Chaotropic Effect-Induced Sol-Gel Transition and Radical Stabilization for Bacterially Sensitive Near-Infrared Photothermal Therapy.

Deep-seated bacterial infections (DBIs) are stubborn and deeply penetrate tissues. Eliminating deep-seated bacteria and promoting tissue regeneration remain great challenges. Here, a novel radical-containing hydrogel (SFT-B Gel) cross-linked by a chaotropic effect was designed for the sensing of DBIs and near-infrared photothermal therapy (NIR-II PTT). A silk fibroin solution stained with 4,4',4″-(1,3,5-triazine-2,4,6-triyl)tris(1-methylpyridin-1-ium) (TPT3+) was employed as the backbone, which could be cross-linked by a closo-dodecaborate cluster (B12H122-) through a chaotropic effect to form the SFT-B Gel. More interestingly, the SFT-B Gel exhibited the ability to sense DBIs, which could generate a TPT2+• radical with obvious color changes in the presence of bacteria. The radical-containing SFT-B Gel (SFT-B★ Gel) possessed strong NIR-II absorption and a remarkable photothermal effect, thus demonstrating excellent NIR-II PTT antibacterial activity for the treatment of DBIs. This work provides a new approach for the construction of intelligent hydrogels with unique properties using a chaotropic effect.

Impact of wrist-ankle acupuncture on propofol dosage under the dual monitoring of density spectrum array and anesthesia consciousness index in elderly patients undergoing urologic surgery: a sham-controlled randomized clinical trial.

Background: Propofol is a widely used intravenous anesthetic in clinic. However, it is easy to cause serious circulatory fluctuation in elderly patients, so the dose should be reduced as appropriate. Studies have shown that wrist-ankle acupuncture (WAA) can reduce the dosage of propofol in patients undergoing painless endoscopy. Unfortunately, there is no report on whether WAA will reduce the dosage of propofol when used for anesthesia in elderly patients. The purpose of this study is to observe the effect of WAA on propofol dosage in elderly patients, and to provide a new method for maintaining circulatory stability in elderly patients under general anesthesia. Methods: From October 2022 to December 2022, Hebei Provincial Hospital of Traditional Chinese Medicine was selected. Forty-four elderly patients undergoing general anesthesia in urology department were randomly divided into two groups according to the complete random method with WAA group, consisting of 22 individuals, and non-WAA (NWAA) group, also consisting of 22 individuals. Both groups were treated with WAA or false needle acupuncture at the same site before anesthesia, respectively, and the needle was kept until the operation was finished. During the operation, the dosage of propofol was adjusted according to the depth of field monitoring density spectrum array (DSA) and anesthesia consciousness index (Ai) with anesthesia monitor. Results: A total of 44 patients participated in this study, and all of them completed the experiment. There were no significant difference in sex, age, height, weight, duration of anesthesia, liver and kidney function, score of Fried frailty scale, activity of daily living (ADL), age-adjusted Charlson comorbidity index (aCCI) and mini-cognitive test (Mini-Cog) between the two groups (P>0.05), but the total dose of propofol (WAA =121.5, NWAA =170.5) mg and maintenance dose (WAA =1.02±0.55, NWAA =1.76±0.67) mg/kg/h, utilization rate of vasoactive drugs during operation, recovery time after anesthesia (WAA =2, NWAA =3) min and surgeon satisfaction (WAA =9, NWAA =8.5) had significant differences (P<0.05). Conclusions: Compared with NWAA group, WAA group could reduce the dosage of propofol in anesthesia for elderly patients with exocrine secretion and was beneficial to circulatory stability. Trial Registration: Chinese Clinical Trial Registry (ID: ChiCTR2100054132).

Nano-Photosensitizer Directed Targeted Phototherapy Effective Against Oral Cancer in Animal Model.

