Method for evaluating melatonin for relieving attenuation toxicity of environmental pollutants

Abstract

The invention relates to the technical field of environmental health risk evaluation, in particular to a method for evaluating melatonin to relieve attenuation toxicity of environmental pollutants. The method comprises the following steps: by taking caenorhabditis elegans as an animal model and 6: 2 chloro polyfluoroalkyl ether sulfonate as an environmental pollutant, setting a blank control group, a 10ng / L environmental pollutant exposure liquid group, a 10ng / L environmental pollutant + 0.1 mu mol / L melatonin exposure liquid group, a 10ng / L environmental pollutant + 1 mu mol / L melatonin exposure liquid group and the like to carry out a subacute exposure experiment, and after the subacute exposure experiment is finished, carrying out a subacute exposure experiment on the caenorhabditis elegans model. Measuring lipofuscin level, motor ability, abnormal motor behavior, alpha-synuclein protein expression and aggregation, DAT-1 expression quantity and active oxygen level of the caenorhabditis elegans, and observing and counting dopaminergic neurons of the caenorhabditis elegans, so as to assess that the melatonin relieves the senescence toxicity of environmental pollutants. By constructing a behavior characteristic and molecular characteristic scheme and combining caenorhabditis elegans data, an aging and PD toxicity evaluation model is generated, and comprehensive aging and PD toxicity evaluation from behaviors to micromolecules is realized.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of environmental health risk assessment, and particularly relates to a method for evaluating melatonin to alleviate the senolytic toxicity of environmental pollutants. BACKGROUND

[0002] Senescence refers to the irreversible process of physiological function progressive decline of organisms with age, and is also an important pathogenic factor for the occurrence and development of neurodegenerative diseases. Parkinson's disease (PD) is a neurodegenerative disease that often occurs in the elderly population. In recent years, the risk of environmental pollutants inducing organism senescence and PD has gradually become a research hotspot in environmental toxicology. Studies have shown that environmental pollutants can induce nematode senescence and PD-like phenotype, and have potential senolytic and PD-inducing risks for human populations. Melatonin (MT) is an endogenous multifunctional molecule, and its antioxidant, anti-inflammatory and mitochondrial protection properties show unique potential in anti-aging and neurodegenerative diseases, and its research in the treatment of neurodegenerative diseases has attracted much attention in recent years. Although animal experiments and some clinical trials have shown the safety and efficacy of MT in delaying aging, existing trials are still limited. The alleviating effect of MT on the senolytic toxicity of environmental pollutants is still unknown. In this context, there is an urgent need to explore a method for alleviating the senolytic toxicity of environmental pollutants using melatonin, which has significant practical significance.

[0003] The traditional method for alleviating the senolytic toxicity of environmental pollutants has limitations: 1. The single dimension of senolytic toxicity evaluation - relying on isolated indicators (such as behavioral changes or dopaminergic neuron degeneration) makes it difficult to capture the multiple induction effects of pollutants on aging and PD; 2. The molecular mechanism of alleviating senolytic toxicity is not clear - some studies have shown that MT has the effect of delaying aging, but existing trials are still limited, and the molecular mechanism of delaying aging is still unclear. 3. The concentration of MT for alleviating senolytic effect is not clear - MT has different physiological effects at different concentrations, and the optimal alleviating effect concentration when co-exposed with pollutants needs to be screened. SUMMARY

[0004] To solve the above technical problems, the present application provides a method for evaluating the alleviating effect of melatonin on the senolytic toxicity of environmental pollutants, which generates a senolytic and PD toxicity evaluation model by constructing a behavior characteristic and molecular characteristic scheme and combining multi-strain C. elegans data, achieving comprehensive senolytic and PD toxicity evaluation from behavior to micro-molecules. Using C. elegans as a model animal, the concentration-toxicity dose-effect model is used to evaluate the ability of MT to alleviate the senolytic toxicity of environmental pollutants, and the molecular mechanism of MT to alleviate senolytic toxicity and the optimal alleviating concentration are determined.

[0005] To this end, the present application provides the following technical solutions,

[0006] In a first aspect, the present application provides, in optional embodiments, a method for evaluating melatonin in alleviating the senolytic toxicity of environmental pollutants, comprising the following steps:

[0007] The animal model is C. elegans, the environmental pollutant is 6:2 chlorinated polyfluoroalkyl ether sulfonate, and the subacute exposure experiment is performed on the blank control group, the 10 ng / L environmental pollutant exposure liquid group, the 10 ng / L environmental pollutant + 0.1 μmol / L melatonin exposure liquid group, the 10 ng / L environmental pollutant + 1 μmol / L melatonin exposure liquid group, the 10 ng / L environmental pollutant + 10 μmol / L melatonin exposure liquid group, and the 1 μmol / L melatonin exposure liquid group. After the experiment, the lipofuscin level, movement ability, abnormal movement behavior, α-synuclein protein expression and aggregation, DAT-1 expression amount, reactive oxygen species level of C. elegans are measured, and the dopamine neurons of C. elegans are observed and counted, so as to evaluate the senolytic toxicity of melatonin in alleviating the environmental pollutants.

