Intervention and analysis method of terahertz radiation on Alzheimer's disease model nematodes

Abstract

The invention discloses an intervention and analysis method of terahertz radiation on Alzheimer's disease model nematodes, which comprises the following steps: directly performing radiation intervention on the Alzheimer's disease model nematodes by adopting terahertz waves, and performing behavioral analysis, fluorescence analysis and data analysis on the nematodes after intervention. The behavioral analysis comprises, but is not limited to, analysis of life, exercise ability and ingestion ability of the nematodes, and the fluorescence analysis comprises, but is not limited to, oxidative stress and aging condition analysis. According to the intervention and analysis method for the Alzheimer's disease model nematodes by the terahertz radiation, a brand-new analysis method is provided for evaluation of terahertz wave intervention and biological effects on the animal level.

Description

Technical Field

[0001] The present invention relates to the technical field of Alzheimer's disease regulation, and in particular to a method for intervening and analyzing terahertz radiation on Alzheimer's disease model nematodes. Background Art

[0002] Alzheimer's Disease (AD) is a neurodegenerative disease caused by impaired brain function, which is mainly manifested by a significant decline in key brain functions such as memory, movement and cognition. According to data collected by the World Health Organization between 2017 and 2020, more than 55 million people in the world suffer from dementia, and AD patients are estimated to account for more than 60%. It is expected that the number of patients will reach a staggering 139 million by 2050. The Aβ cascade hypothesis has always been the mainstream theory to explain the pathogenesis of AD. At the same time, studies have shown that the risk of AD in occupational groups who are exposed to high-intensity electromagnetic fields for a long time is significantly increased. Therefore, more and more studies have gradually focused on the impact of electromagnetic waves on Aβ aggregation.

[0003] Terahertz waves are considered to be the most promising electromagnetic wave frequency band in the biological field. Traditional electromagnetics defines the terahertz band as 0.1~10 THz, which is exactly in the weak interaction energy level range that maintains the stability of biological macromolecules. Based on this particularity, people have concerns about the biological safety of terahertz waves. Studies have shown that terahertz radiation can affect the aggregation process of Aβ. The relevant effects have not yet been verified and evaluated in animal experiments.

[0004] Caenorhabditis elegans is a classic model organism, which is favored by researchers for its simple nervous system, short life cycle, and high homology with human genes. In particular, the β-amyloid protein (Aβ) gene related to AD can be expressed in nematodes through transgenic technology, making it very suitable as a model organism for AD. The biological safety of terahertz waves is verified by intervening terahertz radiation on nematodes. Summary of the invention

[0005] The purpose of the present invention is to provide a method for the intervention and analysis of terahertz radiation on Alzheimer's disease model nematodes, and to fill the gaps in the current research strategies and evaluation methods of terahertz biological effects at the animal level.

[0006] To achieve the above object, the present invention provides the following technical solutions: The intervention and analysis method of terahertz radiation on Alzheimer's disease model nematodes provided by the present invention includes the following steps: directly irradiating the Alzheimer's disease model nematodes with terahertz waves, and after the intervention, performing behavioral analysis, fluorescence analysis and data analysis on the nematodes. The behavioral analysis includes, but is not limited to, analyzing the lifespan, locomotor ability and feeding ability of the nematodes, and the fluorescence analysis includes, but is not limited to, analyzing oxidative stress and aging conditions.

[0007] Further, the feeding conditions of the Alzheimer's disease model nematodes are as follows: a nematode growth medium is prepared with 5-fluorouracil, agar powder, yeast powder, tryptone, calcium chloride and magnesium sulfate, and the nematodes are fed with Escherichia coli OP50 at 16-25 °C.

[0008] Further, the radiation intervention on the Alzheimer's disease model nematodes is specifically as follows: after the terahertz wave source emits terahertz waves, they are converged and then diverged by two off-axis parabolic mirrors, and are reflected by a plane mirror to the upper surface of the culture medium so that the nematodes in the culture medium are subjected to radiation intervention. The intervention period is irradiation once every 48 hours, and each time lasts for 2 hours.

