CN114984046B - Hydrogen-rich water composition and application thereof in relieving animal temperature stress - Google Patents
Hydrogen-rich water composition and application thereof in relieving animal temperature stress Download PDFInfo
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Abstract
The invention discloses a hydrogen-rich water composition and application thereof in relieving animal temperature stress, and belongs to the technical field of medicines. The hydrogen-rich water composition contains H 2 1.2-1.5ppm and 0.5-1g/L gamma-aminobutyric acid. The research of the invention discovers that H for animal stress response caused by temperature 2 The synergistic effect can be achieved by matching with gamma-aminobutyric acid, and animal stress caused by high temperature or low temperature can be effectively relieved.
Description
Technical Field
The invention relates to the technical field of medicines, in particular to a hydrogen-rich water composition and application thereof in relieving animal temperature stress.
Background
The temperature stress of animals comprises heat stress and cold stress, and is a compensation process that when the effective environmental temperature is higher than the upper limit critical temperature or lower than the lower limit critical temperature, the environmental heating power destroys the thermal constancy of the organism, and further generates corresponding changes in aspects of physiology, biochemistry, nutrient allocation, production and behavior. Wherein:
heat stress is the sum of nonspecific systemic reactions that occur when animals are stimulated by excessive temperatures beyond their ability to self-regulate. Summer hot weather often results in animals being in a heat stressed state. Heat stress not only reduces the productivity of livestock, resulting in economic loss, but also endangers the health of livestock and causes various diseases. Studies have shown that livestock exhibit reduced appetite, mental retardation, elevated body temperature and shortness of breath when exposed to heat stress. Long-term heat stress can lead to endocrine disorders, reduced digestive function, reduced immunity, extensive inflammation and other pathological processes. Previous studies showed that milk yield in cows under heat stress was 20% lower than that in cows without heat stress. Some research reports indicate that the content of metabolites such as creatine, lactic acid and the like in the blood of heat-stressed cows is obviously different from that of non-heat-stressed cows, which indicates that heat stress can cause metabolic disorder of the cows. Recent studies have shown that heat stress alters the expression levels of proteins in the small intestine of pigs, which are related to intestinal integrity and function, suggesting that heat stress may compromise intestinal integrity. Therefore, the search for drugs capable of relieving heat stress has important significance for ensuring the production performance and health of livestock and poultry.
Cold stress is the sum of non-specific stress responses that occur when animals are stimulated at too low a temperature. Because of the influence of low temperature in winter, cold stress becomes one of the non-negligible problems in the livestock and poultry breeding process. The influence of cold stress on livestock and poultry cultivation mainly comprises two aspects: the economic loss of farmers and the health level of livestock and poultry are reduced. In the aspect of economy, firstly, cold stress can cause the reduction of the production performance of livestock and poultry; secondly, cold stress can cause the increase of energy consumption of livestock and poultry, so that the feed intake is actively increased, the feed consumption is increased, most of feeds are not used for increasing the development speed of the livestock and poultry, but are only energy sources for cold resistance of the livestock and poultry, so that the feed weight ratio is increased, the feed conversion rate is reduced, and the cultivation cost is increased. In the aspect of livestock health, cold stress can cause neuroendocrine system disorder, immune function decline and oxidation resistance damage of livestock, especially livestock young animals, and various diseases of livestock and even death of livestock and poultry can be caused. Studies have shown that cold stress can disrupt the balance between oxidation and antioxidation in the quail bursa tissue and induce cell damage, reducing the quail's immunity. And it has been found that cold stress reduces the cell viability of bovine PBMC and MDBK cells, inhibiting cell proliferation. The cold stress experiment on weaned pigs shows that the low-temperature stimulation can cause activation of HSP70/TLR4/NF- κB channels in the pigs, induce innate immune response and inflammatory response of the pigs, and obviously reduce the immune function of the pigs along with the extension of stress time. Cold stress can cause great harm to livestock and poultry, so that the search for a medicine capable of effectively relieving the cold stress of the livestock and poultry has great significance for ensuring the health of the livestock and poultry.
