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.

Immobilization of 6-O-α-maltosyl-ß-cyclodextrin on the surface of black phosphorus nanosheets for selective chiral recognition of tyrosine enantiomers.

A novel chiral sensing platform, 6-O-α-maltosyl-ß-cyclodextrin (Mal-ßCD)-based film, is proposed for selective electrochemical recognition of tyrosine (Tyr) enantiomers. Black phosphorus nanosheets (BP NSs) and Mal-ßCD modified glassy carbon electrode (Mal-ßCD/BP NSs/GCE) were prepared by a layer-to-layer drop-casting method, and the platform was easy to fabricate and facile to operate. It is proposed that the amino and hydroxyl groups of the Tyr enantiomers and the chiral hydroxyl groups of Mal-ßCD selectively form intermolecular hydrogen bonds to dominate effective chiral recognition. Two linear equations of Ip (µA) = 11.40 CL-Tyr (mM) + 0.28 (R2 = 0.99147) and Ip (µA) = 7.96 CD-Tyr (mM) + 0.22 (R2 = 0.99583) in the concentration range 0.01-1.00 mM have been obtained. The limits of detection (S/N=3) for L-Tyr and D-Tyr were 4.81 and 6.89 µM, respectively. An interesting phenomenon was that the value of IL-Tyr/ID-Tyr (1.51) in this work was slightly higher than the value of IL-Trp/ID-Trp (1.49) reported in our previous study, where tryptophan (Trp) enantiomers were electrochemically recognized by Nafion (NF)-stabilized BPNSs-G2-ß-CD composite. The two similar sensors fabricated by different methods showed different recognition ability toward either Tyr or Trp enantiomers, and the underlying mechanism was discussed in detail. More importantly, the proposed chiral sensor enables prediction of the percentages of D-Tyr in racemic Tyr mixtures. The chiral sensor may provide a novel approach for the fabrication of novel chiral platforms in the practical detection of L- or D-enantiomer in racemic Tyr mixtures.Graphical abstract.

[Effects of iron supplement intake on gestational diabetes mellitus in early and middle pregnancy in Chengdu City in 2017].

OBJECTIVE: To explore the effect of iron supplement intake on gestational diabetes mellitus(GDM) in early and middle pregnancy. METHODS: From February to April 2017, a prospective study was conducted among 807 early pregnant women in a prenatal clinic of a maternal and child medical institution in Chengdu City through purposive sampling. Data on maternal demographic characteristics was collected through questionnaire in early pregnancy. In early and middle pregnancy, the information of iron supplement intake were collected with questionnaire, 3-day 24 hour dietary recall method was used to assess maternal diet. According to the WHO recommendation, 60 mg/d iron supplementation during pregnancy was used as the dividing point, <60 mg/d iron supplementation was used as the low level group, and ≥60 mg/d iron supplementation was used as the high level group. At the 24 th to 28 th pregnant week, the oral glucose tolerance test(OGTT) was conducted, and GDM was diagnosed according to the Guidelines for the Diagnosis and Treatment of Pregnancy Diabetes in China(2014). Multivariate unconditional Logistic regression model was used to analyze the effect of iron supplement intake on gestational diabetes mellitus(GDM) in early and middle pregnancy. RESULTS: A total of 739 valid samples were followed up, the age was(28. 22±3. 75) years old. In early and middle pregnancy, the rate of taking iron supplementation was 5. 0% and 67. 9%, 3. 8% and 47. 1% of them iron supplement intake was more than 60 mg/d. After adjustmenting for body mass index, age, dietary iron, etc. Multivariate unconditional Logistic regression analysis showed that there was a positive correlation between the average intake of iron supplement and the occurrence of GDM in women during the second trimester of pregnancy(OR=1. 059, 95%CI 1. 016-1. 104). Compared with the lower iron supplement intake(<60 mg/d) women in midpregnancy, the risk of GDM was 1. 406 times(95%CI 1. 019-1. 939)in the higher iron supplement intake(≥60 mg/d) women. No correlation was found between iron intake in early pregnancy and the occurrence of GDM. CONCLUSION: Iron supplement intake during pregnancy may increase the risk of GDM. Appropriate intake of iron supplement for pregnant women is worth discussing.

A Small-Molecule Compound Selectively Activates K2P Channel TASK-3 by Acting at Two Distant Clusters of Residues.