[0008] Preferably, the C. elegans varieties include wild type strain N2, transgenic strain BZ555, and transgenic strain NL5901; and / or,

[0009] The method of the subacute exposure experiment is as follows: the synchronized L1 stage larvae are evenly distributed into the blank control group, the 10 ng / L environmental pollutant exposure liquid group, the 10 ng / L environmental pollutant + 0.1 μmol / L melatonin exposure liquid group, the 10 ng / L environmental pollutant + 1 μmol / L melatonin exposure liquid group, the 10 ng / L environmental pollutant + 10 μmol / L melatonin exposure liquid group, and the 1 μmol / L melatonin exposure liquid group, and an equal amount of E. coli OP50 is added as food in each group, and the culture is performed at 18-22℃ for 2.5-3.5 days; and / or,

[0010] The exposure liquid volumes of the blank control group, the 10 ng / L environmental pollutant exposure liquid group, the 10 ng / L environmental pollutant + 0.1 μmol / L melatonin exposure liquid group, the 10 ng / L environmental pollutant + 1 μmol / L melatonin exposure liquid group, the 10 ng / L environmental pollutant + 10 μmol / L melatonin exposure liquid group, and the 1 μmol / L melatonin exposure liquid group are the same;

[0011] In the blank control group, the solution added is dimethyl sulfoxide solution;

[0012] In the 10 ng / L environmental pollutant + 0.1 μmol / L melatonin exposure liquid group, the mass ratio of the environmental pollutant and melatonin is 1:2322.8;

[0013] In the 10 ng / L environmental pollutants + 1 μmol / L melatonin exposure solution group, the mass ratio of the environmental pollutants and melatonin is 1:23228;

[0014] In the 10 ng / L environmental pollutants + 10 μmol / L melatonin exposure solution group, the mass ratio of the environmental pollutants and melatonin is 1:232280.

[0015] Preferably, the method for determining the lipofuscin level of the C. elegans is as follows:

[0016] After the subacute exposure experiment, the wild type N2 nematodes are collected and anesthetized, then a fluorescence photo is taken, and finally the average fluorescence intensity of each nematode is calculated to determine the lipofuscin level of the nematodes.

[0017] The number of wild type N2 nematodes collected in the blank control group, the 10 ng / L environmental pollutants exposure solution group, the 10 ng / L environmental pollutants + 0.1 μmol / L melatonin exposure solution group, the 10 ng / L environmental pollutants + 1 μmol / L melatonin exposure solution group, the 10 ng / L environmental pollutants + 10 μmol / L melatonin exposure solution group and the 1 μmol / L melatonin exposure solution group is not less than 30, and each group is repeated for 3 times.

[0018] Preferably, the method for determining the movement ability of the C. elegans is as follows:

[0019] After the subacute exposure experiment, the wild type N2 nematodes are collected, washed and placed in NGM medium, and then microscopic tracking determination is performed. After the nematodes are fully crawled, a 20 s video of the movement of the nematodes is taken by using the Wormlab nematode tracking system. After the taking is completed, the center point speed, wavelength and movement amplitude are determined by using the Wormlab analysis system. The movement trajectory of the nematodes is in a sinusoidal curve shape, and the movement direction is taken as the X axis and the body swing direction is taken as the Y axis.

[0020] The number of wild type N2 nematodes collected in the blank control group, the 10 ng / L environmental pollutants exposure solution group, the 10 ng / L environmental pollutants + 0.1 μmol / L melatonin exposure solution group, the 10 ng / L environmental pollutants + 1 μmol / L melatonin exposure solution group, the 10 ng / L environmental pollutants + 10 μmol / L melatonin exposure solution group and the 1 μmol / L melatonin exposure solution group is not less than 30.

[0021] Preferably, the method for determining the abnormal movement behavior of the C. elegans is as follows:

[0022] The wild-type N2 nematodes after subacute exposure experiment were collected, washed and placed in NGM medium, and microscopic tracking measurement was performed. After the nematodes fully crawled out, 20 s of nematode movement video was taken using the Wormlab nematode tracking system, and abnormal behaviors including curling, head shaking or stillness were observed, and the proportion of nematodes with abnormal behaviors in each group was recorded;

[0023] The blank control group, 10 ng / L environmental pollutant exposure liquid group, 10 ng / L environmental pollutant + 0.1 μmol / L melatonin exposure liquid group, 10 ng / L environmental pollutant + 1 μmol / L melatonin exposure liquid group, 10 ng / L environmental pollutant + 10 μmol / L melatonin exposure liquid group and 1 μmol / L melatonin exposure liquid group were repeated 3 times.

[0024] Preferably, the method for determining the expression and aggregation of α-synuclein protein of C. elegans is:

[0025] The transgenic strain NL5901 nematodes after subacute exposure experiment were collected, washed and anesthetized, and about 25-35 nematodes were placed on a 2% agarose pad, and the integrity of dopaminergic neurons and fluorescence intensity were observed under a microscope;

[0026] The blank control group, 10 ng / L environmental pollutant exposure liquid group, 10 ng / L environmental pollutant + 0.1 μmol / L melatonin exposure liquid group, 10 ng / L environmental pollutant + 1 μmol / L melatonin exposure liquid group, 10 ng / L environmental pollutant + 10 μmol / L melatonin exposure liquid group and 1 μmol / L melatonin exposure liquid group were repeated 3 times.

[0027] Preferably, the method for determining the expression amount of DAT-1 of C. elegans is:

[0028] The transgenic strain BZ555 nematodes after subacute exposure experiment were collected and anesthetized, and 35-45 nematodes were placed on a 2% agarose pad, and the fluorescence intensity of dopaminergic neurons was observed under a microscope, and the expression amount of dopamine transporter DAT-1 was evaluated according to the fluorescence intensity.

[0029] Preferably, the method for determining the level of reactive oxygen species of C. elegans is:

[0030] The wild type N2 nematodes after subacute exposure experiment are collected, washed and then frozen and broken, and then a corresponding dopamine level is detected by using an enzyme-linked immunosorbent assay (ELISA) kit, an absorbance at 450 nm wavelength is measured, a sample activity is calculated, a standard curve is drawn, and concentration values of a blank control group, a 10 ng / L environmental pollutant exposure liquid group, a 10 ng / L environmental pollutant + 0.1 μmol / L melatonin exposure liquid group, a 10 ng / L environmental pollutant + 1 μmol / L melatonin exposure liquid group, a 10 ng / L environmental pollutant + 10 μmol / L melatonin exposure liquid group and a 1 μmol / L melatonin exposure liquid group are calculated according to a curve equation.