[0009] Further, the evaluation method of the lifespan of the nematodes is to take the average value after all the nematodes in this group die. The standard for nematode death is to stop all activities and have no response after being gently touched with a platinum wire for 10 s; the evaluation method of the locomotor ability of the nematodes is to count the head swing frequency of the nematodes per unit time, and the definition of one swing is specifically that the head of the nematode swings from one side to the other side and then returns to the original position; the evaluation method of the feeding ability of the nematodes is to count the pharyngeal pump vibration frequency of the nematodes per unit time, and the definition of one vibration is specifically that the pharyngeal pump returns to the same position twice.

[0010] Further, the oxidative stress analysis is specifically to analyze the content of reactive oxygen species; the aging condition analysis is specifically to analyze the content of lipofuscin.

[0011] Further, the content of reactive oxygen species is labeled with H2DCFDA fluorescent probe and observed with an inverted fluorescence microscope; the content of lipofuscin is directly observed with an inverted fluorescence microscope.

[0012] Further, the data analysis uses SPSS 25.0 software to perform statistical analysis on the input data, and the data results are expressed as , when the input data meets the normal distribution condition, the comparison of the statistical analysis results between the two groups of data uses the t-test, and the survival curve analysis uses the Kaplan-Meier test. p <0.05 represents that the difference is statistically significant.

[0013] The present invention also constructs a terahertz radiation intervention system for agar culture medium, and the system includes: an optical platform, a terahertz wave source, an off-axis parabolic mirror, and a plane mirror.

[0014] In the radiation intervention system, terahertz radiation intervention is performed on nematodes, and dry air is introduced to keep the environmental humidity not higher than 5% to reduce the environmental attenuation of terahertz radiation. The intervention period of the experimental group is irradiation once every 48 hours for 2 hours each time. The control group and the experimental group are in the same environment, and the distance between the centers of the culture dishes is greater than 20 cm.

[0015] Based on the above technical solutions, the embodiments of the present invention can at least produce the following technical effects: The method for intervening and analyzing the terahertz radiation on Alzheimer's disease model nematodes provided by the present invention provides a new analysis method for the evaluation of terahertz wave intervention and biological effects at the animal level through the radiation intervention on Alzheimer's disease model nematodes and the behavioral analysis, fluorescence analysis, and data analysis of nematodes. At the same time, the present invention also provides a reference for the early diagnosis, intervention, and treatment strategies of Alzheimer's disease, helps to expand the research in the field of terahertz biomedicine, and promotes its further application in neuroscience and disease treatment. In addition, the related methods involved in the present invention can also be extended to the research of other neurodegenerative disease models, thereby accelerating the R & D process of related disease treatment methods. Description of the Drawings

[0016] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the following drawings are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on the structures shown in these drawings.

[0017] Figure 1 shows the influence of terahertz radiation on the lifespan of Alzheimer's model nematodes in the embodiments of the present invention; Figure 2 shows the influence of terahertz radiation on the locomotor ability of Alzheimer's model nematodes in the embodiments of the present invention; Figure 3 shows the influence of terahertz radiation on the feeding ability of Alzheimer's model nematodes in the embodiments of the present invention; Figure 4 shows the influence of terahertz radiation on the oxidative stress of Alzheimer's model nematodes in the embodiments of the present invention; Figure 5 shows the influence of terahertz radiation on the aging of Alzheimer's model nematodes in the embodiments of the present invention. Detailed Embodiments

[0018] The following will describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the scope of protection of the present invention.

[0019] In the following examples, the Caenorhabditis elegans strain CL4176 was used as the Alzheimer's disease model nematode in this embodiment (hereinafter referred to as CL4176 nematode). The 5-fluorouracil, agar powder, yeast powder, tryptone, calcium chloride, and magnesium sulfate used were all raw materials for preparing the nematode culture medium and were purchased from Beijing Solarbio Science & Technology Co., Ltd. The Escherichia coli E.coli OP50 used for feeding nematodes was purchased from the Caenorhabditis Genetics Center (CGC) of the University of Minnesota, USA.

