Metabolomic profiling combined with network analysis of serum pharmacochemistry to reveal the therapeutic mechanism of Ardisiae Japonicae Herba against acute lung injury.

Introduction: Acute lung injury (ALI) is a common and devastating respiratory disease associated with uncontrolled inflammatory response and transepithelial neutrophil migration. In recent years, a growing number of studies have found that Ardisiae Japonicae Herba (AJH) has a favorable anti-inflammatory effect. However, its serum material basis and molecular mechanism are still unknown in ALI treatment. In this study, metabolomics and network analysis of serum pharmacochemistry were used to explore the therapeutic effect and molecular mechanism of AJH against lipopolysaccharide (LPS)-induced ALI. Methods: A total of 12 rats for serum pharmacochemistry analysis were randomly divided into the LPS group and LPS + AJH-treated group (treated with AJH extract 20 g/kg/d), which were administered LPS (2 mg/kg) by intratracheal instillation and then continuously administered for 7 days. Moreover, 36 rats for metabolomic research were divided into control, LPS, LPS + AJH-treated (5, 10, and 20 g/kg/d), and LPS + dexamethasone (Dex) (2.3 × 10-4 g/kg/d) groups. After 1 h of the seventh administration, the LPS, LPS + AJH-treated, and LPS + Dex groups were administered LPS by intratracheal instillation to induce ALI. The serum pharmacochemistry profiling was performed by UPLC-Orbitrap Fusion MS to identify serum components, which further explore the molecular mechanism of AJH against ALI by network analysis. Meanwhile, metabolomics was used to select the potential biomarkers and related metabolic pathways and to analyze the therapeutic mechanism of AJH against ALI. Results: The results showed that 71 serum components and 18 related metabolites were identified in ALI rat serum. We found that 81 overlapping targets were frequently involved in AGE-RAGE, PI3K-AKT, and JAK-STAT signaling pathways in network analysis. The LPS + AJH-treated groups exerted protective effects against ALI by reducing the infiltration of inflammatory cells and achieved anti-inflammatory efficacy by significantly regulating the interleukin (IL)-6 and IL-10 levels. Metabolomics analysis shows that the therapeutic effect of AJH on ALI involves 43 potential biomarkers and 14 metabolic pathways, especially phenylalanine, tyrosine, and tryptophan biosynthesis and linoleic acid metabolism pathways, to be influenced, which implied the potential mechanism of AJH in ALI treatment. Discussion: Our study initially elucidated the material basis and effective mechanism of AJH against ALI, which provided a solid basis for AJH application.

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.

Peimine ameliorates pulmonary fibrosis via the inhibition of M2-type macrophage polarization through the suppression of P38/Akt/STAT6 signals.

Peimine, a bioactive substance isolated from Chinese medicine Fritillaria, can potentially suppress pulmonary fibrosis (PF); however, its therapeutic mechanism remains unclear. Recent evidence suggests the participation of M2-type macrophages in the pathogenesis of PF. The present study aimed to investigate the effect of peimine on a bleomycin (BLM)-induced PF rat model and the underlying mechanism of this effect. After BLM administration, peimine was administered to rats from day 29 to day 42, with pirfenidone (PFD) as a positive control. H&E and Masson's trichrome stain were used to analyze histological changes. Q-PCR and western blotting were used to measure mRNA levels and protein levels, respectively. High-throughput RNA sequencing (RNA-seq) technology detected the differentially expressed genes (DEGs) regulated by peimine. Our results revealed that peimine treatment significantly ameliorated BLM-induced PF by suppressing histological changes and collagen deposition. In addition, peimine decreased the number of M2 macrophages and the expression of profibrotic factors. RNA-seq results showed that DEGs regulated by peimine in IL-4-induced macrophages were mainly associated with immune system processes, the PI3K/Akt pathway, and the MAPKs pathway. Then, immunofluorescence assay and western blot results demonstrated that peimine treatment suppressed the expression of p-p38 MAPK and p-Akt (s473) and also inhibited the nuclear translocation of p-STAT6. In conclusion, the present study demonstrated that peimine has a protective effect on PF through the suppression of M2 polarization of macrophages by inhibiting the STAT6, p38 MAPK, and Akt signals.

