CN112043828A - Application of porphyrin substances in prolonging of nematode life under illumination - Google Patents
Application of porphyrin substances in prolonging of nematode life under illumination Download PDFInfo
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- RKCAIXNGYQCCAL-UHFFFAOYSA-N porphin Chemical compound N1C(C=C2N=C(C=C3NC(=C4)C=C3)C=C2)=CC=C1C=C1C=CC4=N1 RKCAIXNGYQCCAL-UHFFFAOYSA-N 0.000 description 1
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
- A61K41/0057—Photodynamic therapy with a photosensitizer, i.e. agent able to produce reactive oxygen species upon exposure to light or radiation, e.g. UV or visible light; photocleavage of nucleic acids with an agent
- A61K41/0071—PDT with porphyrins having exactly 20 ring atoms, i.e. based on the non-expanded tetrapyrrolic ring system, e.g. bacteriochlorin, chlorin-e6, or phthalocyanines
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P39/00—General protective or antinoxious agents
- A61P39/06—Free radical scavengers or antioxidants
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- Public Health (AREA)
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- General Health & Medical Sciences (AREA)
- Animal Behavior & Ethology (AREA)
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Abstract
The invention discloses application of porphyrin substances in prolonging the service life of nematodes under illumination. The nematode is caenorhabditis elegans. The nematode diet is uracil-deficient Escherichia coli (E.coli) OP 50. The administration mode of the nematode is as follows: is added before preparing OP50 culture plates, and the final concentration of the drug in OP50 culture medium is 0.4-0.8 mu M; after shaking and mixing well, pour into a 35mm sterile petri dish. The invention identifies the anti-aging and life prolonging functions of porphyrin under illumination through in vivo experiments, provides a theoretical basis for potential application of the porphyrin in anti-aging, excludes the porphyrin and has no obvious adverse effect on the activity of the nematode.
Description
Technical Field
The invention relates to application of porphyrin substances in prolonging the service life of nematodes under illumination, belonging to newly found application of anti-aging small molecule drugs.
Technical Field
According to the sixth census result in the country, the population over 65 years old in China already accounts for 8.87% of the country and enters the aging society of the population. As the aging of the social population is getting worse, senile diseases such as diabetes, coronary heart disease, etc. are remarkably increased (Mandula, etc., 2017), and aging is an important factor causing the increase of these diseases. Aging is also called programmed cell death, and in a broad sense, aging occurs because the self-repair function of cells declines with age, which leads to the decline of the resistance of the human body and the increase of the incidence of diseases. At present, no specific molecular mechanism is described for the occurrence mechanism of aging, and the nine characteristics of aging are proposed by Carlos et al in 2013: (1) genomic instability (Genomic instability); (2) shortening of telomeres (telomee anchorage); (3) alterations in epigenetics (Epigenetic alterations); (4) protein homeostasis imbalance (Loss of dystasis); (5) dystrophy (delayed μ related nutrient-sensing); (6) mitochondrial dysfunction (Mitochondrial dysfunction); (7) cell senescence (Cell μ lar senescence); (8) stem cell exhaustion (Stem cell exhaustion); (9) the intercellular communication changes (alternate intercellular μ lar communication). These nine senescence features are not all detrimental, such as severe mitochondrial DNA mutations, telomere loss, and body damage; while the slight mitochondrial dysfunction is beneficial to the body's resistance, it follows that the damage caused by different degrees of injury varies, and aging does not occur due to a single factor but due to the combined action of multiple complex factors. According to the nine major features of aging proposed by researchers, several measures for aging have been proposed, including anti-inflammatory drugs, stem cells for senescence elimination, chaperone activation, dietary restriction, activation of pathways related to senescence, and the like. This means that aging can be artificially regulated.