Background: Photodynamic therapy (PDT) has emerged as a promising strategy for oral cancer treatment. Verteporfin is a powerful photosensitizer and widely used in the treatment of macular degeneration. However, rare work has reported its potential in the treatment of oral cancer. Methods: In this study, we introduce an innovative approach of nano-photosensitizer based on Verteporfin, which was prepared by utilizing macrophage membrane to coat Verteporfin-loaded zeolitic imidazolate framework 8 (ZIF-8) for effective photodynamic therapy against oral cancer. Nanoparticle characteristics were assessed including size, zeta potential, and PDI. Cellular uptake studies were conducted using CAL-27 cells. Furthermore, inhibitory effects in both in vitro and in vivo settings were observed, ensuring biosafety. Assessment of anticancer efficacy involved tumor volume measurement, histological analyses, and immunohistochemical staining. Results: In vitro experiments indicated that the nano-photosensitizer showed efficient cellular uptake in the oral cancer cells. Upon the laser irradiation, the nano-photosensitizer induced the generation of reactive oxygen species (ROS), leading to cancer cell apoptosis. The in vivo experiments indicated that the coating with cell membranes enhanced the circulation time of nano-photosensitizer. Moreover, the specificity of the nano-photosensitizer to the cancer cells was also improved by the cell membrane-camouflaged structure in the tumor-bearing mouse model, which inhibited the tumor growth significantly by the photodynamic effect in the presence of laser irradiation. Conclusion: Overall, our findings demonstrate the potential of macrophage membrane-coated ZIF-8-based nanoparticles loaded with Verteporfin for effective photodynamic therapy in oral cancer treatment. This nano-system holds promise for synergistic cancer therapy by combining the cytotoxic effects of PDT with the activation of the immune system, providing a novel therapeutic strategy for combating cancer.

Association Between Omega-3 Fatty Acid Intake and Dyslipidemia: A Continuous Dose-Response Meta-Analysis of Randomized Controlled Trials.

Background Previous results provide supportive but not conclusive evidence for the use of omega-3 fatty acids to reduce blood lipids and prevent events of atherosclerotic cardiovascular disease, but the strength and shape of dose-response relationships remain elusive. Methods and Results This study included 90 randomized controlled trials, reported an overall sample size of 72 598 participants, and examined the association between omega-3 fatty acid (docosahexaenoic acid, eicosapentaenoic acid, or both) intake and blood lipid changes. Random-effects 1-stage cubic spline regression models were used to study the mean dose-response association between daily omega-3 fatty acid intake and changes in blood lipids. Nonlinear associations were found in general and in most subgroups, depicted as J-shaped dose-response curves for low-/high-density lipoprotein cholesterol. However, we found evidence of an approximately linear dose-response relationship for triglyceride and non-high-density lipoprotein cholesterol among the general population and more evidently in populations with hyperlipidemia and overweight/obesity who were given medium to high doses (>2 g/d). Conclusions This dose-response meta-analysis demonstrates that combined intake of omega-3 fatty acids near linearly lowers triglyceride and non-high-density lipoprotein cholesterol. Triglyceride-lowering effects might provide supportive evidence for omega-3 fatty acid intake to prevent cardiovascular events.

Suo Quan Wan ameliorates bladder overactivity and regulates neurotransmission via regulating Myosin Va protein expression.

BACKGROUND: Ancient prescriptions of Suo Quan Wan (SQW) have therapeutic effects on diabetic bladder dysfunction. However, the underlying mechanism remains unclear. Here, we hypothesized that SQW ameliorates bladder overactivity and regulates neurotransmission via regulating Myosin Va protein expression. METHODS: After diabetic rats were induced by streptozotocin (65 mg/kg), the model of diabetic bladder dysfunction was established by detecting fasting blood glucose, urodynamic test, in vitro muscle strip experiments, and histological examination. One week after induction, SQW was given to observe the therapeutic effect. The expression levels of Myosin Va in control, Model, SQW L and SQW H groups were detected by RT-qPCR, RNAscope and immunofluorescence assay. The expression levels of ChAT, SP, nNOS and VIP proteins were observed by immunofluorescence assay. After knockdown and overexpression of Myosin Va, the expression changes of ChAT, SP, nNOS and VIP and the regulatory role of SQW were observed. RESULTS: STZ-induced DM rats had significantly higher serum glucose levels and lower body weight. Compared with the diabetic rats, SQW treatment significantly improved urination function with decreased residual volume (RV), bladder compliance (BC), non-voiding contractions (NVCs), and increased voided efficiency (VE). In addition, contractile responses of muscle strips to electrical-field stimulation (EFS), carbachol (CCh), KCl were significantly lower in the SQW H and SQW L groups than those in the model group. RT-qPCR found that the expression of Myosin Va in the bladder tissue or bladder neurons in model group was significantly increased compared with the control group, and SQW treatment significantly decreased the levels of Myosin Va. In DM rats, ChAT and SP expression were significantly increased, while nNOS and VIP expression were significantly decreased, and SQW improved this phenomenon. Interestingly, SQW ameliorated the abnormal expression of ChAT, SP, nNOS and VIP caused by myosin Va knockdown, and Myosin Va overexpression results are consistent with these. CONCLUSIONS: SQW ameliorates overactive bladder and regulate neurotransmission via regulating Myosin Va mRNA and protein expression.

