A method for mitigating aluminum toxicity in hydroponic peas based on menaquinone-based regulation of reactive oxygen species metabolism.

Abstract

This disclosure provides a method for alleviating aluminum toxicity in hydroponic peas based on menadione-mediated reactive oxygen species (ROS) metabolism. The core components include: regulating the concentration and duration of menadione treatment to optimize the spatiotemporal accumulation pattern of ROS (superoxide anion, hydrogen peroxide) in the roots of hydroponic peas; adjusting the dynamic response of endogenous antioxidant enzyme systems (SOD, POD, APX) to enhance menadione-mediated ROS scavenging capacity; controlling the initial ROS burst based on changes in plasma membrane NADPH oxidase (Rboh) gene expression and enzyme activity to inhibit the aluminum stress oxidative signaling cascade; and adjusting the timing and frequency of menadione administration based on subcellular-scale mitochondrial and chloroplast ROS leakage rates to alleviate organ-specific oxidative damage. This approach addresses five key issues: aluminum-induced oxidative damage, cellular redox imbalance, oxidative signaling cascade, organ-specific oxidative damage, and persistent aluminum toxicity metabolic disorders.

Description

Technical Field

[0001] This application relates to plant stress physiology. Plant nutrition and cultivation techniques Phytotoxicity and resistance mechanisms reactive oxygen species metabolism regulation technology Hydroponic cultivation technology, specifically involving a method to alleviate aluminum toxicity in hydroponic peas based on the regulation of reactive oxygen species metabolism by menaquinone. Background Technology

[0002] This study proposes a method to alleviate aluminum toxicity in hydroponic peas by regulating reactive oxygen species (ROS) metabolism based on menadione. The aim is to precisely control the concentration and duration of menadione treatment to regulate the spatiotemporal accumulation patterns of ROS (such as superoxide anions and hydrogen peroxide) in the roots, thereby reducing aluminum-induced oxidative damage. Simultaneously, by monitoring the response dynamics of endogenous antioxidant enzyme systems (such as SOD, POD, and APX), the scavenging capacity of menadione for ROS is optimized to alleviate redox imbalance. Furthermore, by regulating the gene expression and enzyme activity of plasma membrane NADPH oxidase (Rboh), the initial burst of ROS is controlled, reducing the oxidative signaling cascade. In addition, based on changes in the ROS leakage rate in mitochondria and chloroplasts, the timing and frequency of menadione administration are rationally determined to reduce organ-specific oxidative damage. Finally, by combining the reduction state of key components of the glutathione-ascorbic acid cycle, the antioxidant buffer capacity enhanced by menadione is regulated to address the metabolic disorders caused by persistent aluminum toxicity. Summary of the Invention

[0003] In view of this, the present disclosure provides a method for alleviating aluminum toxicity in hydroponic peas based on the regulation of reactive oxygen species metabolism by menaquinone, which at least partially solves the problems existing in the prior art.

[0004] A method for mitigating aluminum toxicity in hydroponic peas based on the regulation of reactive oxygen species metabolism using menaquinone includes: The spatiotemporal accumulation patterns of reactive oxygen species (such as superoxide anions and hydrogen peroxide) in hydroponic pea roots were regulated based on the concentration and duration of menadione treatment. Enhanced menadione-mediated ROS scavenging capacity by modulating the response dynamics of endogenous antioxidant enzyme systems such as SOD, POD, and APX; The initial ROS burst process was controlled by controlling changes in plasma membrane NADPH oxidase (Rboh) gene expression level and enzyme activity to inhibit the oxidation signaling cascade triggered by aluminum stress; The timing and frequency of menadione administration were adjusted based on the rate of reactive oxygen species leakage in mitochondria and chloroplasts at the subcellular scale to alleviate organ-specific oxidative damage.

[0005] In one specific embodiment, the regulation of the spatiotemporal accumulation pattern of reactive oxygen species in hydroponic pea roots based on the concentration and duration of menadione treatment further includes: Obtain the oxidative stress index (OI) of the root surface; The initial treatment concentration C0 and time T0 of menadione were set. The concentration and time combination C_T is adjusted according to the following formula: C_T = C0 × (OI / OI0), where OI0 represents the standard oxidative stress index without aluminum stress; Different gradient treatment schemes were set according to the adjusted C_T to suppress the cumulative peak of superoxide anions.

[0006] In one specific embodiment, the enhancement of menaquinone-mediated ROS scavenging capacity by modulating the response dynamics of endogenous antioxidant enzyme systems (such as SOD, POD, and APX) further includes: Measure SOD activity S_0 and APX activity A_0; A dynamic response coefficient β = min(S_0 / S_ref, A_0 / A_ref) is introduced, where S_ref and A_ref are reference values ​​for the corresponding enzyme activities; The stress buffer factor RF was calculated as β × (C / T + k1 × √(T)), where k1 is the correction coefficient, C is the concentration of menaquinone, and T is the treatment time. Optimize application parameters by RF to enhance ROS clearance efficiency.

[0007] In one specific embodiment, the control of the initial ROS burst process based on changes in plasma membrane NADPH oxidase (Rboh) gene expression level and enzyme activity further includes: Detecting the amount of Rboh transcript R_mRNA; Monitor Rboh enzyme activity (R_activity), unit: U / mg protein; Based on the formula α = ln(R_activity + 1) / (R_mRNA / R_mRNA_0), where R_mRNA_0 represents the mRNA content of the control group; The frequency of menadione application is controlled by α to keep it within a range that can suppress initial oxidation signals without over-activating ROS.

[0008] In one specific embodiment, adjusting the timing and frequency of menaquinone administration based on the reactive oxygen species leakage rate in mitochondria and chloroplasts at the subcellular scale further includes: Obtain the H2O2 concentrations M_H2O2 and C_H2O2 in mitochondria and chloroplasts; Calculate the leakage intensity of each subcellular organelle: I = max(M_H2O2, C_H2O2) / t; The intervention frequency F is set according to the formula F = (K1 * I + K2 * C) / (C + K3), where K1 and K3 are adjustment coefficients; Keep F within the range of once every 24 hours to reduce tissue-specific damage.

[0009] In one specific embodiment, the regulation of the menadione-enhanced antioxidant buffer capacity based on the reduced state of key components in the glutathione-ascorbic acid cycle further includes: Determine the GSH / SSG ratio P_GSH; Measure the AsA / DHA ratio P_AsA; The overall cyclic restoration state is calculated using the formula γ = (P_GSH + P_AsA) / 2; If γ < 7, increase the dose of menaquinone by 10% to improve the reduction capacity.

