阅读说明：本技术 用于预防/治疗仔猪肠道损伤的egf/mmt复合物 (EGF/MMT compound for preventing/treating intestinal tract injury of piglets ) 是由 刘淑杰 徐子伟 齐珂珂 吴杰 于 2021-11-09 设计创作，主要内容包括：本发明提供一种用于预防/治疗仔猪肠道损伤的EGF/MMT复合物,该复合物通过如下方法制备获得：向pH值小于8的缓冲液中加入EGF和MMT或者金属基蒙脱石反应,取沉淀物,烘干；本发明制备EGF/MMT复合制剂,使之增强抵抗消化道不利因素的能力,并随吸附剂MMT“聚集”肠道黏膜损伤部位靶向结合受体,提高损伤部位EGF的稳定性和浓度,达到快速修复损伤肠道的目的,有效发挥EGF及MMT对肠道双重保护作用。(The invention provides an EGF/MMT compound for preventing/treating intestinal tract injury of piglets, which is prepared by the following method: adding EGF and MMT or metal-based montmorillonite into buffer solution with pH value less than 8 for reaction, taking precipitate, and drying; the EGF/MMT composite preparation prepared by the invention enhances the capability of resisting adverse factors of the digestive tract, and gathers' the targeting binding receptor of the injured part of the intestinal mucosa along with the MMT adsorbent, thereby improving the stability and concentration of the EGF at the injured part, achieving the aim of quickly repairing the injured intestinal tract and effectively playing the dual protection role of the EGF and the MMT on the intestinal tract.)

1. The EGF/MMT compound for preventing/treating intestinal tract injury of piglets is prepared by the following method: adding EGF and MMT or metal-based montmorillonite into buffer solution with pH value less than 8, reacting, collecting precipitate, and oven drying.

2. The EGF/MMT complex of claim 1, wherein said buffer solution is a disodium hydrogen phosphate-citric acid buffer or a glycine-hydrochloric acid buffer.

3. The EGF/MMT complex of claim 2, wherein the buffer has a pH of less than 4.6.

4. The EGF/MMT complex of claim 2, wherein the metal-based montmorillonite is Na-MMT and the mass ratio of EGF to Na-MMT is 1:1 x 10³-1×10⁶(ii) a Preferably, the mass ratio of EGF to Na-MMT is 1:2 × 10⁴-4×10⁴。

5. The EGF/MMT complex of claim 1, wherein the disodium hydrogen phosphate-citric acid buffer is prepared at a concentration of 0.05mol/LpH value of 3.8, EGF and Na-MMT are added to the buffer respectively, the mixture is shaken for 1 hour, centrifuged, and the precipitate is dried and ground into powder.

6. A feed additive for preventing/treating intestinal tract injury of piglets, which comprises the EGF/MMT complex of any one of claims 1-5.

7. A feed for preventing/treating intestinal injury of piglets, which comprises the EGF/MMT complex of any one of claims 1 to 5.

8. The EGF/MMT complex of any one of claims 1 to 5, for use in increasing gut length, modulating cytokine concentration in mice, inhibiting gut inflammation and maintaining gut functional barrier integrity response.

Technical Field

The invention belongs to the technical field of animal husbandry and veterinary medicine, and particularly relates to preparation of an EGF/MMT compound and application of the EGF/MMT compound in preventing/treating intestinal injury of weaned piglets.

Background

The early weaning technology of piglets is a key technology commonly adopted in modern large-scale pig farms, and has increasingly significant importance. However, the digestive function and immune system of the early weaned piglets are not completely developed, and the early weaned piglets cannot adapt to the sudden change of nutrition and external environmental factors, so that the intestinal health of the early weaned piglets is firstly harmed. Weaning stress easily causes intestinal tract injury of piglets, intestinal villus is broken off, crypt depth is increased, digestive enzyme activity is reduced, secretion changes of cytokines, antibodies and the like in an intestinal mucosa immune system finally cause reduction of production performance and disease resistance, diarrhea is often caused, even piglets die, and great economic loss is brought to pig raising production. The intestinal tract of animals is not only the place where nutrients are digested and absorbed, but also an important immune organ of the body. Therefore, the feed has the advantages of improving the intestinal structure and function, promoting the intestinal development of weaned pigs, improving the utilization rate of the feed and having important significance for solving the problem of early weaning stress syndrome of the piglets.

