CN115837067A

CN115837067A - Application of spleen aminopeptide in preparation of medicine for improving intestinal flora condition

Info

Publication number: CN115837067A
Application number: CN202211411197.9A
Authority: CN
Inventors: 郑新越; 王瑾; 王月丹
Original assignee: Beijing First Biochemical Pharmaceutical Co ltd
Current assignee: Beijing First Biochemical Pharmaceutical Co ltd
Priority date: 2019-05-31
Filing date: 2019-05-31
Publication date: 2023-03-24
Also published as: CN112007140A

Abstract

The invention discloses an application of spleen aminopeptide in a medicament for improving intestinal flora conditions. The spleen aminopeptide is an animal spleen extract, and can remarkably adjust the intestinal flora structure of the chemotherapy mice and improve the intestinal flora condition of the chemotherapy mice.

Description

Application of spleen aminopeptide in preparation of medicine for improving intestinal flora condition

The application is a divisional application of patent application No. 201910468415.4, which has the application date of 2019, 5 and 31 and is named as application of spleen aminopeptide in preparation of medicines for treating intestinal barrier function deficiency.

Technical Field

The invention belongs to the field of biochemical pharmacy, relates to a new application of spleen aminopeptide, and particularly relates to an application of the spleen aminopeptide in preparing a medicine for improving the condition of intestinal flora.

Background

The malignant tumor is a disease seriously threatening the health of human beings, and data show that the mortality rate of the malignant tumor in China is at a higher level in the world and is in a continuously increasing trend, so that the malignant tumor becomes the first cause of death of urban residents. Chemotherapy is one of the important means for treating malignant tumors and is also accepted by patients. However, the currently used chemotherapeutic drugs of different types, such as epirubicin, cisplatin, vincristine, etc., all have different degrees of side effects on patients, resulting in poor survival status of patients.

For tumor patients, the drug can inhibit or kill cancer cells and damage normal cells, tissues or organs to a certain extent, thereby causing adverse reactions of different types and degrees. Wherein, the intestinal dysfunction is reflected by the dose toxicity of some anti-cancer drugs and is also a common complication when the bone marrow of a patient is inhibited by large-dose chemotherapy, and can induce and/or aggravate systemic inflammatory reaction and multi-organ dysfunction, thereby having important influence on the occurrence, development and prognosis of diseases. The intestinal function state has become one of the most important indicators for determining the prognosis of critically ill patients.

The damage of chemotherapy to the intestinal function is mainly reflected in the following aspects: 1. the mucous membrane barrier is damaged, and the normal renewal of intestinal mucous membrane epithelial cells is slowed, villi are shrunk to different degrees, the absorption area is reduced, the intestinal permeability is increased due to the relaxation of cell connection, and a channel is provided for intestinal bacteria and toxin; 2. chemical barriers are broken, and chemical substances in the intestinal cavity aggravate damage of the intestinal cavity due to the reduction of the self-resistance of the mucous membrane; 3. the biological barrier is destroyed, and the chemotherapy drugs can directly kill normal flora in the intestinal cavity, so that the micro-ecological balance is disordered; 4. and (3) breaking an immune barrier: the intestinal mucosa immune response protective agent consists of intestinal mucosa lymphoid tissues (such as Peyer's knot and mesenteric lymph node), cells (such as dendritic cells, macrophages, T cells, B cells and the like) and secretory antibodies (sIgA) on the intestinal mucosa surface, has the functions of taking, processing and presenting antigens on the mucosa surface, timely identifies foreign microorganisms, food and self abnormal antigens, induces local and systemic immune response reaction, and protects the intestinal tract from being damaged by the foreign antigens and abnormal immune response; chemotherapy drugs cause reduction of local sIgA secretion and impaired T cell immune function, which can cause both immune cell number change and immune regulation abnormality, so that intestinal immune dysfunction, even systemic immune regulation is affected, and finally digestive tract and even systemic diseases can be caused.