Hydrogen (H) 2 ) Is a diatomic gas molecule with reducibility, and in recent years, with the intensive research of hydrogen, hydrogen has been found to have the effects of anti-inflammatory, antioxidant, anti-apoptosis, autophagy regulation and the like. In 2007, nature medical first reported H 2 Are used as antioxidants and free radical scavengers for the treatment of oxidative stress. In 2015, people find that the hydrogen-rich water with higher content can obviously inhibit the development of colon cancer of mice, and in 2020, people find that H 2 Can obviously improve the health index of adult non-small cell lung cancer patients. In addition to tumor therapeutic effects, hydrogen has been found in recent years to prevent and treat parkinsonism, gout, and oneThese metabolic diseases have good effects. And in the aspect of new popular coronaries, the oxyhydrogen gas mixture has proved to have good treatment effect. Hydrogen gas is a therapeutic gas that offers significant advantages over other drugs. Firstly, the molecular mass of hydrogen is small, and the hydrogen can pass through a lipid membrane to reach organelles such as mitochondria, cell nucleus and the like to exert biological effects; secondly, the hydrogen has stronger antioxidation and does not influence the normal oxidation-reduction state of the organism; third, hydrogen has very high safety, and no negative effect of hydrogen has been found at present, whether during laboratory experiments or clinical applications of hydrogen.
However, there are few reports on the relief of temperature stress in animals from hydrogen or hydrogen-rich substances.
Disclosure of Invention
The invention aims to provide a hydrogen-rich water composition and application thereof in relieving temperature stress of animals.
In order to achieve the above purpose, the invention adopts the following technical scheme:
in a first aspect of the present invention, there is provided a hydrogen-enriched water composition comprising H 2 1.2-1.5ppm and 0.5-1g/L gamma-aminobutyric acid.
Preferably, the hydrogen-rich water composition contains H 2 1.4ppm and 0.8g/L of gamma-aminobutyric acid.
In a second aspect of the present invention, there is provided a method for preparing the above hydrogen enriched water composition, comprising the steps of:
dissolving gamma-aminobutyric acid in normal saline serving as a solvent to obtain gamma-aminobutyric acid solution; then H is introduced into the gamma-aminobutyric acid solution 2 And let H 2 Fully dissolved in gamma-aminobutyric acid solution.
In a third aspect, the invention provides an application of the hydrogen-rich water composition in preparing a medicine for relieving temperature stress of animals.
In the above application, the temperature stress is heat stress and/or cold stress.
In the above application, the hydrogen-enriched water composition relieves the temperature stress of the animal by at least one of the following (1) - (2):
(1) The damage effect of high temperature or low temperature to cells is reduced, and the reduction of cell activity caused by high temperature or low temperature is alleviated;
(2) Reducing the increase in heat shock protein expression caused by heat or cold stimulation.
The invention has the beneficial effects that:
H 2 can selectively reduce strong oxidant in cells to be used as an antioxidant with therapeutic and protective effects, and can play a role in cytoprotection by resisting oxidative stress.
Gamma-aminobutyric acid is an important nerve inhibitory transmitter, widely exists in animal bodies, and has been researched in recent years to find that gamma-aminobutyric acid has good effects in improving feed intake and daily gain of livestock and poultry and resisting heat stress.
The research of the invention discovers that H for animal stress response caused by temperature 2 The synergistic effect can be achieved by matching with gamma-aminobutyric acid, and animal stress caused by high temperature or low temperature can be effectively relieved.
Drawings
Fig. 1: establishing a heat stress cell model; (a) change in cell viability after high temperature treatment of the cells; (B) Detecting the expression level of HSP70 of the cells by western blotting; (C) protein level analysis of HSP 70; (D) detecting expression of HSP27 in the cell using western blot; (E) protein level analysis of HSP 27; "HS" in the figure indicates the point in time at which the heat treatment is performed; and (3) injection: data all represent mean values (n=3), P <0.05, P <0.01, P <0.001.
