Method for evaluating risk of Parkinson's disease induced by environmental pollutants

Abstract

The invention relates to the technical field of environmental health risk evaluation, in particular to a method for evaluating the risk of Parkinson's disease induced by environmental pollutants. The method for evaluating the risk of inducing the Parkinson's disease by the environmental pollutants comprises the following steps: carrying out a subacute exposure experiment by taking caenorhabditis elegans as a model organism; observing the transport capacity of the caenorhabditis elegans, observing and counting the abnormal movement ability of the caenorhabditis elegans, and determining the lipofuscin level of the caenorhabditis elegans, the damage degree of dopaminergic neurons, the dopamine level and the expression and aggregation conditions of alpha-synuclein protein, so as to evaluate the risk of the Parkinson's disease induced by environmental pollutants; the environmental pollutant is 6: 2 chlorinated polyfluoroalkyl ether sulfonate. According to the method, the PD toxicity evaluation model is generated by constructing a behavior characteristic and molecular characteristic scheme and combining multi-strain nematode data, and the problem that in the prior art, the risk of inducing PD by environmental pollutants is difficult to comprehensively reveal due to the fact that a single dimension is concerned is solved.

Description

Technical Field

[0001] The present invention relates to the technical field of environmental health risk assessment, and in particular to a method for assessing the risk of Parkinson's disease induced by environmental pollutants. Background Art

[0002] The risk of environmental pollutants inducing Parkinson's disease (PD) has become a research hotspot in environmental toxicology in recent years. However, traditional toxicity assessment methods have significant shortcomings. First, traditional studies assess toxicity through only a single dimension (such as behavior or dopaminergic neurons), making it difficult to fully reveal the multi-level impact of pollutants on organisms. Second, epidemiological surveys are susceptible to confounding factors, which not only limits the accurate assessment of pollutant toxicity, makes it difficult to establish a causal chain between environmental pollutants and PD, but also increases human and material costs. Furthermore, risk prediction models ignore the body's self-protective effects on pollutant toxicity, resulting in low accuracy and reliability. In this context, exploring systematic PD risk assessment schemes is of urgent research need and significant practical significance.

[0003] Traditional toxicity assessment methods have significant limitations: 1. The singleness of the assessment dimension - relying on isolated indicators (such as behavioral changes or dopaminergic neuron degeneration) makes it difficult to capture the multifaceted induction effects of pollutants on PD; 2. The breakdown of the epidemiological causal chain - the interference of confounding variables makes it impossible to establish a definitive causal relationship between "pollutants and PD", and the research cost is high; 3. The static defect of risk prediction - ignoring the offsetting effect of endogenous protective mechanisms on toxic effects, resulting in insufficient prediction reliability. Summary of the Invention

[0004] In order to solve the above technical problems, the present invention provides a method for assessing the risk of Parkinson's disease induced by environmental pollutants. This method overcomes the problem that the existing technology focuses on a single dimension and is difficult to fully reveal the risk of environmental pollutants inducing PD.

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

[0006] In a first aspect, the present invention provides, in an optional embodiment, a method for assessing the risk of Parkinson's disease induced by environmental pollutants, comprising the following steps:

[0007] Subacute exposure experiments using Caenorhabditis elegans as a model organism will assess the risk of Parkinson's disease induced by environmental pollutants by observing the worm's motor capacity, observing and counting abnormal movement, and measuring lipofuscin levels, dopaminergic neuron damage, dopamine levels, and α-synuclein protein expression and aggregation.

[0008] Wherein, the environmental pollutant is 6:2 chloropolyfluoroalkyl ether sulfonate (F-53B).

[0009] Preferably, in the subacute exposure experiment, a blank control group, a 2 ng / L environmental pollutant venom group, a 10 ng / L environmental pollutant venom group and a 50 ng / L environmental pollutant venom group are set up respectively, and three parallel groups are set up in each group; and / or, the varieties of Caenorhabditis elegans include wild type N2, transgenic strain BZ555 and transgenic strain NL5901.