Network pharmacology combined with pharmacodynamics revealed the anti-inflammatory mechanism of Tanreqing capsule against acute-exacerbation chronic obstructive pulmonary disease.

Acute-exacerbation chronic obstructive pulmonary disease (AECOPD) is mainly associated with acute respiratory tract infection. In recent years, a growing number of studies have found that Tanreqing capsule (TRQ) has a favorable anti-inflammatory effect. In this study, we used network pharmacology and pharmacodynamics to explore the molecular mechanism and effects of TRQ in AECOPD treatment. To further understand the molecular mechanism of TRQ in AECOPD treatment, we used the network pharmacology to predict components of TRQ, TRQ-related targets, AECOPD-related targets, and pathways. In addition, we used the cigarette-smoke/lipopolysaccharide -induced AECOPD experimental model in Sprague-Dawley rats (72 rats randomly divided into six groups [n = 12 each]: control, model, high-TRQ [TRQ-H], medium-TRQ [TRQ-M], low-TRQ, and dexamethasone [Dex]) to evaluate the therapeutic effects of TRQ and to verify the network pharmacology. We found that 59 overlapping targets based on component-and AECOPD-related targets were frequently involved in the advanced glycation end product-receptor for advanced glycation end product signaling pathway in diabetic complications, the phosphatidylinositol-3-kinase-protein kinase B signaling pathway, and the hypoxia-inducible factor 1 signaling pathway, which might play important roles in the anti-inflammatory mechanism of TRQ in AECOPD treatment. Moreover, TRQ groups exerted protective effects against AECOPD by reducing the infiltration of inflammatory cells. Meanwhile, TRQ-M and TRQ-H groups significantly downregulated or upregulated the expression of tumor necrosis factor, interleukin (IL) 6, C-reactive protein, IL10, and serum amyloid A, as key targets in network pharmacology, in the serum and bronchoalveolar lavage fluid to achieve anti-inflammatory efficacy. Our study showed that TRQ had better anti-inflammatory efficacy against AECOPD, and initially elucidated its molecular mechanism. Moreover, our study also provides a new strategy to explore effective mechanism of TRQ against AECOPD; and further studies are needed to validate the biological processes and pathways of TRQ against AECOPD.

A validated LC-MS/MS method for the determination of hederasaponin C: Application to pharmacokinetics-pharmacodynamics studies in the therapeutic area of acetic acid-induced ulcerative colitis in rats.

Hederasaponin C (HSC), one of the main components of Pulsatilla chinensis, is considered as a potential drug for the treatment of inflammatory bowel disease. In the present research, we developed a pharmacokinetics-pharmacodynamics model to describe the concentration-effect course of drug action following the intraperitoneal injection of HSC in colitis rats. A sensitive UPLC-MS/MS method was established for the the determination of HSC in rat plasma to explore the pharmacokinetics properties. The separation was performed on an Accucore C18 column (2.1 × 100 mm, 2.6 µm) with a flow phase consisting of acetonitrile and 0.1% formic acid water. The assay method was validated and demonstrated good adaptability for application in the pharmacokinetics study. Then the levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and interleukin-1ß (IL-1ß) in colon tissues were measured using an ELISA assay. The levels of TNF-α, IL-1ß and IL-6 were decreased after HSC administration, suggesting that HSC can significantly improve the level of inflammatory syndrome factor. The pharmacokinetics study showed that the time to peak concentration of HSC was 1 h. The concentration-effect curves showed a hysteresis loop. There was also a hysteresis between the peaked concentration and the maximum effect of HSC. The present study established in vivo pharmacokinetics-pharmacodynamics models and the results showed a great potential of HSC for treating ulcerative colitis.

Systematic characterization of the components and molecular mechanisms of Jinshui Huanxian granules using UPLC-Orbitrap Fusion MS integrated with network pharmacology.