Chemical characterization of the antioxidant and α-glucosidase inhibitory active fraction of Malus transitoria leaves.

Chinese Tibetan tea made from the tender leaves of Malus transitoria is a widely consumed health drink, but there are few reports on its chemical composition and biological activity. In this study, we found that a 50% ethanol extract of M. transitoria had good antioxidant and α-glucosidase inhibitory activities in vitro. Guided by in vitro bioassays, chromatographic separation and purification were conducted, and the most active fraction in M. transitoria was determined. UPLC-Orbitrap-MS/MS was used to further quickly and comprehensively characterize the chemical composition. Library searches, MS/MS fragmentation patterns of two isolated reference compounds, and bibliography were used to annotate 81 compounds, of which 2 were new compounds, and 79 were identified from M. transitoria for the first time. This study provides a scientific basis for the development of antioxidant and anti-diabetic functional foods from M. transitoria.

Three new ursane-type triterpenoids from Rosmarinus officinalis and their biological activities.

Three new ursane-type triterpenoids, 3-oxours-12-en-20, 28-olide (1), 3ß-hydroxyurs-12-en-20, 28-olide (2) and 3ß-hydroxyurs-11, 13(18)-dien-20, 28-olide (3), were isolated from a potent anti-inflammatory and antibacterial fraction of the ethanolic extract of Rosmarinus officinalis. Their structures were elucidated by a combination of extensive 1D- and 2D-NMR experiments, MS data and comparisons with literature reports. Compounds 1-3 exhibited significantly inhibitory effects on nitric oxide production in lipopolysaccharide-activated mouse RAW264.7 macrophages, but no antibacterial activity was found at a concentration of 128 µg·mL-1.

Applications of Molecular Imprinting Technology in the Study of Traditional Chinese Medicine.

Traditional Chinese medicine (TCM) is one of the most internationally competitive industries. In the context of TCM modernization and internationalization, TCM-related research studies have entered a fast track of development. At the same time, research of TCM is also faced with challenges, such as matrix complexity, component diversity and low level of active components. As an interdisciplinary technology, molecular imprinting technology (MIT) has gained popularity in TCM study, owing to the produced molecularly imprinted polymers (MIPs) possessing the unique features of structure predictability, recognition specificity and application universality, as well as physical robustness, thermal stability, low cost and easy preparation. Herein, we comprehensively review the recent advances of MIT for TCM studies since 2017, focusing on two main aspects including extraction/separation and purification and detection of active components, and identification analysis of hazardous components. The fundamentals of MIT are briefly outlined and emerging preparation techniques for MIPs applied in TCM are highlighted, such as surface imprinting, nanoimprinting and multitemplate and multifunctional monomer imprinting. Then, applications of MIPs in common active components research including flavonoids, alkaloids, terpenoids, glycosides and polyphenols, etc. are respectively summarized, followed by screening and enantioseparation. Related identification detection of hazardous components from TCM itself, illegal addition, or pollution residues (e.g., heavy metals, pesticides) are discussed. Moreover, the applications of MIT in new formulation of TCM, chiral drug resolution and detection of growing environment are summarized. Finally, we propose some issues still to be solved and future research directions to be expected of MIT for TCM studies.