[0010] Preferably, the method of the subacute exposure experiment is: the synchronized L1 larvae are evenly distributed into a blank control group, a 2ng / L environmental pollutant venom group, a 10ng / L environmental pollutant venom group and a 50ng / L environmental pollutant venom group, an equal amount of E. coli OP50 is added to each group as food, and cultured at 18-22°C for 2.5-3.5 days; the volume of environmental pollutant venom added to the 2ng / L environmental pollutant venom group, the 10ng / L environmental pollutant venom group and the 50ng / L environmental pollutant venom group is the same, and the blank control group is added.

[0011] In the present invention, the L1 larvae after synchronization refer to: when most of the nematodes on the culture medium are in the pregnant period, the nematodes are washed into a sterilized 1.5mL centrifuge tube with K solution, and the supernatant is discarded after centrifugation at 3000rpm for 2min, and the volume of the sediment in all centrifuge tubes is kept consistent as much as possible. After repeated cleaning three times, 1mL of nematode lysate is added to each tube, and the timing is started after the addition of the lysate. After vortexing for 1.5min, the centrifugation is quickly removed for 10s. After discarding the supernatant, 1mL of lysate is added to each tube again. Different centrifuge tubes are selected and observed quickly while vortexing to avoid observing for too long and affecting the cleavage of nematodes. When a large number of pregnant worms in the tube disappear and only a small amount of nematodes remain, the vortex is stopped immediately, the supernatant is discarded by centrifugation to obtain eggs, and K solution is quickly added. After repeated cleaning three times, the eggs are transferred to NGM culture medium without food and cultured in a 20°C constant temperature biochemical incubator for 16h to obtain L1 synchronized larvae for subsequent exposure experiments. The entire lysis time should not exceed 9 minutes to prevent the eggs from staying in the lysis solution for too long and being damaged, making them unable to hatch into larvae normally.

[0012] In the present invention, the solvent of the environmental pollutant venom liquid added to the 2ng / L environmental pollutant venom liquid group, the 10ng / L environmental pollutant venom liquid group and the 50ng / L environmental pollutant venom liquid group is a dimethyl sulfoxide solvent. In the blank control group, the 2ng / L environmental pollutant venom liquid group, the 10ng / L environmental pollutant venom liquid group and the 50ng / L environmental pollutant venom liquid group, the dimethyl sulfoxide solvent needs to be diluted to 0.01%.

[0013] Preferably, the method for observing the transport capacity of Caenorhabditis elegans is as follows: wild-type N2 nematodes are collected after the subacute exposure experiment, cleaned and placed in NGM culture medium, and subjected to microscopic tracking measurement. After the nematodes have fully crawled away, a 20-second video of the nematode movement is captured using the Wormlab nematode tracking system. After the video is captured, the center point speed, wavelength, and movement amplitude are measured using the Wormlab analysis system. The movement trajectory of the nematode is a sinusoidal curve, with the movement direction as the X-axis and the body swinging direction as the Y-axis. The number of wild-type N2 nematodes collected in the blank control group, 2 ng / L environmental pollutant venom group, 10 ng / L environmental pollutant venom group, and 50 ng / L environmental pollutant venom group is not less than 30.

[0014] Preferably, the method for observing and counting the abnormal movement ability of Caenorhabditis elegans is: collecting wild-type N2 nematodes after the subacute exposure experiment, washing them and placing them in NGM culture medium, and performing microscopic tracking measurement. After the nematodes have fully crawled away, a 20-second video of the nematode movement is shot using the Wormlab nematode tracking system to observe abnormal behaviors, including curling, head shaking or stillness, and recording the proportion of nematodes with abnormal behaviors in each group; the blank control group, 2 ng / L environmental pollutant venom group, 10 ng / L environmental pollutant venom group, and 50 ng / L environmental pollutant venom group were all repeated 3 times.

[0015] Preferably, the method for determining the lipofuscin level of Caenorhabditis elegans is: collecting wild-type N2 nematodes after the subacute exposure experiment and anesthetizing them, then taking fluorescent photos, and finally calculating the average fluorescence intensity of each nematode to determine the lipofuscin level of the nematodes; the number of wild-type N2 nematodes collected in the blank control group, 2 ng / L environmental pollutant venom group, 10 ng / L environmental pollutant venom group and 50 ng / L environmental pollutant venom group is not less than 30, and the experiment is repeated 3 times.

