Intervention method for relieving T-2 toxin induced cellular immune aging based on PER2 protein inhibition and application thereof

Abstract

The invention relates to the cross field of toxicology and immunology, in particular to an intervention method for relieving T-2 toxin induced cellular immune aging based on PER2 protein inhibition and application of the intervention method. The method comprises the following steps: S1, providing an exposed object containing immune cells in an in-vitro culture system; s2, firstly applying a PER2 inhibitor; s3, sequentially finishing S2 and T-2 treatment according to a preset initial administration time interval; and S4, characterizing the Hippo pathway MST1 / YAP / p-YAP in 4 hours and / or 12 hours after T-2 treatment, and obtaining p-YAP subcellular localization, thereby determining or adjusting the administration time course. According to the scheme, closed-loop standard operation and parameter setting are carried out according to'administration-detection-time history determination '.

Description

Technical Field

[0001] This invention relates to the interdisciplinary field of toxicology and immunology, and in particular to an intervention method and its application based on inhibiting PER2 protein to alleviate T-2 toxin-induced cellular immune senescence. Background Technology

[0002] T-2 toxin is a trienzyme toxin that can induce phenotypic cellular immunosenescence and related molecular events at the cellular and individual levels. Existing studies have mostly described the changes after T-2 toxin exposure from the perspectives of hypoxia-related factors (such as HIF-1α), DNA damage, and inflammatory signals, but there is still a lack of a unified technical approach for constructing an implementable intervention protocol.

[0003] The circadian rhythm-related protein PER2 exhibits a time-dependent relationship with immune cell function. Existing research primarily focuses on correlational observations or single-point interventions, lacking a systematic approach that directly regulates PER2. On one hand, the timing and parameters for administering PER2 inhibitors before and after T-2 toxin treatment are unclear. On the other hand, a comprehensive detection protocol is lacking to constrain and confirm dosing timing using pathway-level and localization-level indicators (e.g., expression and subcellular localization of MST1, YAP, and p-YAP in the Hippo pathway). Consequently, existing technologies suffer from uncertainties regarding experimental reproducibility, procedural boundaries, and timing determination.

[0004] Meanwhile, existing literature does not provide a unified step-by-step process to connect the "exposure subject provision - inhibitor administration - dosing schedule setting - molecular and localization detection" in a closed loop for different experimental time windows (such as 4 h and 12 h). This results in a lack of correspondence between dosing parameters, detection windows and evaluation indicators used, making it difficult to objectively determine the dosing schedule in actual operation.

[0005] In summary, the existing technologies have at least the following common problems: (1) lack of standardized intervention steps with PER2 as the core target and applicable to T-2 toxin exposure conditions; (2) lack of molecular / pathway / localization integrated detection panels that match the dosing schedule and can be directly used for operational judgment; (3) lack of technical rules that correspond the above steps and detection results to specific time windows in order to determine the dosing schedule accordingly.

[0006] In summary, the technical problem actually solved by this invention is: under T-2 toxin exposure conditions, to provide a complete set of intervention and evaluation methods that take the inhibition of PER2 protein as the core and use the detection results of Hippo pathway-related protein expression and p-YAP subcellular localization as the basis for time course determination, so as to determine the dosing time course and standardize the operation sequence and parameter setting of each step within the same process. Summary of the Invention

[0007] To overcome the aforementioned technical deficiencies, the present invention aims to provide an intervention method and its application based on inhibiting PER2 protein to alleviate T-2 toxin-induced cellular immune senescence. The present invention applies a PER2 protein inhibitor under T-2 toxin exposure conditions and uses the detection results of Hippo pathway (MST1 / YAP / p-YAP) expression and p-YAP subcellular localization as the basis for determining the dosing time, thereby constructing a closed-loop process of "dosing-detection-time determination" to solve the above-mentioned technical problems.

[0008] This invention discloses an intervention method for alleviating T-2 toxin-induced cellular immune senescence based on inhibiting PER2 protein, comprising:

[0009] S1: Provides the subject of exposure in an in vitro culture system;

[0010] S2: Administer a PER2 protein inhibitor to the exposed subject prior to T-2 toxin treatment;

[0011] S3: During the preset initial dosing time, step S2 and the subsequent T-2 toxin treatment are performed sequentially. The initial dosing time includes at least the pretreatment period of the PER2 protein inhibitor and the treatment period of the T-2 toxin.

[0012] S4: At 4 h and / or 12 h after T-2 toxin treatment, pathway characterization was performed on the exposed subjects using Hippo pathway-related proteins as the detection panel. Hippo pathway-related proteins include MST1, YAP and p-YAP. The protein expression of MST1, YAP and p-YAP and the subcellular localization of p-YAP were obtained. The initial dosing time was determined or adjusted based on the detection results.

[0013] The targets of exposure are immune cells or in vitro cell systems containing immune cells.

[0014] Preferably, the PER2 protein inhibitor is KL044 or a pharmaceutically acceptable salt thereof.

[0015] Preferably, the in vitro pretreatment concentration of KL044 is 5–20 μM and the pretreatment time is 0.5–2 h.

[0016] Preferably, the in vitro pretreatment concentration of KL044 is 10 μM and the pretreatment time is 1 h.

[0017] Preferably, the treatment concentration of T-2 toxin is 10–20 nM.

