CN109568335B - Application of porphyra polysaccharide in intervention of caenorhabditis elegans senescence - Google Patents
Application of porphyra polysaccharide in intervention of caenorhabditis elegans senescence Download PDFInfo
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Abstract
The invention discloses a new application of porphyra polysaccharide. The caenorhabditis elegans is adopted as a model organism, the influence of porphyra polysaccharide on the service life of the nematode, the pharyngeal pump movement rate and the aggregation of polyglutamic acid in the nematode is tested, and the result shows that the porphyra polysaccharide with a plurality of concentrations has the tendency of prolonging the service life of the nematode and inhibiting the aggregation of polyglutamic acid in the AM141 nematode. The invention explains the new pharmacological mechanism of the porphyra polysaccharide and provides new ideas and means for the anti-aging of the porphyra polysaccharide and the application of the porphyra polysaccharide in treating Huntington disease.
Description
Technical Field
The invention belongs to the field of marine medicines, and particularly relates to application of porphyra polysaccharide in intervention of caenorhabditis elegans senescence.
Background
Aging is a continuous change process, and international mainstream believes that free radicals damage human cells, thereby causing a plurality of diseases and accelerating human aging. The free radicals from generation to quenching deeply affect the formation of lipofuscin, the mutation of mitochondrial DNA, the induction of apoptosis and the synthesis of protein, the relationship between the free radicals and aging and diseases caused by the free radicals, thereby providing a certain scientific basis for slowing down the aging of human beings. Anti-aging does not mean increasing the life of a person, but is against life-prolonging factors caused by continuous contact of the human body with the outside, including respiration (oxidation reaction), external pollution, radiation irradiation and other factors which continuously generate free radicals in the human body. Dietary guidelines of countries around the world list vegetables and fruits as important content. When the latest edition of dietary guidelines is revised in 2000 in the United states, the method of selecting diets rich in substances, vegetables and fruits is changed into the method of selecting various vegetables and fruits every day, namely, vegetables and fruits contain a large amount of nutrient substances capable of delaying senescence. The development of anti-aging foods and drugs is an important research direction in the medical field of the old at present.
Huntington's Disease (HD) also known as chorea or Huntington's chorea, is an autosomal dominant hereditary neurodegenerative disease. The disease was discovered by george, a american medical specialist, in 1872 and was thus named. The main cause is that the Huntington gene on the fourth chromosome of a patient is mutated to generate a mutated protein, and the protein is gradually gathered together in cells to form a large molecular mass which is accumulated in the brain to influence the function of nerve cells. The disease usually occurs in middle-aged patients, and is manifested by chorea-like movements, and gradually loses the ability to speak, move, think and swallow as the disease progresses, and the disease lasts for about 10 to 20 years, and finally causes the death of the patients. Polyglutamine (polyQ) aggregation is the first strategy for treating the Huntington patient by inhibiting polyglutamine aggregation because the excessive repetition of CAG sequence at the end of htt gene in the Huntington patient leads to polyQ aggregation chain in the encoded htt protein.
The laver is one of seaweed with higher economic value in the world, and the annual output is second to the kelp. In the Song Dynasty, laver is a tribute dedicated to emperors and is called "Shenxiancai" or "Changshoucai". Modern researches show that abundant polysaccharide in laver is a main factor for playing an anti-aging role. At present, numerous reports on oxidation resistance and immunity enhancement of porphyra polysaccharide exist, but no reports on intervention of caenorhabditis elegans aging models and inhibition of caenorhabditis elegans polyglutamine aggregation exist. Caenorhabditis elegans is a common model organism for researching senescence at present, has short life cycle, clear genetic background, easy observation and culture and better homology with human beings, and is very convenient to develop active substances for delaying senescence or treating Huntington disease by utilizing the caenorhabditis elegans.
Disclosure of Invention
Aiming at the problems, the invention provides the application of the porphyra polysaccharide in intervening caenorhabditis elegans senescence and inhibiting polyglutamine aggregation in caenorhabditis elegans bodies, and provides a reference theoretical basis for further development, utilization and popularization of the porphyra polysaccharide.
