Asiatic acid and madecassic acid cause cardiotoxicity via inflammation and production of excessive reactive oxygen species in zebrafish.

Centella asiatica (L.) Urban is a famous Chinese traditional medicine, which is widely used for treating various chronic inflammatory diseases. Although there are reports that Centella total glycosides exhibit heart-protective properties, our previous experiment showed that it has cardiac toxic effects in zebrafish. The components of Centella total glycosides are complex, so we recommend further research to determine their key components and mechanisms. In this study, sample quantification was done using liquid chromatography-tandem mass spectrometry. The cardiotoxicity of Centella total glycosides, asiaticoside, madecassoside, asiatic acid, and madecassic acid was evaluated using zebrafish and cell models. The zebrafish oxidative stress model and myocarditis model were used to explore further the mechanisms through which cardiotoxicity is achieved. Asiatic acid and madecassic acid caused zebrafish cardiotoxicity and H9C2 cell death. However, no toxicity effects were observed for asiaticoside and madecassoside in zebrafish, until the solution was saturated. The results from the cell model study showed that asiatic acid and madecassic acid changed the expression of apoptosis-related genes in myocardial cells. In the zebrafish model, high concentrations of these components raised the levels of induced systemic inflammation, neutrophils gathered in the heart, and oxidative stress injury. Asiatic acid and madecassic acid are the main components causing cardiotoxicity in zebrafish. This may be due to enhanced inflammation and reactive oxygen species injury, which causes myocardial cell apoptosis, which further leads to cardiac toxicity.

Single-cell RNA sequencing reveals the role of mitochondrial dysfunction in the cardiogenic toxicity of perfluorooctane sulfonate in human embryonic stem cells.

Perfluorooctane sulfonate (PFOS), an endocrine-disrupting chemical pollutant, affects embryonic heart development; however, the mechanisms underlying its toxicity have not been fully elucidated. Here, Single-cell RNA sequencing (scRNA-seq) was used to investigate the overall effects of PFOS on myocardial differentiation from human embryonic stem cells (hESCs). Additionally, apoptosis, mitochondrial membrane potential, and ATP assays were performed. Downregulated cardiogenesis-related genes and inhibited cardiac differentiation were observed after PFOS exposure in vitro. The percentages of cardiomyocyte and cardiac progenitor cell clusters decreased significantly following exposure to PFOS, while the proportion of primitive endoderm cell was increased in PFOS group. Moreover, PFOS inhibited myocardial differentiation and blocked cellular development at the early- and middle-stage. A Gene Ontology analysis and pseudo-time trajectory illustrated that PFOS disturbed multiple processes related to cardiogenesis and oxidative phosphorylation in the mitochondria. Furthermore, PFOS decreased mitochondrial membrane potential and induced apoptosis. These results offer meaningful insights into the cardiogenic toxicity of PFOS exposure during heart formation as well as the adverse effects of PFOS on mitochondria.

Isosteviol improves cardiac function and promotes angiogenesis after myocardial infarction in rats.

Isosteviol has been indicated as a cardiomyocyte protector. However, the underlying mechanism remains unclear. Thus, we sought to confirm the protective effect of isosteviol after myocardial infarction in a model of permanent coronary artery occlusion and investigate the potential proangiogenic activity in vitro and in vivo. A 4-week permanent coronary artery occlusion rat model was generated, and the protective effect of isosteviol was evaluated by echocardiographic imaging and hemodynamics assays. The coronary capillary density was tested by immunochemistry and micro-computed tomography (µCT) imaging. The effect of isosteviol on endothelial cells was determined in human umbilical vein endothelial cells (HUVECs) in vitro and Tg (kdrl: EGFP) zebrafish in vivo. We also examined the expression of related transcription factors by real-time polymerase chain reaction (RT-qPCR). Isosteviol increased ejection fraction (EF), fractional shortening (FS), cardiac systolic index (CI), maximum rate of increase of left ventricular pressure (Max dp/dt), and left ventricular systolic pressure (LVSP) by 32%, 40%, 25%, 26%, and 10%, respectively, in permanent coronary artery occlusion rats. Interestingly, it also promoted coronary capillary density by 2.5-fold. In addition, isosteviol promoted the proliferation and branching of HUVECs in vitro. It also rescued intersegmental vessel (ISV) development and improved endothelial cell proliferation by approximately fivefold (4-6) in zebrafish embryos in vivo. Isosteviol also upregulated the expression of hypoxia inducible factor-1α (HIF-1α) and vascular endothelial growth factor A (VEGFA) in zebrafish by fourfold and 3.5-fold, respectively. Our findings suggest that isosteviol is a proangiogenic agent and that this activity is related to its protective effects against myocardial ischemia. After using the permanent coronary artery occlusion model, we demonstrated that isosteviol promotes angiogenesis directly and increases capillary density in myocardial ischemia rats. Isosteviol promotes angiogenesis in zebrafish in vivo and increases vascular endothelial cell proliferation in HUVECs and zebrafish. The angiogenesis activity of isosteviol may be correlated with VEGFA and HIF-1α signaling.

