Application of sodium acetylsalicylate in preparation of chicken heat stress resistance medicine

Abstract

The invention relates to the technical field of heat stress resistance, in particular to application of sodium acetylsalicylate in preparation of heat stress resistance drugs for chickens. According to the invention, the application of the sodium acetylsalicylate in preparation of the heat stress resistant medicine for chickens is provided for the first time, and a certain dosage of sodium acetylsalicylate is added into drinking water for chickens, so that the heart injury of the chickens in the heat stress process is relieved, and the heat stress death rate of the chickens is reduced. Compared with existing heat stress drugs, the sodium acetylsalicylate provided by the invention has small stimulation on animal gastrointestinal mucosa. The sodium acetylsalicylate is fed according to a certain dosage, the irritating pathological injury of the sodium acetylsalicylate to the gastrointestinal tract of the chicken is not abnormal, and the sodium acetylsalicylate can be infinitely mixed and dissolved with water and is convenient to use. The medicine is easily available in preparation raw materials, low in cost and simple in preparation method. The invention provides a new way for the development of medicines for reducing heat stress death and excitation injury of poultry.

Description

Technical Field

[0001] The present invention relates to the technical field of anti - heat stress, and particularly to the application of sodium acetylsalicylate in the preparation of anti - heat stress drugs for chickens. Background Art

[0002] Chickens are covered with feathers and have no sweat glands, and have a lot of subcutaneous and sub - abdominal fat, resulting in very low tolerance to high - temperature environments. Therefore, heat stress (HS) has become one of the important environmental factors leading to a decline in chicken productivity and an increase in mortality. Research shows that for 1 - 2 - month - old broiler chickens at temperatures of 29.4 - 40.6 °C and above 40.6 °C, their mortality rates are 50% and 87% respectively. Heat stress not only affects the development of chicken tissues and organs, but also leads to a significant decline in immunity and production performance, and even causes a large number of chicken deaths. It is reported that the annual economic loss caused by heat stress in the livestock and poultry breeding industry in the United States is as high as $2.4 billion, of which the loss in the poultry breeding industry alone is as high as $128 million. The economic loss caused by heat stress in China's chicken farming industry every year is also very alarming. Therefore, finding an efficient preparation to relieve heat - stress damage in chickens is of great significance.

[0003] The heart is one of the most important organs for the survival of an animal's body, and chickens can show an unusually high sudden - death phenomenon at the initial stage of heat stress. Heat stress can induce pathological changes characterized by cell swelling and acute degeneration in chicken cardiomyocytes both in vivo and in vitro, and even cell apoptosis and necrosis; ultrastructural pathological observations show swelling of sub - cellular structure mitochondria and shedding of cristae, and under a common optical microscope, the myocardial tissue of heat - stressed chickens shows rupture of myocardial fibers.

[0004] Although there are already many anti - heat stress drugs, the existing anti - heat stress drugs have irritation to the gastrointestinal mucosa of animals and are relatively costly. Therefore, there is an urgent need to develop new anti - heat stress drugs. Summary of the Invention

[0005] The purpose of the present invention is to overcome the shortcomings of the prior art and provide the application of sodium acetylsalicylate in the preparation of anti - heat stress drugs for chickens. The synthesis cost and price of sodium acetylsalicylate are low, which can safely and effectively reduce the heat - stress mortality rate of chickens, greatly weaken the irritation of aspirin / sodium acetylsalicylate to the gastrointestinal mucosa of animals, and ultimately achieve the purpose of improving the economic benefits of chicken breeding.

[0006] The purpose of the present invention is achieved through the following technical solutions:

[0007] The application of sodium acetylsalicylate in the preparation of anti - heat stress drugs for chickens, wherein the sodium acetylsalicylate reduces the pathological damage of the myocardial tissue of heat - stressed chickens.

[0008] Sodium acetylsalicylate, with the chemical formula C9H7O4Na, is an organic compound. It is a white crystalline powder and is soluble in glycerol and water. At present, the clinical efficacy of sodium acetylsalicylate has not been comprehensively reported, especially in relieving heat stress in poultry.

[0009] As a preferred technical solution, the sodium acetylsalicylate relieves heat stress injury by upregulating the expression level of autophagy marker proteins and downregulating the expression level of apoptosis marker proteins. The autophagy marker proteins are HSP90 and BIP, and the apoptosis marker proteins are p-JNK, BCL2, and XDH.

