Glutathione (GSH) is indispensable for maintaining redox homeostasis in biological fluids and serves as a key component in cellular defense mechanisms. Accurate assessment of GSH relative to its oxidized counterpart, glutathione disulfide (GSSG), is critical for the early diagnosis and understanding of conditions related to oxidative stress. Despite existing methods for their quantification, the label-free and simultaneous measurement of GSH and GSSG in biological fluid presents significant challenges. Herein, we report the use of an alpha-hederin (Ah) nanopore for the direct measurement of the GSH:GSSG ratio in simulated biological fluid, containing fetal bovine serum (FBS). This system hinges on detecting characteristic relative ion blockades (ΔI/Io) as GSH and GSSG molecules pass through the Ah nanopore under an applied electric field. The distinct current blockage signals derived from the translocation of GSH and GSSG enabled us to determine the molar ratio of GSH and its oxidized form. Notably, the interactions between the hydroxyl groups of the sugar moiety lining the nanopore\'s inner surface and the sulfhydryl group of GSH significantly influence the translocation dynamics, resulting in a longer translocation time for GSH compared to GSSG. The Ah nanopore technology proposed in this study offers a promising approach for real-time, single molecule-level monitoring of glutathione redox status in biological fluids, eliminating the need for labeling or extensive sample preparation.

Background: Small extracellular vesicles (sEVs) mediate intercellular communication in the tumor microenvironment (TME) and contribute to the malignant transformation of tumors, including unrestricted growth, metastasis, or therapeutic resistance. However, there is a lack of agents targeting sEVs to overcome or reverse tumor chemotherapy resistance through sEVs-mediated TME reprogramming. Methods: The paclitaxel (PTX)-resistant A549T cell line was used to explore the inhibitory effect of alpha-hederin on impeding the transmission of chemoresistance in non-small cell lung cancer (NSCLC) through the small extracellular vesicles (sEVs) pathway. This investigation utilized the CCK-8 assay and flow cytometry. Transcriptomics, Western blot, oil red O staining, and targeted metabolomics were utilized to evaluate the impact of alpha-hederin on the expression of signaling pathways associated with chemoresistance transmission in NSCLC cells before and after treatment. In vivo molecular imaging and immunohistochemistry were conducted to assess how alpha-hederin influences the transmission of chemoresistance through the sEVs pathway. RT-PCR was employed to examine the expression of miRNA and lncRNA in response to alpha-hederin treatment. Results: The resistance to PTX chemotherapy in A549T cells was overcome by alpha-hederin through its dependence on sEV secretion. However, the effectiveness of alpha-hederin was compromised when vesicle secretion was blocked by the GW4869 inhibitor. Transcriptomic analysis for 463 upregulated genes in recipient cells exposed to A549T-derived sEVs revealed that these sEVs enhanced TGFβ signaling and unsaturated fatty acid synthesis pathways. Alpha-hederin inhibited 15 types of unsaturated fatty acid synthesis by reducing the signaling activity of the sEVs-mediated TGFβ/SMAD2 pathway. Further, we observed that alpha-hederin promoted the production of three microRNAs (miRNAs, including miR-21-5p, miR-23a-3p, and miR-125b-5p) and the sorting to sEVs in A549T cells. These miRNAs targeted the TGFβ/SMADs signaling activity in sEVs-recipient cells and sensitized them to the PTX therapy. Conclusion: Our finding demonstrated that alpha-hederin could sensitize PTX-resistant NSCLC cells by sEV-mediated multiple miRNAs accumulation, and inhibiting TGFβ/SMAD2 pathways in recipient cells.

Nigella sativa (commonly known as black seed or black cumin), from the family Ranunculaceae, is a plant that grows in countries bordering the Mediterranean Sea. This narrative review discusses the toxicological profile reported by short- to long-term studies that examined different extracts and oils of N. sativa seeds. Scientific databases including Web of Science, PubMed, Scopus, and Google Scholar were searched using appropriate keywords. LD50 for administered N. sativa seed fixed oil varied from 28.8 mL/kg to 3,371 mg/kg in mice, while 21 g/kg of aqueous, methanol, and chloroform extracts of N. sativa did not lead to any mortality. Subacute toxicity evaluations indicated that aqueous, methanol, and chloroform extracts of N. sativa at doses as high as 6 g/kg do not produce toxicity. Investigation of chronic toxicity found that 2 mL/kg of N. sativa fixed oil is slightly toxic. Cytotoxicity studies indicated that N. sativa chloroform and petroleum ether extracts are more cytotoxic than its other extracts. Although studies that assessed N. sativa toxicity generally introduced it as a safe medicinal herb, to draw a more definitive conclusion on its safety, more detailed studies must be conducted.