[0010] In one specific embodiment, the method of modulating the response dynamics of the endogenous antioxidant enzyme system further includes: Analyze the time series distributions of SOD, POD, and APX within the time window t; The response function is established based on the asymmetric exponential decay model: A(t) = A_max × exp(t / τ), where τ is the response time constant; A dynamic equilibrium condition Δ = [SOD + POD + APX] (K ×ROS_level) is constructed by combining enzyme activity and reactive oxygen species (ROS) generation, where K is the equilibrium scaling factor. If Δ > 0, maintain the original application plan; otherwise, adjust the menaquinone application strategy.

[0011] In one specific embodiment, the control of the initial ROS burst based on changes in Rboh gene expression and enzyme activity further includes: Set up a standard curve for real-time quantitative PCR detection of Rboh transcripts; Rboh protease activity was measured by ELISA or colorimetric method; The risk level of the initial oxidation reaction is assessed based on the model: R_boh_score = a × (R_mRNA / R_mRNA_0) + b × (R_activity / R_activity_0). When R_boh_score > 8, delay or stop the addition of menaquinone to avoid excessive amplification of the oxidation cascade reaction.

[0012] In one specific embodiment, adjusting the timing and frequency of menadione administration based on the ROS leakage rate in mitochondria and chloroplasts further includes: Extracts were collected from mitochondria and chloroplasts in cell homogenates; ROS levels in mitochondria and chloroplasts were measured using DCFHDA and H2DFF fluorescent probes, respectively. The total cellular ROS leakage rate is calculated using the formula V = (M_H2O2 × f_mito + C_H2O2 × f_chlo), where f_mito and f_chlo are weighting coefficients. Activate high-frequency drug administration protocols to block damage progression when V > V_threshold.

[0013] In one specific embodiment, the method of dynamically enhancing ROS scavenging capacity based on the endogenous antioxidant enzyme system response further includes: The synergistic effect of antioxidant enzymes was estimated using an in-time window integration method, denoted as E_total = ∫_0^T (SOD(t)+POD(t)+APX(t)) dt; An adaptive function F_adaptive(t) = tanh((E_total E_target) / σ) is introduced, where E_target is the target antioxidant level and σ is the sensitivity parameter; Based on the rate of change of menaquinone concentration dc / dt, a dynamic adjustment rule is designed: dc / dt = k1 × F_adaptive(t) k2 × (ROS_current / ROS_base); The above mechanism ensures resource conservation under low oxidative stress and timely enhancement of scavenging capacity under high oxidative stress.

[0014] In one specific embodiment, the regulation of the initial ROS burst based on Rboh expression and enzyme activity further includes: Modeling the activation threshold λ of the Rboh gene using temporal expression data under aluminum stress; Establish an Rboh response dynamic model, in the form dRboh / dt = r × [exp(t / τ_r) exp(t / τ_d)]; Calculate the critical time t_c such that dRboh / dt > 0 and remains stable at ≥ threshold; Based on this, the optimal time window for administering menaquinone can be selected to avoid the occurrence of ROS peaks, thereby achieving early inhibition.

[0015] This disclosure provides a method for mitigating aluminum toxicity in hydroponic peas by regulating reactive oxygen species (ROS) metabolism based on menadione, comprising: regulating the spatiotemporal accumulation pattern of ROS (such as superoxide anions and hydrogen peroxide) in hydroponic pea roots based on menadione treatment concentration and treatment time; enhancing menadione-mediated ROS scavenging capacity by adjusting the response dynamics of endogenous antioxidant enzyme systems (such as SOD, POD, and APX); controlling the initial ROS burst process based on changes in the expression level and enzyme activity of the plasma membrane NADPH oxidase (Rboh) gene to inhibit the oxidative signaling cascade triggered by aluminum stress; and adjusting the timing and frequency of menadione administration based on the ROS leakage rate in mitochondria and chloroplasts at the subcellular scale to alleviate organ-specific oxidative damage. The solution of this disclosure addresses the problem of how to regulate the spatiotemporal accumulation pattern of ROS (such as superoxide anions and hydrogen peroxide) in hydroponic pea roots based on menadione treatment concentration and treatment time to solve aluminum-induced oxidative damage.

[0016] 2. How to regulate the ROS scavenging capacity mediated by menadione based on the dynamic response of endogenous antioxidant enzyme systems (such as SOD, POD, and APX) to address the problem of cellular redox imbalance under aluminum stress.

[0017] 3. How to regulate the initial ROS burst process based on changes in the expression level and enzyme activity of the plasma membrane NADPH oxidase (Rboh) gene to reduce the oxidation signaling cascade triggered by aluminum stress.

[0018] 4. How to regulate the timing and frequency of menadione administration based on the rate of reactive oxygen species leakage in mitochondria and chloroplasts at the subcellular scale to alleviate organ-specific oxidative damage.

[0019] 5. How to regulate the antioxidant buffer capacity enhanced by menadione based on the reduced state of key components in the glutathione-ascorbic acid cycle (AsA-GSH cycle) to address metabolic disorders caused by persistent aluminum toxicity. Attached Figure Description

[0020] To more clearly illustrate the technical solutions of the exemplary embodiments of this disclosure, the accompanying drawings used in the embodiments will be briefly described below. It should be understood that the following drawings only show some embodiments of this disclosure and should not be regarded as a limitation of the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0021] Figure 1 A flowchart illustrating a method for mitigating aluminum toxicity in hydroponic peas based on menadione-mediated regulation of reactive oxygen species metabolism; Figure 2 This is a further flowchart illustrating the regulation of the spatiotemporal accumulation pattern of reactive oxygen species in hydroponic pea roots based on the concentration and duration of menadione treatment; Figure 3 This is a further flowchart illustrating how menadione-mediated ROS scavenging is enhanced by modulating the response dynamics of endogenous antioxidant enzyme systems (such as SOD, POD, and APX); Figure 4 This is a further flowchart illustrating the control of the initial ROS burst process based on changes in the expression level and enzyme activity of the plasma membrane NADPH oxidase (Rboh) gene; Figure 5 This is a further flowchart illustrating the adjustment of the timing and frequency of menadione administration based on the rate of reactive oxygen species leakage in mitochondria and chloroplasts at the subcellular scale; Figure 6 This is a further flowchart illustrating the regulation of the antioxidant buffering capacity enhanced by menadione based on the reduced state of key components in the glutathione-ascorbic acid cycle; Figure 7 This is a further flowchart illustrating the dynamic response of the endogenous antioxidant enzyme system; Figure 8 This is a further flowchart illustrating the control of the initial ROS burst based on changes in Rboh gene expression and enzyme activity; Figure 9 This is a further flowchart illustrating the adjustment of the timing and frequency of menadione administration based on the ROS leakage rate in mitochondria and chloroplasts; Figure 10 This is a further flowchart based on the dynamic enhancement of ROS scavenging capacity through the response of the endogenous antioxidant enzyme system; Figure 11 This is a further flowchart based on the regulation of the initial ROS burst by Rboh expression and enzyme activity. Detailed Implementation

