Design, synthesis, and in vitro gene transfer efficacy of novel ionizable cholesterol derivatives.

ABSTACTThe medicinal properties of genetic drugs are highly dependent on the design of delivery systems. Ionizable cationic lipids are considered core materials in delivery systems. However, there has not yet been a widespread consensus on the relationship between the wide diversity of lipid structure design and gene delivery efficiency. The aims of the research work were to synthesize ionizable cholesterol derivatives (iChol-lipids) and to evaluate their potential applications as gene delivery vector. A series of iChol-lipids with different head groups were synthesized with carbamate bond spacer. The chemical structures were characterized by 1H NMR, MS, melting range, and pKa. The interactions between iChol-lipids and MALAT1-siRNA were studied by molecular dynamics simulations and compared with market available DC-Chol, which revealed that hydrogen bonds, salt-bridge, and electrostatic interaction were probably involved. The self-assemble behaviors of these lipids were intensively investigated and evaluated by dynamic laser scattering in the presence of different helper lipids and PEGylated lipids. Their plasmid binding ability, transfection efficiency, hemolytic toxicity, and cytotoxicity were fully studied. IZ-Chol-LNPs was proved to be highly potential to effectively complex with DNA, and endosome escape mechanisms mediated by proton sponge effect was verified by pH-sensitive fluorescence probe BCFL.

Pogostemon cablin essential oil affects anxiety- and depressive-like behaviors and the gut microbiota in chronic unpredictable mild stress model rats.

The gut microbiota is thought to be an important factor that influences brain processes and behaviors through the gut-brain axis. Pogostemon cablin is used in traditional Chinese medicine (TCM) to treat gastrointestinal symptoms. Patchouli essential oil (PCO), the main active agent in P. cablin, is used in aromatherapy for stress relief. The aim of our study was to investigate the effects of orally administered PCO on anxiety- and depressive-like behaviors and the gut microbiota. We constructed a rat model of chronic unpredictable mild stress (CUMS) and explored the anxiolytic- and antidepressant-like effects of PCO using the open field test (OFT) and forced swim test (FST). Changes in the abundance of the gut microbiota, short-chain fatty acids (SCFAs), and other related molecules were assessed to determine the role of the gut microbiota. Our results showed that CUMS induced an anxiety-like phenotype in the OFT, which was reversed by PCO, and that PCO also significantly mitigated the depression-like behaviors caused by CUMS in the FST. Furthermore, we found that PCO increased the relative abundances of several probiotics, including Bacteroides and Blautia, and decreased the relative abundances of Ruminococcus_1 and Ruminococcus_2, which were increased by CUMS. Regarding SCFAs, the metabolites of the gut microbiota, PCO increased the concentration of propionic acid and decreased that of caproic acid. Finally, PCO restored the serotonin (5-hydroxytryptamine, 5-HT) level in the hippocampus, which had been decreased by CUMS. The results of this study suggested that PCO can improve stress-related anxiety- and depression-like behaviors and might exert its effects on the central nervous system through interactions with the gut microbiota.

Elucidating the therapeutic mechanism of Hengqing II decoction in Alzheimer's disease using network pharmacology and molecular docking techniques.

PURPOSE: The aim of our research was to investigate the mechanism of the Hengqing II decoction in treating Alzheimer's disease (AD) through network pharmacology and experimental validation methods. METHODS: Firstly, the major chemical compounds of Hengqing II decoction were characterized by ultra-high-performance liquid chromatography-high resolution mass spectrometry (UHPLC-Q-TOF-MS/MS), and the gene sets related to AD treatment by Hengqing II decoction were collected through the database of PubChem, Swiss TargetPrediction, and DisGeNET. Secondly, a multi-level molecular network of "Traditional Chinese medicine (TCM)-compound-target-disease" was constructed and visualized using the STRING platform and Cytoscape 3.9.1 software, and the enrichment analysis based on the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway databases was performed to predict the potential active compounds and targets of Hengqing II decoction for treating AD. Finally, molecular docking simulation was applied to investigate the binding interactions between potential active compounds and key targets, and the western blotting technique was employed to examine the expression levels of AKT1, TNF-α, and NOS2 proteins affected by active compounds. RESULTS: Totally 120 compounds in Hengqing II decoction were characterized by UHPLC-Q-TOF-MS/MS. Network pharmacology results showed that potential active compounds in Hengqing II decoction in treating AD included catalpol, gastrodin, and rehmannioside D, etc., and the main target proteins were TNF-α, NOS2, and AKT1. Further functional enrichment analysis revealed that Hengqing II decoction mainly exerted its therapeutic effects on AD by regulating lipid and atherosclerosis signaling pathways, AD signaling pathways, AKT1 signaling pathways, and PTGS2 signaling pathways. CONCLUSION: Hengqing II decoction exerted therapeutic effects on AD through multi-component, multi-target, and multi-pathway regulation, and its action mechanisms were related to oxidative stress, neuroinflammation, autophagy, and other pathways. Our research laid the data foundation for further exploration of action mechanism and clarification of clinical positioning and provided new ideas and clues in TCM formula research.

