Metabolism of ADB-FUBIATA in zebrafish and pooled human liver microsomes investigated by liquid chromatography-high-resolution mass spectrometry.

RATIONALE: ADB-FUBIATA is one of the most recently identified new psychoactive substance (NPS) of synthetic cannabinoids. The co-use of in vitro (human liver microsomes) and in vivo (zebrafish) models offers abundant metabolites and may give a deep insight into the metabolism of NPS. METHODS: In vivo and in vitro metabolic studies of new synthetic cannabinoid ADB-FUBIATA were carried out using zebrafish and pooled human liver microsome models. Metabilites were structurally characterized by liquid chromatography-high-resolution mass spectrometry. RESULTS: In total, 18 metabolites were discovered and identified in the pooled human liver microsomes and zebrafish, including seventeen phase I metabolites and one phase II metabolite. The main metabolic pathways of ADB-FUBIATA were hydroxylation, dehydrogenation, N-dealkylation, amide hydrolysis, glucuronidation, and combination thereof. CONCLUSION: Hydroxylated metabolites can be recommended as metabolic markers for ADB-FUBIATA because of the structural characteristics and high intensity. These metabolism characteristics of ADB-FUBIATA were useful for its further forensic or clinical related investigations.

TNFSF14/LIGHT promotes cardiac fibrosis and atrial fibrillation vulnerability via PI3Kγ/SGK1 pathway-dependent M2 macrophage polarisation.

BACKGROUND: Tumour necrosis factor superfamily protein 14 (TNFSF14), also called LIGHT, is an important regulator of immunological and fibrosis diseases. However, its specific involvement in cardiac fibrosis and atrial fibrillation (AF) has not been fully elucidated. The objective of this study is to examine the influence of LIGHT on the development of myocardial fibrosis and AF. METHODS: PCR arrays of peripheral blood mononuclear cells (PBMCs) from patients with AF and sinus rhythm was used to identify the dominant differentially expressed genes, followed by ELISA to evaluate its serum protein levels. Morphological, functional, and electrophysiological changes in the heart were detected in vivo after the tail intravenous injection of recombinant LIGHT (rLIGHT) in mice for 4 weeks. rLIGHT was used to stimulate bone marrow-derived macrophages (BMDMs) to prepare a macrophage-conditioned medium (MCM) in vitro. Then, the MCM was used to culture mouse cardiac fibroblasts (CFs). The expression of relevant proteins and genes was determined using qRT-PCR, western blotting, and immunostaining. RESULTS: The mRNA levels of LIGHT and TNFRSF14 were higher in the PBMCs of patients with AF than in those of the healthy controls. Additionally, the serum protein levels of LIGHT were higher in patients with AF than those in the healthy controls and were correlated with left atrial reverse remodelling. Furthermore, we demonstrated that rLIGHT injection promoted macrophage infiltration and M2 polarisation in the heart, in addition to promoting atrial fibrosis and AF inducibility in vivo, as detected with MASSON staining and atrial burst pacing respectively. RNA sequencing of heart samples revealed that the PI3Kγ/SGK1 pathway may participate in these pathological processes. Therefore, we confirmed the hypothesis that rLIGHT promotes BMDM M2 polarisation and TGB-ß1 secretion, and that this process can be inhibited by PI3Kγ and SGK1 inhibitors in vitro. Meanwhile, increased collagen synthesis and myofibroblast transition were observed in LIGHT-stimulated MCM-cultured CFs and were ameliorated in the groups treated with PI3Kγ and SGK1 inhibitors. CONCLUSION: LIGHT protein levels in peripheral blood can be used as a prognostic marker for AF and to evaluate its severity. LIGHT promotes cardiac fibrosis and AF inducibility by promoting macrophage M2 polarisation, wherein PI3Kγ and SGK1 activation is indispensable.

Mutation of histone H3 serine 28 to alanine influences H3K27me3-mediated gene silencing in Arabidopsis thaliana.