[0016] Preferably, the method for determining the degree of damage to dopaminergic neurons in Caenorhabditis elegans is as follows: collecting transgenic strain BZ555 nematodes after the subacute exposure experiment and anesthetizing them, placing 35-45 nematodes on a 2% agarose pad, observing the integrity and fluorescence intensity of dopaminergic neurons under a microscope, and recording the number of neural vesicles and the proportion of neuronal rupture; the blank control group, 2 ng / L environmental pollutant venom group, 10 ng / L environmental pollutant venom group, and 50 ng / L environmental pollutant venom group were all repeated three times.

[0017] Preferably, the method for determining the dopamine level of Caenorhabditis elegans is: collecting wild-type N2 nematodes after the subacute exposure experiment, washing them and freezing and crushing them, then using an enzyme-linked immunosorbent assay (ELISA) kit to detect the corresponding dopamine level, measuring the absorbance at a wavelength of 450 nm, calculating the sample activity, and drawing a standard curve. The concentration values ​​of the blank control group, 2 ng / L environmental pollutant venom group, 10 ng / L environmental pollutant venom group, and 50 ng / L environmental pollutant venom group are calculated according to the curve equation; when determining the dopamine level of Caenorhabditis elegans, the determination process needs to be carried out at 0-6°C.

[0018] Furthermore, the method for detecting the corresponding dopamine level using the enzyme-linked immunosorbent assay (ELISA) kit is as follows: 50 μL of standards of different concentrations are added to the standard sample wells, 10 μL of samples and 40 μL of sample diluent are added to the sample wells, 100 μL of detection antibody-HRP is added to each well, the reaction wells are sealed with a sealing film, and incubated in a 37°C water bath for 60 minutes. After the incubation, the liquid is discarded, each well is filled with washing solution and allowed to stand for 1 minute, then patted dry on absorbent paper, and the plate is washed 5 times. 50 μL of substrates A and B are added to each well, respectively, and 50 μL of stop solution is added after incubation at 37°C in the dark for 15 minutes. The concentrations of the standards are: 0, 30, 60, 120, 240 and 480 U / L.

[0019] Preferably, the method for determining the expression and aggregation of α-synuclein protein in Caenorhabditis elegans is as follows: collecting the transgenic strain NL5901 nematodes after the subacute exposure experiment, washing and anesthetizing them, placing about 25-35 nematodes on a 2% agarose pad, and observing the integrity and fluorescence intensity of dopaminergic neurons under a microscope; the blank control group, 2 ng / L environmental pollutant venom group, 10 ng / L environmental pollutant venom group, and 50 ng / L environmental pollutant venom group were all repeated three times.

[0020] Compared with the prior art, the present invention has one of the following beneficial effects:

[0021] 1. This study establishes behavioral and molecular characterization schemes and combines them with data from multiple nematode strains to generate a PD toxicity assessment model, enabling comprehensive PD toxicity assessment from behavioral to microscopic molecular levels. Using Caenorhabditis elegans as a model organism, the concentration-toxicity dose-effect model predicts the PD-inducing potential of pollutants. This not only improves the accuracy and comprehensiveness of toxicity assessments but also effectively shortens the testing cycle and reduces time costs.

[0022] 2. This invention provides a systematic solution for the health risk assessment of environmental pollutants, and also provides technical support for the toxicity research and environmental supervision of environmental pollutants. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Schematic diagram of the process of the method for assessing the risk of Parkinson's disease induced by environmental pollutants in Example 1 of the present invention;

[0024] Figure 2 : The effect of F-53B on lipofuscin accumulation in nematodes in Example 1 of the present invention; AD are representative images of lipofuscin accumulation in nematodes in each group; E is the test result of lipofuscin accumulation level in nematodes in each group;

[0025] Figure 3 The effect of F-53B on the nematode movement ability in Example 1 of the present invention; wherein A is the test result of the nematode amplitude experiment for each group; B is the test result of the nematode wavelength experiment for each group; C is the test result of the nematode center point velocity experiment for each group;