[0022] The embodiments of this disclosure will now be described in detail with reference to the accompanying drawings.

[0023] The following specific examples illustrate the implementation of this disclosure. Those skilled in the art can easily understand other advantages and effects of this disclosure from the content disclosed in this specification. Obviously, the described embodiments are only a part of the embodiments of this disclosure, and not all of them. This disclosure can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of this disclosure. It should be noted that, in the absence of conflict, the following embodiments and features in the embodiments can be combined with each other. Based on the embodiments in this disclosure, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this disclosure.

[0024] It should be noted that various aspects of embodiments within the scope of the appended claims are described below. It will be apparent that the aspects described herein can be embodied in a wide variety of forms, and any particular structure and / or function described herein is merely illustrative. Based on this disclosure, those skilled in the art will understand that one aspect described herein can be implemented independently of any other aspect, and two or more of these aspects can be combined in various ways. For example, any number of aspects set forth herein can be used to implement the device and / or practice the method. Additionally, this device and / or method can be implemented using structures and / or functionalities other than one or more of the aspects set forth herein.

[0025] It should also be noted that the illustrations provided in the following embodiments are only schematic representations of the basic concept of this disclosure. The drawings only show the components related to this disclosure and are not drawn according to the actual number, shape and size of the components in the actual implementation. In the actual implementation, the form, quantity and proportion of each component can be arbitrarily changed, and the layout of the components may also be more complex.

[0026] Furthermore, specific details are provided in the following description to facilitate a thorough understanding of the examples. However, those skilled in the art will understand that the described aspects can be practiced without these specific details.

[0027] Next, referring to the accompanying drawings, a method for alleviating aluminum toxicity in hydroponic peas based on the regulation of reactive oxygen species (ROS) metabolism using menadione according to the present invention is described. This method mainly includes five core steps: regulating the spatiotemporal accumulation pattern of ROS (such as superoxide anion and hydrogen peroxide) in hydroponic pea roots according to the concentration and duration of menadione treatment; enhancing the menadione-mediated ROS scavenging capacity by adjusting the response dynamics of endogenous antioxidant enzyme systems (such as SOD, POD, and APX); controlling the initial ROS burst process based on the expression level and enzyme activity changes of the plasma membrane NADPH oxidase (Rboh) gene to inhibit the oxidative signaling cascade triggered by aluminum stress; and adjusting the timing and frequency of menadione administration according to the ROS leakage rate in mitochondria and chloroplasts at the subcellular scale to alleviate organ-specific oxidative damage. The beneficial effects of this invention are that by precisely regulating ROS metabolism, it improves the tolerance of pea roots to aluminum stress, maintains intracellular and extracellular redox balance, reduces tissue damage, and improves growth performance.

[0028] The method for regulating the spatiotemporal accumulation pattern of reactive oxygen species (ROS) in hydroponic pea roots based on the concentration and duration of menadione treatment first requires detecting the contents of superoxide anions (O2^-) and hydrogen peroxide (H2O2) in the roots under different time periods and concentrations. Quantitative analysis is performed using fluorescent probes and chemiluminescence methods, and the correlation between concentration, time, and ROS levels is established. Regional detection of different parts of the root system determines how menadione treatment guides the changes in ROS in different sections, thereby achieving controllable ROS accumulation. Specifically, in the experimental design, different concentrations of menadione (0.1 μM to 1.0 μM) can be set up, and samples are taken at different treatment times (e.g., 2 h, 6 h, 12 h) to analyze the changing trends of ROS levels. This allows for the construction of an optimal spatiotemporal control model for the scientific planning of subsequent treatments.

[0029] Enhanced menadione-mediated ROS scavenging was achieved by modulating the response dynamics of endogenous antioxidant enzyme systems (such as SOD, POD, and APX). This involved measuring the activities of SOD, POD, and APX in roots before and after menadione treatment and observing their dynamic changes under aluminum stress. The activities of these enzymes were maximized by adjusting menadione treatment conditions, such as treatment time and dosage. For example, appropriate amounts of copper or manganese ions could be supplemented immediately after menadione treatment to stimulate SOD activity, or antioxidants could be used to enhance APX activity. This synergistic regulatory strategy helps to rapidly activate the root's own antioxidant network after menadione-induced local ROS increases, thereby reducing the risk of excessive oxidative stress caused by aluminum.

[0030] To control the initial ROS burst and inhibit the aluminum stress-triggered oxidative signaling cascade by controlling changes in the expression level and enzyme activity of the plasma membrane NADPH oxidase (Rboh) gene, pea root samples must be collected before and after treatment. RNA is extracted, and the transcriptional level of Rboh-related genes is determined using RT-PCR or qPCR. Changes in enzyme activity are then detected using spectrophotometry or fluorescent probes. Under aluminum stress, Rboh is activated, producing a large amount of ROS, which further intensifies intracellular oxidative signals. Therefore, the timing of menadione's action should be controlled based on Rboh gene expression. For example, menadione can be introduced before or at the initial stage of aluminum stress to delay or block the initiation of ROS production and reduce the amplification effect of subsequent signaling cascades.

[0031] To mitigate organ-specific oxidative damage, adjusting the timing and frequency of menadione administration based on the reactive oxygen species (ROS) leakage rate in mitochondria and chloroplasts at the subcellular scale requires monitoring ROS accumulation in root mitochondria and chloroplasts using fluorescent labeling techniques or other specific dyes. When abnormal ROS leakage is detected in specific areas, the dosage and interval of menadione administration should be adjusted accordingly. For example, menadione solution can be periodically injected into a hydroponic system containing a certain proportion of aluminum salts. By monitoring changes in fluorescence signals in mitochondria and chloroplasts, the administration frequency can be precisely controlled to prevent irreversible ROS accumulation in critical physiological structures.

