Inhibition of TGF-ß1/Smad3 signaling by compound 5aa: A potential treatment for idiopathic pulmonary fibrosis.

The incidence of idiopathic pulmonary fibrosis (IPF) has been steadily increasing each year, posing significant challenges in its treatment. In this study, we conducted the design and synthesis of 23 new inhibitors that specifically target the TGF-ß1/Smad3 pathway. Initially, we employed a cell model of TGF-ß-induced pulmonary fibrosis, using cell survival rate and HYP expression as indicators to identify the potent ingredient 5aa, which demonstrated significant anti-pulmonary fibrosis activity. Subsequently, we induced mice with bleomycin (BLM) to establish an experimental animal model of pulmonary fibrosis, and evaluated the pharmacodynamics of 5aa in vivo against pulmonary fibrosis. The alterations in HYP and collagen levels in BLM-induced pulmonary fibrosis mice were analyzed using ELISA and immunohistochemistry techniques. The results indicated that compound 5aa effectively suppressed the fibrotic response induced by TGF-ß1, inhibited the expression of the fibrotic marker α-SMA, and hindered the EMT process in NIH3T3 cells. Additionally, oral administration of 5aa demonstrated significant therapeutic effects in a mouse model of IPF, comparable to the established drug Nintedanib. Moreover, compound 5aa exhibited higher bioavailability in vivo compared to Nintedanib. These collective outcomes suggest that 5aa holds promise as a potential inhibitor of TGF-ß1/Smad3 signaling for the treatment of IPF.

Extracellular matrix stiffness mediates radiosensitivity in a 3D nasopharyngeal carcinoma model.

PURPOSE: Radiotherapy is one of the essential treatment modalities for nasopharyngeal carcinoma (NPC), however, radioresistance still poses challenges. Three-dimensional (3D) tumor culture models mimic the in vivo growth conditions of cells more accurately than 2D models. This study is to compare the tumor biological behaviors of NPC cells in 2D, On-Surface 3D and Embedded 3D systems, and to investigate the correlation between radioresistance and extracellular matrix (ECM) stiffness. METHODS: The morphology and radioresistance of the human NPC cell line CNE-1 were observed in 2D and 3D systems. The CCK-8 assay, wounding healing assays, flow cytometry, soft agar assays, and western blot analysis were used to evaluate differences in biological behaviors such as proliferation, migration, cell cycle distribution, and stem cell activity. Different ECM stiffness systems were established by co-blending collagen and alginate in varying proportions. ECM stiffness was evaluated by compressive elastic moduli measurement and colony formation assay was used to assess radioresistance of NPC cells in systems with different ECM stiffness after irradiation. RESULTS: Compared to 2D models, the morphology of NPC cells in 3D culture microenvironments has more in common with in vivo tumor cells and 3D cultured NPC cells exhibit stronger radioresistance. Integrin ß1 but not the epithelial-to-mesenchymal transition pathway in 3D models boost migration ability. Cell proliferation was enhanced, the proportion of tumor stem cells was increased, and G1/S phase arrest occurred in 3D models. NPC cells cultured in softer ECM systems (with low alginate proportions) exhibit striking resistance to ionizing radiation. CONCLUSION: The tumor biological behaviors of NPC cells in 3D groups were obviously different from that of 2D. Radioresistance of NPC cells increased with the stiffness of ECM decreasing.

Rapid and Sensitive Differentiating Ischemic and Hemorrhagic Strokes by Dried Blood Spot Based Direct Injection Mass Spectrometry Metabolomics Analysis.

Cerebral infarction (CI) and intracerebral hemorrhage are lethal cerebrovascular diseases, sometimes sharing similar clinical manifestations but with distinct therapeutic strategies. Delayed treatment usually resulted in poor prognosis. A timely diagnosis report is highly warranted especially in emergency. One hundred twenty-nine CI patients, 73 intracerebral hemorrhage (ICH) patients, and 98 controls were enrolled in this study. A direct injection mass spectrometry metabolomics approach was adopted using dried blood spot samples. This targeted metabolomics analysis focused on absolute quantitation of 23 amino acids, 26 carnitine/carnitine esters, and 22 calculated ratios parameters. Compared to the normal control group, CI and ICH showed distinct metabolite changes, respectively. For stroke differentiation, Tyr, C5-OH/C0, Cit, Asn, Pro, Val, Arg/Orn, Leu, and Val/Phe were elevated in the CI group. On the contrary, C5:1, Phe/Tyr, (C0 + C2 + C3 + C16 + C18:1)/Cit, and Met/Leu were of lower levels in the CI group. Using regression model based on some of the above-mentioned parameters, 79.07% of stroke patients from a new set could be definitely confirmed. This study proved the targeted metabolomics analysis was a promising tool for rapid and timely stroke differentiation.

