CN113616773A

CN113616773A - Application of rice bran active peptide in intervention of caenorhabditis elegans aging or muscle injury

Info

Publication number: CN113616773A
Application number: CN202110981286.6A
Authority: CN
Inventors: 梁盈; 吴钇心; 林亲录; 陈忠旭; 蔡洁; 吴伟
Original assignee: Central South University of Forestry and Technology
Current assignee: Central South University of Forestry and Technology
Priority date: 2021-08-25
Filing date: 2021-08-25
Publication date: 2021-11-09
Anticipated expiration: 2041-08-25
Also published as: CN113616773B

Abstract

The invention discloses a new application of rice bran active peptide. The invention adopts caenorhabditis elegans as a model organism, tests the effect of the rice bran active peptide on intervening caenorhabditis elegans aging and muscle damage, and finds that the rice bran active peptide with a plurality of concentrations has the effects of recovering the muscle damage and prolonging the life of the caenorhabditis elegans. The invention explains the new pharmacological mechanism of the rice bran active peptide, and provides a new idea and means for the application of the rice bran active peptide in resisting aging and repairing muscle damage.

Description

Application of rice bran active peptide in intervention of caenorhabditis elegans aging or muscle injury

Technical Field

The invention relates to the field of protein, in particular to application of rice bran active peptide in intervention of caenorhabditis elegans aging or muscle injury.

Background

Rice bran protein has excellent nutritional value, but as food becomes more refined, rice bran becomes a byproduct of the rice processing process. Therefore, the method has very important significance for carrying out deep processing and carrying out value-added recycling on the byproducts. The rice bran active peptide is peptide which is extracted from rice bran by hydrolyzing rice bran protein and has certain biological activity. The rice bran active peptide has better effects on eliminating active oxygen and free radicals in vivo and resisting oxidation.

Caenorhabditis elegans (C.elegans) is the only organism in the world at present, all cells in a body can be classified one by one, and the caenorhabditis elegans has the advantages of short service life, transparent body, easy observation, low feeding cost, easy acquisition of a large number of synchronized samples, long-term storage, strong experiment operability and the like, and has become a common model organism in scientific research and exploration of people.

Muscle damage is a muscle health problem that is commonly seen in the elderly and in athletes. It is characterized by that the cross-sectional area of muscle fibre and the number of muscle cores are reduced; a decrease in protein content, muscle strength, etc., which is also associated with an increased risk of morbidity and mortality. Despite decades of research, there is no effective way to prevent muscle mass loss.

Through reference to data, no document research related to the aging or muscle damage of the caenorhabditis elegans by the rice bran active peptide is found in China, so that research on the aging resistance or muscle damage of the rice bran active peptide is yet to be explored.

Disclosure of Invention

The invention aims to provide the application of the rice bran active peptide in intervention of caenorhabditis elegans aging or muscle injury, and provides a reference theoretical basis for further development, utilization and popularization of the rice bran active peptide.

In order to achieve the purpose, the technical scheme of the invention is as follows:

an application of a rice bran active peptide in intervention of caenorhabditis elegans aging or muscle injury is disclosed, wherein the rice bran active peptide has a sequence of Lys-His-Asn-Arg-Gly-Asp-Glu-Phe.

The specific technical scheme for the intervention of the rice bran active peptide on nematode muscle damage is as follows:

the swimming is mainly characterized by the body wall muscle of caenorhabditis elegans, and the recovery effect of the rice bran active peptide on the body wall muscle damage is judged through the swimming.

The experiment was divided into a blank group, a rice bran bioactive peptide low dose group (0.05 mM), a rice bran bioactive peptide medium dose group (0.1 mM), and a rice bran bioactive peptide high dose group (0.3 mM), each group containing 30 nematodes. C. elegans at stage L4 was picked up on NGM plates containing 10. mu.M dexamethasone (control group did not contain dexamethasone). Dexamethasone belongs to a glucocorticoid. We used dexamethasone as a lesion for the construction of muscle injury models. Dexamethasone is most commonly applied to cell models as a medicament for constructing muscle injury-related models, and is also applied to mice in recently published literatures. In the experimental process, the movement capacity of the nematodes is changed to evaluate the molding effect, and in order to prove the effect, comprehensive judgment is carried out from a plurality of different ethological indexes to determine the success of molding. In the molding process, we did not selectively pick individual nematodes in order to show that the material is causing a population wide generalized lesion. After incubation for 36h at 20 ℃, nematodes were picked into groups on NGM plates supplemented with M9 buffer alone, where the formulation of M9 buffer was as follows: the total content of sodium chloride (5 g), potassium dihydrogen phosphate (3 g), disodium hydrogen phosphate (6 g) and magnesium sulfate (0.12 g) was 1L. And after the nematodes are adapted for 5min, placing the dishes under a fluorescence inverted microscope, recording a video of 30s under a 40-time microscope by using the camera function of the equipment, and manually controlling an objective table control lever to track the swimming of the caenorhabditis elegans. And recording the swimming frequency of the nematodes, and performing data analysis and mapping. The frequency of swimming was counted as one shaking of the first half of the body from left to right and back to the left.

