Application of TrxR1 as a target in the prevention and treatment of PLTX toxicity damage

Abstract

This invention relates to the field of biomedical technology, providing the application of TrxR1 as a target in the prevention and treatment of PLTX toxicity damage. Experimental studies of this invention show that PLTX specifically targets TrxR1, inhibiting or weakening its biological activity. Simultaneously, through the cystine / GSH / GPx4 axis, it leads to a decrease in downstream GSH (reduced glutathione) synthesis, a reduction in glutathione peroxidase 4 (GPX4) activity, and promotes the accumulation of intracellular reactive oxygen species (ROS), Fe... 2+ Elevated levels of PLTX further accelerate lipid peroxidation (LPO), leading to the production of complex compounds such as malondialdehyde (MDA) that mediate ferroptosis. Overexpression of TrxR1, however, can inhibit ferroptosis and reverse PLTX-induced damage. This invention clarifies for the first time the specific molecular damage mechanism of PLTX, contributing to the development of treatment strategies targeting the toxins themselves at both the cellular and molecular levels, and unlocking their potential clinical application value.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and provides a new use for TrxR1, a protein of the pyridine nucleotide-disulfide oxidoreductase family, specifically its application as a target in the prevention and treatment of PLTX toxic damage. Background Technology

[0002] Marine biotoxins are a class of highly active and unique metabolic components found in marine organisms. They are generally extremely toxic and represent the fastest-growing area of ​​research in marine bioactive substances. From a chemical structure perspective, marine biotoxins can be classified into polyethers, alkaloids, macrolides, etc., but most are structurally complex polyether compounds. Palytoxin (PLTX), also known simply as anemone toxin, is a polyether toxin isolated from sea anemones. This toxin is the most potent known non-peptide natural biotoxin. Such poisoning incidents frequently occur among tourists, fishermen, and other people living near the sea. The onset is often rapid, treatment is difficult, and there is a lack of specific antidotes. In addition to the direct toxic damage caused by the toxin, severe hypersensitivity reactions and even shock may occur. However, research on marine biotoxin poisoning has primarily focused on the characteristics of poisoning, symptoms, clinical diagnosis and treatment, and prevention. The mechanism of action of toxins in sea anemones is mainly limited to their specific action on target receptors on membranes, altering the function of related ion channels, hindering their electrical signal transduction, and disrupting the physiological functions of organisms, thereby producing toxicological effects. The specific downstream mechanisms of ion channels are not fully understood, leading to a lack of targeted treatment strategies. Therefore, further exploration of the damaging mechanisms of sea anemone toxins is essential, not only for the prevention and treatment of marine toxin poisoning but also for the potential to transform them into novel "marine drugs."

[0003] Current research on PLTX toxicity primarily suggests that PLTX binds to Na+ on the cell membrane. + -K + -ATPase subunits, turning them into non-selective cation channels, thereby causing drastic changes in the concentration of various ions in the cell, leading to cell death. However, research on the specific molecular damage mechanism of PLTX has not yet been conducted. Summary of the Invention

[0004] Based on the above research, this invention provides an intracellular specific target TrxR1 for the toxic damage caused by the marine toxin PLTX, and provides the application of this target in the prevention and treatment of PLTX toxic damage.

[0005] This invention demonstrates through experimental research that PLTX specifically targets TrxR1 and inhibits or weakens its biological activity. Simultaneously, through the cystine / GSH / GPx4 axis, it leads to reduced downstream GSH (reduced glutathione) synthesis and decreased glutathione peroxidase 4 (GPX4) activity, promoting the accumulation of intracellular reactive oxygen species (ROS) and further accelerating lipid peroxidation (LPO) to produce complex compounds such as malondialdehyde (MDA), which mediate ferroptosis. Overexpression of TrxR1 inhibits ferroptosis and has a reversible effect on PLTX-induced damage. This invention clarifies the specific molecular damage mechanism of PLTX for the first time, contributing to the development of treatment strategies targeting the toxins themselves at both the cellular and molecular levels, and exploring their potential clinical applications.

