Trigonochinene E promotes lysosomal biogenesis and enhances autophagy via TFEB/TFE3 in human degenerative NP cells against oxidative stress.

BACKGROUND: Macroautophagy (henceforth autophagy) is the major form of autophagy, which delivers intracellular cargo to lysosomes for degradation. Considerable research has revealed that the impairment of lysosomal biogenesis and autophagic flux exacerbates the development of autophagy-related diseases. Therefore, reparative medicines restoring lysosomal biogenesis and autophagic flux in cells may have therapeutic potential against the increasing prevalence of these diseases. PURPOSE: The aim of the present study was thus to explore the effect of trigonochinene E (TE), an aromatic tetranorditerpene isolated from Trigonostemon flavidus, on lysosomal biogenesis and autophagy and to elucidate the potential underlying mechanism. METHODS: Four human cell lines, HepG2, nucleus pulposus (NP), HeLa and HEK293 cells were applied in this study. The cytotoxicity of TE was evaluated by MTT assay. Lysosomal biogenesis and autophagic flux induced by 40 µM TE were analyzed using gene transfer techniques, western blotting, real-time PCR and confocal microscopy. Immunofluorescence, immunoblotting and pharmacological inhibitors/activators were applied to determine the changes in the protein expression levels in mTOR, PKC, PERK, and IRE1α signaling pathways. RESULTS: Our results showed that TE promotes lysosomal biogenesis and autophagic flux by activating the transcription factors of lysosomes, transcription factor EB (TFEB) and transcription factor E3 (TFE3). Mechanistically, TE induces TFEB and TFE3 nuclear translocation through an mTOR/PKC/ROS-independent and endoplasmic reticulum (ER) stress-mediated pathway. The PERK and IRE1α branches of ER stress are crucial for TE-induced autophagy and lysosomal biogenesis. Whereas TE activated PERK, which mediated calcineurin dephosphorylation of TFEB/TFE3, IRE1α was activated and led to inactivation of STAT3, which further enhanced autophagy and lysosomal biogenesis. Functionally, knockdown of TFEB or TFE3 impairs TE-induced lysosomal biogenesis and autophagic flux. Furthermore, TE-induced autophagy protects NP cells from oxidative stress to ameliorate intervertebral disc degeneration (IVDD). CONCLUSIONS: Here, our study showed that TE can induce TFEB/TFE3-dependent lysosomal biogenesis and autophagy via the PERK-calcineurin axis and IRE1α-STAT3 axis. Unlike other agents regulating lysosomal biogenesis and autophagy, TE showed limited cytotoxicity, thereby providing a new direction for therapeutic opportunities to use TE to treat diseases with impaired autophagy-lysosomal pathways, including IVDD.

Dietary Supplementation of Cedryl Acetate Ameliorates Adiposity and Improves Glucose Homeostasis in High-Fat Diet-Fed Mice.

Cedryl acetate (CA), also called acetyl cedrene, is approved by the FDA as a flavoring or adjuvant to be added to foods. In this study, we aimed to investigate the preventive benefits of CA on obesity and obesity-related metabolic syndrome caused by a high-fat diet (HFD). Three groups of C57BL/6J mice (ten-week-old) were fed Chow, an HFD, or an HFD with CA supplementation (100 mg/kg) for 19 weeks. We observed that CA supplementation significantly reduced weight gain induced by an HFD, decreased the weight of the visceral fat pads, and prevented adipocyte hypertrophy in mice. Moreover, mice in the CA group showed significant improvements in hepatic lipid accumulation, glucose intolerance, insulin resistance, and gluconeogenesis compared with the mice in the HFD group. Since 16S rRNA analysis revealed that the gut microbiota in the CA and HFD groups were of similar compositions at the phylum and family levels, CA may have limited effects on gut microbiota in HFD-fed mice. The beneficial effects on the metabolic parameters of CA were reflected by CA's regulation of metabolism-related gene expression in the liver (including Pepck, G6Pase, and Fbp1) and the epididymal white adipose tissues (including PPARγ, C/EBPα, FABP4, FAS, Cytc, PGC-1α, PRDM16, Cidea, and COX4) of the mice. In summary, a potent preventive effect of CA on HFD-induced obesity and related metabolic syndrome was highlighted by our results, and CA could be a promising dietary component for obesity intervention.