The current major treatments for impaired intestinal barrier function include: (1) Improving blood perfusion of intestinal mucosa, supplementing blood volume, and optionally applying medicine for improving intestinal blood circulation, including small dose of dopamine, 654-2 and prostaglandin I ₂ To relieve spasm of mesenteric microvessels; (2) Improving enteral nutrition, and the commonly used special nutrients include glutamine (Gln), growth hormone, arginine, omega fatty acid, etc.; (3) The growth factor can directly stimulate the proliferation and differentiation of small intestine stem cells, enhance the repair effect after the intestinal epithelium is damaged, improve the intestinal barrier function and reduce the flora shift, and the commonly used production factors comprise insulin-like growth factor (IGF-1), epidermal Growth Factor (EGF), transforming growth factor (TGF-alpha) and the like; (4) Improving biological barrier, and supplementing beneficial bacteria group to microecological preparation such as Bacillus bifidus and lactobacillus to regulate intestinal flora ecological balance, so that the beneficial bacteria can generate compact membrane flora in digestive tract to form microbial barrier. Optionally, intestinal non-absorbable antibiotics are applied for selective intestinal decontamination treatment to improve intestinal micro-ecological environment.

At present, for chemotherapy patients, plasma glutamine level is not an index for evaluating the chemotherapy effect, the protection effect of oral glutamine on the intestinal barrier function of the chemotherapy patients is further proved, a basis is provided for clinical medication, and more important indexes, such as damage of intestinal mucosa, adverse reaction of chemotherapy drugs and the like, need to be researched. In addition, the indications, timing, duration and optimal effect of glutamine supplementation by oral administration are further discussed.

In addition, there are limited drugs that can regulate the balance of immune functions in chemotherapy patients, and there has been no deep study on whether spleen aminopeptide is effective in improving the defect of intestinal barrier function and its appropriate therapeutic dose.

Therefore, a better way for nursing chemotherapy patients is sought, and the improvement of the survival condition of the patients is urgent.

The spleen aminopeptide is a polypeptide and nucleotide immunomodulator extracted from an animal spleen, has the functions of specifically transferring a certain specific cell immunity of a donor to a receptor, nonspecifically enhancing a cell immune system of the receptor and improving the binding capacity of mononuclear cells and an Ig-containing complex; has the functions of promoting the release of interferon and lymphokine, improving the immune function of an organism, promoting the function of helper T cells, increasing IL-2, IL-6 and IFN-gamma secretion of T cells, promoting PHA to induce lymphocyte transformation and leukocyte induction interferon in vivo, activating the activity of a mononuclear-macrophage system, improving the serum IgG and IgA levels of a patient, and inhibiting the replication of viruses in tissues to a certain extent, namely, the spleen aminopeptide can induce lymphocyte sensitization or activation so as to initiate cellular immune response to target cells, including direct cytotoxic killing effect, synergistic effect, proliferation effect and replication effect, thereby achieving the purpose of enhancing the cellular immune function. Meanwhile, the spleen aminopeptide can improve the phagocytic function of monocytes, promote the release of interferon and lymphokine, improve the non-specific immune function of the body and the like. Multiple researches prove that the spleen aminopeptide has certain effects on resisting infection, resisting allergy, regulating immunity and the like, and has high tolerance degree.

The problem of whether the spleen aminopeptide can be used as an adjuvant treatment drug after chemotherapy to improve the immunity of patients and improve the survival condition of patients is still to be further researched.

Disclosure of Invention

The invention establishes an intestinal dysfunction model by taking an ICR mouse as a target, researches the influence of the spleen aminopeptide on the intestinal dysfunction of the mouse, researches the regulation effect of the spleen aminopeptide on the immune function of the chemotherapy mouse by comparing the pathological conditions of the intestinal tracts of the mice of different treatment groups and the change of intestinal flora, and further researches the related immunological mechanism, thereby completing the invention. Namely, the present invention is as follows:

the invention firstly provides application of spleen aminopeptide in preparing a medicament for treating intestinal barrier function deficiency, which is characterized in that the intestinal barrier function deficiency is intestinal barrier function deficiency caused by a chemotherapeutic medicament; preferably, the chemotherapeutic agent is epirubicin hydrochloride.

In some embodiments, the spleen aminopeptide is a spleen extract of an animal; preferably, the spleen aminopeptide is a liquid preparation of the spleen aminopeptide.

