CN115350204A - Application of inulin in preventing and treating small intestine diseases - Google Patents
Application of inulin in preventing and treating small intestine diseases Download PDFInfo
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Abstract
The invention belongs to the technical field of research on inulin and small intestine diseases, and particularly discloses application of inulin in preventing and treating small intestine diseases. The application of inulin in preparing products for preventing and treating small intestine diseases, wherein the small intestine diseases comprise small intestine barrier disorder and/or small intestine barrier disorder complication. A preparation for preventing and treating small intestine diseases contains inulin. A preparation for preventing and treating diseases induced by small intestine barrier disorder contains inulin. The invention provides a treatment scheme aiming at individual diseases in some concurrent diseases such as small intestine diseases, provides a better solution for pathological changes caused by pathogenic basic diseases and can achieve a treatment effect; meanwhile, the method overcomes the prejudice of inulin application and develops new application of the inulin.
Description
The application is a divisional application of an invention patent named as new application of inulin, with the application number of 2021111311235 and the application date of 2021, 9 months and 26 days.
Technical Field
The invention belongs to the technical field of inulin research, and particularly relates to application of inulin in preventing and treating small intestine diseases.
Background
Inulin (chemical formula C) 228 H 382 O 191 ) It is in colloid form, is contained in cell protoplasm, is different from starch, is easily dissolved in hot water, is separated out from water by adding ethanol, and does not react with iodine. Inulin is also very easily hydrolysed to fructose under dilute acids, which is characteristic of all fructans. Can also be hydrolyzed into fructose by inulase (inulase). Enzymes that break down inulin are lacking in both humans and animals. The inulin molecule is polymerized by 31 beta-D-fructofuranose and 1-2 pyranoinulin residues, and the fructose residues can be connected by beta-2,1-bond. The inulin is linear straight-chain polysaccharide formed by linking D-fructose through beta (1 → 2) glycosidic bonds, the tail end of the linear straight-chain polysaccharide is usually provided with a glucose residue, the polymerization Degree (DP) is 2-60, wherein the inulin with the average polymerization degree DP less than or equal to 9 is also called short-chain inulin, and the inulin extracted from natural plants simultaneously contains long chains and short chains.
Abdominal Aortic Aneurysms (AAA) are tumorous dilatations that occur in the abdominal aorta, most commonly the infrarenal aorta. An aneurysm diameter of 30mm or more or a ratio of the diameter of the infrarenal aorta to the diameter of the unexpanded suprarenal aorta of 1.5 or more can be diagnosed as AAA. Histopathology of human AAA specimens and animal experimental studies reveal that AAA is characterized by vascular smooth muscle cell apoptosis, degradation of middle-membrane elastic fibers, infiltration of local inflammatory cells, and can be accompanied by calcification of tumor wall and formation of endoluminal thrombus (ILT). Clinically, AAA progresses insidiously, most AAAs are asymptomatic before rupture, with larger tumor body diameters and greater risk of aneurysm rupture, with up to 81% of their risk of death once ruptured. AAA poses a great threat to human health. The main treatment modalities for AAA are currently open surgical and endovascular aneurysm repair (EVAR), however, surgery is only applicable to patients with extreme late dilatation (male diameter ≥ 5.5cm, female diameter ≥ 5.0 cm) or AAA diameter > 4.0cm and rapid progression (diameter increase ≥ 1.0cm over the year). For AAA with occult progression, there is no effective agent intervention to prevent further expansion of the tumor mass. In the research of AAA preparation intervention, various preparations have been explored, including statin lipid-lowering drugs, hypotensive drugs (β adrenoceptor blockers, angiotensin converting enzyme inhibitors), anti-inflammatory agents, antiplatelet agents, tetracycline antibiotics, vitamin E, and the like. However, none of these formulations have achieved significant efficacy in clinical trials. Therefore, the research of new preparation and new method for AAA non-surgical treatment has important guiding significance for clinical disease management.
Intestinal barriers can be divided into physical, biochemical and immunological barriers. Intercellular connexins connect the intestinal epithelial cells, regulate cell bypass permeability, and form a physical barrier with the mucus layer; bile salt, gastric acid, antimicrobial peptide, calcium defensin, intestinal microbiota and the like form a chemical biological barrier; immune barriers are formed by the interaction of immune cells such as Treg cells, th17 cells, dendritic cells, 3-type innate lymphoid cells and B lymphocytes and cytokines IL17, IL22, IL-10 secreted by the immune cells, secretory mucosal antibody IgA and the like.
Disclosure of Invention
Aiming at the problems, the invention provides the application of inulin in preventing and treating small intestine diseases, mainly aims at the problems that the treatment scheme for diseases such as small intestine diseases is not comprehensive enough or no proper treatment means is available for some pathogenic reasons, and simultaneously aims at researching some new medical applications of inulin.
In order to solve the problems, the invention adopts the following technical scheme:
the inulin is applied to preparing products for preventing and treating small intestine diseases.
Wherein the small intestine disease comprises small intestine barrier disorder and/or small intestine barrier disorder complication.
Wherein, the inulin has the function of reducing the permeability of the intestinal tract of the small intestine and/or inhibiting the increase of the permeability of the intestinal tract of the small intestine in preparing the preparation for preventing and treating the small intestine diseases.
Wherein the function of the inulin in the preparation for preventing and treating small intestine diseases comprises
(i) Promoting the expression of intestinal barrier-associated protein genes, wherein the intestinal barrier-associated protein at least comprises one of tight junction protein, occludin, mucin and cadherin,
(ii) Promoting the proliferation of the small intestine goblet cells,
(iii) Promoting the expression of an intestinal antibacterial compound, wherein the intestinal antibacterial compound at least comprises one of antibacterial peptide, phospholipase A2, defensin and lysozyme C,
(iv) Inhibit the translocation of LPS in intestinal flora,
(v) Inhibiting CCR2 expression in cells that are at least Ly6Chi monocytes,
(vi) Inhibiting the migration of mononuclear cells of the peripheral blood Ly6Chi,
(vii) Reduce LPS and/or IL-1 beta levels in peripheral blood.
Wherein the small intestine barrier disorder complication comprises one of sepsis, SIRS, MODS and ischemic bowel disease.
Use of inulin, wherein the use is at least one of
(i) Use of inulin for the preparation of a cell expressing an inhibitor of secreted MMP2 and/or MMP9, preferably said cell being a T cell or a macrophage,
(ii) The application of inulin in preparing an inhibitor for infiltration of CD3+ T cells and/or CD68+ macrophages of aneurysms,
(iii) Use of inulin for preparing a polarization inhibitor of aneurysm macrophages towards M1 proinflammatory phenotype and/or a polarization promoter of macrophages towards M2 inhibing phenotype, preferably macrophages are local macrophages of aneurysm,
(iv) The application of inulin in preparing intestinal barrier-associated protein gene expression promoter comprises at least one of claudin, occludin, mucin and cadherin,
(v) The application of inulin in preparing small intestine goblet cell proliferation promoter,
(vi) The application of inulin in preparing intestinal antibacterial compound expression promoter is characterized by that the described intestinal antibacterial compound at least includes one of antibacterial peptide, phosphatidase A2, defensin and lysozyme C,
(vii) The application of inulin in preparing intestinal flora LPS translocation inhibitor,
(viii) The application of inulin in preparing a preparation for inhibiting the local bacterial load of peripheral blood and/or aneurysm,
(ix) Use of inulin for the preparation of an inhibitor of CCR2 expression in cells, including at least Ly6Chi monocytes,
(x) The application of inulin in preparing the peripheral blood Ly6Chi monocyte migration ability inhibitor,
(xi) Use of inulin for the manufacture of a formulation for reducing the level of LPS and/or IL-1 β in peripheral blood.
