[Pharmacokinetics, pharmacodynamics, and tissue distribution of oral co-loaded puerarin/daidzein mixed micelles in rats].

This study investigated the drug delivery performance of oral co-loaded puerarin(PUE) and daidzein(DAZ) mixed micelles(PUE/DAZ-FS/PMMs) from the perspectives of pharmacokinetics, pharmacodynamics, and tissue distribution. The changes in PUE plasma concentration in rats were evaluated based on PUE suspension, single drug-loaded micelles(PUE-FS/PMMs), and co-loaded micelles(PUE/DAZ-FS/PMMs). Spontaneously hypertensive rats(SHR) were used to monitor systolic blood pressure, diastolic blood pressure, and mean arterial pressure for 10 weeks after administration by tail volume manometry. The content of PUE in the heart, liver, spleen, lung, kidney, brain, and testes was determined using LC-MS/MS. The results showed that compared with PUE suspension and PUE-FS/PMMs, PUE/DAZ-FS/PMMs significantly increased C_(max) in rats(P<0.01) and had a relative bioavailability of 122%. The C_(max), AUC_(0-t), AUC_(0-∞), t_(1/2), and MRT of PUE/DAZ-FS/PMMs were 1.77, 1.22, 1.22, 1.17, and 1.13 times higher than those of PUE suspension, and 1.76, 1.16, 1.08, 0.84, and 0.78 times higher than those of PUE-FS/PMMs, respectively. Compared with the model control group, PUE/DAZ-FS/PMMs significantly reduced systolic blood pressure, diastolic blood pressure, and mean arterial pressure in SHR rats(P<0.05). The antihypertensive effect of PUE/DAZ-FS/PMMs was greater than that of PUE suspension, and even greater than that of PUE-FS/PMMs at high doses. Additionally, the distribution of PMMs in various tissues showed dose dependency. The distribution of PMMs in the kidney and liver, which are metabolically related tissues, was lower than that in the suspension group, while the distribution in the brain was higher than that in the conventional dose group. In conclusion, PUE/DAZ-FS/PMMs not only improved the bioavailability of PUE and synergistically enhanced its therapeutic effect but also prolonged the elimination of the drug to some extent. Furthermore, the micelles facilitated drug penetration through the blood-brain barrier. This study provides a foundation for the development of co-loaded mixed micelles containing homologous components.

Dendrobium officinale Polysaccharides Inhibit 1-Methyl-2-Nitro-1-Nitrosoguanidine Induced Precancerous Lesions of Gastric Cancer in Rats through Regulating Wnt/ß-Catenin Pathway and Altering Serum Endogenous Metabolites.

Dendrobium officinale is a herb in traditional Chinese medicine where D. officinale polysaccharides (DOP) are the main active ingredient. This study aimed at evaluating DOP efficiency at inhibiting 1-Methyl-2-nitro-1-nitrosoguanidine (MNNG) induced precancerous lesions of gastric cancer (PLGC) in rats through the Wnt/b-catenin pathway and analyzing the variations of serum endogenous metabolites. PLGC was established in male Sprague-Dawley (SD) rats by administering 150 µg/mL MNNG in drinking water for 7 months and giving 0.1 mL of 10% NaCl once weekly during the initial 20 weeks. Treatment with DOP inhibited the progress of PLGC through decreasing the expression of ß-catenin by immunohistochemical analysis. The futher study indicated DOP downregulated gene expression of Wnt2ß, Gsk3ß, PCNA, CyclinD1, and ß-catenin, as well as protein expression of Wnt2ß, PCNA, and ß-catenin. On the other hand, there were nine endogenous metabolites identified after the DOP treatment. Among these, the most significant one is betaine because of its strong antioxidant activity, leading to an anti-tumor effect. DOP can inhibit MNNG-induced PLGC models via regulating Wnt/ß-catenin pathway and by changing endogenous metabolites.

Casp3/7-Instructed Intracellular Aggregation of Fe3O4 Nanoparticles Enhances T2 MR Imaging of Tumor Apoptosis.

