[Effects of conditioned medium of alveolar epithelial cells on vascular endothelial cell damage].

OBJECTIVE: To observe the effect of lipopolysaccharide (LPS) induced conditioned medium of alveolar epithelial cells on the inflammatory response and cell damage of vascular endothelial cells, and explore its mechanism. METHODS: The LPS induced type II alveolar epithelial cells (A549) conditioned medium was used as a stimulus to induce human umbilical vein endothelial cells (HUVEC) damage. The cell counting kit-8 (CCK-8) was used to detect the effect of 0% (blank group), 12.5%, 25%, 50%, 75% and 100% A549 cell conditioned medium cultured for 6, 12, 24 and 48 hours on the cell viability of HUVEC. Enzyme-linked immunosorbent assay (ELISA) was used to detect the levels of inflammatory factors [interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α)] and vasoactive substances [vascular endothelial growth factor (VEGF), endothelin-1 (ET-1)] in the supernatant. Phalloidin staining was used to observe the effects of A549 cells conditioned medium on cell morphology. The expressions of protein kinase B/nuclear factor-κB (AKT/NF-κB) pathway in HUVEC induced by conditioned medium was detected by Western blotting. RESULTS: Compared with the blank group, A549 cells conditioned medium with concentrations of 12.5%, 25%, and 50% had no significant effects on cell viability of HUVEC after 6, 12, and 24 hours, but the activity of HUVEC decreased significantly after 48 hours. Therefore, 12.5%, 25%, 50% A549 cell conditioned medium stimulated for 24 hours was selected as the induction condition for follow-up experiments. Compared with the blank group, the level of IL-6 was significantly increased in 12.5% and 50% conditioned medium groups (ng/L: 2 438.95±64.89, 3 036.41±96.69 vs. 1 736.75±20.99, both P < 0.05), the level of TNF-α was significantly increased in 12.5% and 25% conditioned medium groups (ng/L: 174.08±11.09, 81.37±8.17 vs. 50.03±0.26, both P < 0.01), the levels of VEGF and ET-1 were significantly increased in 12.5%, 25% and 50% conditioned medium groups [VEGF (ng/L): 173.60±41.44, 192.49±12.38, 318.89±27.90 vs. 66.68±19.65; ET-1 (ng/L): 54.88±1.37, 36.69±0.29, 24.07±0.73 vs. 10.67±0.25, all P < 0.01]. Phalloidin staining showed that HUVEC induced by 25% A549 cells conditioned medium were irregular in shape, uneven in size, disordered in arrangement, widened in gap, dense and unclear in microfilament structure and serrated in cell membrane. Furthermore, the average fluorescence intensity of 25% conditioned medium group significantly increased compared to the blank group (67 205.60±3 430.40 vs. 56 272.67±7 650.95, P < 0.05). Western blotting showed that compared with the blank group, the expression of HUVEC cells phosphonated inhibitor α of NF-κB (p-IκBα) was significantly decreased in the 12.5%, 25%, and 50% conditioned medium groups (p-IκBα/IκBα: 0.38±0.08, 0.67±0.12, 0.31±0.07 vs. 1.00±0.00, all P < 0.01), the expressions of phosphonated-AKT (p-AKT) and VEGF were significantly increased (p-AKT/AKT: 1.50±0.18, 1.42±0.27, 1.61±0.14 vs. 1.00±0.00, VEGF/GAPDH: 1.37±0.10, 1.53±0.22, 1.40±0.12 vs. 1.00±0.00, all P < 0.05), the expression of phosphonated NF-κB p65 (p-P65) was significantly increased in the 25% conditioned medium group (p-P65/P65: 1.45±0.14 vs. 1.00±0.00, P < 0.05). CONCLUSIONS: LPS induced conditional culture medium of alveolar epithelial cells induced endothelial cell damage via activating AKT/NF-κB pathway.

[Research progress on the role of N6-methyladenosine methylation in the pathogenesis and development of respiratory diseases].

N6-methyladenosine (m6A) is one of the most common post-transcriptional modifications of eukaryotic mRNA. The m6A modification accelerates mRNA metabolism and translation, and plays an important role in cell differentiation, embryonic development and stress response. As a reversible epigenetic modification, m6A modification plays an important role in many physiological and pathological processes. The m6A modification is closely related to the occurrence and progression of respiratory diseases, and the m6A modification regulatory factor may be a potential target for regulating respiratory diseases. This article reviews the role of m6A modification in the development of respiratory diseases such as lung cancer, acute lung injury (ALI), asthma, pulmonary fibrosis, and chronic obstructive pulmonary disease (COPD). The purpose of m6A modification is to provide a reference for the pathogenesis of respiratory diseases and the study of targets.