Use of inulin in the preparation of a formulation, wherein the use in the preparation of a formulation is at least one of
(i) The application of inulin in preparing an anti-abdominal aortic aneurysm tumor body expansion preparation,
(ii) The application of inulin in preparing preparation for inhibiting degradation of elastic fiber in outer membrane of blood vessel wall of abdominal aortic aneurysm,
(iii) The application of inulin in preparing a preparation for resisting local inflammation of an abdominal aortic aneurysm tumor body,
(iv) The application of inulin in preparing preparations for preventing and treating small intestine diseases,
(v) The application of inulin in preparing a preparation for reducing intestinal permeability and/or inhibiting intestinal permeability enhancement,
(vi) The application of inulin in preparing a preparation for preventing and treating small intestinal tract barrier disorder,
(vii) The application of inulin in preparing a preparation for preventing and treating vascular chronic inflammatory diseases, preferably, the diseases are abdominal aortic aneurysm,
(viii) Application of inulin in preparing preparation for inhibiting infiltration of proinflammatory monocytes to aneurysm is provided.
A formulation comprising inulin; wherein the preparation is at least one of
(a) The cells expressing and secreting an inhibitor of MMP2 and/or MMP9, preferably said cells are T-cells or macrophages,
(b) An inhibitor of aneurysm CD3+ T cell infiltration and/or CD68+ macrophage infiltration,
(c) An inhibitor of polarization of aneurysm macrophages towards an M1 proinflammatory phenotype and/or an enhancer of polarization of macrophages towards an M2 inhibing phenotype, preferably macrophages are local macrophages of the aneurysm,
(d) An intestinal barrier-associated protein gene expression promoter, wherein the intestinal barrier-associated protein at least comprises one of tight junction protein, occludin, mucin and cadherin,
(e) A small intestine goblet cell proliferation promoter which is capable of promoting the proliferation of small intestine goblet cells,
(f) An intestinal antibacterial compound expression promoter, wherein the intestinal antibacterial compound at least comprises one of antibacterial peptide, phospholipase A2, defensin and lysozyme C,
(g) An intestinal flora LPS translocation inhibitor is provided,
(h) A preparation for inhibiting local bacterial load of peripheral blood and/or aneurysm,
(i) An inhibitor of CCR2 expression in cells comprising at least Ly6Chi monocytes,
(j) A peripheral blood Ly6Chi monocyte migration ability inhibitor,
(k) An agent that reduces the level of LPS and/or IL-1 β in the peripheral blood of the small intestine.
A preparation for preventing and treating chronic inflammatory vascular diseases contains inulin.
A preparation for preventing and treating abdominal aortic aneurysm disease contains inulin; wherein the preparation is at least one of
(a) An anti-abdominal aortic aneurysm tumor body expansion preparation,
(b) A preparation for inhibiting the degradation of the elastic fiber of the outer membrane of the blood vessel wall of the abdominal aortic aneurysm,
(c) An anti-local inflammation preparation for abdominal aortic aneurysm,
(d) An agent that inhibits infiltration of proinflammatory monocytes to an aneurysm, preferably, the proinflammatory monocytes comprise Ly6Chi monocytes.
In some modes, the preparation for preventing and treating the abdominal aortic aneurysm disease is at least one of a medicine, a conditioning health product and a functional food.
A preparation for preventing and treating small intestine related diseases contains inulin; wherein the preparation is at least one of
(a) Preparations for reducing intestinal permeability and/or inhibiting intestinal permeability increase,
(b) A preparation for preventing and treating small intestine intestinal barrier disorder is provided.
In some forms, the preparation for preventing and treating the small intestine related diseases is at least one of a medicine, a conditioning health-care product and a functional food.
In some embodiments, the formulation is an oral formulation.
A preparation for preventing and treating small intestine barrier disorder diseases and/or small intestine barrier disorder induced diseases comprises inulin, preferably the small intestine barrier disorder induced diseases at least comprise one of sepsis, SIRS, MODS and ischemic bowel diseases, and the small intestine barrier disorder diseases comprise acute myocardial infarction and cerebral apoplexy.
The beneficial effects of the invention are:
a targeted treatment conditioning scheme is provided for partial pathogenic basic diseases of diseases such as small intestine diseases, abdominal aortic aneurysm and the like, a better solution is provided for the pathological changes caused by the pathogenic mechanisms, and a treatment effect can be achieved; meanwhile, the method overcomes the prejudice of the inulin application and develops the application of the inulin.
Drawings
FIG. 1 is a flow chart of an experiment;
FIG. 2 is a graph of the results of reduction of AAA tumor volume expansion by a high fiber inulin-rich diet;
FIG. 3 is a graph of the results of a high fiber diet rich in inulin to mitigate AAA spandex degradation;
FIG. 4 is a graph of the results of inulin-rich high fiber diet reducing MMP2 expression in tumor mass tissues;
FIG. 5 is a graph showing the results of inulin-rich high fiber diet reducing MMP9 expression in tumor mass tissues;
FIG. 6 is a graph of the results of inulin-rich high fiber diet reducing CD3+ T cell infiltration in tumor tissue;
FIG. 7 is a graph of the results of inulin-rich high fiber diet reducing CD68+ macrophage infiltration in tumor mass tissues;
FIG. 8 is a graph of the macrophage flow gating strategy and statistics for aneurysms M1, M2;
FIG. 9 is a graph showing the effect of intestinal permeability of AAA mice under different intervention conditions;
FIG. 10 shows the expression of the protein associated with the intestinal barrier function of AAA mice under different intervention conditions;
FIG. 11 shows PAS-AB staining of AAA mouse intestinal tract under different intervention conditions;
FIG. 12 shows the expression of AAA mouse gut chemical barrier associated protein under different conditioning interventions;
FIG. 13 is a graph of AAA mouse intestinal permeability, LPS translocation and their correlation with aneurysm diameter under different conditions;
FIG. 14 shows the DNA content of peripheral blood and local bacteria of aneurysm of AAA mouse;
FIG. 15 is a mouse peripheral blood Ly6Chi monocyte flow-gated strategy;
FIG. 16 is a flow chart of peripheral blood and Ly6Chi monocytes and Ly6G + neutrophils in the aneurysm.
Detailed Description
The first aspect of this section illustrates some of the uses of the invention:
use of inulin, wherein the use is at least one of
(i) Use of inulin for the preparation of a cell expressing an inhibitor of secreted MMP2 and/or MMP9, preferably said cell being a T cell or a macrophage,
(ii) The application of inulin in preparing inhibitor for infiltration of CD3+ T cells and/or CD68+ macrophages of aneurysm,
(iii) Use of inulin for preparing a polarization inhibitor of aneurysm macrophages towards a proinflammatory phenotype M1 and/or a polarization promoter of macrophages towards an inflammation repressing phenotype M2, preferably the macrophages are local macrophages of aneurysm,
(iv) The application of inulin in preparing intestinal barrier-associated protein gene expression promoter comprises at least one of claudin, occludin, mucin and cadherin,
(v) The application of inulin in preparing small intestine goblet cell proliferation promoter,
(vi) The application of inulin in preparing intestinal antimicrobial compound expression promoter comprises at least one of antimicrobial peptide, phospholipase A2, defensin and lysozyme C,
(vii) The application of inulin in preparing intestinal flora LPS translocation inhibitor,
(viii) The application of inulin in preparing a preparation for inhibiting the local bacterial load of peripheral blood and/or aneurysm,
(ix) Use of inulin for the preparation of an inhibitor of CCR2 expression in cells, said cells being at least Ly6Chi monocytes,
(x) The application of inulin in preparing the peripheral blood Ly6Chi monocyte migration ability inhibitor,
(xi) Use of inulin for the manufacture of a formulation for reducing the level of LPS and/or IL-1 β in peripheral blood.
The application of inulin in regulating pathogenic mechanism (basic disease) is disclosed, and when the diseases or indexes caused by the mechanism are abnormal, the inulin can be correspondingly used for treating, and the inulin serving as a main cost and an auxiliary material is within the scope of the invention.
Use of inulin in the preparation of a formulation, wherein the use in the preparation of a formulation is at least one of
(i) The application of inulin in preparing an anti-abdominal aortic aneurysm tumor body expansion preparation,
(ii) The application of inulin in preparing preparation for inhibiting degradation of elastic fiber of outer membrane of blood vessel wall of abdominal aortic aneurysm,
(iii) The application of inulin in preparing a preparation for resisting local inflammation of an abdominal aortic aneurysm tumor body,
(iv) The application of inulin in preparing preparations for preventing and treating small intestine diseases,
(v) The application of inulin in preparing a preparation for reducing intestinal permeability and/or inhibiting intestinal permeability enhancement,
(vi) The application of inulin in preparing a preparation for preventing and treating small intestinal tract barrier disorder,
(vii) The application of inulin in preparing preparations for preventing and treating chronic vascular inflammatory diseases, preferably abdominal aortic aneurysm,
(viii) Application of inulin in preparing preparation for inhibiting infiltration of proinflammatory monocytes to aneurysm is provided.