[0018] Preferably, pathway characterization includes Western blot detection, and the detection targets include PER2, MST1, YAP, p-YAP, p53, p21, p16, and internal reference protein.

[0019] Preferably, pathway characterization includes immunofluorescence detection to obtain the subcellular localization of p-YAP.

[0020] Preferably, the exposed object undergoes circadian rhythm phase synchronization processing before step S2, which is dexamethasone 200 nM treatment for 2 h.

[0021] Preferably, before step S2, the exposed subject is treated with the HIF-1α inhibitor YC-1, with an in vitro treatment concentration of 5–20 μM and a treatment duration of 0.5–2 h.

[0022] In view of this, the present invention includes the use of KL044 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for carrying out the above-described method under T-2 toxin exposure conditions, wherein the medicament may optionally be used in combination with the MST1 inhibitor XMU-MP-1.

[0023] Compared with existing technologies, the above technical solution has the following advantages:

[0024] 1. Establishment of a closed-loop process (drug administration—detection—time schedule determination)

[0025] Existing technologies primarily focus on the correlation between T-2 toxin-induced aging, often targeting HIF-1α, DNA damage, or inflammatory signals. They lack a complete intervention and evaluation process and cannot objectively determine the timing of drug administration.

[0026] The present invention involves administering a PER2 inhibitor (preferably KL044) under T-2 exposure conditions, and using Hippo pathway (MST1 / YAP / p-YAP) expression and p-YAP subcellular localization as supporting detection indicators to determine the dosing time.

[0027] Beneficial effects: It forms a standardized closed-loop process, unifying drug administration and molecular / localization detection onto an operable timeline, significantly improving experimental repeatability and comparability.

[0028] 2. The core target is clearly defined (PER2 is the direct intervention target).

[0029] Current technologies do not prioritize circadian rhythm factors as intervention targets; most studies only observe the association between rhythm and toxic phenotypes.

[0030] The present invention proposes to directly suppress PER2 as the core operating point and use the downstream Hippo path readout as the basis for timing determination.

[0031] Beneficial effects: It provides an actionable target-level intervention path, avoids the limitations of "only observing and not treating", and improves the pertinence of intervention plans and decision-making efficiency.

[0032] 3. A dual-layer determination system combining pathway and location

[0033] Existing technologies often rely solely on a single dimension such as total protein / transcription amount as readout, making it difficult to define the timeline.

[0034] The present invention uses the following approach: the pathway layer observes the expression of MST1 / YAP / p-YAP, the localization layer observes the nucleus / cytoplasm distribution of p-YAP, and the two are combined to determine the time course.

[0035] Beneficial effects: The judgment dimensions are more comprehensive, and the optimal dosing time and sampling window can be determined more accurately in different time windows (such as 4 h / 12 ​​h).

[0036] 4. Clear time window, adaptable to short-term and delayed effects.

[0037] Existing technology: The detection time points are scattered and lack a unified agreement, resulting in incomparable result time sequences.

[0038] The present invention proposes a fixed detection window around 4 hours and 12 hours, allowing for fine-tuning based on the readout results.

[0039] Beneficial effects: It creates a verifiable time coordinate between short-time signals and delayed responses, which is beneficial for horizontal comparison and amplification verification.

[0040] 5. The parameter window and endpoints are fully exposed, facilitating zooming and panning.

[0041] Existing technology: Commonly uses "recommended values" but lacks ranges and endpoints, resulting in poor reproducibility.

[0042] The present invention provides the range (window) and endpoint implementation examples for key parameters such as KL044, T-2, and YC-1.

[0043] Beneficial effects: Provides engineering margins for differences between different batches of cells / reagents and platforms, improving the robustness and transferability of the solution.

[0044] 6. Upstream / parallel nodes are programmable (YC-1, XMU-MP-1)

[0045] Existing technology: Lack of modular combination of upstream inhibitors or bypass pathways.

[0046] The present invention provides a sequence for the combined use of pretreatment with the HIF-1α inhibitor YC-1 and the MST1 inhibitor XMU-MP-1, in addition to core PER2 inhibition.

[0047] Beneficial effects: Enables programmable intervention combinations from upstream to core to bypass, facilitating mechanism verification and therapy optimization.

[0048] 7. Evaluation panel standardization (WB / IF / qPCR triad)

[0049] Existing technology: The detection indicators are scattered and cannot be compared horizontally.

[0050] The present invention provides a unified panel for WB (PER2, MST1, YAP, p-YAP, p53 / p21 / p16) + IF (p-YAP localization) + qPCR (IL-6 / IL-8).

[0051] Beneficial effects: Unified evaluation criteria, supporting systematic comparison and compliant recording of time-course / dosage / combination therapy.

[0052] 8. Complete process records and quality control to meet standardized transformation requirements.

[0053] Existing technology: Missing batch records / source data retention standards, which seriously reflect the insufficient data chain in reality.

[0054] The present invention addresses the following aspects: clearly defining the terms for solution preparation, timeline, testing, original file names, batch records, QC, and hazardous waste disposal.

[0055] Beneficial effects: Controlled processes and traceable data enhance compliance and auditability in research and translation.

[0056] 9. At the application level, it possesses feasible pathways for both formulation and in vivo application.

[0057] Current technologies mostly focus on cellular observation, lacking formulation and in vivo application procedures.

