The major objective of this study was to investigate the anti-chronic nonbacterial prostatitis (CNP) mechanism of T. patula by metabolomics and network pharmacology. The study demonstrated that the flavonoids and polysaccharides of T. patula could alleviate prostatitis by improving the level of DHT, reducing the secretion of PSA and TNF-α. Besides, both could enhance Na+/K+-ATPase activity, decrease the O2 consumption, CO2 production, heat production, energy expenditure of rats and promote respiratory exchange ratio of rats. Up to 28 potential biomarkers and 8 key metabolic pathways related to the treatment of CNP were elucidated by the metabolomics analysis, including phenylalanine metabolism, taurine and hypotaurine metabolism, tryptophan metabolism etc. Network pharmacology prediction also reflected the potential mechanism was associated with tryptophan metabolism and energy pathway. Generally, the potential anti-CNP mechanism of flavonoids and polysaccharides of T. patula might be through reducing the expression of inflammation factors, adjusting the level of hormone and regulating the amino acid metabolism, energy metabolism and glucose and lipid metabolism.

Paeoniflorin is a glycoside compound found in Paeonia lactiflora Pall that is used in traditional herbal medicine and shows various protective effects on the cardio-cerebral vascular system. It has been reported that the pharmacological effects of paeoniflorin might be generated by its metabolites. However, the bioavailability of paeoniflorin by oral administration is low, which greatly limits its clinical application. In this paper, a paeoniflorin-converting enzyme gene (G6046, GenBank accession numbers: OP856858) from Cunninghamella blakesleeana (AS 3.970) was identified by comparative analysis between MS analysis and transcriptomics. The expression, purification, enzyme activity, and structure of the conversion products produced by this paeoniflorin-converting enzyme were studied. The optimal conditions for the enzymatic activity were found to be pH 9, 45 °C, resulting in a specific enzyme activity of 14.56 U/mg. The products were separated and purified by high-performance counter-current chromatography (HPCCC). Two main components were isolated and identified, 2-amino-2-p-hydroxymethyl-methyl alcohol-benzoate (tirs-benzoate) and 1-benzoyloxy-2,3-propanediol (1-benzoyloxypropane-2,3-diol), via UPLC-Q-TOF-MS and NMR. Additionally, paeoniflorin demonstrated the ability to metabolize into benzoic acid via G6046 enzyme, which might exert antidepressant effects through the blood-brain barrier into the brain.

P2Y receptors are G-protein-coupled receptors (GPCRs) for extracellular nucleotides. The P2Y2 receptor subtype is expressed in a variety of cell types and plays an important role in physiological and pathophysiological processes such as inflammatory responses and neuropathic pain. Based on this, the P2Y2 has been identified as an important drug target. The specificity of current P2Y2 receptor modulators is relatively poor, and currently, specific and efficient P2Y2 receptor modulators and efficient screening strategies are lacking. In this study, a cell model based on calcium-activated chloride channels (CaCCs) was established that can detect changes in intracellular calcium concentrations and can be used to high-throughput screen for P2Y2 receptor-specific regulators. This screening strategy is suitable for screening of most G-protein-coupled receptor regulators that mediate increases in intracellular calcium signals. The cell model consists of three components that include the endogenously expressed P2Y2 receptor protein, the exogenously expressed calcium-activated chloride channel Anoctamin-1 (Ano1), and a yellow fluorescent protein mutant expressed within the cell that is highly sensitive to iodine ions. This model will allow for high-throughput screening of GPCR regulators that mediate increased intracellular calcium signaling using the calcium-activated transport of iodide ions by Ano1. We verified the ability of the model to detect intracellular calcium ion concentration using fluorescence quenching kinetic experiments by applying existing P2Y2 agonists and inhibitors to validate the screening function of the model, and we also evaluated the performance of the model in the context of high-throughput screening studies. The experimental results revealed that the model could sensitively detect intracellular calcium ion concentration changes and that the model was accurate in regard to detecting P2Y2 modulators. The resultant value of the Z-factor was 0.69, thus indicating that the model possesses good sensitivity and specificity.

Lipid nanoparticles (LNPs) exhibit high potential as carriers of messenger RNA (mRNA). However, the arduous preparation process of mRNA-loaded LNPs remains a huge obstacle for their widespread clinical application. Herein, we tackled this issue by mRNA PEGylation through hybridization with polyethylene glycol (PEG)-conjugated RNA oligonucleotides (PEG-OligoRNAs). Importantly, mRNA translational activity was preserved even after hybridization of 20 PEG-OligoRNAs per mRNA. The straightforward mixing of the PEGylated mRNA with lipofectamine LTX, a commercial lipid-based carrier, just by pipetting in aqueous solution, allowed the successful preparation of mRNA-loaded LNPs with a diameter below 100 nm, whereas the use of non-PEGylated mRNA provided large aggregates above 100- and 1000-nm. In vivo, LNPs prepared from PEG-OligoRNA-hybridized mRNA exhibited high structural stability in biological milieu, without forming detectable aggregates in mouse blood after intravenous injection. In contrast, LNPs from non-PEGylated mRNA formed several micrometer-sized aggregates in blood, leading to rapid clearance from blood circulation and deposition of the aggregates in lung capillaries. Our strategy of mRNA PEGylation was also versatile to prevent aggregation of another type of mRNA-loaded LNP, DOTAP/Chol liposomes. Together, our approach provides a simple and robust preparation method to LNPs for in vivo application.