Intelligent supervision of PIVAS drug dispensing based on image recognition technology.

Pharmacy Intravenous Admixture Services (PIVAS) are places dedicated to the centralized dispensing of intravenous drugs, usually managed and operated by professional pharmacists and pharmacy technicians, and are an integral part of modern healthcare. However, the workflow of PIVAS has some problems, such as low efficiency and error-prone. This study aims to improve the efficiency of drug dispensing, reduce the rate of manual misjudgment, and minimize drug errors by conducting an in-depth study of the entire workflow of PIVAS and applying image recognition technology to the drug checking and dispensing process. Firstly, through experimental comparison, a target detection model suitable for drug category recognition is selected in the drug-checking process of PIVAS, and it is improved to improve the recognition accuracy and speed of intravenous drug categories. Secondly, a corner detection model for drug dosage recognition was studied in the drug dispensing stage to further increase drug dispensing accuracy. Then the PIVAS drug category recognition system and PIVAS drug dosage recognition system were designed and implemented.

Andrographolide acts with dexamethasone to inhibit the growth of acute lymphoblastic leukemia CEM­C1 cells via the regulation of the autophagy­dependent PI3K/AKT/mTOR signaling pathway.

Acute lymphoblastic leukemia (ALL) is one of the most common malignant tumor types of the circulatory system. Dexamethasone (DEX) acts on the glucocorticoid (GC) receptor (GR) and is a first-line chemotherapy drug for ALL. However, long-term or high-dose applications of the drug can not only cause adverse reactions, such as osteoporosis and high blood pressure, but can also cause downregulation of GR and lead to drug resistance. In the present study, reverse transcription-quantitative PCR, western blotting and LysoTracker Red staining were used to observe the effects of DEX and andrographolide (AND; a botanical with antitumorigenic properties) combined treatment. It was found that AND enhanced the sensitivity of CEM-C1 cells, a GC-resistant cell line, to DEX, and synergistically upregulated GR both at the transcriptional and post-transcriptional level with DEX. The combination of AND with DEX synergistically alkalized lysosomal lumen and downregulated the expression of autophagy-related genes Beclin1 and microtubule-associated 1 protein light chain 3 (LC3), thereby inhibiting autophagy. Knocking down LC3 expression enhanced GR expression, suggesting that GR was regulated by autophagy. Furthermore, compared with the monotherapy group (AND or DEX in isolation), AND interacted with DEX to activate the autophagy-dependent PI3K/AKT/mTOR signaling pathway by enhancing the phosphorylation of PI3K, AKT and mTOR, thereby decreasing GR degradation and increasing the sensitivity of cells to GCs. In conclusion, the present study demonstrated that AND exhibited a synergistic anti-ALL effect with DEX via upregulation of GR, which was orchestrated by the autophagy-related PI3K/AKT/mTOR signaling pathway. The results of the present study therefore provided novel research avenues and strategies for the treatment of ALL.

Development of a prognostic model for anoikis and identifies hub genes in hepatocellular carcinoma.

Considering the high fatality of hepatocellular carcinoma (HCC), current prognostic systems are insufficient to accurately forecast HCC patients' outcomes. In our study, nine anoikis­related genes (PTRH2, ITGAV, ANXA5, BIRC5, BDNF, BSG, DAP3, SKP2, and EGF) were determined to establish a risk scoring model using LASSO regression, which could be validated in ICGC dataset. Kaplan-Meier curves and time-dependent receiver operating characteristic (ROC) curve analysis confirmed the risk score possessed an accurate predictive value for the prognosis of HCC patients. The high-risk group showed a higher infiltration of aDCs, macrophages, T-follicular helper cells, and Th2 cells. Besides, PD-L1 was significantly higher in the high-risk group compared to the low-risk group. Several anoikis­related genes, such as ANX5, ITGAV, BDNF and SKP2, were associated with drug sensitivity in HCC. Finally, we identified BIRC5 and SKP2 as hub genes among the nine model genes using WGCNA analysis. BIRC5 and SKP2 were over-expressed in HCC tissues, and their over-expression was associated with poor prognosis, no matter in our cohort by immunohistochemical staining or in the TCGA cohort by mRNA-Seq. In our cohort, BIRC5 expression was highly associated with the T stage, pathologic stage, histologic grade and AFP of HCC patients. In general, our anoikis-related risk model can enhance the ability to predict the survival outcomes of HCC patients and provide a feasible therapeutic strategy for immunotherapy and drug resistance in HCC. BIRC5 and SKP2 are hub genes of anoikis­related genes in HCC.