Jinshui Huanxian granules (JSHX) is a clinical Chinese medicine formula used for treating pulmonary fibrosis (PF). However, the effective components and molecular mechanisms of JSHX are still unclear. In this study, a combination approach using ultra-high performance liquid chromatography-Orbitrap Fusion mass spectrometry (UPLC-Orbitrap Fusion MS) integrated with network pharmacology was followed to identify the components of JSHX and the underlying molecular mechanisms against PF. UPLC-Orbitrap Fusion MS was used to identify the components present in JSHX. On the basis of the identified components, we performed target prediction using the SwissTargetPrediction database, protein-protein interaction (PPI) analysis using STRING database, and Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis using Metascape and constructed a component-target-pathway network using Cytoscape 3.7.2. Molecular docking technology was used to verify the affinity between the core components and targets. Finally, the pharmacological activities of three potentially bioactive components were validated in transforming growth factor ß1 (TGF-ß1)-induced A549 cell fibrosis model. As a result, we identified 266 components, including 56 flavonoids, 52 saponins, 31 alkaloids, 10 coumarins, 12 terpenoids and 105 other components. Of these, 90 validated components were predicted to act on 172 PF-related targets and they exhibited therapeutic effects against PF via regulation of cell migration, regulation of the mitogen-activated protein kinase (MAPK) cascade, reduction of oxidative stress, and anti-inflammatory activity. Molecular docking showed that the core components could spontaneously bind to receptor proteins with a strong binding force. In vitro, compared to model group, hesperetin, ruscogenin and liquiritin significantly inhibited the increase of α-smooth muscle actin (α-SMA) and fibronectin (FN) and the decrease of e-cadherin (E-cad) in TGF-ß1-induced A549 cells. This study is the first to show, using UPLC-Orbitrap Fusion MS combined with network pharmacology and experimental validation, that JSHX might exert therapeutic actions against PF by suppressing the expression of key factors in PF. The findings provide a deeper understanding of the chemical profiling and pharmacological activities of JSHX and a reference for further scientific research and clinical use of JSHX in PF treatment.

Network pharmacology analysis uncovers the effect on apoptotic pathway by Bu-Fei formula for COPD treatment.

ETHNOPHARMACOLOGICAL RELEVANCE: The Bu-Fei formula (BFF) has a positive effect on chronic obstructive pulmonary disease (COPD). However, its therapeutic mechanisms against COPD remain unknown. AIM OF THE STUDY: To explore BFF's therapeutic effect on COPD and pharmacological mechanisms. MATERIALS AND METHODS: First, the effect of BFF on rats with COPD was studied. Rats were randomly assigned to the blank, COPD, BFF treatment, and aminophylline (APL) treatment groups. From weeks 1-8, the COPD model was established by Klebsiella pneumoniae (KP) and cigarette smoke. Then, rats were given corresponding treatment for 8 weeks. The lung function of the rats was analyzed by whole-body plethysmography and pulmonary function testing, lung histopathology by electron microscopy and hematoxylin and eosin staining, and protein levels by immunohistochemistry. Next, the key components and targets of BFF in COPD were screened by network pharmacology analysis. Finally, the possible mechanism was verified through molecular docking and in vivo experiments. RESULTS: BFF significantly improved lung function and lung histopathology in COPD rats and inhibit inflammation and collagen deposition in lung tissues. Also, 46 bioactive compounds and 136 BFF targets related to COPD were identified; among them, 3 compounds (quercetin, luteolin, and nobiletin) and 6 core targets (Akt1, BCL2, NF-κB p65, VEGFA, MMP9, and Caspase 8) were the key molecules associated with the mechanisms of BFF. The target enrichment analysis suggested that BFF's mechanisms might involve the apoptosis-related pathway; this possibility was supported by the molecular docking data. Lastly, BFF was indicated to increase the expression of core target genes and the production of apoptosis-related proteins. CONCLUSIONS: BFF affects COPD by regulating the apoptosis-related pathways and targets.

The pharmacokinetic study of Tanreqing and the interaction with cefixime in rat model of pneumonia by validated UPLC-MS/MS.