[0010] As a preferred technical solution, the effective dose of the sodium acetylsalicylate is 25 - 38 mg / kg. It is taken by drinking water according to the dosage.

[0011] More preferably, the effective dose of the sodium acetylsalicylate is 34 mg / kg.

[0012] As a preferred technical solution, the sodium acetylsalicylate is prepared by the following method:

[0013] Sodium bicarbonate is dissolved in a mixed solvent of double-distilled water and ethanol. Acetylsalicylic acid is added at a temperature of 0 - 5°C while stirring until no bubbles are generated. The resulting solution is filtered and then freeze-dried or spray-dried to obtain a white powder.

[0014] More preferably, the volume ratio of double-distilled water to ethanol is 2 - 4:1, the mass-volume ratio of sodium bicarbonate to the mixed solvent is 1 g:15 - 20 ml, and the mass ratio of sodium bicarbonate to acetylsalicylic acid is 1:4 - 5.

[0015] Chemical structural formula:

[0016] C9H8O4 + NaHCO3 → C9H7O4Na + CO2↑ + H2O

[0017]

[0018] The present invention has the following advantages: The present invention first proposes the application of sodium acetylsalicylate in the preparation of drugs for chickens to resist heat stress. By adding a certain dose of sodium acetylsalicylate to the drinking water of chickens, the heart injury of chickens during heat stress is relieved, and the heat stress mortality rate of chickens is reduced. Compared with existing heat stress drugs, the sodium acetylsalicylate provided by the present invention has less irritation to the gastrointestinal mucosa of animals. When feeding sodium acetylsalicylate according to a certain dose, no abnormal findings are seen in the detection of pathological damage caused by the irritation of sodium acetylsalicylate to the gastrointestinal tract of chickens, and it can also be infinitely miscible with water, making it convenient to use. The raw materials for preparing this drug are easily available, the cost is low, and the preparation method is simple. The present invention provides a new way for the development of drugs to reduce heat stress death and injury in poultry. Description of the Drawings

[0019] Figure 1 Survival curve of Wenchang chickens in the ASA-Na treatment group and results of Wenchang chicken heart injury detection. Among them, A is the survival curve of Wenchang chickens under heat stress conditions with a simulated environmental temperature of 42 ± 1 °C and humidity of 65% for 5 h; B is the levels of myocardial injury-related enzymes in Wenchang chickens in different treatment groups, and the data are expressed as mean ± SEM. (ns, P > 0.05; *, P < 0.05; **, P < 0.01; the same below); C is the HE staining map of the heart tissue of Wenchang chickens in different treatment groups.

[0020] Figure 2 Immunoblotting images of endoplasmic reticulum stress marker proteins in the myocardial tissue of Wenchang chickens in different treatment groups.

[0021] Figure 3 Immunoblotting images of autophagy marker proteins in the myocardial tissue of Wenchang chickens in different treatment groups.

[0022] Figure 4 Immunoblotting images of apoptosis marker proteins in the myocardial tissue of Wenchang chickens in different treatment groups.

[0023] Figure 5 Morphological observation results of the digestive tract of Wenchang chickens in the Con group and ASA-Na group. Specific implementation manners

[0024] The following further describes the present invention in conjunction with the accompanying drawings and embodiments. The protection scope of the present invention is not limited to the following:

[0025] Example 1: Sodium acetylsalicylate was prepared by the following method:

[0026] Sodium bicarbonate was dissolved in a mixed solvent of double-distilled water and ethanol. Acetylsalicylic acid was added at 0 °C while stirring until no bubbles were generated. The resulting solution was filtered and then freeze-dried or spray-dried to obtain a white powder. Among them, the volume ratio of double-distilled water to ethanol was 2:1, the mass-volume ratio of sodium bicarbonate to the mixed solvent was 1 g:15 ml, and the mass ratio of sodium bicarbonate to acetylsalicylic acid was 1:4.