Histone modifications are essential for chromatin activity and play an important role in many biological processes. Trimethylation of histone H3K27 (H3K27me3) is a repressive modification established by Polycomb Repressive Complex 2 (PRC2). Although the presence of the histone H3 serine 28 phosphorylation (H3S28ph) modification at adjacent amino acid residues has both positive and negative effects on Polycomb silencing in mammals, little is known about the effect of H3S28ph on H3K27me3-mediated gene silencing in plants. In this study, we show that mutating H3S28A in Arabidopsis (Arabidopsis thaliana) causes a dominant-negative effect that leads to an early-flowering phenotype by promoting the expression of flowering-promoting genes independently of abnormal cell division. While H3S28ph levels decreased due to the H3S28A mutation, H3K27me3 levels at the same loci did not increase. Moreover, we observed decreased H3K27me3 levels at some known PRC2 target genes in H3.3S28A transgenic lines, rather than the expected enhanced H3K27me3-mediated silencing. In line with the reduced H3K27me3 levels, the expression of the PRC2 catalytic subunits CURLY LEAF and SWINGER decreased. Taken together, these data demonstrate that H3.3S28 is required for PRC2-dependent H3K27me3-mediated silencing in Arabidopsis, suggesting that H3S28 has a noncanonical function in H3K27me3-mediated gene silencing.

Doubled-haploid induction generates extensive differential DNA methylation in Arabidopsis.

Doubled haploids (DHs) fix traits from hybrids in one generation. DH induction includes two changes in ploidy levels typically associated with variation in DNA methylation. However, DNA methylation patterns in DH plants and their biological significance are largely unknown. We generated three DH lines in Arabidopsis thaliana by crossing a haploid inducer with the accession Col-0, thus removing tissue culture and hybridization as a variable. DH induction produced thousands of differentially DNA methylated regions (DMRs), most of which were stochastic. Both haploidization and colchicine-induced genome duplication produced DMRs; the former mainly yielded DMRs at non-CG contexts, whereas the latter affected differential gene body methylation. Spontaneous genome doubling of haploid plants also induced DMRs in greater numbers than self-propagation. Our results provide the first evidence that haploid induction and genome doubling result in differential DNA methylation, offering a novel approach to induce epialleles.

Utilizing a Combination of Network Pharmacology and Experimental Validation to Unravel the Mechanism by Which Kuanxiongzhuyu Decoction Ameliorates Myocardial Infarction Damage.

Background and Objectives: With the growing incidence and disability associated with myocardial infarction (MI), there is an increasing focus on cardiac rehabilitation post-MI. Kuanxiongzhuyu decoction (KXZY), a traditional Chinese herbal formula, has been used in the rehabilitation of patients after MI. However, the chemical composition, protective effects, and underlying mechanism of KXZY remain unclear. Materials and Methods: In this study, the compounds in KXZY were identified using a high-performance liquid chromatography-mass spectrometry (HPLC-MS) analytical method. Based on the compounds identified in the KXZY, we predictively selected the potential targets of MI and then constructed a protein-protein interaction (PPI) network to identify the key targets. Furthermore, the DAVID database was used for the GO and KEGG analyses, and molecular docking was used to verify the key targets. Finally, the cardioprotective effects and mechanism of KXZY were investigated in post-MI mice. Results: A total of 193 chemical compounds of KXZY were identified by HPLC-MS. In total, 228 potential targets were obtained by the prediction analysis. The functional enrichment studies and PPI network showed that the targets were largely associated with AKT-pathway-related apoptosis. The molecular docking verified that isoguanosine and adenosine exhibited excellent binding to the AKT. In vivo, KXZY significantly alleviated cardiac dysfunction and suppressed AKT phosphorylation. Furthermore, KXZY significantly increased the expression of the antiapoptotic proteins Bcl-2 and Bcl-xl and decreased the expression of the proapoptotic protein BAD. Conclusions: In conclusion, the network pharmacological and experimental evidence suggests that KXZY manifests anti-cardiac dysfunction behavior by alleviating cardiomyocyte apoptosis via the AKT pathway in MI and, thus, holds promising therapeutic potential.

H3K9 demethylases IBM1 and JMJ27 are required for male meiosis in Arabidopsis thaliana.