The application of inulin in certain diseases is disclosed, and inulin can be correspondingly used for treating the diseases, and the main cost and auxiliary materials are both within the scope of the invention.
The second aspect of this section describes some of the formulations or products of the invention:
a formulation comprising inulin; wherein the preparation is at least one of
(a) The cells expressing and secreting MMP2 and/or MMP9 inhibitors, preferably said cells being T cells or macrophages,
(b) An inhibitor of CD3+ T cell infiltration and/or CD68+ macrophage infiltration in abdominal aortic aneurysm,
(c) An inhibitor of the polarization of macrophages towards the M1 pro-inflammatory phenotype and/or an enhancer of the polarization of macrophages towards the M2 anti-inflammatory phenotype of abdominal aortic aneurysm, preferably macrophages that are local to the aneurysm,
(d) An intestinal barrier-associated protein gene expression promoter, wherein the intestinal barrier-associated protein at least comprises one of tight junction protein, occludin, mucin and cadherin,
(e) A small intestine goblet cell proliferation promoter which is capable of promoting the proliferation of small intestine goblet cells,
(f) An intestinal antibacterial compound expression promoter, wherein the intestinal antibacterial compound at least comprises one of antibacterial peptide, phospholipase A2, defensin and lysozyme C,
(g) An intestinal flora LPS translocation inhibitor is provided,
(h) A preparation for inhibiting local bacterial load of peripheral blood and/or aneurysm,
(i) An inhibitor of CCR2 expression in cells comprising at least Ly6Chi monocytes,
(j) An inhibitor of peripheral blood Ly6Chi monocyte migration ability,
(k) An agent that reduces the level of LPS and/or IL-1 β in the peripheral blood of the small intestine.
In the above, at least one or more thereof may be present unless contradictory to each other. The use of the above formulations in the treatment of particular diseases is not limited, and the formulations are within the scope of the present invention as long as they are consistent with or equivalent to the above mechanisms of action.
A preparation for preventing and treating chronic inflammatory vascular diseases contains inulin.
A preparation for preventing and treating abdominal aortic aneurysm disease contains inulin; wherein the preparation is at least one of
(a) An anti-abdominal aortic aneurysm tumor body expansion preparation,
(b) A preparation for inhibiting the degradation of elastic fiber in the outer membrane of the blood vessel wall of abdominal aortic aneurysm,
(c) An anti-local inflammation preparation for abdominal aortic aneurysm,
(d) An agent that inhibits infiltration of proinflammatory monocytes to an aneurysm, preferably the proinflammatory monocytes comprise Ly6Chi monocytes.
In some modes, the preparation for preventing and treating the abdominal aortic aneurysm disease is at least one of a medicine, a conditioning health product and a functional food.
A preparation for preventing and treating small intestine related diseases contains inulin; wherein the preparation is at least one of
(a) Preparations for reducing intestinal permeability and/or inhibiting intestinal permeability increase,
(b) A preparation for preventing and treating small intestine intestinal barrier disorder is provided.
In some forms, the preparation for preventing and treating the small intestine related diseases is at least one of a medicine, a conditioning health-care product and a functional food.
A preparation for preventing and treating small intestine barrier disorder diseases and/or small intestine barrier disorder induced diseases comprises inulin, preferably the small intestine barrier disorder induced diseases at least comprise one of sepsis, SIRS, MODS and ischemic bowel diseases, and the small intestine barrier disorder diseases comprise acute myocardial infarction and cerebral apoplexy.
In some modes, the preparation, the medicine and the conditioning health-care product are oral preparations.
The preparation of the present invention is not limited to the preparation which needs to be approved by the national drug administration for marketing, and other products with health care and conditioning effects should be within the scope of the present invention as long as the target of action is the same as (or equivalent to) the present invention. One of them is not limited to only one, and it is at least one.
Some existing researches consider that inulin has certain carcinogenicity, but the research finds that inulin has a good effect of relieving and treating partial diseases.
The aforementioned inhibitors (accelerators) include preparations which act directly or indirectly on the symptoms of the corresponding disorder or other products (e.g., conditioning foods) with reference to the problems, effects to be achieved, which the preparations or products are used for.
The third part of this section further explains each research project:
(I) Inulin for improving mouse abdominal aortic aneurysm
The method comprises the following steps: the addition of fermentable fiber "inulin" or low-fermented fiber "cellulose" simulates a normal diet (5% cellulose) and two high-fiber diets (15% inulin, 15% cellulose, respectively). After 8-10 weeks old male C57BL/6 mice are given 4 weeks of normal diet or high fiber diet, a PPE-induced AAA model is constructed and fed for 2 weeks to evaluate the AAA disease progression of the mice. Separating a whole aorta after euthanasia of the mouse, measuring the diameter of a tumor body in vitro and evaluating the expansion degree of the AAA tumor body; fixing aneurysm tissues, embedding paraffin, slicing, and evaluating the degradation degree of elastic fibers by elastic fiber staining (EVG); immunohistochemical staining (IHC) to assess the expression of Matrix Metalloproteinases (MMP) 2 and MMP9 in tumor bodies; IHC evaluation of CD3 + T cells and CD68 + Infiltration of macrophages in the tumor mass; flow cytometry is used for detecting the proportions of local macrophages M1, M2 and M1/M2 of the aneurysm.
As a result: the inulin-rich high fiber diet can reduce the dietary intake of inulin compared with the normal diet and the cellulose-rich high fiber dietThe tumor body expansion of the PPE-AAA mouse is light, the expression of matrix metalloproteinases MMP2 and MMP9 of the tumor body tissue of the PPE-AAA mouse and the degradation of elastic fibers are reduced, and the tumor body tissue CD3 of the PPE-AAA mouse is reduced + T cells and CD68 + Macrophage infiltration inhibits local macrophage differentiation to M1 type of tumor body, promotes macrophage differentiation to M2 type, thereby reducing M1/M2 ratio. Compared to the normal diet, a high-fiber diet rich in cellulose does not have these effects.
And (4) conclusion: compared with the common diet and the high-fiber diet rich in cellulose, the high-fiber diet rich in inulin can protect mouse AAA, reduce the expansion of mouse AAA tumor body and the degradation of elastic fiber, improve local inflammation of tumor body, and make local microenvironment of tumor body show the characteristics of anti-inflammatory repair. Whereas a high fiber diet rich in cellulose does not have these AAA protective effects.
(II) inulin improves intestinal barrier function and reduces bacterial translocation and systemic inflammatory response caused thereby
The method comprises the following steps: the addition of fermentable fiber "inulin" or low-fermented fiber "cellulose" simulates a normal diet (5% cellulose) and two high-fiber diets (15% inulin, 15% cellulose, respectively). After 8-10 weeks old male C57BL/6 mice were given either normal diet or high fiber diet for 4 weeks, a PPE-induced AAA model was constructed and fed for 2 weeks, and peripheral blood, aneurysm and intestinal tissue were removed. The intestinal tissue fixed embedded section was subjected to PAS-AB staining, and the number of small intestine and colon goblet cells and mucin content were observed. Real-time PCR detects the mRNA expression level of ZO-1, occludin, muc2, CDH1 and antimicrobial peptide Reg3 gamma III, pla2g2, alpha-defensens and lysozyme C related to barrier function in small intestine and colon tissues. In addition, the mice were fasted overnight the day before sacrifice, gavaged with 4 kDFITC-dextran, and peripheral blood plasma was taken to measure FITC fluorescence intensity for indication of intestinal permeability. Detecting plasma LPS by an end-point color development method; ELISA detects the plasma systemic inflammation index IL-1 beta; detecting the total amount of peripheral blood and aneurysm translocation bacteria by bacterial quantitative PCR; flow cytometry detection of Ly6C in peripheral blood hi 、Ly6C low Monocyte, ly6G + Proportion of neutrophils, chemokine receptor (C-C chemokine receptor, CC) in monocytesR) 2 expression, and Ly6C in part of aneurysm hi Proportion of monocytes.
