Application of the ankle pump method in a sitting position to evaluate calf perforating veins by color Doppler ultrasound.

OBJECTIVE: We investigated the feasibility and efficacy of assessing calf perforating veins (PVs) using the ankle pump in a sitting position (AP-sit) method by color Doppler ultrasound. METHODS: We performed a multicenter prospective clinical trial between November 2022 and October 2023. Eligible patients with chronic venous disease and healthy controls were enrolled. The calf PVs were assessed using three different methods: manual compression in a standing position, manual compression in a sitting position, and AP-sit method. The reflux durations and detection rate of incompetent PVs (IPVs) were compared among the three methods. The number and diameter of calf PVs and distribution of IPVs were analyzed. RESULTS: A total of 50 patients with chronic venous disease and 50 healthy controls were included. There were 173 calves analyzed, including 97 healthy calves and 76 calves with chronic venous disease. The number of PVs per calf was higher in the diseased calves (median, 7.0; interquartile range [IQR], 6.0-8.0) than in the healthy calves (median, 5.0; IQR, 3.0-6.0; P < .001). The diameter of IPVs (median, 2.3 mm; IQR, 2.0-3.1 mm) was larger than that of competent PVs (median, 1.4 mm; IQR, 1.2-1.7 mm). Most of the IPVs (78.8%) were located in the medial and posterior middle of the calf. The reflux duration induced by the AP-sit method was greater than that induced by the manual compression methods (P < .001). Although the AP-sit method had a higher detection rate (92.0%) of IPVs than the manual compression methods (71.7% and 74.3% for standing and sitting, respectively; P < .001), especially in the distal lower leg, the manual compression methods found IPVs not found using the AP-sit method. CONCLUSIONS: Diseased calves with chronic venous disease have more PVs than do healthy calves. IPVs are commonly larger than competent PVs, with most IPVs located in the medial and posterior middle of the calf. Most importantly, the AP-sit method provides a convenient and effective approach for assessing the calf PVs, especially those located in the distal calf, as an alternative or complementary method to traditional manual compression, which is valuable in the daily practice of sonographers.

Brain Lipid Dynamics in Amyloid Precursor Protein/Presenilin 1 Mouse Model of Early Alzheimer's Disease by Desorption Electrospray Ionization and Matrix Assisted Laser Desorption Ionization-Mass Spectrometry Imaging Techniques.

Alzheimer's disease (AD) is closely associated with lipid metabolism dysfunction. However, space distribution and metabolism of aberrant lipids in the brain of early-stage AD mouse remain unclear. In our current work, a novel lipidomics method based on mass spectrometry imaging was developed to visually disclose molecular perturbation and characterize space distribution in the brain of double transgenic amyloid precursor protein/presenilin 1 mouse (2 and 3 months old). Significant changes were detected, including phosphatidylethanolamines, phosphatidylcholines, fatty acids, lysophospholipids, and glycerides in AD mouse brain. The results in this study suggest that these significantly altered lipid metabolic pathways (glycerophospholipid metabolism) may be implicated in early-stage AD. Our work deepens the understanding of the physio-pathologic mechanism of early-stage AD.

Untargeted lipidomics reveals progression of early Alzheimer's disease in APP/PS1 transgenic mice.

Alzheimer's Disease (AD) is closely connected to aberrant lipid metabolism. However, how early AD-like pathology synchronously influences brain and plasma lipidome in AD mice remains unclear. The study of dynamic change of lipidome in early-stage AD mice could be of great interest for the discovery of lipid biomarkers for diagnosis and monitoring of early-stage AD. For the purpose, an untargeted lipidomic strategy was developed for the characterization of lipids (≤ 1,200 Da) perturbation occurring in plasma and brain in early-stage AD mice (2, 3 and 7 months) by ultra-high performance liquid chromatography coupled with quadrupole-time-of-flight mass spectrometry. Significant changes were detected in the levels of several lipid species including lysophospholipids, phosphatidylcholines (PCs), phosphatidylethanolamines (PEs) and Ceramides (Cers), as well as other related lipid compounds such as fatty acids (FAs), diacylglycerols (DGs) and triacylglycerols (TGs) in AD mice. In this sense, disorders of lipid metabolism appear to involve in multiple factors including overactivation of phospholipases and diacylglycerol lipases, decreased anabolism of lysophospholipids in plasma and PEs in plasma and brain, and imbalances in the levels of PCs, FAs and glycerides at different ages. We revealed the changing panels of potential lipid biomarkers with the development of early AD. The study raises the possibility of developing lipid biomarkers for diagnosis of early-stage AD.