The TASK-3 channel is a member of the K2P family that is important for the maintenance of the resting membrane potential. Previous studies have demonstrated that the TASK-3 channel is involved in several physiologic and pathologic processes, including sleep/wake control, cognition, and epilepsy. However, there is still a lack of selective pharmacological tools for TASK-3, which limits further research on channel function. In this work, using a high-throughput screen, we discovered that N-(2-((4-nitro-2-(trifluoromethyl)phenyl)amino)ethyl)benzamide (NPBA) showed excellent potency and selectivity as a novel TASK-3 activator. The molecular determinants of NPBA activation were then investigated by combining chimera and mutagenesis analysis. Two distant clusters of residues located at the extracellular end of the second transmembrane domain (A105 and A108) and the intracellular end of the third transmembrane domain (E157) were found to be critical for NPBA activation. We then compared the essentials of the actions of NPBA with inhalation anesthetics that nonselectively activate TASK-3 and found that they may activate TASK-3 channels through different mechanisms. Finally, we transplanted the three residues A105, A108, and E157 into the TASK-1 channel, which resists NPBA activation, and the constructed mutant TASK-1(G105A, V108A, A157E) showed dramatically increased activation by NPBA, confirming the importance of these two distant clusters of residues. SIGNIFICANCE STATEMENT: TASK-3 channels conduct potassium and are involved in various physiological and pathological processes. However, the lack of selective modulators has hindered efforts to increase our understanding of the physiological roles of TASK-3 channels. By using a high-throughput screen, we identified NPBA as a potent and selective TASK-3 activator, and we show that NPBA is a more potent activator than terbinafine, the only reported TASK-3 selective activator to date. We also show here that NPBA has outstanding selectivity for TAS-3 channels. These characteristics make NPBA a promising pharmacological probe for research focused on defining TASK-3 channel function(s). In addition, we identified two distant clusters of residues as determinants of NPBA activation providing new molecular clues for the understanding of the gating mechanism of K2P channels.

Melatonin receptor activation provides cerebral protection after traumatic brain injury by mitigating oxidative stress and inflammation via the Nrf2 signaling pathway.

Traumatic brain injury (TBI) is a principal cause of death and disability worldwide. Melatonin, a hormone made by the pineal gland, is known to have anti-inflammatory and antioxidant properties. In this study, using a weight-drop model of TBI, we investigated the protective effects of ramelteon, a melatonin MT1/MT2 receptor agonist, and its underlying mechanisms of action. Administration of ramelteon (10 mg/kg) daily at 10:00 a.m. alleviated TBI-induced early brain damage on day 3 and long-term neurobehavioral deficits on day 28 in C57BL/6 mice. Ramelteon also increased the protein levels of interleukin (IL)-10, IL-4, superoxide dismutase (SOD), glutathione, and glutathione peroxidase and reduced the protein levels of IL-1ß, tumor necrosis factor, and malondialdehyde in brain tissue and serum on days 1, 3, and 7 post-TBI. Similarly, ramelteon attenuated microglial and astrocyte activation in the perilesional cortex on day 3. Furthermore, ramelteon decreased Keap 1 expression, promoted nuclear factor erythroid 2-related factor 2 (Nrf2) nuclear accumulation, and increased levels of downstream proteins, including SOD-1, heme oxygenase-1, and NQO1 on day 3 post-TBI. However, in Nrf2 knockout mice with TBI, ramelteon did not decrease the lesion volume, neuronal degeneration, or myelin loss on day 3; nor did it mitigate depression-like behavior or most motor behavior deficits on day 28. Thus, timed ramelteon treatment appears to prevent inflammation and oxidative stress via the Nrf2-antioxidant response element pathway and might represent a potential chronotherapeutic strategy for treating TBI.

Identification and application of anti-inflammatory compounds screening system based on RAW264.7 cells stably expressing NF-κB-dependent SEAP reporter gene.

BACKGROUND: NF-κB is one of the key transcription factors in the inflammatory response, transactivates a series of pro-inflammatory genes and is therefore regarded as an important target for anti-inflammatory drug screening. METHOD: We recombined the reporter gene vector with inserting the "neo" transcript into the vector pNF-κB-SEAP, made the reporter gene vector stable in a eukaryotic cell line. The recombinant reporter gene vector we named pNF-κB-SEAP-Neo was transfected into RAW264.7. We selected the transfected RAW264.7 cell line with G418 for 15 days and then get RAW264.7 cells stably expressing NF-κB-dependent SEAP named as RAW264.7-pNF-κB-SEAP cells. We treated the RAW264.7-pNF-κB-SEAP cells with NF-κB agonists as LPS, PolyI:C and TNF-α, NF-κB inhibitor as PDTC and BAY117085, in different concentrations and time points and tested the expression of the SEAP, constructed the drug screening system on the base of the RAW264.7-pNF-κB-SEAP cell line. 130 chemicals were screened with the drug screening system we constructed and one of these chemicals numbered w10 was found could inhibit the NF-κB significantly. At last, we verified the inhibition of w10 to expression of genes promoted with NF-κB in HepG2 and Hela, and to migration of Hela. RESULT: In this study, we established a drug screening system based on RAW264.7 cells that stably expressed the NF-κB-dependent, SEAP reporter gene. To develop a standard method for drug screening using this reporter-gene cell line, the test approach of SEAP was optimized and basic conditions for drug screening were chosen. This included the initial cell number inoculated in a 96-well plate, the optimum agonist, inhibitor of NF-κB pathway and their concentrations during screening. Subsequently, 130 newly synthesized compounds were screened using the stable reporter-gene cell line. The anti-inflammatory effects of the candidate compounds obtained were further verified in 2 cancer cell lines. The results indicated that compound W10 (methyl 4-(4-(prop-2-yn-1-ylcarbamoyl) phenylcarbamoyl) benzoate) significantly inhibited SEAP production under the screening conditions. Further results confirmed that the precursor compound significantly inhibited the transcription of NF-κB target genes. CONCLUSION: In conclusion, RAW264.7 cells, stably expressing the NF-κB-dependent SEAP-reporter gene, may provide a new, feasible, and efficient cellular drug-screening system.

