CN114569639A

CN114569639A - Application of Alisipes putriedinis bacteria in preparation of medicine for preventing and treating inflammatory bowel disease

Info

Publication number: CN114569639A
Application number: CN202210164642.XA
Authority: CN
Inventors: 王晓艳; 邓敏子; 罗薇薇; 聂凯; 吴兴; 徐嘉豪; 陈学杰; 张超; 马可嘉
Original assignee: Third Xiangya Hospital of Central South University
Current assignee: Third Xiangya Hospital of Central South University
Priority date: 2022-02-23
Filing date: 2022-02-23
Publication date: 2022-06-03

Abstract

The invention provides application of Alisipes putriedinis bacteria in preparation of a medicine for preventing and treating inflammatory bowel disease, and relates to the technical field of biological medicines. The invention adopts a C57BL/6 mouse colitis model, and discovers that the A.putredensis bacteria can improve intestinal inflammation of DSS mice. The invention firstly proves that the A.putredensis can inhibit intestinal inflammation of a DSS colitis mouse, provides a new theory for a CD pathogenesis, provides a new treatment strategy for CD diagnosis and treatment, and has potential clinical application prospects.

Description

Application of Alisipes putriedinis bacteria in preparation of medicine for preventing and treating inflammatory bowel disease

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to application of Alisipes putriedinis bacteria in preparation of a medicine for preventing and treating inflammatory bowel diseases.

Background

Crohn's Disease (CD) is a chronic recurrent inflammatory disease of intestinal tract, the incidence rate of CD in China is remarkably increased in recent years, the diseased people are younger, the curative effect is poor, and CD patients often have complications such as intestinal obstruction, fistula, abdominal abscess and the like, need surgical treatment and have poor prognosis. The pathogenesis of CD is complex and undefined, and the pathogenesis of CD is revealed, so that the method has important significance for searching a new target and a new strategy for treating CD.

The intestinal flora is an important link for mutual regulation and control of host and environmental factors, is closely related to CD morbidity, but records for treating CD related diseases by using the intestinal flora are rarely seen at present.

Disclosure of Invention

In view of the above, the present invention aims to provide an application of Alistipes putriedinis bacteria in preparing a medicament for preventing and treating inflammatory bowel diseases, wherein the Alistipes putriedinis bacteria can resist intestinal mucosa injury and has an effect of preventing and/or treating inflammatory bowel diseases.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides application of Alisipes putriedinis bacteria in preparation of a medicine for preventing and treating inflammatory bowel diseases.

Preferably, the inflammatory bowel disease comprises crohn's disease.

Preferably, the effective component of the medicament comprises supplemented Alisipes putriedinis bacterial liquid.

Preferably, the preparation method of the supplemented Alisipes putriedinis bacterial liquid comprises the following steps: the Alisipes putriedinis bacterial liquid which is activated and cultured for 3d in a culture medium is taken, centrifuged and thallus is collected and then resuspended to the OD value of 1 by using anaerobic PBS.

Preferably, the preparation method of the Alisipes putriedinis bacterial liquid comprises the step of inoculating bacterial dry powder of the Alisipes putriedinis into a culture medium for 3d activation culture, wherein the culture medium takes a BHI culture medium as a basic culture medium, and also comprises 5 mu g/mL of iron-containing heme and 0.5 mu g/mL of vitamin K1.

Preferably, the activation culture is an anaerobic culture in an environment of 37 ℃.

Preferably, the rotation speed of the centrifugation is 4000rpm, and the time of the centrifugation is 10 min.

The invention also provides application of the reagent for improving the content of Alisipes putriedinis bacteria in the intestinal tract in preparing a medicament for resisting intestinal mucosa injury.

Preferably, the reagent comprises supplemented Alisipes putriedinis liquid.

Has the advantages that: the invention provides application of Alisipes putriedinis bacteria in preparation of a medicine for preventing and treating inflammatory bowel diseases. Botrydinis is a gram-negative anaerobic bacillus belonging to the bacteroidetes, another mycobacteria genus. And then adopting a C57BL/6 mouse colitis model, after intervention of A.putrescindiis bacteria, symptoms such as diarrhea and bloody stool of the mouse are relieved, disease activity index is obviously reduced, the colon shortening degree is relieved, and HE staining shows that pathological characteristics such as intestinal mucosa structure damage, goblet cell loss and inflammatory cell infiltration degree are relieved, which suggests that the A.putrescindiis can relieve intestinal mucosa injury and inflammation of the colitis mouse.