LncRNA HOTTIP facilitates the stemness of breast cancer via regulation of miR-148a-3p/WNT1 pathway.

Emerging evidence suggests that dysregulation of long non-coding RNA (lncRNA) plays a key role in tumorigenesis. The lncRNA, HOXA transcript at the distal tip (HOTTIP), has been reported to be up-regulated in multiple cancers, including breast cancer, and is involved in various biological processes, including the maintenance of stemness. However, the biological function and underlying modulatory mechanism of HOTTIP in breast cancer stem cells (BCSCs) remains unknown. In this study, we found that HOTTIP was markedly up-regulated in BCSCs and had a positive correlation with breast cancer progression. Functional studies revealed that overexpression of HOTTIP markedly promoted cell clonogenicity, increased the expression of the stem cell markers, OCT4 and SOX2, and decreased the expression of the differentiation markers, CK14 and CK18, in breast cancer cells. Knockdown of HOTTIP inhibited the CSC-like properties of BCSCs. Consistently, depletion of HOTTIP suppressed tumour growth in a humanized model of breast cancer. Mechanistic studies demonstrated that HOTTIP directly binds to miR-148a-3p and inhibits the mediation of WNT1, which leads to inactivation of the Wnt/ß-catenin signalling pathway. Our study is the first to report that HOTTIP regulates the CSC-like properties of BCSCs by as a molecular sponge for miR-148a-3p to increase WNT1 expression, offering a new target for breast cancer therapy.

Long noncoding RNA ZFAS1 promoting small nucleolar RNA-mediated 2'-O-methylation via NOP58 recruitment in colorectal cancer.

BACKGROUND: Increasing evidence supports the role of small nucleolar RNAs (snoRNAs) and long non-coding RNAs (lncRNAs) as master gene regulators at the epigenetic modification level. However, the underlying mechanism of these functional ncRNAs in colorectal cancer (CRC) has not been well investigated. METHODS: The dysregulated expression profiling of lncRNAs-snoRNAs-mRNAs and their correlations and co-expression enrichment were assessed by GeneChip microarray analysis. The candidate lncRNAs, snoRNAs, and target genes were detected by in situ hybridization (ISH), RT-PCR, qPCR and immunofluorescence (IF) assays. The biological functions of these factors were investigated using in vitro and in vivo studies that included CCK8, trans-well, cell apoptosis, IF assay, western blot method, and the xenograft mice models. rRNA 2'-O-methylation (Me) activities were determined by the RTL-P assay and a novel double-stranded primer based on the single-stranded toehold (DPBST) assay. The underlying molecular mechanisms were explored by bioinformatics and RNA stability, RNA fluorescence ISH, RNA pull-down and translation inhibition assays. RESULTS: To demonstrate the involvement of lncRNA and snoRNAs in 2'-O-Me modification during tumorigenesis, we uncovered a previously unreported mechanism linking the snoRNPs NOP58 regulated by ZFAS1 in control of SNORD12C, SNORD78 mediated rRNA 2'-O-Me activities in CRC initiation and development. Specifically, ZFAS1 exerts its oncogenic functions and significantly up-regulated accompanied by elevated NOP58, SNORD12C/78 expression in CRC cells and tissues. ZFAS1 knockdown suppressed CRC cell proliferation, migration, and increased cell apoptosis, and this inhibitory effect could be reversed by NOP58 overexpression in vitro and in vivo. Mechanistically, the NOP58 protein could be recognized by the specific motif (AAGA or CAGA) of ZFAS1. This event accelerates the assembly of SNORD12C/78 to allow for further guiding of 2'-O-Me at the corresponding Gm3878 and Gm4593 sites. Importantly, silencing SNORD12C or 78 reduced the rRNAs 2'-O-Me activities, which could be rescued by overexpression ZFAS1, and this subsequently inhibits the RNA stability and translation activity of their downstream targets (e.g., EIF4A3 and LAMC2). CONCLUSION: The novel ZFAS1-NOP58-SNORD12C/78-EIF4A3/LAMC2 signaling axis that functions in CRC tumorigenesis provides a better understanding regarding the role of lncRNA-snoRNP-mediated rRNAs 2'-O-Me activities for the prevention and treatment of CRC.

