Indole derivative for promoting development of piglet intestinal stem cells and application thereof

Abstract

The invention discloses an indole derivative for promoting development of piglet intestinal stem cells, and particularly relates to application of indole-3-aldehyde in promoting development and growth performance of piglet intestinal epithelium. The indole-3-aldehyde can be used as a feed additive to be applied to a piglet creep feed and a nursing feed, promote the development of forms and functions of piglet intestinal stem cells and intestinal epithelium, improve the length and the index of the small intestine of the piglet and reduce the feed-weight ratio of the piglet. It is found that indole-3-aldehyde guarantees animal health and improves the growth performance of animals by adjusting proliferation and differentiation of piglet intestinal stem cells, increasing the number of goblet cells, up-regulating expression of tight junction protein, promoting intestinal epithelium development and intestinal barrier functions and promoting absorption and utilization of intestinal epithelium to nutrient substances.

Description

Technical Field

[0001] The invention belongs to the technical field of animal feed, and relates to an indole derivative for promoting the development of piglet intestinal stem cells and an application thereof. Background Art

[0002] As pig farming shows a trend of large-scale and intensive development, the mortality rate of weaned piglets shows a trend of continuous increase, causing serious economic losses to farmers. Domestic pig farms generally choose to wean piglets between 21 and 35 days of age, or when the weight of piglets reaches 7 to 8 kg or more. Piglets at the weaning stage often have physiological characteristics such as incomplete organ development, low resistance, poor body temperature regulation function, high feed and nutrition requirements, and easy stress response.

[0003] After weaning, piglets’ diet changes from breast milk to solid feed, but the tissues, organs, digestive system, etc. of weaned piglets are not fully developed, the secretion of gastric acid and various digestive enzymes is insufficient, and the enzyme activity is low, resulting in insufficient digestive capacity, weak resistance to external stimuli, and very easy to develop indigestion diarrhea. At the same time, the weaning stage is also a critical period for intestinal development and intestinal barrier formation. During this period, the intestine of piglets has strong plasticity, and its development degree has a significant impact on the later growth and health of piglets. If the intestinal development of piglets is not adequately regulated at this critical stage, it is easy to have abnormal proliferation and differentiation of intestinal stem cells and damage to the intestinal barrier, which will eventually induce various health diseases. In addition, the health status of piglets during the weaning stage will directly affect the feed conversion rate and production efficiency of piglets in the later growth period. Therefore, in order to ensure the normal growth and development of piglets, how to regulate the intestinal development of piglets and reduce the feed-to-weight ratio has become one of the urgent problems to be solved in the current pig farming industry.

[0004] Indole-3-aldehyde (IAld) is a tryptophan metabolite produced by intestinal microorganisms and is a type of indole derivative. Indole-3-aldehyde has a wide range of biological functions, such as anti-inflammatory, antioxidant and anti-tumor. In addition, indole-3-aldehyde is a raw material for synthesizing many physiologically and pharmacologically active compounds and an important intermediate in organic synthesis. Currently, many manufacturers at home and abroad are developing APIs using it as a raw material. However, its application in the piglet feed industry has not been reported. Summary of the invention

[0005] Purpose of the invention: The purpose of the present invention is to provide an application of an indole derivative in promoting the development and growth performance of the intestinal epithelium of piglets, so as to solve the problems of impaired intestinal epithelial development and low growth efficiency of weaned piglets. The present invention proposes a new function of an indole derivative (indole-3-aldehyde). Adding indole-3-aldehyde to the piglet feed can effectively promote the proliferation of intestinal epithelial cells in piglets, improve the intestinal barrier function of piglets, and thus reduce the feed-to-weight ratio and improve the growth efficiency of piglets.

[0006] Technical solution: Application of an indole derivative in promoting the development of intestinal stem cells in piglets.

[0007] The invention discloses an application of an indole derivative in the preparation of a feed additive for promoting the development and growth performance of intestinal stem cells in piglets.

[0008] Wherein, the indole derivative is indole-3-aldehyde.

[0009] Furthermore, in the above application, the indole-3-aldehyde is added to piglet feed as a feed additive.

[0010] Furthermore, in the above application, the amount of the feed additive added to the feed is 100 mg / kg.

[0011] Wherein, the piglets are weaning stage piglets.

[0012] Furthermore, the present invention provides a feed additive for promoting the development of intestinal stem cells in piglets, wherein the feed additive includes indole-3-aldehyde.