[0026] Figure 4 The effect of F-53B on abnormal locomotor behavior of nematodes in Example 1 of the present invention; AD are representative images of nematodes with different degrees of abnormal behavior severity; E is the total incidence of abnormal locomotor behavior of nematodes in each group; F is the incidence of abnormal locomotor behavior of different degrees of severity in each group of nematodes;

[0027] Figure 5 : The effect of F-53B on dopaminergic neurons in nematodes in Example 1 of the present invention; AD are representative images of dopaminergic neuron damage in nematodes in each group; E is the ratio of the number of neural vesicles in dopaminergic neurons in nematodes in each group compared with the control group; F is the incidence of neurorupture in dopaminergic neurons in nematodes in each group; G is the dopamine level in nematodes in each group;

[0028] Figure 6 : This is the effect of F-53B on the expression and aggregation of α-synuclein protein in nematodes in Example 1 of the present invention; wherein, AD are representative images of the expression and aggregation of α-synuclein protein in each group of nematodes; E is the expression level of α-synuclein protein in each group of nematodes. DETAILED DESCRIPTION

[0029] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0030] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0031] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0032] Example 1

[0033] This embodiment provides a method for assessing the risk of Parkinson's disease induced by environmental pollutants, comprising the following steps:

[0034] See also Figure 1 A subacute exposure experiment was conducted using wild-type Caenorhabditis elegans N2, transgenic Caenorhabditis elegans BZ555, and transgenic Caenorhabditis elegans NL5901 as model organisms and 6:2-chloropolyfluoroalkyl ether sulfonate (F-53B) as an environmental pollutant. The experimental method was as follows:

[0035] Four experimental groups were set up, including blank control group (0 ng / LF-53B venom), 2 ng / LF-53B venom group, 10 ng / L F-53B venom group, and 50 ng / LF-53B venom group;

[0036] The venom solution was added to the sterilized 6-well plate according to the concentration of each group, and 3 parallel wells were set up for each experimental group. The volume of venom solution added to each well was 5 mL. The synchronized L1 larvae were evenly distributed to each well, and the venom time was recorded. 100 μL of E. coli OP50 was added to each well as food. The cells were cultured in a constant temperature biochemical incubator at 20°C for 3 days, and the same amount of E. coli OP50 was fed at the same time every day.

[0037] After the exposure experiment, the transport capacity of C. elegans was observed, the abnormal movement ability of C. elegans was observed and counted, and the lipofuscin level of C. elegans, the degree of damage to dopaminergic neurons, the dopamine level, and the expression and aggregation of α-synuclein protein were measured.

[0038] The method for observing the motor ability of Caenorhabditis elegans comprises the following steps:

[0039] After subacute exposure, wild-type N2 nematodes were collected, washed at least three times with K solution, centrifuged, and the supernatant discarded. The nematodes were pipetted into six-well plates containing NGM medium and tracked under a microscope. After the nematodes had fully spread out, a 20-second video of their movements was captured using the Wormlab nematode tracking system. After the video was captured, the center velocity, wavelength, and amplitude of the movements 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 sway as the Y-axis. At least 30 wild-type N2 nematodes were collected from the blank control group, the 2 ng / L environmental pollutant venom exposure group, the 10 ng / L environmental pollutant venom exposure group, and the 50 ng / L environmental pollutant venom exposure group.

[0040] Results see Figure 3The results showed that after F-53B exposure, the different concentrations of F-53B did not significantly change the nematode amplitude compared with the control group ( Figure 3 A), but in the high concentration exposure groups (10 and 50 ng / L) of F-53B, the amplitude of nematodes showed a downward trend. On the other hand, compared with the control group, exposure to different concentrations of F-53B significantly reduced the wavelength of nematodes ( Figure 3 B), with a reduction of 7.9%-10.2%. As for the center point velocity of nematodes, the results showed that exposure to different concentrations of F-53B significantly reduced the center point velocity of nematodes and showed a good dose-effect relationship ( Figure 3 C), the F-53B concentrations in the 2, 10, and 50 ng / L groups decreased by 20.6% (p<0.05), 30.5% (p<0.01), and 32.6% (p<0.001), respectively, indicating that F-53B could impair the movement speed of nematodes at a lower concentration (2 ng / L), revealing that F-53B has the potential to induce PD.