A Review of Aspects of Synaptic Plasticity in Hippocampus via mT Extremely Low-Frequency Magnetic Fields.

The subthreshold magnetic modulation technique stimulates cells with mT extremely low-frequency magnetic fields (ELF-MFs), which are insufficient to induce neuronal action potentials. Although they cannot directly induce resting neurons to discharge, mT magnetic stimulation can regulate the excitability of the nervous system, which regulates learning and memory by some unknown mechanisms. Herein, we describe the regulation of mT ELF-MFs with different parameters on synaptic plasticity in hippocampal neurons. Additionally, we summarize the latest research on the possible mechanism of the effect of ELF-MFs on synaptic plasticity. Some studies have shown that ELF-MFs are able to inhibit long-term potentiation (LTP) by increasing concentration of intracellular Ca2+ concentration ([Ca2+ ]i ), as well as concentration of reactive oxygen species. The research in this paper has significance for the comprehensive understanding of relevant neurological mechanisms of learning and memory by mT ELF-MFs stimulation. However, more high-quality research is necessary to determine the regulatory mechanism of mT ELF-MFs on synaptic plasticity in order to optimize this technique as a treatment for neurological diseases. © 2023 Bioelectromagnetics Society.

HDAC5 Loss Enhances Phospholipid-Derived Arachidonic Acid Generation and Confers Sensitivity to cPLA2 Inhibition in Pancreatic Cancer.

HDAC5 is a class IIa histone deacetylase member that is downregulated in multiple solid tumors, including pancreatic cancer, and loss of HDAC5 is associated with unfavorable prognosis. In this study, assessment of The Cancer Genome Atlas pancreatic adenocarcinoma dataset revealed that expression of HDAC5 correlates negatively with arachidonic acid (AA) metabolism, which has been implicated in inflammatory responses and cancer progression. Nontargeted metabolomics analysis revealed that HDAC5 knockdown resulted in a significant increase in AA and its downstream metabolites, such as eicosanoids and prostaglandins. HDAC5 negatively regulated the expression of the gene encoding calcium-dependent phospholipase A2 (cPLA2), the key enzyme in the production of AA from phospholipids. Mechanistically, HDAC5 repressed cPLA2 expression via deacetylation of GATA1. HDAC5 knockdown in cancer cells enhanced sensitivity to genetic or pharmacologic inhibition of cPLA2 in vitro and in vivo. Fatty acid supplementation in the diet reversed the sensitivity of HDAC5-deficient tumors to cPLA2 inhibition. These data indicate that HDAC5 loss in pancreatic cancer results in the hyperacetylation of GATA1, enabling the upregulation of cPLA2, which contributes to overproduction of AA. Dietary management plus cPLA2-targeted therapy could serve as a viable strategy for treating HDAC5-deficient pancreatic cancer patients. SIGNIFICANCE: The HDAC5-GATA1-cPLA2-AA signaling axis regulates sensitivity to fat restriction plus cPLA2 inhibition in pancreatic ductal adenocarcinoma, proposing dietary management as a feasible strategy for treating a subset of patients with pancreatic cancer.

Eugenol, A Major Component of Clove Oil, Attenuates Adiposity, and Modulates Gut Microbiota in High-Fat Diet-Fed Mice.

SCOPE: Eugenol (EU), the major aromatic compound derived from clove oil, is being focused recently due to its potential in preventing several chronic conditions. Herein, this study aims to evaluate the potential of EU in obesity prevention and to delineate the mechanisms involved. METHODS AND RESULTS: Five-week-old male C57BL/6J mice are fed with high-fat diet (HFD) or HFD supplemented with EU (0.2%, w/w) for 13 weeks. EU significantly reduces obesity-related indexes including final body weight, body weight gain, adipocyte size, visceral fat-pad weight, and fasting blood glucose. EU prevents HFD-induced gut dysbiosis, as indicated by the increase of Firmicutes and decrease of Desulfobacterota at phylum level, and the increase of Dubosiella, Blautia, unclassified_f_Oscillospiraceae, and unclassified_f_Ruminococcaceae, and the decrease of Alistipes, Alloprevotella, and Bilophila at genus level. Notably, the obesity-related indexes are positively correlated with the relative abundances of Bacteroides, unclassified_f_Lachnospiraceae, Colidextribacter, and Bilophila, and negatively correlated with the relative abundances of norank_f_Muribaculaceae and Lachnospiraceae_NK4A136_group. Moreover, the preventive effects of EU on obesity are accompanied by the transcriptomic reprogramming of white adipose tissue. CONCLUSION: These findings demonstrate that EU prevents the HFD-induced adiposity and modulates gut dysbiosis, and highlight the potential of EU in obesity intervention as a functional dietary supplement.

