UTR) of Notch3 and vascular endothelial growth factor-A (VEGFA) harbored miR-206-binding sites, and overexpression of miR-206 repressed the luciferase activity of the vectors containing Notch3 and VEGFA 3'UTR. Overexpression of either Notch3 or VEGFA attenuated miR-206-induced cell apoptosis in HPMECs. More importantly, miR-206 expression was upregulated in the lung tissues from COPD patients and was positively corrected with forced expiratory volume 1% predicted in COPD patients, while Notch3 and VEGFA mRNA levels were downregulated and were negatively correlated with the expression level of miR-206 in the lung tissues from COPD patients. In conclusion, our results showed that miR-206 was upregulated in COPD patients and CSE-treated HPMECs, promoted cell apoptosis via directly targeting Notch3 and VEGFA in HPMECs.

Dusuqing granules (DSQ) suppress inflammation in Klebsiella pneumonia rat via NF-κB/MAPK signaling.

BACKGROUND: Dusuqing granules (DSQ) have been used in the treatment of bacterial pneumonia clinically, with remarkable benefits. This study was initiated to explore the effects of DSQ on pulmonary inflammation by regulating nuclear factor (NF)-κB/mitogen-activated protein kinase (MAPK) signaling in bacterial pneumonia rats. METHODS: Rat model was duplicated with Klebsiella pneumonia by a one-time intratracheal injection. Rats were randomized into control, model, DSQ and levofloxacin (LVX) groups. After administrated with appropriate medicines for 7 days, lung tissues were harvested and prepared for pathological analysis, and interleukin (IL)-1, IL-6, monocyte chemotactic protein (MCP)-1and macrophage inflammatory protein (MIP)-2 detections. NF-κB mRNA was measured by real-time qPCR, and the phosphorylation and total proteins of P38MAPK, JNK46/54, ERK42/44 were determined by Western blotting. RESULTS: Marked pathological impairments were observed in model rats, whereas were improved in DSQ group. The cytokines levels, NF-κB mRNA expression and the phosphorylation of P38MAPK, JNK46/54 and ERK42/44 proteins were significantly higher in model group, and were significantly depressed in DSQ group. CONCLUSION: The protective effects of DSQ on Klebsiella pneumonia might be attributed to its inactivative effects of NF-κB/ MAPK pathway.

Bufei Yishen granule combined with acupoint sticking improves pulmonary function and morphormetry in chronic obstructive pulmonary disease rats.

BACKGROUND: The integrated therapy of Bufei Yishen granule and acupoint sticking has been used in the treatment of stable chronic obstructive pulmonary disease (COPD) clinically, with remarkable benefits. This study was initiated to observe the effects of the combination of Bufei Yishen granule and acupoint sticking on pulmonary function and morphormetry in a COPD rat model. METHODS: Rats were randomized into Control, Model, Bufei Yishen (BY), Acupoint sticking (AS), Bufei Yishen + Acupoint sticking (BY + AS) and aminophyline (APL) groups. COPD rats were duplicated by repeated cigarette smoke and bacterial exposures. The rats were treated with normal saline, Bufei Yishen granule, acupoint sticking, Bufei Yishen + Acupoint sticking and aminophylline, respectively, from week 9 through 20. Pulmonary function was measured by using a whole body plethysmograph every 4 weeks. The rats were sacrificed at the end of week 20, and lung tissue histology and ultrastructure was observed under light and electron microscopes. RESULTS: The pulmonary function, including tidal volume (VT), peak expiratory flow (PEF) and expiratory flow at 50 % tidal volume (EF50), was markedly decreased from week 8 in COPD rats (P < 0.05). At week 20, VT, PEF and EF50 were significantly lower in Model group (P < 0.05). Compared with Model group, VT, PEF and EF50 were higher in BY and BY + AS groups (P < 0.05), and EF50 was higher in AS group, while VT was higher in APL group (P < 0.05). Markedly histological and ultrastructural changes, including respiratory membrane thickening, volume density of lamellar corpuscle decreasing, mitochondria reducing in type II alveolar cell, were found in COPD rats and were alleviated in the treated groups, especially in BY and BY + AS groups. CONCLUSION: Bufei Yishen granule and acupoint sticking can improve pulmonary function and lung pathological impairment in COPD rats, the curative effect of the combination is better than acupoint sticking or aminophylline only.

Bufei Jianpi granules improve skeletal muscle and mitochondrial dysfunction in rats with chronic obstructive pulmonary disease.

