Lactoferrin preparation for preventing and treating mycotoxin poisoning and application thereof

Abstract

The application discloses a lactoferrin-prepared drug for preventing and treating mycotoxin poisoning and an application thereof. Through in-vivo (intestinal tract) research on mice, vomitoxin is added in the diet of the mice to simulate the mice eating feed containing mycotoxin, and the mice are given intragastrically a lactoferrin solution containing a fixed concentration, so that the expression of inflammatory factors caused by vomitoxin can be reduced, and the expression of jejunal tight junction protein can be enhanced, thereby relieving the damage of vomitoxin to intestinal function. In addition, the lactoferrin can also regulate the colon microbiota of the vomitoxin-treated mice and improve intestinal microbial metabolism, and through the interaction between the lactoferrin and the vomitoxin, the growth of the intestinal villi is promoted, and the absorption capacity of the intestinal tract to nutrients is strengthened.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of lactoferrin application, and particularly relates to a lactoferrin preparation for preventing and treating mycotoxin poisoning and application thereof. BACKGROUND

[0002] Under natural conditions, molds can contaminate crops such as corn, wheat and barley and further produce secondary metabolites-mycotoxins. Mycotoxins can accumulate in animal organs and tissues after being ingested and cause mycotoxin poisoning. Long-term and low-dose ingestion of mycotoxins by animals can cause a decrease in feed intake, digestion rate, daily weight gain and production performance, which can cause great economic losses to the livestock industry. Therefore, how to prevent and treat the harm caused by mycotoxin poisoning is crucial to the development of the livestock industry.

[0003] Due to the wide range of mycotoxin contamination and strong stability, it is not easy to remove mycotoxins in the process of feed production, so it is impossible to eradicate mycotoxins in feed. Adding specific additives to feed or diet can alleviate the damage to the body caused by mycotoxins and improve the health status of the body.

[0004] The precursor condition for mycotoxins to cause damage to the human and animal body is to enter the body from the environment, and the intestinal tract is the main organ for the body to ingest mycotoxins and the first physiological barrier against mycotoxins. The intestinal barrier is an important structure for preventing harmful substances in the intestine from entering the body. The tight junction of intestinal epithelial cells is a key part of the intestinal barrier. After ingesting feed containing mycotoxins, mycotoxins often enter the intestinal tract through the digestive tract, and mycotoxins can cause changes in the function of intestinal epithelial cells. Therefore, it is necessary to study the damage of mycotoxins to the intestinal tract.

[0005] After animal mycotoxin poisoning, the first thing to do is to stop feeding the feed contaminated by mycotoxins and use some drugs to treat mycotoxin poisoning. The treatment should be based on the principles of improving the immunity of the animal body, neutralizing toxins, protecting the liver, detoxifying and detoxifying. The commonly used treatment methods for mycotoxin poisoning at present mainly include the following three types.

[0006] (1) Montmorillonite can adsorb mycotoxins in the intestinal tract and expel them out of the body. This product is low in price, and it is especially good for adsorbing aflatoxins. However, montmorillonite can also adsorb nutrients in feed, affecting the normal nutrient intake of livestock and poultry.

[0007] (2) Salt laxative can effectively detoxify mycotoxins by adding substances such as glucose, mannose and potassium sorbate to promote the rapid excretion of toxins in the gastrointestinal tract. Symptomatic treatment is also needed, that is, sufficient vitamin A, vitamin C and vitamin E are supplemented. This method is not comprehensive and complete, and has the defect of causing secondary harm.

[0008] (3) Microecological preparation, the method can convert mycotoxin into low-toxic or non-toxic form. But because the antibiotic sterilization is not selective, the microecological preparation cannot be used in combination with the antibiotic. In addition, the preparation has certain inhibition and killing function to part of the beneficial bacteria, and the combination will lead to the decrease of the drug efficacy.

