A method for alleviating the adverse effects of chronic restraint stress on animals by adding sodium isovalerate to the feed

Abstract

The application discloses a method for relieving the adverse effects of chronic restraint stress on animals by adding sodium isovalerate into feed, which comprises taking C57BL / 6J mice as research objects, studying the effects of sodium isovalerate on growth performance, depression behavior and intestinal barrier and inflammation indexes of the mice with chronic restraint stress, recording daily weight gain and feed intake, and performing tail suspension test and Elisa test and other test methods, and proving that sodium isovalerate can relieve the decrease of mouse weight growth rate, the increase of feed to weight ratio and the decrease of organ index caused by CRS; through fluorescence quantitative PCR and Western Blot and other methods, it is proved that sodium isovalerate can relieve the intestinal injury of the CRS mice, reduce the expression of colon pro-inflammatory factors and improve the expression of tight junction proteins, and can also reduce the phosphorylation level of NF-kappa B in the colon of the CRS mice. The application explores the relieving effect of sodium isovalerate on chronic restraint stress of animals, so as to provide some theoretical basis for relieving stress in actual production and the application of sodium isovalerate in feed additives.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of feed additives, in particular to a method for alleviating the adverse effects of chronic restraint stress on animals by adding sodium isovalerate to feed. BACKGROUND

[0002] In recent years, due to the large use of limit stalls, the activity area of fattening pigs cannot be guaranteed, which will cause limit stall stress to the pigs, damage the intestinal barrier and cause intestinal inflammation, affect the growth performance and psychological health of the animals, and further affect the economic benefits of the livestock farm. The chronic restraint stress model can cause non-injurious stimulation to the body, and the damage caused by limit stall stress to the animals is similar in terms of pathogenic process, so the chronic restraint stress model can be used to simulate limit stall stress under laboratory conditions.

[0003] The intestinal barrier is the first barrier of the animal body against the invasion of viruses and harmful substances, can prevent the invasion of pathogens, and has the functions of separating the contents of the intestinal cavity from the outside world and maintaining the ability to absorb nutrients. Tight junction proteins can maintain the function of epithelial barrier, hinder the invasion of toxic macromolecules and microorganisms, and can selectively regulate the entry of small molecule substances and ions into the body, and are an important part of the intestinal barrier. The reduction of tight junction proteins will lead to damage to the intestinal barrier, and further affect the health of the animals and reduce the growth performance. Stress can damage the intestinal barrier of animals and cause intestinal inflammation. Chronic restraint stress increases the oxidative stress level of the body, reduces the number of goblet cells and the expression of tight junction proteins, increases the levels of CRH and LPS, induces apoptosis of intestinal epithelial cells, and damages the intestinal barrier. The damage to the intestinal barrier caused by stress will lead to the invasion of LPS and further activate the NF-κB pathway and release inflammatory factors, thereby aggravating the inflammatory damage to the body. SUMMARY

[0004] In order to solve the above problems, the purpose of the present application is to provide a method for alleviating the adverse effects of chronic restraint stress on animals by adding sodium isovalerate to feed, which is used to explore the improvement effect of isovaleric acid on chronic restraint stress (CRS), and to alleviate the stress state of the mice by adding sodium isovalerate to the mice with chronic restraint stress, improving the intestinal inflammation of the mice, thereby improving the adverse effects of chronic restraint stress on the mice.

[0005] To achieve the above purpose, the technical scheme adopted by the present application is as follows: a method for alleviating the adverse effects of chronic restraint stress on animals by adding sodium isovalerate to feed, comprising the following steps:

[0006] 1) Select 6-week-old male C57BL / 6J mice as test objects, with a body weight of 20±2g, free feeding and drinking water, and a 12-hour light-dark cycle;

[0007] 2), the mice are divided into 5 groups, namely, a blank control group (CON), a chronic restraint stress group (CRS), a low-dose sodium isovalerate relief group (1 mg / kg IVA+CRS), a medium-dose sodium isovalerate relief group (10 mg / kg IVA+CRS), and a high-dose sodium isovalerate relief group (50 mg / kg IVA+CRS), with 8 mice in each group;