Recent advances in the therapeutic potential of nobiletin against respiratory diseases.

BACKGROUND: Nobiletin is a natural polymethoxylated flavonoid widely present in citrus fruit peels. It has been demonstrated to exert the effects of anti-tumor, anti-inflammation, anti-oxidative, anti-apoptotic and improve cardiovascular function. Increasing evidences suggest that nobiletin plays an important role in respiratory diseases (RDs) treatment. OBJECTIVE: This review aimed to investigate the therapeutic potential of nobiletin against RDs, such as lung cancer, COPD, pulmonary fibrosis, asthma, pulmonary infection, acute lung injury, coronavirus disease 2019, and pulmonary arterial hypertension. METHODS: We retrieved extensive literature of relevant literatures in English until June 26, 2023 from the database of PubMed, Web of Science, and Scopus databases. The keywords of "nobiletin and lung", "nobiletin and respiratory disease", "nobiletin and chronic respiratory diseases", "nobiletin and metabolites", "nobiletin and pharmacokinetics", "nobiletin and toxicity" were searched in pairs. A total of 298 literatures were retrieved from the above database. After excluding the duplicates and reviews, 53 were included in the current review. RESULTS: We found that the therapeutic mechanisms are based on different signaling pathways. Firstly, nobiletin inhibited the proliferation and suppressed the invasion and migration of cancer cells by regulating the related pathway or key target, like Bcl-2, PD-L1, PARP, and Akt/GSK3ß/ß-catenin in lung cancer treatment. Secondly, nobiletin treats COPD and ALI by targeting classical signaling pathway mediating inflammation. Besides, the available findings show that nobiletin exerts the effect of PF treatment via regulating mTOR pathway. CONCLUSIONS: With the wide range of pharmacological activities, high efficiency and low toxicity, nobiletin can be used as a potential agent for preventing and treating RDs. These findings will contribute to further research on the molecular mechanisms of nobiletin and facilitate in-depth studies on nobiletin at both preclinical and clinical levels for the treatment of RDs.

Effective-component compatibility of Bufei Yishen formula alleviates chronic obstructive pulmonary disease inflammation by regulating GSK3ß-mediated NLRP3 inflammasome activation.

Glycogen synthase kinase 3ß (GSK3ß) has been associated with sensing many different stimuli to trigger the NLRP3 inflammasome, which plays a crucial role in promoting the inflammatory response in diseases, including chronic obstructive pulmonary disease (COPD). Bufei Yishen formula (BYF), a traditional Chinese herbal medicine, has beneficial effects on COPD. Effective-component compatibility of BYF (ECC-BYF), optimized from BYF, is equally effective as BYF in inhibiting COPD inflammation. However, the exact mechanism by which ECC-BYF regulates the activation of NLRP3 inflammasome to inhibit COPD inflammation remains unclear. Hence, we investigated the mechanisms underlying the alleviation of COPD inflammation by ECC-BYF through the inhibition of GSK3ß-mediated NLRP3 inflammasome activation by experimental rat model of COPD and lipopolysaccharide/adenosine triphosphate (LPS/ATP) induced macrophages. The data showed that ECC-BYF significantly improved the lung function, attenuated histopathological damage, and alleviated inflammatory cell infiltration and alveolar destruction. Further, it significantly inhibited inflammatory cytokine production and downregulated the phosphorylation of GSK3ß by inhibiting the activation of NLRP3 inflammasome in the rat model of COPD. Moreover, ECC-BYF suppressed the activation of the NLRP3 inflammasome by increasing the phosphorylation at serine 9 and decreasing the phosphorylation at tyrosine 216 of GSK3ß, followed by the inhibition of IL-1ß secretion in macrophages. Together, ECC-BYF effectively ameliorates COPD by suppressing inflammation, which is dependent on the regulation of GSK3ß-mediated NLRP3 inflammasome activation.

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.

Network Pharmacology and Experimental Validation to Reveal Effects and Mechanisms of Icariin Combined with Nobiletin against Chronic Obstructive Pulmonary Diseases.