Dimethylation of histone H3 lysine 9 (H3K9me2), a crucial modification for heterochromatin formation and transcriptional silencing, is essential for proper meiotic prophase progression in mammals. We analyzed meiotic defects and generated genome-wide profiles of H3K9me2 and transcriptomes for the mutants of H3K9 demethylases. Moreover, we also identified proteins interacting with H3K9 demethylases. H3K9me2 is usually found at transposable elements and repetitive sequences but is absent from the bodies of protein-coding genes. In this study, we show that the Arabidopsis thaliana H3K9 demethylases IBM1 and JMJ27 cooperatively regulate crossover formation and chromosome segregation. They protect thousands of protein-coding genes from ectopic H3K9me2, including genes essential for meiotic prophase progression. In addition to removing H3K9me2, IBM1 and JMJ27 interact with the Precocious Dissociation of Sisters 5 (PDS5) cohesin complex cofactors. The pds5 mutant shared similar transcriptional alterations with ibm1 jmj27, including meiosis-essential genes, yet without affecting H3K9me2 levels. Hence, PDS5s, together with IBM1 and JMJ27, regulate male meiosis and gene expression independently of H3K9 demethylation. These findings uncover a novel role of H3K9me2 removal in meiosis and a new function of H3K9 demethylases and cohesin cofactors in meiotic transcriptional regulation.

Altered H3K27 trimethylation contributes to flowering time variations in polyploid Arabidopsis thaliana ecotypes.

Polyploidy is a widespread phenomenon in flowering plant species. Polyploid plants frequently exhibit considerable transcriptomic alterations after whole-genome duplication (WGD). It is known that the transcriptomic response to tetraploidization is ecotype-dependent in Arabidopsis; however, the biological significance and the underlying mechanisms are unknown. In this study, we found that 4x Col-0 presents a delayed flowering time whereas 4x Ler does not. The expression of FLOWERING LOCUS C (FLC), the major repressor of flowering, was significantly increased in 4x Col-0 but only a subtle change was present in 4x Ler. Moreover, the level of a repressive epigenetic mark, trimethylation of histone H3 at lysine 27 (H3K27me3), was significantly decreased in 4x Col-0 but not in 4x Ler, potentially leading to the differences in FLC transcription levels and flowering times. Hundreds of other genes in addition to FLC showed H3K27me3 alterations in 4x Col-0 and 4x Ler. LIKE HETEROCHROMATIN PROTEIN 1 (LHP1) and transcription factors required for H3K27me3 deposition presented transcriptional changes between the two ecotypes, potentially accounting for the different H3K27me3 alterations. We also found that the natural 4x Arabidopsis ecotype Wa-1 presented an early flowering time, which was associated with low expression of FLC. Taken together, our results demonstrate a role of H3K27me3 alterations in response to genome duplication in Arabidopsis autopolyploids, and that variation in flowering time potentially functions in autopolyploid speciation.

Natural products from traditional Chinese medicine for the prevention and treatment of heart failure: progress and perspectives.

Heart failure (HF) is the end stage of several cardiovascular diseases with high mortality worldwide; however, current chemical drugs have not beneficial effect on reducing its mortality rate. Due to its properties of multiple targets components with multiple targets, natural products derived from traditional Chinese medicine (TCM) have exerts unique effects on the amelioration of the clinical symptoms of HF, yet, TCM is not widely used in the clinic since the potential therapeutic targets have not been fully investigated. Therefore, in this review, we briefly summarized the pathophysiological mechanism of HF and reviewed the published clinical evaluations of TCM and natural products from Chinese herbs to treat HF. Then, the therapeutic potential and the underlying mechanisms by which the natural products from Chinese herb exert their protective effects were further summarized. We concluded from this review that natural products from Chinese herbs have been shown to be more effective in treating HF by targeting multiple signaling pathways, including anticardiac hypertrophy, antifibrotic, anti-inflammatory, antioxidative and antiapoptotic activities. However, the major limitations of these compounds is that there are a lack of large scale, multicenter, randomized and controlled clinical trials for their use in treatment of HF, and the toxic effects of natural products from Chinese herbs also needed further investigation. Despite these limitations, further clinical trials and experimental studies will provide a better understanding of the mechanism of natural products from Chinese herbs and promote their wide use to treat HF.