Fast degradation of atrazine by nZVI-Cu0/PMS: Re-evaluation and quantification of reactive species, generation pathways, and application feasibility.

Additive metal to zero-valent iron (ZVI) could enhance the reduction ability and the additive Cu0 was incorporated to ZVI to accelerate PMS activation with atrazine (ATZ) as target compound. The efficiencies of ATZ degradation and PMS decomposition climbed up firstly and then declined as Cu0 loading increased from 0.01 to 1.00 wt% with the maximums at 0.10 wt%. SO4•-, HO•, Fe(IV), O2•- and 1O2 were generated by nZVI-Cu0/PMS based on the results of electron paramagnetic resonance (EPR) and simultaneous degradation of nitrobenzene, ATZ, and methyl phenyl sulfoxide (PMSO). The rate constant of Fe(IV) and ATZ was estimated as 7 × 104 M-1∙s-1 via the variation of methyl phenyl sulfone (PMSO2)formation at different ATZ concentrations. However, Fe(IV) contributed negligibly to ATZ degradation due to the strong scavenging of Fe(IV) by PMS. SO4•- and HO• were the reactive species responsible for ATZ degradation and the yield ratio of SO4•- and HO• was about 8.70 at initial stage. Preliminary thermodynamic calculation on the possible activation ways revealed that the dominant production of SO4•- might originate from the atomic H reduction of PMS in the surface layer of nZVI-Cu0. Ten products of ATZ degradation were identified by HPLC/ESI/QTOF and the possible degradation pathways were analyzed combined with theoretical calculation on ATZ structure. The decrease of temperature or increase of solution pH led to the decline of ATZ degradation, as well as the individual addition of common ions (HCO3-, Cl-, SO42-, NH4+, NO3- and F-) and natural organic matters (NOM). In real water, ATZ was still efficiently degraded with the decontamination efficiency decreasing in the sequence of tap water > surface water > simulated wastewater > groundwater. For the treatment of ATZ-polluted continuous flow, nZVI-Cu0 in double-layer layout had a higher capacity than the single-layer mode. Meanwhile, the leaching TFe and TCu were limited. The results indicate nZVI-Cu0/PMS is applicable and the multiple-layer layout of nZVI-Cu0 is suggested for ATZ-polluted ground water and soil remediation.

Construct ceRNA Network and Risk Model of Breast Cancer Using Machine Learning Methods under the Mechanism of Cuproptosis.

Breast cancer (BRCA) has an undesirable prognosis and is the second most common cancer among women after lung cancer. A novel mechanism of programmed cell death called cuproptosis is linked to the development and spread of tumor cells. However, the function of cuproptosis in BRCA remains unknown. To this date, no studies have used machine learning methods to screen for characteristic genes to explore the role of cuproptosis-related genes (CRGs) in breast cancer. Therefore, 14 cuproptosis-related characteristic genes (CRCGs) were discovered by the feature selection of 39 differentially expressed CRGs using the three machine learning methods LASSO, SVM-RFE, and random forest. Through the PPI network and immune infiltration analysis, we found that PRNP was the key CRCG. The miRTarBase, TargetScan, and miRDB databases were then used to identify hsa-miR-192-5p and hsa-miR-215-5p as the upstream miRNA of PRNP, and the upstream lncRNA, CARMN, was identified by the StarBase database. Thus, the mRNA PRNP/miRNA hsa-miR-192-5p and hsa-miR-215-5p/lncRNA CARMN ceRNA network was constructed. This ceRNA network, which has not been studied before, is extremely innovative. Furthermore, four cuproptosis-related lncRNAs (CRLs) were screened in TCGA-BRCA by univariate Cox, LASSO, and multivariate Cox regression analysis. The risk model was constructed by using these four CRLs, and the risk score = C9orf163 * (1.8365) + PHC2-AS1 * (-2.2985) + AC087741.1 * (-0.9504) + AL109824.1 * (0.6016). The ROC curve and C-index demonstrated the superior predictive capacity of the risk model, and the ROC curve demonstrated that the AUC of 1-, 3-, and 5-year OS in all samples was 0.721, 0.695, and 0.633, respectively. Finally, 50 prospective sensitive medicines were screened with the pRRophetic R package, among which 17-AAG may be a therapeutic agent for high-risk patients, while the other 49 medicines may be suitable for the treatment of low-risk patients. In conclusion, our study constructs a new ceRNA network and a novel risk model, which offer a theoretical foundation for the treatment of BRCA and will aid in improving the prognosis of BRCA.

