Cell Biology and Toxicology最新文献

Objective: Tubal factor infertility (TFI), a major cause of female infertility, lacks effective therapies and is closely associated with macrophage-mediated inflammation. Although DDX3X regulates macrophage polarization, its specific contribution to TFI pathogenesis remains unclear, and the potential involvement of E3 ubiquitin ligase-mediated regulation of DDX3X protein stability in this condition has not been reported. Therefore, our study planned to explore the regulation of DDX3X by the E3 ubiquitin ligase TRIM36 and how this axis influences macrophage polarization and TFI pathogenesis.

Methods: The GSE262037 dataset and the STRING platform were analyzed through bioinformatics approaches to identify TRIM36, an E3 ubiquitin ligase of DDX3X. A mixed bacterial inoculation method was employed to establish a TFI model of rats, and the animals were treated with TRIM36 overexpression (oe-TRIM36). Stimulation of LPS in the fallopian tube epithelial cells was applied to establish the in vitro TFI model, which was treated by the conditioned medium (CM) from rat bone marrow-derived macrophages (BMDM) with LPS, silence (si)/oe-DDX3X, and/or oe-TRIM36 treatment. Co-Immunoprecipitation (Co-IP) detection was employed to analyze the regulation of si/oe-TRIM36 on the ubiquitination of DDX3X protein.

Results: DDX3X expression was significantly upregulated in TFI rats and showed a positive correlation with M1 macrophage polarization. Silencing DDX3X in rat BMDM promoted M2 polarization while suppressing M1 polarization, and the CM derived from these macrophages alleviated LPS-induced damage in fallopian tube epithelial cells. Bioinformatics and Co-IP identified TRIM36 as an E3 ubiquitin ligase binding DDX3X, and TRIM36 overexpression promoted the K48-linked polyubiquitination and the proteasomal degradation of DDX3X. Similarly, TRIM36-mediated DDX3X downregulation shifted macrophage polarization towards the M2 phenotype in vitro and protected fallopian tube epithelial cells against LPS-induced damage. Importantly, in vivo oe-TRIM36 therapy downregulated DDX3X, increased M2 macrophages, reduced tubal inflammation, and significantly alleviated infertility phenotypes in TFI model rats.

Conclusion: This study identifies TRIM36 as a novel E3 ligase that targets DDX3X for proteasomal degradation, thereby driving macrophage M2 polarization and ameliorating TFI. The TRIM36/DDX3X axis may provide a promising therapeutic target for TFI treatment.

Humans are continuously exposed to a wide array of exogenous chemicals via dietary intake, environmental sources, and the use of personal care products. This includes per- and polyfluoroalkyl substances (PFAS), a class of highly persistent compounds that have been associated with developmental effects in humans. This study assessed the effects of four legacy PFAS, namely PFOS, PFOA, PFNA and PFHxS, and mixtures thereof in the PluriLum assay, a 3D human induced pluripotent stem cell (hiPSC)-based model for embryotoxicity testing. We established the individual embryotoxic potencies of PFAS, with PFNA exhibiting the highest potency, followed by PFOS, PFOA and PFHxS. The four PFAS were evaluated in three reconstituted mixtures, prepared either to reflect identical potencies ("equipotent mixture") or the average serum concentrations reported for the European adult or child population ("real-life mixtures"). Comparing observed versus predicted mixture responses demonstrated concentration additivity throughout the entire range of tested concentrations. Studies on uptake in 3D embryoid bodies revealed the highest bioaccumulation of PFOS, followed by PFNA, PFOA, and PFHxS. Moreover, less than 2% of the nominally added PFAS could be recovered in the embryoid bodies. RNA sequencing showed that relatively few genes were affected by PFOS, PFNA and PFOA, however expression of genes related to focal adhesion and functional pathways associated with cardiac, cardiomyocyte and muscle tissue development was significantly changed. Notably, PFOS affected the greatest number of embryonic development pathways. In conclusion, the four tested PFAS significantly impaired cardiomyocyte differentiation, indicating embryotoxicity. The combined responses were consistent with the concentration addition principle, supported by shared functional pathways and indicative of common sites of molecular action.