New polymerized sesquiterpene lactones from Ainsliaea yunnanensis and their activity evaluation.

Two new dimeric and trimeric sesquiterpene lactones (1-2), and nine known sesquiterpene lactones (3-11) were isolated from the EtOAc phase of the ethanolic extract of Ainsliaea yunnanensis. Their structures were identified by NMR, IR and HR-ESIMS spectroscopic methods, and compound 1 was confirmed by single crystal X-ray diffraction experiment. All the compounds were tested for their cytotoxic, anti-microbial and anti-inflammatory activities. Compounds 1, 2, 3, 5, 7, 9 and 11 showed very significant selective cytotoxic activities on MDA-MB-468, PANC-1, HEPG2 or A549 cells. Compounds 6 and 11 showed very significant inhibiting effect on Epicoccum sp. (CPCC 400307), Fusarium solani (CPCC 800013) or Bacillus subtilis. Meanwhile, compounds 6 and 7 can inhibit the NLRP3 inflammasome's activation at the concentration of 10 µM.

Norursane-type triterpenoids from Rosmarinus officinalis and their anti-inflammatory activity evaluation.

Five norursane-type triterpenoids, including three novel of 3ß-28-norursa-12,17,19,21-tetraene-3-ol (1), 3ß-28-norursa-12,20(30)-dien-3-ol (2) and 3ß-28-norursa-12,16,20(30)-triene-3-ol (3), as well as two known 3ß-28-norursa-17,19,21-trien-3-ol (4) and 3ß-28-norursa-12-ene-3-ol (5) were isolated from the ethyl acetate dissolved fraction of the ethanol extract from Rosmarinus officinalis. Their structures were elucidated by HR-ESI-MS, IR, 1D- and 2D-NMR spectroscopic methods. Compounds 1-5 exhibited significant inhibitory effect on NO production in LPS-activated RAW264.7 cells, and compounds 2, 3 and 5 shown better anti-inflammatory activity.

Chemical characterization of a PD-1/PD-L1 inhibitory activity fraction of the ethanol extract from Gymnadenia conopsea.

Searching for PD-1/PD-L1 inhibitor from medicinal plants has become a potential method to discover small molecular cancer immunotherapy drugs. Using PD-1/PD-L1 inhibitory activity assay in vitro, a bioactive fraction was obtained from the ethanol extract of Gymnadenia conopsea. A sensitive UPLC-HRMS/MS method was established for the rapid screening and identification of compositions from bioactive fraction. Based on the characteristic fragmentation patterns of standards analysis and extracted ion chromatogram (EIC) method, 46 compounds were rapidly screened and identified (including 35 succinic acid ester glycosides and 11 other compounds), among which 17 compounds were tentatively identified as new compounds.

Chemical characterization of the polar antibacterial fraction of the ethanol extract from Rosmarinus officinalis.

Rosmarinus officinalis L. has been widely used as a spice to extend the shelf life of foods. Most studies in the literature indicate that its essential oil is its major antibacterial component. In this study, a polar fraction from rosemary exhibited considerably stronger antibacterial activity against Bacillus subtilis than its essential oil. Guided by rapid characterization of the chemical compositions based on UPLC-Orbitrap-MS/MS, further investigation resulted in the isolation and identification of sixteen compounds. Among them, two new and six known compounds were identified in rosemary for the first time. Most isolated compounds exhibited significant antibacterial activities with minimum inhibitory concentration values of 2-128 µg/mL; however, these activities were weaker than that of the polar fraction. Thus, the polar fraction demonstrated a promising potential to serve as a food additive, as an alternative to the essential oil, because of its stronger antibacterial activity.

HuangqiGuizhiWuwu Decoction Prevents Vascular Dysfunction in Diabetes via Inhibition of Endothelial Arginase 1.