The epidermal growth factor is a powerful mitogen for cells, can stimulate cell division and proliferation, increase the synthesis of DNA and protein of epithelial tissues, and has the unique functions of promoting the growth and damage repair of gastrointestinal tissues and inducing the secretion of digestive enzymes of small intestine. The porcine epidermal growth factor (pEGF) is a single-chain polypeptide containing 53 amino acids, the molecular mass is 6.05kDa, and breast milk is a main source of intestinal pEGF in the lactation period of animals. The basic biological effects of pEGF are as follows: (1) promoting the proliferation and differentiation of gastrointestinal epithelial cells; (2) increasing blood flow of gastrointestinal mucosa, relieving gastrointestinal tract injury, and inhibiting gastric acid secretion; (3) increase synthesis and secretion of gastric mucosa mucus glycoprotein, promote the repair of damaged epithelial tissue; (4) inducing the expression of digestive enzyme at the brush border of the mucous membrane of the small intestine and improving the digestion and utilization of nutrients.

The weaning at early stage leads the piglet to suddenly interrupt milk-derived EGF, and at the moment, the exogenous EGF is supplied by an exogenous method, so that the deficiency of the endogenous EGF can be effectively made up, and the feed has an important effect on the intestinal development of the piglet. However, at present, EGF is delivered directly to piglets, mainly by the oral route, and the effect is not stable. Some researches show that the ratio of the jejunum villus height/crypt depth of weaned pigs supplemented with EGF and the content of mucosal protein are not changed, and the intestinal structure, function and growth performance of the weaned pigs are not obviously improved. The key reason is that EGF must meet two requirements, namely, EGF is stably present in the digestive tract, and EGF in a sufficient dosage reaches the intestinal tract and is combined with intestinal mucosal receptors to exert the function. However, after oral administration of EGF to piglets, EGF enters the extreme environment of the digestive tract, part of EGF is possibly degraded into an inactive small molecular form by gastric acid, digestive enzymes and the like, or is mixed with a large amount of chyme and then excreted out of the body along with feces, the probability of targeted binding of EGF to intestinal receptors is greatly reduced, and increasing the oral dose of EGF will undoubtedly increase the cost. Therefore, there is a need to find a superior intestinal delivery vehicle, which can protect EGF from the extreme environment of the digestive tract and can deliver EGF to the intestinal tract for release in combination with the receptor, thereby maximizing its efficacy.

MMT is a 2: 1-type layered silicate mineral composed of two sheets of silica tetrahedra and a sheet of aluminum (magnesium) oxy (hydroxy) octahedra sandwiched therebetween. Si in silicon-oxygen tetrahedron⁴⁺Can be coated with Al³⁺Alternatively, Al in the octahedral sheet of aluminum³⁺Can be coated with Mg²⁺、Zn²⁺、Mn²⁺Or Fe²⁺And replacement, thereby generating permanent negative charges between crystal layers and having ion exchange characteristics. The MMT has fine particles (the particle size is less than 2pm), and the MMT is generally formed by stacking thousands of result unit layers, and the special structure ensures that the MMT has larger specific surface area, can uniformly cover the whole intestinal mucosa surface and is combined with mucosal protein, thereby playing the role of protecting and repairing the intestinal mucosa. In addition, the MMT also has excellent adsorption property and good nano-space structure, so that the MMT becomes an excellent drug delivery carrier, can protect the drug from being damaged by bad factors of the digestive tract, particularly can be concentrated and slowly released at a focus part to achieve targeted delivery and improve local deliveryTarget drug concentration. Protein molecules can enter the MMT sheet layer through ion exchange reaction, intercalation reaction and the like depending on negatively charged acidic amino acid groups and positively charged basic amino acid groups, and can also adsorb the surface of MMT particles. The EGF/MMT composite preparation prepared by using the MMT as the carrier can enhance the capability of resisting adverse factors of the digestive tract, realizes targeted delivery, slow release and accurate regulation and control along with the accumulation of the adsorbent MMT on a targeted binding receptor of an intestinal mucosa injury part, and is used for repairing intestinal injury of weaned piglets under the 'banning' background, resisting diarrhea and improving the production performance.