Combining traditional Chinese medicine and chemical drugs with antimicrobial activities has become more popular, but there is insufficient relevant research on such combinations. The Tanreqing injection (TRQI), a Chinese compound medicine, exhibits therapeutic effects in treating upper respiratory tract infections, severe influenza, and pneumonia. This research investigates the pharmacokinetics of TRQI in pneumonia model rats and explores the effect of the antibiotic cefixime on its metabolism. The pneumonia model rats were randomly divided into six groups: low, medium, and high (3, 6, and 12 mL kg-1) dose TRQI group, and a medium dose TRQI combined with cefixime (14.4 mg kg-1) group, with the remainder two groups were control group. Blood samples were collected from the tail vein at different time points between 0 and 24 h after injection. A sensitive and quick method based on ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was established for the simultaneous determination of the 13 TRQI components in the blood samples. The analytes were separated on an XBridge™C18 column (2.1 mm × 150 mm, 5 µm), with the flow phase consisting of methanol and 0.1% formic acid water at a flow rate of 0.3 mL/min. The assay method met the biological sample determination requirements, demonstrating good adaptability and practicability for application in the pharmacokinetic study of TRQI in pneumonia model rats. Moreover, the method was used successfully in the interaction study of TRQI with cefixime. The results indicated that co-administration results in a significant change in the pharmacokinetic parameters of the main TRQI components. However, the changes in the pharmacokinetic characteristics of multiple TRQI components were inconsistent. Thus, the results of this drug combination under different pathological conditions in clinical applications were unpredictable. Therefore, more attention should be paid to the combined use of cefixime and TRQI in clinical applications to avoid the risk of adverse drug reactions in future studies.

Effect of Yangqing Chenfei Formula on epithelial-mesenchymal transition in silicon dioxide-induced silicosis rats / 中国职业医学

@# Objective Yangqing Chenfei Formula - To investigate the effect of (YCF) on epithelial mesenchymal transition (EMT) Methods in lung tissues of silicosis model rats. Specific pathogen free adult male SD rats were randomly divided into control group, model group, tetrandrine group and YCF group, with eight rats in each group. The rats in the model group, tetrandrine group and YCF group were intratracheally injected with 1.00 mL of silica suspension with a mass concentration of 50.0 g/L, and the rats in the control group were given an equal volume of 0.9% sodium chloride solution. On the 15th day after modeling, the tetrandrine group was given tetrandrine at a dose of 27.0 mg/kg body weight, the YCF group was given YCF with a dose of 8.91 g/kg body weight, while both the control group and model group were given 2.00 mL 0.9% sodium chloride solution. Gavage wasperformed twice a day in the morning and evening for 14 days. On day 29 of the experiment, after evaluating the tidal volume, - functional residual volume (FRC) and vital capacity of rats in each group, lung tissues were collected, and hematoxylin eosin staining and Masson staining were performed to examine the histopathological changes, and the fibrosis score was evaluated. - - Hydroxyproline level was detected by colorimetry. The expression of type Ⅰ collagen (COL Ⅰ), type Ⅲ collagen (COL Ⅲ), - - - - - - E cadherin (E Cad), N cadherin (N Cad) and α smooth muscle actin (α SMA) protein was detected by immunohistochemistry. - The expression of epithelial cell adhesion molecule (EpCAM) and fibroblast specific protein 1 (FSP 1) was detected by Results immunofluorescence. The lung structure was intact and the alveolar structure was normal in the control group. The alveolar structure was destroyed, the alveolar wall was thickened, and cellular nodules were observed/n the model group. The lung tissue lesions of rats in the tetrandrine group and YCF group were reduced compared with that in the model group, and there was no difference in the degree of lesions between the two groups. The tidal volume, FRC and vital capacity of rats in model P< - P< group decreased (all 0.05), the relative expression of E Cad protein in lung tissue decreased ( 0.05), the fibrosis score and - - - - the level of hydroxyproline, the protein relative expression of COL Ⅰ, COL Ⅲ, N Cad and α SMA in lung tissue increased (all P< - 0.05), while the fluorescence intensity of EpCAM protein decreased, and that of FSP 1 protein increased compared with the P< control group. The tidal volume, FRC and vital capacity of rats in tetrandrine and YCF groups increased (all 0.05), the fibrosis - - - score and the level of hydroxyproline, the protein relative expression of COL Ⅰ, N Cad and α SMA in lung tissue decreased (all P< - P< 0.05), the relative expression of E Cad protein in lung tissues increased ( 0.05), while the EpCAM protein fluorescence - intensity increased and FSP 1 protein fluorescence intensity decreased compared with the model group. The relative expression - P< Conclusion of N Cad protein in lung tissues of YCF group was lower than that of the tetrandrine group ( 0.05). YCF can - improve the lung function, alleviate collagen deposition in lung tissues, and inhibit the epithelial mesenchymal transition in silicosis model rats, and then attenuates the progression of silicotic fibrosis.

