CN113813369B - EGF/MMT complex for preventing/treating intestinal injury of piglets - Google Patents
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Abstract
The invention provides an EGF/MMT compound for preventing/treating piglet intestinal injury, which is prepared by the following method: adding EGF and MMT or metal-based montmorillonite into buffer solution with pH value less than 8 to react, taking precipitate, and drying; the EGF/MMT composite preparation is prepared, so that the capability of resisting adverse factors of the digestive tract is enhanced, the target binding receptor along with the damaged part of the intestinal mucosa is 'aggregated' by the adsorbent MMT, the stability and concentration of the EGF at the damaged part are improved, the aim of rapidly repairing the damaged intestinal tract is fulfilled, and the dual protection effect of the EGF and the MMT on the intestinal tract is effectively exerted.
Description
Technical Field
The invention belongs to the technical field of livestock 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 pigs.
Background
The early weaning technology of piglets is a key technology commonly adopted in modern large-scale pig farms, and the importance of the technology is increasingly remarkable. However, the digestive function and immune system of early weaned pigs are not yet developed completely, and the early weaned pigs cannot adapt to sudden changes of nutrition and external environmental factors, and the early weaning firstly damages the intestinal health of the early weaned pigs. Weaning stress is very easy to cause intestinal injury of piglets, intestinal villi is broken, crypt depth is increased, digestive enzyme activity is reduced, and cytokines, antibodies and the like in an intestinal mucosa immune system are changed, so that production performance is reduced, disease resistance is reduced, diarrhea is often caused, even piglets die, and great economic loss is brought to pig raising production. The animal intestinal tract is not only the place for digestion and absorption of nutrient substances, but also an important immune organ of the organism. Therefore, the feed has the advantages of improving the structure and the function of intestinal tracts, promoting the intestinal development of weaned pigs, improving the utilization rate of feed and having great significance for solving the problem of early weaning stress syndrome of the pigs.
The epidermal growth factor is a powerful cell mitogen, can stimulate cell division and proliferation, increases DNA and protein synthesis of epithelial tissues, and has the unique effects of promoting gastrointestinal tissue growth and injury repair and inducing small intestine to secrete digestive enzymes. Pig epidermal growth factor (pEGF) is a single-chain polypeptide containing 53 amino acids, with a molecular mass of 6.05kDa, and breast milk is the main source of intestinal pEGF in mammalian stages. The basic biological effects of pEGF are as follows: (1) promoting proliferation and differentiation of gastrointestinal epithelial cells; (2) Increasing gastrointestinal mucosa blood flow, relieving gastrointestinal damage, and inhibiting gastric acid secretion; (3) Increase synthesis and secretion of gastric mucosa mucus glycoprotein, promote repair of damaged epithelial tissue; (4) Inducing the expression of brush-like edge digestive enzyme of small intestinal mucosa, and improving the digestion and utilization of nutrients.
The early weaning makes the piglet suddenly interrupt the milk-derived EGF, and the deficiency of the endogenous EGF can be effectively compensated by the exogenous method supply at the moment, thereby having an important effect on the intestinal development of the piglet. However, EGF is mainly delivered to piglets directly by the oral route at present, and the effect is not stable. Some researches show that the jejunum and the crypt depth ratio and the mucous membrane protein content of the weaned piglet for supplementing EGF are not changed, and the intestinal structure, the function and the growth performance of the weaned piglet are not obviously improved. The key reason is that EGF must meet two requirements, one is that EGF exists stably in the digestive tract, and the other is that EGF with enough dosage reaches the intestinal tract and can be combined with intestinal mucosa receptor to perform the function. However, EGF enters the extreme environment of the digestive tract after being orally taken by piglets, part of EGF can be degraded into an inactive micromolecular form by gastric acid, digestive enzymes and the like, or the EGF is mixed with a large amount of chyme and discharged out of the body along with feces, so that the probability of targeting the EGF to bind to intestinal receptors is greatly reduced, and the increase of the oral dosage of EGF can definitely increase the cost. Therefore, there is a need for an advantageous enteric delivery vehicle that protects EGF from the extreme environment of the digestive tract and delivers EGF to the intestinal tract for release in combination with a receptor to maximize efficacy.