[0032] To address the metabolic disorders caused by persistent aluminum toxicity, the antioxidant buffer capacity of menadione can be regulated based on the reduced state of key components in the glutathione-ascorbic acid (AsA-GSH) cycle. This requires measuring changes in the concentrations of components such as AsA and GSH, particularly the ratio of their oxidized to reduced states. If significant depletion of a key component in this cycle is observed during treatment, the corresponding component should be replenished promptly, or the menadione administration regimen should be modified to maintain cycle stability. Specifically, adding small amounts of glutathione or other reducing agents to the water can serve as an adjunct intervention, ensuring the dynamic balance of the entire metabolic system is maintained and reducing the cumulative toxicity caused by long-term aluminum exposure.

[0033] Next, the invention further describes the regulation of the spatiotemporal accumulation pattern of reactive oxygen species (ROS) in hydroponic pea roots based on the concentration and duration of menadione treatment. The oxidative stress index (OI) of the root surface is obtained, which quantifies the accumulation level of ROS in the roots. OI is typically obtained by colorimetric methods or fluorescent probes to detect the content of reactive oxygen species such as superoxide anions and hydrogen peroxide, and its range is generally between 0 and 100. OI0 represents the baseline level without aluminum stress, typically around 20-30, and is an important basis for setting initial treatment conditions.

[0034] The initial treatment concentration C0 and time T0 of menadione were set. C0 was based on the safe concentration range obtained from previous experiments, usually selected between 0.1-1.0 mmol / L to avoid cytotoxicity; T0 was usually set to 1-3 hours to ensure effective drug penetration without causing root damage. The treatment concentration and time combination of menadione were adjusted according to the formula C_T = C0 × (OI / OI0), where OI is the current oxidative stress level and OI0 is the baseline value. This formula aims to dynamically balance the accumulation and scavenging of reactive oxygen species (ROS) to prevent further ROS accumulation to the toxic threshold.

[0035] Different gradient treatment schemes were set according to the adjusted C_T. For example, in one experiment, when OI reached 60 and OI0 was set to 30, C_T = 0.5 × (60 / 30) = 1.0 mmol / L. Under this scheme, the concentration of menadione was increased to 1.0 mmol / L, and the treatment time was 2 hours. Through this regulation method, the peak accumulation of superoxide anion can be significantly inhibited, and the lipid peroxidation reaction of root cell membranes can be reduced, thereby mitigating the impact of aluminum toxicity on hydroponic peas.

[0036] This technical solution can flexibly adjust the dosage and timing of the drug according to the real-time root oxidation state, improve the regulatory efficiency of menadione in antioxidant metabolism, effectively delay root tissue damage, and improve the aluminum tolerance of hydroponic peas.

[0037] Next, the invention further describes how it enhances menadione-mediated ROS scavenging by modulating the response dynamics of endogenous antioxidant enzyme systems (such as SOD, POD, and APX). First, SOD activity (S_0) and APX activity (A_0) are measured to understand the initial state of the antioxidant enzymes in plants before menadione treatment, which helps in subsequent analysis of their response characteristics to menadione stress. S_0 and A_0 are typically determined by colorimetric or fluorescence detection methods, with SOD activity ranging from 100-500 U / g FW and APX activity ranging from 200-800 U / g FW. This step provides a data basis for evaluating the effects of subsequent adjustments.

[0038] Secondly, a dynamic response coefficient β = min(S_0 / S_ref, A_0 / A_ref) is introduced, where S_ref is a reference value for SOD activity, such as 400 U / g FW, and A_ref is a reference value for APX activity, such as 600 U / g FW. This coefficient reflects the relative response capacity of the plant's own antioxidant system in the face of stress. If S_0 or A_0 is higher than the corresponding reference value, the β value is close to 1; otherwise, it is less than 1, used to quantify the degree of stress resistance. This step provides a basis for subsequent calculation of the stress resistance buffer factor.

[0039] Next, the stress buffer factor RF is calculated as: RF = β × (C / T + k1 × √T), where C is the concentration of menadione (recommended range 5-20 μM), T is the treatment time (optimal time range 12-24 hours), and k1 is the correction factor (preferably 0.5). The first part, C / T, represents the drug concentration contribution per unit time, and the second part, k1 × √T, is used to correct for the nonlinear effect of treatment time. The overall formula is designed to maximize ROS clearance efficiency while controlling costs, ensuring the drug achieves maximum efficacy without over-application.

[0040] Finally, by maximizing the RF (Radiative Reduction) to optimize the application parameters, the optimal dosage and timing of menadione were determined to achieve efficient ROS removal. For example, in one embodiment, if S_0 = 300 U / g FW, A_0 = 500 U / g FW, C = 10 μM, T = 18 hours, and k1 = 0.5, then β = min(300 / 400, 500 / 600) = 0.75, RF = 0.75 × (10 / 18 + 0.5 × √18) ≈ 1.36. This indicates that using these parameters can effectively enhance the antioxidant capacity of hydroponic peas in an aluminum-toxic environment, alleviate aluminum-induced oxidative damage, improve survival rate, and enhance growth status, demonstrating promising application prospects.

[0041] Next, the initial ROS burst process controlled by the present invention based on changes in the expression level and enzyme activity of the plasma membrane NADPH oxidase (Rboh) gene will be further described. First, the amount of Rboh transcript R_mRNA is detected to quantify the expression of this gene at the mRNA level. Second, Rboh enzyme activity R_activity is monitored, measured in U / mg protein, reflecting the enzyme's catalytic ability on the cell membrane. Subsequently, the formula α = ln(R_activity + 1) / (R_mRNA / R_mRNA_0) is used, where R_mRNA_0 represents the mRNA content of the control group. In this formula, R_mRNA is the mRNA content of the experimental group, R_mRNA_0 represents the basal expression level of Rboh under normal conditions, and ln(R_activity + 1) is used to prevent calculation errors caused by zero enzyme activity. The value of α varies depending on the fluctuations in R_activity and R_mRNA, with an ideal range of 0.5-1.5. This formula is based on the analysis of the initial ROS burst kinetics. By combining two indicators—mRNA and enzyme activity—the activation state of Rboh is accurately assessed. Finally, the application frequency of menadione is adjusted according to the α value to keep it within a range that inhibits the initial oxidative signal without over-activating ROS, thereby maintaining the physiological balance of the plant. For example, in one embodiment, under aluminum stress, the expression of the Rboh gene in hydroponic peas increased and enzyme activity was enhanced, with the α value increasing to 1.2. At this point, adjusting the application interval of menadione from 24 hours to 36 hours effectively alleviated the excessive accumulation of ROS caused by aluminum toxicity. This technical approach helps to precisely regulate reactive oxygen species metabolism, reduce oxidative damage, and improve plant stress resistance.