Porphyrins (porphyrins) are a class of alpha-carbon atoms consisting of four pyrrole subunitsMacromolecular heterocyclic compounds formed by the interconnection of molecules through methine bridges (= CH-). The parent compound is porphine as shown in FIG. 5 (porphin, C)20H14N4) (Wiley-VCH., et al, Encyclopedia of organic and biological chemistry, 2011: 10.1002/978), substituted porphines are known as porphyrins (Rayati, Saeed, T., et al, Journal of Experimental nanoscience, 2016.11 (11): 872.). The porphyrin ring has 26 pi electrons, is a highly conjugated system, and therefore appears dark (Boldyrev, Alexander I T., et al., Organic)& Biomolecular Chemistry. 12 (32):p. 6145–6150)。
Chlorophyll is an important pigment present in plants, and chlorophyll mainly includes chlorophyll a (chla) chlorophyll b (chlb). The chlorophyll has a structure that all the chlorophyll contains four pyrrole rings, and the pyrrole rings are externally connected with four methylene groups to form a porphyrin ring. The chlorophyll metabolite pyropheophytin a, is a porphyrin ring formed by chlorophyll in the removal of phytol and magnesium atoms under the action of chlorophyllase and metal chelates (Suzuki, T., et al, Physiol Biochem, 2005.43 (5): p.459-64.). Although pyropheophytin a loses the magnesium atom, a porphyrin ring is still present. The existence of green color of plants is mainly the function of porphyrin ring, and pyropheophytin a still has ultrahigh sensitivity to light. Chlorophyll is widely applied in the field of disease treatment, but so far, no chlorophyll is reported to be related to aging.
Sunlight is the main energy source of organisms, whether plants or animals, and most of them are capable of perceiving the effects of light. Functionally, mammals and humans have two systems for detecting light, the first being the classical vision system responsible for image formation and the second being the Non-image forming system (NIF) (Daneault, v., et al., J physical antropol, 2016.35: p. 9.). The detection of light helps to regulate many basic functions of organisms, plants can produce energy by photosynthesis using sunlight, and higher animals like humans require light to maintain normal development of organisms. Studies have also shown that circadian rhythm imbalances are associated with diseases such as breast cancer, metabolic disorders and psychiatric and behavioral disorders (bedrisian, t.a. and r.j. Nelson, 2013.18 (7): p. 751-7.).
Disclosure of Invention
Aiming at the problems that the aging regulation and control related micromolecular compounds and the luminous environment influence the biorhythm, the invention provides the function of prolonging the life of the porphyrin substances in the nematodes under the illumination condition; and provides the optimum concentration range of chlorophyll metabolite Pyropheophorbide a (Pyrophorbide-a, Pa) for prolonging life.
In order to achieve the purpose of the invention, the technical scheme adopted by the invention is as follows: the porphyrin substances are applied to prolonging the service life of nematodes under illumination.
Further, the nematode is caenorhabditis elegans.
Further, the nematode diet is uracil-deficient escherichia coli (e. coli) OP 50.
Further, the mode of administration of the nematode: is added before preparing OP50 culture plates, and the final concentration of the drug in OP50 culture medium is 0.4-0.8 mu M; after shaking and mixing well, pour into a 35mm sterile petri dish.
Further, OP50 medium was prepared: lysis Broth solid medium Lysogeny Broth, LB being the main food source for OP50 survival, 1000 ml of LB solid medium was prepared: the following reagents were weighed into a 1L Erlenmeyer flask: 10 g of trypsin, 10 g of NaCl, 5 g of yeast extract and 15 g of agar (OP 50 liquid culture medium does not need to be added), using deionized water to fix the volume to 1L, and performing autoclaving to obtain OP50 culture medium;
synchronizing nematodes: culturing the oviposition nematodes capable of laying eggs and developed to day 1 in a culture medium overnight, removing the female nematodes after 10 h to obtain the nematodes in the same period, and culturing in an incubator at 20 ℃ until the nematodes are developed to young adult period for transfer.
Further, the environmental condition settings are: after the nematodes are synchronized, the nematodes are cultured in a nematode culture medium until adults grow, and the nematodes needing exposure are subjected to exposure treatment. After treatment, the culture is carried out in a constant temperature and light-proof incubator at 20 ℃.
Furthermore, the exposure process uses near-external red light, and the wavelength range of the red light is 660 nm.
Further, the nematode life test is divided into four groups: a first group: dark was not subjected to any treatment of nematodes; second group: treating the LWL only for nematode illumination; third group: treating Dark + Pa only for nematode chlorophyll metabolite Pa; and a fourth group: chlorophyll metabolite Pa and light treatment LWL + Pa were given to the nematodes.
