Evaluation of Oxygen Consumption Rates In Situ.

An analysis of the mitochondrial respiration function represented by the oxygen consumption rate is necessary to assess mitochondrial bioenergetics and redox function. This protocol describes two alternative techniques to evaluate mitochondrial respiration function in situ: (1) measure oxygen consumption rates via an electrode; (2) measure oxygen consumption rates via a seahorse instrument. These in situ approaches provide more physiological access to mitochondria to evaluate mitochondrial respiration function in a relatively integrated cellular system.

Thrombospondin 1 and Reelin act through Vldlr to regulate cardiac growth and repair.

Adult mammalian cardiomyocytes have minimal cell cycle capacity, which leads to poor regeneration after cardiac injury such as myocardial infarction. Many positive regulators of cardiomyocyte cell cycle and cardioprotective signals have been identified, but extracellular signals that suppress cardiomyocyte proliferation are poorly understood. We profiled receptors enriched in postnatal cardiomyocytes, and found that very-low-density-lipoprotein receptor (Vldlr) inhibits neonatal cardiomyocyte cell cycle. Paradoxically, Reelin, the well-known Vldlr ligand, expressed in cardiac Schwann cells and lymphatic endothelial cells, promotes neonatal cardiomyocyte proliferation. Thrombospondin1 (TSP-1), another ligand of Vldlr highly expressed in adult heart, was then found to inhibit cardiomyocyte proliferation through Vldlr, and may contribute to Vldlr's overall repression on proliferation. Mechanistically, Rac1 and subsequent Yap phosphorylation and nucleus translocation mediate the regulation of the cardiomyocyte cell cycle by TSP-1/Reelin-Vldlr signaling. Importantly, Reln mutant neonatal mice displayed impaired cardiomyocyte proliferation and cardiac regeneration after apical resection, while cardiac-specific Thbs1 deletion and cardiomyocyte-specific Vldlr deletion promote cardiomyocyte proliferation and are cardioprotective after myocardial infarction. Our results identified a novel role of Vldlr in consolidating extracellular signals to regulate cardiomyocyte cell cycle activity and survival, and the overall suppressive TSP-1-Vldlr signal may contribute to the poor cardiac repair capacity of adult mammals.

A Recombinase-Aided Amplification Assay for the Detection of Chlamydia felis.

Chlamydia felis is an important zoonotic agent for humans and various animals. A recombinase-aided amplification (RAA) assay was developed for detecting C. felis. RAA can be performed in a closed tube at 39°C within 30 min. The detection limit was 10.6 copies of the C. felis plasmid DNA per reaction. No positive signals for other pathogens were detected. The coincidence rate of RAA and conventional PCR was 95.24% (20/21) and 100% (96/96) for positive and negative samples, respectively. The established RAA assay is a simple, rapid, highly sensitive, and specific method for detecting C. felis.

Improved Sleep Affects Epigastric Pain in Functional Dyspepsia by Reducing the Levels of Inflammatory Mediators.

INTRODUCTION: The pathogenesis of epigastric pain in functional dyspepsia (FD) is complex. The study aims to explore the effect of sleep improvement on this symptom. METHODS: In total, 120 patients with FD-associated epigastric pain and insomnia were randomly divided into experimental and control groups using the envelope method. After applying the exclusion criteria, 107 patients were enrolled in the experimental (56 patients) and control (51 patients) groups. Insomnia was graded according to the Pittsburgh Sleep Quality Index (PSQI). In the experimental group, eszopiclone 3 mg, eszopiclone 3 mg + estazolam 1 mg, and eszopiclone 3 mg + estazolam 2 mg were given to patients with mild, moderate, and severe insomnia, respectively. In the control group, patients were given 1, 2, or 3 tablets of vitamin B complex. Patient sleep quality was monitored with Sleepthing. Epigastric pain was evaluated with a Numeric Rating Scale. The serum levels of IL-1ß, IL-6, IL-8, and tumor necrosis factor-α (TNF-α) were measured by enzyme-linked immunosorbent assay. Pain scores, sleep parameters, and serum levels of inflammatory mediators were compared before and after treatment. RESULTS: After treatment, the pain scores, sleep parameters, and TNF-α and IL-6 levels in the experimental group were significantly lower than those in the control group (p < 0.05). PSQI insomnia scores were significantly associated with pain scores, IL-6, and TNF-α (p < 0.05) but not in IL-8 and IL-1ß levels (p > 0.05) among the three groups. CONCLUSIONS: Improving sleep with eszopiclone and/or estazolam alleviates FD-associated epigastric pain, possibly by inhibiting related downstream transmission pathways and reducing the release of inflammatory mediators.