Disclosure of Invention

Aiming at the special environment of the piglet digestive tract, the EGF/MMT composite preparation prepared by the invention enhances the capability of resisting adverse factors of the digestive tract, and is combined with receptors in a targeting way along with the aggregation of the adsorbent MMT on the injured part of the intestinal mucosa, so that the stability and the concentration of the EGF at the injured part are improved, the aim of quickly repairing the injured intestinal tract is fulfilled, and the dual protection effect of the EGF and the MMT on the intestinal tract is effectively exerted.

In a first aspect, the present invention provides an EGF/MMT complex for preventing/treating intestinal tract injury of piglets, which is prepared by the following method: adding EGF and MMT or metal-based montmorillonite into buffer solution with pH value less than 8, reacting, collecting precipitate, and oven drying.

The EGF/MMT compound prepared by the invention has good stability, can be delivered to the injured part of the intestinal mucosa in a targeted manner to play a role, reduces the use dosage of EGF, and is safe and simple to prepare.

In some embodiments, the buffer solution is a disodium phosphate-citric acid buffer.

In some embodiments, the pH of the buffer is less than 4.6.

In some embodiments, the metal-based montmorillonite is Na-MMT, and the mass ratio of EGF to Na-MMT is 1:1 × 10³-1×10⁶(ii) a Preferably, the mass ratio of EGF to Na-MMT is 1:2 × 10⁴-4×10⁴。

In some preferred embodiments, the complex is prepared by the following method: preparing disodium hydrogen phosphate-citric acid buffer solution with concentration of 0.05mol/LpH value of 3.8, adding EGF and Na-MMT into the buffer solution respectively, shaking for 1h, centrifuging, drying precipitate, and grinding into powder.

In a second aspect, the present invention provides a feed additive for preventing/treating intestinal tract injury of piglets, which comprises the EGF/MMT complex as described above.

In a third aspect, the invention provides a feed for preventing/treating intestinal tract injury of piglets, which comprises the EGF/MMT complex as described above.

In a fourth aspect, the EGF/MMT compound is applied to increase the intestinal length of piglets, regulate the concentration of mouse cytokines, inhibit intestinal inflammation and maintain the intestinal function barrier integrity reaction.

The modified montmorillonite is used for adsorbing and preparing the EGF/MMT composite preparation, so that the capability of resisting adverse factors of the digestive tract is enhanced, the targeting binding receptor of the injured part of the intestinal mucosa is gathered along with the adsorbent MMT, the stability and the concentration of the EGF at the injured part are improved, the purpose of preventing and quickly repairing the injured intestinal tract is achieved, and the dual protection effects of the EGF and the MMT on the intestinal tract are effectively exerted.

In addition, the successful research and development of the MMT/EGF compound is expected to solve the problems of intestinal injury, diarrhea, reduction of production performance and the like of weaned piglets in the current 'resistance banning' environment, has important significance and practical value for improving the health condition of the piglets and the economic benefit of a pig farm, and can replace antibiotics to be used for pig intestinal health regulation and control.

Drawings

FIG. 1 shows the adsorption of EGF by MMT at different reaction pH values;

FIG. 2 shows the adsorption amounts of EGF by MMT at different reaction times;

FIG. 3 shows the adsorption of EGF by MMT at different mass ratios;

FIG. 4 is a FT-IR spectrum of MMT and EGF/MMT complex;

FIG. 5 is a TG curve of MMT and EGF/MMT complex;

FIG. 6 is a pathological colon tissue map of mice in each group.

Detailed Description

The following detailed description of the invention is provided in connection with the accompanying drawings. The EGF, MMT and related chemicals, etc., used in the following examples, as well as experimental procedures not specifically noted, were carried out routinely or under conditions recommended by commercial suppliers.