Aquaporins enriched in endothelial vacuole membrane regulate the diameters of microvasculature in hyperglycemia.

BACKGROUND: In patients with diabetic microvascular complications, decreased perfusion or vascular occlusion, caused by reduced vascular diameter, is a common characteristic that will lead to insufficient blood supply. Yet, the regulatory mechanism and effective treatment approach remain elusive. METHODS AND RESULTS: Our initial findings revealed a notable decrease in the expression of human AQP1 in both diabetic human retina samples (49 healthy vs. 54 diabetic samples) and high-glucose-treated human retinal microvascular endothelial cells. Subsequently, our investigations unveiled a reduction in vascular diameter and compromised perfusion within zebrafish embryos subjected to high glucose treatment. Further analysis indicated a significant downregulation of two aquaporins, aqp1a.1 and aqp8a.1, which are highly enriched in ECs and are notably responsive to hyperglycemic conditions. Intriguingly, the loss of function of aqp1a.1 and/or aqp8a.1 resulted in a reduction of intersegmental vessel diameters, effectively mirroring the phenotype observed in the hyperglycemic zebrafish model.The overexpression of aqp1a.1/aqp8a.1 in zebrafish ECs led to notable enlargement of microvascular diameters. Moreover, the reduced vessel diameters resulting from high-glucose treatment were effectively rescued by the overexpression of these aquaporins. Additionally, both aqp1a.1 and apq8a.1 were localized in the intracellular vacuoles in cultured ECs as well as the ECs of sprouting ISVs, and the loss of Aqps caused the reduction of those vacuoles, which was required for lumenization. Notably, while the loss of AQP1 did not impact EC differentiation from human stem cells, it significantly inhibited vascular formation in differentiated ECs. CONCLUSION: EC-enriched aquaporins regulate the diameter of blood vessels through an intracellular vacuole-mediated process under hyperglycemic conditions. These findings collectively suggest that aquaporins expressed in ECs hold significant promise as potential targets for gene therapy aimed at addressing vascular perfusion defects associated with diabetes.

Comparative analysis between high-grade serous ovarian cancer and healthy ovarian tissues using single-cell RNA sequencing.

Introduction: High-grade serous ovarian cancer (HGSOC) is the most common histological subtype of ovarian cancer, and is associated with high mortality rates. Methods: In this study, we analyzed specific cell subpopulations and compared different gene functions between healthy ovarian and ovarian cancer cells using single-cell RNA sequencing (ScRNA-seq). We delved deeper into the differences between healthy ovarian and ovarian cancer cells at different levels, and performed specific analysis on endothelial cells. Results: We obtained scRNA-seq data of 6867 and 17056 cells from healthy ovarian samples and ovarian cancer samples, respectively. The transcriptional profiles of the groups differed at various stages of ovarian cell development. A detailed comparison of the cell cycle, and cell communication of different groups, revealed significant differences between healthy ovarian and ovarian cancer cells. We also found that apoptosis-related genes, URI1, PAK2, PARP1, CLU and TIMP3, were highly expressed, while immune-related genes, UBB, RPL11, CAV1, NUPR1 and Hsp90ab1, were lowly expressed in ovarian cancer cells. The results of the ScRNA-seq were verified using qPCR. Discussion: Our findings revealed differences in function, gene expression and cell interaction patterns between ovarian cancer and healthy ovarian cell populations. These findings provide key insights on further research into the treatment of ovarian cancer.