[0041] The method for observing and counting the abnormal movement ability of Caenorhabditis elegans is:

[0042] After subacute exposure, wild-type N2 nematodes were collected, washed at least three times with K solution, centrifuged, and the supernatant discarded. The nematodes were transferred to six-well plates containing NGM medium using a pipette and tracked under a microscope. After the nematodes had fully spread out, a 20-second video of their movements was captured using the Wormlab Nematode Tracking System. Abnormal behaviors, including curling, head shaking, or immobility, were observed, and the proportion of nematodes exhibiting abnormal behaviors was recorded in each group. The experiment was repeated three times for the blank control group, the 2 ng / L environmental pollutant venom exposure group, the 10 ng / L environmental pollutant venom exposure group, and the 50 ng / L environmental pollutant venom exposure group.

[0043] Results see Figure 4 , according to the severity of abnormal movement behavior, nematodes are divided into three levels. Normal nematodes (level 0) move forward steadily in a sinusoidal curve ( Figure 4 A) Level 1 behavioral abnormalities: Nematodes move forward discontinuously and in a curled-up position ( Figure 4 B) The nematodes with abnormal behavior severity level 2 are almost motionless, with only their heads shaking irregularly ( Figure 4 C). Nematodes with the most severe abnormal behavior (level 3) stopped moving and remained motionless ( Figure 4 D). Compared with the control group (26 / 223, 11.7% incidence of abnormal behavior), F-53B significantly increased the incidence of abnormal behavior in the 2, 10, and 50 ng / L exposure groups, which were 22.8% (45 / 197; x 2 =9.313; p<0.01), 26.1% (43 / 165; x2 =13.452; p<0.001) and 27.9% (63 / 226; x 2 =18.574; p<0.001), showing a good dose-effect relationship ( Figure 4 E). The severity of abnormal behavior also increased with increasing F-53B concentrations, especially for nematodes with abnormal behavior severity level 2, the incidence rate increased from 10.3% (23 / 223) in the control group to 14.2% (28 / 197), 12.7% (21 / 165), and 22.6% (51 / 226; χ2 = 12.242; p < 0.001) in the 2, 10, and 50 ng / L exposure groups ( Figure 4 F) Aging and Parkinson's disease (PD) in nematodes often exhibit abnormal behaviors such as tremors, curling up, and immobility. These results suggest that F-53 can induce PD-like movement disorders.

[0044] The method for measuring lipofuscin levels in Caenorhabditis elegans is:

[0045] After the subacute exposure experiment, wild-type N2 nematodes were collected and anesthetized with 60 μM levamisole solution. The nematodes were placed on a glass slide and fluorescent images were captured using a ZOE™ Fluorescence Cell Imager under the same exposure parameters and conditions (DAPI channel, 405 nm excitation wavelength, 450 nm emission wavelength). The entire imaging process was completed quickly to avoid prolonged nematode movement after the anesthesia wore off and affecting the imaging. After the imaging was completed, the mean fluorescence intensity of each nematode was calculated using Image J software to determine the nematode lipofuscin level. At least 30 wild-type N2 nematodes were collected for the blank control group, the 2 ng / L environmental pollutant venom exposure group, the 10 ng / L environmental pollutant venom exposure group, and the 50 ng / L environmental pollutant venom exposure group. Each experiment was repeated three times.

[0046] Results see Figure 2 The results showed that after exposure to F-53B, the fluorescence intensity of lipofuscin in all exposure groups increased significantly compared with the control group (p < 0.001). Under the exposure of 2, 10, and 50 ng / L F-53B, the fluorescence intensity of lipofuscin in nematodes increased by 48.7%, 44.0%, and 57.5% respectively compared with the control group. Figure 2 E) showed a good dose-effect relationship, indicating that F-53B can induce the accumulation of lipofuscin, a marker of aging in nematodes, revealing that F-53B has a strong toxic effect of accelerating aging in nematodes.