The pharyngeal pump is mainly used for characterizing pharyngeal muscles of caenorhabditis elegans, and the recovery effect of the rice bran active peptide on muscle damage is judged through the pharyngeal pump.

The experiment was divided into a blank group, a rice bran bioactive peptide low dose group (0.05 mM), a rice bran bioactive peptide medium dose group (0.1 mM), and a rice bran bioactive peptide high dose group (0.3 mM), each group containing 30 nematodes. C. elegans at stage L4 was picked up on NGM plates containing 10. mu.M dexamethasone (control group did not contain dexamethasone). We also used dexamethasone as the lesion modeling material, and the modeling process was similar to that of body wall muscle lesion modeling. Pharyngeal muscle injury and body wall muscle injury are different sites from which the applicant evaluated the effects of injury and protection with rice bran-active peptides. After culturing for 36h at 20 ℃, picking up the successfully molded nematodes from food-free NGM flat plates according to groups, wherein each group comprises 30 nematodes, placing the dishes under a fluorescence inverted microscope under a 100-fold lens for 5min, and recording a 10s video by using the camera function of the equipment, wherein the motion of the caenorhabditis elegans is tracked by manually controlling an operating lever of a stage. Recording the up-and-down shaking frequency of the nematode pharyngeal globules, and drawing after data analysis. The frequency of the wobble was recorded once from top to bottom and back up as per the pharyngeal globule. Here, an unged NGM plate was used because nematode food op50 would form a biofilm on the NGM plate. During the experiment, we found that nematode observations on the pharyngeal pumping of food-containing NGM plates had some impact. Therefore, for ease of experimental observation and accuracy of experimental results, food-free NGM plates were used.

The head swing is mainly used for characterizing the head muscles of caenorhabditis elegans, and the recovery effect of the rice bran active peptide on muscle damage is judged through the head swing.

The experiment was divided into a blank group, a rice bran bioactive peptide low dose group (0.05 mM), a rice bran bioactive peptide medium dose group (0.1 mM), and a rice bran bioactive peptide high dose group (0.3 mM), each group containing 30 nematodes. Caenorhabditis elegans at L4 stage is picked into NGM plates containing 10 μ M dexamethasone (control group does not contain dexamethasone), after culturing at 20 ℃ for 36h, the caenorhabditis elegans are picked into NGM plates without food according to groups, and dexamethasone is also used as a damage molding material, and the molding process is similar to body wall muscle damage molding. Pharyngeal muscle injury and body wall muscle injury are different sites from which the applicant evaluated the effects of injury and protection with rice bran-active peptides. On the one hand, to ensure that the time for injury and protection is consistent between groups, nematodes must be transferred to new plates. On the other hand, nematodes can be affected by food when they crawl on plates with food. Therefore, to ensure that the experiment controls as single variable as possible, it was chosen to transfer it to a diet-free plate. And after the nematodes are adapted for 5min, placing the dishes under a fluorescence inverted microscope for 5min, and recording a 10s video by using the camera function of the equipment, wherein the motion of the caenorhabditis elegans is tracked by manually controlling the operating lever of the objective table. Recording the number of times of head swing of the nematode, and drawing after data analysis. The head swing counts once according to the head lightness of the nematode.

The specific technical scheme for the rice bran active peptide to intervene the life of the nematodes is as follows:

experiments were divided into a blank group, a rice bran bioactive peptide low dose group (0.01 mM), a rice bran bioactive peptide medium dose group (0.03 mM), and a rice bran bioactive peptide high dose group (0.1 mM), each group containing 90 nematodes. C. elegans was picked at stage L4 to a NGM plate containing 150. mu.M of Pentafluorouracil (which inhibits egg laying by nematodes) and cultured at 20 ℃. The nematodes are transferred to new dishes for administration every day in the first 5 days (the nematodes need to be transferred in time during the egg-laying period to avoid the influence of the adult larvae on the final experimental results). From day 6, surviving nematodes were transferred every other day to correspondingly labeled new dishes for culture. The new dish was still an NGM plate, and its components were measured in a total volume of 1L, and contained 25mL of 3g of sodium chloride, 2.5g of tryptone, 20g of agar powder, and 1M potassium phosphate buffer (pH = 6.0). The number of nematodes surviving, dead and lost was recorded by daily observation until all nematodes died, ending the experiment. In the experimental process, dead nematodes are transferred due to misoperation, and nematodes climbing to the dish wall are counted as lost. At the later stage of the experiment, the nematodes are usually immobile, and the heads of the nematodes are touched by a nematode picking rod, so that the nematodes are judged to be dead and counted after no response is caused by external stimulation.

The rice bran active peptide provided by the invention can be applied to the intervention of caenorhabditis elegans aging or muscle injury, and can be applied to food, medicines or health products.

When the rice bran active peptide is used for preparing anti-aging products or medicines for treating muscle injuries, the rice bran active peptide 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 needs.

The invention also claims a pharmaceutical composition, the main component of which is the rice bran active peptide.

The invention also claims application of the pharmaceutical composition in preparing a medicament for intervening caenorhabditis elegans aging or muscle damage.