Drawings

Fig. 1 shows that a.putredensis is significantly reduced in abundance in the intestinal tract of CD patients by metagenomic analysis; wherein A: analyzing the LDA value of metagenome differential bacteria; b: comparison of abundance of a.putredensis bacteria in CD and healthy patient feces; c: gram staining results of putredensis bacteria; d: scanning electron micrograph of putredensis strain, P < 0.001;

fig. 2 shows that a.putriedinis bacteria can inhibit intestinal inflammation in DSS colitis mice; wherein A: a mouse intervention pattern diagram; B-C: carrying out statistical analysis on the weight change of the mice and the weight on the seventh day; D-E: mouse Disease Activity Index (DAI) change and day seven DAI statistical analysis; F-G: colon gross morphology change and length analysis; H-I: HE staining and pathological statistical analysis of mouse intestinal mucosa tissues; j: expression of TNF-a in mouse intestinal mucosal tissue (p < 0.05, p < 0.01, p <0.001, ns: no statistical difference); wherein B, C, D, E, I and A.P in J represent A.putredensis.

Detailed Description

The A.putrescininis is an anaerobic bacterium which is fixedly planted in the intestinal tract of a human body, belongs to Bacteroides, and belongs to the genus of corynebacterium, and is a gram-negative bacterium. According to the invention, the difference of intestinal flora in feces of CD patients and healthy controls before treatment is analyzed by a metagenome sequencing technology, and the fact that the abundance of A.putredensis in the intestinal flora of the CD patients is remarkably reduced and is negatively related to intestinal mucosa injury is found, animal experiments prove that the A.putredensis can inhibit intestinal mucositis of mice, and the A.putredensis is suggested to be applied to prevention and/or treatment of inflammatory bowel diseases.

The inflammatory bowel disease of the present invention preferably comprises crohn's disease. The effective component of the medicament preferably comprises supplemented Alisipes putriedinis bacterial liquid. The preparation method of the supplemented Alisipes putriedinis bacterial liquid preferably comprises the following steps: the Alisipes putriedinis bacterial liquid which is activated and cultured for 3d in a culture medium is taken, centrifuged and thallus is collected and then resuspended to the OD value of 1 by using anaerobic PBS.

The preparation method of the Alisipes putriedinis bacterial liquid preferably comprises the step of inoculating bacterial dry powder of the Alisipes putriedinis into a culture medium for 3d activation culture, wherein the culture medium takes BHI (brain-heart infusion broth) culture medium as a basic culture medium, and also comprises 5 mu g/mL of ferriheme and 0.5 mu g/mL of vitamin K1. The preparation method of the culture medium preferably comprises the steps of carrying out autoclaving on a commercial BHI culture medium at 121 ℃ for 20min, cooling to room temperature, and then adding 5 mu g/mL of ferrugineum and 0.5 mu g/mL of vitamin K1. The activated culture of the invention is preferably carried out in an anaerobic culture environment at 37 DEG CCulturing for 3 days to obtain viable bacteria count of about 1 × 10⁸CFU/mL. The source of the dried bacterial powder of Alisipes putriedinis is not particularly limited, and the dried bacterial powder is preferably from an ATCC biological bank and has the following serial number: ATCC 29800.

The Alisipes putriedinis bacterial liquid is centrifuged, the centrifugation is preferably performed at a low temperature of 4 ℃, the rotating speed is preferably 4000rpm, and the centrifugation time is preferably 10 min. In the invention, after the centrifugation, the collected thallus is resuspended by using oxygen-free PBS, preferably to OD value of 1, namely viable count of 1 × 10⁸～1×10¹⁰CFU/mL。

In the embodiment of the invention, after the A.putreinis intervenes in a mouse DSS (direct sequence digital subscriber line) colitis model, the symptoms of diarrhea, bloody stool and the like of the mouse are relieved, the disease activity index is obviously reduced, the colon shortening degree is relieved, and HE (human immunodeficiency virus) staining shows that the pathological characteristics of intestinal mucosa structure damage, goblet cell loss, inflammatory cell infiltration degree and the like of the mouse are relieved, which indicates that the A.putreinis can relieve the intestinal mucosa damage and inflammation of the colitis mouse.

The reagent preferably comprises a supplemented Alisipes botrytis liquid, and the preparation method of the supplemented Alisipes botrytis liquid is preferably the same as that described above, and is not described again.