[0058] The present invention discloses the formulation and aseptic preparation of KL044 monotherapy / combination with XMU-MP-1, and provides the in vivo administration and sampling procedure for mice.

[0059] Beneficial effects: Extending the "in vitro intervention approach" to drug preparation and in vivo implementation enhances the practicality and industrialization feasibility of the technology.

[0060] 10. Time-based decisions can be directly embedded into the R&D process.

[0061] Existing technology: Dosing window selection relies on experience, and retry costs are high.

[0062] The present invention uses the Hippo channel and the objective results read from the positioning to "lock" the timeline.

[0063] Beneficial effects: It shifts key decisions from experience-based to data-driven, reducing trial and error and shortening the R&D cycle.

[0064] 11. Compatible with and expandable other immune cells or primary systems

[0065] Existing technology: The scope of application of the method is not clearly described.

[0066] The present invention uses a parameter window and endpoint examples as a universal template, and the process can be transferred to other immune cell / primary systems.

[0067] Beneficial effects: High scalability, facilitating rapid verification and horizontal application across different objects. Attached Figure Description

[0068] Figure 1 This is a schematic diagram of the process of an intervention method based on inhibiting PER2 protein to alleviate T-2 toxin-induced cellular immune aging and its application. Detailed Implementation

[0074] The advantages of the present invention will be further illustrated below with reference to the accompanying drawings and specific embodiments.

[0075] Exemplary embodiments will now be described in detail, examples of which are illustrated in the accompanying drawings. When the following description relates to the drawings, unless otherwise indicated, the same numerals in different drawings denote the same or similar elements. The embodiments described in the following exemplary embodiments do not represent all embodiments consistent with this disclosure. Rather, they are merely examples of apparatuses and methods consistent with some aspects of this disclosure as detailed in the appended claims.

[0076] The terminology used in this disclosure is for the purpose of describing particular embodiments only and is not intended to be limiting of the disclosure. The singular forms “a,” “the,” and “the” as used in this disclosure and the appended claims are also intended to include the plural forms unless the context clearly indicates otherwise. It should also be understood that the term “and / or” as used herein refers to and includes any and all possible combinations of one or more of the associated listed items.

[0077] It should be understood that although the terms first, second, third, etc., may be used in this disclosure to describe various information, such information should not be limited to these terms. These terms are used only to distinguish information of the same type from one another. For example, without departing from the scope of this disclosure, first information may also be referred to as second information, and similarly, second information may also be referred to as first information. Depending on the context, the word "if" as used herein may be interpreted as "when," "when," or "in response to determination."

[0078] In the description of this invention, it should be understood that the terms "longitudinal", "lateral", "up", "down", "front", "rear", "left", "right", "vertical", "horizontal", "top", "bottom", "inner", "outer", etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings. They are only for the convenience of describing this invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, they should not be construed as limitations on this invention.

[0079] In the description of this invention, unless otherwise specified and limited, it should be noted that the terms "installation", "connection" and "linking" should be interpreted broadly. For example, they can refer to mechanical or electrical connections, or internal connections between two components. They can be direct connections or indirect connections through an intermediate medium. Those skilled in the art can understand the specific meaning of the above terms according to the specific circumstances.

[0080] In the following description, suffixes such as "module," "part," or "unit" used to denote elements are used only for the convenience of the description of the invention and have no specific meaning in themselves. Therefore, "module" and "part" can be used interchangeably.

[0081] See Figure 1 As shown, the following specific embodiments are provided in this embodiment.

[0082] Example 1-A

[0083] I. Materials and Equipment

[0084] RAW264.7 cells; DMEM (high glucose); FBS; penicillin and streptomycin; PBS (1×, pH 7.4); T-2 toxin stock solution; KL044 stock solution; dexamethasone (DEX); RIPA lysis buffer; PMSF; BCA reagent; SDS-PAGE gel; PVDF membrane; ECL chromogenic solution; primary antibody (PER2, MST1, YAP, p-YAP, p53, p21, p16, β-Actin, or GAPDH); HRP secondary antibody; paraformaldehyde (4%); Triton X-100; BSA (5%); DAPI; RNA extraction and reverse transcription, qPCR reagents; CO2 incubator; Class II biosafety cabinet; inverted microscope; refrigerated centrifuge; electrophoresis / transfer system; chemiluminescence imaging system; laser confocal microscope; qPCR instrument; pipettes and sterile consumables.

[0085] II. Preparation of Working Solution

[0086] 1. Culture system: DMEM + 10% FBS + 1% penicillin and streptomycin, stored at 4 ℃ for ≤7 days.

[0087] 2. T-2 working solution: 14 nM (prepared and used on the same day).

[0088] 3. KL044 working solution: 5 μM (prepared and used on the same day).

[0089] 4. DEX working fluid: 200 nM.

[0090] 5. Solvent control: Maintain the same DMSO volume fraction as each treatment group (≤0.1% v / v).

[0091] III. Cell Preparation

[0092] Add 2 mL of culture medium to each well of a 6-well plate and inoculate with RAW264.7 (2 × 10⁻⁶) 5 –4×10 5 Incubate at 37°C and 5% CO2 until 60%–80% confluence. Phase synchronization: Add 200 nM (2 mL) of DEX to each well, incubate for 2 h, discard, wash once with PBS, and add 2 mL of fresh culture medium.