Fig. 2: effect of hydrogen-rich compositions on heat-treatment induced changes in cell viability.
Fig. 3: effect of the hydrogen-enriched composition on heat shock proteins in cells after heat treatment.
Fig. 4: effect of the hydrogen-enriched composition on liver heat shock protein expression in heat stressed mice.
Fig. 5: effect of hydrogen-rich compositions on low temperature induced changes in cell viability.
Fig. 6: effect of hydrogen-enriched composition on heat shock proteins in cells after low temperature treatment.
Detailed Description
It should be noted that the following detailed description is illustrative and is intended to provide further explanation of the present application. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.
In order to enable those skilled in the art to more clearly understand the technical solutions of the present application, the technical solutions of the present application will be described in detail below with reference to specific embodiments. If experimental details are not specified in the examples, the conditions are generally conventional or recommended by the reagent company; reagents, consumables, etc. used in the examples described below are commercially available unless otherwise specified.
Example 1: preparation of hydrogen-rich water composition
Dissolving gamma-aminobutyric acid in normal saline serving as a solvent to obtain gamma-aminobutyric acid solution; transferring the gamma-aminobutyric acid solution into a stainless steel reaction kettle, and introducing H into the gamma-aminobutyric acid solution 2 Sufficiently stirring to make H 2 Fully dissolved in gamma-aminobutyric acid solution to prepare the hydrogen-rich water composition. The prepared hydrogen-rich water composition contains H 2 1.4ppm and 0.8g/L of gamma-aminobutyric acid.
Example 2: application of hydrogen-rich water composition in relieving heat stress reaction caused by high temperature
1. Materials and methods
(1) Test materials
DMEM medium was purchased from seville biosystems. Fetal bovine serum was purchased from Shanghai Xiao Peng Biotech Co. The CCK-8 kit was purchased from New Saimei Biotechnology. BCA assay kit was purchased from vitamine company. HSP70 and Tubulin antibodies were purchased from Proteintech, and HSP27 from Affinity.
(2) Cell culture
MAC-T cells (bovine mammary epithelial cell line) were cultured in DMEM supplemented with 10% serum and the cells were cultured to 80% confluence for further experiments.
(3) Establishment of heat stress model
To explore the most suitable time points for heat treatment of MAC-T cells, cells were exposed to 42.5℃for 1 hour and recovered at 37℃for various times (3 h, 6h, 9h, 12 h), cell viability and expression of heat shock proteins in the cells were examined.
To investigate the role of hydrogen-rich water compositions in heat stress, MAC-T cells were randomly divided into the following treatments:
treatment 1: the MAC-T cells were inoculated in DMEM medium (DMEM dry powder medium was dissolved in physiological saline, 50mL of physiological saline was used per 1g of DMEM dry powder medium, and NaHCO was added 3 3.7g NaHCO was added to 1L DMEM medium 3 ) In (3) culturing at 37 ℃.
Treatment 2: MAC-T cells were inoculated into a hydrogen rich medium (DMEM dry powder medium was dissolved with the hydrogen rich water composition prepared in example 1, 50mL of the hydrogen rich water composition per 1g of DMEM dry powder medium was dissolved, and NaHCO was added 3 3.7g NaHCO is added into each 1L hydrogen-rich culture medium 3 ) In (3) culturing at 37 ℃.
Treatment 3: inoculating MAC-T cells into DMEM medium (dissolving DMEM dry powder medium with distilled water, dissolving per 1g DMEM dry powder medium with 50mL distilled water, and adding NaHCO 3 3.7g NaHCO was added to 1L DMEM medium 3 ) In the above step, the culture was carried out at 42.5℃for 1 hour and at 37℃for 6 hours.
Treatment 4: MAC-T cells were inoculated into a hydrogen rich medium (DMEM dry powder medium was dissolved with the hydrogen rich water composition prepared in example 1, 50mL of the hydrogen rich water composition per 1g of DMEM dry powder medium was dissolved, and NaHCO was added 3 3.7g NaHCO is added into each 1L hydrogen-rich culture medium 3 ) In the above step, the culture was carried out at 42.5℃for 1 hour and at 37℃for 6 hours.