[0009] And lactoferrin is a kind of natural multifunctional protein, which has broad-spectrum antibacterial, antiviral infection effect, can regulate the balance of iron in the body, regulate the generation of bone marrow cells, promote the growth of cells, regulate the immune function of the body, enhance the disease resistance of the body, inhibit the effect of human tumor cells, and can also synergize with various antibiotics and antifungal preparations, and more effectively treat diseases. SUMMARY

[0010] This section is intended to summarize some aspects of the embodiments of the present application and briefly introduce some preferred embodiments. Some simplifications or omissions may be made in this section and the abstract of the specification and the title of the application in order to avoid obscuring the purpose of this section, the abstract of the specification and the title of the application, and such simplifications or omissions cannot be used to limit the scope of the present application.

[0011] In view of the above and / or problems existing in the prior art, the present application is proposed.

[0012] Therefore, the purpose of the present application is to overcome the deficiencies in the prior art, and to provide a lactoferrin preparation for preventing and treating mycotoxin poisoning.

[0013] To solve the above technical problems, the present application provides the following technical scheme: the preparation method of the lactoferrin medicine is to dissolve lactoferrin powder in a physiological saline solution to obtain a lactoferrin-physiological saline solution.

[0014] As a preferred scheme of the present application, wherein: the mass of the lactoferrin powder is 5-20 mg, and the physiological saline is 100 μL.

[0015] As a preferred scheme of the present application, wherein: the mass of the lactoferrin powder is 10 mg, and the physiological saline is 100 μL.

[0016] Another purpose of the present application is to overcome the deficiencies in the prior art, and to provide an application of a lactoferrin preparation for preventing and treating mycotoxin poisoning.

[0017] As a preferred scheme of the present application, wherein: the application comprises simulating a diet environment of a mouse feeding on mycotoxin, and then performing gavage treatment of the mouse with the lactoferrin-physiological saline solution.

[0018] As a preferred scheme of the present application, wherein: the simulated feeding on mycotoxin is to add vomitoxin to the commercial feed eaten by the mouse to obtain feed granules.

[0019] As a preferred solution of the present application, wherein: the vomitoxin concentration of the feed particles is 12mg / kg.

[0020] As a preferred solution of the present application, wherein: the simulated mouse mycotoxin diet environment includes pouring the prepared feed particles into the feeding trough for the mice to freely eat, and the treatment lasts for 35 days.

[0021] As a preferred solution of the present application, wherein: the lactoferrin medicine can promote the colonization of beneficial bacteria in the intestine and reduce the proliferation of harmful bacteria, thereby improving the content of volatile fatty acids in the intestine by changing the intestinal flora structure, and thereby enhancing the intestinal anti-mycotoxin ability.

[0022] As a preferred solution of the present application, wherein: the lactoferrin medicine reduces the intestinal damage caused by malnutrition by up-regulating the expression of tight junction proteins and reducing the level of pro-inflammatory cytokines.

[0023] As a preferred solution of the present application, wherein: the lactoferrin promotes the growth of small intestinal villi and enhances the absorption capacity of the intestine for nutrients through interaction with vomitoxin.

[0024] The present application has the following advantages:

[0025] (1) The present application uses lactoferrin to be administered intragastrically while the mice are eating daily, which provides a new idea for preventing and treating mycotoxins in the daily life of livestock. On the one hand, lactoferrin can weaken the activation of MAPK, thereby inhibiting intestinal inflammation and maintaining intestinal integrity. On the other hand, lactoferrin also has a "probiotic" effect, which promotes the colonization of beneficial bacteria (lactobacilli) and the production of short-chain fatty acids (SCFAs). These fatty acids can be transported from the intestinal lumen to the host blood and exhibit a variety of biological functions.

[0026] (2) The present application uses lactoferrin to be administered intragastrically to prevent and treat mycotoxins, and the optimal intragastric concentration is selected. This method can effectively counteract the adverse effects caused by vomitoxin in the intestine. Moreover, lactoferrin is an endogenous substance in the animal body, which is safe and non-toxic, does not need to be added in large doses, and has very high prevention and treatment efficiency. BRIEF DESCRIPTION OF DRAWINGS

[0027] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the following will briefly introduce the drawings needed to be used in the embodiment description. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can also be obtained by those skilled in the art without creative labor. Among them:

[0028] Figure 1Figure for the effect of different concentrations of lactoferrin drug on the body weight of mice exposed to vomiting in Example 3 of the present application.