Functional polymorphisms of the lncRNA H19 promoter region contribute to the cancer risk and clinical outcomes in advanced colorectal cancer.

BACKGROUND: The long non-coding RNA H19 plays critical roles in cancer occurrence, development, and progression. The present study is for the first time to evaluate the association of genetic variations in the H19 promoter region with advanced colorectal cancer (CRC) susceptibility, environmental factors, and clinical outcomes. METHODS: 16 single-nucleotide polymorphisms (SNPs) were identified in the H19 gene promoter by DNA sequencing, and 3 SNPs among which including rs4930101, rs11042170, and rs2735970 further expanded samples with 572 advanced CRC patients and 555 healthy controls. RESULTS: We found that harboring SNP [rs4930101 (P = 0.009), rs2735970 (P = 0.003), and rs11042170 (P = 0.003)] or carrying more than one combined risk genotypes significantly increased the risk for CRC [P < 0.0001, adjusted OR (95% CI) 6.48 (2.97-14.15)]. In the correlation analysis with environmental factors, rs2735970 and gender, combined risk genotypes (> 1 vs. ≤ 1) and family history of cancer demonstrated significant interactions. Furthermore, a remarkably worse clinical outcome was found in combined risk genotypes (> 1 vs. ≤ 1), especially in CRC patients with body weight ≥ 61 kg, smoking, and first-degree family history of cancer (Log-rank test: P = 0.006, P = 0.018, and P = 0.013, respectively). More importantly, the multivariate Cox regression analyses further verified that combined risk genotypes > 1 showed a prognostic risk factor for CRC patients with body weight ≥ 61 kg (P = 0.002), smoking (P = 0.008), and family history of cancer (P = 0.006). In addition, MDR analysis consistently revealed that the combination of selected SNPs and nine known risk factors showed a better prediction prognosis and represented the best model to predict advanced CRC prognosis. CONCLUSION: 3 SNPs of rs4930101, rs11042170, and rs27359703 among 16 identified SNPs of H19 gene remarkably increased CRC risk. Furthermore, the combined risk genotypes had a significant impact on environmental factors and clinical outcomes in the advanced CRC patients with body weight ≥ 61 kg, ever-smoking, and first-degree family history of cancer. These data suggest that H19 promoter SNPs, especially these combined SNPs might be more potentially functional biomarkers in the prediction of advanced CRC risk and prognosis.

Synthesis of Isosteviol analogues as potential protective agents against Doxorubicin-induced cardiomyopathy in zebrafish embryos.

Doxorubicin (DOX) is a powerful anthracycline antibiotic agent which is widely used to treat various types of cancers. Despite efficacy, it displays severe cardiotoxic side effects. Discovery of novel and effective protective agents against DOX-induced cardiotoxicity has been a subject of great interest. Herein, we report the synthesis of two series of analogues of Isosteviol (ISV) 1 with modifications at C-16, C-19 positions as the first series and at C-15, C-16 positions as the other series. Interestingly second series analogues have shown a potential protective effect against DOX-induced cardiotoxicity in zebrafish embryos in vivo. Further, we have demonstrated that the synthesized new analogues of ISV, prevented the morphological distortions caused due to DOX cardiotoxicity in zebrafish heart and the associated cardiac impairments.

Lanthanide Hydroxide Nanoparticles Induce Angiogenesis via ROS-Sensitive Signaling.