[0027] Example 2: Sodium acetylsalicylate was prepared by the following method:

[0028] Sodium bicarbonate was dissolved in a mixed solvent of double-distilled water and ethanol. Acetylsalicylic acid was added at 5 °C while stirring until no bubbles were generated. The resulting solution was filtered and then freeze-dried or spray-dried to obtain a white powder. Among them, the volume ratio of double-distilled water to ethanol was 4:1, the mass-volume ratio of sodium bicarbonate to the mixed solvent was 1 g:20 ml, and the mass ratio of sodium bicarbonate to acetylsalicylic acid was 1:5.

[0029] Example 3: Sodium acetylsalicylate was prepared by the following method:

[0030] Sodium bicarbonate is dissolved in a mixed solvent of double-distilled water and ethanol. Acetylsalicylic acid is added at 3°C while stirring until no bubbles are generated. The resulting solution is filtered and then freeze-dried or spray-dried to obtain a white powder. Among them, the volume ratio of the double-distilled water to ethanol is 3:1, the mass-volume ratio of sodium bicarbonate to the mixed solvent is 1 g:18 ml, and the mass ratio of sodium bicarbonate to acetylsalicylic acid is 1:4.5.

[0031] Example 4: Sodium acetylsalicylate is prepared by the following method:

[0032] Dissolve 4 g of sodium bicarbonate (NaHCO3) in a mixed solvent of 50 mL of double-distilled water and 20 mL of ethanol. Slowly add 18 g of acetylsalicylic acid (C9H8O4) under the condition of an ice-water bath (0 - 5°C) while stirring to ensure full contact between the two until no bubbles are generated. The solution is filtered and then freeze-dried or spray-dried to obtain a white powder with a purity of about 99%.

[0033] Example 5: The effect of sodium acetylsalicylate on heat stress in Wenchang chickens

[0034] 1. Construction of the heat stress model for the test chickens

[0035] 72 Wenchang chickens at 70 days old, with a body weight of 800 - 1000 g, are purchased from Longquan Wenchang Chicken Industry Company (Wenchang, China). Under standard feeding conditions, the feeding temperature is 22 ± 2°C, the relative humidity is 50 ± 10%, and they are fed with a conventional diet and water. After one week of adaptive feeding, the formal experiment is carried out. Take 36 Wenchang chickens and pre-feed them with ASA-Na for 7 days. Randomly select 24 chickens for HS for 5 h (20 of them die, which is the ASA-Na + HSD group; 4 survive, which is the ASA-Na + HSS group), and the remaining 12 chickens are fed at normal temperature, which is the ASA-Na group. Among the 36 chickens that are not pre-fed, randomly select 24 chickens for HS for 5 h (all 24 die, which is the HSD group), and the remaining 12 chickens are the Con group. Collect blood from the jugular vein and / or carotid artery of the chickens in each group. After centrifugation, take an appropriate amount of serum and store it in a -80°C ultra-low temperature freezer for later use. After sacrificing the test chickens, separate their hearts. Fix 1 / 2 of the ventricular tissue with neutral formalin, and put the other 1 / 2 into a cryopreservation tube, place it in a liquid nitrogen tank for 24 h, and then transfer it to a -80°C ultra-low temperature freezer for storage. In addition, collect the gizzard, proventriculus, duodenum, jejunum, and ileum of the test chickens in the Con group and ASA-Na group, fix them with neutral formalin, and store them at room temperature for later use.

[0036] 2. Determination of the survival rate of heat-stressed Wenchang chickens

[0037] Figure 1 It is a survival curve of Wenchang chickens in the ASA-Na treatment group and a detection result diagram of heart injury in Wenchang chickens. The survival curve results of chickens in different treatment groups are as Figure 1As shown in Figure A. During acute heat stress (temperature: 42 ± 1°C, humidity: 65%), at 2 h of heat stress, chickens in the HSD group and the ASA-Na + HS group began to die, and the death peak was reached at 2 - 3 h. By 4 h of HS, all chickens in the HSD group had died. By 5 h of HS, 4 chickens in the ASA-Na + HS group were still alive. Finally, the survival rate of chickens in the HSD group was 0%, while the survival duration of chickens fed with ASA-Na during heat stress increased significantly, and the survival rate was 16.7%. The above results indicate that pre-feeding ASA-Na can effectively improve the survival rate of Wenchang chickens under heat stress.

