New application of sodium butyrate

Abstract

The invention discloses a new application of sodium butyrate, and belongs to the field of reproductive biology in developmental biology or animal reproductive science in animal husbandry or animal nutrition and feed science in animal husbandry. The invention relates to an application in preparation of a preparation or a feed additive for rescuing small intestine injury and progeny primordial follicle injury caused by OTA exposure during pregnancy. Compared with other detoxification methods, the method has the advantages that operation is easy and convenient, sodium butyrate (NaB) is good in fluidity, the sodium butyrate (NaB) can be easily mixed with other feed, the pH value of the feed cannot be changed, the sodium butyrate (NaB) can be rapidly absorbed in the body, and the method has the advantages of being free of pollution and harmful residues and can be widely applied to livestock and poultry feed addition.

Description

Technical Field

[0001] The present invention belongs to the field of animal nutrition and feed science in animal husbandry or animal reproduction in animal husbandry or reproductive biology in developmental biology, and specifically relates to a new use of sodium butyrate (NaB). Background Art

[0002] Ochratoxins are mainly found in moldy grains and feeds, including a variety of compounds with similar structures. Among them, ochratoxin A (OTA) is identified as a Class IIB carcinogen by the International Agency for Research on Cancer because of its strong toxicity, wide distribution range, and close relationship with humans. OTA can endanger the health of humans and animals through hepatotoxicity, nephrotoxicity, reproductive toxicity, enterotoxicity, immunotoxicity, carcinogenicity, and teratogenicity. The follicle is the basic unit of ovarian structure and function. It is composed of a central oocyte and surrounding follicular cells. According to the different developmental states, it can be divided into primordial follicles, primary follicles, secondary follicles, and mature follicles. The primordial follicle consists of an oocyte surrounded by a single layer of squamous granulosa cells. Its number is fixed and non-renewable. Therefore, the primordial follicle is the only source of fertilizable eggs in the entire reproductive life of female mammals. Our previous research results show that OTA can inhibit the meiotic process of female mice by inducing oxidative stress and apoptosis in female mouse oocytes, causing damage to the formation of primordial follicles and reducing the reproductive capacity of female mice. In addition, the intestine is also one of the target organs of OTA. OTA can damage the tight junctions between intestinal epithelial cells and cause serious damage to the intestinal barrier of humans and animals. At present, scientists have developed a variety of OTA detoxification methods, including physical detoxification, chemical detoxification and biological detoxification. However, safe, convenient, efficient and environmentally friendly detoxification methods for food and feed mycotoxins need further research and development. At the same time, the idea of ​​"focusing on detoxification and light on detoxification" of mycotoxins needs further discussion. Summary of the invention The present invention aims to provide a new use of sodium butyrate, which is a method for detoxifying mycotoxins and is used to reduce the harm of OTA to humans or animals. Compared with other detoxification methods, the method is simple to operate and can effectively alleviate the symptoms of intestinal barrier damage and offspring reproductive abnormalities caused by animal mycotoxins. Sodium butyrate (NaB) can be widely used as a feed additive for livestock and poultry.

[0004] The object of the present invention is achieved by the following technical solutions: A new use of sodium butyrate, which is the use in preparing a preparation or feed additive for rescuing small intestinal damage and offspring primordial follicle damage caused by ochratoxin A exposure during pregnancy.

[0005] Preferably, the preparations include: oral dosage forms, injection dosage forms, cavity dosage forms, sublingual dosage forms, inhalation dosage forms, and sprays.

[0006] Preferably, the oral administration agent includes tablets, capsules, granules, pills, oral liquids, and oral powders.

[0007] Preferably, the preparation and use of the oral powder comprises the following steps: packing the sodium butyrate into 0.0201 g / portion, encapsulating, and adding 3 mL of ultrapure water to make it 6.7 mg / mL when using.

[0008] Preferably, sodium butyrate is added to the feed to alleviate the intestinal barrier damage and abnormal assembly of primordial follicles in offspring caused by ochratoxin A exposure in livestock and poultry.