Further, the porphyrin-like substance is chlorophyll metabolite Pyropheophorbide-a, Pa.
Has the advantages that: the method has simple steps and good effect. The invention identifies the anti-aging and life prolonging functions of porphyrin under illumination through in vivo experiments, provides a theoretical basis for potential application of the porphyrin in anti-aging, excludes the porphyrin and has no obvious adverse effect on the activity of the nematode. The effect of chlorophyll metabolite Pyropheophorbide a (Pa) on prolonging life of nematode under illumination condition and the optimum concentration range are identified through in vivo experiments, and the application of the chlorophyll metabolite Pyropheophorbide a (Pa) in prolonging life of nematode is provided.
Drawings
FIG. 1 is a graph showing the effect of different concentrations of the chlorophyll metabolite Pa of the present invention on the longevity of C.elegans.
FIG. 2 is a graph showing lifetime of each control group, Dark (no drug administration, no light), LWL (no drug administration, light), Dark +0.8 μ M (drug administration, no light), LWL +0.8 μ M (drug administration, light) at the optimum concentration of 0.8 μ M according to the present invention.
FIG. 3 is the effect of the chlorophyll metabolite Pa of the present invention on nematode motility, left for swimming rate; the right is a graph of the effect of feed rate.
FIG. 4 is a graph showing the results of heat stress, oxidative stress, and osmotic stress life, respectively, of the effects of chlorophyll metabolite Pa and light under stress conditions according to the present invention.
FIG. 5 shows the structural diagram of the porphyrin parent compound, which is the simplest porphyrin-based substance of the present invention.
Detailed Description
In order to facilitate an understanding of the invention, the invention will now be described more fully hereinafter with reference to the accompanying drawings, in which several embodiments of the invention are shown, but which may be embodied in different forms and not limited to the embodiments described herein, but which are provided so as to provide a more thorough and complete disclosure of the invention.
Examples
Experimental materials
Reagent: sodium chloride, dipotassium phosphate, potassium dihydrogen phosphate, disodium phosphate, magnesium sulfate, cholesterol, agar powder, tryptone, yeast powder, peptone, citric Acid monohydrate, tripotassium citrate monohydrate, ethylenediaminetetraacetic Acid (EDTA), potassium citrate, ferrous sulfate heptahydrate, manganese chloride tetrahydrate, zinc sulfate heptahydrate, copper sulfate pentahydrate, 5-Fluorodeoxyuridine (FUDR) (Sigma), Pyrophorbide-a (frontier scientific).
Experimental and insect strains: uracil-deficient escherichia coli (e. coli) OP 50; wild type strain N2.
An experimental instrument:
AL 104-IC-electronic balance (mettler-toledo instruments), SMZ-168-dissecting microscope (Motic), DK-420-electric heating constant temperature water tank (shanghai forest communication laboratory instruments has (harbingo eastern union), ZXSD-81430-biochemical culture (shanghai intelligent honesty analysis instruments ltd.), D-1-automatic steam sterilizer (beijing hair cokor ltd.).
The porphyrin substances are applied to prolonging the service life of nematodes under illumination.
The nematode is caenorhabditis elegans.
The nematode diet is uracil-deficient Escherichia coli (E. coli) OP 50.
1 nematode culture method:
freshly prepared NGM medium containing OP50 was removed in a clean room sterile environment and placed in a clean room to cool for 5 minutes. Several nematodes of stage L1 or L2 were picked by a dissecting microscope into NGM medium, and the medium was incubated in an incubator at 20 ℃.
The experimental steps of the influence of different concentrations of chlorophyll metabolites Pa on the life of the nematode under illumination treatment are as follows:
1. preparation of dishes of chlorophyll metabolite Pa OP50 at different concentrations:
chlorophyll metabolite Pa was added to the OP50 medium before the medium was dispensed into the petri dish so that the final concentrations were 0.4. mu.M, 0.6. mu.M, 0.8. mu.M, 1.0. mu.M, and 1.5. mu.M, respectively.