[0006] Based on the above research, the technical solution to be protected by this invention is as follows:

[0007] In a first aspect, the present invention provides the use of TrxR1 as a target in the prevention or treatment of PLTX toxic injury, including any of the following applications: (1) the use of agonists targeting TrxR1 in the preparation of drugs for the prevention and / or treatment of PLTX toxic injury; (2) the use of in vitro screening of drugs for the treatment of PLTX toxic injury based on the target TrxR1; and (3) the use of in vitro construction of PLTX toxic injury cell engineering platforms and animal models based on the target TrxR1.

[0008] Based on the first use, the present invention first provides the use of TrxR1 agonists in the preparation of medicaments for treating PLTX toxicity.

[0009] Preferably, the TrxR1 agonist is selected from any one or more of the following: exogenous TrxR1 protein or nucleic acid encoding it, substances that promote TrxR1 expression or activity, substances that promote TrxR1 nucleic acid overexpression, liposomes encapsulating TrxR1 nucleic acid, nanomaterials or recombinant expression vectors, precursor proteins or conjugates or complexes thereof that can be converted into TrxR1 in vivo.

[0010] Further preferably, the nucleic acid sequence of TrxR1 is shown in SEQ ID NO.1. In addition to this sequence, the following also fall within the scope of protection of this invention:

[0011] (i) A molecule that hybridizes to the nucleotide sequence defined by SEQ ID NO.1 under stringent conditions;

[0012] (ii) Nucleic acid molecules that are homologous to or have the same sequence as the nucleotide sequence shown in SEQ ID NO.1 and have similar functions;

[0013] (iii) A nucleic acid molecule that has one or more nucleotides substituted, deleted or added in the nucleotide sequence shown in SEQ ID NO.1 and has a similar function.

[0014] Second, this invention provides the use of the TrxR1 recombinant expression vector in the preparation of drugs for treating PLTX toxicity. The TrxR1 recombinant expression vector comprises an expression vector and a TrxR1 nucleic acid molecule inserted onto the expression vector, the sequence of which is described above.

[0015] The expression vectors include viral vectors and non-viral vectors.

[0016] The term "viral vector" includes adenovirus, adeno-associated virus, lentivirus, Coxsackievirus, herpes simplex virus, measles virus, Newcastle disease virus, parvovirus, poliovirus, reovirus, vaccinia virus, and vesicular stomatitis virus, etc. Suitable viral vectors are well known to those skilled in the art.

[0017] The term "non-viral carrier" includes liposomes or lipid complexes, cationic polymers, chitosan polymers, and nanoparticle carriers. Suitable non-viral carriers are well known to those skilled in the art.

[0018] The mechanism of PLTX toxicity is ferroptosis mediated by the cystine / GSH / GPX4 axis. TrxR1 regulates cellular redox homeostasis through the cystine / GSH / GPX4 axis: it can also convert cystine to cysteine ​​by reducing Trx1; intracellular cysteine ​​is the rate-limiting substrate in GSH biosynthesis; and GPX4 is a GSH-dependent enzyme that converts reduced GSH to oxidized GSH, while simultaneously reducing lipid peroxides to their corresponding lipid alcohols or reducing free hydrogen peroxide to water, thereby regulating cellular redox homeostasis and participating in the PLTX toxicity process.

[0019] Ferraphobia is an iron-dependent necrotizing cell death characterized by the rupture of the cell membrane due to the peroxidation of long-chain phospholipids containing unsaturated fatty acids after the inactivation of the intracellular reducing system. Ferraphobia is involved in the occurrence and development of various diseases, and intervention in ferrophobia can effectively address these diseases. As a crucial factor in cell death caused by cell membrane rupture, redox homeostasis imbalance plays a significant role in the mechanism of ferrophobia, and correcting redox homeostasis has been developed as a major pathway to block ferrophobia. Ferraphobia is subject to numerous negative regulatory mechanisms. These mechanisms prevent ferrophobia by inhibiting lipid peroxidation, maintaining redox homeostasis, or regulating iron metabolism through different molecular pathways. Disruption of this homeostasis leads to ferrophobia, which is the role of TrxR1 agonists.

[0020] Cellular experiments confirmed that HaCaT cells overexpressing TrxR1 exhibited significantly reduced intracellular MDA content, significantly increased GSH levels, and significantly enhanced GPX4 activity. These results indicate that TrxR1 overexpression inhibits ferroptosis. After PLTX exposure, HaCaT cells overexpressing TrxR1 showed significantly reduced intracellular MDA content, significantly increased GSH levels, and significantly enhanced GPX4 activity. These results demonstrate that TrxR1 overexpression has a reversible effect on PLTX toxicity.