Soluble polymer facilely self-grown in situ on conducting substrates at room temperature towards electrochromic applications.

Electrochromic polymer film preparation methods such as spin coating, spray coating, and electrochemical polymerization, are commonly used. At present, developing new film preparation technology is an important aspect in the field of electrochromics. Herein, a continuous in situ self-growing method based on the chemical reaction occurring on the surface of an ITO glass between a metal oxide and organic acid groups was successfully applied to prepare electrochromic polymer films at a mild room temperature. SEM, FT-IR spectroscopy, XPS, and XRD characterization methods were combined to reveal the process and mechanism of film formation. The following notable electrochromic properties were observed: switching time within 6 s, contrast reached 35%, and minimal decrease of stability after 600 cycles. Finally, the patterned films were obtained through the directional growth of polymers in solution. This study provides an effective strategy for designing and preparing electrochromic films by self-growing methods in future applications.

Visualizing Mitophagy with Fluorescent Dyes for Mitochondria and Lysosome.

Mitochondria, being the powerhouses of the cell, play important roles in bioenergetics, free radical generation, calcium homeostasis, and apoptosis. Mitophagy is the primary mechanism of mitochondrial quality control and is generally studied using microscopic observation, however in vivo mitophagy assays are difficult to perform. Evaluating mitophagy by imaging live organelles is an alternative and necessary method for mitochondrial research. This protocol describes the procedures for using the cell-permeant green-fluorescent mitochondria dye MitoTracker Green and the red-fluorescent lysosome dye LysoTracker Red in live cells, including the loading of the dyes, visualization of the mitochondria and the lysosome, and expected outcomes. Detailed steps for the evaluation of mitophagy in live cells, as well as technical notes about microscope software settings, are also provided. This method can help researchers observe mitophagy using live-cell fluorescent microscopy. In addition, it can be used to quantify mitochondria and lysosomes and assess mitochondrial morphology.

Inhibition of mitochondrial superoxide promotes the development of hiPS-CMs during differentiation.

The redox state is a crucial determinant of the maturation transition of cardiomyocytes in vivo. Mitochondria, the primary site of superoxide generation, are very sensitive to various stimulations, including oxygen and nutrient supply. How mitochondrial superoxide affects the differentiation and development of induced pluripotent stem cell (iPSC)-derived cardiac myocytes (iPS-CMs) is not completely clear. To address the questions, we monitored the superoxide level during the differentiation and development of human iPS-CMs using MitoSOX. Mitochondria-targeted antioxidant Mito-TEMPO was used to treat hiPS-CMs in the differentiation period. We found that mitochondrial superoxide generation was dramatically enhanced during the differentiation and early development of iPS-CMs. Increased oxidative stress induced oxidative damage to macromolecules in iPS-CMs, such as lipids, proteins, and DNA. Mito-TEMPO protected mitochondrial functions, alleviated oxidative damage to lipids, proteins, and DNA and improved cellular structure and fatty acid utilization. Our findings confirmed that iPS-CM suffered from oxidative stress during differentiation and that mitochondrial-targeted antioxidant is beneficial for the maturation of iPS-CMs.

CircUBR5 acts as a ceRNA for miR-1179 to up-regulate UBR5 and to promote malignancy of triple-negative breast cancer.