[0029] Figure 2 Figure A for the body weight change trend of mice in different experimental groups in Example 4 of the present application within 5 weeks;

[0030] Figure 2 Figure B for the final body weight of mice in different experimental groups in Example 4 of the present application in the 5th week;

[0031] Figure 2 Figure C for the average daily feed intake of mice in different experimental groups in Example 4 of the present application within 5 weeks.

[0032] Figure 3 Figure for the key indicators of intestinal morphology of different experimental groups in Example 4 of the present application.

[0033] Figure 4 Figure for HE staining of different experimental groups in Example 4 of the present application.

[0034] Figure 5 A- Figure 5 Figure E for the protein levels of IL-1β, IL-6, IL-8, TNF-α and IL-10 in the plasma of different experimental groups in Example 4 of the present application;

[0035] Figure 5 F- Figure 5 Figure J for the gene levels of IL-1β, IL-6, IL-8, TNF-α and IL-10 in the jejunum of different experimental groups in Example 4 of the present application.

[0036] Figure 6 A- Figure 6 Figure B for the Western blot results of DAO (diamine oxidase) levels, Claudin-1, Occludin, ITLN1 and GAPDH in the plasma of different experimental groups in Example 4 of the present application;

[0037] Figure 6 C- Figure 6 Figure G for the protein expression levels of Claudin-1 in the jejunum, the protein expression levels of Occludin in the jejunum, the protein expression levels of ITLN1 in the jejunum, the phosphorylation levels of p38 in the jejunum, and the phosphorylation levels of ERK1 / 2 in the jejunum of different experimental groups in Example 4 of the present application.

[0038] Figure 7 A- Figure 7H represents the ACE index, Chao index, principal coordinate analysis, Shannon index, Simpson index, average relative abundance of bacterial groups in the phylum-level colonic microbiota, relative abundance of Firmicutes in colonic digestive fluid, and relative abundance of Bacteroides in colonic digestive fluid in different experimental groups in Example 4 of this invention.

[0039] Figure 8 This is a diagram showing the bacterial species richness of different experimental groups in Example 4 of the present invention.

[0040] Figure 9 This is a correlation analysis diagram showing the significant differences in plasma index, colonic microbiota, and short-chain fatty acids among different experimental groups in Example 4 of this invention. Detailed Implementation

[0041] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to the examples in the specification.

[0042] Many specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be practiced in other ways different from those described herein, and those skilled in the art can make similar extensions without departing from the spirit of the invention. Therefore, the invention is not limited to the specific embodiments disclosed below.

[0043] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The phrase "in one embodiment" appearing in different places in this specification does not necessarily refer to the same embodiment, nor is it a single or selective embodiment that is mutually exclusive with other embodiments.

[0044] Raw materials involved in this invention:

[0045] Lactoferrin was purchased from Ingredia Dairy Experts, a French company.

[0046] Vomitoxin was purchased from Qingdao Puribang Biotechnology Co., Ltd. (product number MSS1011);

[0047] Unless otherwise specified, all other ingredients are commercially available.

[0048] Example 1

[0049] This embodiment prepares drugs of different concentrations for preventing and treating mycotoxin poisoning.

[0050] Put the sterile EP tube on the balance to zero, put the lactoferrin (LF) pure powder into the EP tube with a medicine spoon, respectively to 5, 10, 20 mg of lactoferrin powder in the capsule, add 100 μL of normal saline in the EP tube and mix well to prepare lactoferrin-normal saline solution of different concentrations.

[0051] Example 2

[0052] This example provides a diet environment for simulating mice to feed on mycotoxins.