The construction of a novel ferroptosis-related lncRNA model to predict prognosis in colorectal cancer patients.

Colorectal cancer (CRC) is the most common gastrointestinal tumor with poor prognosis. Ferroptosis is a pivotal form of programmed iron-dependent cell death different from autophagy and apoptosis, and long noncoding RNA (lncRNA) can influence the prognosis of CRC via regulating ferroptosis. To explore the role and prognostic value of the constructed ferroptosis-related lncRNA model in CRC, a prognostic model was constructed and validated by screening ferroptosis-related lncRNAs associated with prognosis based on the transcriptome data and survival data of CRC patients in The Cancer Genome Atlas database. Regarding the established prognostic models, differences in signaling pathways and immune infiltration, as well as differences in immune function, immune checkpoints, and N6-methyladenosine-related genes were also analyzed. A total of 6 prognostic ferroptosis-related lncRNAs were obtained, including AP003555.1, AC010973.2, LINC01857, AP001469.3, ITGB1-DT and AC129492.1. Univariate independent prognostic analysis, multivariate independent prognostic analysis and receiver operating characteristic curves showed that ferroptosis-related lncRNAs could be recognized as independent prognostic factors. The Kaplan-Meier survival curves and the risk curves showed that the survival time of the high-risk group was shorter. Gene set enrichment analysis enrichment analysis showed that ATP-binding cassette transporters, taste transduction and VEGF signaling pathway were more active in high-risk groups that than in low-risk groups. However, the citrate cycle tricarboxylic acid cycle, fatty acid metabolism and peroxisome were significantly more active in the low-risk group than in the high-risk group. In addition, there were also differences in immune infiltration in the high-low-risk groups based on different methods, including antigen-presenting cell co-stimulation, chemokine receptor, parainflammation, and Type II IFN Response. Further analysis of Immune checkpoints showed that most of the Immune checkpoints such as TNFRSF18, LGALS9 and CTLA4 in the high-risk group were significantly higher than those in the low-risk group, and the expressions of N6-methyladenosine related genes METTL3, YTHDH2 and YTHDC1 were also significantly different in the high-risk group. Ferroptosis-related lncRNAs are closely related to the survival of colorectal cancer patients, which can be used as new biomarkers and potential therapeutic targets for the prognosis of colorectal cancer.

TRIOL Inhibits Rapid Intracellular Acidification and Cerebral Ischemic Injury: The Role of Glutamate in Neuronal Metabolic Reprogramming.

As one of the key injury incidents, tissue acidosis in the brain occurs very quickly within several minutes upon the onset of ischemic stroke. Glutamate, an excitatory amino acid inducing neuronal excitotoxicity, has been reported to trigger the decrease in neuronal intracellular pH (pHi) via modulating proton-related membrane transporters. However, there remains a lack of clarity on the possible role of glutamate in neuronal acidosis via regulating metabolism. Here, we show that 200 µM glutamate treatment quickly promotes glycolysis and inhibits mitochondrial oxidative phosphorylation of primary cultured neurons within 15 min, leading to significant cytosolic lactate accumulation, which contributes to the rapid intracellular acidification and neuronal injury. The reprogramming of neuronal metabolism by glutamate is dependent on adenosine monophosphate-activated protein kinase (AMPK) signaling since the inhibition of AMPK activation by its selective inhibitor compound C significantly reverses these deleterious events in vitro. Moreover, 5α-androst-3ß,5α,6ß-TRIOL (TRIOL), a neuroprotectant we previously reported, can also remarkably reverse intracellular acidification and alleviate neuronal injury through the inhibition of AMPK signaling. Furthermore, TRIOL remarkably reduced the infarct volume and attenuated neurologic impairment in acute ischemic stroke models of middle cerebral artery occlusion in vivo. In summary, we reveal a novel role of glutamate in rapid intracellular acidification injury resulting from glutamate-induced lactate accumulation through AMPK-mediated neuronal reprogramming. Moreover, inhibition of the quick drop in neuronal pHi by TRIOL significantly reduces the cerebral damages, suggesting that it is a promising drug candidate for ischemic stroke.