Background: Chronic obstructive pulmonary disease (COPD) is a long-term respiratory disorder marked by restricted airflow and persistent respiratory symptoms. According to previous studies, icariin combined with nobiletin (I&N) significantly ameliorates COPD, but the therapeutic mechanisms remain unclear. Purpose: The aim of the study is to investigate the therapeutic mechanisms of I&N against COPD using network pharmacology and experimental validation. Methods: The targets of I&N and related genes of COPD were screened and their intersection was selected. Next, the protein-protein interaction (PPI) networks, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed. Further, a COPD rat model was established to validate the effect and mechanisms of I&N. Results: 445 potential targets I&N were obtained from SwissTargetPrediction, STITCH 5.0, and PharmMapper databases. 1831 related genes of COPD were obtained from GeneCards, DrugBank, and DisGeNet databases. 189 related genes were screened via matching COPD targets with I&N. 16 highest score targets among 189 targets were obtained according to PPI networks. GO and KEGG pathway enrichment analyses of 16 highest score targets suggested that these key genes of I&N were mostly enriched in the tumor necrosis factor (TNF) pathway, mitogen-activated protein kinase (MAPK) pathway, and phosphatidyl inositol 3-kinase (PI3K)-protein kinase B (AKT) pathway. Therefore, the treatments of I&N for COPD were connected with inflammation-related pathways. In in vivo experiments, the studies indicated that I&N improved the lung function and alleviated the damage of pulmonary histopathology. Moreover, I&N reduced levels of interleukin (IL)-6, IL-1ß, and TNF-α in lung tissues of COPD rats and inhibited the activation of the MAPK pathway and PI3K-Akt pathway. Conclusions: Icariin combined with nobiletin has therapeutic effects on COPD by inhibiting inflammation. The potential mechanisms of I&N may relate to the MAPK pathway and PI3K-Akt pathway.

GSK3ß mediates the spatiotemporal dynamics of NLRP3 inflammasome activation.

Subcellular machinery of NLRP3 is essential for inflammasome assembly and activation. However, the stepwise process and mechanistic basis of NLRP3 engagement with organelles remain unclear. Herein, we demonstrated glycogen synthase kinase 3ß (GSK3ß) as a molecular determinant for the spatiotemporal dynamics of NLRP3 inflammasome activation. Using live cell multispectral time-lapse tracking acquisition, we observed that upon stimuli NLRP3 was transiently associated with mitochondria and subsequently recruited to the Golgi network (TGN) where it was retained for inflammasome assembly. This occurred in relation to the temporal contact of mitochondria to Golgi apparatus. NLRP3 stimuli initiate GSK3ß activation with subsequent binding to NLRP3, facilitating NLRP3 recruitment to mitochondria and transition to TGN. GSK3ß activation also phosphorylates phosphatidylinositol 4-kinase 2 Α (PI4k2A) in TGN to promote sustained NLRP3 oligomerization. Our study has identified the interplay between GSK3ß signaling and the organelles dynamics of NLRP3 required for inflammasome activation and opens new avenues for therapeutic intervention.

m6A mRNA methylation-directed myeloid cell activation controls progression of NAFLD and obesity.

N6-methyladenosine (m6A) RNA modification is a fundamental determinant of mRNA metabolism, but its role in innate immunity-driven non-alcoholic fatty liver disease (NAFLD) and obesity is not known. Here, we show that myeloid lineage-restricted deletion of the m6A "writer" protein Methyltransferase Like 3 (METTL3) prevents age-related and diet-induced development of NAFLD and obesity in mice with improved inflammatory and metabolic phenotypes. Mechanistically, loss of METTL3 results in the differential expression of multiple mRNA transcripts marked with m6A, with a notable increase of DNA Damage Inducible Transcript 4 (DDIT4) mRNA level. In METTL3-deficient macrophages, there is a significant downregulation of mammalian target of rapamycin (mTOR) and nuclear factor κB (NF-κB) pathway activity in response to cellular stress and cytokine stimulation, which can be restored by knockdown of DDIT4. Taken together, our findings identify the contribution of METTL3-mediated m6A modification of Ddit4 mRNA to macrophage metabolic reprogramming in NAFLD and obesity.

The Anti-Inflammatory Effect of a Combination of Five Compounds From Five Chinese Herbal Medicines Used in the Treatment of COPD.