[0053] Weigh 12 mg of vomitoxin pure powder and 1 kg of commercial feed powder, mix evenly and granulate to prepare feed granules with a vomitoxin (DON) concentration of 12 mg / kg. Pour the prepared feed granules into the feeding trough for the mice to freely eat.

[0054] Example 3

[0055] This example investigates the effect of lactoferrin drug concentration on the prevention and treatment of mycotoxin poisoning.

[0056] Fix the mice treated as in Example 2, extract 100 μL of lactoferrin-normal saline solution of different concentrations prepared in Example 1, put the needle into the back of the mouse tongue, and use the daily gavage method for continuous treatment for 35 days. Observe the mice and the results are shown in Figure 1 .

[0057] As can be seen Figure 1 , the effect of different concentrations of lactoferrin drugs on the body weight of mice exposed to vomit is shown. The results show that when the concentration of lactoferrin is 10 mg, the effect on the recovery of mouse body weight is most obvious. When the content of lactoferrin is 20 mg, the recovery effect of mouse body weight is weaker than 10 mg. This is because when the content of lactoferrin is high, it can cause iron poisoning in mice and increase the burden on the kidneys of mice, so the content of lactoferrin cannot be too high.

[0058] Example 4

[0059] This example verifies the effect of lactoferrin drugs on the prevention and treatment of mycotoxin poisoning.

[0060] Four groups of experiments are set up for verification, respectively:

[0061] Blank group: feed mice with ordinary commercial feed, daily gavage with 100 μL of normal saline, and observe the mice after 35 days;

[0062] Lactoferrin group: feed mice with ordinary commercial feed, while daily gavage with 100 μL of lactoferrin-containing normal saline solution, and observe the mice after 35 days;

[0063] Vomitoxin group: according to the processing method of example 2, the mouse was fed with feed particles with the concentration of 12 mg / kg of vomitoxin (DON), and the mouse was allowed to freely eat, and after 35 days, the mouse was observed;

[0064] Vomitoxin + lactoferrin group: according to the processing method of example 2, the mouse was fed with feed particles with the concentration of 12 mg / kg of vomitoxin (DON), and the mouse was allowed to freely eat, and at the same time, 100 μL of lactoferrin-containing saline solution was administered intragastrically every day, and after 35 days, the mouse was observed.

[0065] Referring to Figures 2 to 8 , the results of the above experiments are shown.

[0066] As shown in Figure 2 , Figure 2 A, B, C in the above table are respectively the weight change trend of the mice in the blank group, the lactoferrin group, the vomitoxin group and the vomitoxin + lactoferrin group in example 4 of the present application within 5 weeks (A), the final weight of the mice in the 5th week (B) and the average daily feed intake of the mice within 5 weeks (C); it can be seen that the lactoferrin treatment effectively improves the growth performance of the mice treated with vomitoxin, and has good performance in terms of weight and feed intake.

[0067] Figure 3 Table 2 is the key index of intestinal morphology in the blank group, the lactoferrin group, the vomitoxin group and the vomitoxin + lactoferrin group in example 4 of the present application, wherein each letter represents the duodenal villus height (A), the duodenal villus width (B), the duodenal crypt depth (C), the duodenal villus height / duodenal crypt depth (D), the jejunal villus height (E), the jejunal villus width (F), the jejunal crypt depth (G), the jejunal villus height / jejunal crypt depth (H), the ileal villus height (I), the ileal villus width (J), the ileal crypt depth (K), the ileal villus height / ileal crypt depth (L); Figure 4 Figure 2 is the HE staining diagram of the four experimental groups in example 4.

