Continuous positive airway pressure affects mitochondrial function and exhaled PGC1-α levels in obstructive sleep apnea.

Purpose: Subjects with obstructive sleep apnea (OSA) exhibit systemic and upper airway oxidative stress and inflammation, which cause mitochondrial dysfunction. The intend of this study is to estimate mitochondrial function (mitochondrial DNA/nuclear DNA [Mt/N] ratio) and protein levels of peroxisome proliferator-coactivated receptor gamma co-activator 1-alpha (PGC1-α) in the exhaled breath condensate (EBC) and plasma before and after continuous positive airway pressure (CPAP) treatment. Materials and methods: Twenty healthy individuals (control) and 40 subjects with severe or moderate OSA were recruited to undergo CPAP treatment and evaluation in a sleep study. The Mt/N ratio in the EBC and blood were assayed by quantitative real-time polymerase chain reaction. Enzyme-linked immunosorbent assay was used to measure the protein concentration of PGC1-α in the EBC and plasma. All experiments were performed after 3 months of CPAP treatment in subjects with OSA. Results: We observed no noteworthy differences between the control and treatment groups. Moreover, there were no differences in the Mt/N ratio in the blood and plasma levels of PGC1-α in subjects with OSA before and after treatment. However, the Mt/N ratio and protein levels of PGC1-α in the EBC of OSA subjects were higher than those in the control group and returned to normal levels after CPAP treatment. Conclusions: We successfully treated subjects with OSA by CPAP, which restored the Mt/N ratio and levels of PGC1-α in the EBC.

Mitochondrial replication, transcription, and function in obstructive sleep apnea.

We assessed mitochondrial replication, transcription, and function in the upper airways of obstructive sleep apnea (OSA) patients and the effects of uvulopalatopharyngoplasty. Twenty subjects with mild and 40 with moderate to severe OSA requiring uvulopalatopharyngoplasty were included. Mitochondrial transcription factor A (TFAM) and peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) in uvula specimens were assessed by immunohistochemical staining, and their mRNA and protein expression was examined using reverse-transcription polymerase chain reaction and western blotting, respectively. The mitochondrial to nuclear DNA (Mt/N) ratio in the blood, exhaled breath condensate (EBC), and uvula was measured using quantitative reverse-transcription polymerase chain reaction. TFAM and PGC-1α protein concentrations in the plasma and EBC were determined using enzyme-linked immunosorbent assay. All tested parameters were higher in the OSA group than in the control. Three months later, 21 uvulopalatopharyngoplasty-responsive patients with OSA showed decreased TFAM and PGC-1α concentrations and EBC Mt/N ratio while these remained high in 19 uvulopalatopharyngoplasty-unresponsive patients. The OSA group showed severe inflammation, increased mitochondrial replication and transcription-related signaling, and mitochondrial dysfunction in the uvula. Successful OSA treatment using uvulopalatopharyngoplasty restored the TFAM and PGC-1α levels and EBC Mt/N ratio.

[Effects of ligustrazine on the mitochondrial structure and functions in the process myocardial hypertrophy].

OBJECTIVE: To explore changes of mitochondrial structure and functions, as well as the protection of ligustrazine in the process of myocardial hypertrophy. METHODS: Neonatal myocardial cells were isolated and cultured with angiotensin II (Ang II) for 72 or 96 h. The total protein content was detected using BCA method. The cell diameter was measured by inverted microscope, by which to reflect the proliferation situation of cardiomyocytes. The mitochondrial membrane potential (MMP) was measured by fluorescence microscope. The mitochondrial monoamine oxidase (MAO) activity was detected by spectrophotometer. The mitochondrial cytochrome oxidase (COX) activity and the mitochondrial damage percentage were detected by microplate reader, by which to reflect the damage of mitochondrial outer membrane's structure and the membranes' function. Also, cells were treated with ligustrazine and losartan and then the pharmacological effects on the mitochondrial structure and functions in the myocardial cells treated with Ang II were observed. RESULTS: At 72 h and 96 h, when compared with the blank group, cells treated with Ang II had increased total protein content (P < 0.01) and enlarged diameter (P < 0.01). Treated with Ang II, the MAO activity and the outer membrane damage percentage of myocardial cells significantly increased (P < 0.01), and mitochondrial COX activity and the mitochondrial MMP significantly decreased (P < 0.01). Compared with the model group at the same time period, ligustrazine significantly reduced myocardial cells' total protein content and myocardial cell diameter, and significantly decreased myocardial cells' MAO activity, increased mitochondrial COX activity, improved the outer membrane damage percentage and inner membrane MMP at 72 and 96 h, all showing statistical difference (P < 0.01, P < 0.05). CONCLUSIONS: During the process of myocardial hypertrophy existed the damage to the mitochondrial structure and functions. Ligustrazine protected the mitochondrial structure and functions of the myocardial cells in reversing Ang II induced myocardial cell hypertrophy.