[0031] In the determination of the activity oxygen level of the Caenorhabditis elegans, the determination process needs to be performed at 0-6℃.

[0032] Preferably, the method for detecting the corresponding activity oxygen level by using the enzyme-linked immunosorbent assay (ELISA) kit is as follows:

[0033] Different concentrations of standard samples are added to standard sample wells, 10 μL of a sample and 40 μL of a sample diluent are added to sample wells, 100 μL of detection antibody-HRP is added to each well, the reaction wells are sealed with a sealing film, and then incubated at 37℃ in a water bath for 60 min; after the incubation, the liquid is discarded, each well is filled with a washing liquid, and then left to stand for 1 min, and then dried on a water absorption paper; the plate is washed for 5 times, 50 μL of substrate A and 50 μL of substrate B (the substrate A is a buffer solution containing hydrogen peroxide, and the substrate B is a buffer solution containing tetramethyl benzidine) are added to each well, and then incubated at 37℃ in the dark for 15 min; and then 50 μL of a termination liquid is added.

[0034] The concentrations of the standard samples are as follows: 0, 30, 60, 120, 240 and 480 U / L.

[0035] Preferably, the method for observing and counting the dopamine neurons of the Caenorhabditis elegans is as follows:

[0036] The transgenic line BZ555 nematodes after the subacute exposure experiment are collected and anesthetized, 35-45 nematodes are placed on an agarose pad with a concentration of 2%, and then the integrity and fluorescence intensity of the dopaminergic neurons are observed under a microscope, and the number of nerve vesicles and the proportion of neuron rupture are recorded.

[0037] The blank control group, the 10 ng / L environmental pollutant exposure liquid group, the 10 ng / L environmental pollutant + 0.1 μmol / L melatonin exposure liquid group, the 10 ng / L environmental pollutant + 1 μmol / L melatonin exposure liquid group, the 10 ng / L environmental pollutant + 10 μmol / L melatonin exposure liquid group and the 1 μmol / L melatonin exposure liquid group are repeated for 3 times.

[0038] Compared with the prior art, the present application has one of the following beneficial effects:

[0039] 1. The present application realizes the full range of aging and PD toxicity evaluation from behavior to micro molecule by constructing the behavior characteristics and molecular characteristics scheme, and combining with multi-strain C. elegans data to generate aging and PD toxicity evaluation model. Using C. elegans as a model animal, the ability of MT to alleviate the senolytic toxicity of environmental pollutants is evaluated through the concentration-toxicity dose-effect model, and the molecular mechanism of MT to alleviate the senolytic toxicity and the optimal alleviating concentration are determined.

[0040] 2. The present application provides an alleviating scheme for the senolytic toxicity induced by environmental pollutants, and also provides potential measures and schemes for protecting the health of the human population, especially the health of the elderly population. BRIEF DESCRIPTION OF DRAWINGS

[0041] Figure 1 A flowchart of the method for evaluating the ability of melatonin to alleviate the senolytic toxicity of environmental pollutants in embodiment 1 of the present application;

[0042] Figure 2 The effect of melatonin on the level of lipofuscin in C. elegans after F-53B exposure in embodiment 1 of the present application; wherein, A-F are representative images of the accumulation of lipofuscin in each group of C. elegans; and G is the detection result of the accumulation level of lipofuscin in each group of C. elegans;

[0043] Figure 3 The recovery effect of melatonin on the impairment of the movement ability of C. elegans after F-53B exposure in embodiment 1 of the present application; wherein, A is the detection result of the amplitude experiment of each group of C. elegans; B is the detection result of the wavelength experiment of each group of C. elegans; and C is the detection result of the center point speed experiment of each group of C. elegans;

[0044] Figure 4 The recovery effect of melatonin on the abnormal movement behavior of C. elegans after F-53B exposure in embodiment 1 of the present application; wherein, A-D are representative images of C. elegans with different severity of abnormal behavior; E is the total incidence of abnormal movement behavior in each group of C. elegans; and F is the incidence of different severity of abnormal movement behavior in each group of C. elegans;

[0045] Figure 5 The recovery effect of melatonin on the impairment of dopamine neurons of C. elegans after F-53B exposure in embodiment 1 of the present application; wherein, A-F are representative images of the impairment of dopamine neurons in each group of C. elegans; G is the proportion of the number of nerve axon vacuoles in the dopamine neurons of each group of C. elegans compared with the control group; and H is the incidence of nerve axon fracture in the dopamine neurons of each group of C. elegans;

[0046] Figure 6The recovery effect of melatonin on the expression and aggregation of α-synuclein protein of the nematode after F-53B exposure in Example 1 of the present application; wherein A-F are representative images of the expression and aggregation of α-synuclein protein of the nematode in each group; and G is the expression amount of α-synuclein protein in the nematode in each group;

[0047] Figure 7 The effect of melatonin on the expression of DAT-1 of the nematode after F-53B exposure in Example 1 of the present application;

[0048] Figure 8 The effect of melatonin on the level of reactive oxygen species of the nematode after F-53B exposure in Example 1 of the present application. DETAILED DESCRIPTION

[0049] In order to make the objectives, technical solutions and advantages of the present application clearer, the present application will be further described in detail below with specific examples. It should be understood that the specific examples described herein are only used to explain the present application and do not limit the present application.

[0050] In the following examples, the experimental methods used are conventional methods unless otherwise specified.

[0051] In the following examples, the materials, reagents, etc. used are commercially available unless otherwise specified.