Nondestructive Testing for Wheat Quality with Sensor Technology Based on Big Data.

Nondestructive testing with sensor technology is one of the fastest growing and most promising wheat quality information analysis technologies. Nondestructive testing with sensor technology benefits from the latest achievement of many disciplines such as computer, optics, mathematics, chemistry, and chemometrics. It has the advantages of simplicity, speed, low cost, no pollution, and no contact. It is widely used in wheat quality analysis and testing research. This article summarizes nondestructive testing with sensor technology for wheat quality, including the mechanical model, hyperspectral technology, Raman spectroscopy, and near-infrared techniques for wheat mechanical properties, storage properties, and physical and chemical properties (such as moisture, ash, protein, and starch) in the past decade. Based on the current research progress, big data technology needs a lot of research in spectral data mining, modeling algorithm optimization, model robustness, etc. to provide more data support and method reference for the research and application of wheat quality.

Effective-constituent compatibility-based analysis of Bufei Yishen formula, a traditional herbal compound as an effective treatment for chronic obstructive pulmonary disease.

OBJECTIVE: Critical effective constituents were identified from Bufei Yishen formula (BYF), a traditional herbal compound and combined as effective-constituent compatibility (ECC) of BYF I, which may have potential bioactive equivalence to BYF. METHODS: The active constituents of BYF were identified using four cellular models and categorised into Groups 1 (Bufeiqi), 2 (Bushen), 3 (Huatan) and 4 (Huoxue) according to Chinese medicinal theory. An orthogonal design and a combination method were used to determine the optimal ratios of effective constituents in each group and the ratios of "Groups 1 to 4" according to their pharmacological activity. We also comprehensively assessed bioactive equivalence between the BYF and the ECC of BYF I in a rat model of chronic obstructive pulmonary disease (COPD). RESULTS: We identified 12 active constituents in BYF. The numbers of constituents in Groups 1 to 4 were 3, 2, 5 and 2, respectively. We identified the optimal ratios of effective constituents within each group. In Group 1, total ginsenosides:Astragalus polysaccharide:astragaloside IV ratio was 9:5:2. In Group 2, icariin:schisandrin B ratio was 100:12.5. In Group 3, nobiletin:hesperidin:peimine:peiminine:kaempferol ratio was 4:30:6.25:0:0. In Group 4, paeoniflorin:paeonol ratio was 4:1. An orthogonal design was then used to establish the optimal ratios of Group 1, Group 2, Group 3 and Group 4 in ECC of BYF I. The ratio for total ginsenosides:Astragalus polysaccharide:astragaloside IV:icariin:schisandrin B:nobiletin:hesperidin:peimine:paeoniflorin:paeonol was determined to be 22.5:12.5:5:100:12.5:4:30:6.25:25:6.25. A comprehensive evaluation confirmed that ECC of BYF I presented with bioactive equivalence to the original BYF. CONCLUSION: Based on the ECC of traditional Chinese medicine formula method, the effective constituents of BYF were identified and combined in a fixed ratio as ECC of BYF I that was as effective as BYF itself in treating rats with COPD.

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 granules combined with acupoint sticking therapy suppress oxidative stress in chronic obstructive pulmonary disease rats: Via regulating peroxisome proliferator-activated receptor-gamma signaling.