Suppression of CHOP Reduces Neuronal Apoptosis and Rescues Cognitive Impairment Induced by Intermittent Hypoxia by Inhibiting Bax and Bak Activation.

Our previous study showed that growth arrest- and DNA damage-inducible gene 153 (GAD153/CHOP) plays an important role in intermittent hypoxia- (IH-) induced apoptosis and impaired synaptic plasticity. This study is aimed at determining which signaling pathway is activated to induce CHOP and the role of this protein in mitochondria-dependent apoptosis induced by IH. In the in vivo study, mice were placed in IH chambers for 8 h daily over a period of 2 weeks; the IH chambers had oxygen (O2) concentrations that oscillated between 10% and 21%, cycling every 90 s. In the in vitro study, PC12 cells were exposed to 21% O2 (normoxia) or 8 IH cycles (25 min at 21% O2 and 35 min at 0.1% O2 for each cycle). After 2 weeks of IH treatment, we observed that the expression levels of phosphorylated protein kinase-like endoplasmic reticulum kinase (p-PERK), activating transcription factor 4 (ATF-4) and phosphorylated eukaryotic initiation factor 2 alpha (p-elf2α), were increased, but the levels of activating transcription factor 6 (ATF-6) and inositol-requiring enzyme 1 (IRE-1) were not increased. GSK2606414, a specific chemical inhibitor of the PERK pathway, reduced the expression of p-PERK, ATF-4, p-elf2α, and CHOP and rescued ER structure. In addition, Bax and Bak accumulated in the mitochondria after IH treatment, which induced cytochrome c release and initiated apoptosis. These effects were prevented by GSK2606414 and CHOP shRNA. Finally, the impaired long-term potentiation and long-term spatial memory in the IH group were rescued by GSK2606414. Together, the data from the in vitro and in vivo experiments indicate that IH-induced apoptosis and impaired synaptic plasticity were mediated by the PERK-ATF-4-CHOP pathway. Suppressing PERK-ATF-4-CHOP signaling pathway attenuated mitochondria-dependent apoptosis by reducing the expression of Bax and Bak in mitochondria, which may serve as novel adjunct therapeutic strategy for ameliorating obstructive sleep apnea- (OSA-) induced neurocognitive impairment.

Identifying potential DNA methylation markers in early-stage colorectal Cancer.

Colorectal cancer (CRC) is the second leading malignancy worldwide. Accurate screening is pivotal to early CRC detection, yet current screening modality involves invasive colonoscopy while non-invasive FIT tests have limited sensitivity. We applied a DNA methylation assay to identify biomarkers for early-stage CRC detection, risk stratification and precancerous lesion screening at tissue level. A model of biomarkers SFMBT2, ITGA4, THBD and ZNF304 showed 96.1% sensitivity and 87.0% specificity in CRC detection, with 100.0% sensitivity for advanced precancerous lesion and stage I CRC. Performances were further validated with TCGA data set, which showed a consistent AUC of 0.99 and exhibited specificity against other cancer types. KCNJ12, VAV3-AS1 and EVC were further identified for stage stratification (stage 0-I versus stage II-IV), with AUC of 0.87, 83.0% sensitivity and 71.2% specificity. Additionally, dual markers of NEUROD1 and FAM72C showed 83.2% sensitivity and 77.4% specificity in differing non-advanced precancerous lesions from inflammatory bowel diseases.

Oridonin inhibits the migration and epithelial-to-mesenchymal transition of small cell lung cancer cells by suppressing FAK-ERK1/2 signalling pathway.