Reaction System optimization and preparation of EGF/MMT composite the following examples used montmorillonites which were all sodium based montmorillonites (Na-MMT).

Example 1

Determining the appropriate pH value of the reaction system: respectively preparing disodium hydrogen phosphate-citric acid buffer solutions with the concentration of 0.05mol/LpH and the values of 3.0, 3.8, 4.6, 6.2, 7.0 and 8.0, respectively adding 6mg of EGF and 100mg of Na-MMT into 20mL of the buffer solutions, then oscillating for 1.5h, centrifuging, taking out supernate, washing precipitates with deionized water, combining the supernate, measuring the protein content in the supernate by using a BCA kit, and determining the optimum pH of the reaction to be 3.8 according to the complete reaction and the stomach environment of piglets.

To illustrate, referring to FIG. 1, under the conditions of the reaction system pH3.0-4.6, Na-MMT reacts almost completely with EGF, and the adsorption amount of Na-MMT to EGF is about 100%; the adsorption amount of Na-MMT to EGF is reduced under the condition of pH6.2-8.0.

Example 2

Determination of the appropriate reaction time: preparing a disodium hydrogen phosphate-citric acid buffer solution with the concentration of 0.05mol/L and the pH value of 3.8, adding 6mg of EGF and 100mg of Na-MMT into 20mL of the buffer solution, then shaking for 0.5, 1, 2, 3, 4, 5, 6 and 22 hours respectively, centrifuging, taking out a supernatant, washing precipitates with deionized water, combining the supernatants, and determining the protein content in the supernatant by using a BCA kit. An appropriate reaction time was determined to be 1 h.

It is shown that, referring to FIG. 2, both EGF and Na-MMT can be completely reacted at 0.5-22h, and the amount of adsorbed Na-MMT to EGF is about 100%, considering the reaction completeness and process simplification, 1h is selected.

Example 3

Determining the appropriate mass ratio of EGF to Na-MMT: respectively preparing disodium hydrogen phosphate-citric acid buffer solution with the concentration of 0.05mol/LpH and the value of 3.8, adding 100mg of Na-MMT into 20mL of buffer solution, respectively adding EGF according to the mass ratio of 1:10, 1:20, 1:40, 1:60, 1:80 and 1:100 of EGF to Na-MMT, then shaking for 1h, centrifuging, taking out supernate, washing precipitates with deionized water, combining the supernate, and measuring the protein content in the supernate by using a BCA kit, wherein the concentration ratio can completely react.

It is shown that, referring to FIG. 3, EGF and Na-MMT were completely reacted at a mass ratio of 1:10, 1:20, 1:40, 1:60, 1:80 and 1:100, and adsorbed amount of Na-MMT to EGF was about 100%.

EXAMPLE 4EGF/MMT Complex preparation

Through the optimization of the conditions of examples 1-3, a disodium hydrogen phosphate-citric acid buffer solution with the concentration of 0.05mol/LpH value of 3.8 is prepared, 10mg of EGF and 100mg of Na-MMT are respectively added into the buffer solution, then the solution is shaken for 1h, centrifuged, the precipitate is washed by deionized water, and after the precipitate is dried for 36h at 55 ℃, the precipitate is ground to the particle size of less than 0.074mm, and the EGF/MMT compound is obtained.

The FT-IR test of the EGF/MMT complex shows that the FT-IR spectrum of the EGF/MMT complex has an amide band characteristic peak with a wave number of 2000-1000/cm and has characteristic peaks of MMT and EGF as shown in FIG. 4.

The EGF/MMT compound is subjected to TG test, as shown in figure 5, in a TG curve, the weight loss ratio of the EGF is larger, the weight loss of the EGF is improved by the EGF/MMT compound, the EGF stability is improved, and the EGF/MMT compound is not easily degraded in the preparation and drying process.