Effective compound combination (ECC; i.e, 20-S-ginsenoside Rh1, astragaloside, icariin, nobiletin, and paeonol), derived from Chinese herbal medicine, significantly ameliorates chronic obstructive pulmonary disease (COPD) in rats; however, the underlying mechanisms of ECC remain largely unclear. In this study, network pharmacology analysis integrated with experimental validation was used to explore the therapeutic mechanisms of ECC against COPD. ECC targets and COPD genes and targets were identified from multiple databases, and then used for an analysis of protein-protein interaction (PPI) networks, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, and biological functioning. BisoGenet was used to comprehensively analyze the hub-network. We validated the therapeutic effect and mechanisms of ECC both in vivo and in vitro. We identified 45 ECC targets, which were mainly related to inflammatory processes, such as the NOD-like and NF-kappa B signaling pathways, hematopoietic cell lineage, Th17 cell differentiation, cellular response to lipopolysaccharide, and interleukin-8 secretion. In addition, 1180 COPD genes and 70 COPD targets were identified as being involved in the biological functions associated with COPD development, such as cytokine-cytokine receptor interaction, the TNF signaling pathway, the mitogen-activated protein kinase (MAPK) signaling pathway, regulation of lymphocyte proliferation, and positive regulation of leukocyte migration. Integrative analysis of COPD genes and targets and ECC target networks revealed that 54 genes were mainly involved in the inflammatory process, such as IL-17 signaling, NF-kappa B signaling, innate immune response-activating signal transduction, and macrophage cell differentiation. Six targets (AR, ESR1, HNRNPA1, PAPR1, TP53, and VCAM1) contained in the hub-network and their four related compounds were obtained and recognized as the key molecules associated with the effects of ECC. Molecular docking validation demonstrated that four compounds could bind to six targets that interact with COPD genes. Finally, in vivo and in vitro experiments verified that ECC treatment ameliorated the symptoms of COPD in rats by improving their lung function, reducing pathological changes, and suppressing oxidative responses and pro-inflammatory cytokine secretion, while inhibiting inflammation in LPS-induced macrophages, which may be associated with NF-kappa B and MAPK signaling regulation. This study demonstrates the therapeutic mechanisms and effects of ECC on COPD via regulation of the underlying inflammatory process.

Effective-components combination improves airway remodeling in COPD rats by suppressing M2 macrophage polarization via the inhibition of mTORC2 activity.

BACKGROUND: In chronic obstructive pulmonary disease (COPD), M2 macrophages release multiple tissue repair-related factors, leading to airway remodeling, a significant pathological characteristic. Meanwhile, effective-components combination (ECC), derived from Bufei Yishen formula (BYF), is an effective treatment for COPD. PURPOSE: To determine the potential mechanisms of ECC in airway remodeling in COPD by suppressing M2 macrophage polarization. METHODS: We established a rat COPD Model using exposure to cigarette smoke and bacterial infection to investigate the efficacy of ECC. We also treated macrophages with IL-4 for 12 h to explore the in vivo effect of ECC on M2 macrophage polarization and mTORC2 signals. RESULTS: The disease severity of COPD rats could be alleviated by ECC treatment, which improved pulmonary function and alleviated pathological injuries in lung tissue and the inflammatory cytokine levels. Meanwhile, ECC could ameliorate airway remodeling by reducing collagen deposition, hindering airway mucus hypersecretion and smooth muscle cell proliferation, and reducing the number of M2 macrophages in the lung tissues of COPD rats. Furthermore, with IL-4-induced macrophages, we found that ECC could suppress M2 macrophage polarization by decreasing the levels of M2 macrophage markers. Finally, we discovered that ECC inhibited mTORC2 activity by examining p-mTOR2481 and its downstream protein p-Akt473. CONCLUSIONS: ECC exerts beneficial effects on airway remodeling in COPD rats, likely by suppressing M2 macrophage polarization via the inhibition of mTORC2 activity.

Tiaobu Feishen therapy inhibits inflammation induced by cigarette smoke extracts in a human monocyte/macrophage cell line.