The application of the Alisipes putriedinis bacteria in preparing the medicament for preventing and treating inflammatory bowel disease provided by the invention is described in detail with reference to the following examples, but the application is not to be construed as limiting the scope of the invention.

Example 1

Significantly reduced abundance of putredininis in the gut of CD patients

The midkine newlife company was entrusted with a macrogenomics analysis of fecal specimens from 30 pre-treatment CD patients and 30 healthy controls.

Results as shown in figure 1, the abundance of a.putredensis in CD patients was significantly reduced compared to healthy controls (a-B in figure 1). Gram staining of a.putrescindiis bacteria was also negative (C in fig. 1). Scanning electron microscope observation showed that the a.putrescindiis strain is a typical bacillus (D in fig. 1).

Example 2

Putredinis capable of inhibiting intestinal inflammation of mice with DSS (Severe Sdss) colitis

The experimental design was performed as shown in a in fig. 2: a.pultredinis gastric perfusion intervention (1X 10 is selected) is carried out by using the C57BL/6 mouse DSS colitis model (Wirtz S, Popp V, Kindermann M, Gerlach K, Weidmann B, Fichtner-Feigl S, neuron MF.chemical induced mouse models of acid and cyclic endogenous insulin Nat Protoc.2017 Jul; 12(7):1295-⁹CFU/mL bacterial solution 4000rpm, 10min, 4 ℃ centrifugation bacterial precipitation and then use anaerobic PBS heavy suspension to OD value of 1, 200 u L/only lavage).

As a result, as shown in tables 1 to 5 and fig. 2, DSS colitis mice exhibited weight loss to various degrees, while a. putredensis could inhibit weight loss at a rate lower than that of DSS colitis mice from day 4 (B-C in fig. 2). In addition, the A.putriedinis strain can remarkably reduce symptoms of diarrhea, bloody stool and the like of DSS colitis mice, the disease activity index is reduced (D-E in figure 2), and the colon shortening degree is reduced (F-G in figure 2). HE staining showed that pathological features such as destruction of intestinal mucosal structure, goblet cell loss and degree of inflammatory cell infiltration were alleviated in the a.putriedinis bacterial intervention group compared to the DSS building block (H-I in fig. 2). Putredensis was found to significantly down-regulate DSS-induced TNF-a down-regulation using ELISA to detect the expression of the inflammatory factor TNF-a in colon tissue (J in fig. 2). The above results suggest that a.putredini can reduce intestinal mucosal injury and inflammation in colitis mice.

TABLE 1 statistical table of weight loss percentages

TABLE 2 DAI analysis Table

TABLE 3D 7(12/01) spleen index statistics

TABLE 4 Colon Length statistics (cm)

TABLE 5 TNF-. alpha.expression statistics

Control	DSS	DSS+A.P
			1.79	4.34	2.95
2.91	4.93	3.92
			1.5	6.35	3.33
2.02	5.57	2.55
			3.2	5.75	3.38

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Claims

Application of Alisiprescutedinis in preparation of medicines for preventing and treating inflammatory bowel diseases.
2. The use of claim 1, wherein the inflammatory bowel disease comprises crohn's disease.
3. The use of claim 1, wherein the active ingredient of the medicament comprises a supplemented Alisipesputtredinis fluid.
4. The use of claim 3, wherein the preparation method of the supplemented Alisiprescuntriedinis bacterial liquid comprises the following steps: the Alisiprescutdeninis liquid which is activated and cultured for 3d in the culture medium is taken, centrifuged and thallus is collected and then resuspended to the OD value of 1 by using anaerobic PBS.
5. The use of claim 4, wherein the preparation method of Alisiprescutedinis bacterial liquid comprises inoculating the dry powder of Alisiprescutedinis bacterial into a culture medium for 3d activation culture, wherein the culture medium takes BHI culture medium as a basic medium, and further comprises 5 μ g/mL of iron-containing heme and 0.5 μ g/mL of vitamin K1.
6. The use according to claim 4 or 5, wherein the activated culture is an anaerobic culture in an environment at 37 ℃.
7. The use according to claim 4, wherein the rotation speed of the centrifugation is 4000rpm and the time of the centrifugation is 10 min.
8. Application of a reagent for improving Alisiprescutedinis bacteria content in intestinal tracts in preparation of medicines for resisting intestinal mucosa injury.
9. The use of claim 8, wherein the reagent comprises supplemented Alisipes putriedinis fluid.