Molecular evolution of cystine-stabilized miniproteins as stable proteinaceous binders.

Small cystine-stabilized proteins are desirable scaffolds for therapeutics and diagnostics. Specific folding and binding properties of the proteinaceous binders can be engineered with combinatorial protein libraries in connection with artificial molecular evolution. The combinatorial protein libraries are composed of scaffold variants with random sequence variation, which inevitably produces a portion of the library sequences incompatible with the parent structure. Here, we used artificial molecular evolution to elucidate structure-determining residues in a smallest cystine-stabilized scaffold. The structural determinant information was then applied to designing cystine-stabilized miniproteins binding to human vascular endothelial growth factor. This work demonstrated a general methodology on engineering artificial cystine-stabilized proteins as antibody mimetics with simultaneously enhanced folding and binding properties.

Global Analysis the Potential Medicinal Substances of Shuangxia Decoction and the Process In Vivo via Mass Spectrometry Technology.

Shuangxia decoction is an effective traditional Chinese medicine formula for treating insomnia. Up to now, there has not been any report about the effective substances. An omics data processing method based on mass spectrometry technology is used to explore the chemical composition changes of Shuangxia decoction, the components absorbed into the blood and brain, and to explore the anti-insomnia mechanism based on molecular docking technology. Forty-nine chemical components in Shuangxia decoction have been identified, and 51 new components generated by co-decoction have been discovered. It was found that 7,404 compounds of Shuangxia decoction were absorbed into the blood. Forty kinds of known compounds were quickly identified, and 15 new compounds generated by co-decoction were also found to be absorbed into the blood. By using UPLC-MS/MS method, it was confirmed that 10 compounds were absorbed into the blood and 9 compounds were absorbed into the brain. Furthermore, it is found that rosmarinic acid is mainly distributed in the hypothalamus and striatum, and caffeic acid is mainly distributed in the hypothalamus, striatum, and hippocampus. Molecular docking results showed rosmarinic acid, danshensu, and HMLA with GABAA receptor have excellent binding characteristics, even surpassing the proligand. Danshensu and HMLA with dopamine D2 receptor also showed good binding energy. Our findings will help to further confirm the mechanism of Shuangxia decoction for relieving insomnia, and we also establish a novel data processing method for supplementing the mechanism of the efficacy of other traditional Chinese medicine formula.

Effect of high-frequency low-intensity pulsed electric field on protecting SH-SY5Y cells against hydrogen peroxide and ß-amyloid-induced cell injury via ERK pathway.

As the most common type of neurodegenerative diseases (NDDs), Alzheimer's disease (AD) is thought to be caused mainly by the excessive aggregation of ß-amyloid protein (Aß). However, a growing number of studies have found that reactive oxygen species (ROS) play a key role in the onset and progression of AD. The present study aimed to probe the neuroprotective effect of high-frequency low-intensity pulsed electric field (H-LIPEF) for SH-SY5Y cells against hydrogen peroxide (H2O2) and Aß-induced cytotoxicity. By looking in a systematic way into the frequency- and amplitude-dependent neuroprotective effect of pulsed electric field (PEF), the study finds that H-LIPEF at 200 Hz produces the optimal protective effect for SH-SY5Y cells. The underlying mechanisms were confirmed to be due to the activation of extracellular signal-regulated kinase (ERK) pathway and the downstream prosurvival and antioxidant proteins. Because the electric field can be modified to focus on specific area in a non-contact manner, the study suggests that H-LIPEF holds great potential for treating NDDs, whose effect can be further augmented with the administering of drugs or natural compounds at the same time.

Effects of continuous positive airway pressure on resolvin and matrix metalloproteinase-9 in patients with obstructive sleep apnea.