[0068] As can be seen from Figure 3 and Figure 4 , the lactoferrin can regulate the intestinal morphology of the mice exposed to vomitoxin, the duodenal villus height (VH) and the duodenal villus width (VW) are significantly affected by the interaction between vomitoxin and lactoferrin, the VH and VW of the vomitoxin group are lower than those of the control group and the vomitoxin + lactoferrin group, the VH of the jejunum is affected by the interaction between vomitoxin and lactoferrin, the VH of the vomitoxin group is lower than that of the control group and the vomitoxin + lactoferrin group, and the ratio of VH / CD (the ratio of intestinal villus height to crypt depth) is also reduced due to the treatment of vomitoxin. And from Figure 3 , especially Figure 3 A,Figure 3 B、 Figure 3 F、 Figure 3 It can be seen from G that the duodenum and jejunum villus height and width of the lactoferrin group under these three indicators were significantly lower than those of the DON + lactoferrin group, which was due to the fact that the growth-promoting effect of lactoferrin alone on small intestinal villi was lower than that of DON + lactoferrin combined action, which was because this indicator affected the digestive and absorptive capacity of mice, and the interaction between DON and lactoferrin could stimulate mice to absorb external nutrients.

[0069] Figure 5 A-E in the figure show the protein levels of IL-1β, IL-6, IL-8, TNF-α and IL-10 in the plasma in the blank group, lactoferrin group, DON group and DON + lactoferrin group in the fourth embodiment of the present application, and F-J show the mRNA levels of IL-1β, IL-6, IL-8, TNF-α and IL-10 in the jejunum. It can be seen from the figure that the addition of lactoferrin can reduce the secretion of pro-inflammatory cytokines in mycotoxin-treated mice, and the pro-inflammatory cytokines (IL-1β, IL-6, IL-8 and TNF-α) in the plasma are statistically significantly affected by the interaction between DON and LF, and the levels of IL-1β, IL-8 and TNF-α in the DON group are higher than those in the control group and the DON + lactoferrin group. In addition, the anti-inflammatory cytokine IL-10 in the plasma is significantly affected by the interaction between DON and lactoferrin, and the level of IL-10 in the DON group is lower than that in the control group and the DON + lactoferrin group.

[0070] Figure 6Figures A, G, and G respectively show the plasma DAO (diamine oxidase) level (A), Claudin-1, Occludin (blocking protein), ITLN1, and GAPDH Western blot results (B), Claudin-1 protein expression level (C), Occludin protein expression level (D), ITLN1 protein expression level (E), p38 phosphorylation level (F), and ERK1 / 2 phosphorylation level (G) in the jejunum of the four experimental groups (blank group, lactoferrin group, vomitoxin group, and vomitoxin + lactoferrin group) in Embodiment 4 of this invention. As can be seen from the figures, LF reduces intestinal permeability by enhancing the expression of tight junction proteins in the jejunum. The plasma DAO level is affected by the interaction between vomitoxin and lactoferrin, with the level in the vomitoxin group being significantly lower than that in the control group and the vomitoxin + lactoferrin group. Furthermore, the expression of sealing proteins in the jejunum also shows the same changes as described above. Simultaneously, lactoferrin enhanced ITLN1 protein expression and inhibited MAPK pathway activation. The level of jejunal ITLN1 protein expression was influenced by the interaction between vomitoxin and lactoferrin; the protein level in the vomitoxin group was significantly lower than that in the control group and the vomitoxin + lactoferrin group. Furthermore, p38 phosphorylation showed higher levels in the vomitoxin group than in the control group and the vomitoxin + lactoferrin group. Compared to the control group and the vomitoxin + lactoferrin group, vomitoxin treatment significantly upregulated ERK1 / 2 phosphorylation levels.

[0071] Figure 7 The figures A and H represent the ACE index (A), Chao index (B), principal coordinate analysis (PCoA) (C), Shannon index (D), Simpson index (E), mean relative abundance of bacterial groups in the phylum-level colonic microbiota (F), relative abundance of Firmicutes in the colonic digestive fluid (G), and relative abundance of Bacteroides in the colonic digestive fluid (H) in the four experimental groups (blank group, lactoferrin group, vomitoxin group, and vomitoxin + lactoferrin group) of this invention, respectively. The figures show that lactoferrin can regulate the colonic microbiota of mice treated with vomitoxin. The Shannon and Simpson indices are affected by the interaction between vomitoxin and lactoferrin. Compared with the control group and the vomitoxin + lactoferrin group, the mice in the vomitoxin group had a lower Shannon index and a higher Simpson index. Furthermore, the PCoA of the colonic microbiota revealed that the microbiota of the vomitoxin and lactoferrin groups were differentiated. At the phylum level, Bacteroides is the most prevalent phylum in the colon, and the relative abundance of Bacteroides and Firmicutes is significantly affected by vomitoxin treatment.