Patient-derived organoid elucidates the identical clonal origin of bilateral breast cancer with diverse molecular subtypes.

Bilateral breast cancer (BBC), an infrequent breast cancer subtype, has primarily been studied in terms of incidence, prognosis, and through comparative analysis of synchronous (SBBC) and metachronous (MBBC) manifestations. The advent and application of organoid technology hold profound implications for tumor research and clinical management. This study represents the pioneering use of organoid models in BBC research. We established organoid lines from two surgical tumor specimens of a BBC patient, with one line undergoing detailed pathological and genomic analysis. The BBC organoid from the right breast demonstrated a marker expression profile of ER (-), PR (-), HER-2 (0), and Ki67 index 10%, indicating that it may derived from the TNBC tissue. Whole Exome Sequencing (WES) displayed consistent set of Top10 cancer driver genes affected by missense mutations, frameshift mutation, or splice site mutations in three tumor tissues and the organoid samples. The organoids' single nucleotide polymorphisms (SNPs) were more closely aligned with the TNBC tissue than other tumor tissues. Evolutionary analysis suggested that different tumor regions might evolve from a common ancestral layer. In this case, the development of BBC organoids indicated that simultaneous lesions with diverse molecular profiles shared a high degree of consistency in key tumor-driving mutations. These findings suggest the feasibility of generating BBC organoids representing various molecular types, accurately replicating significant markers and driver mutations of the originating tumor. Consequently, organoids serve as a valuable in vitro model for exploring treatment strategies and elucidating the underlying mechanisms of BBC.

Differentiation of bone marrow mesenchymal stem cells into Leydig-like cells with testicular extract liquid in vitro.

Differentiation of Leydig cells plays a key role in male reproductive function. Bone marrow mesenchymal stem cells (BMSCs) have emerged as a potential cell source for generating Leydig-like cells due to their multipotent differentiation capacity and accessibility. This study aimed to investigate the morphological and genetic expression changes of BMSCs during differentiation into Leydig-like cells. Testicular extract liquid, which simulates the microenvironment in vivo, induced the third passage BMSCs differentiated into Leydig-like cells. Changes in cell morphology were observed by microscopy, the formation of lipid droplets of androgen precursor was identified by Oil Red Staining, and the expression of testicular specific genes 3ß-HSD and SF-1 in testicular stromal cells was detected by RT-qPCR. BMSCs isolated from the bone marrow of Sprague-Dawley (SD) rats were cultured for 3 generations and identified as qualified BMSCs in terms of morphology and cell surface markers. After 14 days of induction with testicular tissue lysate, lipid droplets appeared in the cytoplasm of P3 BMSCs by Oil Red O staining. RT-qPCR detection was performed on BMSCs on the 3rd, 7th, 14th, and 21st day after induction. Relative expression levels of 3ß-HSD mRNA significantly increased after 14 days of induction, while the relative expression of SF-1 mRNA increased after 14 days of induction but was not significant. BMSCs can differentiate into testicular interstitial cells with reserve androgen precursor lipid droplets after induction by testicular tissue lysate. The differentiation ability of BMSCs provides the potential to reconstruct the testicular microenvironment and is expected to fundamentally improve testicular function and provide new treatment options for abnormal spermatogenesis diseases.

Potential protective benefits of Schisandrin B against severe acute hepatitis in children during the COVID-19 pandemic based on a network pharmacology analysis.