In some embodiments, the spleen aminopeptide is administered in a dose of 1.5 mg/kg/day to 7.5 mg/kg/day in the mouse; preferably, the dose administered is 7.5 mg/kg/day. In addition, the spleen aminopeptide can obviously improve the pathological condition of the intestinal tract of mice after chemotherapy and effectively relieve the intestinal barrier function defect caused by chemotherapy drugs.

The invention also provides the application of a composition in preparing a medicament for treating the defect of the intestinal barrier function, wherein the composition comprises the spleen aminopeptide and one or more pharmaceutically acceptable carriers. Wherein the carrier comprises more than 1 of the group consisting of pharmaceutically acceptable diluents, excipients, fillers, binders, absorption enhancers, surfactants and synergists. In addition, the spleen aminopeptide can obviously improve the pathological condition of the intestinal tract of mice after chemotherapy and effectively relieve the intestinal barrier function defect caused by chemotherapy drugs.

In some embodiments, the intestinal barrier function defect is an intestinal barrier function defect caused by a chemotherapeutic drug, preferably, the chemotherapeutic drug is epirubicin hydrochloride.

The invention also provides a method of treating a defect in intestinal barrier function, comprising administering a spleen aminopeptide to a subject having a defect in intestinal barrier function, thereby treating the defect in intestinal barrier function. Further, the spleen aminopeptide is an animal spleen extract; preferably, the spleen aminopeptide is a liquid preparation of the spleen aminopeptide. Further, the intestinal barrier function impairment is intestinal barrier function impairment caused by a chemotherapeutic drug; preferably, the chemotherapeutic agent is epirubicin hydrochloride. When the spleen aminopeptide is administered on an ICR mouse chemotherapy model, the spleen aminopeptide is administered in the mouse at a dose of 1.5 mg/kg/day to 7.5 mg/kg/day; preferably, the dose administered is 7.5 mg/kg/day.

ADVANTAGEOUS EFFECTS OF INVENTION

The test results show that the results of the invention not only prove that the chemotherapy drugs can damage the intestinal tracts of mice to a certain extent, but also preliminarily prove that the spleen aminopeptide has a certain treatment effect on the intestinal tracts. Compared with the prior art, the technical scheme of the invention has the following beneficial effects:

1) The spleen aminopeptide can not only obviously improve the pathological condition of the intestinal tract of the mouse after chemotherapy, but also effectively improve the survival condition of the mouse; can also repair the intestinal mucosa structure of the mice after chemotherapy, relieve inflammatory reaction and maintain the integrity of the intestinal mucosa and cells of the mice.

2) The spleen aminopeptide can effectively relieve bacterial infection caused by chemotherapeutic drugs in chemotherapy mice, and improve intestinal flora conditions of the chemotherapy mice.

In order to make the aforementioned and other objects, features and advantages of the present invention comprehensible, preferred embodiments accompanied with figures are described in detail as follows:

drawings

In FIG. 1, a-f are graphs showing the results of general observations of intestinal specimens of mice in a placebo group, a chemotherapy model group, a low-dose spleen aminopeptide treatment group, a normal-dose spleen aminopeptide treatment group, a high-dose spleen aminopeptide treatment group, and a glutamine treatment group, respectively.

In FIG. 2, a-f are graphs showing the results of HE staining of intestinal specimens of mice in a placebo group, a chemotherapy model group, a low dose spleen aminopeptide treatment group, a normal dose spleen aminopeptide treatment group, a high dose spleen aminopeptide treatment group and a glutamine treatment group, respectively.

In FIG. 3, a-f are graphs showing immunohistochemical results of intestinal specimens of mice in a placebo group, a chemotherapy model group, a low-dose spleen aminopeptide treatment group, a normal-dose spleen aminopeptide treatment group, a high-dose spleen aminopeptide treatment group, and a glutamine treatment group, respectively.

FIG. 4 shows the endotoxin content in serum of each group of mice at day 30 of the experiment. Wherein CK represents a blank control group; EPI represents chemotherapy model group; LD-SAOS represents the low dose spleen aminopeptide treatment group; ND-SAOS represents normal dose spleen aminopeptide treatment group; HD-SAOS represents the high dose spleen aminopeptide treatment group; gln represents glutamine treatment group.