MCCS: a novel recognition pattern-based method for fast track discovery of anti-SARS-CoV-2 drugs.

Given the scale and rapid spread of the coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2, or 2019-nCoV), there is an urgent need to identify therapeutics that are effective against COVID-19 before vaccines are available. Since the current rate of SARS-CoV-2 knowledge acquisition via traditional research methods is not sufficient to match the rapid spread of the virus, novel strategies of drug discovery for SARS-CoV-2 infection are required. Structure-based virtual screening for example relies primarily on docking scores and does not take the importance of key residues into consideration, which may lead to a significantly higher incidence rate of false-positive results. Our novel in silico approach, which overcomes these limitations, can be utilized to quickly evaluate FDA-approved drugs for repurposing and combination, as well as designing new chemical agents with therapeutic potential for COVID-19. As a result, anti-HIV or antiviral drugs (lopinavir, tenofovir disoproxil, fosamprenavir and ganciclovir), antiflu drugs (peramivir and zanamivir) and an anti-HCV drug (sofosbuvir) are predicted to bind to 3CLPro in SARS-CoV-2 with therapeutic potential for COVID-19 infection by our new protocol. In addition, we also propose three antidiabetic drugs (acarbose, glyburide and tolazamide) for the potential treatment of COVID-19. Finally, we apply our new virus chemogenomics knowledgebase platform with the integrated machine-learning computing algorithms to identify the potential drug combinations (e.g. remdesivir+chloroquine), which are congruent with ongoing clinical trials. In addition, another 10 compounds from CAS COVID-19 antiviral candidate compounds dataset are also suggested by Molecular Complex Characterizing System with potential treatment for COVID-19. Our work provides a novel strategy for the repurposing and combinations of drugs in the market and for prediction of chemical candidates with anti-COVID-19 potential.

Binding Characterization of GPCRs-Modulator by Molecular Complex Characterizing System (MCCS).

Increasing attention has been devoted to allosteric modulators as the preferred therapeutic agents for their colossal advantages such as higher selectivity, fewer side effects, and lower toxicity since they bind at allosteric sites that are topographically distinct from the classic orthosteric sites. However, the allosteric binding pockets are not conserved and there are no cogent methods to comprehensively characterize the features of allosteric sites with the binding of modulators. To overcome this limitation, our lab has developed a novel algorithm that can quantitatively characterize the receptor-ligand binding feature named Molecular Complex Characterizing System (MCCS). To illustrate the methodology and application of MCCS, we take G protein coupled receptors (GPCRs) as an example. First, we summarized and analyzed the reported allosteric binding pockets of class A GPCRs using MCCS. Sequentially, a systematic study was conducted between cannabinoid receptor type 1 (CB1) and its allosteric modulators, where we used MCCS to analyze the residue energy contribution and the interaction pattern. Finally, we validated the predicted allosteric binding site in CB2 via MCCS in combination with molecular dynamics (MD) simulation. Our results demonstrate that the MCCS program is advantageous in recapitulating the allosteric regulation pattern of class A GPCRs of the reported pockets as well as in predicting potential allosteric binding pockets. This MCCS program can serve as a valuable tool for the discovery of small-molecule allosteric modulators for class A GPCRs.

Natural product-derived icaritin exerts anti-glioblastoma effects by positively modulating estrogen receptor ß.