Recent studies suggest that the nanorods consisting of europium hydroxide could promote angiogenesis. In this study, it is sought to determine if additional types of nanoparticles are capable of enhancing angiogenesis and in addition, understand the underlying mechanisms. For this reason, a method is employed that combines a high throughput in vitro cell based screen coupled with an in vivo validation using vascular specific green fluorescent protein reporter transgenic zebrafish for examining proangiogenesis activity. After screening multiple types of nanoparticles, it is discovered that four of them, Eu(III) (OH)3 rods (Eu rods), Eu(III) (OH)3 spheres (Eu spheres), Tb(III) (OH)3 rods (Tb rods), and Tb(III) (OH)3 spheres (Tb spheres), are the most effective in promoting angiogenesis. It is also showed that ionic forms of europium nitrate [Eu(NO3 )3 ] (Eu) and terbium nitrate [Tb(NO3 )3 ] (Tb), the two lanthanide elements for these four nanoparticles, are also capable of enhancing angiogenesis. However, this effect is further enhanced by nanoparticle synthesis. Finally, it is demonstrated that reactive oxygen species H2 O2 is a key factor in the process of proangiogenesis by lanthanide elemental nanoparticles.

HDAC2 overexpression is a poor prognostic factor of breast cancer patients with increased multidrug resistance-associated protein expression who received anthracyclines therapy.

OBJECTIVE: Previous studies have revealed the association of multidrug resistance with histone deacetylases inhibitors treatment in cancer cells. But little data were available for the correlation of histone deacetylases and drug-resistant-related proteins in breast cancer tissue. This study aimed to exploring the association of histone deacetylases expression with clinicopathological features, drug-resistant-related proteins, prognosis and therapeutic responses in breast cancer patients. METHODS: We performed immunohistochemistry to study the expression of HDAC1 and HDAC2 in 226 breast cancer and 34 breast fibroadenoma patients, and the expression of breast cancer resistance protein, P-glycoprotein, lung resistance protein and multidrug resistance protein in 226 breast cancer. RESULTS: In breast cancer, HDAC2 expression was significantly increased than in fibroadenoma (P = 0.015), and correlated with lymph node metastasis (P = 0.002), advanced clinical stages (P = 0.016) and high histological grade (P = 0.001). Significant positive correlations were found between HDAC2 and Ki67, HDAC1 and multidrug resistance protein, HDAC2 and breast cancer resistance protein, HDAC2 and multidrug resistance protein. HDAC2 positive expression was associated with shorter overall survival (P = 0.035) of breast cancer patients. In addition, HDAC2-positive expression was significantly associated with shorter overall survival in multidrug resistance protein-positive patients (P = 0.034), but not in multidrug resistance protein-negative patients (P = 0.530). HDAC2-positive expression was associated with shorter survival in patients who received chemotherapy containing anthracyclines (overall survival, P = 0.041; disease-free survival, P = 0.084), but not in patients who received chemotherapy without anthracyclines (overall survival, P = 0.679; disease-free survival, P = 0.708). CONCLUSIONS: HDAC2 overexpression correlated with the metastasis, progression and the increased Ki67, multidrug resistance protein expression in breast cancer, and HDAC2 could be a prognostic factor of breast cancer patients, especially the patients who received anthracyclines therapy.

The Hedgehog signalling pathway mediates drug response of MCF-7 mammosphere cells in breast cancer patients.

BCSCs (breast cancer stem cells) have been shown to be resistant to chemotherapy. However, the mechanisms underlying BCSC-mediated chemoresistance remain poorly understood. The Hh (Hedgehog) pathway is important in the stemness maintenance of CSCs. Nonetheless, it is unknown whether the Hh pathway is involved in BCSC-mediated chemoresistance. In the present study, we cultured breast cancer MCF-7 cells in suspension in serum-free medium to obtain BCSC-enriched MCF-7 MS (MCF-7 mammosphere) cells. We showed that MCF-7 MS cells are sensitive to salinomycin, but not paclitaxel, distinct from parent MCF-7 cells. The expression of the critical components of Hh pathway, i.e., PTCH (Patched), SMO (Smoothened), Gli1 and Gli2, was significantly up-regulated in MCF-7 MS cells; salinomycin, but not paclitaxel, treatment caused a remarkable decrease in expression of those genes in MCF-7 MS cells, but not in MCF-7 cells. Salinomycin, but not paclitaxel, increased apoptosis, decreased the migration capacity of MCF-7 MS cells, accompanied by a decreased expression of c-Myc, Bcl-2 and Snail, the target genes of the Hh pathway. The salinomycin-induced cytotoxic effect could be blocked by Shh (Sonic Hedgehog)-mediated Hh signalling activation. Inhibition of the Hh pathway by cyclopamine could sensitize MCF-7 MS cells to paclitaxel. In addition, salinomycin, but not paclitaxel, significantly reduced the tumour growth, accompanied by decreased expression of PTCH, SMO, Gli1 and Gli2 in xenograft tumours. Furthermore, the expression of SMO and Gli1 was positively correlated with the expression of CD44+ / CD24-, and the expression of SMO and Gli1 in CD44+ / CD24- tissues was associated with a significantly shorter OS (overall survival) and DFS (disease-free survival) in breast cancer patients receiving chemotherapy.