ETHNOPHARMACOLOGICAL RELEVANCE: Traditional Chinese medicine (TCM) is clinically used under the guidance of its unique theory system. Bufei Yishen (BY) granules, an oral Chinese herbal formula, is confirmed effective for treating the syndrome of lung-kidney qi deficiency in chronic obstructive pulmonary disease (COPD) patients. Shu-Fei Tie ointment is another prescription for acupoint sticking (AS) therapy based on the theory of treating an internal disease by external treatment on proper acupoints. The beneficial effects of BY granules combined with Shu-Fei Tie have been proved in previous clinical trials. However, the underlying mechanism remains unclear. The present study was initiated to explore the antioxidative mechanism of the integrated therapy of BY granules and acupoint sticking via regulating by peroxisome proliferator activated receptor-gamma (PPARγ) signaling in a cigarette-smoke/bacterial exposure induced COPD rat model. MATERIALS AND METHODS: Rats were randomized into Control, Model, BY, AS, BY+AS and aminophylline (APL) groups. COPD rats were induced by cigarette-smoke and bacterial exposures, and were administrated with normal saline, BY granules, AS, BY+AS or aminophylline from week 9 and sacrificed at week 20. Activity of superoxide dismutase (SOD) and levels of methane dicarboxylic aldehyde (MDA) in peripheral blood and bronchoalveolar lavage fluid (BALF) were determined by hydroxylamine and thiobarbituric acid methods. The gene and protein expressions of PPARγ in the lung tissues were analyzed by quantitative polymerase chain reaction and western blot. RESULTS: Serum and BALF SOD decreased significantly in Model group (P<0.01), while MDA increased (P<0.01). Compared to COPD rats, serum SOD was higher in all treatment groups (P<0.01), and BALF SOD was higher in BY and BY+AS groups (P<0.01); serum and BALF MDA was lower in all treatment groups (P<0.01). Serum and BALF SOD was higher in BY+AS group than in AS group, while MDA was lower (P<0.05). BALF SOD increased in BY+AS group compared with APL group, while MDA decreased (P<0.05). PPARγ mRNA and protein and the phosphorylation of PPARγ (p-PPARγ) decreased in COPD rats (P<0.01), and increased in all treatment groups (P<0.01). PPARγ mRNA was higher in BY+AS group than in AS group (P<0.05), PPARγ and p-PPARγ were higher in BY+AS group than in AS and APL groups (P<0.05, P<0.01); PPARγ protein was higher in BY group than in APL group (P<0.05). CONCLUSION: Bufei Yishen granules, Shu-Fei Tie and their combination have beneficial effects in stable COPD, and can attenuate the oxidative stress, and the activation of PPARγ signaling might be involved in the underlying mechanisms, but there are no obvious synergistic effect of Bufei Yishen granules and Shu-Fei Tie.

[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.

[Impact and long-term effect of three prescriptions regulating and tonifying lung and kidney on JAK/STAT signaling in rats].

OBJECTIVE: To evaluate the impact and long-term effect of three prescriptions regulating and tonifying lung and kidney (prescription tonifying lung and spleen, prescription tonifying lung and kidney, and prescription tonifying Qi and kidney) on JAK/STAT signaling of COPD rats. METHOD: Rats were randomly divided into the control group, the model group, the Bufeijianpi group, the Bufeiyishen group, the Yiqizishen group and the aminophyline group. The COPD rat model was established by smoke inhalations and bacterial infections. In the 9th week, the control group and the model group were administered with normal saline, while the remaining groups are orally given corresponding medicines. In the 20th and 32nd week, the rats were sacrificed in batches to observe the pathology in their lung tissues, protein expressions of JAK2, STAT1, STAT3, STAT5, and expressions of JAK2 and SOCS3 mRNA. RESULT: In the 20th and 32nd week, protein expressions of JAK2 mRNA and phosphorylation-JAK2, STAT1, STAT3 and STAT5 in the model group were higher than the control group (P < 0.01), whereas the three traditional Chinese medicine (TCM) (Bufeijianpi, Bufeiyishen and Yiqizishen) groups and the aminophyline group were significantly lower (P < 0.05, P < 0.01). The expression of SOCS3 mRNA in the model group was higher than the control group (P < 0.01), whereas the level was notably higher in the three TCM groups and the aminophylline group (P < 0.01). The three TCM groups were remarkably higher than the aminophylline group (P < 0.05, P < 0.01). Compared with the figures in the 20th week, JAK2 mRNA and phosphorylation-JAK2, STAT3 and STAT5 were significantly lower in the Bufeijianpi group in the 32nd week (P < 0.05, P < 0.01), and so did phosphorylation-STAT3 in Bufeiyishen group (P < 0.01) and phosphorylation-STAT3 and STAT5 in the Yiqizishen group (P < 0.05, P < 0.01). However, the aminophylline group showed no significant difference in above indicators. CONCLUSION: The three medicines regulating and tonifying lung and kidney can effectively relieve injury of lung tissues, and have long-term effect, which may be related to the regulation of JAK/ STAT signaling. Specifically, prescription tonifying lung and spleen shows good effect in reducing JAK2, STAT3 and STAT5, prescription tonifying lung and kidney shows good effect in reducing p-STAT3, and prescription tonifying Qi and kidney shows good effect in reducing p-STAT3 and p-STAT5.