[0008] 3), the method for constructing the chronic restraint stress model of the mice is that the mice in the chronic restraint stress group (CRS), the low-dose sodium isovalerate relief group (1 mg / kg IVA+CRS), the medium-dose sodium isovalerate relief group (10 mg / kg IVA+CRS), and the high-dose sodium isovalerate relief group (50 mg / kg IVA+CRS) are put into the restraint stress device for restraint stress between 9:00 and 15:00 every day, for a total of 28 days, and the mice in the control group are normally bred;

[0009] 4), the mice in the low-dose sodium isovalerate relief group (1 mg / kg IVA+CRS), the medium-dose sodium isovalerate relief group (10 mg / kg IVA+CRS), and the high-dose sodium isovalerate relief group (50 mg / kg IVA+CRS) are fed with the corresponding dose (1 mg / kg, 10 mg / kg, and 50 mg / kg) of sodium isovalerate added feed every day, the mice in the blank control group (CON) and the chronic restraint stress group (CRS) are fed with ordinary daily ration every day, for a total of 28 days;

[0010] 5), the tail suspension test is performed after the chronic restraint stress model is constructed for 28 days, and the depression state of the mice in each group is evaluated;

[0011] 6), the serum, heart, liver, spleen, lung, kidney, brain tissue (cerebrum and hypothalamus), and intestinal tissue (jejunum and colon) of the mice in each group are sampled after the chronic restraint stress model is constructed for 29 days;

[0012] 7), the serum of the mice in each group is taken respectively, and the satiety hormone secretion is detected by Elisa;

[0013] 8), the heart, liver, spleen, lung, and kidney of the mice in each group are taken respectively, and the organ index is calculated;

[0014] 9), the brain tissue of the mice in each group is taken respectively, and the 5-HT secretion is detected by Elisa;

[0015] 10), part of the intestinal tissue of the mice in each group is taken respectively, paraffin sections are prepared, and H&E staining is performed, so as to observe the intestinal barrier damage condition;

[0016] 11), part of the intestinal tissue of the mice in each group is taken respectively, and the inflammation index is detected by fluorescence quantification and Western Blot.

[0017] Optionally, the restraint stress device is a 50ml plastic centrifuge tube.

[0018] Furthermore, the centrifuge tube has fine holes distributed on its wall, and a small hole is provided in the center of the tube cap.

[0019] Optionally, the method for the tail suspension test includes: placing the mouse in the laboratory for 30 minutes before the experiment, and during the experiment, fixing the tail with tape 1-2 cm from the tip of the tail in a quiet, windless room, so that the mouse is suspended with its head down, taking care to avoid mechanical damage. The suspension point of the mouse's tail is 50 cm from the experimental table surface. Record the cumulative immobility time of the mouse in the last 4 minutes within 6 minutes. The criterion for immobility is that the mouse stops struggling, its body is in a vertical upside-down state, and it is still.

[0020] Optionally, the ELISA detection method includes: collecting serum and brain tissue homogenates. Adding 350 μL of 1× wash buffer to each well, allowing it to stand for 40 seconds, then discarding the liquid, repeating 3 times. Adding 100 μL of sample diluent to the blank wells. Adding 100 μL of standards at different concentrations (the concentrations of standards in the ELISA kits for different hormones are different) to the standard wells. Adding 50 μL of sample and 50 μL of sample diluent to each well of the antibody-coated plate, sealing the wells with a sealing film, and incubating at 37°C for 2 hours. Then removing the liquid from the microplate. Filling each well of the microplate with washing buffer, and patting the microplate dry after draining the washing buffer, repeating the above operation five times. Adding 100 μL of biotinylated antibody working solution to each well, and covering with a new sealing film, incubating at 37°C for 1 hour. Discarding the liquid in the wells, washing 5 times, adding 100 μL of streptavidin-HRP working solution to each well, and covering with a new sealing film, incubating at 37°C for 30 minutes. Discard the liquid in the wells, wash 5 times, add 100 μL of TMB substrate to each well, and incubate at room temperature in the dark for 20 min. Then add 50 μL of stop solution to each well and measure the absorbance of each well at 450 nm.