UBR5 is an E3 ubiquitin ligase and an oncogene in a panel of human cancers. However, little is known on its impacts in triple-negative breast cancer (TNBC) and even less on its relationship to circUBR5 (hsa_circ_0001819), a circular RNA derived from exons 2, 3, 4, and 5 of UBR5 gene. In this study, we detected higher expressions of both circUBR5 and UBR5 in TNBC tissues, which were associated with worse prognosis, and also in a panel of breast cancer cells, particularly in TNBC cells. Functionally, circUBR5 was crucial for sustaining the malignant growth and metastasis of TNBC cells both in vitro and in vivo. Mechanistically, the oncogenic phenotypes of circUBC5 were mediated through sponging miR-1179 and up-regulating UBR5. Concomitant silencing circUBR5 and miR-1179 abolished the anti-tumor effects of targeting circUBR5 alone. Therefore, targeting circUBR5/miR-1179/UBR5 axis may benefit the treatment of TNBCs.

Ethanol extracts of Rhaponticum uniflorum (L.) DC inflorescence ameliorate LPS-mediated acute lung injury by alleviating inflammatory responses via the Nrf2/HO-1 signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Rhaponticum uniflorum (L.) DC is a member of the Compositae family. Loulu flowers (LLF) is the inflorescence of this plant, which is a commonly used Mongolian medicine for the treatment of inflammatory diseases due to its heat-clearing and detoxifying properties. It is used caused by. However, its anti-inflammatory mechanisms are not clear. AIM OF THIS STUDY: We investigated whether ethanol extracts of LLF can alleviate LPS-induced acute lung injury and explored the mechanism involved. MATERIAL AND METHODS: BALB/C mice were intragastrically administered with sodium carboxymethyl cellulose (0.5%, 1 mL/100 g) or ethanol extracts of LLF at a dose of 100, 200, and 400 mg/kg, once daily, for 3 days. Subsequently, mice models of acute lung injury were established by LPS and used for the determination of anti-inflammatory effects of LLF. After 6 h of treatment, mice were sacrificed to collect lung tissues and bronchoalveolar lavage fluid (BALF). H&E staining assay was performed on the tissues for pathological analysis. The ELISA test was conducted to measure NO, IL-6, TNF-α, MPO, SOD, CAT, MDA and GSH-PX levels. The expression level of proteins associated with the Nrf2/HO-1 and MAPK/NF-κB signaling pathways were determined using Western blot analysis. Levels of F4/80 and Nrf2 in lungs were quantified using immunohistochemistry. RESULTS: Oral administration of LLF extracts alleviated LPS-induced pathological alterations, reduced lung W/D weight ratio, decreased levels of TP, pro-inflammatory factors (TNF-α and IL-6), and NO in BALF. Pretreatment with LLF extract downregulated F4/80 expression in lung tissue and suppressed LPS-induced elevations in BALF and lung tissue levels of MPO. Moreover, treatment with LLF extract reduced the expression level of proteins associated with the MAPK signaling pathway (p-p38, p-JNK, p-ERK) and TLR4/NF-κB signaling pathways (TLR4, Myd88, p-IκB, p-p65). Moreover, LLF extract upregulated Nrf2, HO-1 and NQO1 protein levels, downregulated Keap1 protein level. Immunohistochemical analysis revealed that LLF reduced the LPS-induced increase in Nfr2 expression in lung tissues. CONCLUSION: Ethanol extracts of LLF ameliorated LPS-induced acute lung injury by suppressing inflammatory response and enhancing antioxidation capacity, which correlated with the MAPK/NF-κB and Nfr2/HO-1 signaling pathways.

PGC-1α-Mediated Mitochondrial Quality Control: Molecular Mechanisms and Implications for Heart Failure.

