Hypoxia adaptation can partially improve and restore the composition, structure, and function of blood vessels, thereby enhancing the prognosis of related diseases such as stroke. However, the underlying molecular mechanisms of hypoxia adaptation remain elusive. In this study, multi-omics analysis was performed across yeast, zebrafish, and mouse, revealing that sphingolipid metabolism is crucial for hypoxic adaptation. We found that the expression of the key protein ceramide synthase 2 (CerS2) in mammals and its yeast ortholog very-long-chain ceramide synthase LAC1 was up-regulated. CerS2 and LAC1 are key enzymes that promote hypoxic angiogenesis by increasing the synthesis of C24:1, and this function is highly conserved across species and throughout evolution. CerS2 and LAC1 biosynthesize the long-chain unsaturated ceramide C24:1, which, via its double-bond structure, specifically binds to ROCK2 to activate HIF-1α, thereby up-regulating the expression of vascular endothelial growth factor (VEGF) and increasing AKT phosphorylation. Consequently, this process significantly enhances angiogenesis and improves blood flow perfusion in a mouse model of ischemic stroke. This study deepens our understanding of the function of unsaturated ceramides and reveals the unique mechanism of C24:1 in angiogenesis and protection under hypoxic conditions.

In this paper, the effect of hypoxia on the content of active ceramides in tissues was investigated for the first time, and the pharmacokinetic changes of ceramide C24:1 with hypoxia-protective effects were compared under both normoxic and hypoxic conditions. A UPLC-MS/MS method was developed to determine five ceramides in mouse tissues and plasma. The performance criteria for sensitivity, linearity, matrix effect, recovery, stability, precision, and accuracy were evaluated and found to be within the FDA-recommended guidelines. This method was successfully employed to quantify both endogenous and exogenous ceramides in tissues and plasma. The results revealed tissue-specific changes in endogenous ceramide levels under hypoxia and showed that hypoxia increased the systemic exposure and prolonged the retention of exogenous C24:1 while reducing its clearance. These findings support further investigation of ceramides as potential biomarkers or therapeutic targets for hypoxia-related diseases and provide important in vivo pharmacokinetic data for the development of novel neuroprotective drugs.

Phosphate-containing metabolites serve as critical regulators of energy homeostasis and signal transduction under hypoxic stress. However, their simultaneous quantification is technically challenging due to high polarity and non-specific adsorption. In this study, a sensitive UHPLC-MS/MS method was established for the simultaneous quantification of eight key phosphate metabolites, including adenosine triphosphate (ATP), phosphoenolpyruvate (PEP) and glucose-1-phosphate (P1G), in biological matrices. By compressing the analytical run time to <4 min per sample, this high-throughput platform demonstrated excellent linearity (r2 > 0.990) for all analytes. The method satisfied rigorous bioanalytical validation standards, exhibiting intra- and inter-day precision (RSD) of 2.2%-11.5% and accuracy of 87.0%-109.5%. Application of this method to in vitro and in vivo hypoxia models demonstrated its capability to capture distinct, context-dependent metabolic adaptations. Specifically, the platform differentiated the energetic trajectories of neuronal cells and cardiomyocytes under hypoxia and profiled metabolic shifts in mouse brain tissue. This study provides a reliable analytical platform for quantifying highly polar phosphate metabolites, offering a robust tool for monitoring metabolic pool dynamics under physiological and pathological stress.

The potential effects of Puerariae Lobatae Radix (Gegen in Chinese) water extract (GWE) on overactive bladder (OAB) were previously demonstrated through ex vivo examination of detrusor contraction. However, the mechanisms were not fully understood. The current aim was to investigate the therapeutic mechanisms of GWE against OAB in spontaneously hypertensive rats (SHR) with bladder ischemia. The therapeutic effect of GWE against OAB was evaluated by urodynamics. Hematoxylin & eosin (H&E) staining, Masson staining, and Doppler ultrasonic blood stream detector were utilized to observe bladder structures and local blood flow, respectively. To elucidate the mechanisms, an integrated omics approach was employed. The key proteins and metabolites were validated using Western Blotting and ELISA. A 3-week treatment of GWE demonstrated a significant improvement in urodynamic parameters. The results from Doppler detector, H&E staining, and Masson staining indicated that GWE improved vasodilation of bladder microvessels. Transcriptomic analysis revealed changes in genes such as Ptgfr and Ntsr1, which were involved in regulating intracellular Ca2+ concentration. Proteomic analysis suggested that the downregulation of epoxide hydrolase 2 (EPHX2), maintaining the balance of epoxyeicosatrienoic acids (EETs), was responsible for GWE-induced vasodilation. Metabolomic analysis further supported alterations in arachidonic acid (AA) metabolism. It is concluded that GWE treated OAB in SHR rats by improving bladder blood flow through the inhibition of EPHX2 and upregulation of EETs. This inhibition resulted in the improvement of bladder structure and the suppression of AA metabolism-mediated PTGES/PTGFR/PLCβ1/phospho-MLC signaling pathway.