[0021] Optionally, the quantitative real-time PCR method includes: extracting total RNA from tissues using a Trizol Reagent (Invitrogen) kit; preparing cDNA from the extracted RNA using a First Strand cDNA Synthesis Kit (Thermo Scientific); and using a SYBR Green assay kit (TaKaRa) to detect the RNA. Quantitative real-time PCR was performed on a Roche 480II. 0.5 μL of cDNA template was added to a 10 μL SYBR Green mixture, along with 0.2 μL of either forward or reverse primer. The PCR was then performed using a 2-phase induction phase. -ΔΔCt The method is used for data analysis.

[0022] Optionally, the Western Blot detection method comprises: placing the colon tissue into RIPA lysis buffer added with PMSF, homogenizing after adding magnetic beads in a homogenizer, condition 50Hz, 30s / time, homogenizing 8 times, interval 10s. After homogenization, centrifuge at 12000r / min for 10min at 4℃ to recover the supernatant. The protein concentration is detected using a BCA protein assay kit. The protein sample is electrophoresed by SDS-PAGE gel, 85v electrophoresis for 150min. Then transfer to PVDF membrane by transfer solution. After blocking with 5% skim milk or BSA in TBST for 2 hours, the membrane is incubated with the primary antibodies of anti-NF-κB p65 (6956, CST), Phospho-NF-κB p65 (3033, CST) and β-actin (T40104, abmart) at 4℃ overnight. The results are observed using horseradish peroxidase-conjugated secondary antibodies and chemiluminescent developing solution.

[0023] Compared with the prior art, the present application has at least the following beneficial effects:

[0024] (1) The method proves that isovaleric acid can alleviate the decrease of body weight gain rate, the increase of feed conversion ratio and the decrease of organ index of mice caused by CRS, improve the depression behavior of CRS mice, and promote the secretion of satiety hormone and hedonic hormone 5-HT, by recording the daily weight gain and feed intake, tail suspension test and Elisa test method. It is shown that isovaleric acid can improve the growth performance and depression state of mice caused by chronic restraint stress.

[0025] (2) The method proves that isovaleric acid can alleviate the intestinal injury of CRS mice, reduce the expression of colon pro-inflammatory factors and increase the expression of tight junction proteins, and can also reduce the phosphorylation level of NF-κB in the colon of CRS mice, by H&E staining, fluorescence quantitative PCR and Western Blot methods. It is shown that isovaleric acid has anti-inflammatory effect and can improve the intestinal inflammation and intestinal barrier damage of chronic restraint stress mice.

[0026] (3) The method can effectively solve the problems of easy volatilization and strong odor of isovaleric acid by adding isovaleric acid in the form of sodium salt to the feed. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 is the schematic diagram of the molecular structure of isovaleric acid;

[0028] Figure 2 is the schematic diagram of the effect of isovaleric acid on the body weight gain of chronic restraint stress mice;

[0029] Figure 3A schematic diagram of the effect of isovaleric acid on the food intake of chronic restraint stress mice and serum satiety hormones;

[0030] Figure 4 A schematic diagram of the effect of isovaleric acid on the organ index of chronic restraint stress mice;

[0031] Figure 5 A schematic diagram of the effect of isovaleric acid on the depression state of chronic restraint stress mice;

[0032] Figure 6 A schematic diagram of the effect of isovaleric acid on the intestinal injury of chronic restraint stress mice. DETAILED DESCRIPTION

[0033] The technical solutions in the embodiments of the present application will be described in more detail below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative work fall within the scope of the present application.

