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Abstract Background Bleomycin, a potent antitumor agent, is limited in clinical use due to the potential for fatal pulmonary toxicity. The accelerated DNA damage and senescence in alveolar epithelial cells (AECs) is considered a key factor in the development of lung pathology. Understanding the mechanisms for bleomycin-induced lung injury is crucial for mitigating its adverse effects. Methods Human lung epithelial (A549) cells were exposed to bleomycin and subsequently assessed for cellular senescence, DNA damage, and double-strand break (DSB) repair. The impact of Rad51 overexpression on DSB repair and senescence in AECs was evaluated in vitro. Additionally, bleomycin was intratracheally administered in C57BL/6 mice to establish a pulmonary fibrosis model. Results Bleomycin exposure induced dose- and time-dependent accumulation of senescence hallmarks and DNA lesions in AECs. These effects are probably due to the inhibition of Rad51 expression, consequently suppressing homologous recombination (HR) repair. Mechanistic studies revealed that bleomycin-mediated transcriptional inhibition of Rad51 might primarily result from E2F1 depletion. Furthermore, the genetic supplement of Rad51 substantially mitigated bleomycin-mediated effects on DSB repair and senescence in AECs. Notably, decreased Rad51 expression was also observed in the bleomycin‐induced mouse pulmonary fibrosis model. Conclusions Our works suggest that the inhibition of Rad51 plays a pivotal role in bleomycin-induced AECs senescence and lung injury, offering potential strategies to alleviate the pulmonary toxicity of bleomycin.

Background and aims: Preeclampsia (PE) is the leading cause of maternal and fetal morbidity and mortality worldwide. Apoptosis of trophoblast cells induced by oxidative stress is a principal reason of placental injury in PE. 6-Gingerol, an antioxidant from ginger, plays an important role in many disease models, but its effect on obstetric diseases has not been elucidated. In this study, we investigated the protective effect of 6-gingerol against placental injury.Methods:In vitro hypoxia/reoxygenation (H/R) model of HTR8/Svneo cells and preeclamptic mice model were established to simulate PE. The effects of 6-Gingerol on PE were evaluated by morphological detection, biochemical analysis, and Western blot.Results: We found that H/R treatment induced cell apoptosis, increased the production of reactive oxygen species, malondialdehyde and lactate dehydrogenase, and decreased superoxide dismutase in trophoblast. In addition, the polarization of mitochondrial membrane potential and the cellular calcium flux were also destroyed under H/R condition, which also activated BCL2-interacting protein 3 (BNIP3) and provoked excessive mitophagy. Importantly, 6-Gingerol reversed these corrosive effects. Furthermore, the placenta damage in PE-like mouse caused by the cell apoptosis, oxidative stress and mitophagy was mitigated by 6-Gingerol.Conclusion: These findings suggest that 6-Gingerol exerts a protective effect against placental injury in PE by reducing oxidative stress and inhibiting excessive mitophagy caused by mitochondrial dysfunction.

Objective To investigate the X-ray-specific sensitive genes and potential signaling pathways involved in the latent period of radiation-induced lung injury (RILI) in mouse models. Method Mice were randomized into groups for whole thoracic irradiation with a single fraction of 20 Gy X-ray or 12.5 Gy carbon heavy ion. Lungs were harvested 3 weeks after the irradiation, whole RNA was extracted and detected with the genome-wide transcriptional microarrays. Differentially expressed genes (DEGs) were calculated for each group and the X-ray-specific sensitive genes were determined, followed by the gene enrichment analysis of those DEGs exploring the potentially relevant signaling pathways and biological processes in latent RILI. Results Three weeks after irradiation, gene expression levels varied between groups. 76 up-regulated DEGs were determined with mice in the X-ray group and gene ontology enrichment analysis for biological process (GO-BP) obtained several processes which were associated with radiation reaction, mitotic, immune cell chemotaxis or metastasis, immune factors, p53 apoptosis, and tissue remodeling. KEGG signaling pathway enrichment analysis showed that those 76 up-regulated DEGs were enriched in p53, IL-17, FoXO, melanoma, and non-small-cell lung cancer signaling pathways. By comparing the DEGs in X-ray and heavy ion groups, X-ray-specific sensitive genes were determined, the top 10 genes were Adamts9, Aacs, Col6a2, Fdps, Mdk, Mcam, Stbd1, Lbh, Ak3, and Emid1. The expression level of the top 10 genes was found to be significantly higher in the X-ray group than in the control and heavy ion groups. Conclusion Our research determined the X-ray-specific sensitive gene set in mice lungs after exposure to radiation. The gene set could be used as a genetic marker to suggest the latency of RILI. The enrichment analysis results suggested that the relevant signaling pathways were potentially involved in the development of RILI. Further validation of those genes and signaling pathways is needed to confirm these findings.