OBJECTIVE: To study the mechanistic effects of Tiaobu Feishen therapy (TBFS) on inflammation induced by cigarette smoke extract (CSE) in a human monocyte/macrophage cell line. METHODS: The human monocyte/macrophage cell line THP-1 was stimulated with 10 % CSE in the presence or absence of Bufei Yishen formula (BYF), Bufei Jianpi formula (BJF) and Yiqi Zishen formula (YZF). All formulations contained serum. Pro-inflammatory cytokines were measured in the supernatants using enzyme-linked immunosorbent assay. The activity of STAT3 DNA binding was detected using electrophoretic mobility shift assay and janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway activation was assessed using Western blotting. RESULTS: The results showed that BYF, BJF and YZF treatment strongly decreased the CSE-induced secretion of interleukin (IL)-6, IL-8, tumor necrosis factor-α and matrix metalloproteinase-9 by THP-1 cells. Furthermore, BYF, BJF and YZF treatment attenuated STAT3 DNA binding capacity and JAK2 and STAT3 were shown to be phosphorylated. CONCLUSION: The data revealed that BYF, BJF and YZF effectively inhibited a CSE-induced inflammatory response in THP-1 cells by limiting activation of the JAK2/STAT3 pathway.

A chinese herbal formula ameliorates COPD by inhibiting the inflammatory response via downregulation of p65, JNK, and p38.

BACKGROUND: Bufei Yishen formula (BYF), a traditional Chinese medicine (TCM), is an effective therapeutic strategy for patients with chronic obstructive pulmonary disease (COPD). PURPOSE: To evaluate the efficacy of BYF and investigate its therapeutic mechanisms. METHODS: A total of 134 patients completed the study: 68 patients treated by BYF combined with conventional Western medicine in the trial group; and 66 patients treated using conventional Western medicine in the control group. The efficacy of BYF was evaluated by a subgroup analysis of data obtained from a four-center, open-label, randomized controlled trial of comprehensive TCM interventions. A rat model of COPD was treated with the key active molecules (KAM) of BYF for 8 weeks. An in vitro model of COPD was also treated with KAM. RESULTS: Patients treated with BYF had reduced frequency of acute exacerbation of COPD (p < 0.001) and duration (p = 0.028), dyspnea scale (p = 0.007), 6-min walking distance (p = 0.048). There were no differences observed in forced vital capacity in one second (FVC), forced expiratory volume in one second (FEV1), and FEV1 percentage of the predicted value (FEV1%). The five KAM of BYF (KAM-BYF) improved lung function, including tidal volume, minute ventilation, peak expiratory flow, FVC, FEV0.1, and FEV0.3, and pathological changes in COPD rats. Treatment with KAM-BYF markedly decreased the levels of interleukin 6 (IL6), tumor necrosis factor-α (TNF-α), matrix metalloproteinase 9 (MMP9), and MMP12 in serum and bronchial alveolar lavage fluid. In airway epithelial cells, KAM-BYF decreased the levels of TNF-α-induced IL8 and IL6. Finally, we discovered that the anti-inflammatory effects of KAM-BYF in COPD rats and BEAS-2Bs were mediated through inhibition of nuclear factor-kappaB (NF-κB) p65, c-Jun NH2-terminal kinase (JNK), and p38 mitogen-activated protein kinase signaling. CONCLUSIONS: BYF exerts beneficial effects in patients with COPD via inhibition of inflammation.

[Compatibility characteristics of Bufei Yishen formula III in regulating chronic obstructive pulmonary disease mucus hypersecretion].