[0009] The present invention also provides a technical method for using sodium butyrate to rescue small intestinal damage and primordial follicle damage of offspring caused by exposure to ochratoxin A during pregnancy, comprising the following steps: NaB is packaged into 0.0201 g / tube in 5 mL centrifuge tubes and wrapped with sealing film. When used, 3 mL of ultrapure water is added to make 6.7 mg / mL, and the volume of NaB for gavage is 3 times the weight of the mouse; 7-8 week old male mice and female mice are caged together, and E12.5 pregnant mice are divided into 4 groups, namely CON, OTA, OTA+NaB, and NaB. The CON group is gavaged with 100 μL of physiological saline containing DMSO once a day, the OTA group is gavaged with 100 μL of 35 μg / mL OTA once a day, the OTA+NaB group is treated with OTA for 6 hours and then gavaged with 20 mg / kg NaB once a day, and the NaB group is gavaged with 20 mg / kg NaB once a day; gavage is continued until the offspring are 3 days old.

[0010] The advantage of the present invention over the prior art is that it is a detoxification method for OTA, which is used to reduce the harm caused by OTA to humans or animals. Compared with other detoxification methods, the present method is simple to operate, sodium butyrate (NaB) has good fluidity, is easy to mix with other feeds, and does not change the pH value of the feed. It is quickly absorbed in the body and has the characteristics of no pollution and no harmful residues. It can be widely used in livestock and poultry feed additives.

[0011] NaB can detoxify by destroying the structure of OTA. Sodium butyrate is the sodium salt form of butyric acid. Studies have found that it has a wide range of physiological effects after being hydrolyzed into butyric acid. Butyric acid reacts with OTA to change the structure of OTA, thereby eliminating its toxicity. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 This is the immunofluorescence staining analysis of ovaries of 3dpp newborn mice; Figure 2 This is the statistics of the number of ovarian germ cells in newborn mice at 3dpp; Figure 3 The percentage of primordial follicles in newborn mice at 3 dpp; Figure 4Western blot analysis of genes related to primordial follicle formation in ovaries of newborn mice at 3dpp; Figure 5 HE staining and PAS staining of the small intestine of maternal mice; Figure 6 The height of villi, crypt depth, villus-crypt ratio and number of goblet cells in the small intestine of maternal mice were counted; Figure 7 This is WB detection of tight junction proteins in the small intestine of maternal mice. DETAILED DESCRIPTION

[0013] The present invention is further described below through the accompanying drawings and specific embodiments.

[0014] Example 1 This embodiment provides a new use of sodium butyrate, which is the use in preparing a preparation or feed additive for rescuing small intestinal damage and offspring primordial follicle damage caused by OTA exposure during pregnancy.

[0015] Example 2 This embodiment provides a technical method for using sodium butyrate to rescue small intestinal damage and offspring primordial follicle damage caused by OTA exposure during pregnancy, comprising the following steps: NaB was dispensed into 5mL centrifuge tubes at 0.0201g / tube and wrapped with sealing film. When used, 3mL of ultrapure water was added to make it 6.7mg / mL. The volume of NaB administered intragastrically was 3 times the weight of the mouse. The E12.5 pregnant mice were divided into 4 groups, namely CON, OTA, OTA+NaB, and NaB. The CON group was administered intragastrically with 100μL of normal saline containing DMSO once a day, the OTA group was administered intragastrically with 100μL of 35μg / mL OTA once a day, the OTA+NaB group was treated with OTA for 6h and then administered with 20mg / kg NaB once a day, the NaB group was administered with 20mg / kg NaB once a day; the administration of intragastrically was continued until the pups were 3 days old.