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. Experiments on the service life of the caenorhabditis elegans show that the rice bran active peptide can intervene caenorhabditis elegans aging, and provides a powerful research and development model for further development and utilization of biological functions and molecular mechanisms of other natural source active peptides and other anti-aging traditional Chinese medicine resources in the field of anti-aging and muscle disease treatment medicines.

Drawings

FIG. 1 is a diagram showing the results of a caenorhabditis elegans swimming experiment, in which the one-way analysis of variance, ####: p <0.001 compared to group con; ***: p <0.001 compared to dex group;

FIG. 2 is a diagram showing the result of the caenorhabditis elegans pharyngeal pumping experiment, one-way anova, ###: p <0.001 compared to group con; ***: p <0.001 compared to dex group;

FIG. 3 is a diagram showing the results of the experiment on the head swing of caenorhabditis elegans, one-way anova, ###': p <0.001 compared to group con; ***: p <0.001 compared to dex group;

FIG. 4 is a plot of the survival curves of C.elegans, one-way anova: p <0.05 compared to con group; **: p <0.01 compared to group con;

fig. 5 is a plot of caenorhabditis elegans life results, one-way anova,: p <0.01 compared to group con; ***: p <0.001 compared to group con;

fig. 6 is a graph of the results of reactive oxygen species ROS assays performed on nematodes in independent samples, t-test: p <0.05 compared to con group; fig. 7 is a graph of the results of lipofuscin assays performed on nematodes, independent samples t-test: p <0.01 compared to group con;

FIG. 8 is a graph showing the results of ROS measurement of nematodes by one-way analysis of variance, ###: p <0.001 compared to group con; *: p <0.05 compared to dex group

FIG. 9 is a graph of the KEGG metabolic pathway for down-regulated genes obtained after transcriptome analysis of C.elegans fed with rice bran bioactive peptides and normally fed C.elegans;

FIG. 10 is a graph of the KEGG metabolic pathway for up-regulated genes obtained after transcriptome analysis of C.elegans fed with rice bran bioactive peptides and normally fed C.elegans;

fig. 11 is a graph of relative expression of klo-1 gene mRNA, from independent samples, t-test: p <0.01 compared to group con;

FIG. 12 is a graph showing the relative expression level of mRNA of klo-1 gene, one-way analysis of variance, ### -: p <0.001 compared to group con; ***: p <0.001 compared to dex group;

FIG. 13 is a graph showing the results of the survival curves of the klo-1(ok2925) genotype C.elegans;

FIG. 14 is a graph of the results of an klo-1(ok2925) genotype C.elegans reactive oxygen species ROS assay, independent sample t-test: p <0.01 compared to con group.

Detailed Description

Example 1

Extraction of rice bran active peptide

And (3) squeezing oil from rice bran to obtain residual waste residues, drying the waste residues, and crushing to obtain defatted rice bran powder. After the defatted rice bran powder is sieved by a 60-mesh sieve, water is added according to the feed-liquid ratio of 1:10, the pH value is adjusted to 9, and the mixture is subjected to constant temperature water bath at 40 ℃ for 4 hours. Centrifuging at 8000r/min for 15min, collecting supernatant, adjusting pH to 4, centrifuging under the same conditions, and collecting precipitate. Washing with water for three times, adjusting pH to 7, freeze drying for 48 hr, and extracting to obtain gluten. Dissolving glutelin in water to obtain 5% glutelin suspension, adjusting pH, and performing enzymolysis at constant temperature. Inactivating enzyme at 85 deg.C for 10min after enzymolysis, centrifuging at 3500r/min for 15min, vacuum concentrating supernatant, and freeze drying to obtain testa oryzae protease hydrolysate. Further eluting the rice bran protease hydrolysate with SP-Sephadex G-25 ion exchange chromatography to obtain 7 fractions (PA-PG), and separating the selected PF fraction with Sephadex G-15 gel chromatography to obtain PF1, PF2, and PF 3. PF3 is further divided into 5 components by RP-HPLC to obtain PF 3-gamma, and the substance obtained after freeze drying is the rice bran active peptide used in the subsequent experiment. Specific parameters refer to patent documents already granted by the inventors: china, CN106636274B [ P ] 2021-06-11.

Example 2

Recovery and conventional culture of nematode

Taking out the nematode freezing tube from a-80 ℃ ultra-low temperature refrigerator, and naturally thawing at room temperature. After completely thawing, transferring the cells to a centrifuge tube, centrifuging for 2min at 2000r/min, discarding the supernatant, mixing the rest liquid uniformly, adding the mixture onto an NGM flat plate coated with E.coli OP50 by using a pipette gun, rotating the flat plate to uniformly distribute the mixture, and culturing in an incubator at 20 ℃ at constant temperature with the humidity controlled at 40-60%. Passages were performed according to the diet on the plate and the experimental requirements.