Treatment 5: MAC-T cells were seeded in Medium A (DMEM dry powder Medium with H-containing 2 1.4ppm physiological saline was dissolved, and 50mL of the medium containing H was used per 1g of DMEM dry powder medium 2 1.4ppm physiological saline was dissolved and addedNaHCO 3 3.7g NaHCO was added per 1L of medium A 3 ) In the above step, the culture was carried out at 42.5℃for 1 hour and at 37℃for 6 hours.
Treatment 6: inoculating MAC-T cells into culture medium B (DMEM dry powder culture medium is dissolved in physiological saline containing gamma-aminobutyric acid 0.8g/L, 50mL physiological saline containing gamma-aminobutyric acid 0.8g/L is dissolved in per 1g DMEM dry powder culture medium, and NaHCO is added 3 3.7g NaHCO was added per 1L of medium B 3 ) In the above step, the culture was carried out at 42.5℃for 1 hour and at 37℃for 6 hours.
After the treatment, the cells were collected and analyzed to determine the cell viability and the expression level of heat shock proteins (HSP 27 and HSP 70).
(4) Mouse model establishment and sample collection
Male Kunming mice (8 week old, purchased from Jinan Pengyue laboratory animal Breeding Co., ltd.) were randomly divided into 5 groups (10 animals per group) of:
treatment a: male Kunming mice were fed in a standard environment (constant temperature 25 ℃, humidity 50%,12h light, 12h dark) without any treatment.
Treatment B: the hydrogen enriched water composition of example 1 was consumed by male Kunming mice in a standard environment (constant temperature 25 ℃, humidity 50%,12h light, 12h dark).
Treatment C: male Kunming mice were treated at 42℃for one hour each day at noon, and the rest was recovered in a standard environment (constant temperature 25℃humidity 50%,12h light, 12h dark) and heat treated for seven consecutive days.
Treatment D: male Kunming mice were treated every day at 42℃for one hour at noon, and the rest was recovered in a standard environment (constant temperature 25℃humidity 50%,12h light, 12h dark) under conditions of drinking the hydrogen rich water composition of example 1, and continuously heat-treated for seven days.
The test mice were concentrated with full drinking water three times a day in the morning, in the middle and at night, with a drinking time of 40 minutes each time. At the end of day 7, mice were sacrificed and serum and liver tissue were collected and stored at-80 ℃ until use.
(5) Cell viability assay
Cell viability was measured using the CCK-8 method. Cells were incubated with 10% CCK-8 for 2 hours and OD at 450nm was measured with a microplate reader.
(6) Western blot analysis
Cells of each treatment group were washed three times with PBS and then lysed with RIPA lysate at 4 ℃ for 10 minutes. Cell lysates were then isolated by centrifugation at 12,000Xg for 10 minutes at 4 ℃. The protein concentration of the sample was determined by BCA assay kit. An equal amount of protein sample was diluted in 5 XSDS-PAGE loading buffer and boiled for 15 minutes. Equivalent proteins were electrophoresed in a 12% SDS-PAGE gel and transferred to 0.45 μm PVDF membrane. Blocking with 5% skim milk was performed for 2 hours at room temperature and incubated with diluted primary antibodies HSP27, HSP70 and Tubulin overnight at 4 ℃. After that, the membrane was washed three times with TBST for 10 minutes. And the membrane was incubated with the appropriate secondary antibody for 1 hour at room temperature. Each protein of interest was measured using an ECL protein detector. Protein levels were then analyzed by software Image J.
Statistical analysis
Statistical significance of differences between groups was analyzed using single factor anova with SPSS software (version 25). A p value of <0.05 is considered statistically significant. All data charts were made by Prism (GraphPad software).