[0021] Third, the present invention provides a pharmaceutical composition for PLTX toxicity, comprising an active ingredient and a pharmaceutically acceptable excipient. The active ingredient is TrxR1 or its recombinant expression vector as described above.

[0022] In terms of drug form, it is selected from drugs or drug combinations administered via the following methods: oral administration, injection, gold-coated gene gun bombardment, plasmid DNA-carrying method using reproduction-deficient bacteria, target DNA-carrying method using replication-deficient adenovirus, transdermal administration, etc.

[0023] When the composition of the present invention is administered to animals, including humans, the dosage varies depending on the patient's age and weight, disease characteristics and severity, and route of administration. The dosage can be determined with reference to the results of animal experiments and various other factors, but the total dosage should not exceed a certain range.

[0024] Furthermore, the pharmaceutical composition of the present invention can be used in combination with other pharmaceutical compositions that cause PLTX toxicity.

[0025] Based on a second application, this invention provides the use of TrxR1 in constructing an in vitro screening platform for drugs that cause PLTX toxicity. After administration of candidate drugs, drug screening is achieved by observing whether they promote TrxR1 expression, protect TrxR1 structure, and / or synergize TrxR1 function, thereby alleviating PLTX-mediated ferroptosis.

[0026] Based on the third application, this invention provides a TrxR1 in vitro construction PLTX toxicity-damaged cell engineering platform and its application in animal models for mechanism research or to construct TrxR1 overexpressing cells to alleviate PLTX toxicity damage.

[0027] The role and effect of invention

[0028] This invention clearly reveals the mechanism by which PLTX targets and inhibits TrxR1-mediated ferroptosis, and establishes that TrxR1 overexpression maintains cellular redox homeostasis through the cystine / GSH / GPX4 axis, inhibiting ferroptosis and thus reversing PLTX toxicity. Therefore, it proposes a new intervention strategy for controlling PLTX toxicity and provides a new approach for the prevention and treatment of PLTX toxicity. It also discloses the use of TrxR1 in the preparation of drugs for treating PLTX toxicity and provides useful insights for related research. Attached Figure Description

[0029] Figure 1 This is a schematic diagram of the technical route of the present invention.

[0030] Figure 2 This diagram illustrates the molecular mechanism of PLTX damage and the mechanism by which TrxR1 overexpression reverses PLTX damage.

[0031] Figure 3 The image shows the qualitative detection results of intracellular reactive oxygen species (ROS) levels after PLTX treatment. (a, d) Morphology and fluorescence detection results of cells in the positive control group; (b, e) Morphology and fluorescence detection results of cells in the experimental group; (c, f) Morphology and fluorescence detection results of cells in the negative control group. The results show that the intracellular ROS level of cells treated with PLTX increased significantly, which is consistent with the trend of change in cells in the positive control group.

[0032] Figure 4 The volcano plot for drug-1_vs_con groups was created by plotting peptides in the comparison group based on two factors: fold change and p-value (T-test). Significantly downregulated peptides were marked in blue (FC < 0.63 and p < 0.05), significantly upregulated peptides were marked in red (FC > 1.5 and p < 0.05), and peptides with no difference were marked in gray. The top 10 proteins with the most significant differences for upregulated and downregulated peptides were also marked. The figure shows the results for some high-concentration drug groups.

[0033] Figure 5This figure presents a statistical graph of gene ontology (GO) annotations for proteins corresponding to differentially abundant peptides in the Drug-1_vs_con group. It includes: quantitative (DIA) analysis of toxin target proteomics; qualitative discussion of differential expression of target proteins bound by PLTX in cells; annotation of protein functions, localization, and involved biological pathways in organisms using GO; and statistical analysis of the number of differentially abundant proteins at the GO Level 2 functional annotation level. Results for some high-concentration drug groups are shown in the figure. The vertical axis represents GO Level 2 functional annotation information, including biological processes, molecular functions, and cellular components, distinguished by blue, red, and orange respectively. The horizontal axis (bottom) represents the number of proteins corresponding to differentially abundant peptides in each functional category, and the horizontal axis (top) represents the percentage of proteins corresponding to differentially abundant peptides in each functional category out of the total number of proteins corresponding to differentially abundant peptides. Generally, the higher the number of proteins corresponding to differentially abundant peptides in a functional category, the more important the function, requiring focused attention or further in-depth mechanistic investigation.