Objectives: Network pharmacology is essential for understanding the multi-target and multi-pathway therapeutic mechanisms of traditional Chinese medicine. This study aims to evaluate the influence of database quality on target identification and to explore the therapeutic potential of rhynchophylline (Rhy) in treating overactive bladder (OAB). Methods: An OAB dataset was constructed through extensive literature screening. Using this dataset, we applied network pharmacology to predict potential targets for Rhy, which is known for its therapeutic effects but lacks a well-defined target profile. Predicted targets were validated through in vitro experiments, including DARTS and CETSA. Results: Our analysis identified Rhy as a potential modulator of the M3 receptor and TRPM8 channel in the treatment of OAB. Validation experiments confirmed the interaction between Rhy and these targets. Additionally, the GeneCards database predicted other targets that are not directly linked to OAB, corroborated by the literature. Conclusions: We established a more accurate and comprehensive dataset of OAB targets, enhancing the reliability of target identification for drug treatments. This study underscores the importance of database quality in network pharmacology and contributes to the potential therapeutic strategies for OAB.

Charcot-Marie-Tooth disease type 2N (CMT2N) is an inherited nerve disorder caused by mutations in the alanyl-tRNA synthetase (AlaRS) gene, resulting in muscle weakness and sensory issues. Currently, there is no cure for CMT2N. Here, we found that all five AlaRS mutations in the aminoacylation domain can interact with neuropilin-1 (Nrp1), which is consistent with our previous findings. Interestingly, three of these mutations did not affect alanine activation activity. We then performed a high-throughput screen of 2000 small molecules targeting the prevalent R329H mutant. Using thermal stability assays (TSA), biolayer interferometry (BLI), ATP consumption, and proteolysis assays, we identified Tanshinone I as a compound that binds to and modifies the conformation of the R329H mutant and other CMT-related AlaRS mutants interacting with Nrp1. Molecular docking and dynamic simulation studies further clarified Tanshinone I’s binding mode, indicating its potential against various AlaRS mutants. Furthermore, co-immunoprecipitation (Co-IP) and pull-down assays showed that Tanshinone I significantly reduces the binding of AlaRS mutants to Nrp1. Collectively, these findings suggest that Tanshinone I, by altering the conformation of mutant proteins, disrupts the pathological interaction between AlaRS CMT mutants and Nrp1, potentially restoring normal Nrp1 function. This makes Tanshinone I a promising therapeutic candidate for CMT2N.

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Adaptation to hypoxia has attracted much public interest because of its clinical significance. However, hypoxic adaptation in the body is complicated and difficult to fully explore. To explore previously unknown conserved mechanisms and key proteins involved in hypoxic adaptation in different species, we first used a yeast model for mechanistic screening. Further multi-omics analyses in multiple species including yeast, zebrafish and mice revealed that glycerophospholipid metabolism was significantly involved in hypoxic adaptation with up-regulation of lysophospholipid acyltransferase (ALE1) in yeast, a key protein for the formation of dipalmitoyl phosphatidylcholine [DPPC (16:0/16:0)], which is a saturated phosphatidylcholine. Importantly, a mammalian homolog of ALE1, lysophosphatidylcholine acyltransferase 1 (LPCAT1), enhanced DPPC levels at the cell membrane and exhibited the same protective effect in mammalian cells under hypoxic conditions. DPPC supplementation effectively attenuated growth restriction, maintained cell membrane integrity and increased the expression of epidermal growth factor receptor under hypoxic conditions, but unsaturated phosphatidylcholine did not. In agreement with these findings, DPPC treatment could also repair hypoxic injury of intestinal mucosa in mice. Taken together, ALE1/LPCAT1-mediated DPPC formation, a key pathway of glycerophospholipid metabolism, is crucial for cell viability under hypoxic conditions. Moreover, we found that ALE1 was also involved in glycolysis to maintain sufficient survival conditions for yeast. The present study offers a novel approach to understanding lipid metabolism under hypoxia and provides new insights into treating hypoxia-related diseases.