Mitochondria with structural and functional integrity are essential for maintaining mitochondrial function and cardiac homeostasis. It is involved in the pathogenesis of many diseases. Peroxisome proliferator-activated receptor γ coactivator 1 α (PGC-1α), acted as a transcriptional cofactor, is abundant in the heart, which modulates mitochondrial biogenesis and mitochondrial dynamics and mitophagy to sustain a steady-state of mitochondria. Cumulative evidence suggests that dysregulation of PGC-1α is closely related to the onset and progression of heart failure. PGC-1α deficient-mice can lead to worse cardiac function under pressure overload compared to sham. Here, this review mainly focuses on what is known about its regulation in mitochondrial functions, as well as its crucial role in heart failure.

Correction: Ethyl ferulate contributes to the inhibition of the inflammatory responses in murine RAW 264.7 macrophage cells and acute lung injury in mice.

[This corrects the article DOI: 10.1371/journal.pone.0251578.].

Synergistic anti-inflammatory effects of peimine, peiminine, and forsythoside a combination on LPS-induced acute lung injury by inhibition of the IL-17-NF-κB/MAPK pathway activation.

ETHNOPHARMACOLOGICAL RELEVANCE: Forsythia suspensa (Thunb.) Vahl and Fritillaria thunbergii Miq are traditional Chinese medicines that exhibit the ability to clear heat and toxic material effects. In China, the combination of these two medicines is widely used to treat mucopurulent sputum and bloody phlegm, arising due to phlegm-heat obstruction in respiratory diseases. However, very limited information is available regarding the combined anti-inflammatory effect of important effective components of Forsythia suspensa (Thunb.) Vahl and Fritillaria thunbergii Miq, namely peimine, peiminine, and forsythoside A. AIM OF THIS STUDY: To investigate synergistic anti-inflammatory effects of combined administration of peimine, peiminine, and forsythoside A on LPS-induced acute lung injury compared to combined administration of two compounds or individual administration, and unravel the underlying mechanism. MATERIAL AND METHODS: In the present study, male BALB/c mice received an oral dosage of sodium carboxymethylcellulose (CMC-Na) (0.5%, 1 mL/100 g), peimine, peiminine, forsythoside A, peimine + forsythoside A, peiminine + forsythoside A, and peimine + peiminine + forsythoside A (suspended in CMC-Na; 0.5%), once daily for 7 days. Subsequently, intratracheal instillation of LPS was applied to establish acute lung injury model. After 6 h of administration, the mice were sacrificed, and bronchoalveolar lavage fluid (BALF) and lung tissues were collected. These samples were further used to determine lung W/D (wet/dry) weight ratio, total protein (TP) levels, inflammatory cytokines (IL-6, TNF-α, IL-1ß, and IL-17), and expression of proteins involved in TLR4/MAPK/NF-κB pathway and IL-17 pathway. Further, tissue sections were subjected to H&E staining to assess the pathological alterations induced by LPS. The expression of IL-6 and TNF-α proteins in lung tissues was also analyzed using immunohistochemical staining. RESULTS: A synergistic anti-inflammatory effect of peimine, peiminine, and forsythoside A was observed when administered in combination to LPS-induced acute lung injury. The combined administration of peimine, peiminine, and forsythoside A had a strongly inhibitory effects on the W/D weight ratio, total protein (TP) level and the inflammatory cytokines (TNF-α, IL-6, IL-1ß, and IL-17) level in acute lung injury mice, compared to combined administration of two compounds or individual administration. The infiltration of inflammatory cells and thickened bronchoalveolar walls induced by LPS were also ameliorated through the combined administration of peimine, peiminine, and forsythoside A. More importantly, the upregulation of protein related to TLR4/MAPK/NF-κB signaling pathway and the activation of IL-17 were significantly suppressed by pretreatment with each of the three compounds alone, while the effects of individual compounds were synergistically augmented by the combined pretreatment of these three compounds. CONCLUSION: The combined administration of peimine, peiminine, and forsythoside A ameliorated inflammatory response in acute lung injury mice induced by LPS in a synergistic manner, the mechanism may be related to the dampening of the TLR4/MAPK/NF-κB signaling pathway and IL-17 activation.