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. 2-3 culture dishes for caenorhabditis elegans were prepared. When part of nematodes enter the egg-laying period, using M9 buffer solution to wash down the nematodes and transferring the nematodes into a centrifuge tube, centrifuging the nematodes at 2000r/min for 2min, removing supernatant, and then adding 5M sodium hydroxide: the sodium hypochlorite is prepared according to the proportion of 1:2, and is added into a centrifuge tube after being uniformly mixed. The mixture is inverted and mixed up and down to make the polypide fully contact with the lysis solution. During the process, the liquid is observed to be clear from turbidity, and the cracking end point is reached. Then centrifugating at 2000r/min for 2min, discarding supernatant, adding M9 buffer solution, mixing, centrifugating and washing ovum, and repeating the process for 3 times. Sucking out the precipitate, placing on NGM plate containing OP50, and incubating for about 14 hr to obtain homogenized nematode.

Example 4

Caenorhabditis elegans swimming experiment

1. After incubation of the eggs for about 2 days to the L4 stage, C.elegans was picked up on NGM plates containing 10. mu.M dexamethasone (control group without dexamethasone). The inactivated OP50 contains rice active peptide 0, 0.05mM, 0.1mM and 0.3mM in sequence. We used dexamethasone as a lesion for the construction of muscle injury models. Dexamethasone is most commonly applied to cell models as a medicament for constructing muscle injury-related models, and is also applied to mice in recently published literatures. In the experimental process, the movement capacity of the nematodes is changed to evaluate the molding effect, and in order to prove the effect, comprehensive judgment is carried out from a plurality of different ethological indexes to determine the success of molding. During the molding process, the material was found to cause a population wide common lesion.

2. After culturing for 36h at 20 ℃, successfully molded nematodes are selected to NGM plates added with M9 buffer only according to groups, wherein the formula of the M9 buffer is as follows: the total content of sodium chloride (5 g), potassium dihydrogen phosphate (3 g), disodium hydrogen phosphate (6 g) and magnesium sulfate (0.12 g) was 1L. And after the nematodes are adapted for 5min, placing the dishes under a fluorescence inverted microscope, recording a video of 30s under a 40-time microscope by using the camera function of the equipment, and manually controlling an objective table control lever to track the swimming of the caenorhabditis elegans.

3. And after the recording is finished, the slow speed of 0.5 time is used for playing, the swimming frequency of the nematodes is recorded, and the data are analyzed and then are plotted. The frequency of swimming was counted as one shaking of the first half of the body from left to right and back to the left. All data were plotted using a graphipad prism and data analysis using SPSS.

As a result: as shown in fig. 1. Swimming is mainly characterized by the body wall muscles of caenorhabditis elegans. Compared with the injured group, the 0.05mM, 0.1mM and 0.3mM rice bran active peptide protected groups respectively improve the swimming frequency of caenorhabditis elegans by 28.8%, 50.1% and 54.4%. The swimming times of caenorhabditis elegans can be obviously improved by the rice bran active peptide, so that the caenorhabditis elegans has a good recovery effect on muscle damage of the body wall of the caenorhabditis elegans, and the swimming times are increased along with the increase of the concentration of the caenorhabditis elegans, so that the muscle recovery capability is stronger. Compared with a blank control group, the swimming frequency of the rice bran active peptide protected medium-dose group and the swimming frequency of the rice bran active peptide protected high-dose group are improved by 5.84% and 8.84%. The rice bran active peptide can recover the muscle better than that of a normal blank control group, and the effect is obvious.

Example 5

Caenorhabditis elegans pharyngeal pumping experiment

1. After incubation of the eggs for about 2 days to the L4 stage, C.elegans was picked up on NGM plates containing 10. mu.M dexamethasone (control group without dexamethasone). The inactivated OP50 contains rice active peptide 0, 0.05mM, 0.1mM and 0.3mM in sequence.

2. After culturing for 36h at 20 ℃, picking up successfully molded nematodes into food-free NGM plates according to groups, wherein each group comprises 30 nematodes, placing the plates under a fluorescence inverted microscope after the nematodes are adapted for 5min, recording 10s video by using the camera function of the equipment under a 100-time microscope, and manually controlling an objective table control lever to track the movement of the caenorhabditis elegans.

3. And after the recording is finished, 0.25-time slow playing is used, the up-and-down shaking frequency of the nematode pharyngeal globules is recorded, and the data is analyzed and then is plotted. The frequency of the wobble was recorded once from top to bottom and back up as per the pharyngeal globule.

As a result: as shown in fig. 2. The pharyngeal pump is mainly characterized by pharyngeal muscle of caenorhabditis elegans, and compared with the injured group, the pharyngeal pump frequency of the caenorhabditis elegans is respectively improved by 18.0%, 36.6% and 34.6% by the rice bran active peptide protected group with 0.05mM, 0.1mM and 0.3 mM. Three groups increased pharyngeal pumping frequency by 8.3%, 25.3%, and 23.5% in sequence, even as compared to the blank control. The results show that the pharyngeal pumping frequency of caenorhabditis elegans can be obviously improved by the rice bran active peptide, and the effect is better than that of the undamaged blank control group. Proved that the rice bran active peptide has better recovery effect on pharyngeal muscle injury, and the effect is best under the concentration of 0.1 mM. The effect of the 0.3mM rice bran active peptide protective group is reduced compared with that of the 0.1mM rice bran active peptide group, which shows that the action effect of the rice bran active peptide is not continuously increased along with the increase of the concentration for pharyngeal muscle, and after the action effect of a certain concentration reaches the best, the later higher concentration is slightly reduced. This may occur because the binding sites for the rice bran-active peptides are limited, after all available binding sites have been bound. The excess bran-active peptide is not reused and is thus in an excess state.