Isoliquiritigenin (ISL) has excellent neuroprotective effects. However, its limitations, including poor solubility, low bioavailability, and low accumulation in the brain, restrict its clinical promotion. In this study, a novel type of ISL-loaded liposome (ISL-LP) modified with the brain-targeting polypeptide angiopep-2 was prepared to improve these properties. The zeta potential, morphology, particle size, encapsulation efficiency, drug loading, and in vitro release of ISL-LP were evaluated. The pharmacokinetics and tissue distribution of ISL and ISL-LP were also investigated. The results demonstrated that ISL-LP had an average particle size of 89.36 ± 5.04 nm, a polymer dispersity index of 0.17 ± 0.03, a zeta potential of −20.27 ± 2.18 mV, and an encapsulation efficiency of 75.04 ± 3.28%. The in vitro release experiments indicate that ISL-LP is a desirable sustained-release system. After intravenous administration, LPC-LP prolonged the circulation time of ISL in vivo and enhanced its relative brain uptake. In conclusion, ISL-LP could serve as a promising brain-targeting system for the treatment and prevention of central nervous system (CNS) disorders.

Many studies have focused on the effects of small molecules, such as amino acids, on metabolism under hypoxia. Recent findings have indicated that phenylalanine levels were markedly elevated in adaptation to chronic hypoxia. This raises the possibility that phenylalanine treatment could markedly improve the hypoxic endurance. However, the importance of hypoxia-regulated phenylalanine is still unclear. This study investigates the role of phenylalanine in hypoxia adaptation using a hypoxic zebrafish model and multi-omics analysis. We found that phenylalanine-related metabolic pathways are significantly up-regulated under hypoxia, contributing to enhanced hypoxic endurance. Phenylalanine treatment reduced ROS levels, improved mitochondrial oxygen consumption rate (OCR), and extracellular acidification rate (ECAR) in hypoxic cells. Western blotting revealed increased phenylalanine uptake via L-type amino transporters (LAT1), activating the LKB1/AMPK signaling pathway. This activation up-regulated peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α) and the Bcl-2/Bax ratio, while down-regulating uncoupling protein 2 (UCP2), thereby improving mitochondrial function under hypoxia. This is the first comprehensive multi-omics analysis to demonstrate phenylalanine’s crucial role in hypoxia adaptation, providing insights for the development of anti-hypoxic drugs. Multi-omics analysis in hypoxic models indicated that the phenylalanine-related metabolic pathways strongly associated with hypoxia endurance. Phenylalanine uptake obviously alleviated the damage in hypoxic cells. Phenylalanine obviously activated LKB1/AMPK signaling pathway, so that enhanced the energy metabolism and cell survival.

Sojae semen germinatum (SSG) is derived from mature soybean seeds that have been germinated and dried, typically with sprouts measuring approximately 0.5 cm in length. SSG is traditionally known for its properties in clearing heat and moisture. Nevertheless, limited information was reported on the effects and mechanisms of SSG in alleviating urinary symptoms. This study employed urodynamic parameters to investigate the therapeutic effect of SSG water extract on overactive bladder (OAB) in the rat model with benign prostatic hyperplasia. Through a combination of transcriptomic and metabolomic analyses, the pathways and key proteins of the SSG treatment for OAB were identified and validated by ELISA and Western blotting. Furthermore, network pharmacology elucidated the roles of SSG's isoflavones acting on the target which was identified by above-mentioned multi-omics analysis. Our results indicate that SSG water extract significantly mitigated OAB by down-regulating the PGE2/EP1/PLCβ2/p-MLC signaling pathway. It was speculated that the active ingredient in the SSG on EP1 was genistein. This study provided valuable insights into the molecular mechanisms of SSG water extract, emphasizing the multi-target characteristics and critical pathways in improving OAB. Furthermore, this study contributes to the potential utilization of SSG as a functional food.