Glioblastoma is the most common malignancy of the central nervous system, and patients typically have a poor prognosis. Previous studies indicate a gender bias in the development of glioblastoma; women are at a lower risk compared with men, suggesting that estrogen may confer protective effects. Icaritin, a prenylflavonoid derivative from a Chinese herb of the Epimedium genus, selectively regulates the estrogen receptor (ER) and possesses anti-cancer properties. The aim of the present study was to investigate the protective effects of icaritin on glioblastoma and its underlying mechanisms, with a particular focus on its association with the ER. The results demonstrated that icaritin inhibited the growth of C6 and U87-MG glioblastoma cells in a dose- and time-dependent manner. At a concentration of 12.5 µM, icaritin induced apoptosis, which was characterized by the increased expression of the cleaved forms of caspases 3, 7, 8 and 9 and poly (ADP-ribose) polymerase, downregulation of BCL2 apoptosis regulator and upregulation of BCL2-associated X, apoptosis regulator expression. Additionally, icaritin inhibited the migration of C6 and U87-MG cells. The protein expression levels of matrix metalloproteinase (MMP)-2 and MMP-9 were also downregulated following icaritin treatment. Furthermore, icaritin treatment increased the expression of estrogen receptor (ER)ß and the phosphatase and tensin (PTEN) homolog oncoprotein, thus reducing the expression of downstream targets of PTEN; protein kinase B (Akt) and phosphorylated Akt. Subsequent experiments demonstrated that icaritin cooperates with 17ß-estradiol to inhibit the growth of glioblastoma cells, and the inhibition of ERß with the ERß-specific antagonist ICI 182,780, attenuated the anti-glioblastoma effects of icaritin. In conclusion, the results of the present study demonstrate that the anti-glioblastoma effects of icaritin may be mediated by its modulation of ERß.

Development and validation of an immunity-related classifier of nine chemokines for predicting recurrence in stage I-III patients with colorectal cancer after operation.

INTRODUCTION: Chemokines are closely related with tumor immunity, progression, and metastasis. We aimed to construct a multi-RNA classifier of chemokine family genes for predicting tumor recurrence in stage I-III patients with colorectal cancer (CRC) after operation. PATIENTS AND METHODS: By analyzing microarray data, the Cox regression analysis was conducted to determine survival-related chemokine family genes and develop a multi-RNA classifier in the training set. The prognostic value of this multi-RNA classifier was further validated in the internal validation and external independent sets. Receiver operating characteristic curves were used to compare the prediction ability of the combined model of this multi-RNA classifier and stage, and this multi-RNA classifier and stage alone. RESULTS: Nine survival-related chemokines were identified in the training set. We identified a nine-chemokine classifier and classified the patients as high-risk or low-risk. Compared with CRC patients with high-risk scores, CRC patients with low-risk scores had longer disease-free survival in the training (HR=2.353, 95% CI=1.480-3.742, P<0.001), internal validation (HR=2.389, 95% CI=1.428-3.996, P<0.001), and external independent (HR=3.244, 95% CI=1.813-5.807, P<0.001) sets. This nine-chemokine classifier was an independent prognostic factor in these datasets (P<0.05). The combined model of this nine-chemokine classifier and tumor stage may tend to have higher accuracy than stage alone in the training (area under curve 0.727 vs 0.626, P<0.01), internal validation (0.668 vs 0.584, P=0.03), and external independent (0.704 vs 0.678, P>0.05) sets. This nine-chemokine classifier may only be applied in Marisa's C2, C5, and C6 subtypes patients. CONCLUSION: Our nine-chemokine classifier is a reliable prognostic tool for some specific biological subtypes of CRC patients. It might contribute to guide the personalized treatment for high-risk patients.

Bauhinia championii Flavone Attenuates Hypoxia-Reoxygenation Induced Apoptosis in H9c2 Cardiomyocytes by Improving Mitochondrial Dysfunction.