[0020] In this example, the specific preparation method of the culture medium used for nematode culture was as follows: Weigh 17 g of agar, 3 g of NaCl, and 2.5 g of peptone respectively. After dissolving in ultrapure water and making up the volume to 1000 mL, autoclave it. After cooling to room temperature to 55 °C, add 1 mL of sterilized CaCl 2 (1 M), MgSO 4 (1 M), and cholesterol-ethanol solution (5 mg / mL) respectively. Then add 25 mL of PBS buffer (0.8 g NaCl, 0.02 g KCl, 0.142 g Na 2 SO 4 , 0.02 g KH 2 PO 4 / 100 ml). After mixing well, pour it into 3 cm culture dishes in turn. After it solidifies, store it at 4 °C for later use. Keep sterile operation throughout the process after autoclaving. Pick E.coli OP50 colonies to make a bacterial solution, and evenly coat it on the solidified NGM surface in a laminar flow hood, 100 μL per plate. After coating, transfer the NGM medium to a 37 °C incubator. Wait until the bacterial lawn evenly covers the surface of the medium before inoculating nematodes.

[0021] In this example, the specific preparation method of the M9 buffer used for washing nematodes, etc. was as follows: Weigh 5 g of NaCl, 6 g of Na 2 HPO 4 , 3 g of KH 2 PO 4 , and 1 mL of MgSO 4 (1 M) respectively. After dissolving in ultrapure water and making up the volume to 1000 mL. Autoclave it and then dispense it, and store it at 4 °C.

[0022] In this example, the specific implementation of the radiation intervention is to use a terahertz radiation intervention system to intervene in nematodes. By adjusting the optical path, the terahertz light spot just covers the culture medium. The CL4176 nematodes are placed on the upper surface of the agar culture medium and placed at the center of the light spot to receive radiation. The radiation cycle is to receive radiation intervention once every 48 h, and each time it lasts for 2 h.

[0023] In this example, the culture conditions are as follows: the radiation treatment of the sample is carried out at room temperature (about 20 °C), and immediately transferred to a constant temperature incubator at 25 °C for continued culture after radiation. Three parallel groups are set for each radiation experiment.

[0024] In this example, the steps for nematode synchronization are as follows: Wash the nematodes in the egg-laying period into a centrifuge tube with M9 buffer, centrifuge at 3000 rpm for 1 min, collect the nematodes, add nematode lysis solution (1 g NaOH, 5 mL NaClO2 (12 - 15%) / 100 mL), and shake on a mixer for 5 min to fully lyse the worms. After centrifuging at 3000 rpm for 2 min, remove the supernatant to obtain eggs. After resuspending with M9 buffer, centrifuge the eggs again at 3000 rpm for 2 min to wash the eggs, and repeat the washing operation three times. Resuspend the washed eggs in M9 buffer and incubate in an incubator at 16 °C for 24 h. All the hatched nematodes are in the L1 stage and can be transferred to NGM medium coated with OP50 for culture. The experiment can be started when they develop to the L4 stage.

[0025] In this example, an analysis method for the terahertz biological effects of Alzheimer's disease model nematodes is also proposed. It mainly includes the following aspects: Analysis of nematode lifespan: Transfer the synchronized CL4176 nematodes to the prepared agar nematode growth medium for culture, culture them in a constant temperature incubator at 16 °C until the L4 stage, and then transfer them to a 25 °C incubator to induce Aβ expression and start the experiment. Starting from the first day of entering the L4 stage, irradiate once every 48 h. During the irradiation interval, count the number of dead nematodes and remove the dead individuals. Replace the culture medium on the day of counting the number of nematodes to ensure that the nematodes have sufficient food. Alternate irradiation and counting of surviving individuals until all nematodes die. The standard for nematode death is complete cessation of activity and no response after repeatedly touching with a platinum wire for 10 s.