[0093] IV. Grouping and Timeline

[0094] Control group: 2 mL culture medium, up to the sampling time.

[0095] Toxin group: 2 mL T-2 14 nM, incubated for 4 h or 12 h.

[0096] KL044 group: 2 mL KL044 5 μM, 0.5 h; discard and continue incubation for 4 h or 12 h with 2 mL of culture medium.

[0097] KL044+ toxin group: 2 mL KL044 5 μM, 0.5 h; discard and then 2 mL T-2 14 nM, incubate for 4 h or 12 h.

[0098] Each biological replicate should have ≥3 wells.

[0099] V. Testing Items and Procedures

[0100] WB: Wash once with PBS → Add RIPA+PMSF and lyse on ice for 30 min → 12,000×g, 4 ℃, 10 min → BCA quantification → SDS-PAGE (10%) loading 20–30 μg / lane → PVDF wet transfer at 300 mA, 45–50 min → 5% milk powder or BSA blocking at room temperature for 1 h → overnight incubation with primary antibody at 4 ℃ (PER2, MST1, YAP, p-YAP, p53, p21, p16, internal control) → HRP secondary antibody at room temperature for 1 h → ECL imaging; record target / internal control grayscale.

[0101] IF (p-YAP localization): Treat glass plates with the same group → Fix with 4% paraformaldehyde for 10–15 min → Permeabilize with 0.1%–0.3% Triton for 10 min → Block with 5% BSA for 30–60 min → Incubate with p-YAP primary antibody at 4 ℃ overnight → Incubate with fluorescent secondary antibody for 1 h → Incubate with DAPI for 5–10 min → Confocal acquisition; record nuclear / cytoplasmic fluorescence signals.

[0102] qPCR: Total RNA extracted with TRIzol-type reagents → reverse transcription → SYBR Green qPCR, primers: IL-6, IL-8, internal control (β-Actin or GAPDH) → Ct collection → 2^-ΔΔCt calculation; technical replicates ≥3.

[0103] VI. Data Recording

[0104] Register reagent batch number, solution volume and concentration, sample addition / discard / sampling time; save raw WB .tif, raw IF image, and raw qPCR table (Ct table and calculation table).

[0105] VII. Quality Control

[0106] Cells are free from contamination and have uniform morphology; solvent volume fraction is consistent; WB molecular weight is consistent with the instructions; IF exposure / gain / threshold is consistent; biological replicates ≥3, technical replicates ≥3; the entire batch is redone when the key control fails.

[0107] VIII. Safety and Waste

[0108] Liquids containing T-2 and organic solvents are collected in dedicated containers and treated according to the unit's hazardous waste process; disposable consumables are placed in biohazardous waste bins, sterilized under high pressure, and then disposed of according to regulations; protective equipment is worn throughout the operation.

[0109] Example 1-B

[0110] I. Materials and Equipment

[0111] The materials and equipment listed in this embodiment are consistent with those listed in the text: RAW264.7; DMEM; FBS; penicillin and streptomycin; PBS; T-2; KL044; DEX; WB / IF / qPCR related reagents and equipment; safety and consumables are the same as before.

[0112] II. Preparation of Working Solution

[0113] The culture system was the same as above; T-2 working solution: 14 nM; KL044 working solution: 20 μM; DEX working solution: 200 nM; the solvent control was the same.

[0114] III. Cell Preparation

[0115] 6-hole seeding (2×10) 5 –4×10 5 (2 mL / well) until 60%–80% confluence; DEX 200 nM (2 mL / well) for 2 h, phase synchronized; wash once with PBS.

[0116] IV. Grouping and Timeline

[0117] Control group: 2 mL culture medium.

[0118] Toxin group: 2 mL T-2 14 nM, 4 h or 12 h.

[0119] KL044 group: 2 mL KL044 20 μM, 2 h; discard and continue with 2 mL of culture medium for 4 h or 12 h.

[0120] KL044+ toxin group: 2 mL KL044 20 μM, 2 h; discarded and then 2 mL T-2 14 nM, 4 h or 12 h.

[0121] Biological replicates ≥3.

[0122] V. Testing Items and Procedures

[0123] WB / IF / qPCR: The steps, parameters, and recording methods are consistent with those listed in this example: lysis, quantification, sample loading, wet transfer, blocking, primary / secondary antibody, ECL; IF fixation / permeabilization / blocking / staining / imaging; qPCR extraction / reverse transcription / amplification / calculation; save all source data.

[0124] VI. Data Recording

[0125] Completely record the concentration, volume, time point, file name, and storage path; create a batch record table.

[0126] VII. Quality Control

[0127] Cell state, solvent consistency, correct band molecular weight, and consistent IF parameters; number of replicates meets the standard; abnormal batches should be retested.

[0128] VIII. Safety and Waste

[0129] The same safety and disposal process is described in the text of this embodiment.

[0130] Example 1-C

[0131] I. Materials and Equipment

[0132] RAW264.7; DMEM; FBS; Penicillin and Streptomycin; PBS; T-2; KL044; DEX; RIPA; PMSF; BCA; SDS-PAGE; PVDF; ECL; Related Antibodies; Fluorescent Secondary Antibody; DAPI; RNA / Reverse Transcription / qPCR Reagents; CO2 Incubator; Biosafety Cabinet; Electrophoresis and Transfer System; Imaging System; Confocal Microscopy; qPCR Instrument; Sterile Consumables.