We recently developed a combination of four chemiluminescence-based assays for selective detection of different nitric oxide (NO) metabolites, including nitrite, S-nitrosothiols (SNOs), heme-nitrosyl (heme-NO), and dinitrosyl iron complexes (DNICs). However, these NO species (NOx) may be under dynamic equilibria during sample handling, which affects the final determination made from the readout of assays. Using fetal and maternal sheep from low and high altitudes (300 and 3801 m, respectively) as models of different NOx levels and compositions, we tested the hypothesis that sample handling introduces artifacts in chemiluminescence assays of NOx. Here, we demonstrate the following: (1) room temperature placement is associated with an increase and decrease in NOx in plasma and whole blood samples, respectively; (2) snap freezing and thawing lead to the interconversion of different NOx in plasma; (3) snap freezing and homogenization in liquid nitrogen eliminate a significant fraction of NOx in the aorta of stressed animals; (4) A “stop solution” commonly used to preserve nitrite and SNOs leads to the interconversion of different NOx in blood, while deproteinization results in a significant increase in detectable NOx; (5) some reagents widely used in sample pretreatments, such as mercury chloride, acid sulfanilamide, N-ethylmaleimide, ferricyanide, and anticoagulant ethylenediaminetetraacetic acid, have unintended effects that destabilize SNO, DNICs, and/or heme-NO; (6) blood, including the residual blood clot left in the washed purge vessel, quenches the signal of nitrite when using ascorbic acid and acetic acid as the purge vessel reagent; and (7) new limitations to the four chemiluminescence-based assays. This study points out the need for re-evaluation of previous chemiluminescence measurements of NOx, and calls for special attention to be paid to sample handling, as it can introduce significant artifacts into NOx assays.

Berberine (BBR) ameliorates cellular oxidative stress, apoptosis and autophagy induced by lipid metabolism disorder, however, the molecular mechanism associated with it is not well known. To study the mechanism, we started with m6A methylation modification to investigate its role in lipid deposition zebrafish hepatocytes (ZFL). The results showed that BBR could change the cellular m6A RNA methylation level, increase m6A levels of Camk1db gene transcript and alter Camk1db gene mRNA expression. Via knockdown of the Camk1db gene, Camk1db could promote cellular ERK phosphorylation levels. Berberine regulated the expression level of Camk1db mRNA by altering the M6A RNA methylation of the Camk1db gene, which further affected the synthesis of calmodulin-dependent protein kinase and activated ERK signaling pathway resulting in changes in downstream physiological indicators including ROS production, cell proliferation, apoptosis and autophagy. In conclusion, berberine could regulate cellular oxidative stress, apoptosis and autophagy by mediating Camk1db m6A methylation through the targeting of the Camk1db/ERK pathway in zebrafish-hepatocyte.

The mammalian fetus thrives at oxygen tensions much lower than those of adults. Gestation at high altitude superimposes hypoxic stresses on the fetus resulting in increased erythropoiesis. We hypothesized that chronic hypoxia at high altitude alters the homeostasis of iron and bioactive nitric oxide metabolites (NOx) in gestation. To test for this, electron paramagnetic resonance was used to provide unique measurements of iron, metalloproteins, and free radicals in the blood and aorta of fetal and maternal sheep from either high or low altitudes (3801 or 300 m). Using ozone-based chemiluminescence with selectivity for various NOx species, we determined the NOx levels in these samples immediately after collection. These experiments demonstrated a systemic redistribution of iron in high altitude fetuses as manifested by a decrease in both chelatable and total iron in the aorta and an increase in non-transferrin bound iron and total iron in plasma. Likewise, high altitude altered the redox status diversely in fetal blood and aorta. This study also found significant increases in blood and aortic tissue NOx in fetuses and mothers at high altitude. In addition, gradients in NOx concentrations observed between fetus and mother, umbilical artery and vein, and plasma and RBCs demonstrated complex dynamic homeostasis of NOx among these circulatory compartments, such as placental generation and efflux as well as fetal consumption of iron-nitrosyls in RBCs, probably HbNO. In conclusion, these results may suggest the utilization of iron from non-hematopoietic tissues iron for erythropoiesis in the fetus and increased NO bioavailability in response to chronic hypoxic stress at high altitude during gestation.