Hippocampus Proteomics and Brain Lipidomics Reveal Network Dysfunction and Lipid Molecular Abnormalities in APP/PS1 Mouse Model of Alzheimer's Disease.

Alzheimer's disease (AD) is closely associated with protein dysfunction and aberrant lipid metabolism in the brain. Our study could be conducive to the discovery of lipid and protein biomarkers for early diagnosis and therapy. In our current work, novel quantitative proteomic and lipidomic methods were developed for the characterization of molecular perturbation occurring in the brain in early stage AD mice. For this purpose, we performed a proteomic and lipidomic screening by liquid chromatography with mass spectrometry. Significant changes were detected, including 231 proteins and 11 lipid compounds in the AD mouse brain. Early stage AD disturbed biological pathways implicated in neuroactive ligand-receptor, complement and coagulation cascades, PI3K-Akt signaling and metabolic pathways, and glycerophospholipid metabolism. The results in the current study suggest that these significantly altered proteins and lipids may be implicated in early stage AD. Our work raises the possibility of AD diagnosis and therapy by providing new protein targets and lipid biomarkers.

Isosteviol Sodium Protects Neural Cells Against Hypoxia-Induced Apoptosis Through Inhibiting MAPK and NF-κB Pathways.

BACKGROUND: Stevioside, isolated from the herb Stevia rebaudiana, has been widely used as a food sweetener all over the world. Isosteviol Sodium (STV-Na), an injectable formulation of isosteviol sodium salt, has been proved to possess much greater solubility and bioavailability and exhibit protective effects against cerebral ischemia injury in vivo by inhibiting neuron apoptosis. However, the underlying mechanisms of the neuroprotective effects STV-Na are still not completely known. In the present study, we investigated the effects of STV-Na on neuronal cell death caused by hypoxia in vitro and its underlying mechanisms. METHODS: We used cobalt chloride (CoCl2) to expose mouse neuroblastoma N2a cells to hypoxic conditions in vitro. RESULTS: Our results showed that pretreatment with STV-Na (20 µM) significantly attenuated the decrease of cell viability, lactate dehydrogenase release and cell apoptosis under conditions of CoCl2-induced hypoxia. Meanwhile, STV-Na pretreatment significantly attenuated the upregulation of intracellular Ca2+ concentration and reactive oxygen species production, and inhibited mitochondrial depolarization in N2a cells under conditions of CoCl2-induced hypoxia. Furthermore, STV-Na pretreatment significantly downregulated expressions of nitric oxide synthase, interleukin-1ß, tumor necrosis factor-α, interleukin-6, nuclear factor kappa B (NF-κB), and mitogen-activated protein kinase (MAPK) signalings in N2a cells under conditions of CoCl2-induced hypoxia. CONCLUSIONS: Taken together, STV-Na protects neural cells against hypoxia-induced apoptosis through inhibiting MAPK and NF-κB pathways.

Effect of type 2 diabetes mellitus on flavonoid pharmacokinetics and tissue distribution after oral administration of Radix Scutellaria extract in rats.

Radix Scutellaria is widely applied to the treatment of diabetes mellitus in China. Its main bioactive constituents contain baicalin, wogonoside, oroxyloside, and their aglycones. To investigate the effect of type 2 diabetes mellitus on both pharmacokinetics and tissue distribution of these flavonoid compounds, the six flavonoids in plasma and tissues from the normal and type 2 diabetic rats after oral administration of Radix Scutellaria extract were simultaneously measured by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method. The results showed that baicalin, wogonoside, and oroxyloside had higher Cmax and AUC values (P < 0.05) in type 2 diabetic rats than that in normal rats and the tissue-distribution behaviors of the six flavonoid compounds in hearts, livers, spleens, lungs, kidneys, brains, pancreas, fat and muscle of the type 2 diabetic rats showed obviously differences from the normal rats (P < 0.05). In conclusion, the differences in the pharmacokinetics of oroxyloside and tissue distribution of the six flavanoids in Radix Scutellaria extract between diabetic and normal rats were found for the first time. The results from the present study provided a crucial basis for a better understanding of in vivo anti-diabetic mechanism of action of the six flavonoids from Radix Scutellaria.