Example 6

Caenorhabditis elegans head swing experiment

1. After incubation of the eggs for about 2 days to the L4 stage, C.elegans was picked up on NGM plates containing 10. mu.M dexamethasone (control group did not contain dexamethasone). The inactivated OP50 contains rice active peptide 0, 0.05mM, 0.1mM and 0.3mM in sequence.

2. After culturing for 36h at 20 ℃, picking up successfully molded nematodes into food-free NGM flat plates according to groups, wherein each group comprises 30 nematodes, placing the dishes under a fluorescence inverted microscope after the nematodes are adapted for 5min, recording a video of 30s by using a camera function of the equipment under a 40-time microscope, and controlling an operating lever of an objective table to track the head swing of the caenorhabditis elegans during the period.

3. And after the recording is finished, the slow-speed playing is carried out by 0.5 time, the number of times of head swing of the nematode is recorded, and the data is analyzed and then the drawing is carried out. The head swing counts once according to the head lightness of the nematode.

As a result: as shown in fig. 3. Head wobble was mainly characterized for the head muscles of caenorhabditis elegans. Compared with the injured group, the rice bran active peptide protected groups at 0.05mM, 0.1mM and 0.3mM respectively increase the head swing frequency of caenorhabditis elegans by 23.9%, 52.7% and 37.2%. Compared with the blank control group, the head swing frequency of the rice bran active peptide protected medium-dose group and the head swing frequency of the rice bran active peptide protected high-dose group are improved by 13.8% and 2.24%. The head swing frequency of caenorhabditis elegans can be obviously improved by the rice bran active peptide, the rice bran active peptide has a good recovery effect on head muscle damage, and the rice bran active peptide has the best effect under the concentration of 0.1mM and is better than that of an undamaged blank control group. The effect of the 0.3mM rice bran active peptide protective group is reduced compared with that of the 0.1mM rice bran active peptide group, which shows that the effect of the rice bran active peptide is not continuously increased along with the increase of the concentration for the head muscle like the pharyngeal muscle, and after the effect of a certain concentration reaches the optimum, the higher concentration is slightly reduced.

Example 7

Experiment on the Life of caenorhabditis elegans

1. Experiments were divided into a blank group, a rice bran bioactive peptide low dose group (0.01 mM), a rice bran bioactive peptide medium dose group (0.03 mM), and a rice bran bioactive peptide high dose group (0.1 mM), each group containing 90 nematodes. C. elegans was picked at stage L4 to a NGM plate containing 150. mu.M of Pentafluorouracil (which inhibits egg laying by nematodes) and cultured at 20 ℃.

2. Each group had 60 strips, and the culture was carried out at a constant temperature of 20 ℃. The nematodes are transferred to new dishes for administration every day in the first 5 days (the nematodes need to be transferred in time during the egg-laying period to avoid the influence of the adult larvae on the final experimental results). From day 6, the surviving nematodes were transferred every other day to correspondingly labeled new dishes, which were still NGM plates, containing 25mL of 3g of sodium chloride, 2.5g of tryptone, 20g of agar powder and 1M potassium phosphate buffer (pH = 6.0) in a total volume of 1L. The number of nematodes surviving, dead and lost was recorded by daily observation until all nematodes died, ending the experiment.

3. In the experimental process, dead nematodes are transferred due to misoperation, and nematodes climbing to the dish wall are counted as lost. At the later stage of the experiment, the nematodes are usually immobile, and the heads of the nematodes are touched by a nematode picking rod, so that the nematodes are judged to be dead and counted after no response is caused by external stimulation.

As a result: as shown in fig. 4. The life of the caenorhabditis elegans is the representation of the healthy physiological state of the caenorhabditis elegans, and the survival rate of the caenorhabditis elegans treated by the rice bran active peptide can be obviously improved under the same day by protecting the caenorhabditis elegans rice bran active peptide. Taking day 18 as an example, the survival rate of the blank control group is 71%, the survival rate of the rice bran active peptide low dose group is consistent with that of the rice bran active peptide low dose group, and the survival rate of the medium dose group and the survival rate of the rice bran active peptide high dose group are 76% and 91% respectively. Compared with the blank control group, the medium and high dose groups are respectively improved by 5 percent and 20 percent. And the life span of the nematodes is extended, and the life spans of the nematodes in the blank group, the rice bran bioactive peptide low dose group (0.01 mM), the rice bran bioactive peptide medium dose group (0.03 mM), and the rice bran bioactive peptide high dose group (0.1 mM) are 24 days, 25 days, 27 days, and 28 days, respectively, according to the longest life span of the nematodes in each group. The half survival rates of all the groups of nematodes are 20, 21 and 22 in sequence through data analysis, and the increase of the survival days of the nematodes is verified again in an intensified manner.