[0034] Figure 6 A bubble chart of GO functional enrichment (Biological Process, BP) is presented for the Drug-1_vs_con group under the biological process classification. This chart assesses the significance level of protein enrichment for a specific GO functional entry to reveal the overall functional enrichment characteristics of proteins corresponding to all differentially abundant peptides, identifying the most significant enriched GO entries of interest to researchers. Fisher's Exact Test is used to perform GO functional enrichment analysis on proteins corresponding to differentially abundant peptides. The bubble chart displays the GO entry enrichment under the three major GO categories, with results for some high-concentration drug groups shown in the figure. The horizontal axis represents the enrichment factor (Rich Factor ≤ 1), which indicates the proportion of proteins corresponding to differentially abundant peptides annotated to a specific GO functional category out of all identified proteins annotated to that GO functional category. The vertical axis represents the protein statistics corresponding to the differentially expressed peptides under each GO functional category; the bubble color represents the significance of the enriched GO functional category, i.e., the P-value is calculated based on Fisher's exact test, and the color gradient represents the size of the P-value (taken as -log10). The closer the color is to red, the smaller the P-value, and the higher the significance level of the corresponding GO functional category enrichment.

[0035] Figure 7 Venn diagrams of characteristic protein sequences from different toxin concentration treatment groups, where drug1 is represented by a 1×10⁻⁶ m².-11 M PLTX experimental group, drug2 was 1×10 -9 In the M PLTX experimental group, the target proteins screened were located in the intersection.

[0036] Figure 8 The results show the simulated docking of PLTX and TrxR1 molecules.

[0037] Figure 9 Based on BLI detection results, analysis using Bio-Layer Interferometry (BLI) revealed that the equilibrium response values ​​of PLTX and TrxR1 binding dissociation increased in a concentration-dependent manner within the concentration range of 0.25–4 μM. Global fitting and calculation yielded an average equilibrium dissociation constant (KD) of 5.415 * E⁻⁷ M, indicating a medium-to-high affinity between PLTX and TrxR1.

[0038] Figure 10 This is a confocal fluorescence imaging image, showing that the cell membrane is marked with green fluorescence, the cell nucleus with blue fluorescence, and TrxR1, marked with red fluorescence, is mainly distributed in the cytoplasm, while the yellow fluorescent aptamer that specifically binds to PLTX is diffusely distributed in the cytoplasm. The red and yellow fluorescence overlap to form an orange color. Image J analysis of the image shows that TrxR1 and PLTX have significant spatial co-localization within the cell.

[0039] Figure 11 The graph shows the activity detection results of TrxR1 after toxin treatment, where: Figure 11 PLTX treatment significantly reduced TrxR1 activity, while treatment with OA toxin, which is also a polyether, did not significantly change TrxR1 activity, further demonstrating that PLTX specifically targets and inhibits TrxR1. Figure 11 B indicates that the activity of TrxR1 decreases with increasing PLTX concentration, showing a concentration-dependent change.

[0040] Figure 12 To determine the IC50 of PLTX on HaCaT cells after 4 hours using CCK-8 assay. 50 The results are shown in the figure, where cell viability decreases significantly with increasing PLTX concentration. The IC50 value was calculated using SPSS. 50 Value 5.00 × 10 −5 μM (1.34×10⁻ 4 μg / ml).

[0041] Figure 13To illustrate the oxidative stress effects of PLTX through the cystine / GSH / GPX4 axis, the cystine / GSH / GPX4 system is a classic ferroptosis inhibitory system that effectively inhibits lipid peroxidation, thereby combating ferroptosis. GSH is an important intracellular antioxidant, and intracellular cysteine ​​is the rate-limiting substrate in GSH biosynthesis. Endogenous cysteine ​​is mainly produced through TrxR1-mediated cystine reduction. Glutathione peroxidase (GPX4) is a GSH-dependent enzyme that converts reduced GSH to oxidized GSH, while simultaneously reducing lipid hydroperoxides to their corresponding lipid alcohols or reducing free hydrogen peroxide to water. Specifically: Figure A shows the inhibition of TrxR1 by PLTX targeting, resulting in suppressed cystine-to-cysteine ​​conversion and reduced GSH biosynthesis; Figure B shows the decrease in GSH leading to decreased GPX4 activity; Figure C shows the increase in intracellular ROS levels due to decreased GPX4 activity; and Figure D shows the accumulation of intracellular ROS leading to lipid peroxidation and MDA generation.