Comprehensive investigation of in-vivo ingredients and action mechanism of iridoid extract from Gardeniae Fructus by liquid chromatography combined with mass spectrometry, microdialysis sampling and network pharmacology.

Gardeniae Fructus is a widely used Traditional Chinese Medicines in treating various diseases. However, the absorbed components and metabolites of its main bioactive iridoid ingredients from iridoid extract of the fruits of Gardeniae Fructus in rat plasma need further study. In this study, a systematic method based on ultra-performance liquid chromatography-quadrupole-time-of-flight/mass spectrometry (UPLC-Q-TOF/MS) technique was developed to speculate the absorbed components and metabolites of iridoid extract in rat plasma after oral administration. A total of 19 compounds, including 9 prototype components and 10 metabolites were identified in plasma. 5 metabolites containing 4 new metabolites (M1, M2, M7, M10) were tentatively determined in rat plasma. Besides, Microdialysis-intensity-fading mass spectrometry (MD-IF-MS) method was originally employed to reveal the binding affinities with α-glucosidase for in-vivo prototype components and their metabolites. Finally, the absorbed constituents and the corresponding target proteins were used to generate compound-target network to find the related diseases and action pathways by a network pharmacology method. The results provide useful information for further study of pharmacology and in vivo mechanism of action of iridoid extract from the fruits of Gardeniae Fructus.

Systematic study on metabolism and activity evaluation of Radix Scutellaria extract in rat plasma using UHPLC with quadrupole time-of-flight mass spectrometry and microdialysis intensity-fading mass spectrometry.

Radix Scutellaria is a widely used traditional Chinese medicine in the treatment of various diseases. However, the activities of the absorbed components and metabolites of its main flavones in rat plasma need further investigation. In this study, a systematic method based on ultra-high performance liquid chromatography with quadruple time-of-flight mass spectrometry was developed to speculate the absorbed components and metabolites of the main flavonoids in Radix Scutellaria extract in rat plasma sample after oral administration of the extract. Twelve compounds, including four prototype components and eight metabolites, were confirmed in drug-containing plasma. In these metabolites, five were originally detected in rat plasma. The possible metabolic pathways of these polyhydroxy flavones in vivo were described and clarified. Microdialysis with intensity-fading mass spectrometry was originally employed to investigate the binding affinities of the absorbed components and metabolites with α-glucosidase. The order of their binding affinities was P4 > P3 > P2 > P1≥M5 > M3 > M1. The research result is helpful to deepen the understanding of the absorbed components and metabolic pathways of main flavones from Radix Scutellaria, and provide a new approach to screen potential inhibitors from in vivo components originated from Chinese herb.

Online microdialysis-ultra performance liquid chromatography-mass spectrometry method for comparative pharmacokinetic investigation on iridoids from Gardenia jasminoides Ellis in rats with different progressions of type 2 diabetic complications.

Iridoid glycosides consist the main bioactive constituents of Gardenia jasminoides Ellis (G. jasminoides), which is used alone or in combination with other medicine herbs for treatment of diabetes mellitus in China. This paper investigated for the first time comparative pharmacokinetics (PKs) of four unbound iridoids (including genipin, geniposide, gardenoside and geniposidic acid) in rat blood between healthy and type 2 diabetic groups with different disease progressions (9 or 13 weeks). Online microdialysis-ultra performance liquid chromatography-mass spectrometry (MD- UPLC-MS/MS) method was used after oral administration of iridoid extracts obtained from the fruits of G. jasminoides. Student's t-test was used for statistical comparison of PK parameters. Results showed that genipin, geniposide and geniposidic acid feature higher Cmax, larger area under the curve (AUC), lower clearance (CL), longer Tmax and mean residence time (MRT) in type 2 diabetic rats than those in healthy rats (p<0.05). However, no significant difference was observed in PK parameters between the two diabetic groups with different complication progressions (p>0.05). Type 2 diabetic rats showed significantly altered PK behaviors of genipin, geniposide and geniposidic acid (especially systemic exposure, AUCs of genipin, geniposide and geniposidic acid). Online MD-UPLC-MS/MS provides real-time sampling and monitoring, these functions can be applied to PKs of iridoids from G. jasminoides.