Large magnetic nanoparticles or aggregates are advantageous in their magnetic resonance properties over ultrasmall superparamagnetic iron oxide (USPIO) nanoparticles (NPs), but the former are cleared faster from the blood pool. Therefore, the "smart" strategy of intracellular aggregation of USPIO NPs is required for enhanced T2-weighted MR imaging. Herein, employing an enzyme-instructed condensation reaction, we rationally designed a small molecule Ac-Asp-Glu-Val-Asp-Cys(StBu)-Lys-CBT (1) to covalently modify USPIO NPs to prepare monodispersive Fe3O4@1 NPs. In vitro results showed that Fe3O4@1 NPs could be subjected to caspase 3 (Casp3)-instructed aggregation. T2 phantom MR imaging showed that the transverse molar relaxivity (r2) of Fe3O4@1 NPs with Casp3 or apoptotic HepG2 cells was significantly larger than those of control groups. In vivo tumor MR imaging results indicated that Fe3O4@1 NPs could be specifically applied for enhanced T2 MR imaging of tumor apoptosis. We propose that the enzyme-instructed intracellular aggregation of Fe3O4 NPs could be a novel strategy for the design of "smart" probes for efficient T2 MR imaging of in vivo biomarkers.

Erjingpill bionic cerebrospinal fluid alleviates LPS-induced inflammatory response in BV2 cells by inhibiting glycolysis via mTOR.

ETHNOPHARMACOLOGICAL RELEVANCE: Erjingpill, a well-known prescription documented in the classic Chinese medical text "Shengji Zonglu," has been proven to have effective alleviating effects on neuroinflammation in Alzheimer's disease (AD). Although the alterations in microglial cell glycolysis are known to play a crucial role in the development of neuroinflammation, it remains unclear whether the anti-neuroinflammatory effects of Erjingpill are associated with its impact on microglial cell glycolysis. AIM OF THE STUDY: This study aims to determine whether Erjingpill exerts anti-neuroinflammatory effects by influencing microglial cell glycolysis. MATERIALS AND METHODS: Firstly, Erjingpill decoction was prepared into an Erjingpill bionic cerebrospinal fluid (EBCF) through a process of in vitro intestinal absorption, hepatocyte incubation, and blood-brain barrier (BBB) transcytosis. Subsequently, UPLC/Q-TOF-MS/MS technology was used to analyze the compounds in Erjingpill and EBCF. Next, an in vitro neuroinflammation model was established by LPS-induced BV2 cells. The impact of EBCF on BV2 cell proliferation activity was evaluated using the CCK-8 assay, while the NO release was assessed using the Griess assay. Additionally, mRNA levels of pro-inflammatory factors (IL-1ß, IL-6, TNF-α, and COX-2), anti-inflammatory factors (IL-10, IL-4, Arg-1, and TGF-ß), M1 microglial markers (iNOS, CD86), M2 microglial markers (CD36, CD206), and glycolytic enzymes (HK2, GLUT1, PKM, and LDHA) were measured using qPCR. Furthermore, protein expression of microglial activation marker Iba-1, M1 marker iNOS, and M2 marker CD206 were identified through immunofluorescence, while concentrations of pro-inflammatory cytokines IL-1ß and TNF-α were measured using ELISA. Enzymatic activity of glycolytic enzymes (HK, PK, and LDH) was assessed using assay kits, and the protein levels of pro-inflammatory factors (IL-1ß, iNOS, and COX-2), anti-inflammatory factors (IL-10 and Arg-1), and key glycolytic proteins GLUT1 and PI3K/AKT/mTOR were detected by Western blot. RESULTS: Through the analysis of Erjingpill and EBCF, 144 compounds were identified in Erjingpill and 40 compounds were identified in EBCF. The results demonstrated that EBCF effectively inhibited the elevation of inflammatory factors and glycolysis levels in LPS-induced BV2 cells, promoted polarization of M1 microglial cells towards the M2 phenotype, and suppressed the PI3K/AKT/mTOR inflammatory pathway. Moreover, EBCF alleviated LPS-induced BV2 cell inflammatory response by modulating mTOR to inhibit glycolysis. CONCLUSIONS: EBCF exhibits significant anti-neuroinflammatory effects, likely attributed to its modulation of mTOR to inhibit microglial cell glycolysis. This study furnishes experimental evidence supporting the clinical utilization of Erjingpill for preventing and treating AD.