MMT is a 2:1 layered silicate mineral consisting of two layers of silicon oxygen tetrahedral sheets and one layer of aluminum (magnesium) oxygen (hydroxy) octahedral sheet sandwiched therebetween. Si in silicon oxygen tetrahedra 4+ Can be coated with Al 3+ Replacement of Al in AlO octahedral sheets 3+ Can be coated with Mg 2+ 、Zn 2+ 、Mn 2+ Or Fe (Fe) 2+ And replaced, thereby generating permanent negative charges between crystal layers and having ion exchange characteristics. MMT particles are tiny (particle size is smaller than 2 pm), and are usually stacked by thousands of result unit layers, and the special structure enables MMT to have a larger specific surface area, so that MMT can uniformly cover the whole intestinal mucosa surface and combine with mucosa proteins, and the intestinal mucosa is protected and repaired. In addition, MMT has excellent adsorption property and nano space structure, so that the MMT becomes excellent drug deliveryThe carrier can protect the medicine from being damaged by digestive tract bad factors, and especially can concentrate and release the medicine slowly at focus positions, thereby achieving the purposes of targeted delivery and improving local medicine concentration. Protein molecules can enter MMT sheets by means of ion exchange reaction, intercalation reaction and the like depending on negatively charged acid amino acid groups and positively charged basic amino acid groups, and can also adsorb the surfaces of MMT particles. The EGF/MMT composite preparation prepared by taking the MMT as a carrier can enhance the capability of resisting adverse factors of the digestive tract, and can be targeted to bind with a receptor along with the aggregation of the MMT as an adsorbent at the damaged part of the intestinal mucosa, thereby realizing targeted delivery, slow release and precise regulation, and being used for repairing the intestinal damage of weaned pigs, resisting diarrhea and improving the production performance under the 'forbidden' background.
Disclosure of Invention
Aiming at the special environment of the digestive tract of piglets, the EGF/MMT composite preparation is prepared, so that the capability of resisting adverse factors of the digestive tract is enhanced, the EGF/MMT composite preparation is combined with a receptor in a targeted manner along with the injured part of intestinal mucosa by the MMT of an adsorbent, the stability and concentration of the EGF at the injured part are improved, the aim of rapidly 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 use in the prevention/treatment of intestinal injury in piglets, the complex being prepared by the following method: adding EGF and MMT or metal-based montmorillonite into buffer solution with pH value less than 8 to react, taking precipitate, and drying.
The EGF/MMT compound prepared by the method disclosed by the invention has good stability, can be delivered to the damaged part of intestinal mucosa in a targeted manner to play a role, reduces the dosage of EGF, and is safe and simple to prepare.
In some embodiments, the buffer solution is disodium hydrogen phosphate-citrate 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 1:1×10 3 -1×10 6 The method comprises the steps of carrying out a first treatment on the surface of the Preferably, the mass ratio of EGF to Na-MMT is 1:2×10 4 -4×10 4 。
In some preferred embodiments, the complex is prepared by the following method: preparing disodium hydrogen phosphate-citric acid buffer solution with the concentration of 0.05mol/LpH value of 3.8, respectively adding EGF and Na-MMT into the buffer solution, oscillating for 1h, centrifuging, drying the precipitate, and grinding into powder.
In a second aspect, the present invention provides a feed additive for preventing/treating intestinal lesions of piglets, comprising the EGF/MMT complex as described above.
In a third aspect, the present invention provides a feed for preventing/treating intestinal damage in piglets, the feed comprising an EGF/MMT complex as described above.
In a fourth aspect, the invention applies the EGF/MMT complex described above to increase piglet gut length, regulate mouse cytokine concentration, inhibit gut inflammation and maintain gut functional barrier integrity response.
According to the invention, the EGF/MMT composite preparation is prepared by modified montmorillonite adsorption, so that the capability of resisting adverse factors of digestive tracts is enhanced, and the EGF/MMT composite preparation is targeted to bind with a receptor along with the damaged part of intestinal mucosa by the MMT adsorbent, so that the stability and concentration of the EGF at the damaged part are improved, the purposes of preventing and rapidly repairing damaged intestinal tracts are achieved, and the dual protection effect of the EGF and the MMT on the intestinal tracts is effectively exerted.
