Tumor-repopulating cells evade ferroptosis via PCK2-dependent phospholipid remodeling.

Whether stem-cell-like cancer cells avert ferroptosis to mediate therapy resistance remains unclear. In this study, using a soft fibrin gel culture system, we found that tumor-repopulating cells (TRCs) with stem-cell-like cancer cell characteristics resist chemotherapy and radiotherapy by decreasing ferroptosis sensitivity. Mechanistically, through quantitative mass spectrometry and lipidomic analysis, we determined that mitochondria metabolic kinase PCK2 phosphorylates and activates ACSL4 to drive ferroptosis-associated phospholipid remodeling. TRCs downregulate the PCK2 expression to confer themselves on a structural ferroptosis-resistant state. Notably, in addition to confirming the role of PCK2-pACSL4(T679) in multiple preclinical models, we discovered that higher PCK2 and pACSL4(T679) levels are correlated with better response to chemotherapy and radiotherapy as well as lower distant metastasis in nasopharyngeal carcinoma cohorts.

Phospholipase iPLA2ß averts ferroptosis by eliminating a redox lipid death signal.

Ferroptosis, triggered by discoordination of iron, thiols and lipids, leads to the accumulation of 15-hydroperoxy (Hp)-arachidonoyl-phosphatidylethanolamine (15-HpETE-PE), generated by complexes of 15-lipoxygenase (15-LOX) and a scaffold protein, phosphatidylethanolamine (PE)-binding protein (PEBP)1. As the Ca2+-independent phospholipase A2ß (iPLA2ß, PLA2G6 or PNPLA9 gene) can preferentially hydrolyze peroxidized phospholipids, it may eliminate the ferroptotic 15-HpETE-PE death signal. Here, we demonstrate that by hydrolyzing 15-HpETE-PE, iPLA2ß averts ferroptosis, whereas its genetic or pharmacological inactivation sensitizes cells to ferroptosis. Given that PLA2G6 mutations relate to neurodegeneration, we examined fibroblasts from a patient with a Parkinson's disease (PD)-associated mutation (fPDR747W) and found selectively decreased 15-HpETE-PE-hydrolyzing activity, 15-HpETE-PE accumulation and elevated sensitivity to ferroptosis. CRISPR-Cas9-engineered Pnpla9R748W/R748W mice exhibited progressive parkinsonian motor deficits and 15-HpETE-PE accumulation. Elevated 15-HpETE-PE levels were also detected in midbrains of rotenone-infused parkinsonian rats and α-synuclein-mutant SncaA53T mice, with decreased iPLA2ß expression and a PD-relevant phenotype. Thus, iPLA2ß is a new ferroptosis regulator, and its mutations may be implicated in PD pathogenesis.

ALOX5 inhibition protects against dopaminergic neurons undergoing ferroptosis.

Oxidative disruption of dopaminergic neurons is regarded as a crucial pathogenesis in Parkinson's disease (PD), eventually causing neurodegenerative progression. (-)-Clausenamide (Clau) is an alkaloid isolated from plant Clausena lansium (Lour.), which is well-known as a scavenger of lipid peroxide products and exhibiting neuroprotective activities both in vivo and in vitro, yet with the in-depth molecular mechanism unrevealed. In this study, we evaluated the protective effects and mechanisms of Clau on dopaminergic neuron. Our results showed that Clau directly interacted with the Ser663 of ALOX5, the PKCα-phosphorylation site, and thus prevented the nuclear translocation of ALOX5, which was essential for catalyzing the production of toxic lipids 5-HETE. LC-MS/MS-based phospholipidomics analysis demonstrated that the oxidized membrane lipids were involved in triggering ferroptotic death in dopaminergic neurons. Furthermore, the inhibition of ALOX5 was found to significantly improving behavioral defects in PD mouse model, which was confirmed associated with the effects of attenuating the accumulation of lipid peroxides and neuronal damages. Collectively, our findings provide an attractive strategy for PD therapy by targeting ALOX5 and preventing ferroptosis in dopaminergic neurons.

13-8-13-Membered Tricyclic Ladder-Type Siloxanes Hybridized with BINOLs: Synthesis, Characterization, and Fluorescence Sensing of Fluorides.