In order to achieve the above objects, the porphyra polysaccharide provided by the invention comprises the applications of prolonging the life of nematodes and accelerating the movement rate of a pharyngeal pump, and also provides the application of the porphyra polysaccharide in inhibiting polyglutamine aggregation in caenorhabditis elegans so as to treat Huntington disease.
The thallus Porphyrae includes Porphyra yezoensis, Porphyra haitanensis, Porphyra tenera, and specifically includes RhodophytaRhodophyta) Otteliaceae (A. borreri)Bangiaceae) The following two genera of red vegetable (A)Pyropia) And the genus Porphyra (Porphyra) The laver variety of (5).
The porphyra polysaccharide is galactan with the molecular weight of 3000-10000 Da. Specifically, the high molecular weight polysaccharide obtained by water extraction and alcohol precipitation is obtained by chemical degradation or oxidation reduction degradation, and can also be obtained by directly extracting the laver with hydrochloric acid or citric acid. Can be prepared by the following method: weighing 100g of dried laver algae, adding 30-40 times of water, extracting at 100 ℃ and 120 ℃ for 3-4h, filtering with gauze and diatomite, concentrating and drying to obtain high molecular weight laver polysaccharide. Weighing above polysaccharides, dissolving in water, adding H2O2And ascorbic acid to a final concentration of 20-40 mM. Stirring at room temperature for 2 hr, dialyzing in dialysis bag, desalting, concentrating, and freeze drying to obtain the desired porphyra polysaccharide.
The caenorhabditis elegans is subjected to conventional culture after recovery, and a specific experiment is carried out after homogenization.
The specific technical scheme for prolonging the service life of the nematodes by using the porphyra polysaccharide is as follows:
the experimental groups were blank, polysaccharide low dose (250. mu.g/mL), polysaccharide medium dose (500. mu.g/mL), and polysaccharide high dose (1000. mu.g/mL), with 50 nematodes per group. Homogenizing N2 nematode strain, collecting eggs, culturing in S-culture medium, grouping at adult stage d1 days, transferring nematodes to new culture medium every other day, eliminating intercropped nematodes, and counting the number of nematodes. The experiment continued until the last nematode died.
The specific technical scheme for accelerating the pharyngeal pump movement rate by using the porphyra polysaccharide is as follows:
n2 homogeneity is collected and cultured in S-medium, and transferred to coated medium after d1 days of adultE.coliOP50 on NGM plate, administration group inE.coliOP50 dish was dosed. The nematodes were transferred to new dishes every day and observed under the stereoscope for the number of pharyngeal pumping movements of the nematodes at d5, d8, d10 after adulthood, 1min for each nematode and at least 10 in each group. The pharyngeal pumping rate is calculated.
Polyglutamine (polyQ) aggregation is the polyQ aggregation chain in the encoded htt protein caused by excessive duplication of CAG sequences at the end of the htt gene in patients with Huntington. Huntington's disease is a genetic neurodegenerative disease, and inhibition of polyglutamine accumulation is the first strategy for treating the disease, so that research on inhibition of polyglutamine accumulation by porphyra polysaccharide is of great significance for exploring treatment of Huntington's disease.
The specific technical scheme for inhibiting the aggregation of polyglutamine in the nematode by using the porphyra polysaccharide is as follows:
AM141 homogenizations are collected to S-culture medium for culturing, and divided into three groups at stage L1, and the drug action is 48 h. Nematodes were transferred to slides, anesthetized with 10mM sodium azide, photographed by fluorescent microscope observation, and quantified as ployQ40:: YFP fluorescence accumulation.
The solid culture medium (NGM) for the growth of the caenorhabditis elegans is prepared as follows: weighing 3g of NaCl, 2.5g of peptone and 17g of agar, adding 1000mL of distilled water, mixing uniformly, sterilizing at 120 ℃ for 20 min, and quickly transferring to a 55 ℃ water bath for cooling. The following solutions which had been sterilized were added: 1mol/LCaCl2（1mL）、1mol/LMgSO4(1 mL), 5mg/L cholesterol solution (1 mL), 1mol/L phosphoric acidBuffer (25 mL).