[Effect of invigorating lung and kidney prescription and its components on secretion of cytokines induced by CSE and LPS].

OBJECTIVE: To observe effect of Invigorating Lung and Kidney Prescription (ILKP) on secretion of cytokines induced by Lipopolysaccharide (LPS) and cigarette smoke extract (CSE) in vitro and discuss prescription regularity of ILKP and reveal molecular mechanism of ILKP treatment on COPD. METHODS: Alveolar epithelial cells A549, airway epithelial cells H292 and monocyte macrophage THP-1 were stimulated with LPS and CSE, in which samples were divided into normal group (20% normal rat serum), model group (20% normal rat serum and 5% CSE with 75 pg/mL LPS), ILKP group and its Components [20% Replenishing qi group (1), Replenishing Lung and Kidney group (2), Activating blood group (3), Reduce phlegm group (4), Replenishing qi with Replenishing Lung and Kidney group (5), Replenishing qi with Activating blood group (6), Replenishing qi with Reduce phlegm group (7), Replenishing qi with Replenishing Lung and Kidney and Activating blood group (8), Replenishing qi with Replenishing Lung and Kidney and Reduce phlegm group (9), Replenishing qi with Activating blood and Reduce phlegm group (10), ILKP serum group (11)]. The cytokines were detected by ELISA and the transcription factors of cytokines were analyzed with Network tool. RESULTS: Compared with the normal group,the secretion of MMP-9, GM-CSF and TGF-beta by THP-1 cells,TIMP by A549 cells,TNF-alpha, MMP-9, GM-CSF, CD36 and TIMP-1 by H292 cells all were increased, while secretion of TNF-alpha, PPAP2B, IL-3 and SOD decreased by THP-1 cells, MMP-9, TNF-alpha, TGF-beta and IL-3 by A549 cells, IL-8 by H292 cells all were decreased. Compared with model group, for THP-1 cells, the secretion of GM-CSF in 3, 6, 10 and 11 prescription groups,TGF-beta in 10 and 11 prescription groups all were decreased. MMP-9 decreased in 9 and 5 prescription groups while it increased in 1, 2, 3, 4, 7, 8, 10 and 11 prescription groups. TNF-alpha was increased in 1, 2, 3, 4, 6, 8, and 9 prescription groups, as it decreased in 11 prescription group. Except 2 prescription group, IL-3 was increased in all other drugs groups. SOD in 2, 4, 5, 8, 9, 11 prescription groups, and PPAP2B in 1, 2, 3, 4, 5, 6, 7 prescription groups were increased while PPAP2B was decreased in 11 prescription group. For A549 cells, the secretion of TIMP-1 was increased in 1, 2, 3, 4, and 5 prescription groups, while it was decreased in 11 prescription groups; Except 1 and 4 prescription group, MMP-9 was increased in other groups; Except TGF-beta was increased in 11 prescription groups, it was increased in other groups; IL-3 was increased in 1, 2, 4, 5, 11 and 3 prescription groups. For H292 cells, the secretion of GM-CSF in 5,6,7 and 2 prescription groups,TNF-alpha in 1, 2, 4, 6, 7, 8, 9 and 10 prescription groups, MMP-9 in 4, 8, 7 and 9 prescription groups all were decreased. CONCLUSION: The effect of ILKP and its components on secretion of cytokines induced by LPS and CSE and its target cells are different, in which Activating blood prescription focuses on inflammation cytokines, Replenishing Lung and Kidney prescription on Lipid metabolism and redox regulation. The effect of ILKPA is the most widely, followed by Activating blood Replenishing Lung and Kidney, and Reduce phlegm prescription on Protease regulation.

[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.

Long-term effects of Tiaobu Feishen therapies on systemic and local inflammation responses in rats with stable chronic obstructive pulmonary disease.