[0042] Figure 14 The IC50 of PLTX against TrxR1 overexpression group and negative control group was determined using CCK-8 assay after 4 h. 50 The results are shown in the figure, where TrxR1 overexpression significantly improved cell viability after PLTX treatment. SPSS calculations yielded the IC50 values ​​for the experimental group cells. 50 The value is 3.08 × 10 −4 μM (8.26×10⁻ 4 μg / ml), IC50 in control cells 50 The value is 2.58 × 10 −5 μM (6.91×10) ⁻5 μg / ml).

[0043] Figure 15 The results show that TrxR1 overexpression has a reversing effect on PLTX damage. The left figure shows that TrxR1 overexpression significantly upregulates GSH content; the middle figure shows that TrxR1 overexpression significantly increases GPX4 activity; and the right figure shows that TrxR1 overexpression significantly reduces MDA content.

[0044] Figure 16 To illustrate the results of HaCaT cells transduced with TrxR1 overexpressing and negative control lentiviruses, screening was performed using complete culture medium containing 2 μg / mL puromycin. After 48 hours, the cells were observed under a microscope, and the successfully transduced cells showed green fluorescence.

[0045] Figure 17 The image shows the validation results of TrxR1 overexpression in transduced cells. The TrxR1 expression level in HaCaT cells transduced with TrxR1-overexpressing lentivirus was significantly higher than that in the negative control group. Figure 17A and 17B are the qPCR validation results; Figure 17 C is the WB verification result graph. Detailed Implementation

[0046] Experimental methods in the following examples, unless otherwise specified, are generally performed under standard conditions as described in Sambrook et al., Molecular Cloning: A Laboratory Guide (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of skill in the art. Furthermore, any methods and materials similar or equivalent to those described herein may be used in this invention. The preferred methods and materials described herein are for illustrative purposes only.

[0047] Figure 1 A schematic diagram of the technical route of the present invention is shown. Figure 2 This diagram illustrates the molecular mechanism of PLTX damage and the mechanism by which TrxR1 overexpression reverses PLTX damage. It reveals the molecular mechanism by which PLTX targets and inhibits TrxR1-mediated ferroptosis, and establishes that TrxR1 overexpression regulates cellular redox homeostasis through the cystine / GSH / GPX4 axis, thereby reversing PLTX toxicity. Therefore, a novel intervention strategy for the prevention and / or treatment of PLTX toxicity is proposed.

[0048] The following specific examples illustrate the role of TrxR1 in cells, thereby verifying its effectiveness as a target for preventing and / or treating PLTX toxicity damage.

[0049] Example 1: TrxR1 is a potential target protein of PLTX.

[0050] Starting from the cellular and molecular level, and combining high-throughput proteomics analysis of Lip-SMAP protein-toxin interactions, we explored potential targets of anemone toxins in rock sand anemones.

[0051] (1) Changes in intracellular reactive oxygen species (ROS) levels were detected in HaCaT cells after treatment with PLTX toxin. (See results below.) Figure 3 The fluorescence intensity of the experimental group cells was significantly higher than that of the negative control group, and similar to that of the positive control group cells, indicating that the level of reactive oxygen species in the cells was increased due to the effect of toxins.

[0052] (2) Using the Lip-SMAP toxin-target proteomics experiment, to demonstrate the significant differences in peptides between comparison groups, volcano plots were created using fold change and p-value (Ttest) as criteria. Significantly downregulated peptides were marked in blue (FC < 0.63 and p < 0.05), significantly upregulated peptides were marked in red (FC > 1.5 and p < 0.05), and peptides with no difference were marked in gray. The top 10 proteins with the most significant differences in expression for the upregulated and downregulated peptides were also marked. Results for some high-concentration drug groups are shown below. Figure 4 As shown.

[0053] To further investigate the specific target proteins bound by PLTX, drug target proteomics (DIA) analysis was performed to qualitatively discuss the differential expression of PLTX-bound target proteins in cells. To comprehensively understand the function, localization, and biological pathways involved in proteins in organisms, proteins were annotated using Gene Ontology (GO). Simultaneously, the number of differentially expressed proteins was statistically analyzed at the GO secondary functional annotation level. Results for some high-concentration drug groups are shown below. Figure 5 .