The porphyra polysaccharide provided by the invention can be applied to the intervention of caenorhabditis elegans aging, and can be applied to food, medicines or health products.
When the polysaccharide is used for preparing anti-aging products or medicaments for treating Huntington disease, the polysaccharide with effective dose and pharmaceutically acceptable carriers can be prepared into various dosage forms, such as tablets, capsules, oral liquid, injection, powder and the like according to actual requirements.
The caenorhabditis elegans provided by the invention has 60-80% of human homologous genes and at least 42% of human disease related genes, and is an ideal model for searching a neurodegenerative disease generating mechanism and a treatment method. Caenorhabditis elegans life test shows that the porphyra polysaccharide can intervene caenorhabditis elegans aging, provides a powerful research and development model for the biological function and molecular mechanism of other natural source polysaccharides and the further development and utilization of other anti-aging traditional Chinese medicine resources in the field of late-onset neurological disease drugs, and simultaneously discovers that the porphyra polysaccharide can inhibit the aggregation of polyglutamine in caenorhabditis elegans bodies, thereby providing a research and development model for the drug development for treating Huntington disease.
Drawings
FIG. 1, effect of Z-HV (1000. mu.g/mL) on the survival time of N2 C.elegans.
FIG. 2, effect of Z-HV on polyglutamine accumulation in AM 141.
Detailed Description
Example 1: preparation process of porphyra polysaccharide
Weighing 100g of dried thallus Porphyrae, adding 40 times of water, extracting at 110 deg.C for 3-4 hr, filtering with gauze and diatomite, concentrating, and drying to obtain high molecular weight thallus Porphyrae polysaccharide. Weighing above polysaccharides, dissolving in water, adding H2O2And ascorbic acid to a final concentration of 20 mM. Stirring at room temperature for 2 hr, dialyzing in dialysis bag, desalting, concentrating, and freeze drying to obtain desired porphyra polysaccharide Z-HV.
Example 2: recovery and conventional culture of nematode
Taking out the nematode freezing tube from an ultra-low humidity refrigerator at-80 ℃, and carrying out freezing at room temperatureAnd (5) naturally thawing. Centrifuging for 3min at 1500 deg.C after completely thawing, discarding supernatant, and adding the remaining nematode suspension with 4 times of spray headE.coliOP50, on NGM plate, rotating plate to make it uniformly distributed, culturing at 20 deg.C in incubator with humidity of 40-60%.
Example 3: nematode homogenization procedure
To ensure the accuracy of the experiment, the nematodes need to be homogenized before each batch of experiments are performed to ensure the growth stages are consistent. When the nematodes enter an L4 stage (egg laying period), completely washing the nematodes into a 10mL glass centrifuge tube by using 4.5mL of M9 solution, adding 250 μ L of sodium hypochlorite and 250 μ L of sodium hydroxide (5 mol/L), uniformly mixing, centrifuging for 3min at 1500, discarding supernatant, adding 5mL of M9 solution, uniformly shaking, cleaning the larvae, wherein the step is required to be completely completed within 10min to avoid damaging the eggs), centrifuging for 3min at 1500, discarding supernatant, sucking egg suspension liquid to drop into an NGM empty dish, waiting to hatch in a 20 ℃ incubator, and transferring 24h hatched larvae to an NGM plate coated with OP50 for culture.
Example 4: laver polysaccharide life prolonging experiment
The experimental groups were blank, polysaccharide low dose (250. mu.g/mL), polysaccharide medium dose (500. mu.g/mL), and polysaccharide high dose (1000. mu.g/mL), with 50 nematodes per group. Homogenizing N2 nematode strain, collecting eggs, culturing in S-culture medium, grouping at adult stage d1 days, transferring nematodes to new culture medium every other day, eliminating intercropped nematodes, and counting the number of nematodes. The experiment continued until the last nematode died. The experimental results show that compared with the Control group, Z-HV 1000. mu.g/mL can obviously prolong the life of N2 caenorhabditis elegans, and p is less than 0.05 (figure 1). Both the Z-HV 250. mu.g/mL and 500. mu.g/mL dose groups tended to extend the mean and maximum life of C.elegans.