Inducing aortic aneurysm/dissection in zebrafish: evaluating the efficacy of ß-Aminopropionic Nitrile as a model.

Aortic aneurysm/dissection (AAD) poses a life-threatening cardiovascular emergency with complex mechanisms and a notably high mortality rate. Zebrafish (Danio rerio) serve as valuable models for AAD due to the conservation of their three-layered arterial structure and genome with that of humans. However, the existing studies have predominantly focused on larval zebrafish, leaving a gap in our understanding of adult zebrafish. In this study, we utilized ß-Aminopropionic Nitrile (BAPN) impregnation to induce AAD in both larval and adult zebrafish. Following induction, larval zebrafish exhibited a 28% widening of the dorsal aortic diameter (p < 0.0004, n = 10) and aortic arch malformations, with a high malformation rate of 75% (6/8). Conversely, adult zebrafish showed a 41.67% (5/12) mortality rate 22 days post-induction. At this time point, the dorsal aortic area had expanded by 2.46 times (p < 0.009), and the vessel wall demonstrated significant thickening (8.22 ± 2.23 µM vs. 26.38 ± 10.74 µM, p < 0.05). Pathological analysis revealed disruptions in the smooth muscle layer, contributing to a 58.33% aneurysm rate. Moreover, the expression levels of acta2, tagln, cnn1a, and cnn1b were decreased, indicating a weakened contractile phenotype. Transcriptome sequencing showed a significant overlap between the molecular features of zebrafish tissues post-BAPN treatment and those of AAD patients. Our findings present a straightforward and practical method for generating AAD models in both larval and adult zebrafish using BAPN.

MYC overexpression and poor prognosis in sporadic breast cancer with BRCA1 deficiency.

Breast cancer is a complex disease; the molecular mechanisms involved in sporadic breast carcinogenesis remain to be elucidated. The present study aimed to explore the deficiency of breast cancer susceptibility gene 1 (BRCA1), including protein loss expression, promoter hypermethylation and gene copy deletion, its correlationship with other tumor markers expression (TP53, MYC, etc.), and clinical significance in sporadic breast cancer. BRCA1 protein expression was negative in 226 of 374 (60.4%) cases of this study. Cases negative for BRCA1 protein were more often with pathological tumor-node-metastasis stage III, positive for lymph node metastasis and MYC overexpression than BRCA1-positive tumors. BRCA1 hypermethylation was detected in 16.4% (31 of 189) breast cancers, which correlated with BRCA1 negative, ER negative, MYC overexpression, and triple-negative phenotype. In addition, the percentage of cells with BRCA1 gene copy deletion was significantly increased in BRCA1-methylated tumors. Kaplan-Meier survival analysis showed that patients with BRCA1-negative expression showed a worse overall survival (OS) than those with BRCA1-positive expression, and patients with BRCA1-methylated tumors had a significantly worse disease-free survival than did patients with unmethylated tumors. Furthermore, BRCA1 hypermethylation showed an inverse association with OS in LN-positive or p53-negative subgroup patients. Importantly, uni- and multivariate Cox regression analyses revealed that BRCA1 was an independent prognostic indicator of OS in sporadic breast cancer. Thus, we found MYC overexpression and poor prognosis in sporadic breast cancer with BRCA1 deficiency. The targeting of BRCA1 deficiency in combination with MYC-pathways inhibitors may provide a promising strategy for sporadic breast cancer care, the triple-negative subtype in particular.

Rapid screening of novel tyrosinase inhibitory peptides from a pearl shell meat hydrolysate by molecular docking and the anti-melanin mechanism.