In addition, the successful research and development of the MMT/EGF compound is expected to solve the problems of intestinal injury, diarrhea, production performance reduction and the like of weaned pigs in the current 'forbidden' environment, has important significance and practical value for improving the health condition of the pigs and the economic benefit of a pig farm, and can replace antibiotics for regulating and controlling the intestinal health of the pigs.
Drawings
FIG. 1 shows the adsorption amount of MMT to EGF at different reaction pH values;
FIG. 2 shows the adsorption amount of EGF by MMT at different reaction times;
FIG. 3 shows the adsorption amount of MMT to EGF at different mass ratios;
FIG. 4 is a FT-IR spectrum of MMT and EGF/MMT complexes;
FIG. 5 shows TG curves of MMT and EGF/MMT complexes;
FIG. 6 is a graph showing colon histopathology in each group of mice.
Detailed Description
The invention will be described in detail with reference to the following specific examples and figures. EGF, MMT and related chemicals, etc. used in the examples below were carried out as usual or as recommended by commercial suppliers, without specifying the specific conditions.
Reaction System optimization and preparation of EGF/MMT Complex the montmorillonite used in the following examples was sodium montmorillonite (Na-MMT).
Example 1
Determining the proper pH value of the reaction system: disodium hydrogen phosphate-citric acid buffer solutions with the concentration of 0.05mol/LpH value of 3.0, 3.8, 4.6, 6.2, 7.0 and 8.0 are respectively prepared, 6mg EGF and 100mgNa-MMT are respectively added into 20mL buffer solution, then oscillation is carried out for 1.5 hours, centrifugation is carried out, supernatant is taken out, precipitate is washed by deionized water, supernatant is combined, protein content in the supernatant is measured by adopting a BCA kit, and the optimal pH value of the reaction is determined according to the complete reaction and the stomach environment of piglets.
Illustratively, referring to FIG. 1, na-MMT reacts almost completely with EGF at a reaction system pH of 3.0-4.6, and the adsorption amount of Na-MMT to EGF is about 100%; the adsorption amount of the Na-MMT to EGF is reduced at pH 6.2-8.0.
Example 2
Determination of the appropriate reaction time: disodium hydrogen phosphate-citric acid buffer solution with the concentration of 0.05mol/LpH value of 3.8 is prepared, 6mg EGF and 100mg Na-MMT are added into 20mL buffer solution, then 0.5, 1, 2, 3, 4, 5, 6 and 22 hours are respectively oscillated, the supernatant is taken out after centrifugation, the precipitate is washed by deionized water, the supernatant is combined, and the protein content in the supernatant is measured by using a BCA kit. The appropriate reaction time was determined to be 1h.
Illustratively, referring to FIG. 2, EGF and Na-MMT can be completely reacted at a reaction time of 0.5 to 22 hours, and the adsorption amount of Na-MMT to EGF is about 100%, and 1 hour is selected in consideration of reaction completeness and process simplification.
Example 3
Determining the appropriate mass ratio of EGF and Na-MMT: disodium hydrogen phosphate-citric acid buffer solution with the concentration of 0.05mol/LpH value of 3.8 is respectively prepared, 100mgNa-MMT is added into 20mL of buffer solution, EGF is respectively added according to the mass ratio of EGF to Na-MMT of 1:10, 1:20, 1:40, 1:60, 1:80 and 1:100, then vibration is carried out for 1h, centrifugation is carried out, supernatant is taken out, precipitate is washed by deionized water, supernatant is combined, protein content in the supernatant is measured by adopting a BCA kit, and the concentration ratios can be fully reacted.
Illustratively, referring to FIG. 3, EGF and Na-MMT may be fully reacted at mass ratios of 1:10, 1:20, 1:40, 1:60, 1:80, and 1:100, with the Na-MMT adsorbing about 100% of EGF.
Example 4EGF/MMT Complex preparation
Through condition optimization of examples 1-3, 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 vibration is carried out for 1h, centrifugation is carried out, precipitate is washed by deionized water, and after the precipitate is dried at 55 ℃ for 36h, the precipitate is ground to the particle size of less than 0.074mm, and EGF/MMT compound is obtained.
The EGF/MMT complex was subjected to FT-IR test, and the result is shown in FIG. 4, wherein the EGF/MMT complex has FT-IR spectrum, and the wave number of the EGF/MMT complex is 2000-1000/cm, and has amide band characteristic peaks with characteristic peaks of MMT and EGF.