This study aimed to determine the effects of Bauhinia championii flavone (BCF) on hypoxia-reoxygenation (H/R) induced apoptosis in H9c2 cardiomyocytes and to explore potential mechanisms. The H/R model in H9c2 cardiomyocytes was established by 6 h of hypoxia and 12 h of reoxygenation. Cell viability was detected by CCK-8 assay. Apoptotic rate was measured by Annexin V/PI staining. Levels of mitochondria-associated ROS, mitochondrial transmembrane potential (∆Ψm) and mitochondrial permeability transition pores (MPTP) opening were assessed by fluorescent probes. ATP production was measured by ATP assay kit. The release of cytochrome c, translocation of Bax, and related proteins were measured by western blotting. Our results showed that pretreatment with BCF significantly improved cell viability and attenuated the cardiomyocyte apoptosis caused by H/R. Furthermore, BCF increased ATP production and inhibited ROS-generating mitochondria, depolarization of ΔΨm, and MPTP opening. Moreover, BCF pretreatment decreased Bax mitochondrial translocation, cytochrome c release, and activation of caspase-3, as well as increased the expression of p-PI3K, p-Akt, and the ratio of Bcl-2 to Bax. Interestingly, a specific inhibitor of phosphatidylinositol 3-kinase, LY294002, partly reversed the anti-apoptotic effect of BCF. These observations indicated that BCF pretreatment attenuates H/R-induced myocardial apoptosis strength by improving mitochondrial dysfunction via PI3K/Akt signaling pathway.

Na+-sensitive elevation in blood pressure is ENaC independent in diet-induced obesity and insulin resistance.

The majority of patients with obesity, insulin resistance, and metabolic syndrome have hypertension, but the mechanisms of hypertension are poorly understood. In these patients, impaired sodium excretion is critical for the genesis of Na(+)-sensitive hypertension, and prior studies have proposed a role for the epithelial Na(+) channel (ENaC) in this syndrome. We characterized high fat-fed mice as a model in which to study the contribution of ENaC-mediated Na(+) reabsorption in obesity and insulin resistance. High fat-fed mice demonstrated impaired Na(+) excretion and elevated blood pressure, which was significantly higher on a high-Na(+) diet compared with low fat-fed control mice. However, high fat-fed mice had no increase in ENaC activity as measured by Na(+) transport across microperfused cortical collecting ducts, electrolyte excretion, or blood pressure. In addition, we found no difference in endogenous urinary aldosterone excretion between groups on a normal or high-Na(+) diet. High fat-fed mice provide a model of metabolic syndrome, recapitulating obesity, insulin resistance, impaired natriuresis, and a Na(+)-sensitive elevation in blood pressure. Surprisingly, in contrast to previous studies, our data demonstrate that high fat feeding of mice impairs natriuresis and produces elevated blood pressure that is independent of ENaC activity and likely caused by increased Na(+) reabsorption upstream of the aldosterone-sensitive distal nephron.

Role of Receptor Protein Tyrosine Phosphatase γ in Sensing Extracellular CO2 and HCO3.

Regulation of blood pH-critical for virtually every facet of life-requires that the renal proximal tubule (PT) adjust its rate of H(+) secretion (nearly the same as the rate of HCO3 (-) reabsorption, JHCO3 ) in response to changes in blood [CO2] and [HCO3 (-)]. Yet CO2/HCO3 (-) sensing mechanisms remain poorly characterized. Because receptor tyrosine kinase inhibitors render JHCO3 in the PT insensitive to changes in CO2 concentration, we hypothesized that the structural features of receptor protein tyrosine phosphatase-γ (RPTPγ) that are consistent with binding of extracellular CO2 or HCO3 (-) facilitate monitoring of blood CO2/HCO3 (-) concentrations. We now report that PTs express RPTPγ on blood-facing membranes. Moreover, RPTPγ deletion in mice eliminated the CO2 and HCO3 (-) sensitivities of JHCO3 as well as the normal defense of blood pH during whole-body acidosis. Thus, RPTPγ appears to be a novel extracellular CO2/HCO3 (-) sensor critical for pH homeostasis.

Naturally occurring marine steroid 24-methylenecholestane-3ß,5α,6ß,19-tetraol functions as a novel neuroprotectant.