Metabolic dysfunction-associated steatohepatitis (MASH) drives hepatic stellate cell (HSC) activation and extracellular matrix deposition, leading to liver fibrosis, for which effective treatments remain lacking. Here, we report that 8-hydroxyoctacosatrienoic acid (8-HETrE), an arachidonic acid metabolite generated through cytochrome P450 or lipoxygenase pathways, significantly ameliorates MASH-related fibrosis by targeting sphingosine kinase 1 (SPHK1) and restoring mitochondrial function. Clinical observations revealed markedly reduced circulating 8-HETrE levels in patients with MASH fibrosis. In vivo studies demonstrated that 8-HETrE administration improved liver function, enhanced expression of mitochondrial fusion proteins (Mfn1, Mfn2, Opa1), and attenuated fibrosis in Gubra-Amylin-NASH (GAN)-diet-induced MASH models. In TGF-β1-activated human HSCs cell line (LX-2 cells), 8-HETrE treatment suppressed fibrotic markers (α-SMA, COL1A1) and improved mitochondrial dynamics. Mechanistic investigations revealed that 8-HETrE exerted its anti-fibrotic effects primarily through SPHK1 inhibition: SPHK1 knockdown moderately reduced HSC activation, decreased sphingosine-1-phosphate (S1P), lactate, and nitrite levels, enhanced glucose uptake, and promoted mitochondrial fusion, while completely abolishing 8-HETrE's therapeutic effects. Conversely, SPHK1 overexpression exacerbated fibrotic and metabolic abnormalities, which were effectively reversed by 8-HETrE treatment. Critically, HSC-specific Sphk1 knockout independently improved MASH fibrosis, mitochondrial function, and metabolic parameters, while completely blocking 8-HETrE's benefits. Our findings identify 8-HETrE as a novel mediator that targets the SPHK1-mitochondrial dynamics axis in HSCs, providing both mechanistic insights and therapeutic potential for MASH-related fibrosis treatment.

Nanoplastics (NPs, < 1 µm) are emerging environmental contaminants capable of crossing biological barriers and interacting at the cellular and subcellular level. Despite evidence of microplastics in human kidney tissue and urine, the renal effects of NPs remain poorly understood. This study investigated the short-term effects of NPs polymer type, size, and concentration on human kidney proximal tubule cells (HK-2). Cells were exposed for 24-h to carboxylated polystyrene (PS), poly(methyl methacrylate) (PMMA), and polyethylene (PE) NPs (15-100 nm) at concentrations from 0.1 to 200 µg/mL. NPs morphology, size, and charge were characterised by scanning electron microscopy, dynamic light scattering, and zeta potential. Cell morphology, viability, cell cycle distribution, and NPs internalisation were assessed by microscopy and flow cytometry. Low-concentration exposures had minimal effects, whereas 100 and 200 µg/mL induced marked morphological changes, including cytoplasmic granularity. Viability decreased significantly at 200 µg/mL for several NPs types, with PE NPs causing the largest reduction (79.4%). Polymer type influenced outcomes, with PE and PMMA NPs causing greater morphological disruption than PS. Size effects were most evident in cell cycle analysis: 15 nm and 20 nm PS NPs and 100 nm PMMA NPs induced phase arrest without major viability loss. NPs internalisation increased with concentration but varied with polymer type, with PE NPs showing preferential perinuclear localisation. These findings demonstrate that NPs effects on kidney cells depend on polymer chemistry, particle size, concentration, and highlight the need for long-term studies using environmentally relevant NPs to better assess kidney toxicity risk.

TsRNAs (tRNA-derived small RNAs) also known as tRNA-derived small RNAs, are a relatively new type of non-coding RNAs that have demonstrated promising effect in treating various liver diseases. However, the function of small extracellular vesicles (sEVs) secreted by human bone marrow mesenchymal stem cells (BMSCs) in safeguarding against metabolic-associated fatty liver disease (MAFLD) is still uncertain. In this research, we explored the effects of BMSCs sEVs on lipid metabolism using Palmitic Acid (PA)-induced HepG2 cells, both in the presence and absence of the sEVs inhibitor GW4869. Pandora sequencing and RNA sequencing were utilized to identify differentially expressed genes in sEVs and hepatocytes in vitro. Furthermore, we carried out in vivo studies involving male C57BL/6J mice that fed with high-fat diet (HFD) and either treated with an AAV 5'-tRF-GlyCCC mimic or not, through tail vein injection. Our findings revealed that BMSC-sEVs can relieve lipid accumulation in PA-caused HepG2 cells by inhibiting the formation of de novo fatty acid. We found that 5'-tRF-GlyCCC forms a direct connection with the 3' UTR of FoxO3, thereby decreasing the level of gluconeogenic genes PEPCK and G6Pase. Tail vein administration of the 5'-tRF-GlyCCC AAV alleviated liver gluconeogenesis and lipid metabolism issues in MAFLD mice by enhancing hepatic insulin sensitivity. The results imply that the 5'-tRF-GlyCCC/FoxO3 gluconeogenesis-signaling pathway could be crucial in the therapeutic benefits of BMSC sEVs on MAFLD.