PURPOSE: Resolvin is a checkpoint controller in inflammation. Matrix metalloproteinase-9 (MMP-9) is an airway remodeling regulator. We evaluated the levels of resolvin and MMP-9 protein in the serum and exhaled breath condensate (EBC) before and after continuous positive airway pressure (CPAP) treatment. METHOD: We enrolled 20 non-OSA snorers and 40 patients with moderate to severe OSA scheduled for CPAP treatment. ELISA was used to assess resolvin and MMP-9 levels in the serum and EBC. All patients underwent sleep assessment at baseline and 3 months after CPAP. RESULTS: There was no between-group difference; moreover, there were no differences in the pre- and post-treatment serum levels of resolvin and MMP-9 in patients with OSA. Compared with non-OSA snorers, patients with OSA had lower resolvin and higher MMP-9 levels in the EBC. After CPAP treatment, the EBC levels of resolvin and MMP-9 in patients with OSA returned to normal. CONCLUSIONS: Successful OSA treatment by CPAP can normalize EBC levels of resolvin and MMP-9.

Xinglou Chengqi Decoction improves neurological function in experimental stroke mice as evidenced by gut microbiota analysis and network pharmacology.

The current study was designed to explore the brain protection mechanism of Xinglou Chengqi Decoction (XCD) based on gut microbiota analysis and network pharmacology. A transient middle cerebral artery occlusion (MCAO) model of mice was established, followed by behavioral evaluation, TTC and TUNEL staining. Additionally, to investigate the effects of gut microbiota on neurological function after stroke, C57BL/6 mice were treated with anti-biotic cocktails 14 days prior to ischemic stroke (IS) to deplete the gut microbiota. High-throughput 16S rDNA gene sequencing, metabonomics technique, and flow multifactor technology were used to analyze bacterial communities, SCFAs and inflammatory cytokines respectively. Finally, as a supplement, network pharmacology and molecular docking were applied to fully explore the multicomponent-multitarget-multichannel mechanism of XCD in treating IS, implicated in ADME screening, target identification, network analysis, functional annotation, and pathway enrichment analysis. We found that XCD effectively improved neurological function, relieved cerebral infarction and decreased the neuronal apoptosis. Moreover, XCD promoted the release of anti-inflammatory factor like IL-10, while down-regulating pro-inflammatory factors such as TNF-α, IL-17A, and IL-22. Furthermore, XCD significantly increased the levels of short chain fatty acids (SCFAs), especially butyric acid. The mechanism might be related to the regulation of SCFAs-producing bacteria like Verrucomicrobia and Akkermansia, and bacteria that regulate inflammation like Paraprevotella, Roseburia, Streptophyta and Enterococcu. Finally, in the network pharmacological analysis, 51 active compounds in XCD and 44 intersection targets of IS and XCD were selected. As a validation, components in XCD docked well with key targets. It was obviously that biological processes were mainly involved in the regulation of apoptotic process, inflammatory response, response to fatty acid, and regulation of establishment of endothelial barrier in GO enrichment. XCD can improve neurological function in experimental stroke mice, partly due to the regulation of gut microbiota. Besises, XCD has the characteristic of "multi-component, multi-target and multi-channel" in the treatment of IS revealed by network pharmacology and molecular docking.

Thermal cycling protects SH-SY5Y cells against hydrogen peroxide and ß-amyloid-induced cell injury through stress response mechanisms involving Akt pathway.

Neurodegenerative diseases (NDDs) are becoming a major threat to public health, according to the World Health Organization (WHO). The most common form of NDDs is Alzheimer's disease (AD), boasting 60-70% share. Although some debates still exist, excessive aggregation of ß-amyloid protein (Aß) and neurofibrillary tangles has been deemed one of the major causes for the pathogenesis of AD. A growing number of evidences from studies, however, have suggested that reactive oxygen species (ROS) also play a key role in the onset and progression of AD. Although scientists have had some understanding of the pathogenesis of AD, the disease still cannot be cured, with existing treatment only capable of providing a temporary relief at best, partly due to the obstacle of blood-brain barrier (BBB). The study was aimed to ascertain the neuroprotective effect of thermal cycle hyperthermia (TC-HT) against hydrogen peroxide (H2O2) and Aß-induced cytotoxicity in SH-SY5Y cells. Treating cells with this physical stimulation beforehand significantly improved the cell viability and decreased the ROS content. The underlying mechanisms may be due to the activation of Akt pathway and the downstream antioxidant and prosurvival proteins. The findings manifest significant potential of TC-HT in neuroprotection, via inhibition of oxidative stress and cell apoptosis. It is believed that coupled with the use of drugs or natural compounds, this methodology can be even more effective in treating NDDs.