[0034] A method for alleviating the adverse effects of chronic restraint stress on animals by adding sodium isovalerate to feed, comprising the following steps:

[0035] 1) Select 6-week-old male C57BL / 6J mice as test objects, weighing 20±2g, free feeding and drinking water, 12-hour light-dark cycle;

[0036] 2) The mice are divided into 5 groups, namely blank control group (CON), chronic restraint stress group (CRS), low-dose sodium isovalerate alleviation group (1mg / kg IVA+CRS), medium-dose sodium isovalerate alleviation group (10mg / kg IVA+CRS), and high-dose sodium isovalerate alleviation group (50mg / kg IVA+CRS), with 8 mice in each group;

[0037] 3) The method for constructing a chronic restraint stress model of mice is that the mice in the chronic restraint stress group (CRS), the low-dose sodium isovalerate alleviation group (1mg / kg IVA+CRS), the medium-dose sodium isovalerate alleviation group (10mg / kg IVA+CRS), and the high-dose sodium isovalerate alleviation group (50mg / kg IVA+CRS) are put into the restraint stress device for restraint stress between 9:00 and 15:00 every day, for a total of 28 days, and the control group is normally raised;

[0038] The restraint stress device is a 50ml plastic centrifuge tube, wherein fine holes are distributed on the tube wall of the centrifuge tube, and a small hole is provided in the center of the tube cover for ventilation.

[0039] 4) The low-dose sodium isovalerate relief group (1 mg / kg IVA+CRS), the medium-dose sodium isovalerate relief group (10 mg / kg IVA+CRS) and the high-dose sodium isovalerate relief group (50 mg / kg IVA+CRS) mice were respectively fed with 1 mg / kg, 10 mg / kg, 50 mg / kg sodium isovalerate added feed (i.e. sodium isovalerate was added to the ordinary daily feed fed to the blank control group and the chronic restraint stress group), and the blank control group (CON) and the chronic restraint stress group (CRS) mice were fed with ordinary daily feed every day, for a total of 28 days;

[0040] 5) After the chronic restraint stress model was constructed for 28 days, the tail suspension test was performed to evaluate the depression state of the mice in each group; the tail suspension test method was as follows: before the experiment, the mice were placed in the laboratory for 30 min to adapt, and during the experiment, the tail of the mouse was fixed with adhesive tape at a distance of 1-2 cm from the tail tip in a quiet and windless room, the mouse was suspended with its head downward, and attention was paid to avoid mechanical damage; the suspension point of the mouse tail was 50 cm from the experimental table surface, and the cumulative immobility time of the mouse in the last 4 min was recorded within 6 min; the criterion for immobility was that the mouse stopped struggling, the body was vertically inverted, and the mouse was motionless.

[0041] 6) After the chronic restraint stress model was constructed for 29 days, the serum, heart, liver, spleen, lung, kidney, brain tissue (cerebrum and hypothalamus) and intestinal tissue (jejunum and colon) of the mice in each group were sampled;

[0042] 7) The serum of the mice in each group was taken respectively to detect the satiety hormone secretion by Elisa; the Elisa method was as follows: the serum and brain tissue homogenate were collected. 350 μL of 1x washing buffer was added to each well, and after standing for 40 s, the liquid was discarded, and the operation was repeated 3 times. 100 μL of sample diluent was added to the blank well. 100 μL of standard sample of different concentrations (different concentrations of standard samples in different hormone Elisa kits) was added to the standard well. 50 μL of sample and 50 μL of sample diluent were added to each sample well of the antibody-coated plate, the plate wells were sealed with sealing film, and incubated at 37°C for 2 h. Then the liquid was removed from the microplate. Fill each well of the microplate with washing solution, and after draining the washing solution, tap the microplate dry. Repeat the above operation five times. Add 100 μL of biotinylated antibody working solution to each well, and cover with a new sealing film, incubate at 37°C for 1 h. Discard the liquid in the well, wash 5 times, and add 100 μL of streptavidin-HRP working solution to each well, and cover with a new sealing film, incubate at 37°C for 30 min. Discard the liquid in the well, wash 5 times, and add 100 μL of TMB substrate to each well, incubate at room temperature for 20 min. Then add 50 μL of stop solution to each well, and measure the absorbance of each well at 450 nm.