Small cell lung cancer (SCLC) is a severe malignant with high morbidity; however, few effective and secure therapeutic strategy is used in current clinical practice. Oridonin is a small molecule from the traditional Chinese herb Rabdosia rubescens. This study mainly aimed to investigate the role of oridonin on inhibiting the process of H1688, a kind of small cell lung cancer cells from human. Oridonin could suppress H1688 cell proliferation and induce their apoptosis in a high dosage treatment (20 µmol/L). Meanwhile, cell migration was suppressed by oridonin (5 and 10 µmol/L) that did not affect cell proliferation and apoptosis. The expression level of E-cadherin was significantly increased, and the expression of vimentin, snail and slug was reduced after administration of oridonin. These expression changes were associated with the suppressed integrin ß1, phosphorylation of focal adhesion kinase (FAK) and ERK1/2. In addition, oridonin (5 and 10 mg/kg) inhibited tumour growth in a nude mouse model; however, HE staining revealed a certain degree of cytotoxicity in hepatic tissue after treatment oridonin (10 mg/kg). Furthermore, the concentration of alanine aminotransferase (ALP) was significantly increased and lactate dehydrogenase (LDH) was reduced after oridonin treatment (10 mg/kg). Immunohistochemical analysis further revealed that oridonin increased E-cadherin expression and reduced vimentin and phospho-FAK levels in vivo. These findings indicated that oridonin can inhibit the migration and epithelial-to-mesenchymal transition (EMT) of SCLC cells by suppressing the FAK-ERK1/2 signalling pathway. Thus, oridonin may be a new drug candidate to offer an effect of anti-SCLC with relative safety.

Attenuation of intermittent hypoxia-induced apoptosis and fibrosis in pulmonary tissues via suppression of ER stress activation.

BACKGROUND: Obstructive sleep apnea (OSA) is associated with pulmonary fibrosis and endothelial apoptosis in pulmonary tissues. Chronic intermittent hypoxia (IH) is considered to be the primary player in OSA, but the mechanisms underlying its effect on pulmonary tissues are unknown. Endoplasmic reticulum (ER) stress induced by IH treatment plays an important role in accelerating the process of fibrosis and induction of apoptosis. METHODS: Mice were placed in IH chambers for 4 weeks with an oscillating oxygen (O2) concentration between 5 and 21%, cycling every 90s for 8 h daily. Mice were randomly divided into four groups: control group (normal oxygen), tauroursodeoxycholic acid (TUDCA) group (normal oxygen intraperitoneally injected with TUDCA), IH group and IH + TUDCA group. After 4 weeks, the proteins in three branch signaling pathways of ER stress, including protein kinase RNA (PKR)-like/Pancreatic ER kinase (PERK), activating transcription factor 6 (ATF-6) and inositol-requiring enzyme 1 (IRE-1), were evaluated. The cleaved caspase-3, caspase-12 and TUNNEL staining was assessed. Furthermore, the expression of transforming growth factor-ß1 (TGF-ß1) and thrombospondin-1(TSP-1), two extracellular matrix proteins that play critical role in fibrosis, were examined. Finally, Masson's trichrome staining was performed to detect the expression of collagen. RESULTS: After 4 weeks of IH treatment, the expressions of two ER stress markers, glucose regulated protein-78 (Grp78) and transcription factor C/EBP homologous protein (CHOP) were increased which was prevented by administration of the ER stress attenuator, TUDCA. The expressions of PERK, but not those of ATF-6 and IRE-1, were increased. The effects of IH were accompanied by an increased number of apoptotic cells and increased expressions of cleaved caspase-3 and caspase-12 in pulmonary tissues. In addition, histological examination suggested the presence of fibrosis after chronic IH treatment, indicated by increased expression of collagen, which was associated with the up-regulation of TGF-ß1 and TSP-1 that are known to promote fibrosis. Similarly, TUDCA could reduce the extent of fibrotic area and the expression levels of these proteins. CONCLUSIONS: It reveals the roles of ER stress, especially the PERK pathway, in IH induced apoptosis and fibrosis in pulmonary tissues that might underlie the pulmonary complications observed in OSA.

Endoplasmic reticulum stress/autophagy pathway is involved in diabetes-induced neuronal apoptosis and cognitive decline in mice.