Preparation of OP50 medium: lysis Broth solid medium Lysogeny Broth, LB being the main food source for OP50 survival, 1000 ml of LB solid medium was prepared: the following reagents were weighed into a 1L Erlenmeyer flask: 10 g of trypsin, 10 g of NaCl, 5 g of yeast extract and 15 g of agar (OP 50 liquid culture medium does not need to be added), using deionized water to fix the volume to 1L, and performing autoclaving to obtain OP50 culture medium;
2. synchronizing nematodes: culturing the oviposition nematodes capable of laying eggs and developed to day 1 in a culture medium overnight, removing the female nematodes after 10 h to obtain the nematodes in the same period, and culturing in an incubator at 20 ℃ until the nematodes are developed to young adult period for transfer.
Preparing a nematode culture medium:
preparation of NGM (Neocode Growth Media, NGM) culture medium: the following reagents were weighed into a 1L Erlenmeyer flask: 2.5 g of peptone, 3 g of NaCl, 20 g of agar and deionized water to reach the constant volume of 1L. Autoclaving and adding 1M CaCl2 and 1M MgSO 1, respectively41 ml each, 1 ml of 5 mg/ml cholesterol and 25 ml of phosphate buffer (KPI). Mixing, and packaging into culture dish. After cooling at room temperature, fresh OP50 was added, and the mixture was incubated overnight in a 37 ℃ incubator and stored in a 4 ℃ freezer for further use.
Setting environmental conditions: after the synchronization of the nematodes, culturing the nematodes in a nematode culture medium until adults grow, exposing the group to be exposed for 20 minutes on the 1 st day and the 3 rd day respectively, and culturing the nematode in a constant-temperature and light-proof incubator at the temperature of 20 ℃.
The exposure treatment uses near-external red light, and the wavelength range of the red light is 660 nm.
The administration mode of the nematode is as follows: is added before configuring OP50 culture plates, and the medicine is in OP50 culture medium; after shaking and mixing well, pour into a 35mm sterile petri dish.
3. Nematode longevity experiment:
the synchronized nematodes were transferred to the above medium supplemented with FUDR or chlorophyll metabolite Pa, ensuring that 30 nematodes were transferred per medium.
Culturing to adult, and exposing each group. Nematodes were transferred every 2-3 days to new dishes containing the corresponding FUDR or chlorophyll metabolite Pa to prevent media contamination. Death data were not taken into account when drilling to the bottom of the culture dish, climbing on the dish wall to lose water and die, no care for loss, etc., but they were recorded as experimental subjects and statistically analyzed. Analysis of life tests statistical analysis was performed using an on-line application (OASIS; sbi. postch. ac. kr/OASIS), and the above tests were independently repeated three times.
The porphyrin substances are chlorophyll metabolite Pyropheophorbide-a, Pa.
The experimental result shows that the life prolonging effect of the caenorhabditis elegans is most obvious when the concentration is 0.8 mu M Pa under the illumination condition, the average life reaches 18.99 days, and the life is prolonged by nearly 18 percent. As also shown in the tables of figures 1 and 2. The effect of light at different concentrations Pa on the longevity of wild type C.elegans is shown in Table 1:
TABLE 1
| Pa concentration | Whether it is illuminated | |
| 0 μM | Nothing (Dark) | 17.2 |
| 0 μM | Illumination (LWL) | 17.7 |
| 0.4 μM | Nothing (Dark) | 15.83 |
| 0.4 μM | Illumination (LWL) | 15.63 |
| 0.6μM | Nothing (Dark) | 17.21 |
| 0.6μM | Illumination (LWL) | 17.67 |
| 0.8μM | Nothing (Dark) | 16.55 |
| 0.8μM | Illumination (LWL) | 18.99 |
| 1.0 μM | Nothing (Dark) | 16.78 |
| 1.0 μM | Illumination (LWL) | 16.71 |
| 1.5 μM | Nothing (Dark) | 16.32 |
| 1.5 μM | Illumination (LWL) | 15.86 |
Experimental procedure for Effect of chlorophyll metabolite Pa on nematode Activity
Swimming speed
1. Synchronizing nematodes: culturing the oviposition nematodes capable of laying eggs and developed to day 1 in a culture medium overnight, removing the female nematodes after 10 h to obtain the nematodes in the same period, and culturing in an incubator at 20 ℃ until the nematodes are developed to young adult period for transfer.