[0026] As can be seen from Figure 1 and Table 1, in this example, after terahertz radiation intervention, the lifespan of CL4176 nematodes is significantly shortened, with an overall shortening of about 21.4%. This indicates that under terahertz radiation intervention, the Aβ toxicity in nematodes is enhanced, thereby increasing the mortality rate of CL4176 nematodes.

[0027] Table 1 Effects of Metformin on the Lifespan of CL4176 Nematodes

[0028] Analysis of the locomotor ability of nematodes: The pre-treatment and lifespan analysis methods are the same. Radiation intervention starts on the first day when the nematodes enter the L4 stage. Irradiation is carried out every 48 h, and the locomotor ability of the nematodes is measured during the irradiation interval until the 6th day, that is, the head swing frequency is statistically analyzed on the 2nd, 4th, and 6th days of the start of the intervention. A certain number of nematodes are randomly selected and transferred to 20 μL of M9 buffer respectively. Two people record the number of head swings of the nematodes within 1 min and take the average value. If the difference between the two statistics is greater than 10, it is recorded again. A head swing is regarded as the head of the nematode swinging from one side to the other side and then back to the original position.

[0029] As Figure 2 shown, terahertz electromagnetic radiation will affect the locomotor ability of nematodes. The specific manifestation in this example is that the head swing frequency of the irradiated group of CL4176 nematodes decreases.

[0030] Analysis of the feeding ability of nematodes: The pre-treatment is the same as the lifespan experiment. Radiation intervention starts on the first day when the nematodes enter the L4 stage. Irradiation is carried out every two days for a total of 8 days, and statistics are carried out on the 3rd, 6th, and 9th days of the radiation intervention. The specific operation is to randomly select a certain number of nematodes in the petri dish and count the vibration frequency of the pharyngeal pump within 1 min. The definition of one vibration is specifically that the pharyngeal pump returns to the same position twice. It is recorded by two people respectively. If the difference between the two statistics is greater than 10, it is recorded again.

[0031] As Figure 3 shown, terahertz electromagnetic radiation will affect the feeding ability of nematodes. The specific manifestation in this example is that the vibration frequency of the pharyngeal pump of the irradiated group of CL4176 nematodes decreases.

[0032] Analysis of the oxidative stress of nematodes: The pre-treatment is the same as the lifespan experiment. Radiation intervention starts on the first day when the nematodes enter the L4 stage. Irradiation is carried out every two days. After the 3rd radiation experiment, that is, on the 6th day, lipofuscin fluorescence detection is carried out. The experimental group and the control group of CL4176 nematodes are respectively transferred to M9 buffer containing 100 μM H2DCFDA fluorescent probe and incubated in a 20 °C incubator for 30 min. Then they are transferred to a glass slide containing a solution of levamisole at a concentration of 1 mmol / L again, and fluorescence photos are taken using an inverted fluorescence microscope and the fluorescence intensity is quantitatively analyzed in combination with Image J1.51 software.

[0033] The results of this example are as Figure 4As shown, it can be clearly seen that the fluorescence intensity of ROS in CL4176 nematodes after 3 irradiations is significantly higher than that of the control group. Figure 4 (c) shows the significance of the increase in ROS. The conclusion confirms the enhancing effect of terahertz electromagnetic radiation on Aβ toxicity in this example.

[0034] Analysis of nematode aging: The pretreatment method and radiation method are the same as those for ROS detection. The nematodes to be tested after 3 interventions and the control group of CL4176 nematodes are respectively picked onto slides containing levamisole solution with a concentration of 1 mmol / L. An inverted fluorescence microscope is used to directly observe the fluorescence intensity of lipofuscin in the worm body and take pictures. Image J1.51 software is used to quantitatively analyze the lipofuscin fluorescence intensity value.