Diabetes mellitus is a significant global public health problem depicting a rising prevalence worldwide. As a serious complication of diabetes, diabetes-associated cognitive decline is attracting increasing attention. However, the underlying mechanisms are yet to be fully determined. Both endoplasmic reticulum (ER) stress and autophagy have been reported to modulate neuronal survival and death and be associated with several neurodegenerative diseases. Here, a streptozotocin-induced diabetic mouse model and primary cultured mouse hippocampal neurons were employed to investigate the possible role of ER stress and autophagy in diabetes-induced neuronal apoptosis and cognitive impairments, and further explore the potential molecular mechanisms. ER stress markers GRP78 and CHOP were both enhanced in diabetic mice, as was phosphorylation of PERK, IRE1α, and JNK. In addition, the results indicated an elevated level of autophagy in diabetic mice, as demonstrated by up-regulated expressions of autophagy markers LC3-II, beclin 1 and down-regulated level of p62, and increased formation of autophagic vacuoles and LC3-II aggregates. Meanwhile, we found that these effects could be abolished by ER stress inhibitor 4-phenylbutyrate or JNK inhibitor SP600125 in vitro. Furthermore, neuronal apoptosis of diabetic mice was attenuated by pretreatment with 4-phenylbutyrate, while aggravated by application of inhibitor of autophagy bafilomycin A1 in vitro. These results suggest that ER stress pathway may be involved in diabetes-mediated neurotoxicity and promote the following cognitive impairments. More important, autophagy was induced by diabetes possibly through ER stress-mediated JNK pathway, which may protect neurons against ER stress-associated cell damages.

Protective effects of Danshen injection against erectile dysfunction via suppression of endoplasmic reticulum stress activation in a streptozotocin-induced diabetic rat model.

BACKGROUND: Erectile dysfunction (ED) is a common complication of diabetes. This study aimed to explore the beneficial effect of Danshen injection on ED in a streptozotocin (STZ)-induced diabetic rat model and the underlying mechanism. METHODS: The diabetic rat model was established by an intraperitoneal injection of 60 mg/kg STZ in male Sprague-Dawley rats. The diabetic rats were intraperitoneally injected with Danshen solution (0.5 or 1 mL/kg/day) or the same volume of saline for 6 weeks. Age-matched rats served as controls. After 6 weeks, erectile function and histological morphology of the corpora cavernosum were assessed. Oxidative stress indicators, including superoxide dismutase (SOD) activity, malondialdehyde (MDA) content, and reactive oxygen species (ROS) levels, were measured in penile tissues. The expression levels of glucose-regulated protein 78 (Grp78), growth arrest and DNA damage-inducible gene 153 (GADD153/CHOP) were determined by immunohistochemistry, immunoblotting, and RT-PCR. Apoptosis was detected by a TUNEL assay. RESULTS: The erection times of diabetic rats were significantly less than those of control rats. Danshen injection could improve erectile function via increased erection times. Danshen injection was also found to ameliorate the morphological abnormalities of the corpora cavernosum, to reduce the number of apoptotic cells, and to suppress caspase-3 activation in penile tissue, accompanied by downregulation of the endoplasmic reticulum stress biomarkers Grp78 and CHOP. Danshen injection could increase SOD activity as well as reduce ROS and MDA levels in diabetic rats, indicating suppression of oxidative stress. CONCLUSION: Danshen injection could rescue diabetes-associated ED, possibly via suppressing the oxidative stress and endoplasmic reticulum (ER) stress-induced apoptosis pathways.

Magnesium sulphate can alleviate oxidative stress and reduce inflammatory cytokines in rat placenta of intrahepatic cholestasis of pregnancy model.

PURPOSE: In our study, we try to investigate whether magnesium sulphate (MgSO4) could provide protection against oxidative damage and inflammatory response in rat placenta of intrahepatic cholestasis of pregnancy (ICP) model. METHODS: The rat model of ICP was established by injecting s.c. 17α-ethinyl estradiol (EE) daily for 5 days. MgSO4, as an therapeutic drug for ICP, was injected i.p. daily for 3 days. Age-matched pregnant rats served as controls. The level of serum total bile acid (TBA) was measured. The data including the number and weight of offsprings on day 20 of pregnancy were collected. We observed ultrastructural changes of mitochondria and endoplasmic reticulum (ER) in placenta by transmission electron microscope. The antioxidant proteins peroxiredoxin-6 (Prdx6) and nuclear factor erythroid 2-related factor-2 (Nrf2) were analyzed by Western Blot. The inflammatory cytokines including IL-1ß, TNF-α and IFN-γ were investigated by real-time PCR (RT-PCR) and enzyme-linked immune-sorbent assay (ELISA). RESULTS: The weight of offsprings on day 20 of pregnancy increased in ICP rats treated with MgSO4 (ICP + MG group) compared with that in ICP rats (ICP group). However, the level of TBA was not reduced. The damage of mitochondria and ER was observed in placenta, which was much more slighter in ICP + MgSO4 group as compared with that in ICP group. Prdx6 and Nrf2 were increased, while the inflammatory cytokines including IL-1ß, TNF-α and IFN-γ were decreased in ICP + MgSO4 group compared with that in ICP group. CONCLUSIONS: MgSO4 had beneficial effect on improving growth of offsprings in rat model of ICP. The protective effect of MgSO4 on alleviating oxidative damage and inflammatory response in placenta may play an important role in the process. MgSO4 may improve the function of placenta.