As a result: a high-fiber diet rich in inulin but not cellulose can reduce the intestinal permeability of AAA mice, increase the number of small intestine goblet cells, the expression of intestinal barrier function-related proteins and antibacterial peptide genes, but has no obvious influence on the colon. A high fiber diet rich in inulin, but not cellulose, reduced levels of LPS in the peripheral blood and IL-1 β, a systemic inflammatory marker in AAA mice. The intestinal permeability FITC of the AAA mouse is positively correlated with the diameters of LPS and aneurysm, and the diameters of LPS and aneurysm are also positively correlated. The inulin-rich high fiber diet reduced the bacterial load in the peripheral blood and in the local aneurysm of AAA mice. A high fiber diet rich in inulin rather than cellulose can reduce the peripheral blood proinflammatory Ly6C of AAA mice hi Monocyte proportion, while inhibiting local migration to the aneurysm by downregulating CCR2 expression, but for Ly6C low Monocytes and Ly6G + Neutrophils had no significant effect. Furthermore, local Ly6Chi monocyte infiltration of aneurysms was reduced in AAA mice fed a high fiber diet rich in inulin, but not cellulose.
And (4) conclusion: the inulin-rich high-fiber diet can promote small intestine goblet cell increase and intestinal epithelial barrier function-related protein and antibacterial peptide expression increase, thereby protecting intestinal barrier function, which is not possessed by the cellulose-rich high-fiber diet. Inulin-rich high fiber diets inhibit Ly6C by protecting intestinal barrier function, reducing bacterial translocation and LPS translocation as a component thereof hi The monocyte-mediated systemic inflammatory reaction and its infiltration into the local part of the aneurysm delay the progression of abdominal aortic aneurysm.
In the fourth aspect of this section, the association of inulin with abdominal aneurysm and the like is described in conjunction with an experimental project:
content of the experiment
1 materials of the experiment
1.1 Experimental animals: male mice were housed in SPF-grade laboratory animal centers at the institute of coyohan medicine, wuhan, at week 8-10, and given different custom diets according to experimental groups. Animal treatment was carried out according to the guidelines for laboratory animal feeding and use issued by the national institutes of health and the regulations of laboratory animal management of Tongji medical college of university of science and technology in Huazhong. Approved by the subsidiary of the college of science and technology of Huazhong university and the ethical committee of hospitals.
1.2 mouse feeding and feed formula: the feed for feeding mice with common diet is common feed (D10012M) containing 5% cellulose. The high fiber diet mice were fed on two groups, one group with high fiber diet feed containing 15% cellulose (RD 18013004) and the other group with high fiber diet feed containing 15% inulin (RD 18013005). The feed was changed every three days. AAA molding was performed 4 weeks after the custom feed feeding, see fig. 1. After irradiation sterilization, the obtained product enters an SPF animal experiment center for feeding mice. The feed formula is as follows:
1.3 Main Experimental instruments
1.4 Primary reagents and antibodies
1.5 solution preparation
1.5.1 pentobarbital sodium solution: the concentration of the sodium pentobarbital-physiological saline solution used in the experiment was 1%. Weighing 100mg sodium pentobarbital powder, dissolving with 10ml sterile normal saline, mixing well, and storing at room temperature. The dosage is 10 mul/g when anaesthetizing and 20 mul/g when euthanasia, and the injection is carried out in the abdominal cavity. 1.5.2 Phosphate Buffered Saline (PBS)
Mixing, adjusting pH to 7.4 to obtain 0.01M PBS, autoclaving, packaging, and storing at 4 deg.C.
1.5.3 heparin sodium solution: 1L sterile physiological saline dissolved 100mg heparin sodium powder.
1.5.4 solutions required for staining tissue sections
| VanGieseson's liquid | 1ml of 1% acid fuchsin and 45ml of saturated picric acid. |
| Alcohol hematoxylin | 50ml of absolute ethyl alcohol and 2.5g of hematoxylin are heated slightly. |
| Verhoeff's hematoxylin | 40ml of alcohol hematoxylin, 16ml of 10% ferric trichloride and 16ml of Lugol iodine solution are sequentially added, and the mixture is prepared immediately before use. |
| Lugol iodine liquid | I210g/L and KI20g/L, and storing in dark. |
| 1% hydrochloric acid alcohol | Concentrated hydrochloric acid (1ml) and 70% ethanol (99 ml). |
| Sodium citrate repair liquid | 21.01g/L of citric acid 9ml,29.41g/L of sodium citrate 41ml and H2O450ml, and adjusting the pH value to 6.0. |
| PBST buffer solution | Twenn 201.5ml, PBS500ml,4 ℃. |
| 5% BSA solution | BSA5g, PBST100ml,4 ℃ storage. |
2 Experimental methods
2.1PPE elastase induced mouse AAA model
1) Pentobarbital sodium anesthesia;
2) Fixing in supine position, removing hair from abdomen after alcohol disinfection, disinfecting skin of abdomen again, and cutting skin and muscle layer along abdomen median line;
3) Clamping a sterile cotton ball by using forceps, gently poking the intestine of the abdominal cavity from the lower right to the upper left, and stripping the adipose tissues around the abdominal aorta by using microscopic forceps under a body type microscope to expose the abdominal aorta;
4) Soaking cotton piece with PPE solution, wrapping exposed artery, and changing PPE into normal saline in sham surgery group;
5) Taking out the cotton piece after 40min, washing abdomen with physiological saline for 2 times, suturing abdomen intermittently, and placing the mouse on the electric blanket until it is awake;
6) Mice were euthanized at day 14 post-molding and subjected to various evaluations.
2.2 measurement of tumor body diameter in mice
1) After the mouse is euthanized, the syringe sucks heparin, and a needle is inserted from the apex of the heart to flush the artery;
2) Separating the whole aorta with a microscopy instrument under a body type microscope;
3) Single shot (with scale);
4) image J measurement of maximum aneurysm diameter
2.3 tissue fixation, paraffin embedding, sectioning
1) Fixing a sample by taking 4% Paraformaldehyde (PFA);
2) Washing away the PFA: after the fixation is finished, flushing for 3 times with running water, 5min each time;
3) Alcohol gradient dehydration: 50% -70% -80% -95% -95% of alcohol for 2h respectively; 40min 100% ethanol, 2 times;
4) And (3) transparency: 5min-30min of 50% ethanol and 50% dimethylbenzene, observing the tissue form in time in the process of transparency, adjusting the transparency time, and observing whether the tissue is successfully transparent under illumination, wherein the transparency is moderate;
5) Wax dipping: softening wax and then hardening wax at 60 deg.C for 2 times, each time for 50min;
6) Embedding: quickly putting the tissue block into a mould containing wax liquid, wherein the section of the required tissue is parallel to the bottom, so that the wax liquid is prevented from being solidified in a cold environment;
7) Slicing: the thickness of each slice is 4 μm; AAA was performed by cutting 8 sections per tumor at 100 μm intervals.
2.4EVG staining
1) Fixing, embedding, slicing, dewaxing by xylene for 10min,
2) Hydration: 5min of absolute ethyl alcohol and 2 times; 95% -90% -80% -70% of alcohol for 5min respectively; washing with water;
3) Staining verhoeff's hematoxylin at room temperature for 30min until the color is dark black, and washing with water;
4) Differentiating by using a ferric trichloride solution (2 percent), and washing by using water;
5) Sodium thiosulfate (5%) for 1min, and washing with water;
6) Counter-dyeing with Van Gieseon's solution for 5min, washing with water, dehydrating, and sealing with resin.