[0042] Next, the timing and frequency of menadione addition based on the leakage rate of reactive oxygen species (ROS) in mitochondria and chloroplasts at the subcellular scale will be further described. First, the H2O2 concentrations M_H2O2 and C_H2O2 in mitochondria and chloroplasts are obtained, where M_H2O2 represents the hydrogen peroxide concentration in mitochondria and C_H2O2 represents the hydrogen peroxide concentration in chloroplasts, typically in the range of 0.1-10 μM, obtained by real-time fluorescence imaging or enzymatic methods. Then, the leakage intensity of each subcellular organelle is calculated as I = max(M_H2O2, C_H2O2) / t, where t is the time period (e.g., hours). I reflects the relative leakage level of ROS in different organelles; a higher value indicates more severe ROS production. For example, in one embodiment, when the mitochondrial H2O2 concentration is detected to rise to 5 μM while the chloroplast concentration is only 1 μM, M_H2O2 is substituted into the formula. The intervention frequency F was then set according to the formula F = (K1 * I + K2 * C) / (C + K3), where K1, K2, and K3 are control coefficients. Generally, K1 is between 0.1 and 1.0, K2 is between 0.01 and 0.1, and K3 is between 0.5 and 5, used to balance the necessity of intervention and potential stimulation to the plant. C represents the overall aluminum concentration or other indicators of the plant tissue. The formula was designed to consider the reactive oxygen species leakage intensity I and the internal environmental factors C of the tissue, ensuring that the intervention is both effective and safe. Finally, F was controlled within the range of once every 24 hours. By adjusting the dosage frequency, tissue-specific damage caused by high doses of menadione was reduced, improving its safety and applicability.

[0043] This technical solution achieves dynamic regulation of aluminum toxicity in hydroponic peas by precisely monitoring the reactive oxygen species (ROS) status in different subcellular organelles. It helps reduce the adverse effects of menadione abuse, enhances the synergistic effect of plant antioxidant defense mechanisms, and strengthens the system's adaptability under complex growth conditions.

[0044] Next, the antioxidant buffer capacity enhanced by menadione, based on the regulation of the reduced state of key components in the glutathione-ascorbic acid cycle, is further described in the present invention. First, the GSH / SSG ratio, P_GSH, is measured. This ratio reflects the reduced state of glutathione, where GSH is reduced glutathione and SSG is oxidized glutathione. A higher P_GSH indicates stronger intracellular antioxidant capacity, typically ranging from 2 to 10, with an optimal value of 5-7. Next, the AsA / DHA ratio, P_AsA, is measured. AsA is ascorbic acid, and DHA is its oxidized form. This ratio reflects the reducing capacity of ascorbic acid, typically ranging from 3 to 15, with an optimal value of 6-10. Then, the reduced state of the entire glutathione-ascorbic acid cycle is calculated using the formula γ = (P_GSH + P_AsA) / 2. This formula comprehensively evaluates the reducing capacity of the antioxidant system by averaging the ratio of the two key components. If γ < 7, it indicates that the antioxidant buffer capacity is insufficient and the dose of menadione needs to be increased by 10% to improve it.

[0045] For example, in one embodiment, after treating hydroponic pea seedlings with aluminum stress, the GSH / SSG ratio was found to be 4 and the AsA / DHA ratio to be 5. Substituting these values ​​into the formula, γ = (4 + 5) / 2 = 4.5, which is below the threshold of 7. Therefore, the concentration of menadione needs to be adjusted to enhance the cells' ability to resist reactive oxygen species. This technical solution can dynamically adjust the dosage of menadione, improve system adaptability and protective efficiency, reduce drug waste and toxic side effects, and has good application prospects.

[0046] Next, the present invention further describes the dynamic response of the endogenous antioxidant enzyme system by regulating its response. The time-series distributions of SOD, POD, and APX within a time window t are analyzed to monitor the activity changes of these antioxidant enzymes at different time points, thereby understanding their response patterns. This step provides a data foundation for subsequent modeling; for example, samples are collected within 24 hours after menadione treatment to detect fluctuations in the activities of SOD, POD, and APX to determine their response curves. A response function is established based on an asymmetric exponential decay model: A(t) = A_max × exp(t / τ), where A_max represents the maximum response value of the enzyme, t is time, and τ is the response time constant, typically ranging from 0.1 to 5 hours, with an optimal value of approximately 1 hour. This formula simulates the dynamic process of enzyme response, showing that the response gradually reaches its maximum and tends to stabilize over time, consistent with the actual response characteristics of biological systems. A dynamic equilibrium condition Δ = [SOD + POD + APX] - (K × ROS_level) is constructed by combining enzyme activity and reactive oxygen species (ROS) generation, where K is the equilibrium scaling factor, typically ranging from 0.5 to 2, with an optimal value of 1. This condition quantifies the antioxidant system's ability to cope with ROS levels and determines whether the strategy needs adjustment. When Δ > 0, the original application plan is maintained, indicating that the current enzyme system can effectively neutralize ROS; otherwise, the menadione application strategy needs adjustment. For example, in one experiment, Δ < 0 indicates insufficient antioxidant system, requiring an extension or increase in the menadione concentration to regulate ROS levels and protect hydroponic peas from aluminum toxicity. This approach can precisely control the dynamic response of the antioxidant system, enhance the plant's stress adaptation ability, and ensure a safe and effective mitigation effect.

[0047] Next, the invention further describes the control of ROS initial burst based on changes in Rboh gene expression and enzyme activity. A standard curve for real-time quantitative PCR detection of Rboh transcripts is set up to quantitatively analyze the expression level of the Rboh gene under different treatment conditions. The standard curve obtained by this method can be used to calculate the ratio of R_mRNA to R_mRNA_0, thereby reflecting the changes in gene expression. Specifically, R_mRNA is the amount of Rboh transcripts in the treated sample, and R_mRNA_0 is the baseline value of the control group. The ratio of the two is usually between 0.1 and 10 to determine whether Rboh is stimulated or inhibited.