Bidirectional regulation of i-type lysozyme on cutaneous wound healing.

OBJECTIVE: This study aimed to assess the effect and mechanism of i-type lysozyme on cutaneous wound healing animal model and Multiple cell models both in vivo and in vitro. METHODS: Therefore, to evaluate its regenerative efficacy on wound healing process, we daily applied i-type lysozyme on murine full-thickness excisional wounds. After sacrifice on indicated days, skin tissues around surgical defects were harvested and assessed for re-epithelialization, granulation tissue formation, neovascularization and remodeling. To elucidate the underlying mechanisms, i-type lysozyme was analyzed for its tissue regenerative potency on the proliferation, invasion, migration and tube formation against keratinocytes, fibroblasts and endothelial cells. Antioxidant and antimicrobial experiments were also conducted to elucidate protective ability of i-type lysozyme to wound bed. RESULTS: It displayed excellent bi-directional regulation in wound repair, with significant acceleration of epidermal and dermal regeneration as well as the efficient attenuation of excessive collagen deposition and fibrosis in the surgical lesion. I-type lysozyme treatment augmented the proliferation and migration of HaCaT, NIH 3T3 and HUVECs, enhanced the invasion of HaCaT and HUVECs as well as accelerated tube formation of HUVECs. Additionally, it significantly recovered the proliferation of H2O2-damaged cells, whereas represented no microbicidal effect under effective concentration of wound healing. CONCLUSION: Our findings demonstrate the bi-directional regulation of i-type lysozyme in wound healing process through promoting tissue regeneration while hampering scar formation, implying that it is a promising therapeutic agent for wound repair.

Protease substrate site predictors derived from machine learning on multilevel substrate phage display data.

MOTIVATION: Regulatory proteases modulate proteomic dynamics with a spectrum of specificities against substrate proteins. Predictions of the substrate sites in a proteome for the proteases would facilitate understanding the biological functions of the proteases. High-throughput experiments could generate suitable datasets for machine learning to grasp complex relationships between the substrate sequences and the enzymatic specificities. But the capability in predicting protease substrate sites by integrating the machine learning algorithms with the experimental methodology has yet to be demonstrated. RESULTS: Factor Xa, a key regulatory protease in the blood coagulation system, was used as model system, for which effective substrate site predictors were developed and benchmarked. The predictors were derived from bootstrap aggregation (machine learning) algorithms trained with data obtained from multilevel substrate phage display experiments. The experimental sampling and computational learning on substrate specificities can be generalized to proteases for which the active forms are available for the in vitro experiments. AVAILABILITY: http://asqa.iis.sinica.edu.tw/fXaWeb/

Thermal cycling as a novel thermal therapy to synergistically enhance the anticancer effect of propolis on PANC­1 cells.

Hyperthermia (HT) has shown potential in cancer therapy. In particular, it appears to sensitize cancer cells to chemotherapy. However, a major concern associated with HT is that the thermal dosage applied to the tumor cells may also harm the normal tissue cells. Besides, the drugs used in HT are conventional chemotherapy drugs, which may cause serious side effects. The present study demonstrated a novel methodology in HT therapy called thermal cycle (TC)­HT. With this strategy, a therapeutic window with a maximum synergistic effect was created by combining TC­HT with natural compounds, with minimal unwanted cell damage. The natural compound propolis was selected, and the synergistic anticancer effect of TC­HT and propolis was investigated in pancreatic cancer cells. The present results demonstrated for the first time that TC­HT could enhance the anticancer effect of propolis on PANC­1 cancer cells through the mitochondria­dependent apoptosis pathway and cell cycle arrest. Combined treatment greatly suppressed mitochondrial membrane potential, which is an important indicator of damaged and dysfunctional mitochondria. Furthermore, the cell cycle­regulating protein cell division cycle protein 2 was downregulated upon combined treatment, which prevented cellular progression into mitosis. The present study offers the first report, to the best of our knowledge, on the combination of TC­HT with a natural compound for pancreatic cancer treatment. It is anticipated that this methodology may be a starting point for more sophisticated cancer treatments and may thereby improve the quality of life of many patients with cancer.

[Non-apoptotic programmed cell death induced by extract of Spatholobus suberctus in human lung cancer A549 cells].