[0043] 8), the heart, liver, spleen, lung and kidney of each group of mice were weighed, and the organ index was calculated;

[0044] 9), the brain tissue of each group of mice was taken respectively, and the 5-HT secretion was detected by Elisa;

[0045] 10), part of the intestinal tissue of each group of mice was taken respectively to make paraffin sections and perform H&E staining, and the intestinal barrier damage condition was observed;

[0046] 11), part of the intestinal tissue of each group of mice was taken respectively to detect the expression of inflammatory factors and tight junction proteins by fluorescence quantification. The fluorescence quantification PCR method is as follows: total RNA of the tissue was extracted by Trizol Reagent (Invitrogen) kit. The extracted RNA was used to prepare cDNA by First Strand cDNA Synthesis Kit (Thermo Scientific). Quantitative real-time PCR was performed on 480II (Roche) using SYBR Green detection kit (TaKaRa). 0.5 μL cDNA template was added to the SYBR Green mixture with a total volume of 10 μL, and 0.2 μL forward or reverse primer was added. Data analysis was performed by 2-ΔΔCt method.

[0047] 12), part of the intestinal tissue of each group of mice was taken respectively to detect the phosphorylation level of NF-κB by Western Blot; the Western Blot detection method is as follows: the colon tissue was placed in RIPA lysis buffer containing protease inhibitor phenylmethylsulfonyl fluoride (PMSF), and the magnetic beads were added and homogenized in a homogenizer, with the condition of 50 Hz, 30 s / time, 8 times of homogenization, and 10 s interval. After homogenization, the supernatant was recovered by centrifugation at 12000 r / min for 10 min at 4℃. The protein concentration was detected by BCA protein assay kit. The protein sample was electrophoresed by SDS-PAGE gel, and the electrophoresis was performed at 85v for 150 min. Then the membrane was transferred to PVDF membrane by transfer solution. After blocking in 5% skim milk or BSA in TBST for 2 hours (skim milk incubation for general protein, BSA incubation for phosphorylated protein), the membrane was incubated with primary antibodies of anti-NF-κB p65 (6956, CST), Phospho-NF-κB p65 (3033, CST) and β-actin (T40104, abmart) at 4℃ overnight. The results were observed using horseradish peroxidase-conjugated secondary antibody and chemiluminescent developing solution.

[0048] The following test results were obtained by the above operation method, and the statistical analysis is as follows:

[0049] Figure 1 It is a schematic diagram of the molecular structure of isovaleric acid.​

[0050] Figure 2 It can be seen that compared with the control group, the weight gain and weight gain rate of CRS mice were significantly reduced (P<0.01), and the growth curves showed that the growth of CRS mice was slow. For CRS mice fed sodium isovalerate, except for the low-dose isovalerate group (1 mg / kg), the medium-dose group (10 mg / kg) showed significantly higher weight gain and weight gain rate from 1 to 28 days than the CRS group (P<0.05); the high-dose group (50 mg / kg) showed extremely significantly higher weight gain and weight gain rate from 1 to 14 days than the CRS group (P<0.01), and significantly higher weight gain and weight gain rate from 1 to 28 days (P<0.05). Furthermore, according to the growth curves, the growth rate of all three sodium isovalerate treatment groups was higher than that of the CRS group. Figure 2 E, F). The above results indicate that feeding sodium isovalerate can improve weight loss in mice caused by chronic restraint stress.

[0051] Figure 3 It can be seen that compared with the control group, the feed intake of CRS mice was significantly increased (P<0.01), possibly due to the influence of the hypothalamus-related regions. Although the feed intake of CRS mice increased, their feed conversion ratio was significantly increased (P<0.01), indicating that their energy allocation was affected and energy consumption increased. Only a small portion of the ingested feed may have been converted into body weight, with the majority likely used to maintain survival. For CRS mice fed three doses of sodium isovalerate, their feed intake at 1-14 days and 1-28 days was significantly lower than that of the normal CRS group (P<0.01), and the feed conversion ratio of the medium- and high-dose sodium isovalerate treatment groups was significantly decreased (P<0.01), indicating that the effects of chronic restraint stress on energy allocation and energy consumption in mice were improved. Figure 3 AC). ELISA analysis revealed that serum leptin levels in mice treated with low and medium doses of sodium isovalerate were significantly higher than those in the CRS group (P<0.05). Serum leptin levels also increased somewhat in the high-dose sodium isovalerate treatment group, but the difference was not statistically significant. Figure 3 D). In addition, such as Figure 3 As shown in Figure E, compared to the CRS group, the serum PYY levels in mice treated with medium and high doses of sodium isovalerate were significantly increased (P<0.01). However, there was no significant difference in GLP-1 hormone levels among the groups. Figure 3 F). This indicates that feeding sodium isovalerate leads to an increase in serum leptin and PYY levels in mice, and the resulting decrease in feed intake in mice may be due to the increased secretion of leptin and PYY in the body.