Developing fluorescent chemosensors with sensitivity and high specificity for recognizing fluorides is still challenging. Herein, four innovative compounds based on 13-8-13-membered tricyclic ladder-type siloxanes hybridized with BINOLs (abbreviated as TLS-BINOLs) were prepared through the B(C6F5)3-catalyzed Piers-Rubinsztajn reaction. The well-defined ladder-type structure of the TLS-BINOLs was determined by X-ray crystallographic analysis. Additionally, the fluorescent sensing ability of the TLS-BINOLs toward anions was studied. Our finding revealed that all four ladder-type compounds (TLS-BINOLs) exhibited high specificity in recognizing fluorides through fluoride-triggered structural decomposition. The detection limits for fluorides were determined to be 0.37, 0.35, 0.39, and 0.48 µM for the respective TLS-BINOLs. The nonemissive product induced by the fluorides was also determined using single-crystal X-ray diffraction analysis.

Respiratory depression driven by membrane damage as a mechanism for linalool to inhibit Pseudomonas lundensis and its preservation potential for beef.

AIMS: This study aimed to investigate the mechanism of linalool against Pseudomonas lundensis and its application on beef. METHODS AND RESULTS: Field emission scanning electron microscopy found that linalool exerted antibacterial activity with a minimum inhibitory concentration (MIC) of 1.5 ml l-1 by disrupting cell structure. Loss of cell membrane integrity was monitored due to leakage of nucleic acids and K+. In addition, respiratory depression appeared in Ps. lundensis based on inhibition of enzyme activities including hexokinase (HK), glucose 6-phosphate dehydrogenase (G6PDH), phosphofructokinase (PFK), pyruvate kinase (PK), pyruvate dehydrogenase (PDH), citrate synthase (CS), succinate dehydrogenase (SDH), and malate dehydrogenase (MDH). Subsequently, energy limitation also occurred according to the decrease in ATP content and ATPase activity. Molecular docking confirmed that linalool can combine with enzymes in cell wall (ddlB) and energy synthesis (AtpD) pathways to exert antibacterial effect. Of note, linalool has advantages for beef preservation by delaying quality changes including pH, total volatile basic nitrogen (TVB-N) and total viable count (TVC). CONCLUSIONS: Linalool has significant inhibitory effect on Ps. lundensis, and respiratory depression driven by membrane damage is the main inhibitory mechanism.

Hybridization of amantadine with gardenamide A enhances NMDA antagonism and in vivo anti-PD effects.

Eight hybrids of amantadine (ATD) with a natural modulator gardenamide A (GA) via an alkylene carbonyl bridge or alkylene bridge have been designed and synthesized. Evaluated by electrophysiological assay, compound 5b was confirmed an enhanced NMDAR antagonist compared to ATD with IC50 value of 10.2 ± 1.2 µM. 5b has been demonstrated to reverse the damages of behavioral performance, the loss of dopaminergic neurons, the reduction of TH positive, and the increase of α-synuclein in both MPTP-treated mice and zebrafish models. In both ethological and ecological experiments, the activity of 5b was confirmed better than ATD or ATD/GA combination, and was almost equal to the positive selegiline. In vivo and in vitro, 5b is shown to reverse the ascend of NR1 and i-NOS levels. This candidate was also demonstrated the activity to down-regulated MPTP-increased Ca2+ influx in SH-SY5Y cells in a steep and sharp mode. It is displayed that 5b exerts neuroprotective effect partly by activating the PI3K/Akt signaling pathway. Taken all together, our data support that 5b is a more promising agent against PD than ATD.

BL-918, a small-molecule activator of ULK1, induces cytoprotective autophagy for amyotrophic lateral sclerosis therapy.

Amyotrophic lateral sclerosis (ALS) is one of the most common fatal neurodegenerative diseases in adults. ALS pathogenesis is associated with toxic SOD1 aggregates generated by mutant SOD1. Since autophagy is responsible for the clearance of toxic protein aggregates including SOD1 aggregates, autophagy induction has been considered as a potential strategy for treating ALS. Autophagic signaling is initiated by unc-51 like autophagy activating kinase 1 (ULK1) complex. We previously identified that BL-918 as a specific ULK1 activator, which exerted cytoprotective effect against Parkinson's disease in vitro and in vivo. In this study we investigated whether BL-918 exerted a therapeutic effect against ALS, and characterized its pharmacokinetic profile in rats. In hSODG93A-NSC34 cells, treatment with BL-918 (5, 10 µM) dose-dependently induced ULK1-dependent autophagy, and eliminated toxic SOD1 aggregates. In SODG93A mice, administration of BL-918 (40, 80 mg/kg, b.i.d., i.g.) dose-dependently prolonged lifespan and improved the motor function, and enhanced the clearance of SOD1 aggregates in spinal cord and cerebral cortex through inducing autophagy. In the pharmacokinetic study conducted in rats, we found BL-918 and its 2 metabolites (M8 and M10) present in spinal cord and brain; after intragastric and intravenous administration, BL-918 reached the highest blood concentration compared to M8 and M10. Collectively, ULK1 activator BL-918 displays a therapeutic potential on ALS through inducing cytoprotective autophagy. This study provides a further clue for autophagic dysfunction in ALS pathogenesis.