The EGF/MMT complex was subjected to TG test, as shown in FIG. 5, in which the EGF weight loss ratio was larger, and the EGF/MMT complex improved the EGF weight loss and the EGF stability, so that the EGF/MMT complex was not easily degraded during the preparation and drying process.
Test example 1EGF/MMT in vitro Release test
Weighing 100mg of EGF/MMT complex obtained in example 4, adding 3mL of buffer solution with pH 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, quickly supplementing new medium, and measuring the EGF content of the supernatant by using a BCA kit; as shown in Table 1 below, the EGF/MMT complex releases little EGF at pH3.8 and releases EGF slowly at pH 7-9.
Table 1 shows the in vitro release of EGF/MMT complexes in different pH systems
As is well known, the pH in the stomach of piglets is about 3.8, the pH in the intestine is about 7, 8, or between 7 and 8; the EGF/MMT compound of example 4 can release little EGF at pH value of 3.8, and release EGF slowly at pH value of 7-9, which shows that EGF is hardly released in stomach of piglet and reaches intestinal tract; solves the technical problem that the existing EGF is degraded into an inactive micromolecular form by gastric acid, digestive enzymes and the like after entering the extreme environment of the digestive tract after being orally taken by piglets.
Test example 2 repair of injured intestinal tract by MMT/EGF Complex
Experimental materials and methods:
female healthy BALB/C mice 72 (clean grade, provided by animal experiment center, national academy of sciences of medicine, zhejiang); magnetic stirrer (IKA company, germany); paraffin microtomes (Leica company, germany), fluorescence microscope cameras (OLYMPUS company BX20 type japan), automatic dehydrators (Thermo shandon company, uk), enzyme labelling machines (biotech company, usa).
Female healthy BALB/C mice were 72 and divided into 6 groups. Blank (group I), DSS (group II), MMT (group III), EGF/MMT low dose (group IV), EGF/MMT medium dose (group V), EGF/MMT high dose (group VI); whereas the EGF/MMT complex preparation method for each dose group was as described in example 4, the difference was only that the EGF/MMT mass ratio was different, and the EGF/MMT mass ratios in the EGF/MMT complexes corresponding to the EGF/MMT low dose group (group IV), the EGF/MMT dose group (group V), and the EGF/MMT high dose group (group VI) are shown in Table 1; the treatments for each group in this example are shown in table 2.
Table 2 mice test grouping and treatment
| Group of | Treatment of drinking water | Gastric lavage treatment | MMT/EGF complex ratio |
| Group I | Water and its preparation method | Water and its preparation method | — |
| Group II | 3％DSS | Water and its preparation method | — |
| Group III | 3％DSS | 1g/kg MMT aqueous solution | — |
| Group IV | 3％DSS | 1g/kg EGF/MMT aqueous solution | EGF:MMT＝1:3.3×10 5 |
| Group V | 3％DSS | 1g/kg EGF/MMT aqueous solution | EGF:MMT＝1:3.3×10 4 |
| Group VI | 3％DSS | 1g/kg EGF/MMT aqueous solution | EGF:MMT＝1:8.3×10 3 |
Group II-group VI all mice were given 3% DSS daily on days 1-9 and purified water on days 10-16; group I mice were free to drink purified water on days 1-16. On days 1-9, groups I and II were filled with 100ul purified water every 1 day, group III was filled with 100ul MMT aqueous solution every 1 day, and groups IV-VI were filled with 100ul different doses of EGF/MMT aqueous solution every 1 day. On days 10-16 of the test, each group of mice was changed to be perfused daily with the respective lavage treatments. On day 0, 16 morning, all mice were weighed on an empty stomach. On day 16, blood was collected from the infraorbital vein, serum was isolated, and serum DAO activity and D-LAC concentration were determined; killing a mouse by cervical dislocation, opening an abdominal cavity along an abdominal midline, taking out colon tissues, and measuring the colon length from the cecum end to the anus; taking 1cm colon tissue, placing the colon tissue into 10% neutral formaldehyde solution, embedding a sample in paraffin, slicing, HE staining, and performing histopathological analysis and colitis scoring; and in addition, accurately weighing the residual colon weight of the mouse, adding sterilized normal saline, mechanically homogenizing, centrifuging to obtain supernatant, and analyzing the level of intestinal cytokines and the concentration of the tight junction protein.