Aims: Reports of hepatitis in children during the coronavirus disease 2019 (COVID-19) pandemic garnered worldwide attention. The most probable culprits are adenovirus and severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). At present, the optimal symptomatic treatment consists of a combination of anti-COVID-19 and hepatitis symptom alleviators. Schisandrin B (SchB) has been known to have liver-protective properties for a long time, whereas anti-COVID-19 properties only recently have been discovered. In the case of COVID-19 with hepatitis of unknown origin, we used network pharmacology to explore the symptomatic therapy and protective effects of SchB. Main methods: The most probable protein targets of SchB were predicted in the SwissTargetPrediction database. The GeneCards, National Center for Biotechnology Information, and Online Mendelian Inheritance in Man databases were used to compile information on the diseases hepatitis, adenovirus, and SARS-CoV-2. Following the use of a Venn diagram viewer to identify intersection genes, we constructed a protein-protein interaction network and identified the core genes. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment, as well as molecular docking, were employed to highlight the mechanisms of SchB on hepatitis. Key findings: SchB contains 27 targets on adenovirus_hepatitis and 16 targets on SARS-CoV-2_hepatitis, with 12 shared genes. Both target populations clustered in viral infection and cancer pathways, as well as in processes such as kinase activity phosphatase, cell adhesion, and ATPase binding. These genes might be closely related to liver damage and membrane binding from adenovirus or SARS-CoV-2 infections. In addition, epidermal growth factor receptor, HSP90AA1, and MAPK1 were among the top five targets of both SchB SARS-CoV-2 hepatitis and SchB adenovirus hepatitis. Significance: SchB may target common protective targets and mechanisms against acute hepatitis caused by adenovirus or by SARS-CoV-2 in children during the COVID-19 pandemic. These findings indicate SchB's potential as a treatment for hepatitis of unknown origin.

Long non-coding RNA small nucleolar RNA host gene 1 alleviates the progression of recurrent spontaneous abortion via the microRNA-183-5p/ZEB2 axis.

Long non-coding RNAs (lncRNAs) have been elucidated to play vital roles in the phenotype of trophoblast cells. Nevertheless, the effect of SNHG1 has not been investigated on trophoblast cells in recurrent spontaneous abortion (RSA). We aim to investigate the effect of SNHG1 on the phenotype of trophoblast cells during RSA. The RSA mice were established by mating female CBA/J mice with male DBA/2 mice. Microarray analysis was applied in RSA mice, and SNHG1 was identified as a significantly downregulated lncRNA. SNHG1 improved pregnancy outcome and reduced embryo resorption in RSA mice. Trophoblast cell proliferation, apoptosis, migration, and invasion were investigated by CCK8, EdU, TUNEL, wound healing, and Transwell assays. SNHG1 promoted proliferation, migration, and invasion of trophoblast cells, and reduced apoptosis. Mechanistically, SNHG1 bound to miR-183-5p in trophoblast cells. Moreover, miR-183-5p directly targeted ZEB2. Rescue experiment showed that ZEB2 silencing reversed the ameliorative effect of SNHG1 on pregnancy outcome and the promotion of trophoblast activity in RSA mice by impaired the Wnt/ß-catenin pathway. In conclusion, we found that SNHG1 plays a critical role in the progression of RSA via miR-183-5p/ZEB2 and Wnt/ß-catenin signaling. It has potential to be a therapeutic marker of RSA.

Fine particulate matter and vasoactive 20-hydroxyeicosatetraenoic acid: Insights into the mechanisms of the prohypertensive effects of particulate air pollution.