[0133] II. Preparation of Working Solution

[0134] The culture system was the same as above; T-2 working solution: 10 nM; KL044 working solution: 10 μM; DEX working solution: 200 nM; the solvent control was the same.

[0135] III. Cell Preparation

[0136] 6-hole seeding (2×10) 5 –4×10 5 (2 mL / well); 37 °C, 5% CO2 to 60%–80% confluence; DEX 200 nM 2 h; wash once with PBS.

[0137] IV. Grouping and Timeline

[0138] Control group: 2 mL culture medium.

[0139] Toxin group: 2 mL T-2 10 nM, 4 h or 12 h.

[0140] KL044 group: 2 mL KL044 10 μM, 1 h; discard the remaining 2 mL culture medium and continue for 4 h or 12 h.

[0141] KL044+ toxin group: 2 mL KL044 10 μM, 1 h; discarded and then 2 mL T-2 10 nM, 4 h or 12 h.

[0142] Biological replicates ≥3.

[0143] V. Testing Items and Procedures

[0144] WB / IF / qPCR: Perform according to the steps in this embodiment; Target proteins: PER2, MST1, YAP, p-YAP, p53, p21, p16; Localization: p-YAP; Transcription: IL-6, IL-8; Save source data.

[0145] VI. Data Recording

[0146] Establish three types of record sheets: solution preparation, time axis, and testing; record the batch number, volume, concentration, time point, and file name.

[0147] VII. Quality Control

[0148] The quality control requirements and acceptance criteria are the same as those listed in the text of this embodiment.

[0149] VIII. Safety and Waste

[0150] The same safety and disposal process is described in the text of this embodiment.

[0151] Example 1-D

[0152] I. Materials and Equipment

[0153] The materials and equipment listed in this embodiment are consistent with those listed in the text (cells, culture system, PBS, T-2, KL044, DEX, WB / IF / qPCR related equipment).

[0154] II. Preparation of Working Solution

[0155] T-2 working solution: 20 nM; KL044 working solution: 10 μM; DEX working solution: 200 nM; solvent control was consistent.

[0156] III. Cell Preparation

[0157] Seeds were grown in 6-well plates until 60%–80% confluence was achieved; DEX 200 nM 2 h; wash once with PBS.

[0158] IV. Grouping and Timeline

[0159] Control group: 2 mL culture medium.

[0160] Toxin group: 2 mL T-2 20 nM, 4 h or 12 h.

[0161] KL044 group: 2 mL KL044 10 μM, 1 h; discard the remaining 2 mL culture medium and continue for 4 h or 12 h.

[0162] KL044+ toxin group: 2 mL KL044 10 μM, 1 h; discarded and then 2 mL T-2 20 nM, 4 h or 12 h.

[0163] Biological replicates ≥3.

[0164] V. Testing Items and Procedures

[0165] WB / IF / qPCR: Follow the steps and parameters outlined in this embodiment; save all raw data and images.

[0166] VI. Data Recording

[0167] Record all sample addition and sampling times, concentrations, and volumes, and save the WB / IF / qPCR source data tables and images.

[0168] VII. Quality Control

[0169] Cell condition is acceptable; band molecular weight is correct; IF parameters are consistent; number of replicates meets requirements.

[0170] VIII. Safety and Waste

[0171] Same as the text listed in this embodiment.

[0172] Example 2-A

[0173] I. Materials and Equipment

[0174] RAW264.7; DMEM; FBS; Penicillin and Streptomycin; PBS; T-2; KL044; YC-1; DEX; RIPA; PMSF; BCA; SDS-PAGE; PVDF; ECL; Antibodies (PER2, MST1, YAP, p-YAP, p53, p21, p16, Internal Control); Fluorescent Secondary Antibody; DAPI; RNA / Reverse Transcription / qPCR Reagents; CO2 Incubator; Biosafety Cabinet; Imaging / Electrophoresis / Confocal / qPCR Equipment; Consumables.

[0175] II. Preparation of Working Solution

[0176] T-2 working solution: 14 nM; KL044 working solution: 10 μM; YC-1 working solution: 5 μM; DEX working solution: 200 nM; solvent control was consistent.

[0177] III. Cell Preparation

[0178] 6-hole seeding (2×10) 5 –4×10 5 (2 mL / well), until 60%–80% confluence; DEX 200 nM 2h; wash once with PBS.

[0179] IV. Grouping and Timeline

[0180] Control group: 2 mL culture medium.

[0181] Toxin group: 2 mL T-2 14 nM, 4 h or 12 h.

[0182] YC-1+KL044+toxin group: 2 mL YC-1 5 μM, 0.5 h; after discarding, 2 mL KL044 10 μM, 1 h; after discarding, 2 mL T-2 14 nM, 4 h or 12 h.

[0183] KL044+ toxin group: 2 mL KL044 10 μM, 1 h; discarded and then 2 mL T-2 14 nM, 4 h or 12 h.

[0184] Biological replicates ≥3.

[0185] V. Testing Items and Procedures

[0186] WB: PER2, MST1, YAP, p-YAP, p53, p21, p16, internal control; lysis, quantification, loading, wet transfer, blocking, antibody incubation and imaging shall be performed according to the parameters in this embodiment.