Liver transcriptome response to hyperthermic stress in three distinct chicken lines.

BACKGROUND: High ambient temperatures cause stress in poultry, especially for broiler lines, which are genetically selected for rapid muscle growth. RNA-seq technology provides powerful insights into environmental response from a highly metabolic tissue, the liver. We investigated the effects of acute (3 h, 35 °C) and chronic (7d of 35 °C for 7 h/d) heat stress on the liver transcriptome of 3-week-old chicks of a heat-susceptible broiler line, a heat-resistant Fayoumi line, and their advanced intercross line (AIL). RESULTS: Transcriptome sequencing of 48 male chickens using Illumina HiSeq 2500 technology yielded an average of 33.9 million, 100 base-pair, single-end reads per sample. There were 8 times more differentially expressed genes (DEGs) (FDR < 0.05) in broilers (n = 627) than Fayoumis (n = 78) when comparing the acute-heat samples to the control (25 °C) samples. Contrasting genetic lines under similar heat treatments, the highest number of DEGs appeared between Fayoumi and broiler lines. Principal component analysis of gene expression and analysis of the number of DEGs suggested that the AIL had a transcriptomic response more similar to the Fayoumi than the broiler line during acute heat stress. The number of DEGs also suggested that acute heat stress had greater impact on the broiler liver transcriptome than chronic heat stress. The angiopoietin-like 4 (ANGPTL4) gene was identified as differentially expressed among all 6 contrasts. Ingenuity Pathway Analysis (IPA) created a novel network that combines the heat shock protein family with immune response genes. CONCLUSIONS: This study extends our understanding of the liver transcriptome response to different heat exposure treatments in distinct genetic chicken lines and provides information necessary for breeding birds to be more resilient to the negative impacts of heat. The data strongly suggest ANGPTL4 as a candidate gene for improvement of heat tolerance in chickens.

Effect of Dendrobium officinale Extraction on Gastric Carcinogenesis in Rats.

Dendrobium officinale (Tie Pi Shi Hu in Chinese) has been widely used to treat different diseases in China. Anticancer effect is one of the important effects of Dendrobium officinale. However, the molecular mechanism of its anticancer effect remains unclear. In the present study, gastric carcinogenesis in rats was used to evaluate the effect of Dendrobium officinale on cancer, and its pharmacological mechanism was explored. Dendrobium officinale extracts (4.8 and 2.4 g/kg) were orally administered to the rats of the gastric carcinogenesis model. Compared with the cancer model group, the high dose of Dendrobium officinale extracts significantly inhibited the rate of carcinogenesis. Further analysis revealed that Dendrobium officinale extracts could regulate the DNA damage, oxidative stress, and cytokines related with carcinogenesis and induce cell apoptosis in order to prevent gastric cancer.

The Natural Flavonoid Pinocembrin: Molecular Targets and Potential Therapeutic Applications.

Pinocembrin is a natural flavonoid compound extracted from honey, propolis, ginger roots, wild marjoram, and other plants. In preclinical studies, it has shown anti-inflammatory and neuroprotective effects as well as the ability to reduce reactive oxygen species, protect the blood-brain barrier, modulate mitochondrial function, and regulate apoptosis. Considering these pharmaceutical characteristics, pinocembrin has potential as a drug to treat ischemic stroke and other clinical conditions. In this review, we summarize its pharmacologic characteristics and discuss its mechanisms of action and potential therapeutic applications.

Upregulated protein arginine methyltransferase 1 by IL-4 increases eotaxin-1 expression in airway epithelial cells and participates in antigen-induced pulmonary inflammation in rats.