[Tauroursodeoxycholic acid suppresses endoplasmic reticulum stress in pulmonary tissues of intermittent hypoxia mice].

OBJECTIVE: To explore the mechanism of tauroursodeoxycholic acid (TUDCA) in suppressing apoptosis in pulmonary tissues of intermittent hypoxia (IH) mice model.
 METHODS: A total of 32 C57 mice were randomly divided into a control group, a TUDCA group, an IH group and an IH+TUDCA group (8 mice per group). The mice were put in specially designed chambers and exposed to IH treatment for 4 weeks. In the chambers, oxygen levels repeatedly decreased from 21% to 10% and recovered from 10% to 21%, lasting for 8 hours in every day. After 4 weeks of IH exposure, the expression levels of caspase-12 and cleaved caspase-3 in pulmonary tissues were detected by Western blot. Meanwhile, the expression levels of glucose regulated protein-78 (GRP78) and CCAAT/enhancer-binding protein homologous protein (CHOP) were quantified by Western blot, immunochemistry and real-time PCR.
 RESULTS: Compared with the control group, the expression levels of caspase-12, cleaved caspase-3, GRP78 and CHOP were increased in the IH group (all P<0.01). TUDCA treatment could reduce these proteins expression (all P<0.05).
 CONCLUSION: Endoplasmic reticulum stress-mediated apoptosis can be activated in pulmonary tissues after chronic IH exposure, and TUDCA can reduce the cellular apoptosis via suppressing endoplasmic reticulum stress.

In vitro and in vivo metabolism of 3-Methoxyeticyclidine in human liver microsomes, a zebrafish model, and two human urine samples based on liquid chromatography-high-resolution mass spectrometry.

3-Methoxyeticyclidine (3-MeO-PCE), a phencyclidine-type substance, has a higher N-methyl-D-aspartate receptor binding affinity than phencyclidine and an involvement in fatal intoxication cases. The aim of this study was to identify new biomarkers and biotransformation pathways for 3-MeO-PCE. In vitro models were established using zebrafish and human liver microsomes for analysis of the phases I and II metabolites of 3-MeO-PCE by liquid chromatography-high-resolution mass spectrometry. Urine samples of known 3-MeO-PCE consumers in forensic cases were then subjected to analysis. Overall, 14 metabolites were identified in zebrafish and human liver microsomes, allowing postulation of the following metabolic pathways: hydroxylation, O-demethylation, N-dealkylation, dehydrogenation, combination, and glucuronidation or sulfation. 3-MeO-PCE and three metabolites (M2, M3, and M6) were detected in urine. We recommended M2 (the hydroxylation product) as a potential biomarker for documenting 3-MeO-PCE intake in clinical and forensic cases.

In vitro and in vivo metabolic study of three new psychoactive ß-keto-arylcyclohexylamines.

Since the 2000s, an increasing number of new psychoactive substances have appeared on the illicit drug market. ß-Keto-arylcyclohexylamine compounds play important pharmacological roles in anesthesia; however, because these new psychoactive substances have rapidly increasing illicit recreational use, the lack of detailed toxicity data are of particular concern. Therefore, analysis of their metabolites can help forensic personnel provide references and suggestions on whether a suspect has taken an illicit new psychoactive ß-keto-arylcyclohexylamine. The present study investigated the in vitro and in vivo metabolism and metabolites of three ß-keto-arylcyclohexylamines: deschloro-N-ethyl-ketamine, fluoro-N-ethyl-ketamine and bromoketamine. In vitro and in vivo models were established using zebrafish and human liver microsomes for analysis of Phase I and Phase II metabolites by liquid chromatography-high-resolution mass spectrometry. Altogether, 49 metabolites were identified. The results were applied for the subject urine samples of known fluoro-N-ethyl-ketamine consumer screen analysis in forensic cases. Hydroxy-deschloro-N-ethyl-ketamine, hydroxy-fluoro-N-ethyl-ketamine and hydroxy-bromoketamine were recommended as potential biomarkers for documenting intake in clinical and forensic cases.