[0047] The method for determining the degree of damage to dopaminergic neurons in Caenorhabditis elegans is as follows:

[0048] After subacute exposure, transgenic BZ555 nematodes were collected and rinsed three times with K buffer. The nematodes were anesthetized with 60 μM levamisole solution, and approximately 40 nematodes were placed on a 2% agarose pad. The integrity and fluorescence intensity of dopaminergic neurons were observed under a microscope. The number of neural vesicles and the proportion of neuronal ruptures in each exposure group were recorded, and fluorescence intensity was analyzed using Image J. The experiment was repeated three times for the blank control group, the 2 ng / L environmental pollutant venom group, the 10 ng / L environmental pollutant venom group, and the 50 ng / L environmental pollutant venom group.

[0049] Results see Figure 5 A- Figure 5 F, F-53B exposure caused significant damage to dopaminergic neurons. Normal dopaminergic neurons showed intact neuronal cell bodies and processes ( Figure 5 A). In contrast, the exposed group frequently showed axonal blebs (indicated by arrows) and broken axons (highlighted by circles) ( Figure 5 BD). Compared with the control group, the number of neural vesicles in dopaminergic neurons in the 2, 10, and 50 ng / L exposure groups increased significantly by 2.7 times (p<0.05), 4.1 times (p<0.001), and 5.5 times (p<0.001), respectively ( Figure 5 E). Dopaminergic neuron axonal fragmentation also showed a dose-response relationship with increasing exposure concentration, increasing from 16.0% (4 / 25) in the control group to 27.3% (6 / 22) in the 2 ng / L exposure group, 36.8% (7 / 19) in the 10 ng / L exposure group, and 50.0% (11 / 20; χ2=7.605; p<0.001) in the 50 ng / L exposure group ( Figure 5 F).

[0050] The method for measuring dopamine levels in C. elegans is:

[0051] After the subacute exposure experiment, wild-type N2 nematodes were collected. Frozen aluminum blocks were prepared in advance. Enzyme-free centrifuge tubes corresponding to the sample size were pre-chilled at 4°C. Samples were placed on the frozen aluminum blocks throughout the experiment to maintain low temperatures. After the exposure, the nematodes were collected into enzyme-free centrifuge tubes, washed three times with K solution, centrifuged, and the supernatant discarded. The tubes were frozen in liquid nitrogen and allowed to thaw at room temperature. Once the tubes were smooth, they were ready for disruption. During disruption, the nematodes were placed on the pre-frozen aluminum blocks and disrupted using a cell disruptor for 1 minute using an intermittent disruption cycle. This was done to avoid prolonged disruption, which could cause the tube temperature to rise and affect dopamine quality. After disruption, the supernatant was discarded and the nematode dopamine levels were determined using an enzyme-linked immunosorbent assay (ELISA) kit. The kit utilizes a double-antibody one-step sandwich method, and the standard concentrations are 0, 30, 60, 120, 240, and 480 U / L. Add 50 μL of standard sample at various concentrations 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 wells with sealing film and incubate in a 37°C waterbath for 60 minutes. After incubation, discard the liquid, fill each well with wash solution, let it sit for 1 minute, and then pat dry on absorbent paper. Repeat the wash five times. Add 50 μL each of substrates A and B to each well, incubate at 37°C in the dark for 15 minutes, and then add 50 μL of stop solution. Measure absorbance at 450 nm within 15 minutes to calculate sample activity. The test results are used to construct a standard curve, and the sample concentration values ​​are calculated according to the curve equation.

[0052] Results see Figure 5 Compared with the control group, the mean DA concentration in the 2ng / LF-53B exposure group decreased significantly from 183.7pg / mL to 155.8pg / mL (p<0.05). At 10ng / L, the mean dopamine concentration continued to decrease compared to the 2ng / L group, reaching 141.4pg / mL, significantly lower than the control group (p<0.01), a decrease of 15.2%. When the F-53B exposure concentration reached 50ng / L, the DA concentration reached a minimum of 132.1pg / mL, which was extremely significantly lower than the mean concentration in the control group (p<0.001), a decrease of 28.1%. Subacute exposure to more than 2ng / LF-53B can damage dopaminergic neurons, leading to decreased DA secretion.