[0052] Example 1

[0053] The present embodiment provides a method for evaluating the alleviation of the senolytic toxicity of environmental pollutants by melatonin, comprising the following steps:

[0054] Reference Figure 1 Wild-type C. elegans N2, C. elegans transgenic strain BZ555 and C. elegans transgenic strain NL5901 were used as model organisms, and 6:2 chlorinated polyfluoroalkyl ether sulfonate (F-53B) was used as an environmental pollutant. The blank control group (0 ng / L F-53B exposure solution), the 10 ng / L environmental pollutant exposure solution group, the 10 ng / L environmental pollutant + 0.1 μmol / L melatonin exposure solution group, the 10 ng / L environmental pollutant + 1 μmol / L melatonin exposure solution group, the 10 ng / L environmental pollutant + 10 μmol / L melatonin exposure solution group and the 1 μmol / L melatonin exposure solution group were set up, and a subacute exposure experiment was carried out. The experimental method is as follows:

[0055] The exposure solution was added to the 6-well plate after sterilization according to the concentration of each group, and 3 parallel holes were set for each experimental group, the volume of the exposure solution added to each hole was 5 mL, the synchronized L1 stage larvae were evenly distributed into each hole, the exposure time was recorded, 100 μL of E. coli OP50 was added to each hole as food, and the culture was incubated in a constant temperature biochemical incubator at 20℃ for 3 days, and the same amount of E. coli OP50 was fed at the same time every day during the period.

[0056] In the blank control group, the solution added was dimethyl sulfoxide solution;

[0057] In the 10 ng / L environmental pollutant + 0.1 μmol / L melatonin exposure solution group, the mass ratio of environmental pollutant and melatonin was 1:2322.8;

[0058] In the 10 ng / L environmental pollutant + 1 μmol / L melatonin exposure solution group, the mass ratio of environmental pollutant and melatonin was 1:23228;

[0059] In the 10 ng / L environmental pollutant + 10 μmol / L melatonin exposure solution group, the mass ratio of environmental pollutant and melatonin was 1:232280.

[0060] After the exposure experiment, the lipofuscin level, movement ability, abnormal movement behavior, α-synuclein protein expression and aggregation, DAT-1 expression amount, active oxygen level of C. elegans were determined, and the dopamine neurons of C. elegans were observed and counted.

[0061] The method for determining the movement ability of C. elegans is as follows:

[0062] After the subacute exposure experiment, the wild type N2 nematodes were collected, the nematodes were anesthetized with 60 μM of levamisole solution, placed on a glass slide, and the fluorescence photos were taken under the same exposure parameters and conditions (DAPI channel, 405 nm excitation wavelength, 450 nm emission wavelength) using ZOETM fluorescence cell imager, the whole shooting process should be completed quickly to avoid the influence of movement of nematodes after anesthetization failure on shooting. After shooting, the average fluorescence intensity of each nematode was calculated using Image J software to determine the lipofuscin level of the nematode. The blank control group, 10 ng / L environmental pollutant exposure solution group, 10 ng / L environmental pollutant + 0.1 μmol / L melatonin exposure solution group, 10 ng / L environmental pollutant + 1 μmol / L melatonin exposure solution group, 10 ng / L environmental pollutant + 10 μmol / L melatonin exposure solution group and 1 μmol / L melatonin exposure solution group each contained at least 30 effective nematodes, and at least 3 repeated experiments were performed.

[0063] The results are shown in Figure 2, results showed that F-53B single factor exposure group (10 ng / L) compared with the control group, lipofuscin fluorescence intensity increased about 2.3 times (p < 0.01). Joint processing group (F-53B+MT) in, showed concentration-dependent alleviating effect, 0.1 μM MT showed a significant improvement effect, 1 μM joint processing makes lipofuscin levels returned to the control group 108% (p < 0.05), 10 μM joint processing group compared with F-53B alone exposure group, significantly reduced lipofuscin deposition 23.3% (p < 0.001), indicating that MT has a certain alleviating effect on F-53B toxicity. While the MT alone exposure group, the lipofuscin deposition decreased to 68.5% of the control group (p < 0.05), indicating that MT itself also has a certain anti-aging effect on nematodes.

[0064] The method for determining the movement ability of C. elegans is as follows:

[0065] After the subacute exposure experiment, wild type N2 nematodes were collected, washed at least 3 times with K solution, centrifuged and discarded the supernatant, and then transferred to a 6-well plate coated with NGM medium using a pipette gun, and placed under a microscope for tracking determination. After the nematodes were fully crawled, a 20 s video of the movement of the nematodes was taken using the Wormlab nematode tracking system. After the shooting was completed, the center point speed, wavelength and movement amplitude were determined using the Wormlab analysis system. The movement trajectory of the nematodes was sinusoidal, with the movement direction as the X axis and the body swing direction as the Y axis. The data of the blank control group, 10 ng / L environmental pollutant exposure group, 10 ng / L environmental pollutant+0.1 μmol / L melatonin exposure group, 10 ng / L environmental pollutant+1 μmol / L melatonin exposure group, 10 ng / L environmental pollutant+10 μmol / L melatonin exposure group and 1 μmol / L melatonin exposure group were determined by selecting at least 30 nematodes.

[0066] The results are shown in Figure 3Compared with the blank control group, the amplitude of nematodes in the F-53B single-factor exposure group (10 ng / L) did not change significantly, but the center point velocity and wavelength were significantly reduced (p < 0.01), indicating that this pollutant significantly accelerated the aging process and induced a decline in nematode motility. In the combined treatment group (F-53B+MT), a concentration-dependent mitigation effect was observed. In the amplitude index, 0.1 μM MT showed a significant improvement effect on F-53B. 1 μM combined treatment could restore the amplitude to near the control level. The 10 μM combined treatment group showed a significant increase in nematode motility compared to the F-53B single-exposure group, indicating that MT has a certain mitigating effect on the aging toxicity of F-53B. In the MT single-exposure group, compared with the control group, MT significantly increased the center point velocity and amplitude, indicating that MT also has a certain mitigating effect on the motility loss caused by nematode aging.