2. Test results
(1) Selection of cell model and processing time
To explore the most suitable time point for processing MAC-T cells, cells were exposed to 42.5 ℃ for 1 hour and recovered at 37 ℃ for different times. As shown in fig. 1A, cell viability was significantly reduced at 3 hours after heat treatment and maintained at a low level up to 12 hours. Then, we examined the expression of heat shock proteins in cells. As shown in FIGS. 1B-D, the expression levels of HSP70 and HSP27 were elevated differently in cells after high temperature treatment. Considering the changes in cell activity and heat shock proteins, we selected recovery at 37 ℃ for 6 hours after high temperature stimulation as experimental conditions.
(2) Hydrogen-rich water composition for alleviating cell viability decline caused by high temperature treatment
As a result, as shown in FIG. 2, the MAC-T cell viability was significantly reduced after the high temperature treatment, and the cell viability of treatment 3 was reduced to 50%. The hydrogen-rich water composition (treatment 4) can effectively relieve the decrease of cell viability caused by high temperature, and the cell viability is kept at 95%. The hydrogen-enriched water composition (treatment 4) had a significant synergistic effect in alleviating the decrease in cell viability caused by high temperature compared to hydrogen alone (treatment 5, cell viability 70%) and gamma-aminobutyric acid alone (treatment 6, cell viability 60%).
(3) Effect of Hydrogen-rich Water composition treatment on HSP27 and HSP70 protein expression
The cellular heat stress response is an intracellular stress response generated by cells under high temperature stimulation, and is beneficial to the adaptation and survival of the cells under high temperature. The production of heat shock proteins is the most accepted cellular response during heat stress. As shown in fig. 3, after heat treatment, the heat stress response of the cells was activated, the protein expression of HSP27 and HSP70 was significantly enhanced compared to the control group, and the hydrogen-rich water composition treatment significantly reduced the expression level of HSP27 and HSP 70. This suggests that the hydrogen-enriched water composition reduces the heat stress response of MAC-T cells.
(4) Hydrogen-enriched water composition for reducing increase of heat stress protein in liver of heat stress mice
We examined the expression of heat shock proteins in the liver of heat stressed mice. As shown in fig. 4, after heat treatment, protein expression of HSP27 and HSP70 was significantly improved compared to the control group, and the hydrogen-rich water composition treatment significantly reduced the expression level of HSP27 and HSP 70. It is demonstrated that the hydrogen-enriched water composition relieves the heat stress response in mice.
Example 3: application of hydrogen-rich water composition in relieving heat stress reaction caused by low temperature
1. Materials and methods
(1) Reagents and antibodies
DMEM medium was purchased from seville biosystems and fetal bovine serum was purchased from shanghai Xiao peng biosciences. The CCK-8 kit was purchased from New Saimei Biotechnology. BCA assay kit was purchased from vitamine company. HSP70 and Tubulin antibodies were purchased from Proteintech, and HSP27 from Affinity.
(2) Cell culture
PK-15 cells (porcine kidney cell line) were cultured in DMEM medium supplemented with 10% fetal bovine serum, medium was changed every 24 hours, and cells were cultured to 80% confluence for further experiments.
(3) Establishment of cold stress model
To investigate the role of hydrogen-rich water compositions in cold stress, PK-15 cells were randomly divided into the following treatments:
treatment 1: PK-15 cells were inoculated in DMEM medium (DMEM dry powder medium was dissolved in physiological saline, 50mL of physiological saline was used per 1g of DMEM dry powder medium, and NaHCO was added 3 3.7g NaHCO was added to 1L DMEM medium 3 ) In (3) culturing at 37 ℃.
Treatment 2: PK-15 cells were inoculated in a hydrogen-rich medium (DMEM dry powder medium was dissolved with the hydrogen-rich water composition prepared in example 1, 50mL of the hydrogen-rich water composition per 1g of DMEM dry powder medium was dissolved, and NaHCO was added 3 3.7g NaHCO is added into each 1L hydrogen-rich culture medium 3 ) In (3) culturing at 37 ℃.