Incorporation of Epoxy Carbon onto CeO2-Supported Pt to Tackle the CO Self-Poisoning Issue.

The catalytic oxidation of carbon monoxide (CO) under ambient conditions plays a crucial role in the abatement of indoor CO, which poses risks to human health. Despite the notable activity exhibited by Pt-based catalysts in CO oxidation, their efficacy is usually diminished by the CO self-poisoning issue. In this work, three different Pt/CeO2-based catalysts, which have distinct coordinative environments of Pt but an identical Pt/CeO2 substrate structure, were synthesized by activating the catalyst with CO using different temperatures and durations. Compared with clean and graphite-covered Pt on CeO2, the one modified by epoxy carbon showed higher activity and stability. The combination of characterizations and density functional theory modeling demonstrated that the clean Pt on CeO2 rapidly deactivated due to the CO self-poisoning albeit high initial activity, and conversely, low initial activity was observed for the more stable graphite-covered catalyst due to the obstruction of the Pt site. In contrast, epoxy carbon species on Pt shifted the d-band of Pt to lower energy, weakening the Pt-CO binding strength. Such a modification mitigated the self-poisoning effect while maintaining ample active sites and enabling the complete oxidative removal of CO under ambient conditions. This work may provide a general approach to tackling the self-poisoning issue.

Acute and subacute hepatotoxicity of genipin in mice and its potential mechanism.

Gardenia, as a medicinal and edible herb, has the pharmacological activity of protecting the liver and cholagogue, but the hepatotoxicity induced by the chemical component genipin (GP) limits its application. The aim of this study was to evaluate the acute and subacute hepatotoxicity of genipin in normal mice and mice with α-naphthalene isothiocyanate (ANIT)-induced liver injury. The results of the acute study showed that the LD50 of genipin was 510 mg/kg. Genipin exhibited hepatotoxicity in normal and jaundiced mice at doses of 125 mg/kg, 250 mg/kg, and 500 mg/kg, which increased with dose. In a 28-day subacute study, the 50 mg/kg and 100 mg/kg dose groups showed some pharmacodynamic effects at 7 days but exhibited hepatotoxicity that increased with time and improved after drug withdrawal. In addition, based on proteomics, the mechanism of liver injury induced by genipin may be related to the disruption of the UDP-glucuronosyltransferase system and cytochrome P450 enzyme activity. In conclusion, this study showed that genipin hepatotoxicity was time- and dose dependent, but it is worth mentioning that hepatotoxicity was reversible. It is hoped that this study will provide a scientific basis for circumventing the adverse effects of genipin.

Mitochondrial MICOS complex genes, implicated in hypoplastic left heart syndrome, maintain cardiac contractility and actomyosin integrity.

Hypoplastic left heart syndrome (HLHS) is a severe congenital heart disease (CHD) with a likely oligogenic etiology, but our understanding of the genetic complexities and pathogenic mechanisms leading to HLHS is limited. We performed whole genome sequencing (WGS) on 183 HLHS patient-parent trios to identify candidate genes, which were functionally tested in the Drosophila heart model. Bioinformatic analysis of WGS data from an index family of a HLHS proband born to consanguineous parents prioritized 9 candidate genes with rare, predicted damaging homozygous variants. Of them, cardiac-specific knockdown (KD) of mitochondrial MICOS complex subunit dCHCHD3/6 resulted in drastically compromised heart contractility, diminished levels of sarcomeric actin and myosin, reduced cardiac ATP levels, and mitochondrial fission-fusion defects. These defects were similar to those inflicted by cardiac KD of ATP synthase subunits of the electron transport chain (ETC), consistent with the MICOS complex's role in maintaining cristae morphology and ETC assembly. Five additional HLHS probands harbored rare, predicted damaging variants in CHCHD3 or CHCHD6. Hypothesizing an oligogenic basis for HLHS, we tested 60 additional prioritized candidate genes from these patients for genetic interactions with CHCHD3/6 in sensitized fly hearts. Moderate KD of CHCHD3/6 in combination with Cdk12 (activator of RNA polymerase II), RNF149 (goliath, E3 ubiquitin ligase), or SPTBN1 (ß-Spectrin, scaffolding protein) caused synergistic heart defects, suggesting the likely involvement of diverse pathways in HLHS. Further elucidation of novel candidate genes and genetic interactions of potentially disease-contributing pathways is expected to lead to a better understanding of HLHS and other CHDs.