2.5 immunohistochemical staining (IHC)
1) High-pressure antigen retrieval: 0.01M sodium citrate buffer;
2)3％H 2 O 2 incubating for 10min;
3) PBST washing for 5min, shaking table, washing for 3 times;
4) 5% BSA blocking for 30min;
5) Primary antibody was incubated overnight at 4 ℃ (antibodies used in this study were diluted against mouse CD3, CD68, MMP2, MMP9, 1;
6) Washing with PBST on a shaking table for 5min,3 times;
7) Incubating a secondary antibody, namely IgG-HRP horse radish peroxidase, at 37 ℃ for 30min;
8) PBST washing for 5min, shaking table, washing for 3 times;
9) Developing with DAB developing solution for 2min, and washing with water;
10 Hematoxylin counterstain for 10s, and washing with water;
11 Bluing, transparent, mounting.
2.6 tissue section statistics: sections were observed microscopically and photographed (with a ruler), adobe Photoshop and Image Pro Plus to calculate the positive area ratio.
EVG staining rate is expressed as the percentage of positive staining area to total aortic cross-sectional area.
Number of CD68+ macrophages and CD3+ T cells: by calculating the average number of positively stained cells on each cross section of the aneurysm.
Content of matrix metalloproteinases MMP2, MMP 9: the ratio of the area of positive staining to the total cross-sectional area, and the average of 4-8 sections per mouse.
2.7 flow cytometry
2.7.1 preparation of mouse arterial Single cell suspension
1) Separating the whole aorta under a microscope, and thoroughly shearing the aorta by using a pair of micro scissors;
digesting for 2min at 37 ℃ for 3 times by using vascular digestive enzyme, wherein 800 mu l of enzyme is used for digesting each blood vessel;
3) Collecting digestive juice, filtering with a filter screen, washing with PBS, and then resuspending to obtain a vascular single cell suspension;
2.7.2 flow cytostaining and flow analysis
1) Transferring the single cell suspension to a flow tube, and reserving 100 mu l of liquid;
2) Adding a cell membrane surface labeled antibody, and incubating for 30min at 4 ℃ in a dark place: arterial macrophages (anti-CD 45-APCcy7, anti-CD 11b-PerCp/cy5.5, anti-F4/80-BV 421, anti-CD 206-PEcy7, anti-I-A/I-E-APC);
3) Washing the antibody with PBS, adding 200 μ l of IC fixative for fixation, and incubating at room temperature in dark for 30min;
4) Washing the fixed solution with PBS, and adding 100 μ l PBS for resuspension;
5) Adding 5 mul of flow Counting microspheres Counting Beads before loading;
6) Flow cytometry analysis detected total number of cells =5 x 1.03 x 10^3 x number of flow detection cells/number of flow detection beads. 3. The statistical test method comprises the following steps: data are expressed as mean ± SEM. Statistical analysis was performed using GraphPad Prism software (GraphPad Prism 8.0software Inc.). Normal test was analyzed by Kolmogorov-Smirnov and Shapiro-Wilk test. If the data fit to a normal distribution, one-way analysis of variance and Tukey multiple comparison tests are used in the group 3 statistics. For non-normally distributed data, a nonparametric Mann-WhitneyU test was used. P <0.05 is statistically significant and the sample sizes are labeled in the respective graphs.
Analysis of results
Inulin for reducing PPE-AAA mouse tumor body expansion
Separating the whole aorta under a body type microscope, photographing and measuring the maximum diameter of an aneurysm body; the elastase PPE-induced AAA model was established after C57 mice were fed with normal diet (CD), 15% cellulose diet (HFD-Cell), 15% inulin diet (HFD-In) for 4 weeks, and after feeding with the custom diet for 14 days, images were taken and aneurysm body diameter (infrarenal maximum diameter) was measured. The results showed that the AAA tumor body diameter of the inulin-rich high-fiber diet group mice was significantly smaller than that of the normal diet group and the cellulose-rich high-fiber diet group, and the AAA tumor body diameter of the normal diet group mice was not significantly statistically different from that of the cellulose-rich high-fiber diet group (fig. 2). * P <0.05, P <0.01, P <0.001, by one-way analysis of variance and Tukey multiple comparison test (same below). This indicates that inulin-rich high fiber diet can slow AAA disease progression, whereas cellulose-rich high fiber diet does not slow AAA, with different ingredients of high fiber diet having different effects on AAA.
(II) inulin can reduce PPE-AAA mouse tumor body elastic fiber degradation and tissue matrix metalloproteinases MMP2 and MMP9 expression
Further analysis of whether AAA tumor body expansion was accompanied by characteristic pathological changes of mesangial elastic fiber degradation by EVG staining revealed that the degradation of outer membrane elastic fiber was reduced in the mouse AAA in the inulin-rich high-fiber diet group and was not significantly improved in the cellulose-rich high-fiber diet group compared to the normal diet and the cellulose-rich high-fiber diet group (fig. 3). C57 mice were fed with normal feed (CD), 15% cellulose feed (HFD-Cell), and 15% inulin feed (HFD-In). Since cells express and secrete matrix metalloproteinases MMP2 and MMP9 are important mechanisms for degrading the extracellular matrix, the expression of MMP2 and MMP9 in tumor body tissues was examined by IHC. The results were consistent with the degree of degradation of the adventitial elastic fibers, i.e., the expression of matrix metalloproteinases MMP2 (FIG. 4) and MMP9 (FIG. 5) was reduced in the inulin-rich high-fiber diet group mouse tumor body compared to the normal diet and the cellulose-rich high-fiber diet, and there was no statistical difference between the normal diet group and the cellulose-rich high-fiber diet group. C57 mice were fed with normal feed (CD), 15% cellulose feed (HFD-Cell), and 15% inulin feed (HFD-In).
(III) inulin reduces infiltration of PPE-AAA mouse tumor tissue CD3+ T cells and CD68+ macrophages
T cells and macrophages are the most highly enriched cells in AAA and have the ability to secrete various inflammatory cytokines and matrix metalloproteinases MMP2, MMP9, thereby promoting the inflammatory response and exacerbating AAA progression. Evaluation of local CD3 in tumor tissue by immunohistochemical staining + T cells and CD68 + Infiltration of macrophages. The results showed that the local CD3+ T cells (fig. 6) and CD68+ macrophages (fig. 7) were significantly less infiltrated by AAA tumor bodies in the inulin-rich high-fiber diet group compared to the normal diet and the cellulose-rich high-fiber diet group. C57 mice were fed with common feed (CD), 15% cellulose feed (HFD-Cell) and 15% inulin feed (HFD-In) for 4 weeks, and then AAA model induced by elastase PPE was established, and after feeding with the custom feed for 14 days, AAA tumor body was fixed and embedded, and CD3 and CD68 were immunohistochemically stained. Statistical analysis of the number of positive-stained cells in the cross-section under a 10-fold microscope, each point representing the average of 3-4 sections of the same tumor mass. Inulin-rich high-fiber diets can ameliorate local inflammation of AAA nodules, whereas cellulose-rich high-fiber diets do not.
(IV) inulin inhibits local macrophage polarization to pro-inflammatory phenotype in aneurysms
In view of the central role of macrophage proinflammatory M1 and inflammation-suppressive M2 balance in AAA, the M1 and M2 phenotypes of local macrophages of the aneurysm are analyzed in a flow mode (A in figure 8), and a graph A is a flow gate diagram of the local M1 and M2 macrophages of the aneurysm. Compared with the ordinary diet and the high-fiber diet rich in cellulose, the AAA local high-fiber diet group rich in inulin has reduced M1 type (I-A/I-E + CD 206-) macrophages and increased M2 type (I-A/I-E-CD 206 +) macrophages, and M1/M2 is obviously reduced (B in figure 8), and a B picture in figure 8 is a statistical chart of the proportions of M1, M2 and M1/M2 in local aneurysms. n =5, <0.05, <0.01, <0.001. The results show that inulin polarizes macrophages towards an M2 type inflammation-inhibiting phenotype, inhibits the positive feedback formed by inflammatory reaction in the AAA dilatation process and plays a role in AAA protection.
Animal experiments show that inulin can reduce the degradation of the tunica elastica fibers in the blood vessel wall of AAA through reducing the expression of matrix metalloproteinases MMP2 and MMP9, relieve the infiltration of local CD3+ T cells and CD68+ macrophages of aneurysm, inhibit the inclination of the local macrophages of the aneurysm to a proinflammatory M1 phenotype, and improve the expansion of AAA tumor body, but high fibers rich in cellulose do not have the effect.