BACKGROUND: Emerging evidence suggests that biological intermediates play an important role in initiating fine particulate matter (PM2.5)-associated prohypertensive pathways, but sensitive biomarkers for this pathway are lacking. AIM: To explore whether short-term exposure to PM2.5 is associated with the concentration of 20-hydroxyeicosatetraenoic acid (20-HETE), a potent vasoactive lipid relevant to the pathophysiology of hypertension. METHODS: In this longitudinal panel study, we repeatedly (up to seven times) measured the blood concentrations of 20-HETE in 120 adults living in Beijing, China. Ambient exposure metrics included the concentrations of hourly PM2.5 mass and daily PM2.5 constituents, including three carbonaceous components, eight water-soluble ions, and 16 trace elements. Linear mixed-effects models were used to examine the associations between the change in the 20-HETE concentration and short-term exposure to ambient PM2.5 metrics after adjustment for age, sex, body mass index, behavioral exposure, socioeconomic characteristics, and meteorological factors. RESULTS: The interquartile range (IQR) increase in the 7-15-hour-lag exposure to PM2.5 (80 µg/m3) was associated significantly with a 5.3% (95% confidence interval [CI], 0.1-10.7%) to 6.5% (95% CI, 1.7-11.6%) increase in the blood concentration of 20-HETE. The magnitude of the association differed by age, sex, prediabetic status, obesity, and hypertensive status, with a significantly greater increase in 20-HETE observed among those with fasting plasma glucose concentrations ≥ 6.1 mmol/L. In addition to the PM2.5 mass, the 20-HETE concentration was associated consistently with IQR increases in the 1-day lag exposure to organic carbon (5.7%), black carbon (9.5%), nitrate (3.9%), chloride (2.9%), copper (5.5%), zinc (4.7%), barium (4.1%), and lead (6.2%). The organic carbon estimate was robust in the two-pollutant models. Furthermore, increased 20-HETE correlated with elevated blood pressure (BP), although no mediation of 20-HETE on PM2.5-associated BP change was found. CONCLUSIONS: The 20-HETE blood concentration increased significantly in response to short-term exposure to ambient PM2.5, which may be partly responsible for the prohypertensive effects of PM2.5.

Investigation of the chemical components of ambient fine particulate matter (PM2.5) associated with in vitro cellular responses to oxidative stress and inflammation.

Fine particulate matter (PM2.5) poses a significant risk to human health worldwide, by promoting oxidative stress and inflammation; however, the components responsible for these effects have not been fully evaluated. In this study, we investigated the cellular response of a macrophage cell line exposed to PM2.5 extracts in vitro. We obtained a dataset of chemical components of PM2.5 and determined those associated with the generation of reactive oxygen species (ROS) and secretion of inflammatory cytokines through an orthogonal partial least-squares (OPLS) regression. The results indicated that after water extracts exposure, both ROS and interleukin (IL)-1ß levels were positively correlated with transition metals. In cells exposed to dichloromethane extracts, IL-1ß secretion was significantly correlated with polycyclic aromatic hydrocarbons (PAHs); meanwhile, tumor necrosis factor (TNF)-α secretion was negatively associated with secondary nitrated PAHs, suggesting that atmospheric nitration process might modify the biological effects of PM2.5 components. We also performed source apportionment using a positive matrix factorization (PMF) model to explore the relative influence of different sources of components on cells. It was found that components from vehicle emissions promoted both ROS and TNF-α, while IL-1ß secretion was induced mainly by those from coal combustion. This study provides information regarding PM2.5 components having biological effects, and the sources thereof, which could inform effective measures for controlling this type of air pollution.

Association of internal exposure to polycyclic aromatic hydrocarbons with inflammation and oxidative stress in prediabetic and healthy individuals.

Polycyclic aromatic hydrocarbons (PAHs) are key air pollutants that may contribute to the risk of numerous diseases by inducing inflammation and oxidative stress. Individuals with metabolic disorders may be more susceptible to PAH-induced inflammation and oxidative stress. To test this hypothesis, we designed a panel study involving 60 patients with pre-type 2 diabetes (pre-T2D) and 60 reference participants, and conducted up to seven repeated clinical examinations. Urinary metabolites of PAHs (i.e., OH-PAHs), measured as indicators of total PAH exposure, showed significant associations with markers of respiratory and systemic inflammation, including exhaled nitric oxide, interleukin (IL)-6 in exhaled breath condensate, and blood IL-2 and IL-8 levels and leucocyte count. The most significant effect was on urinary malondiadehyde (MDA), a marker of lipid peroxidation; a onefold increase of OH-PAHs was associated with 9.2-46.0% elevation in MDA in pre-T2D participants and 9.8-31.2% increase in healthy references. Pre-T2D participants showed greater increase in MDA, suggesting that metabolic disorder enhanced the oxidative damage induced by PAH exposure. This study revealed the association between PAH exposure and markers of inflammation and oxidative stress, and the enhanced responses of pre-T2D patients suggested that individuals with metabolic disorders were more susceptible to the adverse health effects of PAH exposure.

Effects on IL-1ß signaling activation induced by water and organic extracts of fine particulate matter (PM2.5) in vitro.

