CCK-8细胞增殖及毒性检测试剂盒

Oxidative stress is a major factor leading to inflammation and disease occurrence, and superoxide dismutase (SOD) is a crucial antioxidative metalloenzyme capable of alleviating oxidative stress. In this study, a novel thermostable SOD gene is obtained from the Hydrogenobacter thermophilus strain (HtSOD), transformed and efficiently expressed in Escherichia coli with an activity of 3438 U mg −1 , exhibiting excellent thermal stability suitable for scalable production. However, the activity of HtSOD is reduced to less than 10% under the acidic environment. To address the acid resistance and gastrointestinal stability issues, a biomimetic mineralization approach is employed to encapsulate HtSOD within the ZIF-8 (HtSOD@ZIF-8). Gastrointestinal simulation results show that HtSOD@ZIF-8 maintained 70% activity in simulated gastric fluid for 2 h, subsequently recovering to 97% activity in simulated intestinal fluid. Cell and in vivo experiments indicated that HtSOD@ZIF-8 exhibited no cytotoxicity and do not impair growth performance. Furthermore, HtSOD@ZIF-8 increased the relative abundance of beneficial microbiota such as Dubosiella and Alistipes , mitigated oxonic stress and intestinal injury by reducing mitochondrial and total reactive oxygen species (ROS) levels in diquat-induced. Together, HtSOD@ZIF-8 maintains and elucidates activity in the intestine and biocompatibility, providing insights into alleviating oxidative stress in hosts and paving the way for scalable production.

Transfer RNA-derived fragments (tRFs) are a recently discovered class of short noncoding RNAs widely distributed in various tissues and cell types. They are involved in the regulation of gene expression and play important roles in both physiological and pathological processes, garnering growing attention. However, the functions and underlying mechanisms of most tRFs in tumorigenesis and progression remain largely unclear. Through small RNA sequencing of nasopharyngeal carcinoma (NPC) and adjacent tissues, we found that among the top 30 highly expressed tRFs in NPC tissues, 13 were derived from lysine tRNAs, forming the 5′-tRF-Lys cluster. This cluster was found to promote NPC cell proliferation, invasion, and migration. Mechanistically, 5′-tRF-Lys binds to the 3′-untranslated region (3′-UTR) of YPEL3 messenger RNA (mRNA), suppressing its expression and thereby activating the Hippo/YAP signaling pathway to drive tumor progression. The elevated expression of pseudouridine synthases PUS1 and PUS7 in NPC tissues catalyzes pseudouridine modification of tRNA-Lys, facilitating its cleavage into 5′-tRF-Lys and accounting for its upregulation. Notably, the PUS1 -targeting small-molecule inhibitor mogroside IV-e effectively reversed malignant phenotypes in both in vitro and in vivo NPC models. This study uncovers a novel mechanism in which pseudouridine synthases PUS1 and PUS7 drive the biogenesis of the tRF-Lys cluster, promoting NPC malignancy by suppressing YPEL3 and activating the Hippo/YAP signaling pathway. These findings highlight the therapeutic potential of targeting pseudouridine synthases to reduce tRF-Lys production as a novel strategy for NPC treatment.

As an alternative plasticizer to conventional phthalates, di(2-ethylhexyl) terephthalate (DEHTP) has attracted considerable concerns, given its widespread detection in the environment and humans. However, the potential toxicity, especially liver toxicity, posed by DEHTP remains unclear. In this study, based on the 2017–2018 National Health and Nutrition Examination Survey, two metabolites of DEHTP, i.e., mono(2-ethyl-5-hydroxyhexyl) terephthalate (MEHHTP) and mono(2-ethyl-5-carboxypentyl) terephthalate (MECPTP), were found to be present in the urine samples of nearly all representative U.S. adults. Moreover, a positive linear correlation was observed between the concentrations of the two metabolites and the risk of nonalcoholic fatty liver disease (NAFLD) in the population. Results of weighted quantile sum and Bayesian kernel machine regression indicated that MEHHTP contributed a greater weight to the risk of NAFLD in comparison with 12 conventional phthalate metabolites. In vitro experiments with hepatocyte HepG2 revealed that MEHHTP exposure could increase lipogenic gene programs, thereby promoting a dose-dependent hepatic lipid accumulation. Activation of liver X receptor α may be an important regulator of MEHHTP-induced hepatic lipid disorders. These findings provide new insights into the liver lipid metabolism toxicity potential of DEHTP exposure in the population.