OBJECTIVE: To evaluate the compatibility laws of effective-component compatibility of Bufei Yishen formula III (ECC-BYF III) in regulating mucus hypersecretion of chronic obstructive pulmonary disease (COPD). METHODS: According to the efficacy of the original Chinese medicine, the components of ECC-BYF III were divided into four categories: Buqi (Ginsenoside Rh1+Astragaloside), Bushen (Icariin), Huatan (Nobiletin), and Huoxue (Paeonol). The four categories were divided into 14 groups based on the method of mathematical permutation. (1) The rats were divided into control group, model group, ECC-BYF III, and different components compatibility groups according to the random number table, totaling 17 groups. COPD rat model in stable phase was established by cigarette smoke exposure combined with repeated bacterial infections. The corresponding drugs were given by gavage at the 9th week of modeling, and the samples were collected at the end of the 16th week. The levels of matrix metalloproteinase-9 (MMP-9) and tissue inhibitors of metalloproteinase 1 (TIMP-1) in serum and bronchoalveolar lavage fluid (BALF), and the levels of mucin (MUC) 5AC in lung tissue and BALF were detected by enzyme linked immunosorbent assay (ELISA). (2) Human lung epithelial cells BEAS-2B were divided into blank group, model group, and different components compatibility groups. Hypoxia-induced mucus hypersecretion model of human lung epithelial cells BEAS-2B was established 4 hours after corresponding drug pretreatment. The mRNA expressions of MUC5AC, MUC5B, and MUC1 were detected by quantitative polymerase chain reaction (PCR). The mucus secretion indexes of rats and BEAS-2B cells were evaluated by Region (R) value comprehensive evaluation method. RESULTS: (1) Compared with the control group, MMP-9 in serum and BALF from the model group were significantly increased, the level of TIMP-1 was significantly decreased, and MUC5AC in lung tissue and BALF were significantly increased. The results of R value comprehensive evaluation showed that except for the Buqi and Bushen groups, ECC-BYF III and other components compatibility groups significantly corrected mucus hypersecretion in COPD rats, ECC-BYF III, Bushen Quxie, Fuzheng Huatan, and Quxie groups were much better (R values were 2.15±0.42, 2.11±0.23, 2.16±0.23 and 2.16±0.55, respectively), compared with the model group (R value: 3.00±0.00), the differences were statistically significant (all P < 0.05). (2) Compared with the blank group, the mRNA expressions of MUC5AC, MUC5B, and MUC1 increased in the model group. But different components compatibility groups had no significant effects on the mucus secretion of BEAS-2B cells. (3) The comprehensive evaluation results of R value about each in vivo and in vitro index showed that ECC-BYF III, Huoxue, Quxie, Bushen Huoxue, Fuzheng Huatan, Buqi Quxie groups significantly corrected the mucus hypersecretion (R values were 2.30±0.43, 2.33±0.44, 2.12±0.68, 2.27±0.64, 2.24±0.27 and 2.29±0.47, respectively), compared with the model group (R value: 3.00±0.00), the difference was statistically significant (all P < 0.01). The order was: Quxie > Fuzheng Huatan > Bushen Huoxue > Buqi Quxie > ECC-BYF III > Huoxue. CONCLUSIONS: Different components compatibility of ECC-BYF III had different effects on COPD mucus secretion. The components containing Huatan (Nobiletin) or Huoxue (Paeonol) showed a better inhibitory effect on mucus secretion.

Mechanisms of the lipopolysaccharide-induced inflammatory response in alveolar epithelial cell/macrophage co-culture.

The interaction between alveolar epithelial cells (EpCs) and macrophages (MPs) serves an important role in initiating and maintaining inflammation in chronic pulmonary diseases. The aim of the present study was to investigate the molecular mechanisms of the inflammatory response in co-cultured EpCs and MPs. Briefly, a co-culture system of A549 (EpCs) and THP-1 (monocyte/MPs) cells was established in a filter-separated Transwell plate to evaluate the inflammatory response. Following lipopolysaccharide (LPS) treatment, cytokine levels were measured using ELISAs, NF-κB transcription factor activity was detected using EMSA and protein expression levels were analyzed using Western blot assays subsequently in EpCs and MPs. Co-cultured EpCs/MPs were found to secrete increased levels of interleukin (IL)-6, IL-1ß, IL-8 and tumor necrosis factor (TNF)-α following LPS exposure for 6, 12, 24 and 48 h compared with either EpC or MP monocultures. Concurrently, NF-κB was revealed to be activated in MPs at 6 and 12 h, and in EpCs at 24 h. NF-κB DNA binding, Toll-like receptor 4 expression levels and the p65 phosphorylation status were also increased, which may contribute to the inflammatory response in the EpC/MP co-cultures. Notably, cytokine levels decreased following the inhibition of NF-κB expression with pyrrolidinedithiocarbamate. In conclusion, the present study successfully established an EpC/MP co-culture system using LPS, which may be a useful model for studying chronic inflammation in vitro.

Three Tiaobu Feishen formulae reduces cigarette smoke-induced inflammation in human airway epithelial cells.