Test example 1EGF/MMT in vitro Release test

Weighing 100mg of the EGF/MMT compound obtained in example 4, adding 3mL of buffer solution with the pH values of 3.8, 7, 8 and 9 respectively, placing the sample in a constant-temperature shaking table at 37 ℃ and 150r/min, taking out the sample at 1, 3 and 5h respectively, centrifuging at 3000r/min for 10min, taking 200uL of supernatant, rapidly supplementing a new medium, and determining the content of EGF in the supernatant by using a BCA kit; as shown in Table 1 below, the EGF/MMT complex releases almost no EGF at pH3.8 and releases EGF at pH7-9 with sustained release.

TABLE 1EGF/MMT complex in vitro release at different pH systems

As is well known, the pH in the stomach of piglets is about 3.8, and the pH in the intestine is about 7, 8, or between 7 and 8; in contrast, in the release system with the pH value of 3.8, the EGF/MMT complex in the example 4 hardly releases EGF, and can release EGF in a sustained manner at the pH value of 7-9, which indicates that the EGF/MMT complex hardly releases EGF in the stomach of the piglet and can release EGF in a sustained manner when reaching the intestinal tract; solves the technical problem that the EGF is degraded into an inactive micromolecule form by gastric acid, digestive enzyme and the like after entering the extreme environment of the digestive tract after being orally taken by piglets.

Test example 2 repair of the MMT/EGF Complex on injured intestinal tract

Experimental materials and methods:

72 female healthy BALB/C mice (clean grade, provided by animal experiments center of the institute of medical sciences, Zhejiang); magnetic stirrers (IKA, germany); paraffin microtomes (Leica, germany), fluorescence microscope cameras (BX 20, OLYMPUS, japan), automatic dehydrators (Thermo shandon, uk), and microplate readers (biotech, usa).

Female healthy BALB/C mice 72, divided into 6 groups. Blank control group (group I), DSS group (group II), MMT group (group III), EGF/MMT low dose group (group IV), EGF/MMT medium dose group (group V), EGF/MMT high dose group (group VI); the EGF/MMT complex preparation method of each dose group is as shown in the table 1, except that the EGF/MMT mass ratio is different, and the EGF/MMT mass ratio of the EGF/MMT low dose group (group IV), the EGF/MMT medium dose group (group V) and the EGF/MMT high dose group (group VI) is shown in the table 1; the treatment of each group in this example is shown in Table 2.

Table 2 mouse trial grouping and handling

Group of	Drinking water treatment	Gavage treatment	MMT/EGF Complex ratio
				Group I	Water (W)	Water (W)	—
Group II	3％DSS	Water (W)	—
				Group III	3％DSS	1g/kg of aqueous MMT solution	—
Group IV	3％DSS	1g/kg EGF/MMT aqueous solution	EGF:MMT＝1:3.3×105
				Group V	3％DSS	1g/kg EGF/MMT aqueous solution	EGF:MMT＝1:3.3×104
Group VI	3％DSS	1g/kg EGF/MMT aqueous solution	EGF:MMT＝1:8.3×103

Group II-group VI all mice drunk 3% DSS daily on days 1-9 and purified water on days 10-16; mice in group I had free access to purified water on days 1-16. On days 1-9, group I and group II were infused with 100ul of purified water every 1 day, group III was infused with 100ul of MMT aqueous solution every 1 day, and group IV-group VI were infused with 100ul of different doses of EGF/MMT aqueous solution every 1 day. On days 10-16 of the experiment, each group of mice was administered their respective gavage treatments daily. On the morning of day 0, 16, all mice were weighed on an empty stomach. On day 16, sampling blood from the infraorbital vein, separating serum, and measuring the DAO activity and D-LAC concentration of the serum; the mice were sacrificed by cervical dislocation, the abdominal cavity was opened along the midline of the abdomen, colon tissue was taken out, and the length of the colon from the cecum end to the anus was measured; taking 1cm of colon tissue, putting the colon tissue into 10% neutral formaldehyde solution, carrying out paraffin embedding on the sample, then slicing, carrying out HE staining, and carrying out histopathological analysis and colon inflammatory scoring; and accurately weighing the weight of the residual colon of the mouse, adding sterilized normal saline, mechanically homogenizing, centrifuging to obtain supernatant, and analyzing the level of the intestinal tract cell factor and the concentration of the tight junction protein.

