CN111658710A

CN111658710A - Application of tea polyphenol in preparing medicine for relieving heat stress injury of chicken cardiac muscle cells

Info

Publication number: CN111658710A
Application number: CN202010571355.1A
Authority: CN
Inventors: 殷斌; 李桂明; 刘月月; 杨世发; 李颖; 林树乾; 赵增成; 黄中利
Original assignee: Poultry Research Institute Shandong Academy of Agricultural Sciences
Current assignee: Poultry Research Institute Shandong Academy of Agricultural Sciences
Priority date: 2020-06-22
Filing date: 2020-06-22
Publication date: 2020-09-15

Abstract

The invention relates to the technical field of heat stress resistant medicine application, in particular to application of tea polyphenol in preparing a medicine for relieving heat stress injury of chicken myocardial cells. The application realizes effective alleviation of heat stress injury of the cardiac muscle cells of the chickens by using the tea polyphenol with effective dose for effect observation and detection of the heat stress injury of the cardiac muscle cells of the chickens, changes the action target of treatment compared with the existing heat stress therapeutic agent, not only prolongs the survival time of the chickens under the heat stress state, obviously reduces the death rate of the chickens, improves the integral physiological function of the chickens, and provides a new idea and mode for the existing medicine for treating the heat stress of the chickens.

Description

Application of tea polyphenol in preparing medicine for relieving heat stress injury of chicken cardiac muscle cells

Technical Field

The invention relates to the technical field of heat stress resistant medicine application, in particular to application of tea polyphenol in preparing a medicine for relieving heat stress injury of chicken myocardial cells.

Background

Poultry has the characteristics of plump feathers, no sweat glands on skin, vigorous metabolism, high body temperature and the like, and the production performance of the poultry is very easily influenced by the environmental temperature. Along with the development of scientific and normative breeding, an automatic environment control system and various cooling systems are popularized and applied, however, in the time period of high temperature and high humidity in summer, the cooling effect of the cooling system is not ideal, the temperature in a henhouse is still 32-34 ℃, for a free-ranging farmer who does not put into environment control equipment, the temperature in the henhouse even exceeds 35 ℃, and the chicken flock is in a heat stress sub-health state. The research finds that: the temperature of the chicken house is higher than 30 ℃, the body temperature regulating capability of the chicken per se is reduced, the appetite is reduced, and the laying rate and the egg weight are obviously reduced; when the temperature of the chicken house reaches 33-34 ℃, the laying rate of the chicken is reduced by 8.6 percent, and the average egg weight in the week is reduced by 7.21 percent at most; when the average temperature of the henhouse reaches 34 ℃, the death and culling rate of the chicken flocks in the week is as high as 0.66 percent.

The heat stress stimulation seriously affects the production performance and organism metabolism of the chickens, such as the reduction of feed intake and weight gain rate, the reduction of egg yield, egg quality and fertility, the increase of death rate, the acceleration of heart rate, the aggravation of in vivo oxidation process and the like. The heat stress has the most serious influence on the metabolism of chicken organisms, the myocardial cells ARE sensitive to heat and become one of the important organs suffering from damage, the heat stress can induce the imbalance of oxidation reduction and protein homeostasis to damage the myocardial cells, the cells can activate two important endogenous protection mechanisms of Keap1-Nrf2-ARE and HSF1-HSE, and the dual protection effects on the adaptation and survival of the cells ARE respectively realized by regulating and controlling the expression of cell antioxidant enzymes and Hsps, but the internal regulation of the chicken is difficult to deal with along with the continuation of the heat stress, and the damage of the myocardial cells seriously influences the whole normal physiological function of the chicken.

The existing medicines for chicken heat stress injury comprise a sedative, a traditional Chinese medicine, aspirin, bacitracin zinc and the like, wherein the sedative belongs to a limited use type, and can increase the weight of chicken and prolong the survival time of the chicken under a high-temperature condition after being added; the traditional Chinese medicine components contain various nutritional components, so that the traditional Chinese medicine components can play a role in clearing away heat and toxic materials, sterilizing and resisting diseases in the heat stress injury of the chicken, can reduce the heat stress hazard, but have a slow treatment process; after the aspirin is added, the heat stress can be relieved to a certain extent; the bacitracin zinc as antibiotic can obviously reduce the maintenance requirement of chicken in heat stress, enhance the metabolic rate and improve the egg yield, but is easy to generate drug resistance. In summary, the existing anti-stress injury medicines generally aim at improving the growth and production performance of the chickens and prolonging the survival time of the chickens at high temperature, but the actual treatment effect on heat stress is limited, and the death rate is still increased.