2. Treating nematodes: nematodes are divided into four groups: 1. no treatment of the nematode (control); 2. line worm light treatment only (LWL); 3. treatment of only nematode chlorophyll metabolite Pa (Dark + Pa); 4. chlorophyll metabolite Pa and light treatment (LWL + Pa) were given to the nematodes.
The addition of FUDR to all four groups of media prevented the progeny from affecting the life-span experiments. The synchronized nematodes were transferred to the above medium supplemented with FUDR or chlorophyll metabolite Pa, ensuring that 30 nematodes were transferred per medium. Culturing to adult, and exposing each group. The nematode was transferred every 2-3 days to prevent medium contamination.
3. And (3) counting nematodes: to measure the swimming rate in a liquid, the nematodes on day 1 and day 11 were transferred to M9 buffer. And recording the times of body swinging within 30 seconds, wherein the change of the distortion direction of the middle part of the nematode body is one-time swinging, and each group of nematodes need to detect 20 nematodes. The experiments were repeated 3 times.
Feeding rate:
1. nematode synchronization and processing is shown in steps 1 and 2 in (a).
2. And (3) counting nematodes: observing the peristalsis of the euproctis 1 and 11 days by using a dissecting microscope, and recording the peristalsis times of the euproctis within 10 s. Each group of nematodes required 20 nematodes to be detected. The experiments were repeated 3 times.
The experimental results are as follows: as shown in FIG. 3, the swimming rate and feeding rate of nematodes were not significantly different (p > 0.05) between the chlorophyll metabolite Pa/light-treated day and the treatment day eleven compared with the control group, and the swimming and feeding rate of the light-treated group or the chlorophyll metabolite Pa group alone was also not significantly different (p > 0.05) compared with the control group. It follows that the chlorophyll metabolite Pa is unable to slow down the decline in muscle function caused by aging, indicating that the chlorophyll metabolite Pa has no significant effect on activity.
Experimental step for influence of chlorophyll metabolite Pa on nematode stress capacity
Resistance to heat stress
1. Synchronizing nematodes: for the chlorophyll metabolite Pa treated group (Dark + Pa) alone and the chlorophyll metabolite Pa and light treated group nematodes (LWL + Pa), the administration was started From hatch, so that the Day-1 mother worms were picked to lay eggs in a medium containing the chlorophyll metabolite Pa; nematodes were not dosed in the untreated group (Dark) and the light-treated group (LWL) alone, so the Day-1 mother worms were picked to lay eggs in normal medium (medium without chlorophyll metabolite Pa). After 10 h, the female worms are removed to obtain the nematodes in the same period.
2. Treating nematodes: culturing to adult, and exposing each group. Each group of 120 nematodes was transferred from the 20 ℃ incubator to a 37 ℃ incubator for 7 hours of heat treatment. And then checking the survival condition of the nematodes every 2 hours, lightly touching the nematodes with platinum wires, and counting the non-reacted nematodes as dead after waiting for 5 seconds.
3. And (3) service life statistics: statistical analysis was performed using OASIS and this experiment was repeated at least twice.
Resistance to oxidative stress
1. Synchronizing nematodes: for the chlorophyll metabolite Pa treated group (Dark + Pa) alone and the chlorophyll metabolite Pa and light treated group nematodes (LWL + Pa), the administration was started From hatch, so that the Day-1 mother worms were picked to lay eggs in a medium containing the chlorophyll metabolite Pa; nematodes were not dosed in the untreated group (Dark) and the light-treated group (LWL) alone, so the Day-1 mother worms were picked to lay eggs in normal medium (medium without chlorophyll metabolite Pa). After 10 h, the female worms are removed to obtain the nematodes in the same period.
2. Treating nematodes: culturing to adult, and exposing each group. All nematodes were finally transferred to medium containing 10 mM t-BOOH for 24 hours and nematode survival was monitored every 4 hours.
3. And (3) service life statistics: nematode survival was analyzed by (OASIS; sbi. postech. ac. kr/OASIS) and the assay was repeated two more times.
Osmotic stress resistance
1. Synchronizing nematodes: for the chlorophyll metabolite Pa treated group (Dark + Pa) alone and the chlorophyll metabolite Pa and light treated group nematodes (LWL + Pa), the administration was started From hatch, so that the Day-1 mother worms were picked to lay eggs in a medium containing the chlorophyll metabolite Pa; nematodes were not dosed in the untreated group (Dark) and the light-treated group (LWL) alone, so the Day-1 mother worms were picked to lay eggs in normal medium (medium without chlorophyll metabolite Pa). After 10 h, the female worms are removed to obtain the nematodes in the same period.