[0013] Furthermore, the present invention provides a feed for promoting the development of intestinal stem cells in piglets, wherein the feed is composed of basic piglet feed and the above-mentioned feed additive.

[0014] Furthermore, the present invention provides a method for raising weaned piglets, comprising feeding the weaned piglets with the above-mentioned feed.

[0015] In order to deal with the above problems faced by the weaned piglet breeding industry, the present invention has previously studied the growth performance of piglets and found that adding 100 mg / kg of indole-3-aldehyde to the feed can significantly reduce the feed-to-weight ratio of piglets and improve the growth efficiency of piglets. After the piglets were slaughtered, the weight of the small intestine, the length of the small intestine, the expression of the small intestinal tight junction marker proteins Occludin and Claudin 1, the expression of the goblet cell marker protein MUC2, the expression of the intestinal stem cell marker proteins Olfm4 and Lgr5, the expression of the differentiated cell marker protein KRT20, the expression of the intestinal epithelial proliferation-related genes and proteins PCNA, β-catenin, c-Myc and Cyclin D1 were analyzed, and it was found that supplementing 100 mg / kg of indole-3-aldehyde to the feed can significantly promote the intestinal barrier function of piglets and the proliferation and differentiation of intestinal stem cells.

[0016] Design principle: By adding 100 mg / kg indole-3-aldehyde to the piglet feed, the role of 100 mg / kg indole-3-aldehyde in improving the growth performance of piglets, promoting intestinal barrier function and the proliferation and differentiation of intestinal stem cells was determined. At the same time, in order to further verify the proliferation-promoting effect of indole-3-aldehyde on intestinal stem cells, intestinal organoids were used to simulate the cell growth environment in the small intestine, and intestinal organoids were treated with 15 μM indole-3-aldehyde. It was found that indole-3-aldehyde can significantly promote the growth of intestinal organoids, especially the proliferation of intestinal stem cells.

[0017] The present invention proposes for the first time the application of indole-3-aldehyde in promoting the development and growth performance of the intestinal epithelium of piglets. For weaned piglets, the digestion and absorption of nutrients mainly occurs in the small intestine, so promoting the development of small intestinal epithelial cells, especially the development of intestinal stem cells, can effectively improve the intestinal physiological environment and promote growth performance.

[0018] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: 1. The indole-3-aldehyde used in the present invention is an economical and effective additive. Adding indole-3-aldehyde to piglet feed can significantly reduce the feed-to-weight ratio, improve feed remuneration, effectively reduce costs, and improve economic benefits;

[0019] 2. The present invention proposes a new function of indole-3-aldehyde, which can promote the development of intestinal epithelial cells, especially the proliferation of intestinal stem cells, improve the intestinal barrier function, maintain the intestinal health of piglets, reduce the occurrence of diseases, and improve the production level of animals. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The bar graph shows the effect of dietary indole-3-aldehyde on feed-to-weight ratio and small intestine relative weight in piglets.

[0021] Figure 2Immunofluorescence images and bar graphs showing the effects of dietary indole-3-aldehyde on intestinal barrier function in piglets;

[0022] Figure 3 This is an immunofluorescence image showing the effect of dietary indole-3-aldehyde on the development of the intestinal epithelium in piglets.

[0023] Figure 4 This is a statistical chart showing the effect of dietary indole-3-aldehyde on the proliferation of intestinal epithelial cells in piglets on mRNA and protein expression.

[0024] Figure 5 This is a statistical graph showing the effect of indole-3-aldehyde addition on the expression of proliferation proteins in piglet intestinal stem cells. DETAILED DESCRIPTION

[0025] In order to facilitate the understanding of the present invention, the present invention will be described more comprehensively below in conjunction with specific embodiments.

[0026] Comparative Example 1

[0027] The piglet feed for the control group was prepared, including a basal diet, wherein the basal diet formula was designed according to the nutritional requirements of piglets, and its composition and nutritional level are shown in Table 1.

[0028] Table 1: Composition and nutrient levels of basal diets

[0029]

[0030] Note: 1 Each kilogram of premix contains: 32000KIU, vitamin A; 8000KIU, vitamin D3; 200000mg, vitamin E; 10000mg, vitamin K3; 10094mg, vitamin B1; 20000mg, vitamin B2; 12054mg, vitamin B6; 80mg, vitamin B12; 5985mg, folic acid; 99960mg, D-calcium pantothenate; 1000mg, biotin; 50490mg, niacinamide; 10000mg, copper; 50100mg, iron; 32205mg, zinc; 14628mg, manganese; 150mg, selenium; 85mg, iodine; 78mg, chromium.