Reducing lipid peroxidation attenuates stress-induced susceptibility to herpes simplex virus type 1.

Psychological stress increases the susceptibility to herpes simplex virus type 1 (HSV-1) infection. There is no effective intervention due to the unknown pathogenesis mechanisms. In this study we explored the molecular mechanisms underlying stress-induced HSV-1 susceptibility and the antiviral effect of a natural compound rosmarinic acid (RA) in vivo and in vitro. Mice were administered RA (11.7, 23.4 mg·kg-1·d-1, i.g.) or acyclovir (ACV, 206 mg·kg-1·d-1, i.g.) for 23 days. The mice were subjected to restraint stress for 7 days followed by intranasal infection with HSV-1 on D7. At the end of RA or ACV treatment, mouse plasma samples and brain tissues were collected for analysis. We showed that both RA and ACV treatment significantly decreased stress-augmented mortality and alleviated eye swelling and neurological symptoms in HSV-1-infected mice. In SH-SY5Y cells and PC12 cells exposed to the stress hormone corticosterone (CORT) plus HSV-1, RA (100 µM) significantly increased the cell viability, and inhibited CORT-induced elevation in the expression of viral proteins and genes. We demonstrated that CORT (50 µM) triggered lipoxygenase 15 (ALOX15)-mediated redox imbalance in the neuronal cells, increasing the level of 4-HNE-conjugated STING, which impaired STING translocation from the endoplasmic reticulum to Golgi; the abnormality of STING-mediated innate immunity led to HSV-1 susceptibility. We revealed that RA was an inhibitor of lipid peroxidation by directly targeting ALOX15, thus RA could rescue stress-weakened neuronal innate immune response, thereby reducing HSV-1 susceptibility in vivo and in vitro. This study illustrates the critical role of lipid peroxidation in stress-induced HSV-1 susceptibility and reveals the potential for developing RA as an effective intervention in anti-HSV-1 therapy.

Potential of Tamarind Shell Extract against Oxidative Stress In Vivo and In Vitro.

Tamarind shell is rich in flavonoids and exhibits good biological activities. In this study, we aimed to analyze the chemical composition of tamarind shell extract (TSE), and to investigate antioxidant capacity of TSE in vitro and in vivo. The tamarind shells were extracted with 95% ethanol refluxing extraction, and chemical constituents were determined by ultra-performance chromatography-electrospray tandem mass spectrometry (UPLC-MS/MS). The free radical scavenging activity of TSE in vitro was evaluated using the oxygen radical absorbance capacity (ORAC) method. The antioxidative effects of TSE were further assessed in 2,2-azobis (2-amidinopropane) dihydrochloride (AAPH)-stimulated ADTC5 cells and tert-butyl hydroperoxide (t-BHP)-exposed zebrafish. A total of eight flavonoids were detected in TSE, including (+)-catechin, taxifolin, myricetin, eriodictyol, luteolin, morin, apigenin, and naringenin, with the contents of 5.287, 8.419, 4.042, 6.583, 3.421, 4.651, 0.2027, and 0.6234 mg/g, respectively. The ORAC assay revealed TSE and these flavonoids had strong free radical scavenging activity in vitro. In addition, TSE significantly decreased the ROS and MDA levels but restored the SOD activity in AAPH-treated ATDC5 cells and t-BHP-exposed zebrafish. The flavonoids also showed excellent antioxidative activities against oxidative damage in ATDC5 cells and zebrafish. Overall, the study suggests the free radical scavenging capacity and antioxidant potential of TSE and its primary flavonoids in vitro and in vivo and will provide a theoretical basis for the development and utilization of tamarind shell.

Enhanced antioxidation capacity endowed to a mixed type aldose reductase inhibitor leads to a promising anti-diabetic complications agent.