Fig. 5 shows the relative abundance of the flora at the family level in the intestines of each group of mice at day 30 of the experiment. Wherein, A represents a blank control group; b represents a chemotherapy model group; c represents a low-dose spleen aminopeptide treatment group; d represents a normal dose spleen aminopeptide treatment group; e represents a high dose spleen aminopeptide treatment group; f represents the glutamine treatment group. O, L and R respectively represent mouse individuals with different marks in each group.

FIG. 6 shows the results of the bacterial population diversity in the intestinal tract of the groups of mice on day 30 of the experiment. Wherein CK represents a blank control group; EPI represents chemotherapy model group; LD-SAOS represents the low dose spleen aminopeptide treatment group; ND-SAOS represents normal dose spleen aminopeptide treatment group; HD-SAOS represents the high dose spleen aminopeptide treatment group; gln represents glutamine treatment group. The Chao1 diversity index on the ordinate is an index that measures the abundance of a population, i.e. the number of populations.

Detailed Description

The raw material of the spleen aminopeptide used in the embodiment of the invention is extracted from fresh bovine spleen, and the medicines, reagents and main experimental instruments used in the embodiment can be purchased from the market.

Drugs and reagents: spleen aminopeptide (Beijing, first Biochemical pharmaceutical Co., ltd., specification of 10mg/ml, sample extracted from fresh healthy bovine spleen, spray filtered by molecular sieve to colorless to yellowish clear liquid, bottled by glass-made oral liquid, sealed and stored in 4 deg.C cool and dark place, national drug Standard WS1- (X-451) -2003Z); epirubicin hydrochloride (T0125, 10mg × 3 bottles in standard, shanghai pottery arts biology Co., ltd.); glutamine powder (550-065-EG, 100 g/bottle, adult dose 2 g/person/day); sodium citrate anticoagulant (R-10269, 100 ml/bottle, bao Yi (Beijing) Biotechnology Limited); PBS buffer (AC 13716274, 500 ml/bottle standard, hyclone Co.); tissue fixative solution (MA 0192, 500ml, EPSILON); hematoxylin staining solution (Cat No. T1686, specification 50ml, shanghai pottery art); eosin dye liquor (cargo number C0109, specification 100ml, shanghai Bin Yuntian Biotechnology Co., ltd.); anti-Claudin 1antibody (abcam, cat No. ab15098, 500. Mu.l); endotoxin detection kit (Genescript, cat # L00447-20, 1kit).

The main experimental apparatus: a mouse holder (DM 10171, nanchu Biotechnology Ltd.); upright photo microscope (OLYMPUS, model BX 53F); microplate reader (Thermo, model Multiskan Mk 3).

Example 1 establishment and treatment of mouse model for intestinal dysfunction

1. Materials: female ICR mice, 6-8 weeks old, were provided by the department of medicine, university of Beijing.

2. Establishment and treatment of intestinal dysfunction mouse model:

30 female ICR mice (provided by the department of medicine, university of Beijing) 6-8 weeks old were randomly divided into 6 groups of 5 mice each.

The first group is a blank control group (CK for short), and 1ml of normal saline is perfused every day; the other five groups are experimental groups, and chemotherapeutic drug epirubicin hydrochloride solution (10 mg/kg body weight) is injected in equal dose on the 0 th, 5 th and 10 th days respectively. Wherein, the second group is chemotherapy model group (EPI for short), the third group to the sixth group are treatment groups, and the mice after chemotherapy are treated by respectively taking low-dose spleen aminopeptide (gavage dose is 1.5 mg/kg/day, LD-SAOS for short), normal-dose spleen aminopeptide (gavage dose is 4.5 mg/kg/day, ND-SAOS for short), high-dose spleen small molecular weight polypeptide (gavage dose is 7.5 mg/kg/day, HD-SAOS for short) and glutamine solution (gavage dose is 3.0 mg/kg/day, gln for short) (the dose for the mice experiment is converted according to the currently accepted pharmacological experiment methodology compiled by professor of xu Tertiary cloud, and the coefficient is 0.0026).