BACKGROUND: Bufei Jianpi granules has been confirmed effective in improving pulmonary function, alleviating acute exacerbations, improving six-minute walk distance and quality of life, and benefited in 12-month follow-up in chronic obstructive pulmonary disease (COPD) patients with syndrome of lung-spleen qi deficiency. Skeletal muscle dysfunction (SMD), an important extrapulmonary complication, occurs in the very initiation of COPD and is closely related to morbidity and mortality. To evaluate the efficacy of Bufei Jianpi granules on SMD, we observed skeletal muscular function and histomorphology, mitochondrial morphormetry and proteins in COPD rats induced by cigarette-smoke and Klebsiella pneumoniae. METHODS: Seventy-two Sprague-Dawley rats were randomized into Control + Saline, Control + Bufei Jianpi, Control + Aminophylline, COPD + Saline, COPD + Bufei Jianpi and COPD + Aminophylline groups. From week 9 to 20, rats were administrated intragastricly by normal saline, Bufei Jianpi granules and aminophylline, respectively. Muscular tension and fatigue index of intercostal muscle, quadriceps, biceps and soleus were detected by using electrophysiological technology. Pathological and ultrastructural changes and expressions of mitochondrial Bcl-2 nineteen-kilodalton interacting protein 3 (Bnip3) and cytoplasm cytochrome C (Cyto C) in the four skeletal muscles were observed by using optical and electron microscope and western blotting. RESULTS: There was no statistical difference among the control rats treated with saline, Bufei Jianpi granules or aminophylline in above-mentioned parameters. Muscular tension, mitochondria volume density (Vv) and compared membrane surface (δm) of the four muscles were significantly lower in COPD + Saline group compared to Control + Saline group, while fatigue index, mitochondria surface area (δ), Bnip3 and Cyto C were higher (P < 0.05). COPD rats showed more morphological changes in muscle tissues than controls, such as atrophy, degeneration, necrosis and matrix hyperplasia. Utrastructurally, mitochondria populations decreased significantly in the four muscles, and were shrunken and even cavitation changed. The up-mentioned parameters were improved in Bufei Jianpi group (P < 0.05) in the four muscles. CONCLUSIONS: Bufei Jianpi granules can improve skeletal muscle function via improving mitochondria population and function, reducing apoptotic factors such as Bnip3 and Cyto C, and is more effective than aminophylline.

[R-value comprehensive evaluation of effect of three methods for lung-kidney comprehensive evaluation study on R value of three methods for regulating and invigorating lung and kidney in regulating lung inflammation signaling pathways in COPD rats].

Previous studies showed that three methods for regulating and invigorating lung and kidney (lung invigorating and spleen strengthening, lung invigorating and kidney tonifying, and Qi supplementing and kidney nourishing) could regulate inflammatory signaling pathways of chronic obstructive pulmonary disease (COPD) in rats, so as to alleviate inflammation. In the present study, R-value comprehensive evaluation method was used to evaluate the comprehensive effect of three methods for regulating and invigorating lung and kidney on inflammatory signaling pathways. Rats were randomly divided into control, model, lung invigorating and spleen strengthening, lung invigorating and kidney tonifying, Qi supplementing and kidney nourishing and aminophylline groups. The COPD rat models were established by cigarette smoking combined with bacterial infection, and orally administered with drugs between the 9th and 20th week. Afterwards, efforts were made to observe the long-term effects between the drug withdrawal and the 32rd week and detect indicators in two batches in the 20th week and 32th week. Specifically, (1) Linking JAK/STAT signaling pathway: JAK2 mRNA, and protein expressions of STAT-1, STAT-3, STAT-5, JAK-2; (2) NF-kappaB signaling pathway: Smad2 mRNA and protein expressions of I-kappaB, NF-kappaB, TGF-beta1; (3) PPARgamma and antioxidant signaling pathway: SOD, PGE mRNA, PPARgamma protein. According to the results, 5 indicators in JAK/STAT pathway, 4 indicators in NF-kappaB pathway, and 3 indicators in PPARgamma pathway were significantly rectified by three methods for regulating and invigorating lung and kidney in between the 20th week and 32nd week. Between the 20th and 32nd week, the recipes for rectifying JAK/STAT pathway with intensity from high to low were recipes for lung invigorating and spleen strengthening, Qi supplementing and kidney nourishing, lung invigorating and kidney tonifying, aminophylline, particularly those for lung invigorating and spleen strengthening; The recipes for rectifying NF-kappaB pathway with intensity from high to low were recipes for lung invigorating and spleen strengthening, lung invigorating and kidney tonifying, Qi supplementing and kidney nourishing and aminophylline, particularly the first three types of drugs. The recipes for rectifying PPARgamma and antioxidant signaling pathway with intensity from high to low were recipes for lung invigorating and kidney tonifying, Qi supplementing and kidney nourishing, lung invigorating and spleen strengthening and aminophylline. Therefore, three methods for regulating and invigorating lung and kidney showed better long-term effects in regulating COPD lung inflammation signaling pathways. Specifically, recipe for lung invigorating and spleen strengthening showed a better effect in JAK/STAT and NF-kappaB pathways, while recipe for lung invigorating and kidney tonifying and Qi supplementing and kidney nourishing showed better effects in PPARgamma and antioxidant signaling pathways. In conclusion, R-value comprehensive evaluation method can evaluate the comprehensive effect of medicines and define the ranking of multiple drugs and their main targets.