Pearls are an edible and medicinal resource with whitening activity and nutritional value in China. In the previous study, we found that the pearl shell meat hydrolysate showed dual activities of antioxidation and tyrosinase inhibition, which were similar to the activities of pearls. In this research, a pearl shell meat hydrolysate was isolated, identified and screened by molecular docking, and three peptides FLF, SPSSS and WLL with high tyrosinase inhibitory activities were obtained. The results indicated that FLF, SPSSS and WLL could effectively inhibit tyrosinase activities and the inhibition rates (1.0 mg mL-1) were 54.32%, 65.26% and 57.50%, respectively. The results of a zebrafish whitening experiment showed that the tyrosinase activities of zebrafish treated with FLF, SPSSS and WLL decreased by 75.41%, 62.87% and 64.99% (p < 0.05), respectively, and the melanin content decreased by 37.34%, 38.52% and 40.39% (p < 0.05), respectively. In a B16F10 cell whitening experiment, compared with a control group, FLF, SPSSS and WLL also showed a significant whitening effect, the tyrosinase activities decreased by 84.08%, 79.08% and 77.45% (p < 0.05), respectively, and the melanin content decreased by 42.23%, 34.37% and 34.02% (p < 0.05), respectively. Moreover, the active peptides could act on three signal pathways including Wnt/ß-catenin, MAPK and MC1R/α-MSH and significantly downregulated the expressions of the signaling factors WNT4, MITF, ß-catenin, ERK, JNK, TRP1 and TRP2 (p < 0.05). The results demonstrated that the whitening active peptides were edible natural antioxidants, tyrosinase inhibitors and skin anti-melanin agents, which could be added to functional foods as food ingredients.

[Research on EGCG improving the degenerative changes of the brain in AD model mice induced with chemical drugs].

OBJECTIVE: To study the effect and mechanism of (-)-Epigallocatechin-3-gallate (EGCG) on the degeneretive changes of the brain in Alzheimer's disease (AD) model mice induced with chemical drugs. METHODS: AD model mice were established by subcutaneously injecting with 3% D-gal at the dose of 150 mg/kg body weight once daily for 6 weeks. From the third week, the mice of D-gal + V(E) 280 U/kg group, D-gal + EGCG 2 mg/(kg x d) group and D-gal + EGCG 6 mg/(kg x d) group were intragastricly given with 5.6% V(E) at the dose of 280 IU/kg and EGCG at the dose of 2 mg/kg x d or 6 mg/kg x d respectively after injection of D-gal. The mice of control group, D-gal + dd H2O group and D-gal + oil group were administered with same volume vehicle distilled water and soybean oil respectively. The pathological changes of the brain in AD model mice were observed by HE staining analysis, the immunohistochemical analysis of beta-amyloid (Abeta) and evaluating the expression of amyloid precursor protein (APP) in the hippocampus of mice by Western blot analysis. RESULTS: EGCG 2 mg/(kg x d) or 6 mg/(kg x d) 4 weeks, ig evidently released neuronal injury in the hippocampus of the AD mice induced by D-gal, and significantly reduced the express of Abeta and APP in the hippocampus of AD model mice induced by D-gal (P < 0.01). CONCLUSION: EGCG has a protective effect on AD model mice induced by D-gal by decreasing the expression of APP and beta-Amyloid in the hippocampus of mice.

Effects of long-term and low-concentration exposures of benzene and formaldehyde on mortality of Drosophila melanogaster.

Single-chemical thresholds cannot comprehensively evaluate the risk of chemical mixture exposure in indoor air. Moreover, a large number of researches have focused on short-term and high-concentration co-exposure scenarios related to different species, based on diverse endpoints, which hampers the application and improvement of existing risk evaluation models of chemical mixture exposures. More importantly, current risk evaluation models are not user-friendly for construction practitioners who do not have sufficient toxicological knowledge. Therefore, in this study, an inhalation experiment system and a hazard index (HI) were developed to investigate the risks associated with low-concentration and long-term inhalation exposure scenarios of formaldehyde and benzene, individually and combined, based on Drosophila melanogaster mortality. The results showed that the system exhibited good reproducibility in providing stable exposure concentrations during D. melanogaster life cycle. Furthermore, in a range of experimental concentrations, the interaction between formaldehyde and benzene was additive or synergistic, which was concentration- and ratio-dependent. This study is of great significance in harmonising and providing toxicity data under long-term and low-concentration exposure scenarios, which is beneficial for establishing a new user-friendly risk evaluation model for indoor chemical mixture exposures. It should be noted that the proposed HI value could indicate the hazard degrees of long-term inhalation exposures of formaldehyde and benzene, individually and combined, to D. melanogaster. However, the applicability of this index requires further experiments to evaluate the exposure risks of other volatile organic compounds (VOCs) to D. melanogaster.