OBJECTIVE: To investigate the therapeutic efficacy of Tiaobu Feishen formulae (TBFS) on cigarette smoke-induced inflammation in vitro using lipopolysaccharide (LPS)-induced and cigarette smoke extract (CSE)-induced NCI-H292 cells. METHODS: We evaluated the inhibitory effects of Bufei Jianpi formula (BJF), Bufei Yishen formula (BYF), and Yiqi Zishen formula (YZF) on the expressions of inflammatory cytokines including tumor necrosis factor (TNF)-α and interleukin (IL)-8, matrix metalloproteinase (MMP)-9, tissue inhibitor of matrix metalloprotease (TIMP)-1, and superoxide dismutase (SOD) in H292 cells stimulated with LPS or CSE. Their related transcription factors and signaling pathways were also analyzed. RESULTS: BJF, BYF, and YZF significantly inhibited the LPS- or CSE-induced expressions of TNF-α, IL-8, MMP-9, TIMP-1, and SOD in H292 cells, and suppressed the activation of transcription factors including nuclear transcription factor (NF)-κB, activator protein (AP)-1, and signal transducers and activators of transcription (STAT) 3 and their corresponding pathways, including NF-κB, mitogen-activated protein kinase (MAPK), STAT3, and peroxisome proliferator-activated receptor (PPAR). CONCLUSION: BJF, BYF, and YZF effectively suppressed inflammatory responses, protease-antiprotease imbalance, and oxidative stress induced by LPS and CSE, an effect that was closely associated with the inhibition of the NF-κB, MAPK, STAT3, and PPAR pathways.

Digoxin improves steatohepatitis with differential involvement of liver cell subsets in mice through inhibition of PKM2 transactivation.

The cardiac glycoside digoxin was identified as a potent suppressor of pyruvate kinase isoform 2-hypoxia-inducible factor-α (PKM2-HIF-1α) pathway activation in liver injury mouse models via intraperitoneal injection. We have assessed the therapeutic effects of digoxin to reduce nonalcoholic steatohepatitis (NASH) by the clinically relevant oral route in mice and analyzed the cellular basis for this effect with differential involvement of liver cell subsets. C57BL/6J male mice were placed on a high-fat diet (HFD) for 10 wk and started concurrently with the gavage of digoxin (2.5, 0.5, 0.125 mg/kg twice a week) for 5 wk. Digoxin significantly reduced HFD-induced hepatic damage, steatosis, and liver inflammation across a wide dosage range. The lowest dose of digoxin (0.125 mg/kg) showed significant protective effects against liver injury and sterile inflammation. Consistently, digoxin attenuated HIF-1α sustained NLRP3 inflammasome activation in macrophages. We have reported for the first time that PKM2 is upregulated in hepatocytes with hepatic steatosis, and digoxin directly improved hepatocyte mitochondrial dysfunction and steatosis. Mechanistically, digoxin directly bound to PKM2 and inhibited PKM2 targeting HIF-1α transactivation without affecting PKM2 enzyme activation. Thus, oral digoxin showed potential to therapeutically inhibit liver injury in NASH through the regulation of PKM2-HIF-1α pathway activation with involvement of multiple cell types. Because of the large clinical experience with oral digoxin, this may have significant clinical applicability in human NASH.NEW & NOTEWORTHY This study is the first to assess the therapeutic efficacy of oral digoxin on nonalcoholic steatohepatitis (NASH) in a high-fat diet (HFD) mouse model and to determine the divergent of cell type-specific effects. Oral digoxin reduced liver damage, steatosis, and inflammation in HFD mice. Digoxin attenuated hypoxia-inducible factor (HIF)-1α axis-sustained inflammasome activity in macrophages and hepatic oxidative stress response in hepatocytes via the regulation of PKM2-HIF-1α axis pathway activation. Oral digoxin may have significant clinical applicability in human NASH.

Three Tiaobu Feishen therapies protect human alveolar epithelial cells against cigarette smoking and tumor necrosis factor--induced inflammation by nuclear factor-kappa B pathway.

OBJECTIVE: To investigate the efficacy of Tiaobu Feishen formulae (TBFS), including Bufei Jianpi formula (BJF), Bufei Yishen formula (BYF), and Yiqi Zishen formula (YZF), on inflammatory response, protease-anti-protease imbalance and collagen deposition in rats. METHODS: In present work, we used an in vitro model of cigarette smoking extract (CSE)- and tumor necrosis factor-α (TNF-α)-induced A549 cells to examine the efficacy of BJF, BYF and YZF on the production of inflammatory cytokines, including TNF-α and interleukin (IL)-8, IL-6, matrix metalloproteinases (MMP)-9, and IL-10 in CSE or TNF-α-induced A549 cells. And their related transcription factors and signaling pathway were also analyzed. RESULTS: The results showed that BJF, BYF and YZF could significantly decrease the expression levels of the pro-inflammatory cytokines induced by CSE or TNF-α. Furthermore, BJF, BYF and YZF could suppress CSE- or TNF-α-induced activation of nuclear factor-kappa B (NF-κB) transcription factors and its corresponding pathways. Taken together, these data implied that BJF, BYF and YZF effectively inhibited CSE- or TNF-α-induced inflammatory response in alveolar epithelial cell, which was due to their inhibition effect on NF-κB pathways. CONCLUSION: Our findings suggest that the Tiaobu Feishen therapies may protect human alveolar epithelial cells against cigarette smoking and TNF-α-induced inflammation. NF-κB pathway may involve in the actions.