Treatment 3: PK-15 cells were inoculated in DMEM medium (DMEM dry powder medium was dissolved in distilled water, 50mL of distilled water was used per 1g of DMEM dry powder medium, and NaHCO was added 3 3.7g NaHCO was added to 1L DMEM medium 3 ) In the above step, the culture was carried out at 30℃for 1 hour and at 37℃for 6 hours.
Treatment 4: PK-15 cells were inoculated in a hydrogen-rich medium (DMEM dry powder medium was dissolved with the hydrogen-rich water composition prepared in example 1, 50mL of the hydrogen-rich water composition per 1g of DMEM dry powder medium was dissolved, and NaHCO was added 3 3.7g NaHCO is added into each 1L hydrogen-rich culture medium 3 ) In the above step, the culture was carried out at 30℃for 1 hour and at 37℃for 6 hours.
Treatment 5: PK-15 cells were inoculated in Medium A (DMEM dry powder Medium with H 2 1.4ppm physiological saline was dissolved, and 50mL of the medium containing H was used per 1g of DMEM dry powder medium 2 1.4ppm physiological saline was dissolved and NaHCO was added 3 3.7g NaHCO was added per 1L of medium A 3 ) In the first step, at 3Culturing at 0deg.C for 1 hr, and culturing at 37deg.C for 6 hr.
Treatment 6: PK-15 cells were inoculated in Medium B (DMEM dry powder medium was dissolved in physiological saline containing gamma-aminobutyric acid 0.8g/L, 50mL of physiological saline containing gamma-aminobutyric acid 0.8g/L was used per 1g of DMEM dry powder medium, and NaHCO was added 3 3.7g NaHCO was added per 1L of medium B 3 ) In the above step, the culture was carried out at 30℃for 1 hour and at 37℃for 6 hours.
After the treatment, the cells were collected and analyzed to determine the cell viability and the expression level of heat shock proteins (HSP 27 and HSP 70). The measurement method and data processing were the same as in example 2.
2. Test results
(1) Hydrogen-rich water composition for alleviating cell viability decline caused by low temperature treatment
PK-15 cells were treated at 30℃for 1 hour and cultured in hydrogen-rich medium at 37℃for 6 hours. The change in cell viability was then measured using the CCK-8 method. As shown in fig. 5, after the low-temperature treatment, the cell viability is obviously reduced, and the hydrogen-rich water composition can effectively relieve the cell viability reduction caused by low temperature; the hydrogen-enriched water composition (treatment 4) had a significant synergistic effect in alleviating the decrease in cell viability caused by the low temperature treatment compared to the hydrogen gas alone (treatment 5) and the gamma-aminobutyric acid alone (treatment 6).
(2) Hydrogen-enriched water composition for alleviating activation of heat shock proteins caused by low temperature treatment
The production of heat shock proteins is a reaction that commonly occurs by cells during stress. As shown in fig. 6, after the low temperature treatment, the cells developed stress, protein expression of HSP27 and HSP70 was significantly enhanced compared to the control group, and the hydrogen-rich water composition treatment significantly reduced the expression level of HSP27 and HSP 70. This suggests that the hydrogen enriched water composition reduces cold stress response in PK-15 cells.
The foregoing description is only of the preferred embodiments of the present application and is not intended to limit the same, but rather, various modifications and variations may be made by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application should be included in the protection scope of the present application.
Claims (2)
1. Application of hydrogen-rich water composition in preparation of medicines for relieving animal temperature stress, wherein the hydrogen-rich water composition contains H 2 1.4ppm and 0.8g/L gamma-aminobutyric acid;
the hydrogen-rich water composition is prepared by the following steps:
dissolving gamma-aminobutyric acid in normal saline serving as a solvent to obtain gamma-aminobutyric acid solution; then H is introduced into the gamma-aminobutyric acid solution 2 And let H 2 Fully dissolved in gamma-aminobutyric acid solution.
2. Use according to claim 1, wherein the temperature stress is a heat stress and/or a cold stress.
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