Glabrol impurity exacerbates glabridin toxicity in zebrafish embryos by increasing myofibril disorganization.

ETHNOPHARMACOLOGICAL RELEVANCE: Glabridin, extracted from Glycyrrhiza glabra L., is widely used for the treatment of hyperpigmentation because of its anti-inflammatory and antioxidant activities and its ability to inhibit melanin synthesis. This led to the strict regulation of its quality and safety. However, traditional quality control methods used for plant extracts cannot reflect the product quality owing to multiple unknown impurities, which necessitates the further analysis of impurities. AIM OF THE STUDY: The study identified the toxic impurities of glabridin and their toxicological mechanism. MATERIALS AND METHODS: In total, 10 glabridin samples from different sources were quantified using high-performance liquid chromatography. Sample toxicities were evaluated using zebrafish and cell models. To identify impurities, samples with different toxicity were analyzed by ultra-high-performance liquid chromatography coupled with quadrupole-Orbitrap mass spectrometry. The toxicity of related impurities was verified in the zebrafish model. Phalloidin stain was used to evaluate subtle changes in myofibril alignment. RESULTS: Although glabridin content in the samples was similar, there were significant differences in toxicity. The results were verified using four different mammalian cell lines. Higher contents of glabrone and glabrol were identified in the sample with the highest toxicity. In the zebrafish model, the addition of glabrol reduced the LC50 of glabridin to 9.224, 6.229, and 5.370 µM at 48, 72, and 96 h post-fertilization, respectively, whereas glabrone did not have any toxic effect. Phalloidin staining indicated that a glabrol impurity exacerbates the myotoxicity of glabridin in zebrafish embryos. CONCLUSION: Glabrol, but not glabrone, was identified as a key impurity that increased glabridin toxicity. This finding indicates that controlling glabrol content is necessary during glabridin product production.

Evaluation of right ventricular myocardial strain in pulmonary arterial hypertension associated with atrial septal defect by cardiac magnetic resonance feature tracking.

We aimed to research the role of right ventricular strain parameters (RVSP) quantified by cardiac magnetic resonance feature tracking (CMR-FT) in the early assessment of right ventricular (RV) function in patients with pulmonary arterial hypertension associated with atrial septal defect (PAH-ASD). From September 2017 to May 2021, we retrospectively enrolled 41 patients with PAH-ASD and 20 healthy controls. All subjects underwent CMR-FT, and right heart catheterization was conducted in patients with PAH-ASD. The relationship between RVSP and RV functional parameters was subjected to correlation analysis, and intragroup correlation coefficient (ICC) and Bland-Altman plots were used to assess the consistency. The subjects were divided into three groups: Group A (controls; n = 20), Group B (PAH-ASD, RVEF ≥ 45%; n = 14), and Group C (PAH-ASD, RVEF < 45%; n = 27). Compared with healthy controls, the RV global longitudinal strain (GLS) in Group B was significantly decreased (- 19.68 ± 2.72% vs. - 25.21 ± 3.6%, P < 0.05). In RVEF-preserved PAH-ASD patients (Group B), compared with patients with GLS ≤ - 20%, patients with GLS > - 20% also had significantly elevated right ventricular end-diastolic pressure (RVEDP) [8 (6.5-8.25) mmHg vs. 4.5 ± 1.64 mmHg, P < 0.05]. RV GLS had a moderate to strong correlation with RVEF, RVESVi, RVEDVi, RVEDP, and NT-proBNP (P < 0.05). ICC and Bland-Altman plots showed good intragroup and intergroup consistency in radial, circumferential and longitudinal strains of RV. In conclusion, it is feasible to quantify RV strain in patients with PAH-ASD by CMR-FT, and GLS is valuable for the early assessment of RV dysfunction in patients with PAH-ASD.

A novel HIF-2α targeted inhibitor suppresses hypoxia-induced breast cancer stemness via SOD2-mtROS-PDI/GPR78-UPRER axis.