Background: Gliomas are the malignance of a poor prognosis. The current WHO classification remains unable to predict survival outcomes accurately. Novel surrogates are highly required for improved stratification of patients and hence, allowing to delivery of the most appropriate treatment. Methods: Transcriptional profiles of 301 glioma cases on the platform of Chinese Glioma Genome Atlas (CGGA) were retrospectively studied. Results: Extracellular matrix (ECM) scores were established by integrating a panel of most featured gene-signatures, correlating well with pathological tumor stages. Linear regression analysis revealed that the ECM score corroborated with the infiltration status of monocytes, M0 and M1 macrophages. Furthermore, the WHO stage II-IV dependent abundance of those 3 immune cells was determined. Univariate and multivariate analysis of clinicopathological characteristics in the GBM cohort identified M1 enrichment score as an independent risk factor. A high abundance of M1 macrophages was associated with poor survival outcomes and radiotherapy response in IDH-wildtype GBM. Conclusions: Our study demonstrated that M1 macrophages correlated with WHO grades and predicted robustly for the survival performance for GBM patients. Increased infiltration of M1 macrophages was associated with a poor radiation response for IDH-wildtype GBM. Together, it will facilitate more precise stratifications of glioma patients based on molecular and immunological surrogates.

DNA is subject to a range of endogenous and exogenous insults that can impair DNA replication and lead to DNA double-strand breaks (DSBs). The repair capacity of cancer cells mediates their radiosensitivity, but the roles of miR-1587 during radiation resistance are poorly characterized. In this study, we explored whether miR-1587 regulates the growth and radiosensitivity of colorectal cancer (CRC) cells through its ability to regulate DNA Ligase4 (LIG4). We found that CRC cells in which miR-1587 was overexpressed inhibited cell growth and promoted apoptosis through increasing DSBs and promoting cell cycle arrest. We found that overexpression of miR-1587 significantly inhibited LIG4 messenger RNA and protein expression and further revealed the ability of miR-1587 to directly bind to the LIG4-3′-untranslated region through dual-luciferase reporter assays. More notably, miR-1587 mimics increased the radiosensitivity of CRC cells. Taken together, we show that miR-1587 overexpression enhances the formation of DSBs, arrests CRC cell growth, and enhances the radiosensivity of CRC cells through the direct repression of LIG4 expression. These results reveal novel roles for miR-1587 during DNA damage repair and the radiosensivity of CRC cells. This highlights miR-1587 as a novel therapeutic target for CRC.

Background: MiR-664 has been demonstrated to play an important role in dermal diseases. However, the functions of miR-664 in ultraviolet B (UVB) radiation-induced keratinocytes damage remain to be elucidated. Objective: The present study aimed to investigate the molecular mechanisms under the UVB-induced keratinocytes damage and provide translational insights for future therapeutics and UVB protection. Methods: HaCaT cells were transfected with miR-664, either alone or combined with UVB irradiation. Levels of messenger RNA and protein were tested by quantitative real-time polymerase chain reaction and Western blot analyses. Cell proliferation, percentage of apoptotic cells, and expression levels of apoptosis-related factors were measured by Cell Counting Kit-8 assay, flow cytometry assay, and Western blot analysis, respectively. Results: We found that a significant increase in miR-664 was observed in UVB-induced HaCaT cells. Overexpressed miR-664 promoted cell vitalities and suppressed apoptosis of UVB-induced HaCaT cells. Additionally, the loss/gain of armadillo-repeat-containing protein 8 (ARMC8) rescued/blocked the effects of miR-664 on the proliferation of UVB-induced HaCaT cells. Conclusions: Our data demonstrate that miR-664 functions as a protective regulator in UVB-induced HaCaT cells via regulating ARMC8.

Introduction: Acute exposure to ionizing radiation (IR) is hazardous or even lethal. Accurate estimation of the doses of IR exposure is critical to wisely determining the following treatments. Exosomes are nanoscale vesicles harboring biomolecules and mediate the communications among cells and tissues to influence biological processes. Screening out the microRNAs (miRNAs) contained in exosomes as biomarkers can be useful for estimating the IR exposure doses and exploring the correlation between these miRNAs and the occurrence of disease. Methods: We treated mice with 2.0, 6.5, and 8.0 Gy doses of IR and collected the mice sera at 0, 24, 48, and 72 hours after exposure. Then, the serum exosomes were isolated by ultracentrifuge and the small RNA portion was extracted for sequencing and the following bioinformatics analysis. Qualitative polymerase chain reaction was performed to validate the potential dose-specific markers. Results: Fifty-six miRNAs (31 upregulated, 25 downregulated) were differentially expressed after exposure of the above 3 IR doses and may act as common IR exposure miRNA markers. Bioinformatic analysis also identified several dosage-specific responsive miRNAs. Importantly, IR-induced miR-151-3p and miR-128-3p were significantly and stably increased at 24 hours in different mouse strains with distinct genetic background after exposed to 8.0 Gy of IR. Conclusion: Our study shows that miR-151-3p and miR-128-3p can be used as dose-specific biomarkers of 8.0 Gy IR exposure, which can be used to determine the exposure dose by detecting the amount of the 2 miRNAs in serum exosomes.