Comparative analysis of the testes from wild-type and Alkbh5-knockout mice using single-cell RNA sequencing.

The RNA demethylase ALKBH5 is regarded as the "eraser" in N6-methyladenosine modification. ALKBH5 deficiency causes male infertility in mice; however, the mechanisms that confer disruption of spermatogenesis are not completely clear. In this study, we profiled testis samples from wild-type and Alkbh5-knockout mice using single-cell RNA sequencing. We obtained single-cell RNA sequencing data of 5,596 and 6,816 testis cells from a wild-type and a knockout mouse, respectively. There were differences detected between the transcriptional profiles of the groups at various germ cell developmental stages. This ranged from the development of spermatogonia to sperm cells, in macrophages, Sertoli cells, and Leydig cells. We identified the differentially expressed genes related to spermatogenesis in germ cells and somatic cells (Sertoli cells and Leydig cells) and evaluated their functions and associated pathways, such as chromatin-related functional pathways, through gene ontology enrichment analysis. This study provides the first single-cell RNA sequencing profile of the testes of ALKBH5-deficient mice. This highlights that ALKBH5 is an important gene for germ cell development and spermatogenesis and offers new molecular mechanistic insights. These findings could provide the basis for further research into the causes and treatment of male infertility.

Comprehensive Analysis of the Transcriptome-Wide m6A Methylation in Mouse Pachytene Spermatocytes and Round Spermatids.

Spermatogenesis, an efficient and complex system in male germline development, requires a series of elaborately regulated genetic events in which diploid spermatogonia differentiate into haploid spermatozoa. N6-methyladenosine (m6A) is an important epigenetic RNA modification that occurs during spermatogenesis. ALKBH5 is an m6A eraser and knocking out Alkbh5 increases the level of total m6A methylation and causes male infertility. In this study, comprehensive analyses of MeRIP-seq and RNA-seq data revealed differences between wild-type (WT) and Alkbh5 knockout (KO) mice. In pachytene spermatocytes (PA), 8,151 m6A peaks associated with 9,959 genes were tested from WT and 10,856 m6A peaks associated with 10,016 genes were tested from KO mice. In the round spermatids (RO), 10,271 m6A peaks associated with 10,109 genes were tested from WT mice and 9,559 m6A peaks associated with 10,138 genes were tested from KO mice. The peaks were mainly concentrated in the coding region and the stop codon of the GGAC motif. In addition, enrichment analysis showed significant m6A methylation genes in related pathways in spermatogenesis. Furthermore, we conducted joint analyses of the m6A methylome and RNA transcription, suggesting an m6A regulatory mechanism of gene expression. Finally, seven differentially expressed mRNAs from RNA-seq data in both PA and RO were verified using qPCR. Overall, our study provides new information on m6A modification changes between WT and KO in PA and RO, and may provide new insights into the molecular mechanisms of m6A modification in germ cell development and spermatogenesis.

Corrigendum: Comprehensive analysis of the transcriptome-wide m6A methylation in mouse pachytene spermatocytes and round spermatids.

[This corrects the article DOI: 10.3389/fgene.2022.832677.].

Promotion of fracture healing by conjugated linoleic acid in rats.

BACKGROUND: The ability of fracture healing in Tibetans is significantly superior to Chinese Hans, which may attribute to factors from diet, altitude to physical conditions. Conjugated linoleic acid (CLA) is an important ingredient in Tibetan diet, playing a role in antioxidation, antiatherosclerosis, and decrease in body fat accumulation.  Methods: This study further quantified CLA effect in fracture healing in rats using combined structural evaluation (X-ray and micro-computed tomography), biomechanical test, and histological examination. RESULTS: CLA could promote fracture healing with quicker development of trabecular connection, network and thickening and were more active at the stage of bony union and remodeling. The load to failure could reach 78.12 ± 10.03 N, 41.4% stronger than the control by week 6 ( p = 0.0209). CONCLUSIONS: CLA improved the quality and mechanical strength of fracture healing in rats callus. The information may offer insight in development of new therapeutic strategies of fracture healing for general populations beyond Tibetans.