A Chinese Herbal Formula Ameliorates Pulmonary Fibrosis by Inhibiting Oxidative Stress via Upregulating Nrf2.

This study aimed to explore the protective effects of a Chinese herbal formula, Jinshui Huanxian formula (JHF), on experimental pulmonary fibrosis and its underlying mechanisms. After being treated with single dose of bleomycin (5 mg/kg) intratracheally, rats were orally administered with JHF and pirfenidone from day 1 to 42, then sacrificed at 7, 14, 28, or 42 days post-bleomycin instillation. JHF ameliorated bleomycin-induced pathological changes, collagen deposition in the rat lung and recovered pulmonary function at different days post-bleomycin instillation. In lungs of JHF-treated rats, the levels of total superoxide dismutase, catalase and glutathione were higher, and myeloperoxidase and methane dicarboxylic aldehyde were lower than those in vehicle-treated rats, respectively. Additionally, JHF inhibited the expression of NADPH oxidase 4 (NOX4) and increased the Nuclear Factor Erythroid 2-Related Factor 2 (Nrf2) in lung tissues. In vitro, JHF and ruscogenin, a compound of Ophiopogonis Radix contained in JHF, significantly inhibited transforming growth factor ß1 (TGF-ß1)-induced differentiation of fibroblasts. Furthermore, JHF markedly decreased the level of reactive oxygen species in TGF-ß1-induced fibroblast. In line with this, upregulation of NAD(P)H: quinone oxidoreductase 1 and heme oxygenase 1, and downregulation of NOX4 were found in JHF-treated fibroblast induced by TGF-ß1. While on the other hand, Nrf2 siRNA could suppress the JHF-mediated inhibition effect on alpha-smooth muscle actin (α-SMA), and FN1 expression induced by TGF-ß1 in fibroblasts. These results indicated that JHF performed remarkably therapeutic and long-term effects on pulmonary fibrosis in rat and suppressed the differentiation of fibroblast into myofibroblast through reducing the oxidative response by upregulating Nrf2 signaling. It might provide a new potential natural drug for the treatment of pulmonary fibrosis.

LPS­induced proinflammatory cytokine expression in human airway epithelial cells and macrophages via NF­κB, STAT3 or AP­1 activation.

Lipopolysaccharide (LPS), the major outer surface membrane component of Gram-negative bacteria, is one of the main etiological factors in the pathogenesis of several lung diseases, such as chronic obstructive pulmonary disease. The respiratory epithelium and the macrophages comprise the dynamic interface between the outside environment and the host response to bacterial infection via cytokine secretion. In the present study, the mechanisms of LPS induced­inflammatory response in human lung cells and macrophages were investigated. The effects of LPS exposure on cytokine production, inflammation­related transcription factors and intracellular signaling pathway activation were assessed in human lung mucoepidermoid carcinoma H292 cells and human macrophage THP­1 cells. The results demonstrated that LPS markedly increased the expression of interleukin (IL)­6, IL­8, tumor necrosis factor (TNF)­α, matrix metallopeptidase (MMP)­9 and tissue inhibitor of metalloproteinases­1 in H292 cells, while it increased the production of IL­6, IL­8 and TNF­α in differentiated THP­1 cells. In addition, LPS exposure activated nuclear factor (NF)­κB and activator protein (AP)­1 signaling in H292 cells, while it activated NF­κB and signal transducer and activator of transcription (STAT) 3 signaling in THP­1 cells. Furthermore, treatment with NF­κB, AP­1 or STAT3 inhibitors significantly decreased the LPS­mediated expression of IL­8 and TNF­α in these cells, suggesting that these pathways might serve crucial roles in LPS­induced cytokine expression. In conclusion, LPS stimulation of H292 and THP­1 cells induced cytokine expression and NF­κB, mitogen­activated protein kinase and Janus kinase/STAT3 pathway activation with subsequent nuclear translocation of NF­κB, AP­1 and STAT3, which demonstrated potential of the use of NF­κB, AP­1 and STAT3 in therapies for conditions and diseases associated with chronic inflammation.