Strong static magnetic field delayed the early development of zebrafish.

One of the major topics in magnetobiology is the biological effects of strong static magnetic field (SMF) on living organisms. However, there has been a paucity of the comprehensive study of the long-term effects of strong SMF on an animal's development. Here, we explored this question with zebrafish, an excellent model organism for developmental study. In our research, zebrafish eggs, just after fertilization, were exposed to a 9.0 T SMF for 24 h, the critical period of post-fertilization development from cleavage to segmentation. The effects of strong SMF exposure on the following developmental progress of zebrafish were studied until 6 days post-fertilization (dpf). Results showed that 9.0 T SMF exposure did not influence the survival or the general developmental scenario of zebrafish embryos. However, it slowed down the developmental pace of the whole animal, and the late developers would catch up with their control peers after the SMF was removed. We proposed a mechanical model and deduced that the development delaying effect was caused by the interference of SMF in microtubule and spindle positioning during mitosis, especially in early cleavages. Our research data provide insights into how strong SMF influences the developing organisms through basic physical interactions with intracellular macromolecules.

N-Cadherin is Involved in Neuronal Activity-Dependent Regulation of Myelinating Capacity of Zebrafish Individual Oligodendrocytes In Vivo.

Stimulating neuronal activity increases myelin sheath formation by individual oligodendrocytes, but how myelination is regulated by neuronal activity in vivo is still not fully understood. While in vitro studies have revealed the important role of N-cadherin in myelination, our understanding in vivo remains quite limited. To obtain the role of N-cadherin during activity-dependent regulation of myelinating capacity of individual oligodendrocytes, we successfully built an in vivo dynamic imaging model of the Mauthner cell at the subcellular structure level in the zebrafish central nervous system. Enhanced green fluorescent protein (EGFP)-tagged N-cadherin was used to visualize the stable accumulations and mobile transports of N-cadherin by single-cell electroporation at the single-cell level. We found that pentylenetetrazol (PTZ) significantly enhanced the accumulation of N-cadherin in Mauthner axons, a response that was paralleled by enhanced sheath number per oligodendrocytes. By offsetting this phenotype using oligopeptide (AHAVD) which blocks the function of N-cadherin, we showed that PTZ regulates myelination in an N-cadherin-dependent manner. What is more, we further suggested that PTZ influences N-cadherin and myelination via a cAMP pathway. Consequently, our data indicated that N-cadherin is involved in neuronal activity-dependent regulation of myelinating capacity of zebrafish individual oligodendrocytes in vivo.

Exogenous melatonin inhibits neutrophil migration through suppression of ERK activation.

Neutrophil migration to inflammatory sites is the fundamental process of innate immunity among organisms against pathogen invasion. As a major sleep adjusting hormone, melatonin has also been proved to be involved in various inflammatory events. This study aimed to evaluate the impact of exogenous melatonin on neutrophil migration to the injury site in live zebrafish and further investigate whether ERK signaling is involved in this process. Using the tail fin transection model, the fluorescently labeled neutrophil was in vivo visualized in transgenic Tg(lyz:EGFP), Tg(lyz:DsRed) zebrafish. We found that exogenous melatonin administration dramatically inhibited the injury-induced neutrophil migration in a dose-dependent and time-dependent manner. The inhibited effect of melatonin on neutrophil migration could be attenuated by melatonin receptor 1, 2, and 3 antagonists. The ERK phosphorylation level was significantly decreased post injury when treated with melatonin. The blocking of ERK activation with inhibitor PD0325901 suppressed the number of migrated neutrophils in response to injury. However, the activation of ERK with the epidermal growth factor could impair the inhibited effect of melatonin on neutrophil migration. We also detected that PD0325901 significantly suppressed the in vivo neutrophils transmigrating over the vessel endothelial cell using the transgenic Tg(flk:EGFP);(lyz:DsRed) line labeled as both vessel and neutrophil. Taking all of these data together, the results indicated that exogenous melatonin had an anti-migratory effect on neutrophils by blocking the ERK phosphorylation signal, and it led to the subsequent adhesion molecule expression. Thus, the crossing of the vessel endothelial cells of neutrophils became difficult.