As shown in Table 3, the colon length was significantly reduced in each group of mice as compared with the blank control group, but the colon length was higher in the EGF/MMT medium and high dose groups than in the DSS and MMT groups, respectively. The colonic inflammation score results show that the colonic inflammation score of mice in the DSS group and the MMT group is significantly higher than that in the blank control group, while the inflammatory score of EGF/MMT low, medium and high dose groups is significantly lower than that in the DSS group and the MMT group, and is not significantly different compared with that in the blank control group.

Table 3 shows the colon length and inflammatory score of each group of mice

The HE staining result shows (fig. 6) that the colon tissue of the blank control group has complete structure of each layer, the mucosal epithelial cells are complete, and inflammatory cell infiltration, ulcer, tumor and the like are not seen; the structures of all layers of the colon of the DSS group exist, a large amount of inflammatory cells are infiltrated in the inherent layer and the submucosa, even lymph follicles are formed, glands are atrophied to different degrees, and ulcers, tumors and the like are not seen; each layer of the colon in the MMT group has clear structure, no obvious ulcer is seen, and more inflammatory cells in the inherent layer of the mucous membrane are infiltrated; mucosal epithelium is slightly hyperplastic, etc.; the colon layers of EGF/MMT dose groups are clear in structure and free from obvious ulcer, the low dose groups only see a small amount of inflammatory cell infiltration in the mucosa lamina propria, the medium dose groups occasionally see a small amount of inflammatory cell infiltration, and the high dose groups occasionally see a small amount of inflammatory cell infiltration.

In addition, the concentration of IL-10 was very significantly reduced in the DSS group (P <0.01) and the concentrations of TNF- α, IFN- γ and IL-6 were very significantly increased (P <0.01) compared to the blank control group (Table 4). Compared with the DSS group, the MMT group had a significantly increased IL-10 concentration (P <0.05) and a very significantly decreased TNF- α concentration (P <0.01) (table 4). Compared with the DSS group, the concentrations of TNF-alpha, IFN-gamma and IL-6 in the EGF/MMT low and medium dose groups are very significantly reduced (P <0.01), the concentration of IL-10 in the low dose group is very significantly increased (P <0.01), and the concentration of IL-10 in the medium dose group is significantly increased (P < 0.05). The IL-6 concentration was significantly lower in the high dose group than in the DSS group (P <0.05) (table 4). These show that the DSS group mice have shortened intestinal tracts, have different degrees of damage to colon tissues and induce inflammatory response, and the EGF/MMT compound can increase the length of the intestinal tracts, regulate the concentration of cytokines to restore the normal level, inhibit the inflammatory response of the intestinal tracts and have certain protection and damage repair effects on the intestinal tracts.

TABLE 4 Colon cytokine concentrations in groups of mice

The results of colon permeability tests (Table 5) show that compared with the blank control group, the DSS group has a very significant increase in the serum DAO activity and D-LAC concentration (P <0.01), and the colon claudin-1 and occludin concentrations are very significantly reduced (P < 0.01). Compared with the DSS group, the concentration of claudin-1 in the MMT group is obviously increased (P <0.05), and the concentration of occludin is extremely obviously increased (P < 0.01). Compared with the DSS group, the DAO activity and the D-LAC concentration of the EGF/MMT low and medium dose groups are extremely remarkably reduced (P <0.01), and the concentrations of claudin-1 and occludin are extremely remarkably increased (P < 0.01). The colonic occludin concentration was very significantly elevated in the high dose group compared to the DSS group (P < 0.01). These indicate that DSS increases intestinal permeability in mice, disrupting intestinal functional barrier integrity, while EGF/MMT complex significantly decreases intestinal mucosal permeability, protecting intestinal functional barrier integrity. In combination, the dose effect is better in EGF/MMT.

TABLE 5 serum DAO Activity, D-LAC concentration and Colon claudin concentration in groups of mice