Simultaneous quantification method for comparative pharmacokinetics studies of two major metabolites from geniposide and genipin by online mircrodialysis-UPLC-MS/MS.

Genipin-1-o-glucuronic acid and genipin-monosulfate are two major metabolites from geniposide and genipin. Based on diabetic rat model, we developed a simultaneous quantification method to investigate their comparative pharmacokinetics by online mircrodialysis-ultra performance liquid chromatography-mass spectrometry (MD-UPLC-MS/MS) without their standard compounds. Online microdialysis sampling could avoid unexpected contamination or degradation of the analytes during the storage and transfer steps. Combined with good sensitivity, selectivity and selectivity of UPLC-MS/MS, online MD-UPLC-MS/MS method could real-timely monitor metabolites in rat blood for quantitative analysis. Our research found that AUC0→t of genipin-1-o-glucuronic acid and genipin-monosulfate in blood of diabetic group were 17.68 and 7.58 times than those in normal group, respectively, and AUC0→t of genipin-1-o-glucuronic acid was 2.28 times than that of genipin-monosulfate in blood of diabetic group, which revealed the effect of diabetes on the pharmacokinetic properties of the two metabolites. This study not only provides an approach for pharmacokinetic studies for various metabolites from herb medicines, but also can predict druggability of their bioactive metabolites. The insight obtained should facilitate drug development and toxicity research.

A strategy for identification and structural characterization of compounds from Gardenia jasminoides by integrating macroporous resin column chromatography and liquid chromatography-tandem mass spectrometry combined with ion-mobility spectrometry.

In this paper, an analysis strategy integrating macroporous resin (AB-8) column chromatography and high performance liquid chromatography-electrospray ionization-tandem mass spectrometry (HPLC-ESI-MS/MS) combined with ion mobility spectrometry (IMS) was proposed and applied for identification and structural characterization of compounds from the fruits of Gardenia jasminoides. The extracts of G. jasminoides were separated by AB-8 resin column chromatography combined with reversed phase liquid chromatography (C18 column) and detected by electrospray ionization tandem mass spectrometry. Additionally, ion mobility spectrometry (IMS) was employed as a supplementary separation technique to discover previously undetected isomers from the fruits of G. jasminoides. A total of 71 compounds, including iridoids, flavonoids, triterpenes, monoterpenoids, carotenoids and phenolic acids were identified by the characteristic high resolution mass spectrometry and the ESI-MS/MS fragmentations. In conclusion, the IMS-MS technique achieved the separation of isomers in crocin-3 and crocin-4 according to their acquired mobility drift times differing from classical analysis by mass spectrometry. The proposed strategy can be used as a highly sensitive and efficient procedure for identification and separation isomeric components in extracts of herbal medicines.

Bio-mimetic drug delivery systems designed to help the senior population reconstruct melatonin plasma profiles similar to those of the healthy younger population.