[0035] The results of this example are as Figure 5 shown. Figure 5 (c) shows the significance of the lipofuscin fluorescence intensity. It can be clearly seen that the fluorescence intensity of lipofuscin in CL4176 nematodes after 3 irradiations is significantly higher than that of the control group. This once again verifies the promoting effect of terahertz radiation on the pathological process of CL4176 nematodes in this example.

[0036] In summary: The method for intervening and analyzing terahertz radiation on Alzheimer's disease model nematodes proposed by the present invention enables this example to comprehensively evaluate the intervention effect of terahertz radiation on Alzheimer's model nematodes. It provides a new methodology for the evaluation of terahertz wave intervention and biological effects at the animal level.

[0037] Finally, it should be particularly noted that the above-provided embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the present invention. Those skilled in the art should understand that any modification, deformation, or equivalent replacement made to the above specific embodiments without departing from the spirit and scope of the present invention shall be included within the protection scope of the claims of the present invention.

Claims

1. A method for intervening and analyzing Alzheimer's disease model nematodes using terahertz radiation, characterized in that: The following steps are involved: Terahertz waves are used to directly perform radiation intervention on Alzheimer's disease model nematodes, and behavioral analysis, fluorescence analysis and data analysis are performed on the nematodes after the intervention. The behavioral analysis includes but is not limited to analysis of the lifespan, movement ability and feeding ability of the nematodes, and the fluorescence analysis includes but is not limited to analysis of oxidative stress and aging.

2. The method for intervention and analysis of terahertz radiation on Alzheimer's disease model nematodes according to claim 1, characterized in that: The feeding conditions of the Alzheimer's disease model nematodes are as follows: a nematode growth medium is prepared with 5-fluorouracil, agar powder, yeast powder, tryptone, calcium chloride and magnesium sulfate, and Escherichia coli is used at 16-25 °C. E. coli OP50 feeding.

3. The method for intervention and analysis of terahertz radiation on Alzheimer's disease model nematodes according to claim 1, characterized in that: The radiation intervention for the Alzheimer's disease model nematodes is specifically as follows: after the terahertz wave source emits the terahertz wave, it converges through two off-axis parabolic mirrors and then diverges, and is reflected by a plane mirror to the upper surface of the culture medium so that the nematodes in the culture medium are subjected to radiation intervention. The intervention cycle is once every 48 hours, and each time lasts for 2 hours.

4. The method for intervention and analysis of terahertz radiation on Alzheimer's disease model nematodes according to claim 1, characterized in that: The life span of the nematode is evaluated by taking the average value after all the nematodes in the group have died, and the criterion for the death of the nematode is that the nematode stops all activities and has no response when repeatedly touched by the platinum wire for 10 seconds; the movement ability of the nematode is evaluated by counting the frequency of the nematode's head swinging per unit time, and one swing is specifically defined as the nematode's head swinging from one side to the other side and then returning to the original position; the feeding ability of the nematode is evaluated by counting the frequency of the nematode's pharyngeal pump vibration per unit time, and one vibration is specifically defined as the pharyngeal pump returning to the same position twice.

5. The method for intervention and analysis of terahertz radiation on Alzheimer's disease model nematodes according to claim 1, characterized in that: The oxidative stress analysis specifically involves analyzing the content of active oxygen; and the aging condition analysis specifically involves analyzing the content of lipofuscin.

6. The method for intervention and analysis of terahertz radiation on Alzheimer's disease model nematodes according to claim 5, characterized in that: The active oxygen content is labeled with H2DCFDA fluorescent probe and then observed using an inverted fluorescence microscope; the lipofuscin content is directly observed using an inverted fluorescence microscope.

7. The method for intervention and analysis of terahertz radiation on Alzheimer's disease model nematodes according to claim 1, characterized in that: The data analysis was performed using SPSS 25.0 software, and the data results were expressed as ,When the input data satisfied the normal distribution condition, the t test was used to compare the ,statistical analysis results between the two groups of data, and the Kaplan-Meier test was used for ,survival curve analysis.

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