[0067] The method for determining abnormal movement behavior in *Caenorhabditis elegans* is as follows:

[0068] Wild-type N2 nematodes were collected after subacute exposure experiments, washed at least three times with K solution, centrifuged and the supernatant discarded. The nematodes were then transferred by pipette into 6-well plates containing NGM medium and tracked under a microscope. After the nematodes had spread out sufficiently, a 20-second video of their movement was recorded using the Wormlab nematode tracking system. Abnormal behaviors, including curling, head shaking, or stillness, were observed, and the proportion of nematodes exhibiting abnormal behavior in each group was recorded. The blank control group, the 10 ng / L environmental pollutant exposure group, the 10 ng / L environmental pollutant + 0.1 μmol / L melatonin exposure group, the 10 ng / L environmental pollutant + 1 μmol / L melatonin exposure group, the 10 ng / L environmental pollutant + 10 μmol / L melatonin exposure group, and the 1 μmol / L melatonin exposure group were all repeated three times.

[0069] See results Figure 4 Nematodes are classified into three grades based on the severity of their abnormal movement behavior. Normal nematodes (grade 0) move forward steadily in a sinusoidal curve (…). Figure 4 A). Level 1 behaviorally abnormal nematodes move forward discontinuously and exhibit a coiled posture. Figure 4 B). Nematodes exhibiting a severity level of abnormal behavior of 2 are almost completely still, with only their heads trembling and swaying irregularly. Figure 4 C). Nematodes exhibiting the most severe abnormal behavior (level 3) cease movement and remain still. Figure 4 D). Compared with the control group (22 / 207, 10.6% incidence of abnormal behavior), F-53B significantly increased the incidence of abnormal nematode behavior in the 10 ng / L exposure group to 25.8% (51 / 198; χ²). 2=15.678; p < 0.001. In the combined treatment group (F-53B+MT), 1 μM MT initially showed a significant inhibitory effect on abnormal nematode behavior (32 / 187; χ² = 15.678; p < 0.001). 2 =4.251; p < 0.05), the 10 μM combined treatment group showed better remission, with a significantly lower rate of abnormal nematode motility (26 / 189; χ² = 4.251; p < 0.05) compared to the F-53B exposure group alone. 2 =8.738; p<0.01), which is basically close to the abnormality rate of the control group, indicating that MT has a good alleviating effect on F-53B-induced PD. Figure 4 E). Meanwhile, the severity of abnormal behavior also decreased with increasing MT concentration; the incidence of different levels of abnormal behavior in nematodes was significantly lower compared to the F-53B-only exposure group. Figure 4 F).

[0070] The method for observing and statistically analyzing the dopamine neurons in *Caenorhabditis elegans* is as follows:

[0071] Transgenic strain BZ555 nematodes were collected after subacute exposure experiments, washed three times with K buffer, and anesthetized with 60 μM levamisole solution. Approximately 40 nematodes were placed on a 2% agarose mat, and the integrity and fluorescence intensity of dopaminergic neurons were observed under a microscope. The number of neural vesicles and the proportion of neuronal breaks were recorded for each exposure group. The fluorescence intensity was analyzed using ImageJ. The blank control group, the 10 ng / L environmental pollutant exposure group, the 10 ng / L environmental pollutant + 0.1 μmol / L melatonin exposure group, the 10 ng / L environmental pollutant + 1 μmol / L melatonin exposure group, the 10 ng / L environmental pollutant + 10 μmol / L melatonin exposure group, and the 1 μmol / L melatonin exposure group were all repeated three times.

[0072] See results Figure 5 F-53B exposure causes significant damage to dopaminergic neurons. Normal dopaminergic neurons show intact neuronal dendrites ( Figure 5 A). The F-53B exposure group frequently exhibited nerve axonal vesicles (indicated by arrows) and severed nerve axons (highlighted by circles). Figure 5 (B) In the co-exposure group with MT relief, the number of axonal vacuoles and the rate of axonal breakage in dopaminergic neurons showed a dose-response relationship decreasing with increasing MT exposure concentration. Compared with the control group, the F-53B 10 ng / L exposure group showed a significantly increased number of axonal vacuoles and axonal breakage rate in dopaminergic neurons by 4.3-fold (p < 0.001) and 27.91% (p < 0.05), respectively. Figure 5G, H). In the co-exposure group with MT alleviation, the degree of damage to the dopamine neurons of the nematodes gradually improved with the increase in the concentration of MT, and the 10 mM MT had the best alleviating effect. Compared with the F-53B single exposure group, the number of nerve axon vacuoles in the 10 mM MT combined exposure group decreased by 3.3 times (p < 0.001), and the nerve axon fracture rate decreased by 18.8% (p < 0.05), which was basically close to that of the control group, indicating that MT had an effective alleviating and protective effect on the dopamine neuronal damage induced by F-53B. Dopamine neuronal damage is one of the important pathological characteristics of PD, which indicates that MT has a certain alleviating potential for PD.

[0073] The method for determining the expression and aggregation of the alpha-synuclein protein of the C. elegans is as follows:

[0074] After the subacute exposure experiment, the transgenic NL5901 nematodes were collected, rinsed with K buffer for 3 times, and anesthetized with a 60 mM levamisole solution. About 30 nematodes were placed on a 2% agarose pad, and the fluorescence intensity was observed under a microscope. The fluorescence intensity data were analyzed using Image J. The blank control group, the 10 ng / L environmental pollutant exposure group, the 10 ng / L environmental pollutant + 0.1 mM melatonin exposure group, the 10 ng / L environmental pollutant + 1 mM melatonin exposure group, the 10 ng / L environmental pollutant + 10 mM melatonin exposure group, and the 1 mM melatonin exposure group were repeated 3 times.