Steroids have been shown to have multiple effects on the nervous system including neuroprotective activities, and they have the potential to be used for the treatment of neurodegenerative diseases. In this current study, we tested the hypothesis that the marine steroid 24-methylenecholestane-3ß,5α,6ß,19-tetraol (Tetrol) has a neuroprotective effect. (1) We synthesized Tetrol through a multiple step reaction starting from hyodeoxycholic acid (HDCA). (2) We then evaluated the neuroprotective effect of Tetrol with a glutamate-induced neuronal injury model in vitro. Tetrol concentration dependently increased the survival rate of cerebellar granule neurons challenged with toxic concentration of glutamate. Consistently, Tetrol significantly decreased glutamate-induced lactate dehydrogenase (LDH) release with a threshold concentration of 2.5 µM. (3) We further evaluated the neuroprotective effect of Tetrol in a middle cerebral artery occlusion (MCAO)-induced cerebral ischemia model in rat. Tetrol, at a dose of 12 mg/kg, significantly decreased MCAO-induced infarction volume by ∼50%. (4) Finally, we probed the mechanism and found that Tetrol concentration dependently attenuated N-methyl-d-aspartate (NMDA)-induced intracellular calcium ([Ca(2+)]i) increase with an IC50 of 7.8±0.62 µM, and inhibited NMDA currents in cortical neurons with an IC50 of 10.28±0.71 µM. Taken together, we have synthesized and characterized Tetrol as a novel neuroprotectant through negative modulation of NMDA receptors.

Cholesterol metabolite cholestane-3ß,5α,6ß-triol suppresses epileptic seizures by negative modulation of voltage-gated sodium channels.

Imbalance of excitation and inhibition in neurons is implicated in the pathogenesis of epilepsy. Voltage-gated sodium channels, which play a vital role in regulating neuronal excitability, are one of the major targets for developing anti-epileptic drugs. Here we provide evidence that cholestane-3ß,5α,6ß-triol (triol), a major metabolic oxysterol of cholesterol, is an effective state-dependent negative sodium channels modulator. Triol reduced Na(+) current density in a concentration-dependent manner. 10 µM triol shifted steady-state/fast/slow inactivation curves of sodium channels toward the hyperpolarizing direction. Additionally, triol reduced voltage-gated sodium currents in a voltage- and frequency-dependent manner. In a kainic acid-induced seizures mouse model, triol (25 mg/kg) significantly increased the latency of seizure onset and attenuated seizure severity. Our findings provide novel insights for understanding the modulatory role of a small molecular oxysterol on voltage-gated sodium channels and suggest triol may represent a novel and promising candidate for epilepsy intervention.

The major cholesterol metabolite cholestane-3ß,5α,6ß-triol functions as an endogenous neuroprotectant.

Overstimulation of NMDA-type glutamate receptors is believed to be responsible for neuronal death of the CNS in various disorders, including cerebral and spinal cord ischemia. However, the intrinsic and physiological mechanisms of modulation of these receptors are essentially unknown. Here we report that cholestane-3ß,5α,6ß-triol (triol), a major metabolite of cholesterol, is an endogenous neuroprotectant and protects against neuronal injury both in vitro and in vivo via negative modulation of NMDA receptors. Treatment of cultured neurons with triol protects against glutamate-induced neurotoxicity, and administration of triol significantly decreases neuronal injury after spinal cord ischemia in rabbits and transient focal cerebral ischemia in rats. An inducible elevation of triol is associated with ischemic preconditioning and subsequent neuroprotection in the spinal cord of rabbits. This neuroprotection is effectively abolished by preadministration of a specific inhibitor of triol synthesis. Physiological concentrations of triol attenuate [Ca(2+)]i induced by glutamate and decrease inward NMDA-mediated currents in cultured cortical neurons and HEK-293 cells transiently transfected with NR1/NR2B NMDA receptors. Saturable binding of [(3)H]triol to cerebellar granule neurons and displacement of [(3)H]MK-801 binding to NMDA receptors by triol suggest that direct blockade of NMDA receptors may underlie the neuroprotective properties. Our findings suggest that the naturally occurring oxysterol, the major cholesterol metabolite triol, functions as an endogenous neuroprotectant in vivo, which may provide novel insights into understanding and developing potential therapeutics for disorders in the CNS.

Anti-hepatitis B virus activity of Boehmeria nivea leaf extracts in human HepG2.2.15 cells.