According to the scheme, the heat stress injury of the chicken is treated from the aspect of relieving and improving the heat stress injury of the cardiac muscle cells by researching the heat stress injury of the chicken, and a new thought and a new mode are provided for effectively treating the heat stress injury of the chicken.

Disclosure of Invention

The invention provides application of tea polyphenol in preparing a medicine for relieving heat stress injury of chicken myocardial cells, which realizes effective relief of heat stress injury of the chicken myocardial cells by observing and detecting the effect of the effective dose of tea polyphenol on the heat stress injury of the chicken myocardial cells.

The technical scheme adopted by the invention is as follows:

the application of tea polyphenol in preparing a medicine for relieving heat stress injury of chicken myocardial cells.

As previously described, the alleviation of heat stress injury of chicken cardiomyocytes was alleviated by down-regulating LDH, CK-MB and TNF-alpha, and by up-regulating the expression levels of CRYAB, Nrf2, Hsp27 and Hsp70 and down-regulating the expression level of Caspase 3.

As mentioned above, tea polyphenols are administered in a concentration of 0.15g/L-0.24g/L in water.

Further, tea polyphenols are administered in a concentration of 0.2g/L in drinking water.

Further, the tea polyphenol is green tea-derived tea polyphenol.

Further, the green tea-derived tea polyphenol is a sunshine green tea-derived tea polyphenol.

Furthermore, the sunshine green tea-derived tea polyphenol is a poor sunshine green tea-derived tea polyphenol.

The application comprises the construction of a tested chicken heat stress model, the detection of the heat stress pathological damage of heart muscle cells of the tested chicken, the detection of a heart muscle damage zymogram of the tested chicken, the detection of the dynamic expression levels of Hsps, Nrf2 and Caspase3 of the heart muscle cells of the tested chicken, and the feeding of tea polyphenol according to the dosage to detect the effect of relieving the heat stress damage of the heart muscle cells of the chicken by the tea polyphenol group.

The invention has the beneficial effects that:

the application of the tea polyphenol in preparing the medicine for relieving the heat stress injury of the cardiac muscle cells of the chickens treats the heat stress of the chickens from the aspect of relieving and improving the cardiac muscle cell injury, and provides a new treatment mode for treating the heat stress injury of the chickens. By clarifying the action and mechanism of the tea polyphenol on the heat injury of the myocardial cells of the laying hens caused by heat stress, the connection of the action and the internal mechanism of relieving the heat injury of the myocardial cells of the laying hens by the tea polyphenol and the damage of the myocardial cells of the laying hens caused by the heat stress under the condition of the antipyretic stress is facilitated; the method can be used for deeply researching physiological reaction and regulation mechanism of the layer chicken myocardial cells to the high-heat environment, and simultaneously provides an experimental model and data reference for researching and developing the medicine for relieving the layer chicken myocardial cell heat stress injury.

The tea polyphenol is fed to the chicken for treating heat stress stimulation with water at a specific dose, LDH, CK-MB and TNF-alpha can be reduced, the expression level of CRYAB, Nrf2, Hsp27 and Hsp70 can be increased, and the expression level of Caspase3 can be reduced, so that the heat stress injury of heart muscle cells of the chicken can be obviously relieved, the whole physiological function of the chicken is well recovered, the death rate of the chicken is obviously reduced, and the growth performance and the immunity of the chicken are improved. The invention selects the tea polyphenol from green tea for feeding, is more economical and has low cost.