[0075] The transgenic nematode model NL5901 was selected to simulate the pathological formation process of Lewy bodies in the neurons of human PD patients. Alpha-synuclein is a soluble protein composed of 140 amino acids, which is an important pathogenic protein of PD. The results are shown in Figure 6 The quantitative results of the fluorescence intensity show that F-53B exposure has a significant promoting effect on the expression and aggregation of alpha-synuclein. The body wall cells of normal NL5901 nematodes under a fluorescence microscope show uniform and less expression of alpha-synuclein Figure 6 A), while the expression of alpha-synuclein in the body wall cells of the nematodes in the F-53B exposure group significantly increased, and there were diffuse and significant aggregation points (arrowed, Figure 6 B and Figure 6 G) in the body wall cells of the nematodes. In the co-exposure group with MT alleviation, the expression and aggregation of alpha-synuclein showed a dose-effect relationship with the increase in the concentration of MT exposure. Compared with the control group, the expression of alpha-synuclein in the nematodes in the F-53B 10 ng / L exposure group increased by 1.29 times (p < 0.001) Figure 6G). In the co-exposure group of MT alleviation, the expression of nematode α-synuclein protein gradually decreased with the increase of MT concentration, and 10 μM MT had the best alleviating effect. Compared with the F-53B single exposure group, the expression of α-synuclein in the 10 μM MT joint exposure group decreased by 0.8 times (p < 0.01), which was basically close to the control group, indicating that MT had an effective slowing effect on the increase of nematode α-synuclein expression induced by F-53B. Compared with the F-53B single exposure group, the MT joint exposure group also reduced the abnormal aggregation of α-synuclein. As can be seen from the figure, the abnormal aggregation points indicated by the arrows were significantly reduced. The increase and abnormal aggregation of α-synuclein are one of the important pathological characteristics of PD. The experimental results of the present application prove that MT has a certain alleviating effect on PD. The 1 μM MT single exposure group significantly reduced the expression of α-synuclein by 0.1 times (p < 0.05) compared with the control group, and also had a certain alleviating effect on the aggregation of nematode α-synuclein, further proving that MT has a good effect on alleviating PD.

[0076] The method for determining the expression of DAT-1 of C. elegans is as follows:

[0077] The transgenic line BZ555 nematodes after subacute exposure experiment were collected, washed with K buffer for 3 times, anesthetized with 60 μM levodopa solution, about 40 nematodes were placed on a 2% agarose pad, and the fluorescence intensity of dopaminergic neurons was observed under a microscope. The expression of dopamine transporter DAT-1 was evaluated according to the fluorescence intensity.

[0078] F-53B mainly causes oxidative stress by up-regulating the expression of dopamine transporter to transport excessive DA into dopaminergic neurons. The results are shown in Table 1. Figure 7The fluorescence intensity quantitative results showed that F-53B exposure significantly up-regulated DAT-1 expression. In the MT alleviated co-exposure groups, DAT-1 expression showed a dose-effect relationship with the increase of MT exposure concentration. Compared with the control group, DAT-1 expression in the F-53B 10 ng / L exposure group was significantly increased by 1.1 times (p < 0.001). In the MT alleviated co-exposure groups, DAT-1 expression in the nematodes gradually decreased with the increase of MT concentration, and showed a statistical difference in the 10 μM MT joint exposure group. Compared with the F-53B single exposure group, DAT-1 expression in the 10 μM MT joint exposure group was significantly decreased by 1.07 times (p < 0.01), which was basically close to the control group, indicating that MT had an effective down-regulation effect on the DAT-1 expression increase induced by F-53B in nematodes. DAT-1 overexpression, thereby causing oxidative stress damage to dopaminergic neurons by excessive DA uptake, is an important mechanism for F-53B-induced nematode PD. MT reducing DAT-1 expression can reduce DA uptake into dopaminergic neurons and reduce the degree of oxidative damage. The results of the present study further illustrate that MT reduces oxidative stress induced by excessive DA uptake into dopaminergic neurons by DAT, thereby alleviating the senility toxicity of F-53B.

[0079] The method for determining the level of reactive oxygen species in C. elegans is as follows:

[0080] Wild type N2 nematodes after subacute exposure experiment, frozen aluminum block in advance, prepare the corresponding number of samples of enzyme-free centrifuge tube in the refrigerator 4℃ pre-cooling, the whole experiment sample placed in the frozen aluminum block to ensure low temperature operation. Collect nematodes after exposure to enzyme-free centrifuge tube, using K solution washing 3 times, centrifugation, using liquid nitrogen refrigeration, after taking out in room temperature melting, when the ice sand state can be carried out next step of breaking. Breaking the nematodes placed in the pre-frozen aluminum block operation, using cell crusher intermittent crushing way to break the nematodes 1 min, avoid continuous long time during the breakage caused by the temperature rise in centrifuge tube and affect the quality of dopamine. After breaking the end centrifugation get rid of the supernatant, nematode dopamine. Using enzyme-linked immunosorbent assay (ELISA) kit to detect the corresponding dopamine level. Kit using double antibody one step sandwich method, the concentration of standard sample is: 0, 30, 60, 120, 240 and 480 U / L. Standard sample well in different concentrations of standard sample 50 μL, sample well in 10 μL sample and sample diluent 40 μL, each hole in 100 μL detection antibody-HRP, with sealing plate film to seal the reaction well in 37℃ water bath incubator for 60 min. Incubation was completed after the liquid, each hole full of washing liquid, 1 min later in the water paper dry, repeat washing plate 5 times, each hole respectively adding substrate A, B 50 μL, 37℃ avoid light incubation for 15 min after adding stop solution 50 μL, 15 min within the determination of 450 nm wavelength absorbance, calculate the sample activity. The detection results for the preparation of standard curve, according to the curve equation to calculate the concentration of each sample value.