The results are shown in Table 3, where the colon length was significantly reduced in each group compared to the blank, but the colon length was higher in EGF/MMT and in the high dose group than in the DSS group and the MMT group, respectively. The colitis scoring results showed that the colitis scores were significantly higher in the DSS and MMT groups than in the placebo group, whereas the EGF/MMT low, medium and high dose groups were significantly lower in the inflammatory scores than in the DSS and MMT groups, whereas the differences were not significant compared to the placebo group.
Table 3 shows colon length and inflammatory scores for each group of mice
HE staining results show (figure 6), the colon tissue of the blank group has complete structure of each layer, the mucous membrane epithelial cells are complete, and inflammatory cell infiltration, ulcer, tumor and the like are not found; the structure of each layer of colon of DSS group still exists, a large amount of inflammatory cells can infiltrate in the lamina propria and submucosa, even form lymphatic follicles, gland atrophy of different degrees is not seen, ulcer, tumor and the like; the structure of each layer of colon of MMT group is clear, no obvious ulcer is seen, and more inflammatory cells infiltrate in the mucosa intrinsic layer; the mucous epithelium has a little hyperplasia, etc.; the colon of each dose group of EGF/MMT has clear structure, no obvious ulcer is seen, the low dose group only has small inflammatory cell infiltration in the mucosa lamina propria, the medium dose group has small inflammatory cell infiltration occasionally, the high dose group has small inflammatory cell infiltration occasionally, and the like.
In addition, the DSS group showed very significant decrease in IL-10 concentration (P < 0.01) and very significant increase in TNF- α, IFN- γ and IL-6 concentrations (P < 0.01) compared to the placebo group (table 4). Compared to DSS group, MMT group IL-10 concentration was significantly increased (P < 0.05), TNF- α concentration was extremely significantly decreased (P < 0.01) (table 4). EGF/MMT low and medium dose groups showed very significant reductions in TNF-alpha, IFN-gamma and IL-6 concentrations (P < 0.01) compared to the DSS group, low dose groups showed very significant increases in IL-10 concentration (P < 0.01), and medium dose groups showed very significant increases in IL-10 concentration (P < 0.05). The high dose group had significantly lower IL-6 concentration than the DSS group (P < 0.05) (table 4). These show that DSS mice have shortened intestinal tract, colon tissue with different damage degree and inflammatory reaction, and EGF/MMT compound can increase intestinal tract length, regulate cytokine concentration to restore normal level, inhibit intestinal inflammatory reaction and protect intestinal tract and repair intestinal tract.
TABLE 4 colon cytokine concentrations in groups of mice
The results of the colon permeability test showed (Table 5) that the serum DAO activity and D-LAC concentration of DSS group were significantly increased (P < 0.01) and the concentration of colon claudin-1 and occludin were significantly decreased (P < 0.01) compared with the blank group. Compared to DSS group, the MMT group had significantly elevated claudin-1 concentration (P < 0.05) and the occludin concentration (P < 0.01). Compared with the DSS group, the EGF/MMT low and medium dose groups have extremely significantly reduced DAO activity and D-LAC concentration (P < 0.01), and the claudin-1 and occludin concentrations are extremely significantly increased (P < 0.01). The high dose group showed a very significant increase in colonic occludin concentration (P < 0.01) compared to the DSS group. These demonstrate that DSS increases intestinal permeability in mice, destroying barrier integrity in intestinal function, while EGF/MMT complex significantly decreases intestinal mucosal permeability, protecting barrier integrity in intestinal function. Taken together, the dose effect in EGF/MMT was better.
TABLE 5 serum DAO activity, D-LAC concentration and colon-tight junction protein concentration of mice of each group
Claims (1)
1. The application of the EGF/MMT compound in preparing the compound for treating the repair of the intestinal injury of the piglets is characterized in that the compound is prepared by the following method: preparing disodium hydrogen phosphate-citric acid buffer solution with the concentration of 0.05mol/LpH value of 3.8, respectively adding EGF and Na-MMT into the buffer solution, oscillating for 1h, centrifuging, and drying the precipitate to obtain the EGF/MMT compound.
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