Example 2 Effect of oral solution of spleen aminopeptide on the indices of mice treated with chemotherapy

1. Comparison of gross observations of intestinal specimens from chemotherapy mice administered with different doses of oral solution of splenic aminopeptide

The test method comprises the following steps: the 6 groups of mice described in example 1 were sacrificed by cervical dislocation after 30 days of treatment, and gross specimens of the mouse intestinal tracts were observed and recorded by photographing.

And (3) test results: from the results of the intestinal dissection of gross specimens of the mice in each group of fig. 1, it can be seen that: after being treated by chemotherapeutic drugs, the chemotherapy group mice have serious edema of intestinal tracts, have serious adhesion phenomenon and can not distinguish the basic characteristics of the intestinal tracts; compared with the mice in the chemotherapy group, the general results of the intestinal tracts of the mice in the low-dose, normal-dose and high-dose spleen aminopeptide groups show that the intestinal tracts of the mice are treated by the spleen aminopeptide, the intestinal tracts of the mice have clear and visible structures, the edema phenomenon is obviously improved, and the survival condition of the mice is better; the positive drug control group (namely the glutamine solution treatment group) shows that the intestinal tract of the mouse is obviously improved compared with the chemotherapy group. The experimental result shows that the spleen aminopeptide can obviously improve the intestinal pathological condition of chemotherapy mice and improve the survival condition of the mice.

2. Comparison of HE staining results and immunohistochemical results of intestinal tract specimens of chemotherapy mice by administration of spleen aminopeptide oral solution at different doses

2.1 HE staining and immunohistochemical assay methods: the 6 groups of mice described in example 1 were sacrificed by breaking their necks after 30 days of treatment, the mice were dissected and then taken out about 1cm of small intestine organs and placed in a tissue fixing solution to be preserved in the dark, and wax blocks were prepared by treatments such as dehydration and embedding, and then the sections were HE-stained and immunohistochemically sectioned, and finally photographed and observed under a microscope.

2.2 HE staining test results: as can be seen from the HE staining result in FIG. 2, the chemotherapy group mice had damaged intestinal mucosa structure, injured mucosal epithelial cells, edema of mucosa, enlarged intercellular gaps, damaged connective structures, disordered submucosal lymphoid tissue structure, and inflammatory cell infiltration; the intestinal mucosa structure of the mice in the spleen aminopeptide treatment group is complete, the cell arrangement is relatively uniform, no obvious pathological change condition exists, but the effect of different dosage groups cannot be judged only by the HE staining result; the positive drug control group (namely the glutamine solution treatment group) shows that the intestinal mucosa of the mouse is also obviously improved. The experimental result shows that the spleen aminopeptide can repair the structure of the intestinal mucosa, relieve inflammatory reaction and maintain the integrity of the intestinal mucosa and cells.

2.3 Claudins proteins are major backbone proteins constituting tight junctions, and play an important role in maintaining intercellular junctions, proliferation, migration, and the like. Because the calmudins in the epithelial cells of the intestinal mucosa can be used for representing whether the mucosa is complete and healthy, the expression and distribution of the calmudins are usually detected by using anti-claudin protein antibodies for histochemistry or related experiments. In the experiment, anti-claudin 1antibody is adopted to carry out immunohistochemistry on intestinal mucosa to explore the distribution of claudin proteins in the intestinal mucosa of each group.

Immunohistochemical test results: as can be seen from the results of intestinal immunohistochemistry of mice in each group in FIG. 3, the distribution of claudin-1 protein in the mice in the chemotherapy group is obviously reduced compared with that in other groups, the staining is lighter, which indicates that the intestinal mucosa tight junction function is damaged, and the distribution of intestinal mucosa claudins protein in the mice in the spleen aminopeptide treatment group, especially in the mice in the high dose group, is more uniform. It should be noted that immunohistochemical results can only be characterized and qualitatively determined whether the function of the intestinal mucosa is damaged. From the results, the spleen aminopeptide treatment group has a certain treatment effect on intestinal mucosa injury.

3. Effect of administration of various doses of oral solutions of spleen aminopeptide on endotoxin content in sera of chemotherapy mice

In order to further explore the harm of chemotherapeutic drugs to mice and verify the therapeutic effect of spleen low molecular weight polypeptides on chemotherapeutic mice, blood of each group of mice is obtained by an eyeball blood sampling method at the end of an experiment, serum is obtained by centrifugation, and ELISA analysis is carried out on endotoxin in the serum by using an endotoxin detection kit.