Background Despite many new drugs, multiple myeloma (MM) remains an incurable plasma cell malignancy, and drug resistance is a long-standing topic in this field. Characterized by efficient transcription without being limited by the double helix structure and promoter, extrachromosomal circular DNA (EccDNA) has been proven to be widely involved in cancer development and drug resistance. Methods We performed circle-seq and mRNA-seq on samples from three MM patients at the time of complete response and relapse to screen EccDNA candidate molecules. Outward PCR and Sanger sequencing were used to identify EccDNA molecules. RT‒qPCR and WB were performed to detect gene expression levels. Fluorescence in situ hybridization (FISH) was carried out to detect the deletion of chromosome 17p (del (17p)). Transmission electron microscopy (TEM) was conducted to observe autophagosomes. Luciferase reporter assays were performed to validate the binding of microRNAs to target genes. Cell viability assays and apoptosis assays were employed to assess drug resistance. Xenograft tumor mouse models were established for in vivo experiments. Immunohistochemistry (IHC) was used to detect protein expression levels. Results We successfully identified an EccDNA molecule (EccDNA chr17:38719676–38719812 ) in one relapsed MM patient with del(17p) and named it MIR4726 EccDNA . We demonstrated that the overexpression of MIR4726 EccDNA in MM cells can increase bortezomib resistance. We further confirmed that the precursor miRNA carried by MIR4726 EccDNA can be efficiently transcribed in MM cells and that MIR4726 EccDNA drives bortezomib resistance via the MIR4726-5p/NXF1/NKIRAS2 axis. We further revealed that downregulation of NFKB inhibitor interacting Ras like 2 (NKIRAS2) activated the NF-κB pathway and increased autophagy. Moreover, we established a xenograft model of human MM via subcutaneous inoculation. We administered intra-tumoral injection of AgoMIR4726-5p and intraperitoneal injection of bortezomib and found that AgoMIR4726-5p promoted tumor progression and partially drove bortezomib resistance. Conclusions In summary, our findings indicate that artificially synthesized MIR4726 EccDNA is functional in cells and that MIR4726 EccDNA enhances tumor progression and partially mediates drug resistance by enhancing MIR4726-5p/NXF1/NKIRAS2 axis dependent autophagy.

The integrity of blood-brain barrier (BBB) plays a pivotal role in the pathogenesis of Alzheimer’s disease (AD) by regulating Aβ clearance and neurotoxic compound exclusion. Hyperlipidemia exacerbates AD by impairing the BBB function. Inclisiran, a PCSK9-targeting siRNA, reduces cholesterol levels; however, its neuroprotective effects remain unclear. Here, we report the novel discovery that Inclisiran attenuates AD-like changes through the PCSK9-ferroptosis axis in brain microvascular endothelial cells (BMECs). First, integrated bioinformatics analysis and experimental validation of cortical tissues from patients with AD and healthy controls revealed a coordinated upregulation of PCSK9 and β-amyloid (Aβ), accompanied by increased iron deposition and significant activation of the ferroptosis pathway. Interestingly, these changes are located in the BMECs of the blood-brain barrier rather than in the brain parenchyma. Second, in hyperlipidemic ApoE-/- mouse models, integrated application of cerebral microvessel isolation, molecular biology techniques, immunofluorescence co-localization analysis, and behavioral tests demonstrated that Inclisiran significantly reduced AD-like changes by attenuating BBB dysfunction based on the suppression of PCSK9-mediated ferroptosis in BMECs. Third, in vitro studies employing the HCMEC/D3 BBB model with integrated assessments of lipid peroxidation, mitochondrial function, and transwell-based barrier integrity demonstrated that Inclisiran significantly reduced ferroptosis and restored BBB integrity via PCSK9 suppression. Our findings not only establish a novel PCSK9-ferroptosis-BBB regulatory axis in AD pathogenesis but also posit the clinically approved lipid-lowering drug, Inclisiran, as a promising therapeutic candidate for AD, providing new targets and mechanisms for the prevention and treatment of AD.

Background and purpose Research has revealed the involvement of mitochondrial autophagy and iron death in the pathogenesis of myocardial fibrosis. The objective of this study is to investigate whether the mitochondrial-targeted H 2 S donor AP39 inhibits mitochondrial autophagy and antagonizes myocardial cell iron death through the PINK1/Parkin pathway, thereby improving myocardial fibrosis in rats with myocardial infarction. Experimental approach A rat model of myocardial infarction was created by intraperitoneal injection of a high dose of isoproterenol, and H9c2 myocardial cells were subjected to hypoxic injury induced by CoCl 2 . Western blot, RT-PCR, transmission electron microscopy, immunohistochemistry, as well as echocardiography, and studies on isolated hearts were employed. Key results In the hearts of rats with myocardial infarction, there was a significant accumulation of interstitial collagen fibers, accompanied by downregulation of CSE protein expression, activation of the PINK1/Parkin signaling pathway, and activation of mitochondrial autophagy. Intervention with AP39 resulted in a significant improvement of the aforementioned changes, which could be reversed by the addition of PAG. Similar results were observed in vitro experiments. Furthermore, the addition of CCCP reversed the antagonistic effect of AP39 on myocardial cell iron death, while the addition of RSL3 reversed the inhibitory effect of AP39 on collagen production in myocardial cells. Conclusion and implications The mitochondrial-targeted H 2 S donor AP39 can inhibit mitochondrial autophagy through the PINK1/Parkin pathway, antagonize myocardial cell iron death, and improve myocardial fibrosis in rats with myocardial infarction.