新时代创新型医学人才需具备辩证思维能力.药理学作为基础医学课程,其中蕴含丰富的辩证唯物主义元素.寓辩证思维于药理课堂教学,不仅有助于学生深刻理解学科专业知识,还可培养辩证思维能力与科学世界观,使课程思政与专业教学有效联动、交融共振.本文通过教学实例阐释药理学的辩证因素,旨在培养医药专业本科生思辨能力,更好地发挥药理学课程的育人功能.

Background: In the previous study, Puerariae Lobatae Radix (named Gegen in Chinese) water extract attenuated M3 receptor agonist carbachol-induced detrusor contraction after 3-week oral administration in a hypertension-associated OAB (overactive bladder) model. This research aimed to investigate the active ingredients from Gegen water extract against OAB. Methods: Bioassay-guided fractionation was performed by using preparative HPLC for fast isolation of fractions followed by screening their ex vivo activity through carbachol-induced bladder strip contraction assay. Chemicals in each active fraction were analyzed by HPLC-UV. Urine metabolites were quantified by LC-MS/MS after sub-acute administration. Thermal shift assay with the recombinant human M3 receptor protein was performed, and molecular docking analysis was used for molecular modelling of M3 receptor inhibition. Results: Bioassay-guided fractionation results for isolating M3 receptor inhibitors indicated that four compounds were identified as active ingredients of Gegen water extract, and their inhibition potency on carbachol-induced detrusor contraction was ranked in descending order according to their inhibition concentrations as follows: genistein > daidzein > biochanin A >> puerarin. Daidzein in urine reached an ex vivo effective concentration to inhibit detrusor contraction, but others did not. Daidzein concentration-dependently increased the melt temperature (Tm) of recombinant human M3 receptor protein with a positive binding (ΔTm = 2.12 °C at 100 μg/ml). Molecular docking analysis showed that daidzein can potently bind to the ligand binding pocket of the M3 receptor via hydrogen bonding. Conclusion: Puerarin and its derivatives were pro-drugs, and daidzein was their in vivo active form via M3 receptor inhibition for treating OAB.

Purpose We designed a novel isoliquiritigenin (ISL) loaded micelle prepared with DSPE-PEG2000 as the drug carrier modified with the brain-targeting polypeptide angiopep-2 to improve the poor water solubility and low bioavailability of ISL for the treatment of acute ischemic stroke. Methods Thin film evaporation was used to synthesize the ISL micelles (ISL-M) modified with angiopep-2 as the brain targeted ligands. The morphology of the micelles was observed by the TEM. The particle size and zeta potential were measured via the nanometer particle size analyzer. The drug loading, encapsulation and in vitro release rates of micelles were detected by the HPLC. The UPLC-ESI-MS/MS methods were used to measure the ISL concentrations of ISL in plasma and main tissues after intravenous administration, and compared the pharmacokinetics and tissue distributions between ISL and ISL-M. In the MCAO mice model, the protective effects of ISL and ISL-M were confirmed via the behavioral and molecular biology experiments. Results The results showed that the drug loading of ISL-M was 7.63 ± 2.62%, the encapsulation efficiency was 68.17 ± 6.23%, the particle size was 40.87 ± 4.82 nm, and the zeta potential was −34.23 ± 3.35 mV. The in vitro release experiments showed that ISL-M had good sustained-release effect and pH sensitivity. Compared with ISL monomers, the ISL-M could significantly prolong the in vivo circulation time of ISL and enhance the accumulation in the brain tissues. The ISL-M could ameliorate the brain injury induced by the MCAO mice via inhibition of cellular autophagy and neuronal apoptosis. There were no the cellular structural damages and other adverse effects for ISL-M on the main tissues and organs. Conclusion The ISL-M could serve as a promising and ideal drug candidate for the clinical application of ISL in the treatment of acute ischemic stroke.

药理学教学内容繁杂、课程难度大,学生学习兴趣不大.为了提高学生的学习热情和自主学习意识,文献讨论课辅助知识讲授的教学模式被引入药理学教学.这种教学模式可以提高学生对药理学课程的兴趣,加深对专业知识的理解和记忆,同时培养学生的英文文献阅读能力、逻辑思维能力、归纳总结能力和语言表达能力.