[0053] The method for measuring the expression and aggregation of α-synuclein protein in Caenorhabditis elegans is as follows:

[0054] After subacute exposure, transgenic NL5901 nematodes were collected, rinsed three times with K buffer, and anesthetized with 60 μM levamisole. Approximately 30 nematodes were placed on a 2% agarose pad, and fluorescence intensity was observed under a microscope. Fluorescence intensity data were analyzed using Image J. Three replicates were performed for each of the blank control group, the 2 ng / L environmental pollutant venom exposure group, the 10 ng / L environmental pollutant venom exposure group, and the 50 ng / L environmental pollutant venom exposure group.

[0055] Results see Figure 6 α-synuclein is a soluble protein composed of 140 amino acids and is an important pathogenic protein in PD. The expression of α-synuclein protein was quantified by fluorescence intensity. Compared with the control group, the expression of α-synuclein protein in the 2, 10, and 50 ng / L exposure groups increased significantly by 1.25 (p<0.01), 1.30 (p<0.001), and 1.37 times (p<0.001), respectively. ( Figure 6 AE). In addition, a significant increase in the aggregation of α-synuclein protein (arrow) was also observed in nematodes in all exposure groups ( Figure 6 In the F-53B-exposed group, the expression and aggregation of α-synuclein protein in nematodes were significantly increased, further demonstrating the PD-inducing effect of F-53B.

[0056] Although the principles of the present invention have been described in detail above in conjunction with the preferred embodiments of the present invention, those skilled in the art should understand that the above embodiments are merely illustrative of the present invention and are not intended to limit the scope of the present invention. The details in the embodiments do not constitute a limitation on the scope of the present invention. Without departing from the spirit and scope of the present invention, any obvious changes such as equivalent transformations and simple substitutions based on the technical solution of the present invention fall within the scope of protection of the present invention.

Claims

1. A method for assessing the risk of Parkinson's disease induced by environmental pollutants, characterized in that: The following steps are involved: Subacute exposure experiments using Caenorhabditis elegans as a model organism will assess the risk of Parkinson's disease induced by environmental pollutants by observing the worm's motor capacity, observing and counting abnormal movement, and measuring lipofuscin levels, dopaminergic neuron damage, dopamine levels, and α-synuclein protein expression and aggregation. Wherein, the environmental pollutant is 6:2 chloropolyfluoroalkyl ether sulfonate.

2. The method for assessing the risk of Parkinson's disease induced by environmental pollutants according to claim 1, characterized in that: In the subacute exposure experiment, a blank control group, a 2 ng / L environmental pollutant venom group, a 10 ng / L environmental pollutant venom group, and a 50 ng / L environmental pollutant venom group are set up respectively, and three parallel groups are set up in each group; and / or, The varieties of Caenorhabditis elegans include wild type N2, transgenic strain BZ555 and transgenic strain NL5901.

3. The method for assessing the risk of Parkinson's disease induced by environmental pollutants according to claim 2, characterized in that: The method of the subacute exposure experiment is as follows: synchronized L1 larvae are evenly distributed into a blank control group, a 2 ng / L environmental pollutant venom group, a 10 ng / L environmental pollutant venom group, and a 50 ng / L environmental pollutant venom group, and each group is fed with an equal amount of E. coli OP50 as food, and cultured at 18-22°C for 2.5-3.5 days; The volumes of environmental pollutant venom added to the 2ng / L environmental pollutant venom group, the 10ng / L environmental pollutant venom group and the 50ng / L environmental pollutant venom group were all the same; the solution added to the blank control group was dimethyl sulfoxide solution, and the added volume was the same as the volume of environmental pollutant venom added to the 2ng / L environmental pollutant venom group.

4. The method for assessing the risk of Parkinson's disease induced by environmental pollutants according to claim 2, characterized in that: The method for observing the movement ability of Caenorhabditis elegans is: Wild-type N2 nematodes were collected after the subacute exposure experiment, cleaned, and placed in NGM culture medium for microscopic tracking. After the nematodes fully crawled away, a 20-second video of the nematodes' movements was captured using the Wormlab nematode tracking system. The Wormlab analysis system was used to measure the center point velocity, wavelength, and movement amplitude. The nematode's movement trajectory was a sinusoidal curve, with the movement direction as the X-axis and the body swing direction as the Y-axis. The number of wild-type N2 nematodes collected from the blank control group, 2 ng / L environmental pollutant venom group, 10 ng / L environmental pollutant venom group and 50 ng / L environmental pollutant venom group was no less than 30.