Drawings

FIG. 1 is a diagram showing the construction of a heat stress model of a chicken to be tested;

FIG. 2 is a diagram of the examination of the pathological injury of heart muscle cells of a test chicken (HE X400);

FIG. 3 is a diagram showing the results of detecting the contents of LDH, CK-MB and TNF-alpha in the myocardial injury enzymes of tested chickens;

FIG. 4 shows the results of dynamic measurement of the expression level of cardiac myocyte protein in chicken;

wherein: in FIG. 3, A is an LDH detection result, B is a CK detection result in FIG. 3, C is a CK-MB detection result in FIG. 3, and D is a TNF-alpha detection result in FIG. 3; and indicate significant differences at levels 0.05 and 0.01 compared to HS 0d, # and # # indicate significant differences at levels 0.05 and 0.01 compared to HS for the TP + HS group under equivalent conditions;

in FIG. 4, A is the detection result of protein bands of Nrf2, CRYAB, Hsp27, Hsp70 and Caspase-3, and B in FIG. 4 is the detection result of grey scale values of Nrf2, CRYAB, Hsp27, Hsp70 and Caspase-3 bands; and represent significant differences at the 0.05 and 0.01 levels compared to HS 0 d; # and # # indicate significant differences at the 0.05 and 0.01 levels for the TP + HS group versus HS under equivalent conditions.

Detailed Description

In order to clearly explain the technical features of the present invention, the following detailed description of the present invention is provided with reference to the accompanying drawings.

Construction of heat stress model of tested chicken and sample collection

80 300-day-old Hailan brown laying hens were randomly divided into 2 groups, namely a control group and a tea polyphenol group, and model construction was as shown in FIG. 1. The tested chickens were only bred in an environment with a temperature of 22-24 ℃ and a humidity of 60% to adapt for one week. After the acclimatization growth period is finished, the administration period of 7d is entered, the control group of tested chickens are only fed with normal drinking water, and the tea polyphenol group of tested chickens are fed with drinking water added with 0.2g/L tea polyphenol. After the administration, the heat stress treatment period is started, the heat stress temperature is 37 + -1 deg.C, the humidity is maintained at 60%, and the heat stress treatment is carried out for 0d, 0.5d, 2d, and 5d according to the drinking water during the administration period. After the heat stress is finished, blood samples and heart tissues are rapidly collected. Serum was immediately separated from the collected blood. Collected heart tissues were placed in 4% paraformaldehyde and liquid nitrogen, respectively, for histopathological and molecular biological assays.

Instrumentation for detection: automatic tissue hydroextractor (HistoCore PEARL), paraffin embedding machine (HistoCore arcardia H), cold bench (HistoCore arcardia C), automatic dyeing machine (Autostainer XL/ST5010), slicer (Leica RM2235) are all products of german Leica, optical microscope (AXio Cam HRC Imager a2) is a product of german ZEISS, TNF-alpha detection kit (JYM003 0033Ch) is wuhan genetechnology limited, full automatic enzyme mark analyzer (AMR-100) is a product of hangzhou osheng instruments limited.

The kit for detection is anti-Nrf2(1:1000, Abcam), anti-Hsp70(1:1000, Enzo), anti-Hsp27(1:1000, Santa), anti-CRYAB (1:1000, CST), anti-cleared caspase 3(1:500, CST), anti-GAPDH (1:5000, Abcam), anti-mouse IgG HRP conjugated (1:3000, CST), anti-rabbitIgG HRP conjugated (1:3000, CST); the protein electrophoresis apparatus (Power PacTM Basic) is a BioRad product, a biochemical luminometer (LAS 500), a GE ImageQuant product.

Sudden death of the test chickens: the sudden death rate is obtained by counting the death number of the tested chicken caused by heat stress. As in table 1 below.