[0052] Figure 4It can be seen that compared with the control group, the CRS mice showed a significant decrease in cardiac index (P<0.05) and a highly significant decrease in spleen index (P<0.01), while the lung index showed no significant change, but exhibited a decreasing trend. Figure 4 A, C, and D indicate that chronic restraint stress may lead to atrophy or degenerative changes in the heart and spleen of mice, and may also have some impact on the lungs. Feeding sodium isovalerate can antagonize the changes in organ indices caused by CRS. Figure 4 As shown in A, C, and D, feeding low-dose sodium isovalerate significantly increased the spleen index in CRS mice (P<0.05), but had no effect on the heart and lung indices; feeding medium-dose sodium isovalerate significantly increased the heart and spleen indices in CRS mice (P<0.05), and the lung index increased extremely significantly (P<0.01); feeding high-dose sodium isovalerate extremely significantly upregulated the heart and spleen indices in CRS mice (P<0.01), and significantly increased the lung index (P<0.05). These results indicate that feeding sodium isovalerate can alleviate organ damage caused by chronic restraint stress in mice and improve the immune and cardiopulmonary function of CRS mice. However, neither chronic restraint stress nor sodium isovalerate feeding had a significant effect on the liver and kidney indices in mice. Figure 4 (B, E). Additionally, such as Figure 4 As shown in Figure F, compared with the CRS group, the leg muscle index of mice in the medium-dose and high-dose groups was significantly increased (P<0.05), suggesting that feeding sodium isovalerate may also have a certain promoting effect on muscle growth and development.

[0053] Figure 5 A showed that compared with the control group, the behavioral despair time of CRS mice was significantly increased (P<0.01), indicating the presence of depressive symptoms. The behavioral despair time of mice treated with low-, medium-, and high-dose sodium isovalerate was reduced, with the medium-dose group showing a significantly lower behavioral despair time than the CRS group (P<0.05). Changes in 5-HT are considered an important factor in the development of depression. Tryptophan hydroxylase 1 (TPH1) is a key rate-limiting enzyme of 5-HT and can affect 5-HT synthesis. ELISA analysis revealed that chronic restraint stress significantly reduced 5-HT levels in the mouse brain (P<0.01), while the 5-HT levels in the brains of mice in the low- and medium-dose groups were significantly higher than those in the CRS group (P<0.05). Furthermore, the 5-HT levels in the hypothalamus of mice in the medium- and high-dose groups were also significantly increased (P<0.05). Quantitative analysis revealed that TPH1 mRNA expression in the low-dose group was significantly higher than that in the CRS group (P<0.05), while TPH1 mRNA expression in the medium-dose group was significantly increased (P<0.01). Figure 5D). This indicates that the 5-HT synthesis is affected after feeding sodium isovalerate, thus improving the depression of mice induced by CRS.