The secretion of melatonin (MT) is obviously different in the younger and the senior sectors of the population, and the maximum plasma concentration of seniors is only half of that in the younger population group. If exogenous MT can be supplied to senior citizens based on the secretion rate and amount of endogenous MT in the younger population by a bio-mimetic drug delivery system (DDS), an improved therapeutic effect and reduced side effects can be expected. Based upon this hypothesis, the pharmacokinetic parameters of MT, namely, the absorption rate constant (k a), the elimination rate constant (k e), and the ratio of absorption rate (F) to the apparent volume of distribution (V) were obtained by a residual method depending on the plasma concentration curve of immediate release preparations in the healthy younger population. The dose-division method was applied to calculate the cumulative release profiles of MT achieved by oral administration of a controlled release drug delivery system (DDS) to generate plasma MT profiles similar to the physiological level-time profiles. The in vivo release of MT deduced from the healthy younger population physiological MT profiles as the pharmacokinetic output of the bio-mimetic DDS showed a two-phase profile with two different zero order release rates, namely, 4.919 µg/h during 0-4 h (r=0.9992), and 11.097 µg/h during 4-12 h (r=0.9886), respectively. Since the osmotic pump type of DDS generally exhibits a good correlation between in vivo and in vitro release behaviors, an osmotic pump controlled delivery system was designed in combination with dry coating technology targeting on the cumulative release characteristics to mimic the physiological MT profiles in the healthy younger population. The high similarity between the experimental drug release profiles and the theoretical profiles (similarity factor f 2>50) and the high correlation between the predicted plasma concentration profiles and the theoretical plasma concentration profiles (r=0.9366, 0.9163, 0.9264) indicated that a prototype bio-mimetic drug delivery system of MT was established. The similarity factors between the experimental drug release profiles and the theoretical release profile were all larger than 50 both in periods of 0-4 h and 4-12 h, namely, 68.8 and 57.3 for the first batch (Batch No. 20131031), 76.7 and 50.2 for the second batch (Batch No. 20131101), and 73.7 and 51.1 for the third batch (Batch No. 20131126), respectively. The correlation coefficients between the predicted plasma concentration profiles based on the release profiles of the bio-mimetic DDS and physiological profiles were 0.9366 (Batch No. 20131031), 0.9163 (Batch No. 20131101), 0.9264 (Batch No. 20131126), respectively. Since the pharmacokinetic profile of MT in any kind of animal differs markedly from that of human beings, it is impossible to test the bio-mimetic DDS in animals directly. Therefore, the predicted pharmacokinetic profile based upon the in vitro release kinetics is an acceptable surrogate for the conventional animal test. In this research, a bio-mimetic DDS for replacement of MT was designed with in silico evaluation.

Evaluation of calcipotriol transdermal permeation through pig, rat and mouse skin using liquid chromatography-tandem mass spectrometry.

A rapid and sensitive liquid chromatography-tandem mass spectrometric method to evaluate the permeation and retention of calcipotriol in excised samples of pig, rat and mouse skin after application of a calcipotriol ointment has been developed and validated. After sample preparation of ointment, skin homogenate and receptor medium by liquid-liquid extraction, chromatography was performed on an Extend-C18 column using isocratic elution. Detection was by electrospray ionization in the negative ion mode using multiple-reaction monitoring of the precursor to product ion transitions of calcipotriol at m/z 411.1 → 393.5, and of lovastatin (internal standard) at m/z 403.2 → 101.2. The assay was linear in all matrices with LLOQs of 1, 0.5 and 40 ng/mL for skin homogenate, receptor medium and ointment samples respectively. In terms of the permeation profiles, it was found that calcipotriol permeated through all skins to only a limited extent over 20 h after application but was efficiently retained in all skins at a level at 20 h of between 40% (pig) and 60% (rat and mouse) of the applied dose. This indicates that calcipotriol ointment has the potential to provide sustained therapeutic benefit in the treatment of psoriasis with minimal systemic side effects.

Ternary system of dihydroartemisinin with hydroxypropyl-ß-cyclodextrin and lecithin: simultaneous enhancement of drug solubility and stability in aqueous solutions.