[0038] 3. Determination of the levels of enzymes related to myocardial injury in heat-stressed Wenchang chickens

[0039] The serum CK-MB and LDH levels of Wenchang chickens in each group were determined according to the instructions of the Creatine Kinase-MB (CK-MB) and Lactate Dehydrogenase (LDH) kits produced by Nanjing Jiancheng Bioengineering Institute. The results are as Figure 1 shown in Figure B. The activities of CK-MB and LDH enzymes in the serum of the HSD group, the ASA-Na + HSS group, and the ASA-Na + HSD group were significantly higher than those in the Con group. The activities of CK-MB and LDH enzymes in the serum of the ASA-Na + HSS group were significantly lower than those in the HSD group, and the activities of CK-MB and LDH enzymes in the serum of the ASA-Na + HSD group were significantly higher than those in the HSD group. The above results indicate that ASA-Na can significantly reduce the levels of enzymes related to myocardial injury in heat-stressed Wenchang chickens.

[0040] 4. Effects of ASA-Na on the histopathology of the heart of heat-stressed Wenchang chickens

[0041] The heart tissues of Wenchang chickens fixed for 24 h were dehydrated, cleared, embedded, and then serially sectioned with a thickness of 4 μm. After picking up the sections and fixing them on glass slides, they were baked at 58°C. They were dewaxed twice in xylene, 5 - 10 min each time. They were rehydrated with gradient ethanol (100%, 95%, 85%, 75%), stained with hematoxylin for 8 min, and rinsed with running water; differentiated with hydrochloric acid ethanol for 3 s, and rinsed with running water; blued in 1% ammonia water for 1 min, and rinsed with running water; stained with eosin for 2 min, and rinsed with running water. They were observed and photographed under an optical microscope. The results of H&E staining are as Figure 1As shown in Figure C, in the Con group and the ASA-Na group, the myocardial fibers in the H&E-stained sections were arranged tightly, and no obvious pathological changes were observed. In the HSD group, obvious destruction of the cardiac tissue structure was observed in the H&E-stained sections, with disordered arrangement of myocardial fibers, widened gaps, swollen myocardial cells (black vertical arrows), and dissolution and fragmentation of myocardial fibers at multiple sites (white vertical arrows). In the ASA-Na + HSS group, the H&E-stained sections showed disordered arrangement of myocardial fibers and widened gaps (white horizontal arrows), and swollen myocardial cells (black vertical arrows). In the ASA-Na + HSD group, the H&E-stained sections showed disordered arrangement of myocardial fibers and widened gaps (white horizontal arrows), swollen myocardial cells (black vertical arrows), and pyknosis of myocardial cell nuclei (black horizontal arrows). The results showed that pre-feeding with ASA-Na could reduce myocardial injury caused by heat stress in Wenchang chickens.

[0042] 5. Effects of ASA-Na on Protein Expression in the Heart Tissue of Heat-Stressed Wenchang Chickens

[0043] Cut and weigh 100 mg of the heart tissue of heat-stressed Wenchang chickens. After adding RIPA lysis buffer and thoroughly grinding, centrifuge at 12,000 rpm for 10 min. Take the supernatant, measure the protein concentration using a BCA kit, and then adjust the protein concentration to 2 μg / μL. Boil the sample at 100 °C for 5 min. Take the above protein sample (30 μg) and add it to the loading wells of the stacking gel. Electrophorese at 120 v for 100 min using a pre-stained Marker as a control. Determine the position of the protein to be detected according to the position of the protein Marker. Cut the gel into appropriate sizes and place it in a wet transfer apparatus. Transfer at 100 v for 60 min at 4 °C. Place the transferred PVDF membrane in 5% skim milk powder and block it at room temperature for 2 h. Wash it 5 times with TBST, 5 min each time. Add HSP90 antibody (1:5000), GRP78 antibody (1:10,000), PERK antibody (1:1000), p-PERK antibody (1:1000), IRE1 antibody (1:1000), p-IRE1 antibody (1:500), LC3 antibody (1:2500), and GAPDH antibody (1:10,000), and incubate overnight at 4 o °C. After incubation with the primary antibody, wash it 5 times with TBST, 5 min each time. Add goat anti-mouse HRP-labeled secondary antibody (1:10,000) or goat anti-rabbit HRP-labeled secondary antibody (1:10,000). Incubate on a horizontal shaker at room temperature for 2 h. Wash it 5 times with TBST, 5 min each time. Mix solution A and solution B in the ultra-sensitive ECL chemiluminescent ready-to-use substrate kit in a ratio of 1:1 to prepare the luminescent working solution. Add the luminescent reaction solution to the transfer membrane, react in the dark for 30 s, place the transfer membrane in a fully automatic gel imaging analyzer, and automatically expose, image, and take pictures. Use Image J analysis software to perform gray-scale scanning analysis on the protein electrophoresis bands.