Mitochondrial autophagy: molecular mechanisms and implications for cardiovascular disease.

Mitochondria are highly dynamic organelles that participate in ATP generation and involve calcium homeostasis, oxidative stress response, and apoptosis. Dysfunctional or damaged mitochondria could cause serious consequences even lead to cell death. Therefore, maintaining the homeostasis of mitochondria is critical for cellular functions. Mitophagy is a process of selectively degrading damaged mitochondria under mitochondrial toxicity conditions, which plays an essential role in mitochondrial quality control. The abnormal mitophagy that aggravates mitochondrial dysfunction is closely related to the pathogenesis of many diseases. As the myocardium is a highly oxidative metabolic tissue, mitochondria play a central role in maintaining optimal performance of the heart. Dysfunctional mitochondria accumulation is involved in the pathophysiology of cardiovascular diseases, such as myocardial infarction, cardiomyopathy and heart failure. This review discusses the most recent progress on mitophagy and its role in cardiovascular disease.

Extracellular vesicles derived from human bone marrow mesenchymal stem cells protect rats against acute myocardial infarction-induced heart failure.

Extracellular vesicles (EVs) derived from human bone marrow mesenchymal stem cells (BMSCs) are suggested to promote angiogenesis in a rat model of acute myocardial infarction (AMI). This study aimed to explore the underlying mechanism of BMSCs-EVs in AMI-induced heart failure (HF). BMSCs were isolated and verified, and EVs were purified and identified. After establishment of AMI-induced HF models, rats were treated with BMSCs-EVs and/or overexpressing (ov)/knocking down (kd) bone morphogenetic protein 2 (BMP2). Cardiac function, myocardial histopathological changes, angiogenesis, and vascular regeneration density were measured. Levels of pro-angiogenesis factors and cardiomyocyte apoptosis were detected. The viability and angiogenesis of hypoxic human umbilical vein endothelial cells (HUVECs) were measured. After BMSCs-EV treatment, the cardiac function of HF rats was improved, myocardial fibrosis and inflammatory cell infiltration were decreased, angiogenesis was increased, and cardiomyocyte apoptosis was inhibited. BMP2 was significantly upregulated in the myocardium. Ov-BMP2-BMSCs-EVs alleviated myocardial fibrosis and inflammatory cell infiltration, and promoted angiogenesis of HF rats, and improved the activity and angiogenesis of hypoxic HUVECs, while kd-BMP2-BMSCs-EVs showed limited protection against AMI-induced HF. BMSCs-EVs deliver BMP2 to promote angiogenesis and improve cardiac function of HF rats.

Long non-coding RNA Sox2OT promotes coronary microembolization-induced myocardial injury by mediating pyroptosis.

OBJECTIVE: As a common complication of coronary microembolization (CME), myocardial injury (MI) implies high mortality. Long non-coding RNAs (lncRNAs) are rarely studied in CME-induced MI. Herein, this study intended to evaluate the role of lncRNA Sox2 overlapping transcript (Sox2OT) in CME-induced MI. METHODS: The CME rat models were successfully established by injection of microemboli. Rat cardiac functions and MI were observed by ultrasonic electrocardiogram, HE staining, and HBFP staining. Functional assays were utilized to test the inflammatory responses, oxidative stress, and pyroptosis using reverse transcription quantitative polymerase chain reaction, Western blotting, immunohistochemistry, immunofluorescence, and ELISA. Dual-luciferase reporter gene assay and RNA immunoprecipitation were conducted to clarify the targeting relations between Sox2OT and microRNA (miRNA)-23b and between miR-23b and toll-like receptor 4 (TLR4). RESULTS: Rat CME disrupted the cardiac functions and induced inflammatory responses and oxidative stress, and activated the nuclear factor-kappa B (NF-κB) pathway and pyroptosis (all P < 0.05). An NF-κB inhibitor downregulated the NF-κB pathway, reduced pyroptosis, and relieved cardiomyocyte injury and pyroptosis. Compared with the sham group (1.05 ± 0.32), lncRNA Sox2OT level (4.41 ± 0.67) in the CME group was elevated (P < 0.05). Sox2OT acted as a competitive endogenous RNA (ceRNA) of miR-23b to regulate TLR4. Silencing of Sox2OT favoured miR-23b binding to 3'UTR of TLR4 mRNA leading to suppressed TLR4-mediated NFKB signalling and pyroptosis in myocardial tissues harvested from CME rat models. In addition, miR-23b overexpression could supplement the cytosolic miR-23b reserves to target TLR-4 and partially reverse Sox2OT-mediated pyroptosis in LPS-treated H9C2 cells. CONCLUSIONS: This study supported that silencing Sox2OT inhibited CME-induced MI by eliminating Sox2OT/miR-23b binding and down-regulating the TLR4/NF-κB pathway. This investigation may provide novel insights for the treatment of CME-induced MI.