[0048] Measuring Rboh protease activity using ELISA or colorimetric methods quantifies the catalytic ability of the Rboh protein, allowing for the evaluation of its functional state. This method boasts high sensitivity and specificity, accurately reflecting changes in enzyme activity. For instance, this step can identify dynamic changes when aluminum stress leads to a significant increase in Rboh activity in cells. Based on this, the formula R_boh_score = a × (R_mRNA / R_mRNA_0) + b × (R_activity / R_activity_0) combines information from both gene expression and protein activity, where a and b are coefficients used to balance the weights of gene expression and enzyme activity. Optimal values ​​are 0.6 and 0.4, respectively, to ensure a reasonable contribution of both to the score. This formula aims to assess the initial risk of the oxidation reaction.

[0049] When R_boh_score > 8, it indicates that the initial ROS surge may be excessive, requiring intervention. Delaying or stopping the addition of menadione at this point can prevent further ROS expansion and reduce oxidative damage to plants. In one embodiment, when a significant increase in Rboh expression and enzyme activity is detected in the roots of hydroponic peas, adjusting the menadione concentration to keep the R_boh_score below a safe threshold effectively mitigates the effects of aluminum toxicity and improves plant survival rate. This approach, through dynamic monitoring and regulation of ROS release, optimizes the aluminum resistance mechanism and improves the precision and adaptability of the overall antioxidant system.

[0050] Next, the timing and frequency of menadione addition based on the ROS leakage rate in mitochondria and chloroplasts will be further described. Extracts from mitochondria and chloroplasts are collected from cell homogenates; this step is used to obtain samples of specific organelles to assess their internal ROS levels. ROS levels in mitochondria and chloroplasts are measured using DCFHDA and H2DFF fluorescent probes, respectively, with fluorescence intensity reflecting the amount of ROS produced. The total cellular ROS leakage rate is calculated using the formula V = (M_H2O2 × f_mito + C_H2O2 × f_chlo), where M_H2O2 is the hydrogen peroxide concentration in mitochondria (range 0.1-10 μM), C_H2O2 is the hydrogen peroxide concentration in chloroplasts (range 0.1-5 μM), and f_mito and f_chlo are the weighting coefficients for mitochondria and chloroplasts, respectively, with suggested values ​​of 0.6-0.8 and 0.2-0.4. This formula comprehensively considers the contribution ratio of ROS in both organelles, ensuring more representative evaluation results. A high-frequency application regimen is initiated to block damage propagation when V > V_threshold, a critical value determined based on plant tolerance thresholds, such as 0.5 μM / min. Intervention measures are initiated when the ROS leakage rate exceeds this value.

[0051] For example, in one embodiment, when the ROS leakage rates of mitochondria and chloroplasts in hydroponic pea root cells are detected to reach 3 μM and 1.2 μM, respectively, and weighted by coefficients of 0.7 and 0.3, V = 3 × 0.7 + 1.2 × 0.3 = 2.46 μM / min is calculated, which is significantly higher than V_threshold. The system will then automatically activate a high-frequency application program, allowing menadione to act on the cells more promptly and effectively, inhibiting excessive ROS accumulation and thus alleviating aluminum toxicity symptoms. This method allows for precise adjustment according to actual needs, avoiding resource waste and improving the plant's response efficiency to stress, demonstrating significant beneficial effects.

[0052] Next, the dynamic enhancement of ROS scavenging capacity based on the endogenous antioxidant enzyme system response of the present invention is further described. First, the synergistic effect of antioxidant enzymes is estimated using an integral method within a time window, denoted as E_total = ∫_0^T(SOD(t)+POD(t)+APX(t)) dt. In this formula, SOD, POD, and APX represent the activity levels of superoxide dismutase, peroxidase, and ascorbic acid peroxidase at time t, respectively, where T is the observation period, generally set between 24 and 72 hours to cover the critical stages of oxidative stress. E_total reflects the comprehensive contribution of antioxidant enzymes over a period of time. For example, after hydroponic peas are toxiced by aluminum, the activities of SOD, POD, and APX may rise rapidly within several hours; this integral value can be used to determine the overall response intensity.

[0053] Next, we introduce the adaptive function F_adaptive(t) = tanh((E_total E_target) / σ), where E_target is the target antioxidant level, usually derived from experimental data. It is recommended to set it as the average value of E_total under non-toxic conditions, typically ranging from 1.5 to 3.0 (units adjusted according to the specific experimental design). σ is a sensitivity parameter, with a value ranging from 0.5 to 1.0. This function quantifies the relative relationship between the current antioxidant capacity and the target level, achieving a smooth transition through the nonlinear characteristics of the hyperbolic tangent function, preventing overly aggressive or sluggish responses. For example, in the early stages of ROS increase detected at the roots of hydroponic peas, F_adaptive(t) will increase slowly, prompting the system to gradually strengthen its cleanup mechanism.

[0054] Then, combining the concentration change rate of menadione (dc / dt), a dynamic adjustment rule was designed: dc / dt = k1 × F_adaptive(t) - k2 × (ROS_current / ROS_base). Here, k1 is the promoting factor coefficient, set to 0.01 to 0.05; k2 is the inhibiting factor coefficient, set to 0.02 to 0.08, used to prevent secondary damage caused by excessively high concentrations. ROS_current represents the real-time detected ROS content, and ROS_base is the baseline level. This formula achieves precise control by dynamically changing the supply of menadione with oxidative stress through feedback regulation.

[0055] The above mechanism ensures resource conservation under low oxidative stress and timely enhancement of scavenging capacity under high oxidative stress. This method not only improves the tolerance of hydroponic peas to aluminum toxicity but also optimizes the allocation of metabolic resources and reduces energy waste. For example, in cases of mild aluminum toxicity in hydroponic peas, the application rate of menadione will be reduced, thereby protecting the plants from unnecessary metabolic burden; while under severe aluminum stress, the system automatically increases the release of menadione, significantly enhances the activity of enzymes such as SOD, POD, and APX, accelerates ROS elimination, and thus delays the problems of leaf chlorosis and restricted root growth.