TABLE 1 test results of mortality of the tested chickens

Detection of heart myocyte heat stress pathological injury of tested chicken

The myocardial tissue fixed in 4% paraformaldehyde was stained with Hematoxylin-eosin (HE). The specific operation is as follows: firstly, trimming blocks. The myocardial tissue fixed in paraformaldehyde for more than 48h is modified to have the size of 5mm × 5mm × 2mm in length × width × height, and fixed in 4% paraformaldehyde again for 12 h. ② according to the procedures of 1h of 50 percent alcohol, 1h of 75 percent alcohol, 1h of 85 percent alcohol, 1.5h of 95 percent alcohol, 1h of 100 percent alcohol, 20min of benzene alcohol, 20min of dimethylbenzene, 2h of wax and 2h of wax, the dehydration, the transparence and the wax dipping are completed. And embedding. And (5) finishing the paraffin embedding of the tissues by an automatic embedding machine. And fourthly, preparing slices. Slicing with a slicer (4 μm), spreading in water bath at 48-52 deg.C, baking in a 65 deg.C oven for 3 hr, and storing at 4 deg.C. And fifthly, dyeing. Dyeing is carried out by a full-automatic dyeing instrument according to the procedures of 10min of dimethylbenzene, 2min of 100% alcohol, 1.5min of 100% alcohol, 1min of 95% alcohol, 30s of 80% alcohol, 3min of water washing, 5min of hematoxylin dyeing solution, 2min of water washing, 3s of 1% hydrochloric acid alcohol, 30min of water washing, 2min of eosin dyeing solution, 30s of 95% alcohol, 1.5min of 100% alcohol, 2.5min of dimethylbenzene, 2min of dimethylbenzene and 2min of dimethylbenzene. Sixthly, sealing the sheet. A drop of neutral resin was placed in the center of the slide and the slide was covered with a cover slip. Seventhly, observing under a microscope and taking a picture.

The results of the pathological damage examination of the heart muscle cells of the tested chickens due to heat stress are shown in FIG. 2 (HE X400 times). The heart muscle cells of the tested chickens are damaged after heat stress, and the heart damage is serious along with the prolonging of the heat stress time. The manifestations are that the myocardial cells show vacuolar degeneration (→) when the heat stress at 38 ℃ lasts for 0.5d, the vacuolar degeneration is more obvious and visible in capillary vessel expansion (≠) when the heat stress lasts for 2d, and the myocardial cells show necrosis (↓) characterized by deep staining and nuclear compaction when the heat stress lasts for 5 d. The drinking of 0.2g/L tea polyphenol can effectively relieve myocardial cell injury under heat stress conditions, and particularly, no obvious vacuole denaturation and capillary vessel expansion appear when the heat stress is 2 days, and no obvious cell necrosis appears when the heat stress is 5 days.

Detection of zymogram of myocardial injury of tested chicken

Lactate Dehydrogenase (LDH), Creatine Kinase (CK) and creatine kinase isozyme MB (CK-MB) in the blood serum of the tested chicken are sent to a Jinjuan district detection center for detection. The detection of TNF-alpha in chicken serum is carried out by adopting an ELISA method, and the specific operation steps are carried out according to the instruction. Preparing a concentration standard curve according to the specification requirement, setting a blank hole and a sample hole to be detected, sequentially adding various reagents provided in the kit, washing after complete reaction at 37 ℃ for 30min, finally adding a color development solution, reading the light absorption value of each hole by using an ELISA reader at 450nm after full reaction, and calculating the content of the detected sample according to the standard curve.

The results of the detection of LDH, CK-MB and TNF-alpha in the tested chicken serum are shown in FIG. 3. Heat stress can cause the content of LDH, CK-MB and TNF-alpha in the blood serum of the laying hens to rise, wherein the LDH shows a significant difference (P < 0.01) when the heat stress lasts for 2d and 5d, the CK and the CK-MB show a significant difference (P < 0.01) when the heat stress lasts for 0.5d, 2d and 5d, and the TNF-alpha shows a significant difference (P < 0.01) when the heat stress lasts for 5 d. When 0.2g/L of TP is drunk, the up-regulation of LDH, CK-MB and TNF-alpha caused by heat stress can be effectively retarded. Compared with the heat stress group under the same conditions, the tea polyphenol group shows remarkable down-regulation (P < 0.05) to CK-MB at 0.5d, shows remarkable down-regulation (P < 0.01) to CK and CK-MB at 2d, and shows down-regulation trend to CK (P < 0.05) and TNF-alpha (P < 0.01) at 5 d.