The fifth aspect of this section is described with reference to an experimental project, which relates inulin to intestinal function and the like:
content of the experiment
1 materials of the experiment
1.1 Experimental animals: corresponding matters to the fourth aspect
1.2 mouse feeding and feed formula: corresponding matters to the fourth aspect
1.3 Main laboratory instruments: in addition to the apparatus used in the fourth aspect, the following apparatus is also used:
1.4 Primary reagents and antibodies: in addition to the reagents used in the fourth aspect, the following reagents are also required:
1.5 solution preparation:
1.5.1 Mixed solution of vascular digestive enzymes (10X)
Stored at-20 ℃ and diluted 10-fold with PBS before use.
1.5.2 flow cytostaining buffer
Adjusting pH to 7.2-7.4, and storing at 4 deg.C.
2. Experimental methods
2.1 Elastin (PPE) induced Abdominal aneurysm model in mice (see the fourth aspect for details)
2.2 intestinal permeability assay
1) PPE-AAA model was established 4 weeks after feeding mice with plain diet (CD), cellulose-rich high fiber diet (HFD-Cell), inulin-rich high fiber diet (HFD-In). After feeding the custom feed for 13 days, fasting overnight, and gavage with 4kD FITC-dextran;
2) 4 hours after the lavage, the mouse is killed by excessive anesthetic, the peripheral blood is taken out from a pyrogen-free EDTA anticoagulation tube, and hemolysis is avoided as much as possible when the blood is taken out;
3) Centrifuging peripheral blood at 3000rpm for 5min, collecting supernatant as blood plasma, packaging, and storing at-80 deg.C;
4) The FITC fluorescence intensity was measured by pipetting 40. Mu.l of plasma into a clear 96-well plate at 485 excitation/528 nm emission.
2.3Real-time PCR detection of expression of intestinal tissue barrier-associated protein and antibacterial peptide
2.3.1 intestinal Total RNA extraction: phenol-chloroform extraction process
1) Taking intestinal tissues from which contents are removed and which are rinsed clean by ice PBS, shearing 2cm sections of small intestine and colon intestine close to cecum by using high-pressure scissors, putting the small intestine and colon intestine sections into a 1.5ml centrifuge tube, and adding 2 RNase-free grinding beads and 1ml Trizol;
2) Homogenizing at 100rpm for 5 times in a rapid low-temperature homogenizer;
standing on ice for 30-60min;
4) Adding 200 μ l chloroform, covering the tube cover tightly, shaking for 15s, standing on ice for 10min for layering, and pre-cooling the centrifuge;
5) Centrifuging at 4 ℃ at 12000rpm/min for 15min;
6) Transferring the centrifuged upper layer liquid (water phase) into a new 1.5ml centrifuge tube (about 400 μ l), taking care not to absorb the white middle layer liquid and the red lower layer liquid, adding equal volume of isopropanol, shaking and mixing for 10s, and standing on ice for 10min;
7) Centrifuging at 12000rpm/min at 4 ℃ for 15min;
8) Discarding the supernatant, adding 1ml of anhydrous ethanol (precooling), shaking up and down, mixing uniformly, and centrifuging at 7500rpm/min at 4 ℃ for 5min;
9) Discarding the supernatant, air-drying in a superclean bench, adding 20-40 μ l DEPC water in the tube to dissolve the total RNA;
10 Purity and concentration of RNA were measured by NanoDrop, and purity was expressed as the ratio of absorbance at 260nm to absorbance at 280nm, preferably 1.85 to 2.05.
2.3.2 reverse transcription
The extracted total RNA is reversely transcribed into cDNA by a reverse transcription reagent of TaKaRa company, and the operation is carried out according to the instruction, and the specific operation is as follows:
the reverse transcription reaction system was configured as follows (total volume 20. Mu.l):
the reverse transcription reaction conditions were as follows:
the reverse transcription reaction product cDNA was stored at-20 ℃.
2.3.3real-time PCR reaction
1) A Real-time PCR kit of TaKaRa is adopted to detect the expression quantity of a target gene in cDNA, and a 10 mu l system is configured as follows:
2) Performing Real-time PCR reaction on a Real-time fluorescent quantitative PCR instrument under the following reaction conditions;
3) Calculating the relative expression amount of the target gene:
the reference gene is GAPDH, the relative value of the sample to be detected is = 2-delta Ct, delta Ct = Ct target gene-CtGAPDH, and delta Ct = delta Ct reference of the sample to be detected.
2.3.4 primer design
Based on the cDNA sequence of Genebank, the Primer 3.0 webpage is used for design, and the Primer sequences are as follows:
2.4 Alisin Blue-Periodic acid-Schiff dyeing (pharmacin Blue Periodic acid Schiff, PAS-AB)
PAS-AB staining was negative, i.e., non-mucin. ( And (3) red color: glycogen, neutral mucus; blue color: acid mucus, bluish violet: mixing the mucus. )
Cutting intestinal sections of small intestine and colon near cecum, fixing with Ka's stationary liquid overnight, embedding in paraffin, and slicing;
dewaxing, washing (details of the steps are shown in the related content of the fourth aspect)
Dyeing with AB staining solution (PH 2.5 Ali new blue staining solution) for 5-10min, and washing with water until no dye falls off;
PAS staining solution A reagent (periodic acid) stains 5miin, and distilled water is used for rinsing lightly;
dyeing with PAS dyeing liquid B reagent (Schiff reagent) for 5-10min, and washing with water;
dehydrating, transparent and sealing.
2.5LPS detection (end-point visualization)
According to the operation of the limulus kit for detecting endotoxin of BIOENDO company (pyrogen-free consumables are required), the method comprises the following steps:
sample pretreatment:
collecting blood with heparin sodium anticoagulation tube, centrifuging at 3000rpm for 5min, and collecting supernatant (100 μ l plasma); adding 900 mul sample treatment fluid, mixing uniformly to obtain 10X dilution
Heating in 70 deg.C water bath for 10min, sealing to prevent water droplet from flowing in;
cooling with ice water for 3min;
measuring absorbance of blank tube (hemolytic sample need to deduct absorbance of blank tube)
Adding 200 mul endotoxin detection water into 100 mul plasma 10X diluent, sequentially adding 500 mul azotization reagent 1, 500 mul azotization reagent 2 and 500 mul azotization reagent 3, uniformly mixing, and measuring the absorbance of a sample blank tube at the lambda =545 nm;
preparation of bacterial endotoxin standard solution (for use in situ, for use within 4 h)
Taking 1 bottle of bacterial endotoxin standard substance, adding 1ml of water for bacterial endotoxin detection, and violently shaking for 15 minutes on a vortex mixer, wherein attention is paid to the fact that the bacterial endotoxin standard substance cannot splash out in the shaking process;
the above solution was diluted with endotoxin test water to 1.0 EU/ml.
Respectively configuring 0.01U/ml-0.1EU/ml according to the following table; 0.1U/ml-1U/ml standard solution
1) 100 μ l endotoxin test water (negative control)/endotoxin standard solution/test sample (pretreated 10X dilution);
2) Adding 100 mul limulus reagent solution, and mixing;
3) Incubation at 37 ℃;
4) Adding 100 μ l of chromogenic matrix solution, mixing, and incubating at 37 deg.C;
5) Sequentially adding 500 mul of solution 1, solution 2 and solution 3 of the azo reagent, and uniformly mixing;
6) Standing for 5min, measuring absorbance at 545nm wavelength
Data processing: and drawing a standard curve (linear relation should be adopted) according to the absorbance of the standard solution, wherein the ordinate is the absorbance value, and the abscissa is the endotoxin concentration. And solving the concentration of the sample according to the absorbance of the sample to be detected.