The test method comprises the following steps: collecting blood of each group of mice into a sterile 1.5ml EP tube by an eyeball blood-taking method, and centrifuging at 3000 r/min to take upper serum; serum endotoxin content of each group of mice was detected using an endotoxin ELISA kit and recorded.

And (3) test results: as can be seen from FIG. 4, the endotoxin content in the serum of the mice in the chemotherapy group was significantly increased (P < 0.0001) compared to the mice in the normal group (i.e., the blank control group), indicating that there was a serious bacterial infection in the mice in the chemotherapy group; the endotoxin content in the serum of the mice in the spleen amino peptide treatment group is obviously lower than that of the mice in the chemotherapy group, and the endotoxin content in the serum of the mice in the high-dose spleen amino peptide treatment group is lower and almost has no difference with that of the normal group; the content of endotoxin in the serum of the mice of the positive drug control group (namely the glutamine solution treatment group) is reduced. Therefore, the spleen aminopeptide can relieve bacterial infection caused by chemotherapeutic drugs.

4. Effect of administration of various doses of oral solutions of spleen aminopeptide on intestinal flora of chemotherapy mice

In order to explore the influence of chemotherapy on the intestinal flora of mice and the influence of spleen aminopeptide on the intestinal flora of mice, fresh excrement of the mice is taken on the 30 th day of an experiment to carry out intestinal flora sequencing analysis.

And (3) test results: as shown in fig. 5, the relative abundance graph of the result analysis of the bacterial flora of each group of mice at the family level can show that the diversity of the intestinal flora of the mice at the chemotherapy group is reduced, the relative abundance of the bacteroidetes is obviously increased, and the porphyromonas is obviously reduced; the change of the intestinal flora of mice in normal dose and high dose spleen aminopeptide treatment groups tends to a blank control group, and the drug is proved to be capable of remarkably adjusting the intestinal flora structure of the chemotherapy mice and improving the intestinal flora condition.

As can be seen from the comparison of the intestinal flora diversity results of the mice in each group in FIG. 6: the abundance of intestinal flora of the mice in the chemotherapy group is obviously reduced, which indicates that the condition of the intestinal flora is worsened; the relative abundance of the spleen aminopeptide treatment group approached that of the normal group. The results again verify that the spleen aminopeptide can increase the number of intestinal flora of mice and improve the intestinal flora condition of chemotherapy mice.

The embodiments of the present invention have been described in detail, but the embodiments are merely examples, and the present invention is not limited to the embodiments described above. Any equivalent modifications or alterations to this practice will occur to those skilled in the art and are intended to be within the scope of this invention. Accordingly, equivalent changes and modifications made without departing from the spirit and scope of the present invention should be covered by the present invention.

Claims

1. Application of spleen aminopeptide in preparing medicine for improving intestinal flora condition is provided.

2. The use of claim 1, wherein the spleen aminopeptide can significantly modulate the intestinal flora structure of the chemotherapy mice and simultaneously improve the intestinal flora condition of the chemotherapy mice.

3. The use of claim 1, wherein the spleen aminopeptide significantly increases the number of intestinal flora and increases flora diversity in the mice undergoing chemotherapy.

4. The use of claim 1, wherein the spleen aminopeptide is a solid or liquid formulation of an animal spleen extract.

5. The use of claim 1, wherein the spleen aminopeptide is administered in a dose of from 1.5 mg/kg/day to 7.5 mg/kg/day in the mouse.

6. The use of claim 1, wherein the spleen aminopeptide is administered in a dose of 7.5 mg/kg/day in the mouse.

7. Use of a composition comprising spleen aminopeptide and one or more pharmaceutically acceptable carriers for the manufacture of a medicament for improving the condition of the intestinal flora.

8. The use according to claim 7, said carrier comprising at least one of the group consisting of pharmaceutically acceptable diluents, excipients, fillers, binders, absorption-promoting agents, surfactants and synergists.

9. The use of claim 7, wherein the spleen aminopeptide can significantly modulate the intestinal flora structure of the chemotherapy mice and simultaneously improve the intestinal flora status of the chemotherapy mice.