[0054] To reveal the overall functional enrichment characteristics of proteins corresponding to all differentially abundant peptides, and to identify the most significant enriched GO entries by evaluating the significance level of protein enrichment for a specific GO functional entry, Fisher's Exact Test was used to perform GO functional enrichment analysis on proteins corresponding to differentially abundant peptides. Bubble charts are used to display the GO entry enrichment under the three major GO categories (bar charts are available in the output file). Results for some high-concentration drug groups are shown in [link to relevant documentation]. Figure 6 , Figure 7 .

[0055] Based on the above results and combined with the previous cell ROS experiments indicating that PLTX treatment increased intracellular reactive oxygen species, the function-related protein TrxR1 was screened from the candidate differential target proteins as a potential target protein for PLTX treatment.

[0056] Example 2: PLTX and TrxR1 molecules are stably bound together by hydrogen bonds in a concentration-dependent manner.

[0057] To explore the molecular interaction between PLTX and TrxR1, the obtained stable PLTX structure was used as the docking ligand, and the TrxR1 nucleophile was used as the ligand. Semi-flexible molecular docking simulation was performed using HEX (Ver. 8.0). The experimental results are as follows: Figure 8As shown, TrxR1 interacts with PLTX at two sites, forming two hydrogen bonds. Specifically, the carbonyl group on the amide group at 230Q interacts with the hydroxyl group in PLTX to form one hydrogen bond, and the carbonyl group on the amide group at 234N interacts with the hydroxyl group in PLTX to form another hydrogen bond. This demonstrates an affinity between the two molecules, allowing TrxR1 to form a stable bond with PLTX.

[0058] like Figure 9 As shown, BLI analysis yielded 0.25 × 10 -4 The equilibrium response values ​​of PLTX and TrxR1 binding dissociation within the μM concentration range showed a concentration-dependent increase. After global fitting and calculation, the average equilibrium dissociation constant (KD) was found to be 5.415 × 10⁻⁶. -7 M indicates that there is a medium-to-high affinity between PLTX and TrxR1.

[0059] like Figure 10 As shown, after immunohistochemical staining, the cell nucleus showed blue fluorescent labeling. Confocal fluorescence imaging showed that TrxR1, which is labeled with red fluorescence, is mainly distributed in the cytoplasm, while the yellow fluorescent aptamer that specifically binds to PLTX is diffusely distributed in the cytoplasm. The red fluorescence and yellow fluorescence overlap to form an orange color. ImageJ analysis of the images showed that TrxR1 and PLTX have significant spatial colocalization in the cell and exhibit medium to high intensity binding.

[0060] like Figure 11 As shown in Figure A, PLTX treatment significantly reduced TrxR1 activity, while treatment with OA toxin, another polyether, showed no significant change in TrxR1 activity, further demonstrating that PLTX specifically targets and inhibits TrxR1. Figure 11 As shown in Figure B, the activity of TrxR1 decreases with increasing PLTX treatment concentration, exhibiting a concentration-dependent change.

[0061] Example 3: PLTX-targeted inhibition of TrxR1-induced ferroptosis

[0062] This embodiment explores the molecular mechanism by which TrxR1 inhibits ferroptosis, as detailed below:

[0063] The cell viability of HaCaT cells treated with different concentrations of PLTX for 4 hours was determined using the CCK-8 assay to evaluate the specific toxicity of PLTX to HaCaT cells. The results are as follows: Figure 12 As shown, cell viability decreased significantly with increasing PLTX concentration. The IC50 value calculated by SPSS was 5.00 × 10⁻⁶. −5 μM (1.34×10⁻ 4To investigate the differences in cellular material content after PLTX treatment, a concentration of 3.73 × 10⁻⁶ μg / ml was selected. −4 μM (1×10⁻³ μg / ml) and 3.73×10 −6 μM (1×10⁻) 5 The concentration (μg / ml) was used as the treatment group for subsequent experiments.