Fine particulate matter (PM2.5) air pollution poses a major risk to human health worldwide, and absorbed chemicals play a key role in determining the toxicity of PM2.5. After inhalation and entry into the lungs, PM2.5 components induce pro-inflammatory cytokines (e.g., interleukin (IL)-1ß) in pulmonary cells. To test whether PM2.5 components induce IL-1ß through signing pathways that include the toll-like receptor 4 (TLR4)/nuclear factor-κ-gene binding (NF-κB), nucleotide-binding domain and leucine-rich repeat protein 3 (NLRP3), we exposed the mouse macrophage cell-line RAW264.7 to both water and organic extracts of PM2.5 sampled over a 1-year period in Beijing, China. Varying degrees of oxidative stress and inflammatory responses were induced following exposure, while organic extracts of PM2.5 collected during the heating season induced more significant responses. This response is attributed to high concentrations of polycyclic aromatic hydrocarbons (PAHs) originating from coal combustion and biomass burning for domestic heating. The inhibition of signaling molecules suggested that increased IL-1ß was associated with the TLR4/NF-κB pathway and NLRP3 inflammasome activation, with a slightly difference between water and organic extracts exposure groups, which was likely the result of different chemical components. Our study elucidated a potentially important mechanism by which PM2.5 components could trigger pulmonary inflammation, thus improving our understanding of the deleterious effects of this important and prevalent form of air pollution.

Macrophage-Mediated Effects of Airborne Fine Particulate Matter (PM2.5) on Hepatocyte Insulin Resistance in Vitro.

Fine particulate matter (PM2.5) pollution poses significant health risks worldwide, including metabolic syndrome-related diseases with the characteristic feature of insulin resistance. However, the mechanism and influencing factors of this effect are poorly understood. In this serial in vitro study, we aimed at testing the hypothesis that macrophage-mediated effects of PM2.5 on hepatic insulin resistance depend on its chemical composition. Mouse macrophages were exposed to PM2.5 that had been collected during summer or winter in Beijing, which represented different compositions of PM2.5. Thereafter, hepatocytes were treated with macrophage-conditioned medium (CM). PM2.5 induced interleukin-6, tumor necrosis factor-α, and monocyte chemoattractant protein-1 expression and secretion in macrophages, particularly after winter PM2.5 exposure. Correspondingly, winter CM weakened hepatocellular insulin-stimulated glucose consumption. Further investigation revealed that the normal insulin pathway was suppressed in winter CM-treated hepatocytes, with increased phosphorylation of insulin receptor substrate 1 at serine residue 307 (Ser307) and decreased phosphorylation of protein kinase B (PKB/AKT) and forkhead box transcription factor O1 (FoxO1). Moreover, c-Jun N-terminal kinase, a key moderator of the sensitivity response to insulin stimulation, was activated in hepatocytes treated with winter CM. Although further studies are warranted, this preliminary study suggested an association between PM composition and insulin resistance, thus contributing to our understanding of the systemic toxicity of PM2.5.

A dried blood spot mass spectrometry metabolomic approach for rapid breast cancer detection.

OBJECTIVE: Breast cancer (BC) is still a lethal threat to women worldwide. An accurate screening and diagnosis strategy performed in an easy-to-operate manner is highly warranted in clinical perspective. Besides the routinely focused protein markers, blood is full of small molecular metabolites with diverse structures and properties. This study aimed to screen metabolite markers with BC diagnosis potentials. METHODS: A dried blood spot-based direct infusion mass spectrometry (MS) metabolomic analysis was conducted for BC and non-BC differentiation. The targeted analytes included 23 amino acids and 26 acylcarnitines. RESULTS: Multivariate analysis screened out 21 BC-related metabolites in the blood. Regression analysis generated a diagnosis model consisting of parameters Pip, Asn, Pro, C14:1/C16, Phe/Tyr, and Gly/Ala. Tested with another set of BC and non-BC samples, this model showed a sensitivity of 92.2% and a specificity of 84.4%. Compared to the routinely used protein markers, this model exhibited distinct advantage with its higher sensitivity. CONCLUSION: Blood metabolites screening is a more plausible approach for BC detection. Furthermore, this direct MS analysis could be finished within few minutes, which means that its throughput is higher than the currently used imaging techniques.