OBJECTIVE: To evaluate the influence and long-term effects on systemic and local inflammation responses in rat with stable chronic obstructive pulmonary disease (COPD) treated with traditional Chinese medicine (TCM) for regulating and invigorating the lung and kidney, including invigorating the lung and spleen (Bufei Jianpi) therapy, supplementing the lung and kidney (Bufei Yishen) therapy, and nourishing qi and kidney (Yiqi Zishen) therapy. METHODS: Rats were randomly divided into six groups: control, model, aminophyline, Bufei Jianpi, Bufei Yishen and Yiqi Zishen groups. The stable COPD model of rat was duplicated by cigarette smoke inhalations and bacterial infection. From the ninth week, the rats with stable COPD were treated with Bufei Jianpi, Bufei Yishen, Yiqi Zishen granules or aminophyline respectively until the 20th week. Half of the animals were sacrificed at the 20th or 32nd week respectively. The leukocyte count and neutrophil percentage in peripheral blood and bronchoalveolar lavage fluid (BALF) were measured; levels of interleukin (IL)-8, IL-10 and tumor necrosis factor-α (TNF-α) in BALF, and levels of IL-1ß, IL-6, IL-8, IL-10 and TNF-α and soluble tumor necrosis factor receptor II (sTNFR2) in serum and lungs were detected by enzyme-linked immunosorbent assay or immunohistochemical method. RESULTS: There were no statistical differences in leukocyte count and neutrophil percentage in peripheral blood among six groups (P>0.05). At the 20th week, leukocyte count in BALF was higher in the model group than in the control group (P<0.01), and was lower in the three TCM groups and the aminophyline group than in the model group (P<0.05, P<0.01), and that of the Bufei Jianpi group was lower than the aminophyline group (P<0.01); while at the 32nd week, leukocyte count in BALF of the three TCM groups decreased and was lower than that of the aminophyline group (P<0.05, P<0.01). At the 20th and 32nd weeks, levels of IL-1ß, IL-6, IL-8, IL-10, TNF-α and sTNFR2 in serum and lungs, and IL-8, IL-10 and TNF-α in BALF of the model group increased, which were higher than those in the control group (P<0.05, P<0.01); the mentioned cytokines were decreased in the three TCM groups compared with the model group (P<0.05, P<0.01), and were also lower in serum and BALF of the three TCM groups than those of the aminophyline group (P<0.05, P<0.01). Expressions of cytokines in lung tissues were depressed in the three TCM groups as compared to those in the aminophyline group. There was no statistical difference on expressions of the mentioned cytokines either in serum and BALF or in the lungs between week 32 and week 20. CONCLUSION: The Bufei Jianpi, Bufei Yishen and Yiqi Zishen therapies can significantly reduce the systemic and local inflammation responses in rats with stable COPD, and have evident long-term effects.

A rat model for stable chronic obstructive pulmonary disease induced by cigarette smoke inhalation and repetitive bacterial infection.

To develop a stable chronic obstructive pulmonary disease (COPD) model in rats. Sprague-Dawley rats were treated with cigarette-smoke inhalation (CSI) for 12 weeks, repetitive bacterial infection (RBI) for 8 weeks, or the combination of the two (CCR) for 12 weeks and followed up for the additional 20 weeks. Tidal volume (V(T)), peak expiratory flow (PEF) and 50% V(T) expiratory flow (EF(50)), histological changes in the lungs, and levels of the cytokines tumor necrosis factor (TNF)-α, interleukin (IL)-8, and IL-10 in serum and bronchial alveolar lavage fluid (BALF) were examined at intervals during the 32 week study period. The right ventricular hypertrophy index (RVHI) was also determined at the same times. V(T), PEF, and EF(50) were decreased in rats with COPD compared to the control. The expression of TNF-α, IL-8 and IL-10 increased in both serum and BALF with a similar trend. Bronchiole and arteriole wall thickness and the degree of bronchiole stenosis and alveolar size increased in COPD rats. RVHI was reduced gradually following the treatment. All of these changes were more pronounced in the CCR-treatment group than in the other groups. Our results have shown that CSI or RBI alone can induce COPD in rats, but that the combination of CSI with RBI induces a stable COPD that has more similarity to complications seen in patients with COPD. This combination may therefore provide a more appropriate model for study of human COPD.