Protein arginine methyltransferases (PRMTs), catalyzing methylation of both histones and other cellular proteins, have emerged as key regulators of various cellular processes. This study aimed to identify key PRMTs involved in Ag-induced pulmonary inflammation (AIPI), a rat model for asthma, and to explore the role of PRMT1 in the IL-4-induced eosinophil infiltration process. E3 rats were i.p. sensitized with OVA/alum and intranasally challenged with OVA to induce AIPI. The expressions of PRMT1-6, eotaxin-1, and CCR3 in lungs were screened by real-time quantitative PCR. Arginine methyltransferase inhibitor 1 (AMI-1, a pan-PRMT inhibitor) and small interfering RNA-PRMT1 were used to interrupt the function of PRMT1 in A549 cells. In addition, AMI-1 was administrated intranasally to AIPI rats to observe the effects on inflammatory parameters. The results showed that PRMT1 expression was mainly expressed in bronchus and alveolus epithelium and significantly upregulated in lungs from AIPI rats. The inhibition of PRMTs by AMI-1 and the knockdown of PRMT1 expression were able to downregulate the expressions of eotaxin-1 and CCR3 with the IL-4 stimulation in the epithelial cells. Furthermore, AMI-1 administration to AIPI rats can also ameliorate pulmonary inflammation, reduce IL-4 production and humoral immune response, and abrogate eosinophil infiltration into the lungs. In summary, PRMT1 expression is upregulated in AIPI rat lungs and can be stimulated by IL-4. Intervention of PRMT1 activity can abrogate IL-4-dependent eotaxin-1 production to influence the pulmonary inflammation with eosinophil infiltration. The findings may provide experimental evidence that PRMT1 plays an important role in asthma pathogenesis.

A novel naturally occurring salicylic acid analogue acts as an anti-inflammatory agent by inhibiting nuclear factor-kappaB activity in RAW264.7 macrophages.

Methyl salicylate 2-O-ß-D-lactoside (DL0309), is a molecule chemically related to salicylic acid that is isolated from Gaultheria yunnanensis (FRANCH.) REHDER (G. yunnanensis). G. yunnanensis, a traditional Chinese herbal medicine, is widely used for treating rheumatoid arthritis, swelling, pain, trauma, and chronic tracheitis. In the present study, we explored the mechanism whereby DL0309 exerts anti-inflammatory effects, using the model of lipopolysaccharide (LPS)-treated RAW264.7 cells. We examined the effects of DL0309 on LPS-induced nuclear factor-kappaB (NF-κB) activity by Western blot analysis, cell imaging analysis and an electrophoretic mobility shift assay (EMSA). Production of pro-inflammatory cytokines was also measured. Our observations indicate that DL0309 suppressed production of nitric oxide (NO), reactive oxygen species (ROS) and the pro-inflammatory cytokines, such as tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-1ß (IL-1ß), in a concentration-dependent manner. The phosphorylation of IKK-ß and degradation of IκB-α by LPS were both inhibited by DL0309 in the cytoplasm. The increased protein level of NF-κB by LPS in the nucleus was also reduced by DL0309. Consistent with these results, we found that DL0309 prevents the nuclear translocation and DNA binding activity of NF-κB. Finally, our results demonstrate that DL0309 exerts anti-inflammatory effects, by inhibiting the production of pro-inflammatory cytokines and suppressing of the activation of the NF-κB signaling pathway in LPS-treated macrophage cells. Therefore, DL0309 may have therapeutic potential for treating inflammatory diseases by regulating the NF-κB pathway and pro-inflammatory cytokine production.

Luteolin isolated from the medicinal plant Elsholtzia rugulosa (Labiatae) prevents copper-mediated toxicity in ß-amyloid precursor protein Swedish mutation overexpressing SH-SY5Y cells.

Luteolin, a 3',4',5,7-tetrahydroxyflavone, is a plant flavonoid and pharmacologically active agent that has been isolated from several plant species. In the present study, the effects of luteolin obtained from the medicinal plant Elsholtzia rugulosa and the related mechanisms were examined in an Alzheimer's disease (AD) cell model. In this model, copper was used to exacerbate the neurotoxicity in ß-amyloid precursor protein Swedish mutation stably overexpressed SH-SY5Y cells (named "APPsw cells" for short). Based on this model, we demonstrated that luteolin increased cell viability, reduced intracellular ROS generation, enhanced the activity of SOD and reversed mitochondrial membrane potential dissipation. Inhibition of caspase-related apoptosis was consistently involved in the neuroprotection afforded by luteolin. Furthermore, it down-regulated the expression of AßPP and lowered the secretion of Aß1₋42. These results indicated that luteolin from the Elsholtzia rugulosa exerted neroprotective effects through mechanisms that decrease AßPP expression, lower Aß secretion, regulate the redox imbalance, preserve mitochondrial function, and depress the caspase family-related apoptosis.