A series of 5f-based new compounds has been designed and synthesized. In vitro screening demonstrated that the binding affinity and selectivity on aldose reductase (AR) were positively correlated with its antioxidation capacity. Compound 6d was verified the most active candidate, where its IC50, selective index (SI), and EC50 value was 22.3 ± 1.6 nM, 236.2, and 8.7 µM respectively. 6d was confirmed as both an excellent antioxidant and aldose reductase inhibitor (ARI). It was identified as a mixed type ARI with Ki and Kis values of 23.94 and 1.20 nM. When evaluated by a high-glucose impaired chicken embryo model, it was found that 6d attenuated the incidence of neural tube defect (NTD) and death rate in a dose-dependent manner. It significantly improved the hyperglycemia-induced abnormalities of body weight and morphology of chicken embryos. 6d reversed the hyperglycemia-raised AR activity, sorbitol accumulation, reactive oxygen species (ROS) and malondialdehyde (MDA) levels. It restored the high-glucose-reduced Pax3 protein expression. At the same dose (0.5 µM), 6d showed better effects than 5f in all the above detections. By the way, 6d did not affect hyperglycemia-elevated aldehyde reductase (ALR1) activity. This evidence together with its kinetic properties, implicated that 6d is a high selective ARI without the suspicion of promiscuity. 6d was proved here an effective agent to treat diabetic peripheral neuropathy (DPN). Whether 6d has potential to treat other types of diabetic complications (DC) needs to be further investigation.

Respiratory Depression as Antibacterial Mechanism of Linalool against Pseudomonas fragi Based on Metabolomics.

Linalool showed a broad-spectrum antibacterial effect, but few studies have elucidated the antibacterial mechanism of linalool on Pseudomonas fragi (P. fragi) to date. The present study aimed to uncover the antimicrobial activity and potential mechanism of linalool against P. fragi by determining key enzyme activities and metabolites combined with a high-throughput method and metabolomic pathway analysis. As a result, linalool had excellent inhibitory activity against P. fragi with MIC of 1.5 mL/L. In addition, the presence of linalool significantly altered the intracellular metabolic profile and a total of 346 differential metabolites were identified, of which 201 were up-regulated and 145 were down-regulated. The highlight pathways included beta-alanine metabolism, pantothenic acid and CoA metabolism, alanine, aspartate and glutamate metabolism, nicotinate and nicotinamide metabolism. Overall, linalool could cause metabolic disorders in cells, and the main metabolic pathways involved energy metabolism, amino acid metabolism and nucleic acid metabolism. In particular, the results of intracellular ATP content and related enzymatic activities (ATPase, SDH, and GOT) also highlighted that energy limitation and amino acid disturbance occurred intracellularly. Together, these findings provided new insights into the mechanism by which linalool inhibited P. fragi and theoretical guidance for its development as a natural preservative.

Antibacterial mechanism of linalool emulsion against Pseudomonas aeruginosa and its application to cold fresh beef.

Pseudomonas aeruginosa (P. aeruginosa) is the dominant spoilage bacterium in cold fresh beef. The current strategy is undertaken to overcome the low water solubility of linalool by encapsulating linalool into emulsions. The results of field emission scanning electron microscopy and particle size distribution revealed that the appearance of the bacterial cells was severely disrupted after exposure to linalool emulsion (LE) with an minimum inhibitory concentration (MIC) of 1.5 mL/L. Probes combined with fluorescence spectroscopy were performed to detect cell membrane permeability, while intracellular components (protein and ion leakage) and crystal violet staining were further measured to characterize cell membrane integrity and biofilm formation ability. The results confirmed that LE could destroy the structure of the cell membrane, thereby leading to the leakage of intracellular material and effective removal of biofilms. Molecular docking confirmed that LE can interact with the flagellar cap protein (FliD) and DNA of P. aeruginosa, inhibiting biofilm formation and causing genetic damage. Furthermore, the results of respiratory metabolism and reactive oxygen species (ROS) accumulation revealed that LE could significantly inhibit the metabolic activity of P. aeruginosa and induce oxidative stress. In particular, the inhibition rate of LE on P. aeruginosa was 23.03% and inhibited mainly the tricarboxylic acid cycle (TCA). Finally, LE was applied to preserve cold fresh beef, and the results showed that LE could effectively inhibit the activity of P. aeruginosa and delay the quality change of cold fresh beef during the storage period. These results are of great significance to developing natural preservatives and extending the shelf life of cold fresh beef.