[0064] The cystine / GSH / GPX4 system is a classic inhibitory system against ferroptosis, effectively suppressing lipid peroxidation and thus combating ferroptosis. GSH is an important intracellular antioxidant, and intracellular cysteine ​​is the rate-limiting substrate in GSH biosynthesis. Endogenous cysteine ​​is mainly produced through cysteine ​​reduction mediated by GSH thioredoxin reductase 1 (TrxR1). Glutathione peroxidase GPX4 is a GSH-dependent enzyme that converts reduced GSH to oxidized GSH, while simultaneously reducing lipid hydroperoxides to their corresponding lipid alcohols or reducing free hydrogen peroxide to water. Experimental results showed that when TrxR1 was targeted and inhibited by PLTX, the conversion of cysteine ​​to cysteine ​​was inhibited, and GSH biosynthesis decreased. Figure 13 A), which in turn leads to a decrease in GPX4 activity ( Figure 13 B), leading to intracellular ROS accumulation and lipid peroxidation (B), Figure 13 C), generating a series of complex compounds represented by MDA ( Figure 13 D) Disrupts cellular redox homeostasis, induces oxidative stress, and ultimately induces ferroptosis.

[0065] Furthermore, ROS accumulation induces oxidative stress, leading to an increase in ferrous ion levels within HaCaT cells. Figure 14 ), and ferroptosis occurs in the cells.

[0066] Example 4: Overexpression of TrxR1 reverses toxin damage

[0067] The cell viability of TrxR1 overexpression group and negative control group cells treated with different concentrations of PLTX for 4 hours was determined using the CCK-8 assay. The results are as follows: Figure 14 As shown; the IC50 of the experimental group cells was calculated using SPSS. 50 The value is 3.08 × 10 −4 μM (8.26×10⁻ 4 μg / ml), IC50 in control cells 50 The value is 2.58 × 10 −5 μM (6.91×10) ⁻5 (μg / ml) It was found that TrxR1 overexpression could significantly upregulate cell viability after PLTX treatment and reverse toxin damage.

[0068] By comparing the GSH content, GPX4 activity, and MDA content in cells of the TrxR1 overexpression group and the negative control group, it was found that TrxR1 overexpression significantly upregulated GSH content and GPX4 activity, and decreased MDA level. Figure 15 It can reverse the damage caused by toxins. Specifically:

[0069] 1. Construction of TrxR1 overexpression lentivirus

[0070] TrxR1 overexpressing lentivirus and a negative control lentivirus were used as the experimental and control groups, respectively. After transducing HaCaT cells with the lentiviral solution, selection was performed using complete culture medium containing 2 μg / mL puromycin. After 48 hours, cells were observed under a microscope; successfully transduced cells showed green fluorescence. Figure 16 ).

[0071] RT-PCR experiments revealed that the expression level of TrxR1 gene in the experimental group was significantly higher than that in the control group (P<0.0001), and the expression level of TrxR1 gene in the experimental group was approximately 3.2 times that in the control group. Figure 17 A); Western blot results showed that the expression level of TrxR1 protein in the experimental group was significantly higher than that in the control group (P<0.01), and the expression level of TrxR1 protein in the experimental group was approximately 1.49 times that in the control group (A). Figure 17 B) A distinct band is observed at approximately 55 kb, consistent with the expected band location. Figure 17 (C) indicates that the TrxR1 protein was stably overexpressed in HaCaT cells.

[0072] 2. Lentiviral transduction

[0073] 1) On the first day, prepare a complete culture medium with a density of 1×10⁻⁶. 5 A cell suspension of 2 ml per well was seeded into each well of a six-well plate to ensure that the cell density was around 90% by day 5.

[0074] 2) On the second day, remove the culture medium from the six-well plate, wash the cells twice with 1 ml PBS in each well, add 1 ml of complete culture medium and 40 μl of HitransGP infection enhancement solution (25×), and mix by pipetting; add LV-TrxR1 lentivirus and negative control lentivirus respectively, and mix by pipetting.

[0075] The viral volume was calculated as (MOI × cell number) / viral titer. MOI, or re-infection index, refers to the virus's ability to infect cells; a higher MOI indicates greater resistance to infection. The MOI of a cell line is typically defined as the ratio of virus particles to cell number required to achieve 80% infection. The HaCaT cells used in this experiment had an MOI of 20, a viral titer of LV-TrxR1 of 4.4E+08, and a negative control lentivirus of 1.3E+09.