[0081] Results are shown in Figure 8 , F-53B exposure significantly increased ROS levels. In the MT alleviated co-exposure group, ROS levels showed a dose-effect relationship with the increase of MT exposure concentration. Compared with the control group, the ROS level of nematodes in the F-53B 10 ng / L exposure group increased significantly by 30.2% (p < 0.001). In the MT alleviated co-exposure group, with the increase of MT concentration, the ROS level of nematodes gradually decreased, and there was no statistical difference in the 0.1 μM MT joint exposure group, while in the high concentration MT joint exposure group, the ROS level decreased significantly, compared with the F-53B single exposure group, the ROS level in the 10 μM MT joint exposure group decreased by 23.4% (p < 0.01), basically close to the control group, indicating that MT has an effective alleviating effect on the decrease of ROS level induced by F-53B in nematodes. While the ROS level in the 1 μM MT single exposure group decreased significantly by 14.3% (p < 0.05) compared with the control group, indicating that MT also has an eliminating effect on the ROS produced by the metabolism of nematodes itself, further illustrating that MT can alleviate the senescence toxicity of F-53B by reducing the ROS level.

[0082] Although the principles of the present application have been described in connection with the preferred embodiments thereof with reference to the drawings, it should be understood that the application is not limited to the description of the preferred embodiments, and that modifications and variations are possible without departing from the spirit and scope of the application. The details in the embodiments are not intended to limit the scope of the application, and any obvious changes, simple replacements, and the like based on the technical solutions of the present application are within the scope of protection of the present application.

Claims

1. A method for assessing the mitigation of aging toxicity caused by environmental pollutants by melatonin, characterized in that, Includes the following steps: Using *C. elegans* as an animal model and 6:2 chloropolyfluoroalkyl ether sulfonate as an environmental pollutant, subacute exposure experiments were conducted with four groups: a blank control group, a 10 ng / L environmental pollutant exposure group, a 10 ng / L environmental pollutant + 0.1 μmol / L melatonin exposure group, a 10 ng / L environmental pollutant + 1 μmol / L melatonin exposure group, a 10 ng / L environmental pollutant + 10 μmol / L melatonin exposure group, and a 1 μmol / L melatonin exposure group. After the experiment, the lipofuscin level, motility, abnormal motility behavior, α-synuclein protein expression and aggregation, DAT-1 expression level, reactive oxygen species level, and dopamine neurons in *C. elegans* were measured and analyzed to evaluate the catalytic toxicity of melatonin in mitigating the aging effects of environmental pollutants.

2. The method for assessing the aging toxicity of melatonin in mitigating environmental pollutants according to claim 1, characterized in that, The varieties of *Caenorhabditis elegans* include wild-type strain N2, transgenic strain BZ555, and transgenic strain NL5901; and / or, The subacute exposure experiment was conducted as follows: synchronized L1-stage larvae were evenly distributed into a blank control group, a 10 ng / L environmental pollutant exposure group, a 10 ng / L environmental pollutant + 0.1 μmol / L melatonin exposure group, a 10 ng / L environmental pollutant + 1 μmol / L melatonin exposure group, a 10 ng / L environmental pollutant + 10 μmol / L melatonin exposure group, and a 1 μmol / L melatonin exposure group. Each group was supplemented with an equal amount of E. coli OP50 as food, and cultured at 18-22℃ for 2.5-3.5 days; and / or, The blank control group, the 10 ng / L environmental pollutant exposure solution group, the 10 ng / L environmental pollutant + 0.1 μmol / L melatonin exposure solution group, the 10 ng / L environmental pollutant + 1 μmol / L melatonin exposure solution group, the 10 ng / L environmental pollutant + 10 μmol / L melatonin exposure solution group, and the 1 μmol / L melatonin exposure solution group all had the same exposure solution volume; In the blank control group, the added solution was a dimethyl sulfoxide solution; In the 10 ng / L environmental pollutant + 0.1 μmol / L melatonin exposure solution group, the mass ratio of the environmental pollutant to melatonin is 1:2322.8; In the 10 ng / L environmental pollutant + 1 μmol / L melatonin exposure solution group, the mass ratio of the environmental pollutant to melatonin is 1:23228; In the 10 ng / L environmental pollutant + 10 μmol / L melatonin exposure solution group, the mass ratio of the environmental pollutant to melatonin is 1:232280.

3. The method for assessing the aging toxicity of melatonin in mitigating environmental pollutants according to claim 1, characterized in that, The method for determining the lipofuscin level in *C. elegans* is as follows: Wild-type N2 nematodes were collected after subacute exposure experiments and anesthetized. Fluorescence images were then taken, and the average fluorescence intensity of each nematode was calculated to determine the lipofuscin level of the nematodes. The number of wild-type N2 nematodes collected in the blank control group, the 10 ng / L environmental pollutant exposure group, the 10 ng / L environmental pollutant + 0.1 μmol / L melatonin exposure group, the 10 ng / L environmental pollutant + 1 μmol / L melatonin exposure group, the 10 ng / L environmental pollutant + 10 μmol / L melatonin exposure group, and the 1 μmol / L melatonin exposure group was no less than 30, and the experiment was repeated 3 times in each group.