[0187] IF: p-YAP localization; complete execution of fixation, permeabilization, blocking, staining, and imaging steps; recording of nuclear / cytoplasmic fluorescence.

[0188] qPCR: IL-6, IL-8 and internal control; complete execution of extraction, reverse transcription, amplification, calculation and recording.

[0189] VI. Data Recording

[0190] Record the order and time of drug administration, the concentration and volume of each working solution, and the sampling time; save the WB / IF / qPCR source data and images.

[0191] VII. Quality Control

[0192] Solvent consistency; complete references; correct molecular weight of bands; consistent IF exposure / threshold; number of repetitions meets requirements.

[0193] VIII. Safety and Waste

[0194] Waste liquids and consumables containing small molecules and T-2 are recycled in accordance with the hazardous waste process; biohazardous waste is disposed of after high-pressure sterilization; protective equipment is worn during operation.

[0195] Example 2-B

[0196] I. Materials and Equipment

[0197] RAW264.7; DMEM; FBS; penicillin and streptomycin; PBS; T-2; KL044; YC-1; DEX; WB / IF / qPCR related reagents and equipment; safety and consumables are the same as above.

[0198] II. Preparation of Working Solution

[0199] T-2 working solution: 14 nM; KL044 working solution: 10 μM; YC-1 working solution: 20 μM; DEX working solution: 200 nM; solvent control was consistent.

[0200] III. Cell Preparation

[0201] 6-hole seeding (2×10) 5 –4×10 5 (2 mL / well), until 60%–80% confluence; DEX 200 nM 2h; wash once with PBS.

[0202] IV. Grouping and Timeline

[0203] Control group: 2 mL culture medium.

[0204] Toxin group: 2 mL T-2 14 nM, 4 h or 12 h.

[0205] YC-1+KL044+toxin group: 2 mL YC-1 20 μM, 2 h; after discarding, 2 mL KL044 10 μM, 1 h; after discarding, 2 mL T-2 14 nM, 4 h or 12 h.

[0206] KL044+ toxin group: 2 mL KL044 10 μM, 1 h; discarded and then 2 mL T-2 14 nM, 4 h or 12 h.

[0207] Biological replicates ≥3.

[0208] V. Testing Items and Procedures

[0209] WB / IF / qPCR: Perform according to the procedures and parameters given in this example; target protein, localization and transcription panel are the same as before; save all source data.

[0210] VI. Data Recording

[0211] Record the solution preparation / volume / time axis / sampling; save the WB / IF / qPCR file names and paths; create batch records.

[0212] VII. Quality Control

[0213] Cells were in good condition; the results were consistent with controls; the molecular weight of the bands was correct; the IF parameters were consistent; and the replicates met the standards.

[0214] VIII. Safety and Waste

[0215] Dispose of the waste in accordance with hazardous waste and biosafety regulations; wear compliant personal protective equipment.

[0216] Example 3-A

[0217] I. Materials and Equipment

[0218] RAW264.7; DMEM; FBS; penicillin and streptomycin; PBS; T-2 toxin stock solution; KL044 stock solution; XMU-MP-1 stock solution; dexamethasone (DEX); RIPA; PMSF; BCA; SDS-PAGE; PVDF; ECL; primary antibodies (PER2, MST1, YAP, p-YAP, p53, p21, p16, β-Actin / GAPDH); fluorescent secondary antibody; DAPI; RNA / reverse transcription / qPCR reagents; CO2 incubator; Class II biosafety cabinet; centrifuge; electrophoresis / transfer system; chemiluminescence imaging; confocal microscopy; qPCR instrument.

[0219] II. Preparation of Working Solution

[0220] Culture system: DMEM + 10% FBS + 1% antibiotic; T-2: 14 nM; KL044: 10 μM; XMU-MP-1: 0.5 μM; DEX: 200 nM; solvent control at equal volume fraction (DMSO ≤0.1% v / v).

[0221] III. Cell Preparation

[0222] 6-well plate seeding with RAW264.7 (2×10⁻⁶) 5 –4×10 5 Add 2 mL / well (samples / well) until 60%–80% confluence; add DEX 200 nM for 2 h, simultaneously; wash once with PBS.

[0223] IV. Grouping and Timeline

[0224] Control: 2 mL culture medium;

[0225] Toxin: 2 mL T-2 14 nM, incubate for 4 h / 12 ​​h;

[0226] KL044+ toxin: 2 mL KL044 10 μM 1 h → discard → 2 mL T-2 14 nM 4 h / 12 ​​h;

[0227] KL044+XMU-MP-1+toxin: 2 mL KL044 10 μM 1 h → discard → 2 mL (XMU-MP-1 0.5 μM + T-2 14 nM) 4 h / 12 ​​h.

[0228] Each biological group has ≥3 biological replicates.

[0229] V. Testing

[0230] Western blot (WB): RIPA lysis for 30 min, 12,000×g for 10 min; BCA quantification; loading 20–30 μg / lane; wet transfer at 300 mA for 45–50 min; blocking for 1 h; primary antibody overnight at 4 ℃ (PER2, MST1, YAP, p-YAP, p53, p21, p16, internal control); secondary antibody for 1 h; ECL imaging.