[0054] Figure 6 It can be seen that compared with the control group, the intestinal morphology of CRS mice is damaged, the jejunum villi are sparse, and the brush border is diffuse, the colon crypt is reduced and fused. After feeding sodium isovalerate, the intestinal damage of stressed mice is improved. As shown in Figure 6 B, C, quantitative detection found that the expression of inflammatory factors in CRS mice increased, IL-1β mRNA expression was significantly higher than that of the control group (P<0.05) and TNF-α mRNA expression increased significantly (P<0.01); feeding low-dose sodium isovalerate can significantly reduce IL-1β mRNA expression (P<0.05) and reduce TNF-α mRNA expression; feeding medium and high doses of sodium isovalerate can significantly reduce the expression of IL-1β and TNF-α mRNA induced by chronic restraint stress (P<0.01). This indicates that sodium isovalerate may have certain anti-inflammatory effects, can reduce the intestinal inflammation induced by chronic restraint stress, and improve the intestinal morphology. In addition, as shown in Figure 6 D-G, after chronic restraint stress, the expression of tight junction protein in mice colon decreased, among which claudin-3 and Occludin mRNA expression decreased significantly (P<0.05); claudin-7 and ZO-1 mRNA expression decreased significantly (P<0.01). After feeding sodium isovalerate, the mRNA expression of tight junction protein in CRS mice increased significantly, among which feeding low-dose (1 mg / kg) sodium isovalerate can significantly increase the expression of claudin-3 mRNA (P<0.05), and the expression of Occludin mRNA increases significantly (P<0.01); feeding medium-dose (10 mg / kg) sodium isovalerate can significantly increase the expression of tight junction proteins claudin-3 and claudin-7 (P<0.01) and can significantly increase the expression of tight junction proteins Occludin and ZO-1 (P<0.05); feeding high-dose (50 mg / kg) sodium isovalerate can significantly increase the expression of claudin-7, Occludin and ZO-1 in CRS mice (P<0.01), and can significantly increase the expression of claudin-3 (P<0.05). Western Blot detection found that chronic restraint stress treatment can significantly increase the phosphorylation level of NF-κB in mice (P<0.05), and after feeding sodium isovalerate, the phosphorylation level of NF-κB in CRS mice is significantly reduced (P<0.01). This indicates that sodium isovalerate has certain anti-inflammatory effects, can inhibit the activation of NF-κB, reduce the intestinal inflammation induced by chronic restraint stress, and promote the expression of tight junction proteins, improve the intestinal morphology.

[0055] By Figures 1-6It can be seen that the present application can inhibit the decrease of the weight gain rate, the increase of the feed conversion ratio and the decrease of the organ index of the mice caused by chronic restraint stress, and improve the depression behavior and intestinal inflammation of the CRS mice, thereby relieving the chronic restraint stress of the mice.

[0056] While the embodiments of the application have been illustrated and described, it will be understood by those skilled in the art that various changes, modifications, substitutions, and alternatives can be made to the embodiments without departing from the principles and spirit of the application, to form other embodiments which are readily apparent to those skilled in the art. The scope of the present application is defined by the appended claims and their equivalents.

Claims

1. A method for alleviating the adverse effects of chronic restraint stress on animals by adding sodium isovalerate to the feed, characterized in that, It comprises the following steps: 1) Select 6-week-old male C57BL / 6J mice as test objects, weighing 20±2g, free feeding and drinking water, 12-hour light-dark cycle; 2) The mice are divided into 5 groups, namely blank control group, chronic restraint stress group, low-dose sodium iso-valerate relief group, medium-dose sodium iso-valerate relief group, and high-dose sodium iso-valerate relief group, 8 mice in each group; 3) The method for constructing the chronic restraint stress model of mice is that the mice in the chronic restraint stress group, low-dose sodium iso-valerate relief group, medium-dose sodium iso-valerate relief group, and high-dose sodium iso-valerate relief group are put into the restraint stress device for restraint stress between 9:00 and 15:00 every day, for a total of 28 days, and the control group is normally bred; 4) The mice in the low-dose sodium iso-valerate relief group, medium-dose sodium iso-valerate relief group, and high-dose sodium iso-valerate relief group are fed with corresponding doses of sodium iso-valerate added feed every day, and the mice in the blank control group and chronic restraint stress group are fed with ordinary daily ration every day, for a total of 28 days; 5) The tail suspension test is performed after the chronic restraint stress model is constructed for 28 days to evaluate the depression state of the mice in each group; 6) The serum, heart, liver, spleen, lung, kidney, brain tissue, and intestinal tissue of the mice in each group are sampled after the chronic restraint stress model is constructed for 29 days, the brain tissue includes the brain and hypothalamus, and the intestinal tissue includes the jejunum and colon; 7) The serum of the mice in each group is taken for Elisa detection of satiety hormone secretion; 8) The heart, liver, spleen, lung, and kidney of the mice in each group are weighed to calculate the organ index; 9) The brain tissue of the mice in each group is taken for Elisa detection of 5-HT secretion; 10) Part of the intestinal tissue of the mice in each group is taken to make paraffin sections and perform H&E staining to observe the intestinal barrier damage condition; 11) Part of the intestinal tissue of the mice in each group is taken to detect inflammation indicators by fluorescence quantitative PCR and Western Blot.