Quantitative PCR of bacteria
Genomic DNA extraction
The use of the TIANGEN genomic DNA extraction kit:
after weighing, the tissue was treated:
adding erythrocyte lysate with three times volume of peripheral blood, reversing and uniformly mixing, standing at room temperature for 5min, centrifuging at 10000rpm for 1min, and sucking supernatant to obtain precipitate, namely leukocyte; grinding the aneurysm tissue into single cell suspension by using a glass homogenizer, centrifuging at 10000rpm for 1min, and sucking the supernatant; adding 110. Mu.l of TE buffer (20mM Tris, pH8.0, 2mM Na2-EDTA;1.2% Triton) and 70. Mu.l of lysozyme solution (50 mg/ml) to the precipitate, shaking and mixing, and water-bathing at 37 ℃ for 30min;
adding 20 μ l of protease K solution, mixing (mixing aneurysm tissue, placing at 56 deg.C, dissolving tissue, and removing wall water droplet instantly);
adding 200 μ l buffer GB, mixing, standing at 70 deg.C for 10min (the solution becomes clear), and removing water drops on the tube wall instantly;
adding 200 μ l of anhydrous ethanol, shaking and mixing for 15s (flocculent precipitate appears), and removing water drops on the tube wall instantly;
adding the solution and flocculent precipitate into adsorption column CB3 (placing adsorption column into collection tube), centrifuging at 12000rpm for 30s, discarding waste liquid, and placing adsorption column back into collection tube;
centrifuging at 12000rpm for 1min with 500 μ l buffer GD, discarding the waste liquid, and returning the adsorption column to the collection tube;
centrifuging at 12000rpm for 1min with 600 μ l rinsing solution PW, discarding the waste liquid, and placing the adsorption column back to the collecting tube for one time;
centrifuging at 12000rpm for 1min without adding any liquid, discarding the waste liquid, and air-drying the adsorption column at room temperature;
placing the adsorption column into a new centrifuge tube, suspending and dropwise adding 20 μ l DEPC water into the middle part of the adsorption membrane, centrifuging at 12000rpm for 2min, and collecting the liquid in the centrifuge tube as total DNA; the storage is repeated once at-20 ℃.
2.6 drawing a standard curve
Cloning a target gene PCR product to a plasmid vector;
plasmid standards (1 ng/. Mu.l) were diluted in 10-fold gradients and plasmid copy number = (ng/. Mu.l). 10^9 ^ 6.02 ^ 1023/(660. Mu.450bp) = copies/. Mu.l calculated
Ct value was determined by quantitative PCR (specific procedure as follows) and a standard curve was drawn: log (copy number) is linear with Ct value; 2.7 quantitative PCR of bacteria
1) Fluorescent quantitative PCR System establishment (15. Mu.l)
2) Performing Real-time PCR reaction on a Real-time fluorescent quantitative PCR instrument under the following reaction conditions;
3) Substituting Ct value into standard curve equation to obtain copy number
2.8 detection of IL-1. Beta. By ELISA
The IL 1. Beta. Concentration of the mouse plasma stock solution was measured using ELISA kit manufactured by NEOSCIENCE, according to the following protocol:
1) Balancing the lath to room temperature, adding the standard substance after gradient dilution to the blank hole, adding the sample to the other holes, sealing the plate with a membrane seal plate, and incubating for 90min at 37 ℃;
2) Preparing a biotinylation antibody working solution 20min in advance;
3) Washing the plate for 5 times;
4) 100 μ l biotinylated antibody: adding diluent into blank holes, processing other holes as liquid, sealing plates and membrane sealing plates, and incubating at 37 ℃ for 90min;
5) Preparing an enzyme conjugate working solution 20min in advance, and placing the solution at room temperature in a dark place;
6) Washing the plate for 5 times;
7) 100 μ l of enzyme conjugate: adding diluent into blank holes, processing other holes as liquid, sealing plates and membrane sealing plates, and incubating at 37 ℃ for 90min;
8) Washing the plate for 5 times;
9) Adding 100 mul of chromogenic substrate into each hole, incubating for 15min at 37 ℃ in a dark place, and preheating an enzyme labeling instrument;
10 Add 100. Mu.l of reaction stop solution into each well, mix well and measure OD450 value;
11 Drawing a standard curve according to the OD value of the standard sample, substituting the OD value of the measured sample, and calculating the concentration of the sample to be measured.
2.9 flow cytometry: preparation of mouse peripheral blood mononuclear cell suspension
1) After the mice die, 500. Mu.l of peripheral blood is taken and added with 1ml of PBS to be mixed evenly;
2) Slowly adding the above liquid into 1.5ml lymphocyte separation liquid, centrifuging at 2350rpm density gradient for 20min at room temperature, +9/-0;
3) Centrifuging, sucking the middle white cell layer, adding PBS to 15ml, washing, centrifuging at 500G for 10min, and removing the supernatant;
4) Resuspending the cell pellet to obtain peripheral blood mononuclear cells;
2.10 preparation of mouse arterial Single cell suspension (same as described in the fourth aspect)
2.11 flow cytostaining and flow analysis
1) Transferring the single cell suspension to a flow tube, and reserving 100 mu l of liquid;
2) Adding a cell membrane surface labeled antibody, and incubating at 4 ℃ in a dark place for 30min: peripheral blood mononuclear/neutrophils (anti-CD 45-FITC, anti-CD 11b-PerCp/cy5.5, anti-Ly 6C-PEcy7, anti-Ly 6G-APCcy7, anti-CCR 2-BV 605); arterial monocytes (anti-CD 45-APCcy7, anti-CD 11b-PerCp/cy5.5, anti-Ly 6C-FITC), dilution ratio refer to the description;
3) Washing the antibody with PBS, adding 200 μ l of IC fixative, fixing, and incubating at room temperature in dark for 30min;
4) Washing away the fixing solution with PBS, and adding 100. Mu.l of PBS for resuspension;
5) Adding 5 mul of flow Counting microspheres Counting Beads before loading;
6) Flow cytometry analysis detected total number of cells =5 x 1.03 x 10^3 x number of flow detection cells/number of flow detection beads.
3. The statistical test method comprises the following steps: corresponding to the fourth aspect above.
Analysis of results
Inulin for protecting intestinal barrier function
Intestinal permeability was measured in high fiber diet and regular diet AAA mice. AAA mice were fasted overnight before sacrifice, gavaged with 4kD FITC-dextran, and plasma was taken 4 hours after gavage to detect FITC fluorescence intensity. The more fully functioning the intestinal barrier, the lower the FITC-dextran strength in the blood, i.e. in the plasma, of the mice.
The results show that the common diet group has no obvious difference from the cellulose diet group; plasma FITC fluorescence intensity was significantly reduced in AAA mice in the inulin-rich high fiber diet group compared to the normal diet and cellulose diet (fig. 9). This indicates that inulin decreases intestinal permeability in AAA mice.
The intestinal barrier was evaluated in high fiber diet and general diet AAA mice. By examining the gene expression of zonulin (ZO-1), occludin (Occludin), mucin (Muc 2) and cadherin (CDH 1) associated with barrier function in the tissues of the small intestine and colon, we found that inulin can promote the gene expression of proteins associated with barrier function of the small intestine without significant effect on the colon, and that cellulose has no significant improvement in barrier function of the intestinal tract (fig. 10). The number of small intestine and colon goblet cells and mucin content were examined by PAS-AB staining, and as a result, it was found that the number of small intestine Goblet Cells (GC) was increased in AAA mice on inulin-rich high fiber diet and the intracellular mucin content was abundant without a significant increase in the number of colon goblet cells, as compared to ordinary diet and cellulose-rich high fiber diet (fig. 11). Finally, we evaluated the chemical barrier function of AAA mice on high and normal diets by examining the gene expression of intestinal tissue antimicrobial peptide (Reg 3 γ iii), phospholipase A2 (Pla 2g 2), defensin (α -defensins), lysozyme C (lysozyme C). The results show that inulin-rich high-fiber diet can significantly increase the expression of AAA mouse antibacterial complex, including α -defenses in the small intestine, lysozyme C, and Pla2g2 in the colon, whereas cellulose-rich high-fiber diet does not have this effect (fig. 12).
The FITC fluorescence intensity was measured on normal diet (CD), cellulose-rich high-fiber diet (HFD-Cell), inulin-rich high-fiber diet (HFD-In) AAA mice, plasma from 4kD FITC-dextran after intragastric administration. Each dot represents one mouse, P <0.05, P <0.01, P <0.001. The test method comprises single-factor analysis of variance and Tukey multiple comparison test, the same below.