[Research progress of Gan-Yu-Hua-Huo syndrome based on emotional stress-induced latent herpes simplex virus-1 reactivation].

Gan-Yu-Hua-Huo syndrome(Live qi stagnation transforming into fire pattern) is one of the core contents of the theory of emotional diseases in traditional Chinese medicine(TCM). It is the key link of the pathogenesis change of emotion-related diseases and widely exists in the pathological process of various related diseases. However, due to the lack of animal models in line with the characteristics of TCM syndromes, the research on biomedical basis of Gan-Yu-Hua-Huo syndrome and study of Chinese medicines for soothing liver and purging fire have been restricted seriously. This study found that the pathological process of facial fire-heat symptoms of Gan-Yu-Hua-Huo syndrome was similar to the facial symptoms due to the emotional stress-induced latent herpes simplex virus-1(HSV-1) reactivation. Therefore, this study proposed that the emotional stress-induced latent HSV-1 activation be used to establish the animal model of Gan-Yu-Hua-Huo syndrome. In this study, the state-of-art literature in the field of Gan-Yu-Hua-Huo syndrome was summarized, and the experimental animal model of Gan-Yu-Hua-Huo syndrome was established from the perspective of emotional stress-induced latent HSV-1 reactivation to reveal the active substances, potential targets and pathways related to the pathological mechanism of the syndrome. This study was expected to provide reference and basis for the pharmacodynamic characterization of commonly used Chinese medicine for Gan-Yu-Hua-Huo syndrome in clinical practice.

A plant RNA virus hijacks endocytic proteins to establish its infection in plants.

Endocytosis and endosomal trafficking play essential roles in diverse biological processes including responses to pathogen attack. It is well established that animal viruses enter host cells through receptor-mediated endocytosis for infection. However, the role of endocytosis in plant virus infection still largely remains unknown. Plant dynamin-related proteins 1 (DRP1) and 2 (DRP2) are the large, multidomain GTPases that participate together in endocytosis. Recently, we have discovered that DRP2 is co-opted by Turnip mosaic virus (TuMV) for infection in plants. We report here that DRP1 is also required for TuMV infection. We show that overexpression of DRP1 from Arabidopsis thaliana (AtDRP1A) promotes TuMV infection, and AtDRP1A interacts with several viral proteins including VPg and cylindrical inclusion (CI), which are the essential components of the virus replication complex (VRC). AtDRP1A colocalizes with the VRC in TuMV-infected cells. Transient expression of a dominant negative (DN) mutant of DRP1A disrupts DRP1-dependent endocytosis and supresses TuMV replication. As adaptor protein (AP) complexes mediate cargo selection for endocytosis, we further investigated the requirement of AP in TuMV infection. Our data suggest that the medium unit of the AP2 complex (AP2ß) is responsible for recognizing the viral proteins as cargoes for endocytosis, and knockout of AP2ß impairs intracellular endosomal trafficking of VPg and CI and inhibits TuMV replication. Collectively, our results demonstrate that DRP1 and AP2ß are two proviral host factors of TuMV and shed light into the involvement of endocytosis and endosomal trafficking in plant virus infection.

Successive High-Resolution (H2O)n-GCIB and C60-SIMS Imaging Integrates Multi-Omics in Different Cell Types in Breast Cancer Tissue.

The temporo-spatial organization of different cells in the tumor microenvironment (TME) is the key to understanding their complex communication networks and the immune landscape that exists within compromised tissues. Multi-omics profiling of single-interacting cells in the native TME is critical for providing further information regarding the reprograming mechanisms leading to immunosuppression and tumor progression. This requires new technologies for biomolecular profiling of phenotypically heterogeneous cells on the same tissue sample. Here, we developed a new methodology for comprehensive lipidomic and metabolomic profiling of individual cells on frozen-hydrated tissue sections using water gas cluster ion beam secondary ion mass spectrometry ((H2O)n-GCIB-SIMS) (at 1.6 µm beam spot size), followed by profiling cell-type specific lanthanide antibodies on the same tissue section using C60-SIMS (at 1.1 µm beam spot size). We revealed distinct variations of distribution and intensities of >150 key ions (e.g., lipids and important metabolites) in different types of the TME individual cells, such as actively proliferating tumor cells as well as infiltrating immune cells. The demonstrated feasibility of SIMS imaging to integrate the multi-omics profiling in the same tissue section at the single-cell level will lead to new insights into the role of lipid reprogramming and metabolic response in normal regulation or pathogenic discoordination of cell-cell interactions in a variety of tissue microenvironments.