[0031] Example 1

[0032] The piglet feed of the test group was prepared, including a basic diet and indole-3-aldehyde, that is, 100 mg / kg indole-3-aldehyde was added to the basic diet of Comparative Example 1.

[0033] Example 2

[0034] The piglet feed of the test group was prepared, including a basic diet and indole-3-aldehyde, that is, 200 mg / kg indole-3-aldehyde was added to the basic diet of Comparative Example 1.

[0035] Test example:

[0036] Twenty-four healthy 28-day-old weaned piglets (Landrace) of similar weight were randomly divided into three groups. The control group was fed the basic diet of comparative example 1; the IAld100 group was fed the feed of Example 1, i.e., 100 mg / kg indole-3-aldehyde was added to the basic diet; the IAld200 group was fed the feed of Example 2, i.e., 200 mg / kg indole-3-aldehyde was added to the basic diet. The test period was 14 days, during which all piglets were free to eat and drink, and no antibiotics or immunization were performed.

[0037] During the experiment, the feed intake and weight changes of the piglets were recorded. One day before slaughter, all piglets were weighed, and the piglets in the IAld100 group with relatively low feed weight were selected and slaughtered together with the control group for sampling on the 14th day of the experiment.

[0038] Test 1:

[0039] The feed intake and weight changes of the piglets were recorded during the experiment. The piglets were weighed one day before slaughter, and the indole-3-aldehyde (IAld) group with relatively low feed weight was selected. They were slaughtered and sampled together with the control group on the 14th day of the experiment. After the piglets were slaughtered, the abdominal cavity was opened, the front end of the stomach and the front end of the rectum were ligated with cotton thread, and the intestines were dissected out. The lengths of the jejunum, ileum and colon were measured and recorded as the small intestine length. The chyme in the jejunum, ileum and colon was weighed after being drained, and the total weight of the jejunum, ileum and colon was recorded as the total intestinal weight. The total weight of the jejunum and ileum was recorded as the small intestine weight.

[0040] Feed conversion rate generally refers to feed-to-weight ratio (FCR), which refers to the amount of feed consumed by livestock and poultry to gain one kilogram of weight. It is an important indicator for evaluating feed efficiency. A high feed-to-weight ratio means that more feed is used, but less meat is gained; conversely, a low feed-to-weight ratio means that less feed is used, but more meat is gained. Figure 1 It can be seen that compared with the control group, both the IAld100 group and the IAld200 group reduced the feed-to-weight ratio of piglets 0-14 days after weaning. Among them, the IAld100 group, which added 100 mg / kg indole-3-aldehyde to the feed, could significantly reduce the feed-to-weight ratio of piglets 0-14 days after weaning, and significantly increase the small intestine weight / length and small intestine index, with the best effect.

[0041] Test 2:

[0042] (1) The piglets in the IAld100 group with relatively low feed weight were selected for slaughter and sampling together with the piglets in the control group. After the piglets were slaughtered, the 1 cm chyme-free area in the middle of the small intestine was quickly taken and fixed in 4% paraformaldehyde. After embedding in paraffin, the preparation of immunofluorescence staining sections was completed through the steps of sectioning, dewaxing, antigen repair, serum blocking, primary antibody incubation, secondary antibody incubation, DAPI staining solution counterstaining of cell nuclei, autofluorescence quenching, and sealing. The sections were observed using an LSM 900 laser confocal microscope (ZEISS, Germany), and appropriate fluorescent filters and lasers were selected. Five different fields of view were randomly selected for each section, and the average value was taken as the data for a single section. The average fluorescence intensity was measured using ImageJ software.

[0043] (2) Weigh 0.1 g of small intestinal tissue and extract total RNA from the tissue using a total RNA extraction kit (Adlai, China). Then, reverse transcribe the RNA into cDNA using a reverse transcription kit (TaKaRa, China). Detect the expression of the target gene using a quantitative PCR kit (TaKaRa, China).

[0044] (3) Weigh 0.1 g of small intestinal tissue and lyse it with RIPA lysis buffer containing 1% protease inhibitor. After shaking and centrifugation, the supernatant was obtained. The protein concentration in the supernatant was detected using a protein detection kit (Nanjing Jiancheng, China). After unifying the concentration, the protein was denatured. The denatured protein was subjected to electrophoresis, transfer, blocking, primary antibody and secondary antibody incubation, and finally developed using a chemical imager. The grayscale value of the band was measured using ImageJ software.