Controlled intracellular self-assembly and disassembly of 19F nanoparticles for MR imaging of caspase 3/7 in zebrafish.

Compared to (1)H MRI, (19)F MRI provides higher selectivity but lower sensitivity. Therefore, the need to inject high doses of the (19)F probe to improve its sensitivity for in vivo diagnosis remains a challenge. A "smart" strategy is needed that could locally concentrate a low-dose (19)F probe while avoiding the fast transverse relaxation of the probes. Locally self-assembling and disassembling (19)F nanoparticles may be an optimal measure to achieve this goal. Herein, we report a dual-functional probe 1 for glutathione (GSH)-controlled self-assembly and subsequent caspase 3/7 (Casp3/7)-controlled disassembly of formed nanoparticles (i.e., 1-NPs). Consecutive assembly and disassembly of 1-NPs translate to "off" and "on" (19)F magnetic resonance (MR) signal states, respectively. Employing this smart strategy, we successfully used 1 for the consecutive detection of GSH and Casp3/7 activity in vitro and in cells and imaging Casp3/7 activity in cells and in zebrafish at low doses with a 14.1 T magnetic field.

Intracellular Self-Assembly and Disassembly of (19)F Nanoparticles Confer Respective "Off" and "On" (19)F NMR/MRI Signals for Legumain Activity Detection in Zebrafish.

(19)F MRI has higher selectivity but lower sensitivity than (1)H MRI for in vivo diagnosis. Therefore, to avoid using a high injection dose of the (19)F probe while, in the meantime, maintaining the high sensitivity of (19)F MRI has remained challenging. Local self-assembly and disassembly of (19)F nanoparticles could be one of the "smart" strategies to achieve this goal. Herein, we report a dual-functional probe 1 for glutathione (GSH)-controlled self-assembly and subsequent legumain (Lgmn)-controlled disassembly of its nanoparticles (i.e., 1-NPs). Self-assembly and disassembly of 1-NPs confer (19)F magnetic resonance (MR) signals "off" and "on", respectively. Employing this strategy, we successfully applied 1 for consecutive detections of GSH and Lgmn in vitro and in cells, imaging Lgmn activity in HEK 293T tumors in zebrafish at a low dosage under 14.1 T.

Neurogenesis of retinal ganglion cells is not essential to visual functional recovery after optic nerve injury in adult zebrafish.

Zebrafish central nervous system (CNS) possesses a strong neural regeneration ability to restore visual function completely after optic nerve injury (ONI). However, whether neurogenesis of retinal ganglion cell (RGC) contributes to functional recovery remains controversial. Our quantitative analysis of RGCs in different ONI models showed that almost all RGCs survived in optic nerve crush (ONC) model; while over 90% of RGCs survived in the first 2 weeks with 75% remaining after 7 weeks in optic nerve transection (ONT) model. Retrograde labeling from tectum revealed a surprising regeneration rate, with over 90% and over 50% of RGCs regrowing axons to tectum at the first week in ONC and ONT model respectively. In the latter one, the number of regenerative RGCs after 4 weeks had no significant difference from the control group. As for neurogenesis, newborn RGCs were rarely detected either by double retrograde labeling or BrdU marker. Since few RGCs died, microglia number showed a temporary increase at 3 days post injury (dpi) and a decrease at 14 dpi. Finally, myelin structure within retina kept integrity and optomotor response (OMR) test demonstrated visual functional restoration at 5 weeks post injury (wpi). In conclusion, our results have directly shown that RGC survival and axon regrowth are responsible for functional recovery after ONI in adult zebrafish.