Radiation biodosimeters are required urgently for fast and accurate evaluation of absorbed dose for irradiated individuals. Lipidomics has appeared as a credible technique for identification and quantification of lipid for researching biomarker of diseases. We performed a lipidomic profile on mice serum at time points of 6, 24, and 72 hours after 0, 2, 5.5, 7, and 8 Gy irradiation to select radiation-responsive lipids and conducted Kyoto Encyclopedia of Genes and Genome pathway enrichment analysis to recognize the pathways and network changes. Then, Pearson correlation analysis was performed to evaluate the feasibility of radiation-responsive lipids to estimate radiation dose. Seven radiation-responsive lipids including PC (18:2/18:2), PC (18:0/18:2), Lyso PC 18:1, PC (18:0/20:4), SM (D18:0/24:1), PC (16:0/18:1), and Lyso PC 18:2 were identified in which glycerophospholipid metabolism presented as the most significant pathway, and they all presented good linear correlation with the irradiated dose. This study identified 7 radiation-responsive lipids in mice serum and certificate their feasibility of dose estimation as biodosimeters.

In response to large-scale radiological incidents, rapid, accurate, and early triage biodosimeters are urgently required. In this study, we investigated candidate radiation-responsive biomarkers using proteomics approaches in mouse models. A total of 452 dysregulated proteins were identified in the serum samples of mice exposed to 0, 2, 5.5, 7, and 8 Gy at 6, 24, and 72 hours postirradiation. Ninety-eight proteins, including 46 at 6 hours, 36 at 24 hours, and 36 at 72 hours, were identified as radiation-responsive proteins (RRPs). Gene Ontology analysis showed the RRPs were involved in proteolysis, extracellular space, hydrolase activity, and carbohydrate binding. Kyoto Encyclopedia of Genes and Genome enrichment showed the RRPs were regulated in “the pentose phosphate pathway,” “the proteasome,” and “AGE-RAGE signaling in diabetic complications.” There were 3 proteins changed and overlapped at all the 3 time points, 8 proteins changed at 6 and 24 hours, 4 proteins changed at 24 and 72hours, and 2 proteins changed at both 6 and 72 hours. Of these proteins, ORM2, HP, SAA1, SAA2, MBL2, COL1A1, and APCS were identified as candidate biomarkers for biodosimeter-based diagnosis through Pearson correlation analysis.

Successful chemotherapy with paclitaxel (PTX) is impeded by multidrug resistance (MDR) in tumor cells. In this study, lipid-albumin nanoassemblies co-loaded with borneol and paclitaxel (BOR/PTX LANs) were prepared to circumvent MDR in C6 glioma cells. The physiochemical properties including particle size, encapsulation efficiency and morphology were evaluated in vitro. Quantitative and qualitative investigations of cellular uptake were carried out in C6 glioma cells. The cytotoxicity of the BOR/PTX LANs was determined by MTT assay. After that, the tumor targeting was also evaluated in C6 glioma bearing mice by in vivo imaging analysis. BOR/PTX LANs have a higher entrapment efficiency (90.4 ± 1.2%), small particle size (107.5 ± 3.2 nm), narrow distribution (P.I. = 0.171 ± 0.02). The cellular uptake of PTX was significantly increased by BOR/PTX LANs compared with paclitaxel loaded lipid-albumin nanoassemblies (PTX LANs) in quantitative research. The result was further confirmed by confocal laser scanning microscopy qualitatively. The cellular uptake was energy-, time- and concentration-dependent, and clathrin- and endosome/lysosome-associated pathways were involved. The BOR/PTX LANs displayed a higher cytotoxicity agaist C6 glioma cells in comparion with PTX LANs and Taxol. Moreover, the encapsulation of BOR in LANs obviously increased the accumulation of the drug in tumor tissues, demonstrating the tumor targeted ability of BOR/PTX LANs. These results indicated that BOR/PTX LANs could overcome MDR by combination of drug delivery systems and P-gp inhibition, and shown the potential for treatment of gliomas.