2. The method for alleviating the adverse effects of chronic restraint stress on animals by adding sodium isovalerate to feed according to claim 1, characterized by, The restraint stress device is a 50ml plastic centrifugal tube.

3. The method for alleviating the adverse effects of chronic restraint stress on animals by adding sodium isovalerate to feed according to claim 2, characterized by, Fine holes are distributed on the wall of the centrifugal tube, and a small hole is formed in the center of the tube cover.

4. The method for alleviating the adverse effects of chronic restraint stress on animals by adding sodium isovalerate to feed according to claim 1, characterized by, The tail suspension test method comprises: placing the mice in the laboratory for 30 minutes before the experiment, fixing the tail of the mouse 1-2cm away from the tail tip with adhesive tape in a quiet and windless room, suspending the mouse head downward, and avoiding mechanical damage, the suspension point of the mouse tail is 50cm away from the experimental table, and the cumulative immobility time of the mouse in the last 4 minutes is recorded within 6 minutes, and the immobility standard is that the mouse stops struggling, the body is vertically inverted, and is motionless.

5. The method for alleviating the adverse effects of chronic restraint stress on animals by adding sodium isovalerate to feed according to claim 1, characterized by, The Elisa detection method comprises: collecting serum and brain tissue homogenate, adding 1x washing buffer 350ul to each well, discarding the liquid after standing for 40s, repeating 3 times; adding 100ul sample diluent to the blank hole, adding 100ul standard of different concentrations to the standard hole; add 50ul sample, 50ul sample diluent to each sample hole of the antibody coated plate, seal the plate hole with sealing film, incubate at 37 °C for 2h; then remove the liquid from the microplate; fill each well of the microplate with washing solution, and after draining the washing solution, dry the microplate, repeat the above operation five times; add 100ul biotinylated antibody working solution to each well, cover with new sealing film, incubate at 37 °C for 1h; discard the liquid in the hole, wash 5 times, add 100ul streptavidin-HRP working solution to each well, cover with new sealing film, incubate at 37 °C for 30min; discard the liquid in the hole, wash 5 times, add 100ul TMB substrate to each well, incubate at room temperature for 20min; then add 50ul stop solution to each well, measure the absorbance of each well at 450nm.

6. The method for alleviating the adverse effects of chronic restraint stress on animals by adding sodium isovalerate to feed according to claim 1, characterized by, The fluorescent quantitative PCR method comprises: extracting total RNA of the tissue with a Trizol Reagent kit, preparing cDNA by a First Strand cDNA Synthesis Kit using the extracted RNA; performing quantitative real-time PCR on a 480 II using a SYBR Green detection kit; adding 0.5 μL of a cDNA template into a SYBR Green mixture with a total volume of 10 μL, and adding 0.2 μL of forward or reverse primers; performing data analysis by 2 -ΔΔCt Methods.

7. The method for alleviating the adverse effects of chronic restraint stress on animals by adding sodium isovalerate to feed according to claim 1, characterized by, The Western Blot detection method comprises: placing the colon tissue into RIPA lysis buffer added with PMSF, homogenizing after adding magnetic beads in a homogenizer, conditions 50Hz, 30s / time, homogenizing 8 times, with 10s interval; after homogenization, centrifuge at 4℃12000r / min for 10min to recover the supernatant; use BCA protein assay kit to detect the protein concentration; the protein sample is electrophoresed by SDS-PAGE gel, 85v electrophoresis for 150min; then transfer to PVDF membrane by transfer membrane liquid; after blocking with 5% skimmed milk or BSA in TBST for 2h, incubate the membrane with anti-NF-κB p65, Phospho-NF-κB p65 and β-actin primary antibody at 4℃ overnight; use horseradish peroxidase conjugated secondary antibody and chemiluminescent developing solution to observe the results.

Images