[0072] Figure 8A-J are the graphs of the relative abundance of the 20 most abundant genera (A), the relative abundance of Clostridia, Ruminococcus, another branch of bacteria and the relative abundance of the Odoribacter (B-D), the level (concentration unit) of acetic acid, propionic acid, butyric acid and valeric acid (G-J) in the blank group, lactoferrin group, vomitoxin group and vomitoxin + lactoferrin group in the present case 4 of the present application; at the genus level, Clostridia-XIVa and Lactobacillus are the dominant bacteria, the relative abundance of Clostridia-XIVa in the vomitoxin group is lower than that in the control group and the vomitoxin + lactoferrin group, while the relative abundance of Lactobacillus is higher. In addition, compared with the control group, vomitoxin treatment reduces the relative abundance of another branch of bacteria and odoribacter. At the same time, lactoferrin promotes the production of intestinal microbial metabolites in mice exposed to vomitoxin, and the level of propionic acid in the colon is affected by the interaction of vomitoxin and lactoferrin, which is manifested as higher propionic acid level and lower butyric acid level in the vomitoxin group than in the control group and the vomitoxin + lactoferrin group.

[0073] Figure 9 The figure for the correlation analysis of the plasma index, colon microbiota and short-chain fatty acids that are significantly different in the blank group, lactoferrin group, vomitoxin group and vomitoxin + lactoferrin group in the present case 4 of the present application. Blue represents significant positive correlation, and red represents significant negative correlation. As shown in Figure 9 , the correlation analysis of the levels of cytokines and DAO in plasma, microbiota and colon short-chain fatty acids is shown, the IL-1β level is significantly positively correlated with the levels of IL-8, TNF-α and DAO, and the relative abundance of Ruminococcus, and is significantly negatively correlated with IL-10, the level of propionic acid and the relative abundance of Clostridia-XIVa. In addition, the level of IL-10 is positively correlated with the level of colon propionic acid. As for short-chain fatty acids, it is positively correlated with the level of butyric acid, the relative abundance of another branch of bacteria, Clostridia-XIVa and Odoribacter.

[0074] In summary, the addition of lactoferrin as described in the present application can reduce the expression of inflammatory factors caused by vomitoxin, and at the same time can enhance the expression of tight junction proteins in the jejunum, thereby alleviating the damage of vomitoxin to intestinal function. In addition, lactoferrin can also regulate the colon microbiota of mice treated with vomitoxin and improve intestinal microbial metabolism, and through the interaction between lactoferrin and vomitoxin, promote the growth of intestinal villi and strengthen the absorption capacity of the intestine for nutrients.

[0075] It should be noted that the above examples are only used to illustrate the technical solutions of the present application and not to limit it, although the present application has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present application can be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present application, which should be covered in the scope of the claims of the present application.

Claims

1. The application of lactoferrin in the preparation of drugs for the prevention and treatment of mycotoxin poisoning, characterized in that: Lactoferrin powder was dissolved in physiological saline solution to prepare lactoferrin-physiological saline solution; The mycotoxin in question is vomitoxin; The lactoferrin drug can promote the colonization of beneficial bacteria in the intestine and reduce the proliferation of harmful bacteria, thereby enhancing the intestinal antifungal ability by changing the intestinal flora structure and increasing the content of volatile fatty acids in the intestine. The lactoferrin drug alleviates intestinal damage caused by malnutrition by upregulating the expression of tight junction proteins and reducing the level of pro-inflammatory cytokines. The lactoferrin promotes the growth of intestinal villi and enhances the intestine's ability to absorb nutrients through its interaction with vomitoxin.

Images