Antifeedant Mechanism of Dodonaea viscosa Saponin A Isolated from the Seeds of Dodonaea viscosa.

Dodonaea viscosa is a medicinal plant which has been used to treat various diseases in humans. However, the anti-insect activity of extracts from D. viscosa has not been evaluated. Here, we found that the total saponins from D. viscosa (TSDV) had strong antifeedant and growth inhibition activities against 4th-instar larvae of Spodoptera litura. The median antifeeding concentration (AFC50) value of TSDV on larvae was 1621.81 µg/mL. TSDV affected the detoxification enzyme system of the larvae and also exerted antifeedant activity possibly through targeting the γ-aminobutyric acid (GABA) system. The AFC50 concentration, the carboxylesterase activity, glutathione S-transferases activity, and cytochrome P450 content increased to 258%, 205%, and 215%, respectively, and likewise the glutamate decarboxylase activity and GABA content to 195% and 230%, respectively, in larvae which fed on TSDV. However, D. viscosa saponin A (DVSA) showed better antifeedant activity and growth inhibition activity in larvae, compared to TSDV. DVSA also exerted their antifeedant activity possibly through targeting the GABA system and subsequently affected the detoxification enzyme system. Further, DVSA directly affected the medial sensillum and the lateral sensillum of the 4th-instar larvae. Stimulation of Spodoptera litura. with DVSA elicited clear, consistent, and robust excitatory responses in a single taste cell.

Identification of limonoids from Walsura yunnanensis and evaluation of their cytotoxicity against cancer cell lines.

Three new limonoids, walsurauias A-C (1-3), along with four known ones, were isolated from the leaves and twigs of Walsura yunnanensis C. Y. Wu. Their structures were determined on the basis of comprehensive spectroscopic data analysis. The new limonoids were screened for their cytotoxic activity (IC50 0.81-5.73 µM) against four human cancer cell lines, including A549, HepG2, HCT116 p21KO and CNE-2. And α,ß-unsaturated ketone moieties in rings A and B are essential for their cytotoxic activity. Selected compounds were further investigated. Compounds 1-3 effectively induced G2/M cell cycle arrest and apoptosis in a dose-dependent manner in cancer cells. In addition, compounds 1-3 inhibited the colony formation and compounds 2 and 3 suppressed the migration of cancer cells.

l-carnitine supplementation during in vitro culture regulates oxidative stress in embryos from bovine aged oocytes.

Aging oocytes undergo various molecular, cellular, and biochemical changes. Aging of oocytes results in reduced embryo developmental capacity and blastocyst quality, which is thought to be caused partly by the accumulation of reactive oxygen species (ROS). This study aimed to determine the effect of l-carnitine (LC) on the development of embryos formed from aged oocytes in vitro. The development and quality of the blastocysts in the LC-treated group were significantly higher than those in the untreated aged group after in vitro fertilization (IVF). In addition, after LC treatment, the level of intracellular ROS in aged group significantly decreased, and glutathione (GSH) levels significantly increased compared with those in the untreated aged group. There was no significant difference in the mitochondrial membrane potential among the three groups. Moreover, ROS could induce autophagy and LC3 antibody was widely used as a marker for detecting autophagy. The fluorescence intensity of LC3 in the aged group was significantly higher than that of LC3 in the LC-treated group. Furthermore, Real-time reverse transcriptase-polymerase chain reaction showed that the mRNA levels of antioxidation genes GPX4 and SOD1 were significantly higher in embryos from LC-treated group than in those from the untreated aged group. In summary, our results indicated that LC can improve the developmental capacity of embryos from aging oocytes in vitro by reducing oxidative stress.