Age-related decreases in follicle numbers and oocyte quality are major contributors to the decline in female fertility, which is associated with increased infertility rates. Emerging evidence suggests that targeting granulosa cell senescence could delay ovarian aging and depletion of the ovarian reserve, highlighting the potential for therapeutic interventions focused on granulosa cells. Advanced glycation end products (AGEs) accumulate with age and result in oxidative stress in the follicular microenvironment, but their direct impact on human granulosa cell (hGC) senescence and the fundamental processes are still mostly unknown. In this study, we found that AGEs treatment significantly exacerbated hGC senescence, impaired mitochondrial function, and suppressed mitophagy in a concentration-dependent manner. Importantly, these deficits were lessened by urolithin A-induced mitophagy activation, whereas Cyclosporine A-induced mitophagy inhibition had the reverse consequences. In addition, silencing Sirtuin 3 (SIRT3) or PINK1 further aggravated these adverse effects, while SIRT3 overexpression attenuated senescence and restored mitochondrial function by enhancing mitophagy. Furthermore, SIRT3 overexpression promoted the synthesis of estradiol-17β and progesterone, key hormones for ovarian function. Our findings demonstrated that AGEs induced hGC senescence by disrupting mitochondrial function and inhibiting mitophagy, with SIRT3 playing a protective role. Enhancing mitophagy by targeting SIRT3 may be a promising treatment approach to counteract age-related declines in female fertility.

Diabetic patients face delayed wound healing due to angiogenesis dysfunction. This study aims to investigate the function of endothelial progenitor cell (EPC)-derived extracellular vesicles (EVs) in wound healing of diabetic mice, providing a theoretical basis for treating difficult-to-heal diabetic wounds. The full-thickness skin wound model was used as an animal model. After treatment with EPC-EVs, wound healing and histopathological structures were evaluated. Peripheral blood was collected to analyze circulating EPCs. In cell models, EV endocytosis, cell viability, angiogenic capacity, and cell migration were detected. miR-204-5p, lncRNA SNHG1, EIF4A3, and HDAC6 were detected. EVs derived from EPCs with miR-204-5p overexpression were extracted to investigate their effects on wound healing. The bindings between miR-204-5p and SNHG1, SNHG1 and EIF4A3, and EIF4A3 and HDAC6 mRNA were validated. EPC-EVs promoted wound healing in diabetic mice. EPC-EVs enhanced angiogenesis and migration in cell models. EPC-EVs with miR-204-5p overexpression exhibited better therapeutic effects. EPC-EVs delivered miR-204-5p into tissues/cells to lower SNHG1 expression. SNHG1 bound to EIF4A3 to increase HDAC6 expression. SNHG1/HDAC6 overexpression partly reversed the pro-angiogenic effects of EPC-EVs on diabetic wound healing and HG-impaired endothelial cells. In conclusion, EPC-EVs enhance EPC mobilization and angiogenesis to accelerate wound repair in diabetic mice via the miR-204-5p/SNHG1/HDAC6 axis.