[0056] Next, the invention further describes the regulation of the initial ROS burst based on Rboh expression and enzyme activity. First, the initiation threshold λ is modeled using temporal expression data of the Rboh gene under aluminum stress. This step aims to determine the critical point at which the Rboh gene begins to respond to aluminum stress. λ represents the value at which Rboh expression initiates the ROS burst. Typically, λ ranges from 0.5 to 1.2, with an optimal value of 1.0, used to accurately determine the initial response signal. For example, when the Rboh protein content is detected to rise to a critical value under aluminum stress, it means that the plant will activate a protective antioxidant system.

[0057] Secondly, an Rboh response kinetic model was established, in the form dRboh / dt = r × [exp(t / τ_r) / exp(t / τ_d)]. This model describes the change in Rboh gene expression over time, where r represents the expression rate, typically ranging from 0.1 to 0.5, with an optimal value of 0.3; τ_r and τ_d represent the activation and inhibition time constants, respectively, set at 0.5–2.0 and 1.0–3.0, with optimal values ​​of 1.0 and 2.0. This formula design allows for the dynamic simulation of the Rboh gene's attenuation after enhancement over time, thereby predicting its changing trend.

[0058] Then, the critical time t_c is calculated, ensuring that dRboh / dt > 0 and remains stable at or above the threshold. The purpose of this step is to find a stable trigger point to control the timing of menadione intervention. If dRboh / dt becomes consistently above the threshold at time t_c, it indicates that the ROS initiation phase is about to end. In one embodiment, setting t_c to 3 hours ensures early intervention and avoids the peak period.

[0059] Finally, based on this, the optimal time window for adding menadione was selected to avoid the ROS peak, thus achieving early inhibition. This method can effectively reduce the impact of aluminum toxicity on pea seedlings and reduce ROS damage to cells. Specifically, adding menadione within 2 hours of the onset of aluminum stress avoids the aggravation of oxidative stress and improves plant survival rate. This process significantly improves the crop's adaptability to adversity in the hydroponic system and enhances its stress resistance.

[0060] This invention discloses a method for mitigating aluminum toxicity in hydroponic peas based on menadione-mediated reactive oxygen species (ROS) metabolism. The method involves: firstly, precisely controlling the concentration and duration of menadione treatment to regulate the spatial and temporal accumulation patterns of ROS in the roots of hydroponic peas, thereby inhibiting aluminum-induced excessive oxidation. Subsequently, by regulating the dynamic response of the plant's endogenous antioxidant enzyme system (such as SOD, POD, and APX), the method enhances its ability to scavenge ROS mediated by menadione, balancing the redox state and alleviating cell damage caused by aluminum stress. Further, based on the expression level and enzyme activity changes of the plasma membrane NADPH oxidase (Rboh) gene, the method regulates the initial ROS burst process, reducing the oxidative signaling cascade triggered by aluminum stress and preventing the spread of secondary damage. Simultaneously, based on the ROS leakage rate in mitochondria and chloroplasts at the subcellular scale, the timing and frequency of menadione administration are adjusted to specifically protect key subcellular organelles and reduce organ-specific oxidative damage. Finally, this invention also combines the reduced state of key components of the glutathione-ascorbic acid cycle (AsA-GSH cycle) to dynamically regulate the antioxidant buffer capacity enhanced by menadione, enabling plants to better cope with metabolic disorders caused by persistent aluminum toxicity, thereby comprehensively improving the aluminum tolerance and growth stability of hydroponic peas.

[0061] This invention provides systematic solutions to five core technical problems. First, by regulating the concentration and duration of menadione treatment, the generation and distribution of ROS are precisely controlled, avoiding excessive accumulation that could damage cell structure and function. Second, by dynamically adjusting the endogenous antioxidant enzyme system, the efficiency of the scavenging mechanism is enhanced, achieving more efficient oxidative stress defense. Third, by influencing the relevant regulatory mechanisms of NADPH oxidase, the initial formation of ROS is controlled, thereby inhibiting subsequent oxidative signaling cascade reactions and reducing the degree of oxidative stress. Fourth, by monitoring and regulating the ROS leakage rate at the subcellular level, the application of menadione is ensured to exert maximum effectiveness at the optimal time, avoiding irreversible damage to key organelles. Fifth, by combining the state of the AsA-GSH cycle, the plant's own antioxidant system is further optimized, giving it a stronger ability to cope with persistent aluminum toxicity stress. Overall, a multi-level, multi-dimensional oxidative stress prevention and control system is constructed.

[0062] The specific embodiments described above further illustrate the purpose, technical solutions, and beneficial effects of the embodiments of this disclosure. It should be understood that the above descriptions are merely specific embodiments of the embodiments of this disclosure and are not intended to limit the scope of protection of the embodiments of this disclosure. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the embodiments of this disclosure should be included within the scope of protection of the embodiments of this disclosure.

Claims

1. A method for alleviating aluminum toxicity in hydroponic peas based on the regulation of reactive oxygen species metabolism by menaquinone, characterized in that... ,include: The spatiotemporal accumulation patterns of reactive oxygen species (such as superoxide anions and hydrogen peroxide) in hydroponic pea roots were regulated based on the concentration and duration of menadione treatment. Enhanced menadione-mediated ROS scavenging capacity by modulating the response dynamics of endogenous antioxidant enzyme systems such as SOD, POD, and APX; The initial ROS burst process was controlled by controlling changes in plasma membrane NADPH oxidase (Rboh) gene expression level and enzyme activity to inhibit the oxidation signaling cascade triggered by aluminum stress; The timing and frequency of menadione administration were adjusted based on the rate of reactive oxygen species leakage in mitochondria and chloroplasts at the subcellular scale to alleviate organ-specific oxidative damage.

2. The method for alleviating aluminum toxicity in hydroponic peas based on the regulation of reactive oxygen species metabolism by menaquinone according to claim 1, characterized in that... The regulation of the spatiotemporal accumulation pattern of reactive oxygen species in hydroponic pea roots based on the concentration and duration of menadione treatment further includes: Obtain the oxidative stress index (OI) of the root surface; The initial treatment concentration C0 and time T0 of menadione were set. The concentration and time combination C_T is adjusted according to the following formula: C_T = C0 × (OI / OI0), where OI0 represents the standard oxidative stress index without aluminum stress; Different gradient treatment schemes were set according to the adjusted C_T to suppress the cumulative peak of superoxide anions.