2. Treating nematodes: culturing to adult, and exposing each group. Survival was monitored after transferring all nematodes to pre-prepared medium containing 400 mM NaCl for 48 hours of treatment. The mortality rate of the nematodes was recorded every 4 hours and the nematodes were considered dead without responding to a soft touch.
3. And (3) service life statistics: nematode survival was analyzed by (OASIS; sbi. postech. ac. kr/OASIS) and the assay was repeated two more times.
4. The experimental results are as follows: as shown in fig. 4, there was no difference between nematode longevity of the light group or chlorophyll metabolite Pa group and the control group (p > 0.05) compared to the control group; there was also no significant difference between the nematode longevity of chlorophyll metabolite Pa/light group and the control group (p > 0.05), indicating that chlorophyll metabolite Pa could not improve the nematode's resistance to heat stress. Oxidative stress experiments find that the survival rate of chlorophyll metabolite Pa/light group is not significantly different from that of the nematode of a control group (p is greater than 0.05); there was also no significant difference in the chlorophyll metabolite Pa alone or in the light group of nematodes (p > 0.05), indicating that the chlorophyll metabolite Pa did not improve the oxidative resistance of the nematodes. Osmotic pressure experiments show that compared with a control group, the nematode has higher osmotic resistance under the action of the chlorophyll metabolite Pa/light, and the service life of the nematode is prolonged by 19 percent; there was no significant difference in longevity (p > 0.05) for the light alone or the chlorophyll metabolite Pa group compared to the control group, indicating that chlorophyll metabolite Pa/light can improve nematode penetration resistance.
It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the foregoing description only for the purpose of illustrating the principles of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the invention as defined by the appended claims, specification, and equivalents thereof.
Claims (9)
1. Application of porphyrin substances in prolonging service life of nematodes under illumination.
2. Use according to claim 1, characterized in that: the nematode is caenorhabditis elegans.
3. Use according to claim 1, characterized in that: the nematode diet is uracil-deficient Escherichia coli (E. coli) OP 50.
4. Use according to claim 1, characterized in that: the administration mode of the nematode is as follows: is added before preparing OP50 culture plates, and the final concentration of the drug in OP50 culture medium is 0.4-0.8 mu M; after shaking and mixing well, pour into a 35mm sterile petri dish.
5. Use according to claim 4, characterized in that: preparation of OP50 medium: lysis Broth solid medium Lysogeny Broth, LB being the main food source for OP50 survival, 1000 ml of LB solid medium was prepared: the following reagents were weighed into a 1L Erlenmeyer flask: 10 g of trypsin, 10 g of NaCl, 5 g of yeast extract and 15 g of agar, using deionized water to fix the volume to 1L, and performing autoclaving to obtain an OP50 culture medium;
synchronizing nematodes: culturing the oviposition nematodes capable of laying eggs and developed to day 1 in a culture medium overnight, removing the female nematodes after 10 h to obtain the nematodes in the same period, and culturing in an incubator at 20 ℃ until the nematodes are developed to young adult period for transfer.
6. Use according to claim 5, characterized in that: setting environmental conditions: after the nematodes are synchronized, the nematodes are cultured in a nematode culture medium until adults grow, and are cultured in a constant-temperature and light-proof incubator at 20 ℃ after exposure treatment of an illumination group.
7. Use according to claim 6, characterized in that: the exposure treatment uses near-external red light, and the wavelength range of the red light is 660 nm.
8. Use according to claim 7, characterized in that: the nematode longevity test is divided into four groups: a first group: dark was not subjected to any treatment of nematodes; second group: treating the LWL only for nematode illumination; third group: treating Dark + Pa only for nematode chlorophyll metabolite Pa; and a fourth group: chlorophyll metabolite Pa and light treatment LWL + Pa were given to the nematodes.
9. Use according to any one of claims 1 to 8, characterized in that: the porphyrin substances are chlorophyll metabolite Pyropheophorbide-a, Pa.
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