New insights into the effects of caffeine on adult hippocampal neurogenesis in stressed mice: Inhibition of CORT-induced microglia activation.

Chronic stress-evoked depression has been implied to associate with the decline of adult hippocampal neurogenesis. Caffeine has been known to combat stress-evoked depression. Herein, we aim to investigate whether the protective effect of caffeine on depression is related with improving adult hippocampus neurogenesis and explore the mechanisms. Mouse chronic water immersion restraint stress (CWIRS) model, corticosterone (CORT)-established cell stress model, a coculture system containing CORT-treated BV-2 cells and hippocampal neural stem cells (NSCs) were utilized. Results showed that CWIRS caused obvious depressive-like disorders, abnormal 5-HT signaling, and elevated-plasma CORT levels. Notably, microglia activation-evoked brain inflammation and inhibited neurogenesis were also observed in the hippocampus of stressed mice. In comparison, intragastric administration of caffeine (10 and 20 mg/kg, 28 days) significantly reverted CWIRS-induced depressive behaviors, neurogenesis recession and microglia activation in the hippocampus. Further evidences from both in vivo and in vitro mechanistic experiments demonstrated that caffeine treatment significantly suppressed microglia activation via the A2AR/MEK/ERK/NF-κB signaling pathway. The results suggested that CORT-induced microglia activation contributes to stress-mediated neurogenesis recession. The antidepression effect of caffeine was associated with unlocking microglia activation-induced neurogenesis inhibition.

Autophagy-dependent removal of α-synuclein: a novel mechanism of GM1 ganglioside neuroprotection against Parkinson's disease.

GM1 ganglioside is particularly abundant in the mammalian central nervous system and has shown beneficial effects on neurodegenerative diseases. In this study, we investigated the therapeutic effect of GM1 ganglioside in experimental models of Parkinson's disease (PD) in vivo and in vitro. Mice were injected with MPTP (30 mg·kg-1·d-1, i.p.) for 5 days, resulting in a subacute model of PD. PD mice were treated with GM1 ganglioside (25, 50 mg·kg-1·d-1, i.p.) for 2 weeks. We showed that GM1 ganglioside administration substantially improved the MPTP-induced behavioral disturbance and increased the levels of dopamine and its metabolites in the striatal tissues. In the MPP+-treated SH-SY5Y cells and α-synuclein (α-Syn) A53T-overexpressing PC12 (PC12α-Syn A53T) cells, treatment with GM1 ganglioside (40 µM) significantly decreased α-Syn accumulation and alleviated mitochondrial dysfunction and oxidative stress. We further revealed that treatment with GM1 ganglioside promoted autophagy, evidenced by the autophagosomes that appeared in the substantia nigra of PD mice as well as the changes of autophagy-related proteins (LC3-II and p62) in the MPP+-treated SH-SY5Y cells. Cotreatment with the autophagy inhibitor 3-MA or bafilomycin A1 abrogated the in vivo and in vitro neuroprotective effects of GM1 ganglioside. Using GM1 ganglioside labeled with FITC fluorescent, we observed apparent colocalization of GM1-FITC and α-Syn as well as GM1-FITC and LC3 in PC12α-Syn A53T cells. GM1 ganglioside significantly increased the phosphorylation of autophagy regulatory proteins ATG13 and ULK1 in doxycycline-treated PC12α-Syn A53T cells and the MPP+-treated SH-SY5Y cells, which was inhibited by 3-MA. Taken together, this study demonstrates that the anti-PD role of GM1 ganglioside resulted from activation of autophagy-dependent α-Syn clearance.

Hepatic small extracellular vesicles promote microvascular endothelial hyperpermeability during NAFLD via novel-miRNA-7.