4. The method for assessing the aging toxicity of melatonin in mitigating environmental pollutants according to claim 1, characterized in that, The method for determining the locomotion ability of *Caenorhabditis elegans* is as follows: Wild-type N2 nematodes were collected after subacute exposure experiments, washed, and placed in NGM medium. Microscopic tracking was performed. After the nematodes had spread out, a 20-second video of their movement was captured using the Wormlab nematode tracking system. After the video was captured, the velocity, wavelength, and amplitude of their movement were measured using the Wormlab analysis system. The nematode's movement trajectory was sinusoidal, with the direction of movement as the X-axis and the direction of body swaying as the Y-axis. The number of wild-type N2 nematodes collected in the blank control group, the 10 ng / L environmental pollutant exposure group, the 10 ng / L environmental pollutant + 0.1 μmol / L melatonin exposure group, the 10 ng / L environmental pollutant + 1 μmol / L melatonin exposure group, the 10 ng / L environmental pollutant + 10 μmol / L melatonin exposure group, and the 1 μmol / L melatonin exposure group was no less than 30.

5. The method for assessing the aging toxicity of melatonin in mitigating environmental pollutants according to claim 1, characterized in that, The method for determining abnormal movement behavior in *Caenorhabditis elegans* is as follows: Wild-type N2 nematodes were collected after subacute exposure experiments, washed, and placed in NGM medium. Microscopic tracking was performed. After the nematodes had spread out, a 20-second video of nematode movement was captured using the Wormlab nematode tracking system. Abnormal behaviors, including curling, head shaking, or stillness, were observed. The proportion of nematodes exhibiting abnormal behaviors in each group was recorded. The blank control group, the 10 ng / L environmental pollutant exposure solution group, the 10 ng / L environmental pollutant + 0.1 μmol / L melatonin exposure solution group, the 10 ng / L environmental pollutant + 1 μmol / L melatonin exposure solution group, the 10 ng / L environmental pollutant + 10 μmol / L melatonin exposure solution group, and the 1 μmol / L melatonin exposure solution group were all tested three times.

6. The method for assessing the aging toxicity of melatonin in mitigating environmental pollutants according to claim 1, characterized in that, The method for determining the expression and aggregation of α-synuclein protein in Caenorhabditis elegans is as follows: After subacute exposure experiments, transgenic strain NL5901 nematodes were collected, washed, and anesthetized. Approximately 25-35 nematodes were placed on a 2% agarose pad, and the integrity and fluorescence intensity of dopaminergic neurons were observed under a microscope. The blank control group, the 10 ng / L environmental pollutant exposure solution group, the 10 ng / L environmental pollutant + 0.1 μmol / L melatonin exposure solution group, the 10 ng / L environmental pollutant + 1 μmol / L melatonin exposure solution group, the 10 ng / L environmental pollutant + 10 μmol / L melatonin exposure solution group, and the 1 μmol / L melatonin exposure solution group were all tested three times.

7. The method for assessing the aging toxicity of melatonin in mitigating environmental pollutants according to claim 1, characterized in that, The method for determining the expression level of DAT-1 in Caenorhabditis elegans is as follows: Transgenic strain BZ555 nematodes were collected after subacute exposure experiments and anesthetized. 35-45 nematodes were placed on a 2% agarose pad and the fluorescence intensity of dopaminergic neurons was observed under a microscope. The expression level of the dopamine transporter DAT-1 was assessed based on the fluorescence intensity.

8. The method for assessing the aging toxicity of melatonin in mitigating environmental pollutants according to claim 1, characterized in that, The method for determining the reactive oxygen species (ROS) level in *C. elegans* is as follows: Wild-type N2 nematodes were collected after subacute exposure experiments, washed, and then frozen and lysed. The corresponding dopamine levels were detected using an enzyme-linked immunosorbent assay (ELISA) kit. The absorbance at 450 nm was measured, the sample activity was calculated, and a standard curve was plotted. The concentrations of the blank control group, the 10 ng / L environmental pollutant exposure group, the 10 ng / L environmental pollutant + 0.1 μmol / L melatonin exposure group, the 10 ng / L environmental pollutant + 1 μmol / L melatonin exposure group, the 10 ng / L environmental pollutant + 10 μmol / L melatonin exposure group, and the 1 μmol / L melatonin exposure group were calculated according to the curve equation. When determining the reactive oxygen species (ROS) level in Caenorhabditis elegans, the measurement process needs to be carried out at 0-6°C.

9. The method for assessing the aging toxicity of melatonin in mitigating environmental pollutants according to claim 8, characterized in that, The method for detecting the corresponding reactive oxygen species level using the enzyme-linked immunosorbent assay (ELISA) kit is as follows: Add 50 μL of different concentrations of standard to the standard sample wells, 10 μL of sample and 40 μL of sample diluent to the sample wells, and 100 μL of detection antibody-HRP to each well. Seal the reaction wells with sealing film and incubate in a water bath at 37°C for 60 min. After incubation, discard the liquid, fill each well with washing buffer, let stand for 1 min, and pat dry on absorbent paper. Repeat washing 5 times. Add 50 μL of substrate A and 50 μL of substrate B to each well, incubate at 37°C in the dark for 15 min, and then add 50 μL of stop solution. The concentrations of the standards were 0, 30, 60, 120, 240, and 480 U / L, respectively.

10. The method for assessing the mitigating aging toxicity of melatonin from environmental pollutants according to claim 1, characterized in that, The method for observing and statistically analyzing the dopamine neurons in *Caenorhabditis elegans* is as follows: Transgenic strain BZ555 nematodes were collected after subacute exposure experiments and anesthetized. 35-45 nematodes were placed on a 2% agarose pad and the integrity and fluorescence intensity of dopaminergic neurons were observed under a microscope. The number of nerve vesicles and the proportion of neuronal breaks were recorded. The blank control group, the 10 ng / L environmental pollutant exposure solution group, the 10 ng / L environmental pollutant + 0.1 μmol / L melatonin exposure solution group, the 10 ng / L environmental pollutant + 1 μmol / L melatonin exposure solution group, the 10 ng / L environmental pollutant + 10 μmol / L melatonin exposure solution group, and the 1 μmol / L melatonin exposure solution group were all tested three times.

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