Hyperglycemia induces vascular endothelial dysfunction, which contributes to the development of vascular complication of diabetes. A classic prescription of traditional medicine, HuangqiGuizhiWuwu Decoction (HGWWD) has been used for the treatment of various cardiovascular and cerebrovascular diseases, which all are related with vascular pathology. The present study investigated the effect of HGWWD treatment in streptozocin (STZ)-induced vascular dysfunction in mouse models. In vivo studies were performed using wild type mice as well as arginase 1 knockout specific in endothelial cells (EC-A1-/-) of control mice, diabetes mice and diabetes mice treated with HGWWD (60 g crude drugs/kg/d) for 2 weeks. For in vitro studies, aortic tissues were treated with mice serum containing HGWWD with or without adenoviral arginase 1 (Ad-A1) transduction in high glucose (HG) medium. We found that HGWWD treatment restored STZ-induced impaired mean velocity and pulsatility index of mouse left femoral arteries, aortic pulse wave velocity and vascular endothelial relaxation accompanied by elevated NO production in the aorta and plasma, as well as reduced endothelial arginase activity and aortic arginase 1 expression. The protective effect of HGWWD is reversed by an inhibitor of nitric oxide synthesis. Meanwhile, the preventive effect of serum containing HGWWD in endothelial vascular dysfunction is completely blocked by Ad-A1 transduction in HG incubated aortas. HGWWD treatment further improved endothelial vascular dysfunction in STZ induced EC-A1-/- mice. This study demonstrates that HGWWD improved STZ-induced vascular dysfunction through arginase 1 - NO signaling, specifically targeting endothelial arginase 1.

Rapid Characterizaiton of Chemical Constituents of the Tubers of Gymnadenia conopsea by UPLC-Orbitrap-MS/MS Analysis.

Gymnadenia conopsea R. Br. is a traditional Tibetan medicinal plant that grows at altitudes above 3000 m, which is used to treat neurasthenia, asthma, coughs, and chronic hepatitis. However, a comprehensive configuration of the chemical profile of this plant has not been reported because of the complexity of its chemical constituents. In this study, a rapid and precise method based on ultra-high performance liquid chromatography (UPLC) combined with an Orbitrap mass spectrometer (UPLC-Orbitrap-MS/MS) was established in both positive- and negative-ion modes to rapidly identify various chemical components in the tubers of G. conopsea for the first time. Finally, a total of 91 compounds, including 17 succinic acid ester glycosides, 9 stilbenes, 6 phenanthrenes, 19 alkaloids, 11 terpenoids and steroids, 20 phenolic acid derivatives, and 9 others, were identified in the tubers of G. conopsea based on the accurate mass within 3 ppm error. Furthermore, many alkaloids, phenolic acid derivates, and terpenes were reported from G. conopsea for the first time. This rapid method provides an important scientific basis for further study on the cultivation, clinical application, and functional food of G. conopsea.

SPIO Enhance the Cross-Presentation and Migration of DCs and Anionic SPIO Influence the Nanoadjuvant Effects Related to Interleukin-1ß.

Superparamagnetic iron oxide nanoparticles (SPIO) have been synthesized and explored for use as carriers of various nanoadjuvants via loading into dendritic cells (DCs). In our study, homogeneous and superparamagnetic nanoparticles are susceptible to internalization by DCs and SPIO-pulsed DCs showed excellent biocompatibility and capacity for ovalbumin (OVA) cross-presentation. Herein, we found that SPIO-loaded DCs can promote the maturation and migration of DCs in vitro. SPIO coated with 3-aminopropyltrimethoxysilane (APTS) and meso-2,3-dimercaptosuccinic acid (DMSA), which present positive and negative charges, respectively, were prepared. We aimed to investigate whether the surface charge of SPIO can affect the antigen cross-presentation of the DCs. Additionally, the formation of interleukin-1ß (IL-1ß) was examined after treatment with oppositely charged SPIO to identify the nanoadjuvants mechanism. In conclusion, our results suggest that SPIO are biocompatible and can induce the migration of DCs into secondary lymph nodes. SPIO coated with APTS (SPIO/A+) exhibited excellent adjuvant potentials for the promotion of antigen cross-presentation and T cell activation and surpassed that of DMSA-coated nanoparticles (SPIO/D-). This process may be related to the secretion of IL-1ß. Our study provides insights into the predictive modification of nanoadjuvants, which will be valuable in DC vaccine design and could lead to the creation of new adjuvants for applications in vaccines for humans.