Long-term effects of three Tiao-Bu Fei-Shen therapies on NF-κB/TGF-ß1/smad2 signaling in rats with chronic obstructive pulmonary disease.

BACKGROUND: The three Tiao-Bu Fei-Shen (Bufei Jianpi, Bufei Yishen, Yiqi Zishen) granules have been confirmed for their beneficial clinical efficacy in chronic obstructive pulmonary disease (COPD) patients on reducing frequency and duration of acute exacerbation, improving syndromes, pulmonary function and exercise capacity. But the short- or long-term mechanism of them is not fully clear. Nuclear factor (NF)-κB/transforming growth factor (TGF)-ß1/smad2 signaling pathway is involved in the progress of inflammation and remodeling in chronic obstructive pulmonary disease COPD. This study aimed to explore the long-term effects mechanism of Tiao-Bu Fei-Shen granules by regulating NF-κB/TGF-ß/Smads signaling in rats with COPD. METHODS: Sprague Dawley rats were randomized into control, model, Bufei Jianpi, Bufei Yishen, Yiqi Zishen and aminophylline groups. COPD rats, induced by cigarette smoke and bacterial infections exposures, were administrated intragastricly by normal saline, Bufei Jianpi, Bufei Yishen, Yiqi Zishen granules or aminophylline from week 9 through 20, respectively. At week 20 and 32, lung tissues were harvested. Immunohistochemistry was used to detect interleukin (IL)-1ß and tumor necrosis factor (TNF)-α, quantitative real-time polymerase chain reaction (qRT-PCR) was used for TGF-ß1 and Smad2 mRNA analysis, western blotting was used to determine the phosphorylation of NF-κB (p-NF-κB) and IκBα (p-IκBα). RESULTS: COPD rats had marked airway injury, such as chronic airway inflammation and remodeling, emphysema, which were improved in the three traditional Chinese medicines (TCM)-treated animals. The levels of IL-1ß, TNF-α, p-NF-κB, p-IκBα, TGF-ß1 and Smad2 were significantly higher in COPD rats than in controls, while they were dramatically reduced in the three TCM- and aminophylline-treated groups. At the meantime, all these endpoints were significantly lower in three TCM-treated groups than in aminophylline group, especially in Bufei Jianpi and Bufei Yishen groups. Compared to week 20, all endpoints decreased significantly in three TCM groups at week 32. CONCLUSION: The three Tiao-Bu Fei-Shen therapies can reduce pulmonary inflammation and remodeling in COPD and have significant long-term effects. NF-κB/TGF-ß1/smad2 signaling might be involved in the mechanism.

Bufei Yishen formula protects the airway epithelial barrier and ameliorates COPD by enhancing autophagy through the Sirt1/AMPK/Foxo3 signaling pathway.

OBJECT: Bufei Yishen formula (BYF), a traditional Chinese medicine alleviates COPD symptoms and suppresses airway epithelial inflammation. In this study, we determined whether BYF protects the airway epithelial barrier from destruction in COPD rats. METHODS: The protective effects of BYF on the airway epithelial barrier were examined in a rat COPD model. BEAS-2B epithelial cells were exposed to cigarette smoke extract (CSE) to determine the effect of BYF on epithelial barrier function. Transcriptomic and network analyses were conducted to identify the protective mechanisms. RESULTS: Oral BYF reduced the severity of COPD in rats by suppressing the decline in lung function, pathological changes, inflammation, and protected airway epithelial barrier function by upregulating apical junction proteins, including occludin (OCLN), zonula occludens (ZO)-1, and E-cadherin (E-cad). BYF treatment reduced epithelial permeability, and increased TEER as well as the apical junction proteins, OCLN, ZO-1, and E-cad in BEAS-2B cells exposed to CSE. Furthermore, 58 compounds identified in BYF were used to predict 421 potential targets. In addition, the expression of 572 differentially expressed genes (DEGs) was identified in CSE-exposed BEAS-2B cells. A network analysis of the 421 targets and 572 DEGs revealed that BYF regulates multiple pathways, of which the Sirt1, AMPK, Foxo3, and autophagy pathways may be the most important with respect to protective mechanisms. Moreover, in vitro experiments confirmed that nobiletin, one of the active compounds in BYF, increased apical junction protein levels, including OCLN, ZO-1, and E-cad. It also increased LC3B and phosphorylated AMPK levels and decreased the phosphorylation of FoxO3a. CONCLUSIONS: BYF protects the airway epithelial barrier in COPD by enhancing autophagy through regulation of the SIRT1/AMPK/FOXO3 signaling pathway.