Background Neuroblastoma (NB) is the most common solid tumor in children, characterized by high recurrence rates, drug resistance, and significant mortality. Methods In this study, we analyzed the proteomic profiles of NB tissue samples alongside other pathological categories, including ganglioneuroma (GN) and ganglioneuroblastoma (GNB). Using weighted gene co-expression network analysis (WGCNA), the core prognostic gene models associated with histopathology of NB were identified. Furthermore, by mapping our core prognostic gene models onto drug-perturbed transcriptome profiles from the L1000FWD and CMap databases, repurposing drug candidates were screened and validated for NB. Results Our proteomic analysis reveals that pathways associated with the cell cycle and DNA replication are significantly upregulated in NB, while oxidative phosphorylation, pyruvate metabolism, and the TCA cycle are notably downregulated compared to GNB and GN. By applying WGCNA, we identified a core prognostic gene model strongly associated with the unfavorable subtype and high MKI of NB and primarily related to chromatin binding and mRNA metabolic process. Protein–protein interaction network analysis identified 15 hub genes in this core prognostic module: SMARCA4, SMARCA5, SMARCC2, SMARCC1, PBRM1, BRD3, ARID1A, BRD2, ARID1B, KDM1A, TP53BP1, ALYREF, CBX1, SF3B1, and ADNP, which mainly related to chromatin remodeling. Notably, SMARCA4 and ALYREF are also high-risk genes of mortality and validated as potential prognostic biomarkers for NB. Through repurposing drugs screening, mocetinostat and clofarabine were validated as effective treatments in two NB cell lines. Conclusion Mocetinostat and clofarabine offer valuable insights for the development of novel targeted therapies in neuroblastoma.

Bacterial infections associated with metal implants and devices are an urgent challenge in biomedical materials, especially when bacterial biofilms form on the implant surface. Here, we developed a novel strategy to combat infections with bacteria-associated titanium implants. Curcumin molecular modification was carried out on the phenolic hydroxyl group to obtain curcumin carboxylic acid (Cur-COOH) with the β-diketone structure of curcumin retained. Cur-COOH was covalently immobilized on the (3-aminopropyl) triethoxysilane (APTES)-treated titanium surface via an amidation reaction to establish the curcumin-titanium implant interface (Ti- g -Cur). Scanning electron microscopy (SEM), energy dispersive spectrometry (EDS), water contact angle (WCA), and X-ray photoelectron spectroscopy (XPS) proved that Cur-COOH was successfully anchored on the titanium implants surface. Ti- g -Cur showed excellent antibacterial activity against E. coli and S. aureus , with antibacterial rates of 90.53 % and 94.38 %, respectively. Confocal laser scanning microscopy (CLSM) imaging showed that Ti- g -Cur could effectively inhibit bacterial biofilm formation for up to 5 days. In addition, Ti- g -Cur exhibited superior performance on mammalian hemocompatibility and cytocompatibility. Replacing antibiotics with natural pigment curcumin derivatives for application in biological materials can not only result in a high degree of biocompatibility but also fights bacteria and biofilm formation effectively.

Exposure to fine particulate matter (PM 2.5 ) represents a critical environmental health threat, with growing evidence linking it to accelerated chronic kidney disease (CKD) progression. However, the underlying mechanism of this toxicity remains poorly understood. This study investigated whether PM 2.5 exposure induces renal tubular cell senescence and explored the molecular basis of this process. We found that PM 2.5 exposure caused kidney injury in mice and upregulated senescence markers in both mice and human kidney proximal tubule epithelial (HK-2) cells. Mechanistically, PM 2.5 downregulated FOXP1 expression, relieving its transcriptional repression of CDKN1A (encoding P21), leading to P21 upregulation and subsequent cell cycle arrest. Overexpressing FOXP1 or treating with quercetin mitigated PM 2.5 -induced senescence in HK-2 cells. Our findings demonstrate that reduced FOXP1 drives cellular senescence in PM 2.5 -induced renal injury and identify quercetin as a potential therapeutic agent that activates FOXP1 and alleviates PM 2.5 nephrotoxicity.

Hydrophilic thermoplastic polyurethane (TPU) is applicable in the fields of packaging, oil–water separation, and biomedical. Nevertheless, the process of its preparation is still a great challenge. The objective of this study is to develop TPU with excellent wettability and hydrophilic stability by surface modification. Four methods were evaluated: coating with PVP (polyvinylpyrrolidone) solution, immersion in PVP solution, N 2 /Ar plasma treatment, and N 2 /Ar plasma-induced graft polymerization with PVP. Finally, the results indicate that the sample called TPU-g-PVP, which utilizes plasma-induced grafting, exhibits the best performance. The water contact angle (WCA) reduces from 109° to 9.2°, and maintains within 20° after 80 days under atmospheric conditions. Its surface energy (72.4 mN/m) is increased by 2.8 times than untreated TPU. The surface morphology and roughness are almost unchanged when compared to the original TPU. Some characteristic peaks (–OH and C = O) of PVP on the TPU-g-PVP were found by infrared spectroscopy. X-ray photoelectron spectroscopy (XPS) examination revealed a reduction in C on the TPU surface as the content of N and O increases. Experiments on biocompatibility showed that the PVP deposited substrates improved cell adherence without generating new cytotoxicity.