Hypoxic tumor microenvironment (TME) plays critical roles in induction of cancer stem cell-like phenotype in breast cancer and contribute to chemoresistance. However, the mechanism underlying stemness reprogramming of breast cancer cells (BCs) by hypoxic TME remains largely unknown. In the present study, we illustrated that HIF-2α, but not HIF-1α, induces stemness in BCs under hypoxia through SOD2-mtROS-PDI/GRP78-UPRER pathway, linking mitochondrial metabolic state to endoplasmic reticulum (ER) response via mitochondrial reactive oxygen species (mtROS) level. HIF-2α activates endoplasmic reticulum unfolded protein response (UPRER) in drug-sensitive MCF7 and T47D cells to induce drug-resistant stem-like phenotype. Genetic depletion or pharmacological inhibition (YQ-0629) of HIF-2α abolished hypoxia-induced stem-like phenotype in vitro and in vivo. Mechanistically, HIF-2α activates transcription of superoxide dismutase 2 (SOD2) under hypoxia and thereby decreases mtROS level. With less mtROS transported to endoplasmic reticulum, the expression and activity of protein disulfide isomerase (PDI) is suppressed, allowing glucose-regulated protein 78 (GRP78) to dissociate from receptor proteins of UPRER and bind misfolded protein to activate UPRER, which eventually confer chemoresistance and stem-like properties to BCs. Moreover, the increase in mtROS and PDI levels caused by HIF-2α knockdown and the subsequent UPRER inhibition could be substantially rescued by mitoTEMPOL (a mtROS scavenger), 16F16 (a PDI inhibitor), or GRP78 overexpression. Overall, we reported the critical roles of HIF-2α-SOD2-mtROS-PDI/GRP78-UPRER axis in mediating hypoxia-induced stemness in BCs, highlighting the interaction between organelles and providing evidence for further development of targeted HIF-2α inhibitor as a promising therapeutic strategy for chemoresistant breast cancer.

Enhancement of Fear Extinction Memory and Resistance to Age-Related Cognitive Decline in Butyrylcholinesterase Knockout Mice and (R)-Bambuterol Treated Mice.

Butyrylcholinesterase (BChE) is detected in plaques preferentially in Alzheimer's disease (AD) and may be associated with stress disorders. However, the physiological function of BChE in the central nervous system remains to be further investigated. BChE knockout (KO) mice and wild-type (WT) mice with orally or intranasal administration of (R)-bambuterol were used to explore the effect of BChE on behavior changes. (R)-bambuterol is a specific and reversible inhibitor of BChE. The behavior changes were evaluated and compared among 3-10 month old mice. Our finding showed that BChE KO and (R)-bambuterol administration enhanced episodic memory, including fear conditioning memory and fear extinction memory in fear conditioning and fear extinction test. BChE KO and (R)-bambuterol administered mice rescued age-related spatial memory and general activity in the water maze test and open field test. The brain metabolomics were imaged using a desorption electrospray ionization mass spectrometry imaging (DESI-MSI). The image of DESI-MS demonstrated that glutamine content increased in the brain of BChE KO mice. In conclusion, this study found that inhibition of BChE ameliorated episodic and spatial memories. This study also suggested that (R)-bambuterol as a BChE inhibitor has the potential application in the treatment of post-traumatic stress disorder (PTSD) and early cognitive decline.

CD276 (B7H3) improve cancer stem cells formation in cervical carcinoma cell lines.

BACKGROUND: Cancer stem cells (CSCs) have been considered as a potential therapeutic target for cervical carcinoma. CD 276 is a well-known immune check point molecular, but its relationship with cervical CSCs was still unclear. METHODS: HeLa cell lines were obtained as cervical carcinoma in vitro model. HeLa cell Sphere formation culture was performed and CD276, OCT4 and SOX2 expression were determined by RT-qPCR. Transiently transfection and siRNA interference were used to modify CD276 expression. HeLa cell colony has been counted and cell proliferation was assessed by MTT assay. The relationship between CD276 and chemotherapy resistance of HeLa cell were evaluated by cisplatin treatment. Additionally, the mice model of xenograft tumor was established and CD276's function was evaluated in vivo. RESULTS: Here, we demonstrate that the expression of CD276 is positively correlated with the amount of sphere-forming cells in HeLa cell lines. Overexpression of CD276 causes the inhibition of HeLa cells' sphere formation, colony formation and cell viability. Meanwhile, the downregulation of CD276 leads to the other way. We also demonstrate that CD276 contributes to the chemotherapy resistance in the cell line. Furthermore, we verify the CD276's function on HeLa xenotransplantation mice model. CONCLUSIONS: These results suggest that CD276 elevates the self-renewal capacity of HeLa CSCs.