Discovery of Novel 3,4-Dihydro-2(1H)-Quinolinone Sulfonamide Derivatives as New Tubulin Polymerization Inhibitors with Anti-Cancer Activity.

In this paper, a small series of novel quinoline sulfonamide derivatives was synthesized, and their structure of the target compounds were confirmed by 1H NMR and MS. The screening of the news target compounds' in vitro cytotoxic activities against tumor cell lines by the MTT method was performed. Among them, compound D13 (N-(4-methoxybenzyl)-2-oxo-N-(3,4,5-trimethoxyphenyl)-1,2,3,4-tetrahydroquinoline-6-sulfonamide exhibited the strongest inhibitory effect on the proliferation of HeLa (IC50: 1.34 µM), and this value correlated well with the inhibitory activities of the compound against tubulin polymerization (IC50: 6.74 µM). In summary, a new type of quinoline-sulfonamide derivative with tubulin polymerization inhibitory activity was discovered, and it can be used as a lead compound for further modification.

The complete chloroplast genome sequence of Rhaponticum uniflorum, the first of the genus Rhaponticum.

Rhaponticum uniflorum is commonly used as a source for traditional medicines with the main effect of clearing heat. Here, we sequenced the complete chloroplast (cp) genome of R. uniflorum to develop molecular markers for taxonomic classification and species determination of R. uniflorum. It was 152,760 bp in size and has a typical circular structure, including a pair of inverted repeats with 25,205 bp, a large single-copy region with 83,687 bp, and a small single copy region with 18,663 bp. The genome encodes 110 unique genes, including 80 protein-coding, four rRNA and 26 tRNA genes. Phylogenomic analysis shows that R. uniflorum is closely related to the Saussurea. The study is useful for phylogenetic and population genetic studies of Rhaponticum plants.

Synthesis and Evaluation of Bakuchiol Derivatives as Potent Anti-inflammatory Agents in Vitro and in Vivo.

Bakuchiol, a prenylated phenolic monoterpene derived from the fruit of Psoralen corylifolia L. (Buguzhi), is widely used to treat tumors, viruses, inflammation, and bacterial infections. In this study, we designed and synthesized 30 bakuchiol derivatives to identify new anti-inflammatory drugs. The anti-inflammatory activities of the derivatives were screened using lipopolysaccharide-induced RAW264.7 cells. To evaluate the anti-inflammatory activity of the compounds, we measured nitric oxide (NO), interleukin-6, and tumor necrosis factor-α production. Based on the screening results, compound 7a displayed more pronounced activity than bakuchiol and celecoxib. Furthermore, the mechanistic studies indicated that 7a inhibited pro-inflammatory cytokine release, which was correlated with activation of the nuclear factor erythroid 2-related factor 2/heme oxygenase-1 signaling pathway and blockade of the nuclear factor-κB/mitogen-activated protein kinase signaling pathway. The in vivo anti-inflammatory activity in zebrafish indicated that 7a inhibited NO and reactive oxygen species production in a dose-dependent manner. These results indicate that 7a is a potential candidate for development as an anti-inflammatory agent.