BACKGROUND: A recent study has reported that patients with nonalcoholic fatty liver disease (NAFLD) are more susceptible to coronary microvascular dysfunction (CMD), which may predict major adverse cardiac events. However, little is known regarding the causes of CMD during NAFLD. In this study, we aimed to explore the role of hepatic small extracellular vesicles (sEVs) in regulating the endothelial dysfunction of coronary microvessels during NAFLD. RESULTS: We established two murine NAFLD models by feeding mice a methionine-choline-deficient (MCD) diet for 4 weeks or a high-fat diet (HFD) for 16 weeks. We found that the NOD-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome-dependent endothelial hyperpermeability occurred in coronary microvessels during both MCD diet and HFD-induced NAFLD. The in vivo and in vitro experiments proved that novel-microRNA(miR)-7-abundant hepatic sEVs were responsible for NLRP3 inflammasome-dependent endothelial barrier dysfunction. Mechanistically, novel-miR-7 directly targeted lysosomal associated membrane protein 1 (LAMP1) and promotes lysosomal membrane permeability (LMP), which in turn induced Cathepsin B-dependent NLRP3 inflammasome activation and microvascular endothelial hyperpermeability. Conversely, a specific novel-miR-7 inhibitor markedly improved endothelial barrier integrity. Finally, we proved that steatotic hepatocyte was a significant source of novel-miR-7-contained hepatic sEVs, and steatotic hepatocyte-derived sEVs were able to promote NLRP3 inflammasome-dependent microvascular endothelial hyperpermeability through novel-miR-7. CONCLUSIONS: Hepatic sEVs contribute to endothelial hyperpermeability in coronary microvessels by delivering novel-miR-7 and targeting the LAMP1/Cathepsin B/NLRP3 inflammasome axis during NAFLD. Our study brings new insights into the liver-to-microvessel cross-talk and may provide a new diagnostic biomarker and treatment target for microvascular complications of NAFLD.

[Qingre Xiaoyanning Capsules alleviate HSV-1 susceptibility induced by emotional stress].

In this study, emotional stress-induced herpes simplex virus type 1(HSV-1) susceptibility model was employed to simu-late the pathological state of " depression-induced liver fire", and the protection effect of Qingre Xiaoyanning(QX) in clearing liver fire was investigated. BALB/c mice were randomly divided into a normal group, a HSV-1 group, a restraint stress + HSV-1 group,low-(0. 658 g·kg~(-1)) and high-dose(1. 316 g·kg~(-1)) QX groups, and an acyclovir group. Except for the normal group and the HSV-1 group, the mice in other groups received daily restraint stress for 6 h from day 3 of medication. On day 9 of medication, mice were anesthetized by isoflurane and infected intranasally with HSV-1. Survival rate, weight change, encephalitis symptoms, and eye injury of mice were recorded for 14 d after virus infection. Hematoxylin-eosin(HE) staining and immunohistochemical staining were used to detect pathological changes and HSV-1 antigen distribution. Plaque assay was performed to detect the titer of HSV-1. The protein ex-pression of ICP27 in the mouse brain was detected by Western blot. The experimental results showed that QX could increase the survival rate of HSV-1-infected mice loaded with emotional stress(P<0. 001), reduce the titer of HSV-1 in the mouse brain(P<0. 01), relieve brain inflammation(P<0. 05) and eye injury(P<0. 05), down-regulate the expression of ICP27 related to HSV-1(P<0. 05), and decrease the distribution of HSV-1 antigen in the mouse brain. The results demonstrated that QX significantly reduced the susceptibility to HSV-1 induced by emotional stress, which is expected to provide a theoretical basis for the treatment and preven-tion of HSV-1 infection and promote the clinical development and application of Chinese medicine effective in clearing liver fire.

Discovery of Neuritogenic Securinega Alkaloids from Flueggea suffruticosa by a Building Blocks-Based Molecular Network Strategy.

A combined strategy of building blocks recognition and molecular network construction, termed the building blocks-based molecular network (BBMN), was first presented to facilitate the efficient discovery of novel natural products. By mapping the BBMN of the total alkaloid fraction of Flueggea suffruticosa, three Securinega alkaloids (SEAs) with unusual chemical architectures, suffranidines A-C (1-3), were discovered and isolated. Compound 1 characterizes an unprecedented 8/5/6/5/6/6/6/6-fused octacyclic scaffold with a unique cage-shaped 3-azatricyclo[6.4.0.03,11 ]dodecane core. Compounds 2 and 3 are highly modified SEA dimers that incorporate additional C6 motifs. A hypothetical biosynthetic pathway for 1-3 was proposed. In addition, 1 significantly induced neuronal differentiation and neurite extension by upregulating eukaryotic elongation factor 2 (eEF2)-mediated protein synthesis.