A UHPLC-QTOF-MS/MS method for the simultaneous determination of eight triterpene compounds from Poria cocos (Schw.) Wolf extract in rat plasma: Application to a comparative pharmacokinetic study.

Poria cum Radix Pini (PRP), White Poria (WP), Rubra Poria (RP), and Poriae Cutis (PC), different parts of the dried sclerotium of Poria cocos (Schw.) Wolf (PCW), have possessed various pharmacological effects and clinical application. In the present study, a novel ultra-high performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UHPLC-QTOF-MS/MS) method was developed and validated for the simultaneous determination of eight triterpene compounds in rat plasma and then was applied in the comparison of pharmacokinetic characteristics of PRP, WP, RP, and PC extracts. Chromatographic separation was performed on an ACQUITY UPLC® BEH C18 (2.1 × 100 mm, 5 µm) with a mobile phase composed of aqueous solution (containing 0.5‰ formic acid and 0.5 mmol/L ammonium acetate) and acetonitrile in gradient elution. Mass spectrometric of the analytes and internal standard (IS) were conducted in negative electrospray ionization with high-resolution multiple reaction monitoring (MRMHR) mode. The lower limit of quantification (LLOQ) for the eight analytes were in the range of 2.00-20.16 ng/mL. All calibration curves showed good linearity (r > 0.993). The inter- and intra-batch precision and accuracy for the eight triterpene compounds were acceptable. The results indicated that the eight triterpene compounds displayed different pharmacokinetic characteristics in PRP, WP, RP, and PC, and that poricoic acid B, poricoic acid A, pachymic acid, dehydrotrametenolic acid, dehydrotumulosic acid, polyporenic acid C and dehydropachymic acid may be the major bioactive compounds of PCW contributing to the diuretic effect.

Discovery of targetable genetic alterations in advanced non-small cell lung cancer using a next-generation sequencing-based circulating tumor DNA assay.

Next-generation sequencing (NGS)-based circulating tumor DNA (ctDNA) assays have provided a new method of identifying tumor-driving genes in patients with advanced non-small cell lung carcinoma (NSCLC), especially in those whose cancer tissues are unavailable or in those that have acquired treatment resistance. Here, we describe a total of 119 patients with advanced EGFR-TKI-naive NSCLC and 15 EGFR-TKI-resistant patients to identify somatic SNVs, small indels, CNVs and gene fusions in 508 tumor-related genes. Somatic ctDNA mutations were detected in 82.8% (111/134) of patients in the total cohort. Of the 119 patients with advanced NSCLC, 27.7% (33/119) were suitable for treatment with National Comprehensive Cancer Network (NCCN) guideline-approved targeted drugs. Actionable genetic alterations included 25 EGFR mutations, 5 BRAF mutations, and 1 MET mutation, as well as 1 EML4-ALK gene fusion and 1 KIF5B-RET gene fusion. In 19.3% (23/119) of the patients, we also identified genomic alterations with that could be targeted by agents that are in clinical trials, such as mTOR inhibitors, PARP inhibitors, and CDK4/6 inhibitors. Additionally, the EGFR T790M mutation was found in 46.7% (7/15) of the patients with EGFR-TKI-resistant NSCLC, suggesting that the NGS-based ctDNA assay might be an optional method to monitor EGFR-TKI resistance and to discover mechanisms of drug resistance.

Effect of halotolerant rhizobacteria isolated from halophytes on the growth of sugar beet (Beta vulgaris L.) under salt stress.