Identification of idiopathic pulmonary fibrosis hub genes and exploration of the mechanisms of action of Jinshui Huanxian formula.

Idiopathic pulmonary fibrosis (IPF) is a common and heterogeneous chronic disease, and the mechanism of Jinshui Huanxian formula (JHF) on IPF remains unclear. For a total of 385 lung normal tissue samples from the Gene Expression Omnibus database, 37,777,639 gene pairs were identified through microarray and RNA-seq platforms. Using the individualized differentially expressed gene (DEG) analysis algorithm RankComp (FDR < 0.01), we identified 344 genes as DEGs in at least 95 % (n = 81) of the IPF samples. Of these genes, IGF1, IFNGR1, GLI2, HMGCR, DNM1, KIF4A, and TNFRSF11A were identified as hub genes. These genes were verified using quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) in mice with pulmonary fibrosis (PF) and MRC-5 cells, and they were highly effective at classifying IPF samples in the independent dataset GSE134692 (AUC = 0.587-0.788) and mice with PF (AUC = 0.806-1.000). Moreover, JHF ameliorated the pathological changes in mice with PF and significantly reversed the changes in hub gene expression (KIF4A, IFNGR1, and HMGCR). In conclusion, a series of IPF hub genes was identified, and validated in an independent dataset, mice with PF, and MRC-5 cells. Moreover, the abnormal gene expression was normalized by JHF. These findings provide guidance for further exploration of the pathogenesis and treatment of IPF.

[Influence and long-term effects of three methods for regulating and invigorating fei-shen on T lymphocyte subsets and CD4+ CD25+ in COPD rats].

OBJECTIVE: To evaluate the therapeutic and long-term effects of three methods for regulating and invigorating Fei-Shen [reinforcing Fei and invigorating Pi (RFIP), reinforcing Fei and invigorating Shen (RFIS), benefiting qi and nourishing Shen (BQNS)] on T lymphocyte subsets and CD4+ CD25+ in rats with chronic obstructive pulmonary disease (COPD). METHODS: Totally 120 rats were randomly divided into the control group, the model group, the RFIP group, the RFIS group, the BQNS group, and the aminophylline group, 20 in each group. Except those in the control group, the rest rats were exposed to cigarette smoking and bacterial infection to prepare the COPD rat model. Rats in the RFIP group, the RFIS group, the BQNS group, and the aminophylline group were administrated with Bufei Jianpi Recipe, Bufei Yishen Recipe, Yiqi Zishen Recipe, and aminophylline from week 9 to 20. After rats were sacrificed at week 20 and 32, lung pathological impairments and the levels of T lymphocyte subsets (CD3+, CD4+, CD8+, CD4+ / CD8+) and CD4+ CD25+ in the peripheral blood and the bronchoalveolar lavage fluid (BALF) were detected. RESULTS: At week 20 and 32, the impairments in the lungs were obvious in rats of the model group, while the levels of CD3+, CD4+, CD8+, and CD4+ CD25+ were significantly lower in the peripheral blood and the BALF in the model group than in the controls group (P < 0.05, P < 0.01), and they were higher in the four groups than in the model group (P < 0.05, P < 0.01). However, the levels of CD3+ and CD4+ in the peripheral blood and the BALF were higher in the three TCM-treated groups than in the aminophylline group (P < 0.05, P < 0.01). CD4+ in the peripheral blood in the RFIP group was higher than in the RFIS group and the BQNS group (P < 0.01). At week 20, the ratio of CD4+ /CD8+ was higher in the RFIP group than in the aminophylline group (P < 0.01). CD4+ was higher in the three TCM-treated groups than in the aminophylline group (P < 0.05, P < 0.01). At week 32, the ratio of CD4+ / CD+ in the three TCM- and aminophylline-treated groups was higher than that of the model group (P < 0.05). CD4+ in the RFIP group and the RFIS group was higher than that of the aminophylline group (P < 0.05, P < 0.01). Compared with that at week 20, the ratio of CD4+/CD8+ in the BALF group was significantly higher in the RFIP at week 32 (P < 0.05). The CD4+ CD25+ levels in the peripheral blood and BALF of the BQNS group was significantly lower (P < 0.05). CONCLUSIONS: The efficacy and long-term effects of three methods for regulating and invigorating Fei-Shen might be possibly associated with regulating T lymphocyte subsets (CD3+, CD4+, CD8+, CD4+ / CD8+) and CD4+ CD25+ levels. Of them, RFIP showed significant effects in regulating CD4+ and CD4+ / CD8+ in the peripheral blood and BALF.