Suppression of PERK/eIF2α/CHOP pathway enhances oridonin-induced apoptosis by inhibiting autophagy in Small-Cell lung cancer cells.

Chinese herbs have been used to treat small-cell lung cancer (SCLC) due to their low toxicity and significant efficacy. This study focused on oridonin, a natural compound extracted from Rabdosia rubescens, and aimed to investigate its potential antitumor activity on SCLC and to evaluate the synergistic effect of combining oridonin with other small molecules. In this study, oridonin exhibited a dual effect. At lower concentrations, it suppressed the cell viability of SCLC cells (H1688 and H446). At high concentrations, oridonin induced SCLC cell apoptosis, damaged HBE cells in vitro and compromised the function of the liver and heart in vivo. The lower concentration of oridonin induced autophagy by enhancing the expression of p62 and the LC3B-II/LC3B-I ratio. This phenomenon might be associated with the activation of the protein kinase RNA-like ER kinase (PERK)/eukaryotic initiation factor 2 alpha (eIF2α)/growth arrest and DNA damage-inducible gene 153 (CHOP/GAD153) pathway. Therefore, the combined effect of oridonin with GSK2606414 or 3- methyladenine increased apoptosis in SCLC cells and reduced tumor growth. A similar phenomenon was observed after oridonin was combined with p62 or CHOP RNA interference treatment. Simultaneously, the combination of oridonin and GSK2606414 exhibited therapeutic efficacy without manifesting adverse effects. Our findings suggest that oridonin at lower concentrations can induce autophagy by activating the PERK/eIF2α/CHOP signaling pathway. The inhibition of the PERK/eIF2α/CHOP pathway could enhance oridonin therapeutic responses by triggering apoptosis. The novel therapeutic approach of combining oridonin with a PERK inhibitor is promising as a strategy for the treatment of SCLC.

Geriatric nutritional risk index as a prognostic factor in elderly patients with non-muscle-invasive bladder cancer: a propensity score-matched study.

PURPOSE: The Geriatric Nutrition Risk Index (GNRI) is a simple and validated tool used to assess the nutritional status of elderly patients and predict the risk of short-term postoperative complications, as well as the long-term prognosis, after cancer surgery. In this study, we aimed to evaluate the predictive value of GNRI for the long-term postoperative prognosis in elderly patients with primary non-muscle-invasive bladder cancer (NMIBC) who underwent transurethral resection of bladder tumor (TURBT). METHODS: We retrospectively analyzed data from 292 elderly patients with primary NMIBC. Using X-tile software, we divided the cohort into two groups based on GNRI and determined the cut-off value for postoperative recurrence-free survival (RFS). Propensity score matching (PSM) with a ratio of 1:3, Kaplan-Meier analysis, log-rank test, and COX proportional hazards regression were used to assess the correlation between GNRI and prognosis and identify factors predicting recurrence and progression. RESULTS: In the entire cohort, the 3 year recurrence group had significantly lower GNRI compared to the 3 year non-recurrence group (P = 0.0109). The determined GNRI cut-off value was 93.82. After PSM, the low GNRI group had significantly lower RFS (P < 0.0001) and progression-free survival (PFS) (P = 0.0040) than the high GNRI group. Multivariate COX regression showed that GNRI independently predicted RFS (HR 2.108; 95% CI 1.266-3.512; P = 0.004) and PFS (HR 2.155; 95% CI 1.135-4.091; P = 0.019) in elderly patients with primary NMIBC. CONCLUSION: Preoperative GNRI is a prognostic marker for disease recurrence and progression in elderly patients with primary NMIBC undergoing TURBT.