[0045] Depend on Figure 2 It can be seen that the IAld100 group, which fed piglets with feed supplemented with 100 mg / kg indole-3-aldehyde, significantly increased the number of MUC2-positive cells and MUC2 mRNA expression in the small intestine of piglets, as well as the protein expression of Occludin and Claudin 1. This suggests that the addition of 100 mg / kg indole-3-aldehyde to the diet can significantly improve the intestinal barrier function of piglets.

[0046] Depend on Figure 3 It can be seen that the IAld100 group, i.e., the feed supplemented with 100 mg / kg indole-3-aldehyde, can significantly increase the number of positive cells of Olfm4 and KRT20 in the small intestine of piglets. This suggests that the addition of 100 mg / kg indole-3-aldehyde to the diet can promote the proliferation and differentiation of intestinal epithelial cells.

[0047] Depend on Figure 4 It can be seen that the IAld100 group, which feeds piglets with feed supplemented with 100 mg / kg indole-3-aldehyde, can significantly increase the expression of genes and proteins related to intestinal epithelial cell proliferation in piglets.

[0048] Test 3:

[0049] A healthy 7-day-old piglet (Du × Chang × Da) was selected for the experiment, and the intestine was removed immediately after slaughter. An intestinal segment of about 8 cm in length in the middle of the small intestine was placed in a pre-cooled DPBS buffer. After gently scraping off the villi on the intestine, the intestinal segment was cut into 5×5 mm pieces with scissors, and then washed with cold DPBS until the mixture became clear. Then, the tissue fragments were placed in a DPBS solution containing 30mM EDTA-2Na and incubated on ice for 30 minutes. After incubation, the mixture was mixed by shaking and centrifuged, and the supernatant was discarded. The precipitate obtained was the intestinal crypt. The isolated crypts were resuspended in organoid growth medium (Stemcell, Germany), mixed with matrix gel at a ratio of 1:1, and the cell suspension was spotted on a 24-well cell culture plate for culture. After the matrix gel solidified, 500 μL of culture medium was added to each well, and 15 μM indole-3-aldehyde was added to the culture medium in the IAld treatment group. All organoids were cultured at 37°C and a cell culture incubator containing 5% CO2. After 48 hours of culture, cell samples were collected and RNA and protein were extracted. The steps of RNA and protein extraction were similar to those in Example 4.

[0050] Depend on Figure 5 It can be seen that indole-3-aldehyde significantly increased the protein expression of stem cell marker protein Lgr5 and active β-catenin, which suggests that indole-3-aldehyde can promote the proliferation ability of intestinal stem cells.

[0051] Combined with the above embodiments and test results, indole-3-aldehyde can be added to piglet feed to effectively promote the development of intestinal epithelial morphology and function, increase the length and small intestine index of piglets, and reduce the feed-to-weight ratio of piglets. Indole-3-aldehyde can regulate the proliferation and differentiation of intestinal stem cells in piglets, increase the number of goblet cells, upregulate the expression of tight junction proteins, promote intestinal epithelial development and intestinal barrier function, and ensure animal health. Indole-3-aldehyde can be used to prepare feed additives that promote the development and growth performance of intestinal epithelium in piglets.

Claims

1. Application of an indole derivative in promoting the development of intestinal stem cells in piglets.

2. The use of an indole derivative in the preparation of a feed additive for promoting the development and growth performance of intestinal stem cells in piglets.

3. The use according to claim 1 or 2, characterized in that: The indole derivative is indole-3-aldehyde.

4. The use according to claim 3, characterized in that: The indole-3-aldehyde is added to piglet feed as a feed additive.

5. The use according to claim 4, characterized in that: The added amount of the feed additive in the feed is 100 mg / kg.

6. The use according to any one of claims 1 to 5, characterized in that: The piglets are weaned stage piglets.

7. A feed additive for promoting the development of intestinal stem cells in piglets, characterized in that: The feed additive includes indole-3-aldehyde.

8. A feed for promoting the development of intestinal stem cells in piglets, characterized in that: The feed consists of basic piglet feed and the feed additive according to claim 7.

9. A method for raising weaned piglets, characterized in that: The method comprises feeding the feed of claim 8 to the weaned piglets.

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