CSF3 exerts a significant function in the progression of colorectal cancer (CRC). N6-methyladenosine (m6A) modification is now considered the main driving factor of RNA influence for maintaining homeostasis in cancer cells. Nevertheless, how m6A mediates the role of CSF3 and its influence in pathogenesis of CRC is still elusive. After neutrophil isolation from bone marrow, the purity and survival rate of neutrophils were assessed. Azoxymethane (AOM)/Dextran Sodium Sulfate (DSS) was employed to construct the CRC mice model. Both loss-of-function and gain-of-function experiments were conducted to explore the influence of CSF3 on NETosis and tumorigenesis of CRC in vitro and in vivo. The purity and survival rate of neutrophils were 88.07% and 94.84%, respectively. Overexpression of CSF3 (oe-CSF3) markedly enhanced NETosis, while CSF3 knockdown (sh-CSF3) suppressed it. Intriguingly, CSF3 expression positively correlated with relaxin-2 (RLN2) levels in CRC cells, and RLN2 supplementation rescued tumorigenesis and NETosis after sh-CSF3 treatment. Mechanistically, fat mass and obesity-associated protein (FTO)-mediated m6A demethylation of CSF3 mRNA suppressed CRC tumorigenesis in vivo. CSF3 upregulation counteracted the tumor-suppressive effects of FTO overexpression, restoring NETosis and tumor growth. Consistent with this, FTO overexpression in CRC mice alleviated disease severity, as evidenced by improved body weight, reduced tumor burden, and diminished NETosis. Collectively, our findings establish a novel regulatory axis in which FTO-dependent m6A demethylation of CSF3 suppresses NETosis by inhibiting RLN2 expression, offering new insights into therapeutic targeting of the m6A-CSF3-RLN2 pathway in CRC.

Background: Programmed cell death (PCD) patterns play important roles in lung adenocarcinoma (LUAD) development as well as treatment resistance, and in-depth study of PCD is beneficial for improving the therapeutic paradigm for LUAD.

Methods: Fourteen PCD-related patterns were integrated and multiple datasets from TCGA and GEO were collected to develop a PCD signature using 101 machine learning algorithm combinations. Prognosis, immune cell infiltration, and sensitivity to chemotherapy and immunotherapy were compared between different risk groups and validated by multiple bulk RNA-seq and scRNA-seq datasets of patients receiving immunotherapy. CellChat was used to analyze the cellular interactions between patients with different PCD groups. Immune cell infiltration in the tumor tissues of 38 LUAD patients treated with anti-PD-1 therapy was validated by multiplex immunohistochemistry (mIHC).

Results: A PCD signature containing 7 genes was constructed using 101 machine learning algorithm combinations and validated across multiple datasets. High PCD scores in patients are associated with poorer prognosis, lower immune cell infiltration, and reduced responsiveness to immunotherapy. In addition, the PCD signature were comprehensively analyzed by scRNA-seq, and the results showed that the high PCD signature was concentrated mainly in advanced LUAD. Moreover, pathways associated with tumor progression and immune resistance were more strongly promoted in the high PCD signature group. The expression of the key gene NAPSA correlated with immune cell infiltration and immunotherapy response, as confirmed by IHC and mIHC.

Conclusion: The PCD signature confers significant potential to predict prognosis of LUAD in patients, and NAPSA is promising as a new marker for predicting the efficacy of immunotherapy.

Although the effects of noise-induced hearing loss (NIHL) on cognitive functions have been widely investigated, the cognitive effects of noise-induced hidden hearing loss (NIHHL), particularly its impact on memory, remain poorly understood. The Dnah11 gene, which encodes a dynein motor protein involved in synaptic development, may play a role in NIHHL-related cognitive impairment. We aimed to investigate whether NIHHL induces memory impairment and explore the role of Dnah11 expression in this process. Behavioral experiments identified the peak of memory impairment at 1 month following noise exposure. To elucidate molecular changes, hippocampal gene expression was analyzed using transcriptomic sequencing, reverse transcription quantitative polymerase chain reaction (RT-qPCR), and immunofluorescence. RNA sequencing revealed significant Dnah11 upregulation, with immunofluorescence confirming DNAH11 overexpression in hyperactivated CaMKIIα-positive excitatory neurons. Stereotaxic injection of recombinant adeno-associated virus (rAAV) vectors to knock down hippocampal Dnah11 expression improved memory performance in NIHHL mice without improving hearing loss. This cognitive improvement was accompanied by partial restoration of synaptic plasticity-related proteins, including SYN and PSD95. These findings indicate that Dnah11 upregulation in hippocampal excitatory neurons contributes to NIHHL-induced memory impairment, and targeting Dnah11 may offer a therapeutic strategy for memory impairment associated with hidden hearing loss.