Calcium-dependent, neuronal adenylyl cyclase subtype 1 (AC1) is critical for cortical potentiation and chronic pain. NB001 is a first-in-class drug acting as a selective inhibitor against AC1. The present study delineated the pharmacokinetic (PK) properties of human-used NB001 (hNB001) formulated as immediate-release tablet. This first-in-human (FIH) study was designed as randomized, double-blind, placebo-controlled trial. hNB001 showed placebo-like safety and good tolerability in healthy volunteers. A linear dose-exposure relationship was demonstrated at doses between 20 mg and 400 mg. The relatively small systemic exposure of hNB001 in human showed low bioavailability of this compound through oral administration, which can be improved through future dosage research. Food intake had minimal impact on the absorption of hNB001 tablet. Animal experiments further confirmed that hNB001 had strong analgesic effect in animal models of neuropathic pain. In brain slice prepared from the anterior cingulate cortex (ACC), bath application of hNB001 blocked the induction of long-term potentiation (LTP). These results from both rodents and human strongly suggest that hNB001 can be safely used for the future treatment of different types of chronic pain in human patients.

雨课堂是互联网背景下学校教育与信息化技术融合的产物,通过手机追踪可以覆盖课前、课中、课后的每个教学环节,为PBL教学模式的开展提供了平台.这种新型的教学模式突破了传统灌输式教学的弊端,充分调动学生的学习积极性,及时全面地掌握学生的学习动态,做出全面公正的形成性评价.在首都医科大学本科生教学中利用雨课堂融合PBL的教学模式,结合药理学课程的特点,初步探索针对药理学混合式教学模式的可行性.

As a natural flavonoid, kaempferol is widely distributed in natural medicines. Our study was aimed at analyzing and comparing the pharmacokinetic differences of kaempferol between normoxia and hypoxia in rats, to further explore the effect of hypoxia on drug metabolism enzymes. A sensitive UPLC-MS/MS method was established and validated for the determination of kaempferol in rat plasma. The results indicated that AUC, MRT, t1/2 and Cmax of kaempferol significantly increased and the clearance reduced in hypoxic rats. Based on the comparison of pharmacokinetics, the metabolites of kaempferol in hypoxic rats were identified by using UPLC-QTOF-MS and UNIFI 1.8 software. Then we explored the effect of hypoxia on the mRNA and protein expression of CYP1A2 and UGT1A9. The study revealed that hypoxia could markedly reduce the mRNA and protein expression of CYP1A2 and UGT1A9, resulting in the reduction of metabolic rate and enhancement of systematic exposure. Our data also indicated that we should pay attention to adjusting the dosage regimen and reducing drug interactions when drugs metabolized by CYP1A2 and UGT1A9 are used in combination with kaempferol. Our findings suggested the potential requirement for dose adjustment of kaempferol or its structural analogs in hypoxic condition.

转化医学是以临床上的需求来决定基础研究的内容,并将基础研究的成果运用于临床疾病的诊疗;而药理学是基础医学向临床医学的桥梁学科,是转化医学研究的重要领域,因此药理学教学改革需要融入转化医学的思维.通过加强师资力量、更新教学方法、拓展教学内容、扩充教学资源和加强实验教学等方法,克服目前医学教育中培养转化医学人才过程中存在的不足.这不仅能极大地调动学生学习的积极性,而且有利于新型医药人才的培养.

Salidroside (SAL), a major bioactive compound of Rhodiola crenulata, has significant anti-hypoxia effect, however, its underlying molecular mechanism has not been elucidated. In order to explore the protective mechanism of SAL, the lactate dehydrogenase (LDH), reactive oxygen species (ROS), superoxide dismutase (SOD) and hypoxia-induced factor 1α (HIF-1α) were measured to establish the PC12 cell hypoxic model. Cell staining and cell viability analyses were performed to evaluate the protective effects of SAL. The metabolomics and bioinformatics methods were used to explore the protective effects of salidroside under hypoxia condition. The metabolite-protein interaction networks were further established and the protein expression level was examined by Western blotting. The results showed that 59 endogenous metabolites changed and the expression of the hub proteins of CK2, p-PTEN/PTEN, PI3K, p-Akt/Akt, NF-κB p65 and Bcl-2 were increased, suggesting that SAL could increase the expression of CK2, which induced the phosphorylation and inactivation of PTEN, reduced the inhibitory effect on PI3K signaling pathways and activated the PI3K/Akt/NF-κB survival signaling pathway. Our study provided an important insight to reveal the protective molecular mechanism of SAL as a novel drug candidate.