The purpose of this study was to simultaneously improve the solubility and stability of dihydroartemisinin (DHA) in aqueous solutions by a ternary cyclodextrin system comprised of DHA, hydroxypropyl-ß-cyclodextrin (HP-ß-CD) and a third auxiliary substance. Solubility and phase solubility studies were carried out to evaluate the solubilizing efficiency of HP-ß-CD in association with various auxiliary substances. Then, the solid binary (DHA-HP-ß-CD or DHA-lecithin) and ternary systems were prepared and characterized by Fourier transform infrared (FT-IR), differential scanning calorimetry (DSC) and power X-ray diffraction (PXRD). The effect of the ternary system on the solubility, dissolution and stability of DHA in aqueous solutions was also investigated. As a result, the soybean lecithin was found to be the most promising third component in terms of solubility enhancement. For the solid characterization, the disappearance of the drug crystallinity indicated the formation of new solid phases, implicating the formation of the ternary system. The dissolution rate of the solid ternary system was much faster than that of the drug alone and binary systems. Importantly, compared with binary systems, the ternary system showed a significant improvement in the stability of DHA in Hank's balanced salt solutions (pH 7.4). The solubility and stability of DHA in aqueous solutions were simultaneously enhanced by the ternary system, which might be attributed to the possible formation of a ternary complex. For the ternary interactions, results of molecular docking studies further indicated that the lecithin covered the top of the wide rim of HP-ß-CD and surrounded around the peroxide bridging of DHA, providing the possibility for the ternary complex formation. In summary, the ternary system prepared in our study, with simultaneous enhancement of DHA solubility and stability in aqueous solutions, might have an important pharmaceutical potential in the development of a better oral formulation of DHA.

Quantitation of bivalirudin, a novel anticoagulant peptide, in human plasma by LC-MS/MS: Method development, validation and application to pharmacokinetics.

A rapid and sensitive method based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) for the determination of a novel anticoagulant peptide bivalirudin in human plasma has been developed and validated. Plasma samples were precipitated protein with acetonitrile and re-extracted with dichloromethane, after which the analyte and triptorelin as an internal standard (IS) were separated on a 300SB-C18 column (150 mm×4.6 mm i.d., 5 µm particle size) using 0.1% formic acid:methanol (45:55, v/v) as mobile phase. The triple-quadrupole mass spectrometer, equipped with electrospray ionization (ESI) interface, was operated in the positive ion mode, and the multiple-reaction monitoring (MRM) transitions of bivalirudin and IS were at m/z 1091.0→650.4 and m/z 656.5→249.3, respectively. The lower limit of quantification (LLOQ) was 1 ng/mL for 100 µL plasma sample and the assay was linear over the concentration range 1-1000 ng/mL. The accuracy was within a range from -0.4% to 0.5% in terms of relative error (RE) and the intra- and inter-day precisions in terms of relative standard deviation (RSD) were ≤2.92 and ≤3.36, respectively. The method was successfully applied to a pharmacokinetic study involving intravenous administration of bivalirudin (0.5 mg/kg) to Chinese volunteers.

Development of cell-active N6-methyladenosine RNA demethylase FTO inhibitor.

The direct nucleic acid repair dioxygenase FTO is an enzyme that demethylates N(6)-methyladenosine (m(6)A) residues in mRNA in vitro and inside cells. FTO is the first RNA demethylase discovered that also serves a major regulatory function in mammals. Together with structure-based virtual screening and biochemical analyses, we report the first identification of several small-molecule inhibitors of human FTO demethylase. The most potent compound, the natural product rhein, which is neither a structural mimic of 2-oxoglutarate nor a chelator of metal ion, competitively binds to the FTO active site in vitro. Rhein also exhibits good inhibitory activity on m(6)A demethylation inside cells. These studies shed light on the development of powerful probes and new therapies for use in RNA biology and drug discovery.

Expression of endostatin mediated by a novel non-viral delivery system inhibits human umbilical vein endothelial cells in vitro.

Ultrasound (US)-mediated microbubble destruction is recognized to have considerable potential for gene delivery, whereas, there is few report of its effect on enhancing liposomal transfection. In this study, we used pIRES2-EGFP/hES containing human endostatin (hES) cDNA as target gene to test the hypothesis that US exposure with microbubbles could improve liposomal transfection, and to investigate the possibility of intracellular delivery of ES gene using this method. Under the controlled US exposure condition with microbubbles, the plasmid:liposome was transferred into COS-7 cells. The transfection rate, the expression of endostatin and the inhibition effect of transfection-endostatin on endothelial cells were assessed. The results revealed that US-mediated microbubble destruction together with liposome could significantly enhance gene transfection without obvious cell damage. By this means, endostatin gene could be efficiently transferred into COS-7 cells and expressed. The transfection-endostatin could inhibit endothelial proliferation and migration, which suggests that the non-viral method might be useful in anti-angiogenesis therapy in the future.