Dynamic detection of expression levels of Hsps, Nrf2 and Caspase-3 of tested chicken cardiac muscle cells

Accurately weighing 20mg of myocardial tissue at the heart apex of the tested chicken, placing the myocardial tissue in a 1.5mL sterile centrifuge tube, adding 200 mu L of RIPA lysate containing 1% PMSF, homogenizing at 3500rpm at 4 ℃, placing the homogenized mixture in an environment at 4 ℃ for cracking for 30min, centrifuging at 12000rpm for 15min, and taking supernatant as the total protein extracted. Protein samples were assayed for protein concentration using the BCA assay kit and adjusted to the same concentration level. Adding 5 xSDS Loading buffer into the protein sample according to the proportion of 4:1, fully and uniformly mixing, denaturing the protein sample for 10min under the condition of water bath at 98 ℃, cooling the sample, and storing at-80 ℃ for later use in Western blot detection. Western blot detection: in the experiment, 10% of separation gel and 5% of concentrated gel are used for separation. The Western blot detection method comprises the following specific steps: and (4) sampling. And (3) taking about 20 mu g of protein sample to the sample adding hole, adding 3 mu L of pre-dyed protein Marker on two sides of the sample, and marking the sample number. ② electrophoresis. Electrophoresis at 60V for 30min, pressing the protein sample into a line, and increasing the voltage to electrophoresis at 90V for 90 min. And transferring. The gel was cut according to the position indicated by the protein Marker, and the proteins in the separation gel were transferred to a cellulose acetate membrane (PVDF membrane) which had been activated with methanol, and then transferred at 100V for 90 min. And fourthly, sealing. The transferred PVDF membrane was then blocked with 5% skim milk for 2-3h on a shaker. Fifthly, primary incubation and primary antibody treatment. Incubate at 4 ℃ overnight. Sixthly, washing the membrane. TBST membrane washing, 5min each time, repeat 6 times. And incubation of secondary antibody. HRP-labeled secondary antibody, incubated at room temperature for 2-3 h. And eighthly, washing the membrane. TBST membrane washing, 5min each time, repeat 6 times. And ninthly, developing color. Mixing ECL luminescence solution A and B at a ratio of 1:1, adding onto PVDF membrane, reacting for 10min, and exposing in LAS500 luminescence/bioluminescence analyzer for imaging. Analysis of the results in the r. And taking GAPDH as an internal reference, and performing gray scanning analysis by using Quality one image analysis software. Protein expression amount ═ (target protein gray value-gray background value)/(GAPDH gray value-gray background value).

The expression levels of Hsps, Nrf2 and Caspase3 in chicken myocardial cells were measured, and the results are shown in FIG. 4. Activating the redox regulatory pathway of the organism in the early stage of heat stress of the laying hens, wherein the redox regulatory pathway is expressed by up-regulating the level of Nrf2 (P < 0.01) at 0.5d of heat stress; with the continuous increase of the heat stress time, the expression level of Nrf2 shows a descending trend, and shows a very significant decline at 2d (P < 0.01); when the heat stress continued to 5d, the level of Nrf2 was again substantially restored. When 0.2g/L of TP is drunk, the expression level of Nrf2 under normal conditions (P < 0.01) can be induced, the level of Nrf2 under the same heat stress condition is also up-regulated, and a remarkable difference (P < 0.05) is shown at the time point of heat stress 2 d.

The expression levels of CRYAB, Hsp27 and Hsp70 in the heart muscle cells of the laying hens tend to increase, decrease and then increase along with the duration of heat stress time. Hsp27 showed a very significant increase (P < 0.01) at 0.5d heat stress, with a trend for the increase in CRYAB and Hsp70, but with no statistical difference. Both CRYAB and Hsp27 showed a very significant down-regulation (P < 0.01) upon heat stress at 2 d. When the heat stress is continued to 5d, the expression levels of CRYAB, Hsp27 and Hsp70 are increased compared with the expression level of 2d heat stress. The level of expression of Hsp27 and Hsp70 under normal conditions (P < 0.01) was up-regulated with 0.2g/L of TP, and the level of expression of CRYAB, Hsp27 and Hsp70 under heat stress conditions was up-regulated to varying degrees. Tea polyphenols can regulate the expression level of CRYAB at 0.5d (P < 0.01) and 2d (P < 0.05) of heat stress, regulate the level of Hsp27 at 0.5d (P < 0.01) and 5d (P < 0.05) of heat stress, and regulate the level of Hsp70 (P < 0.01) at 0.5d of heat stress.