The results show that in AAA mice, the inulin-rich high fiber diet has an intestinal barrier protective function, whereas the cellulose-rich high fiber diet does not. Inulin can reduce intestinal permeability, and mainly promote intestinal barrier integrity of small intestine, without significant improvement on intestinal barrier of colon.
(II) inulin can improve intestinal permeability of AAA mouse, reduce LPS in peripheral blood and protect AAA mouse from systemic inflammation level
Bacterial endotoxin LPS, the most abundant component of the cell wall of gram-negative bacteria in the intestinal tract, is often used to indicate bacterial translocation. Our experimental results show that LPS and its induced systemic inflammation marker IL-1 β levels are reduced in plasma of inulin-rich high fiber diet AAA mice with more complete intestinal barrier function than both regular diet and cellulose-rich high fiber diet (a, B in fig. 13).
In the correlation analysis of intestinal permeability with LPS, the FITC-dextran correlated positively with endotoxin LPS levels (C in fig. 13, R2=0.7126, p = 0.0011), indicating that increased plasma LPS levels in AAA mice were associated with increased intestinal permeability. Plasma endotoxin LPS levels in AAA mice were positively correlated with AAA diameter (D in fig. 13, R2=0.7658, p = 0.0004), indicating that the higher the LPS level, the more severe the AAA tumor body expansion in mice. AAA mouse intestinal permeability was positively correlated with aneurysm diameter (E, R2=0.8962, p-slas 0.0001 in fig. 13), which is consistent with the trend for high fiber diets to improve AAA, suggesting that inulin-rich high fiber diets exert AAA protective effects by protecting intestinal barrier function. The intestinal permeability FITC, the endotoxin LPS and the AAA diameter are related, which suggests that the high-fiber diet can reduce the translocation of the intestinal flora LPS by improving the intestinal barrier function of mice, thereby protecting the AAA.
(III) inulin reduces the bacterial load of peripheral blood and local aneurysm of AAA mouse
A high fiber diet improves gut barrier function and may affect inflammatory responses and AAA disease progression by reducing the total amount of translocating bacteria.
In order to investigate the presence of translocation bacteria in the circulation of AAA mice and in the focus of aneurysm, and the influence of inulin-rich high-fiber diet on the total amount of translocation bacteria, the bacterial load, i.e.bacterial DNA copy number, in peripheral blood and in the local region of aneurysm was determined by the method of bacterial quantitative PCR. The results indicate that translocating bacteria are present in the blood and aneurysms of AAA mice, and that a high fiber diet rich in inulin can indeed reduce bacterial translocation in AAA mice, restoring them to physiological levels comparable to the general diet sham group (fig. 14), consistent with their role in gut barrier protection and reduction of LPS translocation.
(IV) inulin reduction of peripheral blood pro-inflammatory type Ly6C hi Monocytes and their migratory capacity
The translocated bacterial component, either LPS or bacterial DNA, is a strong stimulator of innate immunity, and we examined AAA mouse peripheral blood innate immune cells (monocytes, neutrophils). Adhesion by flow analysis trapped individual cells, ly6G- (neutrophile-depleted) -CD 11B + monocytes-CD 11B + Ly6Chi monocytes, and finally CCR2, detected CCR2 expression in CD11B + Ly6Chi monocytes (fig. 15), no significant difference in the ratios of CD11B + Ly6G + neutrophils and CD11B + Ly6Clo patrol monocytes in mouse peripheral blood with normal diet and high fiber diet (B, F in fig. 16), but high fiber diet rich in inulin reduced expression of chemokine CCR2 in AAA mouse peripheral blood pro-inflammatory CD11B + Ly6Chi monocytes (a in fig. 16), CD11B + Ly6Clo monocytes, and Ly6Chi monocytes (C, D in fig. 16).
The neutrophils are mainly involved in the acute phase of inflammatory reaction, and the level of the neutrophilic granulocytes is reduced to a lower level after the AAA modeling is carried out for 14 days, so that the level of the neutrophils cannot be obviously reduced even if the inulin can inhibit translocation of components such as LPS, bacterial DNA and the like through protecting the intestinal barrier function. While the mononuclear macrophage system is involved in the chronic stage of the disease through the balanced transformation of proinflammatory and inflammation inhibition, the proinflammatory type Ly6C is also detected in the aneurysm part of the AAA mouse with the inulin-rich high-fiber diet hi Reduction of monocytes (E in fig. 16). Combined with the change of peripheral blood mononuclear cells, the inulin is suggested to reduce the peripheral blood Ly6C of the AAA mouse hi The monocyte and the migration ability to AAA lesion tissue inhibit the local infiltration of proinflammatory monocyte to aneurysm, thereby playing a role in disease protection.
It will be apparent to those skilled in the art that various modifications to the above embodiments can be made without departing from the general spirit and concept of the invention. All falling within the scope of protection of the present invention. The protection scheme of the invention is subject to the appended claims.
Claims (5)
1. The inulin is applied to preparing products for preventing and treating small intestine diseases.
2. The use according to claim 1, wherein the small bowel disease comprises and/or is a complication of a small bowel barrier disorder.
3. The use as claimed in claim 1, wherein the inulin is used for preparing a preparation for preventing and treating intestinal diseases by reducing intestinal permeability and/or inhibiting intestinal permeability increase.
4. The use according to claim 1, wherein the effect of inulin in a formulation for the control of small intestinal diseases comprises
(i) Promoting the expression of intestinal barrier-associated protein genes, wherein the intestinal barrier-associated protein at least comprises one of tight junction protein, occludin, mucin and cadherin,
(ii) Promoting the proliferation of the small intestine goblet cells,
(iii) Promoting the expression of an intestinal antibacterial compound, wherein the intestinal antibacterial compound at least comprises one of antibacterial peptide, phospholipase A2, defensin and lysozyme C,
(iv) Inhibit the translocation of the intestinal flora LPS,
(v) Inhibiting CCR2 expression in cells that are at least Ly6Chi monocytes,
(vi) Inhibiting the migration of mononuclear cells of the peripheral blood Ly6Chi,
(vii) Reduce peripheral blood LPS and/or IL-1 beta levels.
5. The use of claim 2, wherein the complication of small intestinal barrier disorder comprises one of sepsis, SIRS, MODS, ischemic bowel disease.
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| BRPI0721321A2 (en) * | 2007-02-20 | 2013-01-08 | Nutricia Nv | use of colostrum and composition |
| CN108157703A (en) * | 2018-02-01 | 2018-06-15 | 河北朗益生物科技有限公司 | A kind of Cranberry inulin probiotics solid beverage and preparation method thereof |
| US20200078391A1 (en) * | 2017-05-23 | 2020-03-12 | Beijing Ruiqianjing Science And Technology Development Co. Ltd. | Combination and balanced nutritious food for improving intestinal micro-ecology and preventing chronic disease, and application thereof |
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| BRPI0721321A2 (en) * | 2007-02-20 | 2013-01-08 | Nutricia Nv | use of colostrum and composition |
| US20200078391A1 (en) * | 2017-05-23 | 2020-03-12 | Beijing Ruiqianjing Science And Technology Development Co. Ltd. | Combination and balanced nutritious food for improving intestinal micro-ecology and preventing chronic disease, and application thereof |
| CN108157703A (en) * | 2018-02-01 | 2018-06-15 | 河北朗益生物科技有限公司 | A kind of Cranberry inulin probiotics solid beverage and preparation method thereof |
Non-Patent Citations (4)
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| JULIE UERLINGS 等: "Differential effects of inulin or its fermentation metabolites on gut barrier and immune function of porcine intestinal epithelial cells", JOURNAL OF FUNCTIONAL FOODS, vol. 67, pages 1 - 9 * |
| YUE HE 等: "Inulin-Type Fructans Modulates Pancreatic–Gut Innate Immune Responses and Gut Barrier Integrity during Experimental Acute Pancreatitis in a Chain Length- Dependent Manner", FRONTIERS IN IMMUNOLOGY, pages 1 - 14 * |
| 张莉: "日粮纤维对仔猪肠道屏障功能的影响及调控机制的研究", 中国博士学位论文全文数据库农业科技辑, pages 050 - 42 * |
| 赵晓丹: "食物抗营养因子", 中国农业大学出版社, pages: 42 * |
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