[0016] Example 3 Detection of ovarian reserve and primordial follicle assembly process: The 3dpp newborn mouse ovaries of Example 2 were collected and subjected to immunohistofluorescence to detect the ovarian reserve and primordial follicle assembly process by the following steps: the slide was placed in an iron rack, and after being baked in an oven at 60°C for 30 minutes, the sample was rehydrated (xylene I, xylene II for 15 minutes each → anhydrous ethanol, 95% ethanol, 90% ethanol, 80% ethanol, 70% ethanol, 50% ethanol for 5 minutes each → PBS for 10 minutes). Antigen repair solution (3g sodium citrate + 0.4g citric acid, added ultrapure water to 1L) was prepared, heated to 96°C in a water bath, the iron rack with the sample was placed in the antigen repair solution, the lid was tightly closed, and heating was continued for 10 minutes, and then naturally cooled to room temperature. The slide was taken out, TBS and TBST were washed for 5 minutes each, and then transferred to a wet box, 50μL of blocking solution (3% BSA + 10% goat serum, dissolved in TBS) was added to each slide, and the sealing film was covered and blocked at room temperature for 45 minutes. Prepare the primary antibody dilution. After blocking, remove the sealing film, add 50μL of primary antibody dilution to each slide, cover with sealing film, put into a humidified box, seal the humidified box with a transparent bag, and incubate at 4°C overnight. After incubation with the primary antibody, take out the humidified box, rewarm for 30 minutes, and wash with 1×TBST for 3×10 minutes. Prepare the secondary antibody dilution. Add 50μL of secondary antibody dilution to each slide, cover with sealing film, put into a humidified box, seal the humidified box with a transparent bag, and incubate at 37°C for 45 minutes. After incubation with the secondary antibody, wash with 1×TBST for 3×10 minutes, add 50μL Hoechst to each slide, cover with sealing film, and stain the nucleus at room temperature for 5 minutes. Remove the sealing film, wash with 1×PBS for 2×10s and 1×3min. Add an appropriate amount of antiquenching tablets, seal with a coverslip, observe and count, and the results are shown in the table. Figures 1 to 4 .

[0017] Conclusion: From the figure, it can be observed that the ovaries after OTA treatment are significantly smaller than those in the other three groups, while the ovaries after NaB treatment are comparable in size to those in the control group ( Figure 1 ). The number of germ cells was statistically analyzed and it was found that the total number of germ cells in the ovaries was significantly reduced after OTA exposure compared with the control group ( Figure 2 ), and the proportion of primordial follicles was significantly reduced, while the total number of germ cells in the OTA+NaB group increased significantly, and the proportion of primordial follicles was significantly increased ( Figure 3 ). Subsequently, WB was used to detect changes in the expression of genes related to ovarian primordial follicle formation at the protein level. The results showed that OTA exposure during pregnancy led to a significant decrease in the protein level of ovarian reserve-related genes (MVH, LHX8 and SOHLH1) in offspring mice, while NaB could effectively restore the expression of ovarian reserve-related genes caused by OTA ( Figure 4 ).

[0018] Example 4 Detection of intestinal morphology and intestinal barrier status The small intestine of the mother fed in Example 2 was collected, and the small intestine sections were HE stained through the following steps: xylene I, xylene II, 10 min each → anhydrous ethanol I 5 min, anhydrous ethanol II, 5 min each → 95% ethanol I, 95% ethanol II, 3 min each → 80% ethanol 2 min, 70% ethanol, 50% ethanol, 2 min each → distilled water 2 min → hematoxylin 2 min → tap water washing 5 min → distilled water 2 min → 1% hydrochloric acid alcohol 3-5 s → 45 ℃ tap water blueing 5-15 min → distilled water 2 min → 50% y ethanol 2 min, 70% ethanol 2 min, 80% ethanol, 2 min each → eosin 5 s → 95% ethanol I 2-5 min → 95% ethanol II 2-5 min → anhydrous ethanol I5 min → anhydrous ethanol II 5 min → xylene I 5 min → xylene II 5-10 min. After staining, resin was dripped on the specimens to seal the slides and air-dry them, and observation and statistics were performed. The results are shown in Figure 5-Figure 6 .

[0019] Conclusion: In the control group, the villi were closely arranged, the intestinal epithelial cells were in good morphology, and the muscle layer and mucosa layer were closely connected; after OTA treatment, the villi were sparsely arranged, the villi became shorter and thicker, the intestinal epithelial cells were irregular in morphology, and the muscle layer and mucosa layer were not closely connected; after OTA and NaB co-treatment, the villi were closely arranged and the villi became longer; after NaB treatment alone, the villi were similar to the control group ( Figure 5 ). Further statistical analysis found that compared with the control, the height of the villi in the small intestine was significantly reduced and the crypt depth was significantly increased after OTA treatment, which aggravated the decrease in the villus-crypt ratio. The crypt depth was significantly increased, which aggravated the decrease in the villus-crypt ratio. After NaB treatment, the height of the villi and the crypt depth of the small intestine were significantly increased and decreased, respectively, and the villus-crypt ratio was significantly increased ( Figure 6 ).