5. The method for assessing the risk of Parkinson's disease induced by environmental pollutants according to claim 2, characterized in that: The method for observing and counting the abnormal movement ability of Caenorhabditis elegans is: Wild-type N2 nematodes were collected after the subacute exposure experiment, cleaned, and placed in NGM culture medium for microscopic tracking. After the nematodes fully crawled away, a 20-second video of the nematodes' movements was recorded using the Wormlab nematode tracking system. Abnormal behaviors, including curling up, head shaking, or stillness, were observed, and the proportion of nematodes exhibiting abnormal behaviors in each group was recorded. The experiment was repeated three times for the blank control group, 2 ng / L environmental pollutant venom group, 10 ng / L environmental pollutant venom group and 50 ng / L environmental pollutant venom group.

6. The method for assessing the risk of Parkinson's disease induced by environmental pollutants according to claim 2, characterized in that: The method for measuring the lipofuscin level of Caenorhabditis elegans is: Wild-type N2 nematodes were collected and anesthetized after the subacute exposure experiment, and fluorescent photos were taken. Finally, 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 from the blank control group, 2 ng / L environmental pollutant venom group, 10 ng / L environmental pollutant venom group, and 50 ng / L environmental pollutant venom group was no less than 30, and the experiment was repeated three times.

7. The method for assessing the risk of Parkinson's disease induced by environmental pollutants according to claim 2, characterized in that: The method for determining the degree of damage to dopaminergic neurons in Caenorhabditis elegans is as follows: After the subacute exposure experiment, transgenic BZ555 nematodes were collected and anesthetized. 35-45 nematodes were placed on a 2% agarose pad. The integrity and fluorescence intensity of dopaminergic neurons were observed under a microscope, and the number of neural blebs and the proportion of neuronal rupture were recorded. The experiment was repeated three times for the blank control group, 2 ng / L environmental pollutant venom group, 10 ng / L environmental pollutant venom group and 50 ng / L environmental pollutant venom group.

8. The method for assessing the risk of Parkinson's disease induced by environmental pollutants according to claim 2, characterized in that: The method for measuring the dopamine level of Caenorhabditis elegans is: Wild-type N2 nematodes were collected after the subacute exposure experiment, cleaned, and frozen and crushed. The corresponding dopamine levels were then detected using an enzyme-linked immunosorbent assay (ELISA) kit. The absorbance at a wavelength of 450 nm was measured, and the sample activity was calculated. A standard curve was drawn, and the concentration values ​​of the blank control group, 2 ng / L environmental pollutant venom group, 10 ng / L environmental pollutant venom group, and 50 ng / L environmental pollutant venom group were calculated according to the curve equation. When measuring dopamine levels in Caenorhabditis elegans, the measurement process needs to be carried out at 0-6°C.

9. The method for assessing the risk of Parkinson's disease induced by environmental pollutants according to claim 8, characterized in that: The method for detecting the corresponding dopamine level with the enzyme-linked immunosorbent assay (ELISA) kit is as follows: Add 50 μL of standards of different concentrations to the standard sample wells, add 10 μL of samples and 40 μL of sample diluent to the sample wells, add 100 μL of detection antibody-HRP to each well, seal the reaction wells with sealing film and incubate in a 37°C water bath for 60 minutes. After the incubation, discard the liquid, fill each well with washing solution, let it stand for 1 minute, and then pat dry on absorbent paper. Repeat the washing process 5 times, add 50 μL of substrates A and B to each well, incubate at 37°C in the dark for 15 minutes, and then add 50 μL of stop solution; The concentrations of the standards are: 0, 30, 60, 120, 240 and 480 U / L.

10. The method for assessing the risk of Parkinson's disease induced by environmental pollutants according to claim 2, characterized in that: The method for determining the expression and aggregation of α-synuclein protein in Caenorhabditis elegans is as follows: After the subacute exposure experiment, the transgenic NL5901 nematodes were collected, washed, and anesthetized. About 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 experiment was repeated three times for the blank control group, 2 ng / L environmental pollutant venom group, 10 ng / L environmental pollutant venom group and 50 ng / L environmental pollutant venom group.

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