OBJECTIVE: The goal of study is to explore the cytotoxic activity and its underlying mechanisms of the extract of Spatholobus suberctus in human lung cancer A549 cells. METHOD: The inhibitory effects of the extract on proliferation of human lung cancer cell line A549 was measured by MTT cell viability assay and growth curve. Cell cycle was analyzed using flow cytometry. Apoptotic morphological changes was observed by HE staining technique and AO/PI double-staining confocal laser scanning microscope (CLSM). Employing agarose gel electrophoresis and Annexin V-PI assay, we examed the presence of cytoplasmic histone-associated DNA fragments, and membrane phosphatidylserine (PS) externalization as well as caspase-3 activation. RESULT: The extract of S. suberctus shows strong cytotoxic power on A549 cells during 24 hours and IC50 is 25.54 mg x L(-1). The cells in S-phase increase while the cells in G0-G1 and G2-M decrease. These changes recovered after 48 hours. The nucleus became pyknosis between 8 to 12 hours and many vacuoles and granules in cytoplasm can be seen. Membrane phosphatidylserine externalization occurs in a dose-dependent and time-dependent manner afer 12 hours. Caspase-3 activity has no more changes in a converse dose-dependent manner. No cytoplasmic histone-associated DNA fragments was detected by agarose gel electrophoresis. CONCLUSION: The extract of S. suberctus shows a direct anti-tumor activity. The drug acts quickly and causes S delay in one cell cycle. The main cell death feature appears to be non-apoptotic programmed cell death.

Pharmacodynamic study on insomnia-curing effects of Shuangxia Decoction in Drosophila melanogaster.

The present study aimed to establish a pharmacodynamic method using the pySolo software to explore the influence of freeze-dried powders of Shuangxia Decoction (SXD) on the sleep of normal Drosophila melanogaster and the Drosophila melanogaster whose sleep was divested by light. The dose-effect and the time-effect relationships of SXD on sleep were examined. The effect-onset concentration of SXD was 0.25%, the plateau appeared at the concentration of 2.5% and the total sleep time showed a downtrend when the concentration was greater than 2.5%. The sleep time was the longest on the fourth day after SXD was given. The fruit fly sleep deprivation model was repeated by light stimulation at night. The middle dosage group (2.5%) had the best insomnia-curing effect. In conclusion, using the pySolo software, an approach for the pharmacodynamics study was established with Drosophila melanogaster as a model organism to determine the insomnia-curing effects of the traditional Chinese medicine (TCM). Our results demonstrated the reliability of this method. The freeze-dried powders of SXD could effectively improve the sleep quality of Drosophila melanogaster.

[Determination of 2,3,5,4'-tetrahydroxystilbene-2-O-beta-D-glucoside in yangan oral liquid by HPLC].

OBJECTIVE: To establish the determination method of 2,3,5,4'-tetrahydroxystilbene-2-O-beta-D-glucoside in Yangan Oral Liquid. METHOD: 2,3,5,4'-tetrahydroxystilbene-2-O-beta-D-glucoside was determined by HPLC on C18 column, in acetonitrile-water (14:86) solution as a mobile phase, detection wavelength as 320 nm. RESULT: This method showed a good linear relationship. The average recovery was 99.05% and RSD was 1.31%. CONCLUSION: The method was simple with styong specificity and good repriducibility and can be used for quality control for Yangan Oral Liquid.

Factor Xa active site substrate specificity with substrate phage display and computational molecular modeling.

Structural origin of substrate-enzyme recognition remains incompletely understood. In the model enzyme system of serine protease, canonical anti-parallel beta-structure substrate-enzyme complex is the predominant hypothesis for the substrate-enzyme interaction at the atomic level. We used factor Xa (fXa), a key serine protease of the coagulation system, as a model enzyme to test the canonical conformation hypothesis. More than 160 fXa-cleavable substrate phage variants were experimentally selected from three designed substrate phage display libraries. These substrate phage variants were sequenced and their specificities to the model enzyme were quantified with quantitative enzyme-linked immunosorbent assay for substrate phage-enzyme reaction kinetics. At least three substrate-enzyme recognition modes emerged from the experimental data as necessary to account for the sequence-dependent specificity of the model enzyme. Computational molecular models were constructed, with both energetics and pharmacophore criteria, for the substrate-enzyme complexes of several of the representative substrate peptide sequences. In contrast to the canonical conformation hypothesis, the binding modes of the substrates to the model enzyme varied according to the substrate peptide sequence, indicating that an ensemble of binding modes underlay the observed specificity of the model serine protease.