TABLE 1 Effect of Z-HV on the survival time of N2 C.elegans (Mean + -SEM)
P < 0.05 compared to Control group
Example 5: experiment for accelerating nematode pharyngeal pump movement rate by porphyra polysaccharide
N2 homogeneity is collected and cultured in S-medium, and transferred to coated medium after d1 days of adultE.coliOP50 on NGM plate, administration group inE.coliOP50 dish was dosed. The nematodes were transferred to new dishes every day and observed under the stereoscope for the number of pharyngeal pumping movements of the nematodes at d5, d8, d10 after adulthood, 1min for each nematode and at least 10 in each group. The pharyngeal pumping rate is calculated. As can be seen from tables 2 and 3, Z-HV 500. mu.g/mL had significantly higher pharyngeal pumping rates than the control group at both days 5 and 10 of adulthood. The pharyngeal pump movement rate of Z-HV 1000 mug/mL is obviously higher than that of a control group at 5, 8 and 10 days of adulthood.
TABLE 2 influence of Z-HV on the pharyngeal pumping rate of N2 C.elegans (first) (mean + -SEM, N =10)
| Group of | d5 | d8 | d10 |
| Control | 173.0±97.9(n=10) | 205.0±94.9(n=10) | 288.0±40.8(n=10) |
| Z-HV(250μg/mL) | 238.0±76.0(n=10) | 243.6±87.2(n=11) | 315.8±35.3(n=12) |
| Z-HV(500μg/mL) | 285.0±25.9(n=10)** | 270.8±87.5(n=12) | 324.5±23.0(n=11)* |
| Z-HV(1000μg/mL) | 250.0±26.2(n=10)* | 300.0±46.4(n=15)* | 341.0±28.1(n=10)** |
P < 0.05, p < 0.01 compared to Control group
TABLE 3 influence of Z-HV on the pharyngeal pumping rate of N2 C.elegans (second batch) (mean + -SEM, N =10)
| Group of | d5 | d8 | d10 |
| Control | 206.0±44.5(n=100 | 292.0±36.8(n=10) | 271.8±36.6(n=11) |
| Z-HV(250μg/mL) | 251.0±63.0(n=10) | 280.9±36.7(n=11) | 311.8±46.2(n=11)* |
| Z-HV(500μg/mL) | 249.1±49.1(n=11)* | 286.0±22.7(n=10) | 334.5±18.6(n=11)*** |
| Z-HV(1000μg/mL) | 263.6±58.0(n=11)* | 325.0±24.6(n=10)* | 335.6±32.4(n=9)*** |
P < 0.05, p < 0.001 compared to Control group
Example 6: experiment for inhibiting polyglutamine in nematode by porphyra polysaccharide
AM141 homogenizations are collected to S-culture medium for culturing, and divided into three groups at stage L1, and the drug action is 48 h. Nematodes were transferred to slides, anesthetized with 10mM sodium azide, photographed by fluorescent microscope observation, and quantified as ployQ40:: YFP fluorescence accumulation. As can be seen from Table 4 and FIG. 2, Z-HV 250. mu.g/mL significantly inhibited the accumulation of polyglutamine in the AM141 nematode compared to Control.
TABLE 4 Effect of Z-HV on polyglutamine aggregation in AM141 (mean. + -. SEM)
| Group of | n | Number of |
| Control | ||
| 5 | 87.80±7.70 | |
| Z-HV(250μg/mL) | 5 | 63.40±4.51* |
| Z-HV(500μg/mL) | 5 | 71.20±3.89 |
| Z-HV(1000μg/mL) | 5 | 75.00±6.28 |
P < 0.05 compared to Control group
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are included in the scope of the present invention.
Claims (1)
1. The application of porphyra polysaccharide in preparing a medicament for treating Huntington disease, wherein the porphyra polysaccharide is galactan with the molecular weight of 3000-10000 Da.
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