Boehmeria nivea (Linn.) Gaudich of the Urticaceae family is a perennial ratoon herbal plant, the root of which is used in traditional Chinese medicine and possesses a variety of pharmacological properties. The 20% ethanol Boehmeria nivea root extract was shown to exert an anti-hepatitis B virus (HBV) effect in vitro and in vivo; however, whether the Boehmeria nivea leaf (BNL) extract possesses similar properties has not been determined. In this study, we aimed to investigate the anti-HBV effects of the BNL extract in HepG2.2.15 cells transfected with human HBV DNA. Our results demonstrated that the secretion of HBsAg and HBeAg was reduced in HepG2.2.15 cells treated with the BNL extract, without any recorded cytotoxic effects. In addition, the chloroform fraction (CF) and ethyl acetate fraction (EAF) of BNL were shown to be more potent compared to the other fractions: CF (100 mg/l) inhibited the secretion of HBsAg by 94.00±1.78% [inhibitory concentration 50 (IC50) = 20.92 mg/l] and that of HBeAg by 100.19±0.35% (IC50=19.67 mg/l) after 9 days of treatment. Similarly, EAF (200 mg/l) inhibited the secretion of HBsAg by 89.95±2.26% (IC50=39.90 mg/l) and that of HBeAg by 98.90±1.42% (IC50=36.45 mg/l). Furthermore, we observed that the content of HBV DNA in the medium secreted by the HepG2.2.15 cells was significantly decreased under CF (100 mg/l) or EAF (200 mg/l) treatment. Thus, we concluded that the BNL extracts exhibited anti-HBV activity, with CF and EAF being the most potent among the fractions.

Alphaxalone inhibits growth, migration and invasion of rat C6 malignant glioma cells.

Malignant gliomas are the most devastating and aggressive brain tumors affecting the central nervous system. The insidious growth and infiltration are the most prominent characteristics of malignant gliomas, which render the current therapies for malignant gliomas including surgery, radiation and chemotherapy unsuccessful. Inhibition of infiltration as well as proliferation in combination with surgery might be more effective in the treatment of malignant gliomas. In the current study, we demonstrate the alphaxalone (3-hydroxypregnane-11,20-dione) could effectively inhibit the proliferation of C6 glioma cells in a concentration dependent manner. Moreover, this compound could also suppress the migration and invasion of C6 glioma cells at a concentration without causing significant cytotoxicity. Except the in vitro anti-glioma activity, alphaxalone effectively delayed the growth of rat C6 malignant glioma xenografts in vivo. Together, these findings suggest alphaxalone might be a promising candidate for the treatment of malignant gliomas and may also provide helpful clues for anti-glioma drugs development in future.

Diazepam inhibits proliferation of human glioblastoma cells through triggering a G0/G1 cell cycle arrest.

BACKGROUND: Glioblastoma (GBM), the most common primary brain tumor, is the most aggressive malignancy in humans. Its rapid proliferation is a major obstacle to successful treatment. Patients with GBM often suffer from psychological disturbances associated with poor prognosis and physical discomfort. Diazepam is one of the most frequently used benzodiazepines (BZs) in cancer patients for its desirable psychotropic effects. The central effects of BZs are mediated by the activation of central BZ receptors. This study investigates whether diazepam has inhibitory effect on proliferation of GBM cell line T98G and explores its possible mechanism. METHODS: Cell viability and proliferation were respectively determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay and 5-bromo-2'-deoxyuridine (BrdU) incorporation assay. Cell cycle distribution was examined by flow cytometry. Western blot with specific protein antibodies was used to detect regulatory proteins involved in cell cycle regulation. RESULTS: Diazepam significantly decreased the proliferation of T98G cells in a dose-dependent and time-dependent manner. This effect was not reversed by the central BZ receptor antagonist flumazenil or the peripheral BZ receptor antagonist PK11195, indicating that it was not mediated by BZ receptors. Flow cytometry indicated that diazepam caused a cell accumulation in G0/G1 phase, thereby contributing to cell proliferation inhibition. Furthermore, our findings showed that lessened phosphorylation of Rb accounted for diazepam-induced G0/G1 phase arrest. CONCLUSIONS: Diazepam inhibits the proliferation of human GBM T98G cells by inducing G0/G1 phase arrest. Diazepam has potential to be a lead for new drugs in GBM therapy because of its antitumor activity.