Statistics were performed on the life of each nematode in each group and the results are shown in figure 5. The mean life span of the four groups of nematodes was 19.04 days, 19.46 days, 20.39 days and 22.27 days in this order. Compared with a blank control group, the average life of the rice bran active peptide in the low, medium and high dose groups is improved by 2.23%, 7.08% and 16.94% in sequence. The above data fully demonstrate that the rice bran bioactive peptide has a good effect on prolonging the life of caenorhabditis elegans.

Example 8

Reactive Oxygen Species (ROS) assay for nematodes

The synchronized C.elegans was cultured normally to L4 stage. Caenorhabditis elegans were picked into NGM plates. The blank and protected groups contained 0 and 0.1mM rice bioactive peptides, respectively, in inactivated OP50, and after incubation at 20 ℃ for 48h, nematodes were transferred to EP tubes containing 100. mu. LM9 buffer using a spodiophora needle, shaken well up and down, and centrifuged to discard the supernatant. This process was repeated 2 times for cleaning. After discarding the supernatant, 100. mu.L of ROS fluorescent probe DCFH-DA (purchased from Bioharp) was added and incubated at 25 ℃ for 30min with mixing well by inverting during incubation. After the incubation was completed, the supernatant was centrifuged and washed 2 times with M9 buffer. Then, a liquid transfer gun is used for uniformly mixing the worm body and the liquid, the mixture is sucked out and dripped on an agar gasket, and meanwhile, 2% levamisole is dripped in a worm area for anaesthetizing the nematodes. The agar pad was placed under a fluorescent inverted microscope and photographed with the device under a 4-fold microscope. Then, the processing analysis was performed using Image J.

Reactive oxygen species are the most direct indicators of oxidative stress. The active oxygen detection is carried out on the nematodes, and the results are shown in figure 6 by carrying out quantitative analysis on the fluorescence intensity, and the following can be found: compared with the blank control group, the active oxygen production of the group fed with the rice bran active peptide with the concentration of 0.1mM is reduced by 11.4%. The feed of the rice bran active peptide can obviously reduce the active oxygen level in the body of the nematode, and the fact that the rice bran active peptide plays a certain role in the aspect of oxidative stress is proved.

Example 9

Lipofuscin assay for nematodes

The synchronized C.elegans was cultured normally to L4 stage. Caenorhabditis elegans were picked into NGM plates. The blank and protected groups contained 0 and 0.1mM rice bioactive peptides, respectively, in inactivated OP50, and after incubation at 20 ℃ for 48h, nematodes were transferred to EP tubes containing 100. mu. LM9 buffer using a spodiophora needle, shaken well up and down, and centrifuged to discard the supernatant. This process was repeated 2 times for cleaning. Then, a liquid transfer gun is used for uniformly mixing the worm body and the liquid, the mixture is sucked out and dripped on an agar gasket, and meanwhile, 2% levamisole is dripped in a worm area for anaesthetizing the nematodes. The agar pad was placed under a fluorescent inverted microscope and photographed with the device under a 4-fold microscope. Then, the processing analysis was performed using Image J.

Lipofuscin is an index that characterizes aging. Through quantitative analysis after the determination, the result is shown in fig. 7, the lipofuscin production of the rice bran active peptide group with the feeding concentration of 0.1mM is reduced by 20.7% compared with the blank control group, which shows that the rice bran active peptide can remarkably reduce the lipofuscin production, and the rice bran active peptide is proved to have good effect on resisting aging. The results for lipofuscin are also consistent with the effect of nematodes in prolonging senescence.

Example 10

Reactive Oxygen Species (ROS) assay for nematodes

The synchronized C.elegans was cultured normally to L4 stage. Caenorhabditis elegans were picked up into NGM plates containing 10 μ M dexamethasone in sequence (control group did not contain dexamethasone). The inactivated OP50 contains 0, 0 and 0.1mM rice bioactive peptides in sequence, and after culturing at 20 ℃ for 36h, nematodes are transferred to an EP tube containing 100 μ LM9 buffer solution by using a spontoon needle, shaken up and down uniformly, and centrifuged to remove the supernatant. This process was repeated 2 times for cleaning. After discarding the supernatant, 100. mu.L of ROS fluorescent probe DCFH-DA was added, and incubated at 25 ℃ for 30min with the mixture being mixed by reversing the direction of the incubation. After the incubation was completed, the supernatant was centrifuged and washed 2 times with M9 buffer. Then, a liquid transfer gun is used for uniformly mixing the worm body and the liquid, the mixture is sucked out and dripped on an agar gasket, and meanwhile, 2% levamisole is dripped in a worm area for anaesthetizing the nematodes. The agar pad was placed under a fluorescent inverted microscope and photographed with the device under a 4-fold microscope. Then, the processing analysis was performed using Image J.

Similarly, when the active oxygen is detected in the nematode and the fluorescence intensity is quantitatively analyzed, the results are shown in fig. 8, and it can be found that: injury to dexamethasone resulted in a sharp increase in reactive oxygen species with a rise in relative fluorescence intensity from 19.1 to 37.7, compared to the blank control. While the active oxygen production of the nematode group, which was damaged by feeding the rice bran active peptide at a concentration of 0.1mM, was reduced from 37.7 to 31.6, but still much higher than that of the blank group. The rice bran active peptide plays a certain role in oxidative stress caused by dexamethasone injury, but the level of reducing active oxygen is slightly low, so that the rice bran active peptide does not reduce muscle injury by means of antioxidant stress.