[0231] IF (p-YAP localization): Same group in glass dish; fix 4% PFA for 10–15 min → permeate 0.1%–0.3% Triton for 10 min → block 5% BSA for 30–60 min → p-YAP primary antibody overnight → fluorescent secondary antibody for 1 h → DAPI; confocal imaging.

[0232] qPCR: Total RNA was extracted → reverse transcribed → IL-6, IL-8 and internal control were amplified in the SYBR system; Ct was collected and relative expression was calculated.

[0233] VI. Data Recording

[0234] Record the concentration, volume, and time of sample addition / discard / sampling for each group; save the WB source image, IF raw image, qPCR Ct table, and calculation table.

[0235] VII. Quality Control and Safety

[0236] Cells are in good condition; solvent volume is equal; band molecular weight is correct; IF exposure / threshold is consistent; biological repeatability ≥3, technical repeatability ≥3; waste liquid and consumables containing T-2 are disposed of according to hazardous waste procedures, and protective equipment is worn during operation.

[0237] 8. Schedule Confirmation

[0238] The expression of MST1, YAP, and p-YAP, as well as the subcellular localization of p-YAP, were compared at two time points, 4 h and 12 h, and the dosing schedule of the PER2 protein inhibitor was determined accordingly.

[0239] Example 3-B

[0240] I. Materials and Equipment

[0241] Consistent with that listed in Example 3-A: RAW264.7; culture system and all detection / equipment are the same as in Example 3-A; the key difference is the XMU-MP-1 1.0 μM solution.

[0242] II. Preparation of Working Solution

[0243] T-2: 14 nM; KL044: 10 μM; XMU-MP-1: 1.0 μM; DEX: 200 nM; Solvents were consistent with the control.

[0244] III. Cell Preparation

[0245] Inoculate 2 mL / well of 6-well plate with RAW264.7 until 60%–80% confluence; incubate with DEX 200 nM for 2 h; wash once with PBS.

[0246] IV. Grouping and Timeline

[0247] Comparison;

[0248] Toxin: T-2 14 nM 4 h / 12 ​​h;

[0249] KL044+ toxin: KL044 10 μM 1 h → T-2 14 nM 4 h / 12 ​​h;

[0250] KL044+XMU-MP-1+toxin: KL044 10 μM 1 h → (XMU-MP-1 1.0 μM + T-2 14 nM) 4h / 12 h.

[0251] Biological replicates ≥3.

[0252] V. Testing | VI. Record Keeping | VII. Quality Control and Safety

[0253] Perform and record the process exactly as described in text 3-A of this embodiment (parameters are at the same level as in 3-A, and the XMU-MP-1 concentration is 1.0 μM).

[0254] 8. Schedule Confirmation

[0255] The Hippo index and p-YAP localization at 4 h and 12 h were compared using the same method, and the dosing time of the PER2 protein inhibitor was determined accordingly.

[0256] Example 3-C

[0257] I. Materials and Equipment

[0258] Active ingredient: KL044 (active pharmaceutical ingredient or sterile API); Excipients: sodium chloride, water for injection, pH buffer (e.g., phosphate buffer); 0.22 μm filter; sterile glass vials and rubber stoppers; clean dispensing equipment; pH meter.

[0259] II. Prescription (per 10 mL)

[0260] KL044 10 mg; NaCl 90 mg; water for injection to 10 mL; pH 7.2–7.4 (adjusted with buffer).

[0261] III. Preparation Process

[0262] Dissolve KL044 in water for injection → Add NaCl to adjust to isotonicity → Adjust pH to 7.2–7.4 → 0.22 μm aseptic filtration → Aseptically dispense 10 mL / bottle → Stopper and seal → Label and retain sample.

[0263] IV. Quality Control and Storage

[0264] The product should be clear in appearance, have a suitable pH level, and be of the correct fill volume; it should be sterile / endotoxin tested according to the pharmacopoeia; and stored at 2–8 ℃ protected from light.

[0265] V. External use

[0266] RAW264.7 6-well plate; (optional) DEX 200 nM 2 h; add the formulation diluted to equivalent KL044 10 μM (total volume 2 mL / well) and incubate for 1 h → discard → add T-2 14 nM 2 mL and treat for 4 h / 12 ​​h → WB / IF / qPCR. Time course confirmation: compare Hippo markers and p-YAP localization at 4 h and 12 h, and determine the dosing time course of the PER2 protein inhibitor accordingly.

[0267] Example 3-D

[0268] I. Materials and Equipment

[0269] Raw materials: KL044, XMU-MP-1; NaCl; water for injection; pH buffer; 0.22 μm filter; sterile containers and dispensing equipment.

[0270] II. Prescription (per 10 mL)

[0271] KL044 10 mg; XMU-MP-1 2 mg; NaCl 90 mg; water for injection to 10 mL; pH 7.2–7.4.

[0272] III. Preparation Process and Inspection

[0273] Perform according to the process in Example 3-C; Compound compatibility stability: continuously observe and record appearance / clarity / pH for 24–48 h; pass sterility / endotoxin test; store at 2–8 ℃ protected from light.

[0274] IV. External Use

[0275] RAW264.7; (optional) DEX 200 nM 2 h; add compound diluted to equivalent KL044 10 μM + XMU-MP-1 0.5 μM (2 mL / well) and incubate for 1 h → discard → T-2 14 nM 2 mL 4 h / 12 ​​h → WB / IF / qPCR.