The level of Caspase3 in the myocardial cells of the laying hens under the condition of heat stress is detected, and the heat stress induces the expression quantity of Caspase3 and shows a very significant difference (P < 0.01) at 0.5 d. The addition of 0.2g/L TP to the drinking water can effectively inhibit the up-regulation of Caspase3 caused by heat stress, and the inhibition effect is obvious at 0.5d (P is less than 0.01).

The intervention treatment of the tea polyphenol group shows that the dosage of 0.2g/L tea polyphenol is taken along with water, and the tea polyphenol has good relieving effect on myocardial cell injury caused by heat stress of chickens; compared with the existing drug treatment, the drug can further relieve the heat stress survival time of the chicken, and can further remarkably reduce the death rate of the chicken on the basis of improving the growth performance of the chicken and enhancing the immunity; therefore, by relieving the myocardial cell injury caused by heat stress, the treatment of the heat stress myocardial injury can be ensured, the overall physiological function of the chicken is improved, the heat stress of the chicken is effectively treated, and the effect is remarkable.

The heat stress can cause the reduction of the laying rate and the quality of the laying hens, which is shown in that the weight of the eggs is reduced, and the difference is very obvious at 2d (P is less than 0.01); the thickness of the eggshell is reduced, and the eggshell shows very significant difference (P < 0.01) when the eggshell is thermally stressed for 2d and 5 d; the hardness of the eggshell is reduced, and the eggshell has a very significant difference between 2d (P < 0.01) and 5d (P < 0.05) of heat stress. By adopting the scheme of the invention, 0.2g/L of tea polyphenol is added into drinking water for feeding, so that the heat stress injury of chicken myocardial cells is well relieved, and the influence of heat stress on the laying rate, the egg weight, the eggshell thickness and the eggshell hardness can be effectively relieved.

Actual production testing

When the temperature of the chicken house is higher than 30 ℃, the tea polyphenol (the usage amount is disclosed) is fed to the chicken, the appetite is not reduced, and the laying rate per week and the egg weight are not reduced; when the temperature of the chicken house reaches 33-34 ℃, the tea polyphenol with the dosage of the invention is fed to the chicken, the laying rate of the chicken is not reduced, and the average egg weight in the week is not reduced; when the average temperature of the henhouse reaches 34 ℃, the death and culling rate of the chicken flocks in the week is 0.1 percent.

Compared with the treatment effect of the existing heat stress therapeutic agent for the heat stress injury of the chicken, the egg laying rate and the egg weight of the chicken are improved after the existing heat stress therapeutic agent is used, but the effect is certain compared with that of the existing heat stress therapeutic agent under the general condition; the tea polyphenol can maintain the overall production and growth performance of the chicken on the premise of effectively relieving the heat stress injury of the cardiac muscle cells of the chicken, and the death and elimination rate is extremely low.

The above-described embodiments should not be construed as limiting the scope of the invention, and any alternative modifications or alterations to the embodiments of the present invention will be apparent to those skilled in the art.

The present invention is not described in detail, but is known to those skilled in the art.

Claims

1. The application of tea polyphenol in preparing a medicine for relieving heat stress injury of chicken myocardial cells.

2. The use of claim 1, wherein the alleviation of heat stress injuries of chicken cardiomyocytes is achieved by down-regulation of LDH, CK-MB and TNF-a alleviation, and by up-regulation of CRYAB, Nrf2, Hsp27 and Hsp70 expression levels, and down-regulation of Caspase3 expression levels.

3. The use as claimed in claim 1, wherein the tea polyphenols are administered in a concentration of 0.15g/L to 0.24g/L in water.

4. The use as claimed in claim 3, wherein the tea polyphenols are administered in a concentration of 0.2g/L to water.

5. Use according to claim 1, wherein the tea polyphenols are green tea derived tea polyphenols.

6. The use according to claim 5, wherein the green tea-derived tea polyphenols are sun green tea-derived tea polyphenols.