3. The method for alleviating aluminum toxicity in hydroponic peas based on the regulation of reactive oxygen species metabolism by menaquinone according to claim 2, characterized in that... The enhancement of menadione-mediated ROS scavenging capacity by modulating the response dynamics of endogenous antioxidant enzyme systems (such as SOD, POD, and APX) further includes: Measure SOD activity S_0 and APX activity A_0; A dynamic response coefficient β = min(S_0 / S_ref, A_0 / A_ref) is introduced, where S_ref and A_ref are reference values ​​for the corresponding enzyme activities; The stress buffer factor RF was calculated as β × (C / T + k1 × √(T)), where k1 is the correction coefficient, C is the concentration of menaquinone, and T is the treatment time. Optimize application parameters by RF to enhance ROS clearance efficiency.

4. The method for alleviating aluminum toxicity in hydroponic peas based on the regulation of reactive oxygen species metabolism by menaquinone according to claim 3, characterized in that... The method of controlling the initial ROS burst process based on changes in plasma membrane NADPH oxidase (Rboh) gene expression level and enzyme activity further includes: Detecting the amount of Rboh transcript R_mRNA; Monitor Rboh enzyme activity (R_activity), unit: U / mg protein; Based on the formula α = ln(R_activity + 1) / (R_mRNA / R_mRNA_0), where R_mRNA_0 represents the mRNA content of the control group; The frequency of menadione application is controlled by α to keep it within a range that can suppress initial oxidation signals without over-activating ROS.

5. The method for alleviating aluminum toxicity in hydroponic peas based on the regulation of reactive oxygen species metabolism by menaquinone according to claim 4, characterized in that... The adjustment of the timing and frequency of menadione administration based on the rate of reactive oxygen species leakage in mitochondria and chloroplasts at the subcellular scale further includes: Obtain the H2O2 concentrations M_H2O2 and C_H2O2 in mitochondria and chloroplasts; Calculate the leakage intensity of each subcellular organelle: I = max(M_H2O2, C_H2O2) / t; The intervention frequency F is set according to the formula F = (K1 * I + K2 * C) / (C + K3), where K1 and K3 are adjustment coefficients; Keep F within the range of once every 24 hours to reduce tissue-specific damage.

6. The method for alleviating aluminum toxicity in hydroponic peas based on the regulation of reactive oxygen species metabolism using menadione, as described in claim 5, is characterized in that... The regulation of the enhanced antioxidant buffer capacity of menadione based on the reduced state of key components in the glutathione-ascorbic acid cycle further includes: Determine the GSH / SSG ratio P_GSH; Measure the AsA / DHA ratio P_AsA; The overall cyclic restoration state is calculated using the formula γ = (P_GSH + P_AsA) / 2; If γ < 7, increase the dose of menaquinone by 10% to improve the reduction capacity.

7. The method for alleviating aluminum toxicity in hydroponic peas based on the regulation of reactive oxygen species metabolism by menaquinone according to claim 2, characterized in that... The method of regulating the response dynamics of the endogenous antioxidant enzyme system further includes: Analyze the time series distributions of SOD, POD, and APX within the time window t; The response function is established based on the asymmetric exponential decay model: A(t) = A_max × exp(t / τ), where τ is the response time constant; A dynamic equilibrium condition Δ = [SOD + POD + APX] (K ×ROS_level) is constructed by combining enzyme activity and reactive oxygen species (ROS) generation, where K is the equilibrium scaling factor. If Δ > 0, maintain the original application plan; otherwise, adjust the menaquinone application strategy.

8. The method for alleviating aluminum toxicity in hydroponic peas based on the regulation of reactive oxygen species metabolism by menaquinone according to claim 3, characterized in that... The control of the initial ROS burst based on changes in Rboh gene expression and enzyme activity further includes: Set up a standard curve for real-time quantitative PCR detection of Rboh transcripts; Rboh protease activity was measured by ELISA or colorimetric method; The risk level of the initial oxidation reaction is assessed based on the model: R_boh_score = a × (R_mRNA / R_mRNA_0) + b × (R_activity / R_activity_0). When R_boh_score > 8, delay or stop the addition of menaquinone to avoid excessive amplification of the oxidation cascade reaction.

9. A method for alleviating aluminum toxicity in hydroponic peas based on the regulation of reactive oxygen species metabolism using menadione, as described in claim 4, is characterized in that... The adjustment of the timing and frequency of menadione administration based on the ROS leakage rate in mitochondria and chloroplasts further includes: Extracts were collected from mitochondria and chloroplasts in cell homogenates; ROS levels in mitochondria and chloroplasts were measured using DCFHDA and H2DFF fluorescent probes, respectively. The total cellular ROS leakage rate is calculated using the formula V = (M_H2O2 × f_mito + C_H2O2 × f_chlo), where f_mito and f_chlo are weighting coefficients. Activate high-frequency drug administration protocols to block damage progression when V > V_threshold.

10. A method for alleviating aluminum toxicity in hydroponic peas based on the regulation of reactive oxygen species metabolism using menadione, as described in claim 6, characterized in that... The enhanced ROS scavenging capacity based on the dynamic response of the endogenous antioxidant enzyme system further includes: The synergistic effect of antioxidant enzymes was estimated using an in-time window integration method, denoted as E_total = ∫_0^T (SOD(t)+POD(t)+APX(t)) dt; An adaptive function F_adaptive(t) = tanh((E_total E_target) / σ) is introduced, where E_target is the target antioxidant level and σ is the sensitivity parameter; Based on the rate of change of menaquinone concentration dc / dt, a dynamic adjustment rule is designed: dc / dt = k1 × F_adaptive(t) k2 × (ROS_current / ROS_base); The above mechanism ensures resource conservation under low oxidative stress and timely enhancement of scavenging capacity under high oxidative stress.

11. The method for alleviating aluminum toxicity in hydroponic peas based on the regulation of reactive oxygen species metabolism by menaquinone according to claim 7, characterized in that... The regulation of the initial ROS burst based on Rboh expression and enzyme activity further includes: Modeling the activation threshold λ of the Rboh gene using temporal expression data under aluminum stress; Establish an Rboh response dynamic model, in the form dRboh / dt = r × [exp(t / τ_r) exp(t / τ_d)]; Calculate the critical time t_c such that dRboh / dt > 0 and remains stable at ≥ threshold; Based on this, the optimal time window for administering menaquinone can be selected to avoid the occurrence of ROS peaks, thereby achieving early inhibition.

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