[0044] As Figure 2 shown, compared with the Con group, the expression levels of HSP90 and GRP78 in the ASA-Na group were increased, while in the ASA-Na+HSS group and the ASA-Na+HSD group, the expression levels of HSP90, GRP78 and endoplasmic reticulum (ER) stress (ERS)-related proteins were higher than those in the HSD group. The protein expression levels of HSP90 and GRP78 in the HSD group showed an upward trend compared with the Con group, while the expression level of ERS-related proteins was lower than that in the CON group. As Figure 3 shown, the autophagy levels in the ASA-Na+HSS group and the ASA-Na+HSD group were significantly higher than those in the HSD group, and the expression level of autophagy-related proteins in the ASA-Na+HSS group was significantly higher than that in other groups. As Figure 4 shown, compared with the Con group, the expression levels of apoptosis-related proteins in the HSD group were significantly increased, while the expression level of the anti-apoptotic protein Bcl2 showed a downward trend. Compared with the HSD group, the expression level of Bax protein in the ASA-Na+HSS group was significantly down-regulated, while the expression levels of Cleaved-caspase 9 and Cleaved-caspase 3 proteins were significantly increased; the expression level of Bax protein in the ASA-Na+HSD group was significantly up-regulated. The results indicate that under heat stress conditions, ASA-Na can induce autophagy through endoplasmic reticulum stress and reduce the apoptosis level.

[0045] 6. Effects of ASA-Na on the digestive system of chickens

[0046] The fixed gizzard, proventriculus and duodenum tissues were paraffin-embedded and sectioned (4 μm) using a microtome. The cut wax ribbons were placed in the water of a spreading machine, the wax ribbons were fished out and baked in an oven for more than 3 h, and then rehydrated and stained successively in gradient ethanol. The staining rack was placed in hematoxylin staining solution for 8 min and rinsed with running water; it was placed in hydrochloric acid ethanol differentiation solution for 3 s and rinsed with running water; it was blued in 1% ammonia water for 1 min and rinsed with running water; it was placed in eosin staining solution for 2 min and rinsed with running water. Finally, images were obtained under an optical microscope. As Figure 5 shown, no bleeding was observed in the digestive organs of chickens administered ASA-Na for one week in the histopathological sections. The results suggest that the ASA-Na used in the experiment did not affect the digestive organs of chickens.

[0047] The above is only a preferred specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art within the technical scope disclosed by the present invention, according to the technical solution and inventive concept of the present invention, makes equivalent substitutions or changes, and all are covered by the protection scope of the present invention.

Claims

1. Use of sodium acetylsalicylate in the preparation of a drug for chickens against heat stress, wherein the sodium acetylsalicylate reduces the pathological damage of the myocardial tissue of heat-stressed chickens.

2. The application according to claim 1, wherein The sodium acetylsalicylate relieves heat stress injury by up-regulating the expression level of autophagy marker proteins and down-regulating the expression level of apoptosis marker proteins.

3. The application according to claim 1, characterized in that The effective dose of the sodium acetylsalicylate is 25 - 38 mg / kg.

4. The application according to claim 3, wherein The effective dose of the sodium acetylsalicylate is 34 mg / kg.

5. The application according to claim 1, wherein The sodium acetylsalicylate is prepared by the following method: Sodium bicarbonate is dissolved in a mixed solvent of double-distilled water and ethanol. Acetylsalicylic acid is added at a temperature of 0 - 5°C while stirring until no bubbles are generated. The resulting solution is filtered and then freeze-dried or spray-dried to obtain a white powder.

6. The application according to claim 5, wherein The volume ratio of the double-distilled water to ethanol is 2 - 4:1, the mass-volume ratio of sodium bicarbonate to the mixed solvent is 1 g:15 - 20 ml, and the mass ratio of sodium bicarbonate to acetylsalicylic acid is 1:4 - 5.

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