Nanopores have been emerged as a powerful tool for analyzing the structural information and interactional properties of a range of biomolecules. The spatial resolution of nanopore is determined by the diameter and effective thickness of its constriction region, but the presence of vestibule or stem structure in protein-based nanopore could negatively affect the sensitivity of the nanopore when applied for genome sequencing and topological analysis of DNA. Recently, alpha-hederin (Ah) has been reported to form a sub-nanometer scale pore structure in lipid membrane. With the simple structure and extremely small effective thickness, the Ah nanopore was shown to discriminate four different types of nucleotides. However, identification of a certain nucleotide in a strand of DNA, which is essential for genome sequencing, remains challenging. Here, we investigated the resolving capability of Ah nanopore to discriminate few nucleotides in a strand of single-stranded DNA, and the factors determining the sensitivity of Ah nanopore. The Ah nanopore was shown to be able to identify as few as three adenosine nucleotides in a strand of poly cytidine, in which the dwell time of the additional current blockade that represents the adenosine residue was in good agreement with their physical length. We also found that the lateral tension and chain pressure generated around the nanopore were influenced by pore\'s diameter and played as a dependent variables to determine the geometry of nanopore\'s constriction as well as the spatial resolution of the Ah nanopore.

Various types of biological and synthetic nanopores have been developed and utilized for the high-throughput investigation of individual biomolecules. Biological nanopores made with channel proteins are so far superior to solid-state ones in terms of sensitivity and reproducibility. However, the performance of a biological nanopore is dependent on the protein in the channel structure its dimensions are predetermined and are difficult to modify for broader applications. Here inspired by the cytotoxic mechanisms of a saponin derivative, alpha-hederin, we report a nonproteinaceous nanopore that can be formed spontaneously in a lipid membrane. We propose the pore-forming mechanism of alpha-hederin in a cholesterol-rich lipid membrane and a strategy to control the pore-forming rate by a lipid partitioning method. The small diameter and effective thickness of alpha-hederin nanopores enabled us to discriminate ssDNA homopolymers as well as four types of nucleotides, showing its potential as a DNA sequencing tool.

Chemoresistance is a major limiting factor that impairs the outcome of non-small cell lung cancer (NSCLC) chemotherapy. Paclitaxel (Tax) induces protective autophagy in NSCLC cells, leading to the development of drug resistance. We recently identified a new autophagy inhibitor (alpha-hederin) and hypothesized that it may promote the killing effect of Tax on NSCLC cells. We found that alpha-hederin (α-Hed) could block late autophagic flux in NSCLC cells by altering lysosomal pH and inhibiting lysosomal cathepsin D maturation. Combination treatment of α-Hed and Tax synergistically reduced NSCLC cell proliferation and increased NSCLC cell apoptosis compared with treatment with α-Hed or Tax alone. Furthermore, α-Hed plus Tax enhanced the accumulation of intracellular reactive oxygen species (ROS) in NSCLC cells, while the ROS inhibitor N-acetylcysteine reversed the inhibitory effect of the combination treatment. Our findings suggest that α-Hed can increase the killing effect of Tax on NSCLC cells by promoting ROS accumulation, and that combining α-Hed with classical Tax represents a novel strategy for treating NSCLC.

OBJECTIVE: In previous studies the therapeutic effects of Nigella sativa have been demonstrated on asthmatic animals. In the present study, the preventive effect of single dose of alpha-hederin, its active constituent, has been evaluated on lung inflammation and some inflammatory mediators in lungs of ovalbumin sensitized rat in order to elicit its mechanism.
METHODS: Forty rats were randomly grouped in 4 groups; control (C), sensitized (S), sensitized pretreated groups with thymoquinone (3 mg/kg i.p., S+TQ) and alpha-hederin (0.02 mg/kg i.p., S+AH). Levels of IL-13 mRNA and miRNA-126 in lung tissue and its pathological changes in each group were assessed.
RESULTS: Elevated levels of miRNA-126, IL-13 mRNA and pathological changes were observed in the sensitized group compared to the control group (p<0.001 to p<0.05). All of these factors were significantly reduced in S+TQ and S+AH groups in comparison to S group (p<0.001 to p<0.05). Although alpha-hederin decreased the levels of miRNA-126, IL-13 mRNA and pathological changes in comparison with thymoquinone, the results were statistically not significant.
CONCLUSIONS: The results suggested that alpha-hederin had preventive effect on sensitized rats like thymoquinone. It may intervene in miRNA-126 expression, which consequently could interfere with IL-13 secretion pathway leading to a reduction in inflammatory responses.