Example 11

Transcriptome analysis of caenorhabditis elegans fed with rice bran bioactive peptide and of normally fed caenorhabditis elegans

After hatching the eggs and culturing for about 2 days to the L4 stage, the caenorhabditis elegans is washed by M9 buffer solution, centrifuged at 2000r/min for 2min, the supernatant is discarded, the nematodes are suspended in the liquid, the liquid is dripped onto an NGM plate containing 150 mu M of pentafluorouracil (the pentafluorouracil is used for inhibiting the egg laying of the nematodes) by using a pipette, and the eggs are cultured at 20 ℃. The concentration of 0.1mM is found to be the best effect due to the comprehensive consideration of the previous experiments. Therefore, the experiment was divided into a blank group and a rice bran bioactive peptide high dose group (0.1 mM), and after 2 days of culture, nematodes were washed into a centrifuge tube using M9 buffer, centrifuged at 2000r/min for 2min, and the supernatant was discarded. The liquid was transferred to a sterile, enzyme-free tube. The tubes were kept in dry ice and sent out for subsequent testing and analysis reports were completed by wuhan measurements.

The results of the differential expression gene distribution and the centralized pathway analysis are shown in fig. 9-10, and fig. 9 is a KEGG metabolic pathway map of the down-regulated gene. Through comprehensive evaluation of the factor association tightness and the enrichment degree, the pathways enriched from top to bottom are starch and sucrose metabolism, sphingolipid metabolism, retinol metabolism, purine metabolism and porphyrin and chlorophyll metabolism in sequence. And FIG. 10 is a KEGG metabolic pathway map of up-regulated genes. The enrichment pathways from top to bottom are the degradation of valine, leucine and isoleucine, the metabolism of tryptophan, the degradation of RNA, the metabolism of pyruvic acid and the metabolism of phenylalanine in turn. Taken together, the two figures demonstrate that rice-active peptide feeding can prolong nematode longevity, more likely due to changes in factors in metabolic pathways. KEGG pathway enrichment analysis of differentially expressed genes shows that the changed regulatory gene sites such as klo-1 and tre-3 are mainly distributed in metabolic pathways, particularly sugar metabolic pathways.

Example 12

klo-1 Gene mRNA relative expression level

All nematodes in the experimental group were washed with M9 buffer, supernatant was removed after precipitation, and washing was repeated 3 times. The M9 buffer solution in the centrifuge tube was aspirated as far as possible. Adding 1mL of RNAex lysate (RNAex extraction reagent, purchased from Esciurel biological Co., Ltd., Hunan), placing into liquid nitrogen for quick freezing, dissolving at room temperature, repeatedly freezing and thawing for 4-5 times, centrifuging at 8000r/min for 5min after the insect is completely broken, carefully sucking and taking supernatant, and taking out lower organic phase. Following RNAex extraction reagent instructions. The extracted total RNA was subjected to reverse transcription according to the instructions of a reverse transcription kit (purchased from Exkory, Inc. of Hunan). The unused RNA solution may be stored at-80 ℃ and cDNA at-20 ℃. The extracted cDNA was subjected to reverse real-time quantitative analysis according to the SYBR kit (purchased from Esci Bio Inc. of Hunan).

Transcriptome data analysis shows that the rice bran active peptide plays a role mainly in sugar metabolism. Therefore, we verified klo-1 gene by RT-PCR. The nematode treatment was as in example 11. As shown in FIG. 11, quantitative analysis of the blank group and the high dose group (0.1 mM) of the rice bran-active peptide of the intact nematodes showed that the expression level of klo-1 was significantly reduced in the protected group. Compared with an undamaged control group, the protective group is reduced by 30.1%, which shows that the rice bran active peptide has more close relationship with a carbohydrate metabolism pathway for prolonging the service life and resisting aging, and the results are consistent with the sequencing result of a transcriptome.

The active peptide of rice bran is found to play a role mainly in sugar metabolism. Therefore, we also verified the klo-1 gene in dexamethasone-injured nematodes. The nematode treatment was as in example 10. As a result of quantitative analysis, as shown in FIG. 12, it was found that klo-1 expression was significantly elevated in the lesion group. Compared with the control group, the expression level of ko-1 in the injury group is increased by 25.3%, and is reduced by 16.7% after being protected by the rice bran active peptide. The rice bran active peptide protected group was restored to a level that was not different from the control group. This further confirms that the effect of the rice bran active peptide on dexamethasone-induced muscle damage is more likely to be associated with carbohydrate metabolism pathways.

Example 13

Experiment on longevity of klo-1 Gene-knock-out caenorhabditis elegans

The experiment was divided into two groups, a placebo group and a 0.1mM rice bran active peptide protected group, each group containing 90 nematodes. Both groups used klo-1(ok2925) (nematode name VC2175, purchased from the genetic center of C.elegans, USA) knock-out nematodes. C. elegans was picked at stage L4 to a NGM plate containing 150. mu.M of Pentafluorouracil (which inhibits egg laying by nematodes) and cultured at 20 ℃. The subsequent steps were the same as in example 7 above.