[0076] 3) Incubate at 37°C for 16 hours, then replace with complete culture medium and continue incubation.

[0077] 4) Continue culturing on the third and fourth days, and change the medium to maintain cell viability.

[0078] 5) By the fifth day, the infection efficiency was observed using a fluorescence microscope.

[0079] 72 hours after infection (during which time passage and medium changes are possible), when cells reach 70%-80% confluence, cell selection is performed using complete medium containing 2 μg / ml Puromycin. The selection results are observed after 48 hours. Subsequently, continuous selection and passage are performed using medium containing 1 μg / ml Puromycin to ensure cell line stability.

[0080] In summary, TrxR1 plays a crucial role in the mechanism of PLTX toxicity and can serve as a research target for drugs to prevent and / or treat PLTX toxicity. This invention discloses a target TrxR1 for the prevention and / or treatment of PLTX toxicity. Targeting TrxR1, drugs for the prevention and / or treatment of PLTX toxicity can be prepared, thus providing a pharmaceutical composition for the prevention and / or treatment of PLTX toxicity. Furthermore, a method for screening drugs for the treatment of PLTX toxicity based on the target TrxR1 can be provided, by administering drugs targeting TrxR1 to directly or indirectly enhance the biological activity of TrxR1; that is, the drug is a reagent that promotes TrxR1 expression, protects TrxR1 structure, and / or synergizes TrxR1 function.

[0081] In terms of mechanism, it was elucidated that TrxR1 regulates cellular redox homeostasis through the cystine / GSH / GPX4 axis, further demonstrating that TrxR1 can serve as an effective target for the prevention and / or treatment of PLTX toxicity, thereby participating in the regulation of cellular redox homeostasis, finding a new approach for the prevention and treatment of PLTX toxicity, and having extremely important application value in the preparation of drugs for the treatment of PLTX toxicity.

[0082] The TrxR1 gene sequence is as follows:

[0083]

[0084] Among them, [T]: CTC is a synonymous mutation to CTT.

[0085] The undescribed parts of this invention are the same as or implemented using existing technology. The applicant declares that this invention is illustrated through the above embodiments, but the invention is not limited to the above detailed methods, i.e., it does not mean that the invention must rely on the above detailed methods to be implemented. Those skilled in the art should understand that any improvements to this invention, equivalent substitutions of raw materials for the product of this invention, additions of auxiliary components, and selection of specific methods all fall within the protection and disclosure scope of this invention.

Claims

1. The use of TrxR1 agonists in the preparation of drugs to prevent and treat PLTX toxicity.

2. The use according to claim 1, characterized in that, The TrxR1 agonist is selected from any one or more of the following: exogenous TrxR1 protein or nucleic acid encoding it, substances that promote TrxR1 expression or activity, substances that promote TrxR1 nucleic acid overexpression, liposomes encapsulating TrxR1 nucleic acid, nanomaterials, precursor proteins or their conjugates or complexes that can be converted into TrxR1 in vivo.

3. The use according to claim 1, characterized in that, The nucleic acid sequence of TrxR1 is shown in SEQ ID NO.

1.

4. A TrxR1 recombinant vector, characterized in that, The recombinant vector includes an expression vector and a TrxR1 nucleic acid molecule inserted into the expression vector, as described in claim 3.

5. The TrxR1 recombinant vector according to claim 4, characterized in that: The expression vector is a plasmid vector, a granular vector, a bacteriophage vector, or a viral vector, etc., and the viral vector is selected from adeno-associated virus or lentivirus.

6. Use of the TrxR1 recombinant vector according to claim 4 or 5 in the preparation of a drug for treating and preventing PLTX toxicity damage.

7. A pharmaceutical composition for treating PLTX toxicity damage, characterized in that, The pharmaceutical composition includes an active ingredient and pharmaceutically acceptable excipients. The active component is the TrxR1 agonist according to any one of claims 1 to 3 or the TrxR1 recombinant expression vector according to any one of claims 4 to 5.

8. The pharmaceutical composition for treating PLTX toxicity injury according to claim 7, characterized in that, This drug composition is used in combination with other drugs for treating PLTX toxicity.

9. Application of TrxR1 in constructing an in vitro screening platform for PLTX toxic drugs.

10. Application of TrxR1 in the in vitro construction of PLTX toxicity-damaged cell engineering platforms or animal models.

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