Utilization of rhizobacteria that have associated with plant roots in harsh environments could be a feasible strategy to deal with limits to agricultural production caused by soil salinity. Halophytes occur naturally in high-salt environments, and their roots may be associated with promising microbial candidates for promoting growth and salt tolerance in crops. This study aimed to isolate efficient halotolerant plant-growth-promoting rhizobacterial strains from halophytes and evaluate their activity and effects on sugar beet (Beta vulgaris L.) growth under salinity stress. A total of 23 isolates were initially screened for their ability to secrete 1-aminocyclopropane-1-carboxylate deaminase (ACD) as well as other plant-growth-promoting characteristics and subsequently identified by sequencing the 16S rRNA gene. Three isolates, identified as Micrococcus yunnanensis, Planococcus rifietoensis and Variovorax paradoxus, enhanced salt stress tolerance remarkably in sugar beet, resulting in greater seed germination and plant biomass, higher photosynthetic capacity and lower stress-induced ethylene production at different NaCl concentrations (50-125 mM). These results demonstrate that salinity-adapted, ACD-producing bacteria isolated from halophytes could promote sugar beet growth under saline stress conditions.

A pre-classification strategy based on UPLC-Triple-TOF/MS for metabolic screening and identification of Radix glehniae in rats.

Traditional Chinese Medicines (TCMs) have gained increasing popularity in modern society. However, the profiles of TCMs in vivo are still unclear owing to their complexity and low level in vivo. In this study, UPLC-Triple-TOF techniques were employed for data acquiring, and a novel pre-classification strategy was developed to rapidly and systematically screen and identify the absorbed constituents and metabolites of TCMs in vivo using Radix glehniae as the research object. In this strategy, pre-classification for absorbed constituents was first performed according to the similarity of their structures. Then representative constituents were elected from every class and analyzed separately to screen non-target absorbed constituents and metabolites in biosamples. This pre-classification strategy is basing on target (known) constituents to screen non-target (unknown) constituents from the massive data acquired by mass spectrometry. Finally, the screened candidate compounds were interpreted and identified based on a predicted metabolic pathway, well - studied fragmentation rules, a predicted metabolic pathway, polarity and retention time of the compounds, and some related literature. With this method, a total of 111 absorbed constituents and metabolites of Radix glehniae in rats' urine, plasma, and bile samples were screened and identified or tentatively characterized successfully. This strategy provides an idea for the screening and identification of the metabolites of other TCMs.

Tetramethylpyrazine induces SH-SY5Y cell differentiation toward the neuronal phenotype through activation of the PI3K/Akt/Sp1/TopoIIß pathway.

Tetramethylpyrazine (TMP) is an active compound extracted from the traditional Chinese medicinal herb Chuanxiong. Previously, we have shown that TMP induces human SH-SY5Y neuroblastoma cell differentiation toward the neuronal phenotype by targeting topoisomeraseIIß (TopoIIß), a protein implicated in neural development. In the present study, we aimed to elucidate whether the transcriptional factors specificity protein 1 (Sp1) and nuclear factor Y (NF-Y), in addition to the upstream signaling pathways ERK1/2 and PI3K/Akt, are involved in modulating TopoIIß expression in the neuronal differentiation process. We demonstrated that SH-SY5Y cells treated with TMP (80µM) terminally differentiated into neurons, characterized by increased neuronal markers, tubulin ßIII and microtubule associated protein 2 (MAP2), and increased neurite outgrowth, with no negative effect on cell survival. TMP also increased the expression of TopoIIß, which was accompanied by increased expression of Sp1 in the differentiated neuron-like cells, whereas NF-Y protein levels remained unchanged following the differentiation progression. We also found that the phosphorylation level of Akt, but not ERK1/2, was significantly increased as a result of TMP stimulation. Furthermore, as established by chromatin immunoprecipitation (ChIP) assay, activation of the PI3K/Akt pathway increased Sp1 binding to the promoter of the TopoIIß gene. Blockage of PI3K/Akt was shown to lead to subsequent inhibition of TopoIIß expression and neuronal differentiation. Collectively, the results indicate that the PI3K/Akt/Sp1/TopoIIß signaling pathway is necessary for TMP-induced neuronal differentiation. Our findings offer mechanistic insights into understanding the upstream regulation of TopoIIß in neuronal differentiation, and suggest potential applications of TMP both in neuroscience research and clinical practice to treat relevant diseases of the nervous system.