The effect on nematode longevity was verified by using the klo-1 knock-out strain C.elegans klo-1(ok 2925). As shown in FIG. 13, the mean life span of the control group and the 0.1mM rice bran peptide-protected group after klo-1 gene knock-out was 7.99 days and 8.19 days, respectively, and there was no difference after analysis, indicating that the rice bran peptide did not exert a beneficial effect on the life span of nematodes after klo-1 gene knock-out. It was thus confirmed that the rice bran-active peptide exerts a life-prolonging effect under the control of the klo-1 gene.

Example 14

Reactive Oxygen Species (ROS) assay for klo-1 knock-out nematodes

The synchronized klo-1 nematodes were normally cultured to L4. Experiments were conducted with a blank control group and a 0.1mM rice bran-active peptide-protected group, and caenorhabditis elegans was picked up on NGM plates. After incubation at 20 ℃ for 48h, the nematodes were transferred to EP tubes containing 100. mu.L of 9 buffer using a spork needle, shaken well up and down, and centrifuged to discard the supernatant. This process was repeated 2 times for cleaning. The subsequent steps were the same as in example 8 above.

By quantitatively analyzing the fluorescence intensity, as shown in fig. 14, it was found that: compared with a control group of klo-1 nematodes fed normally, the active oxygen of the group fed with rice bran active peptide with the concentration of 0.1mM to klo-1 nematodes is reduced by 7.34%, and the reduction degree of the active oxygen of the group fed with rice bran active peptide is similar to that of nematodes without knockout of klo-1 genes. It is shown that the klo-1 gene is not related to the beneficial effect of rice bran bioactive peptides on oxidative stress.

In conclusion, the rice bran active peptide can have a remarkable effect on the recovery of the movement capacity of the caenorhabditis elegans with muscle injury constructed by dexamethasone, and the service life of the caenorhabditis elegans can be prolonged by only feeding the rice bran active peptide. The rice bran active peptide has good effects on the aspects of intervening muscle injury and prolonging the service life. Feeding of rice bran bioactive peptides was indeed able to significantly reduce their content, as determined by lipofuscin assays on nematodes, which corresponds to increased nematode longevity. Active oxygen measurement under two different conditions shows that the rice bran active peptide can play a certain role in oxidative stress, but the effect is small. The transcriptome analysis shows that the way of feeding the rice bran active peptide to act on the nematode is mainly concentrated on the metabolic pathway. The klo-1 gene related to sugar metabolism is selected from the rice bran active peptide, and further verification shows that the klo-1 gene has very obvious reduction after being protected by the rice bran active peptide, and the result is consistent with the result of transcriptome analysis data. Then, klo-1 gene knockout type line worms are used for verifying the effects of the life and active oxygen, and the results show that after klo-1 knockout, feeding of rice bran active peptide can not prolong the life any more, but klo-1 knockout has no influence on the generation of active oxygen. It is also more strongly suggested that the rice bran active peptides are more likely to act via sugar metabolic pathways rather than oxidative stress in terms of intervening muscle damage and prolonging life.

In conclusion, experimental results show that ros and lipofuscin are detected after the undamaged nematodes are treated by the rice bran active peptide, and the effect of reducing the lipofuscin is obvious but the effect of reducing the ros is not obvious. Meanwhile, after the nematode subjected to damage molding is treated by the rice bran active peptide, the ros reduction effect is not obvious, and the repair effect cannot be obviously achieved. And (3) sample delivery detection, which finds that an expression pathway is concentrated in a metabolic pathway, and selects aging related genes and damage related genes for verification according to results, so that the effect of the rice bran active peptides on recovering muscle damage or prolonging the life is more closely related to a carbohydrate metabolic pathway.

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. The application of the rice bran active peptide in intervention of caenorhabditis elegans aging or muscle injury is characterized in that the rice bran active peptide has a sequence of Lys-His-Asn-Arg-Gly-Asp-Glu-Phe.

2. The use of claim 1, wherein the rice bran bioactive peptide is used in the preparation of a food, pharmaceutical or nutraceutical product for combating caenorhabditis elegans aging or muscle damage.

3. The application of the rice bran active peptide in preparing the anti-aging or anti-muscle injury medicine, food or health care product is characterized in that the rice bran active peptide has the sequence of Lys-His-Asn-Arg-Gly-Asp-Glu-Phe.

4. The use of claim 3, wherein the anti-aging or anti-muscle-injury medicament is a tablet, capsule, oral liquid, injection or powder medicament prepared from an effective amount of the rice bran active peptide and a pharmaceutically acceptable carrier.

5. A pharmaceutical composition is characterized in that the main active ingredient of the pharmaceutical composition is rice bran active peptide, and the rice bran active peptide has a sequence of Lys-His-Asn-Arg-Gly-Asp-Glu-Phe.

6. Use of the pharmaceutical composition of claim 5 for the manufacture of a medicament for the intervention of caenorhabditis elegans senescence or muscle damage.