L-carnitine prevents bovine oocyte aging and promotes subsequent embryonic development.

L-carnitine (LC) is well known for its antioxidant activity. In this study, we explored the potential mechanistic effects of LC supplementation on aged bovine oocytes in vitro. We showed that in-vitro maturation could enhance the subsequent developmental capacity of aging oocytes, when supplemented with LC. After in vitro fertilization, the blastocyst formation rate in the aged oocytes post-LC treatment significantly increased compared to that in untreated aged oocytes (29.23 ± 2.20% vs. 20.90 ± 3.05%). Furthermore, after LC treatment, the level of intracellular reactive oxygen species in aged oocytes significantly decreased, and glutathione levels significantly increased, compared to those in untreated aged oocytes. Mitochondrial membrane potential, the percentage of early apoptotic oocytes, and caspase-3 activity were significantly reduced in LC-treated aged oocytes compared to those in untreated aged oocytes. Furthermore, during in vitro aging, the mRNA levels of the anti-apoptotic genes, Bcl-xl and survivin in LC-treated aged oocytes were significantly higher than those in untreated aged oocytes. Overall, these results indicate that at least in in vitro conditions, LC can prevent the aging of bovine oocytes and improve the developmental capacity of bovine embryo.

Extraction, Purification, and Hydrolysis Behavior of Apigenin-7-O-Glucoside from Chrysanthemum Morifolium Tea.

Apigenin-7-O-glucoside is an active phenolic compound in Asteraceae flowers and possesses remarkable therapeutic applications. However, its high price and low abundance in plants limit its use, meanwhile it would hydrolyze in the purification process. In this study, apigenin-7-O-glucoside extracted with ultrasound and purified with preparative HPLC from Chrysanthemum morifolium 'Huangju' was investigated, as well as its hydrolysis behavior and bioactivities. The optimized extraction conditions were: solid/liquid ratio: 1:20, extraction time: 35 min, temperature: 50 °C, and ultrasound power: 350 W. The content of apigenin-7-O-glucoside was up to 16.04 mg/g. Apigenin-7-O-glucoside was then purified with preparative HPLC from the extract, and confirmed by Q-TOF/MS. Apigenin-7-O-glucoside was partially hydrolyzed in acidic condition, and the hydrolysis rate depended on the pH value and temperature. The antioxidant activity increased as a result of the hydrolysis process. This study provided a green and effective way to obtain apigenin-7-O-glucoside and would be beneficial for further investigations into nutritional and functional aspects apigenin-7-O-glucoside and other glycosides.

ZNF185-derived peptide induces fertility suppression in mice.

Zinc finger protein 185 (ZNF185) belongs to the ZNF family and is involved in male reproduction. However, it is unclear whether ZNF185 may be a target candidate for contraceptive vaccines. In this study, antigenic peptides derived from ZNF185 were prepared, and their immune contraceptive effects were investigated using mice. Results from enzyme-linked immunosorbent assay (ELISAs) showed that peptide immunization induced an antibody titre increase that reached a peak in week 12. Peptide-3 and peptide-4 were then chosen for subsequent experiments. The results of the fertility assays showed that peptide immunization inhibited the mating and fertility rates of the mice, whereas there were no obvious changes in the number of pups per litter. Subsequently, epididymal sperm was analysed. The results demonstrated that the sperm count and sperm motility were significantly decreased in the peptide group, while the amount of abnormal sperm was significantly increased in the peptide-3 group. The male reproductive organs were also evaluated. There were no obvious differences in testis or epididymal weights, in the diameters of the seminiferous tubules, or in the thicknesses of the seminiferous epithelium between the peptide group and the phosphate buffer saline (PBS) group. In addition, histological analysis indicated that there were no obvious pathologic changes in testis and epididymal histology in the peptide group; however, the number of spermatozoa present in the epididymal lumen of the peptide group was significantly decreased when compared with the PBS group. Our study demonstrates for the first time that peptides derived from ZNF185 may induce fertility suppression in mice without damaging reproductive organs. These peptides have the potential to be used as a male contraceptive vaccine.