[0276] Schedule confirmation: As above, determine the dosing schedule accordingly.

[0277] Example 3-E (Sequential Dosing Procedure)

[0278] A. In vitro sequential

[0279] RAW264.7; (optional) DEX 200 nM 2 h; add KL044 equivalent 10 μM 2 mL 1 h → discard → add XMU-MP-1 equivalent 0.5 μM + T-2 14 nM 2 mL 4 h / 12 ​​h → WB / IF / qPCR; record batch number / volume / time point throughout the process; determine the dosing schedule accordingly.

[0280] B. Mouse Sequential

[0281] C57BL / 6; group n≥6; KL044 10 mg / kg ip → 1 h → XMU-MP-1 1 mg / kg ip → 0–15 min → T-2 0.5 mg / kg ip; collect tissue samples (spleen / liver / PBMC) at 4 h / 12 ​​h; perform Western blotting (PER2, MST1, YAP, p-YAP, p53, p21, p16), and qPCR (IL-6, IL-8); record dose / volume / time; dispose of hazardous waste in compliance with regulations; determine the dosing schedule accordingly.

[0282] Example 3-F

[0283] I. Animals and Ethics

[0284] C57BL / 6 mice (18–22 g, male or female), SPF conditions; ethical approval compliant.

[0285] II. Grouping and Dosing

[0286] Grouping (n≥6 / group): Control; T-2; KL044; KL044+T-2; (optional) KL044+XMU-MP-1+T-2.

[0287] Dosage: KL044 10 mg / kg ip; T-2 0.5 mg / kg ip; (optional) XMU-MP-1 1 mg / kg ip.

[0288] Time axis: KL044 → 1 h → T-2; sampling window 4 h / 12 ​​h.

[0289] III. Sampling and Testing

[0290] Collect spleen / liver / PBMCs; homogenize with PBS; Western blotting: load 20–40 μg / lane, detect PER2, MST1, YAP, p-YAP, p53, p21, p16; qPCR: IL-6, IL-8 and internal control; (optional) perform p-YAP localization on tissue IF. Save all source data.

[0291] IV. Records / QC / Safety

[0292] Record animal number, weight, dosage (mg / kg), volume, injection and sampling time, and tissue weight; injection volume ≤10 mL / kg; rapid low-temperature treatment of tissues; and compliant disposal of biohazardous waste.

[0293] V. Schedule Confirmation

[0294] Hippo markers and p-YAP localization were compared at 4 h and 12 h to determine the dosing time of the PER2 protein inhibitor.

[0295] It should be noted that the embodiments of the present invention have better implementability and are not intended to limit the present invention in any way. Any person skilled in the art may use the above-disclosed technical content to change or modify it into equivalent effective embodiments. However, any modifications or equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention shall still fall within the scope of the technical solution of the present invention.

Claims

1. An intervention method for alleviating T-2 toxin-induced cellular immune senescence based on inhibiting PER2 protein, characterized in that, include: S1: Provides the subject of exposure in an in vitro culture system; S2: Prior to T-2 toxin treatment, administer a PER2 protein inhibitor to the exposed subject; S3: Step S2 and the subsequent T-2 toxin treatment are performed sequentially within a preset initial dosing time period, wherein the initial dosing time period includes at least the pretreatment period of the PER2 protein inhibitor and the treatment period of the T-2 toxin. S4: At 4 h and / or 12 h after T-2 toxin treatment, pathway characterization is performed on the exposed subjects using the Hippo pathway-related proteins as the detection panel. The Hippo pathway-related proteins include MST1, YAP, and p-YAP. The protein expression of MST1, YAP, and p-YAP, as well as the subcellular localization of p-YAP, are obtained. The initial dosing schedule is determined or adjusted based on the detection results. The exposed object is an immune cell or an in vitro cell system containing immune cells.

2. The method according to claim 1, characterized in that, The PER2 protein inhibitor is KL044 or a pharmaceutically acceptable salt thereof.

3. The method according to claim 2, characterized in that, The in vitro pretreatment concentration of KL044 was 5–20 μM, and the pretreatment time was 0.5–2 h.

4. The method according to claim 2, characterized in that, The in vitro pretreatment concentration of KL044 was 10 μM and the pretreatment time was 1 h.

5. The method according to claim 1, characterized in that, The treatment concentration of the T-2 toxin is 10–20 nM.

6. The method according to claim 1, characterized in that, The pathway characterization includes Western blot detection, and the detection targets include PER2, MST1, YAP, p-YAP, p53, p21, p16, and internal reference protein.

7. The method according to claim 1, characterized in that, The pathway characterization includes immunofluorescence detection to obtain the subcellular localization of the p-YAP.

8. The method according to claim 1, characterized in that, The exposed object undergoes circadian rhythm phase synchronization processing before step S2, which is dexamethasone 200 nM treatment for 2 h.

9. The method according to claim 1, characterized in that, Prior to step S2, the exposed subjects were treated with the HIF-1α inhibitor YC-1 at an in vitro treatment concentration of 5–20 μM for a duration of 0.5–2 h.

10. An application characterized in that, KL044 or a pharmaceutically acceptable salt thereof, used in the preparation of a medicament for carrying out the method of claim 1 under T-2 toxin exposure conditions, wherein the medicament may optionally be used in combination with the MST1 inhibitor XMU-MP-1.

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