[0020] The small intestine was stained with PAS to observe the goblet cells on the villi of the small intestine: After the small intestine was removed, the sample was processed (xylene I, xylene II for 10 minutes each → anhydrous ethanol for 5 minutes → 90% ethanol, 80% ethanol, 70% ethanol, distilled water for 2 minutes each). Periodic acid (100 μL / slice) was added in a dark room and kept in a wet box away from light for 10 minutes. The slice was washed in a distilled water wash cup on a shaker for 5 minutes. Schiff reagent (100 μL / slice) was added, placed in a wet box away from light, and then placed in a 37 ℃ oven for 30 minutes-1 hour, and the slice was washed in a distilled water wash cup on a shaker for 5 minutes. Hematoxylin (100 μL / slice) was added, stained for 30 seconds, and the staining results were observed under a microscope. The slice was washed with distilled water for more than 2 times, 3 seconds each time, until the floating color was washed away. Hydrochloric acid alcohol differentiation for 30 seconds, distilled water wash 3 times, 2 seconds each time, 45 ℃ tap water anti-blue for 5 minutes. Dehydrate the sample (90% ethanol, anhydrous ethanol, 2 min each → xylene I, xylene II, 5 min each). Add neutral gum to the specimen to seal the slide, observe and count. Figure 6 , Figure 7 .

[0021] Conclusion: The number of goblet cells decreased significantly after OTA treatment, but increased and even exceeded the control level after NaB rescue ( Figure 6 In addition, WB results showed that NaB effectively restored the damaged tight junctions between intestinal epithelial cells caused by OTA treatment ( Figure 7 OTA treatment significantly downregulated the expression levels of tight junction proteins OCCLUDIN and CLAUDIN-1, while NaB reversed this trend.

[0022] The present invention successfully utilizes NaB to rescue the small intestinal damage and primordial follicle damage of offspring mice caused by OTA exposure during pregnancy. This method can also alleviate the intestinal barrier damage and abnormal assembly of primordial follicles of offspring caused by OTA in humans and livestock by adding NaB to feed.

Claims

1. A new use of sodium butyrate, characterized in that: Application in the preparation of preparations or feed additives for rescuing small intestinal damage and offspring primordial follicle damage caused by ochratoxin A exposure during pregnancy.

2. The new use of sodium butyrate according to claim 1, characterized in that: The preparations include: oral administration preparations, injection administration preparations, cavity administration preparations, sublingual administration preparations, inhalation administration preparations, and sprays.

3. The new use of sodium butyrate according to claim 2, characterized in that: The oral administration preparations include tablets, capsules, granules, pills, oral liquids, and oral powders.

4. The new use of sodium butyrate according to claim 3, characterized in that: The preparation and use method of the oral powder comprises the following steps: packing sodium butyrate into 0.0201 g / portion, encapsulating, and adding 3 mL of ultrapure water to make 6.7 mg / mL when using.

5. The new use of sodium butyrate according to claim 1, characterized in that: Sodium butyrate is added to feed to alleviate intestinal barrier damage and abnormal primordial follicle assembly in offspring caused by ochratoxin A exposure in livestock and poultry.

6. A technical method for using sodium butyrate to rescue small intestinal damage and primordial follicle damage in offspring caused by ochratoxin A exposure during pregnancy, characterized in that: The method comprises the following steps: NaB is dispensed into 5 mL centrifuge tubes at 0.0201 g / tube, which are wrapped with sealing film, and 3 mL of ultrapure water is added to make it 6.7 mg / mL when used, and the volume of NaB administered intragastricly is 3 times of the weight of the mouse; pregnant mice at E12.5 are divided into 4 groups, namely CON, OTA, OTA+NaB, and NaB, the CON group is administered intragastricly with 100 μL of normal saline containing DMSO once a day, the OTA group is administered intragastricly with 100 μL of 35 μg / mL OTA once a day, the OTA+NaB group is administered intragastricly with 20 mg / kg NaB after being treated with OTA for 6 hours, and the NaB group is administered intragastricly with 20 mg / kg NaB once a day, and the administration of intragastric administration is continued until the pups are 3 days old.

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