CN112870158A

CN112870158A - Inflammation-inhibiting tryptophan enema preparation as well as preparation method and application thereof

Info

Publication number: CN112870158A
Application number: CN202010786259.9A
Authority: CN
Inventors: 刘杰; 骆菲菲; 宋欢; 陈琳; 罗忠光
Original assignee: Huashan Hospital of Fudan University
Current assignee: Huashan Hospital of Fudan University
Priority date: 2019-11-29
Filing date: 2020-08-06
Publication date: 2021-06-01

Abstract

The invention belongs to the technical field of medicines, and particularly relates to an anti-inflammatory Tryptophan (Tryptophan, Trp) enema preparation and a preparation method and application thereof.

Description

Inflammation-inhibiting tryptophan enema preparation as well as preparation method and application thereof

Technical Field

The invention belongs to the technical field of medicines, relates to a novel inflammation-inhibiting Tryptophan enema, a preparation method and application thereof, and particularly relates to application of Tryptophan (Tryptophan) in preparation of a medicine for inhibiting local intestinal inflammation of inflammatory bowel diseases.

Background

Research has shown that Inflammatory Bowel Disease (IBD) is an Inflammatory disease characterized by disturbance of the intestinal immune system and affecting different parts of the digestive tract, and the disease has the characteristic of recurrent attacks, and the mechanism of the disease is not completely clear. In clinical practice the disorder is mainly divided into two distinct types, Ulcerative Colitis (UC) and Crohn's Disease (CD). In recent years, the incidence of the disease has increased remarkably and the disease tends to be younger. At present, the main clinical treatment modes are mainly nutritional therapy, drug therapy, biological therapy related to monoclonal antibodies and immunosuppressant therapy, but the practical reality is that the therapies can not completely and effectively control the development of inflammation, and part of patients often develop to the stage requiring surgical intervention, which seriously influences the life quality of the patients.

There is evidence that amino acids play an important role in maintaining intestinal health, and Tryptophan (Trp) is an important essential amino acid in the human body and is widely involved in protein synthesis, nucleic acid synthesis, and immune regulation. Studies have shown that tryptophan has mainly 3 decomposition pathways: one is along the metabolic pathway of Kynurenine (kynin); secondly, the metabolic pathway of 5-hydroxytryptamine (5-HT); thirdly, indole metabolic pathway involved by bacteria; tryptophan can be directly metabolized by intestinal microorganisms to produce indole and various derivatives thereof, most of which are activating ligands of aromatic hydrocarbon receptors (ahrs). In recent years, the AhR signaling pathway is considered to be involved in epithelial regeneration and mucosal barrier maintenance by acting directly on intestinal epithelial cells, intraepithelial immune cells, Th17 cells, resident lymphocytes, macrophages, and dendritic cells, and to play an important role in regulation of intestinal immune homeostasis. In the context of IBD disease, a significant change in the gut flora profile is a scientific fact that has been recognized for many years. Researchers have considered the important role of intestinal flora in tryptophan metabolism, and tryptophan metabolic pathways are changed to some extent in the intestinal tract of IBD patients; in fact, there is data indicating a significant decrease in AhR ligand in the blood of patients with UC, and a significant decrease in the expression of AhR itself in the intestinal tract. Based on the current situation of the prior art, the inventor of the application intends to provide a novel inflammation-inhibiting Tryptophan enema preparation and a preparation method and application thereof based on the consideration that the intestinal homeostasis maintaining function of AhR can be exerted or enhanced again by locally supplementing Tryptophan in intestinal tract, and particularly provides the application of Tryptophan (Tryptophan, Trp) in preparing a medicament for inhibiting the local inflammation of the intestinal tract of inflammatory bowel diseases.

Disclosure of Invention

The invention aims to provide a novel inflammation-inhibiting Tryptophan enema preparation and a preparation method and application thereof based on the current situation of the prior art, and particularly provides application of Tryptophan (Tryptophan) in preparation of a medicine for inhibiting local intestinal inflammation of inflammatory bowel diseases.

The invention provides an inflammation-inhibiting Tryptophan enema, which takes Tryptophan (Tryptophan) as an active ingredient, prepares an enema solution with the concentration of 1% by double distilled water, and obtains the Trp enema by filtering and removing impurities and bacteria by using a 0.22 mu m filter; the enema preparation can be prepared on site;

the Tryptophan (Tryptophan, Trp) is purchased from Sigma company in the United states;

in vivo experiments prove that after the tryptophan enema preparation is intervened, the clinical symptoms of colitis such as weight loss, diarrhea, hematochezia and the like of experimental animals can be effectively improved, the pathological damage of colon tissues is relieved, and the infiltration of inflammatory cells of experimental mice to the colon tissues is inhibited; the result proves that the Tryptophan (Tryptophan, Trp) compound has good anti-inflammatory immunosuppressive activity and higher safety, and has good application prospect in the aspect of preparing enema medicines of inflammatory bowel diseases used clinically.

More specifically, the present invention is to provide a novel,

in order to achieve the purpose, the invention adopts the technical scheme that:

1. dextran Sodium Sulfate (DSS) molding and animal grouping

C57BL/6 female mice were modeled after two weeks of acclimatization (six weeks of age). Control group: drinking water normally, and killing after 7 days; model group: 2.5% DSS drinking water, the period lasts for 7 days, and the mice DSS drinking water is replaced every 2-3 days in the water feeding period. Trp intervention group: 2.5% DSS drinking water, lasting for 7 days, replacing mouse DSS drinking water every 2-3 days in the water feeding stage, and performing Trp enema intervention on the 2 nd, 4 th and 6 th days of the experiment (the specific flow is shown in figure 1);

DSS was purchased from MP Biomedical, usa and formulated as a 2.5% DSS solution with double distilled water; filtering with 0.22 μm filter to remove impurities and bacteria, and mixing;

the general conditions of each group of mice, anus blood and diarrhea conditions of the mice are observed and recorded in the experimental process;

2. medicine preparation and enema

Tryptophan is purchased from Sigma company in America, and is prepared into a 1% enema solution by double distilled water, and a 0.22 mu m filter is used for filtering, removing impurities and sterilizing, and the tryptophan is used as the preparation;

the sausage filling operation method comprises the following steps: exposing the anus of the mouse after the mouse is normally held, lightly touching the anus with a cotton swab to stimulate the defecation of the mouse, connecting a stomach tube with the diameter of 2mm to a 1mL syringe, sucking an enema solution and lubricating the stomach tube with glycerol; slowly inserting the stomach tube into the anus of a mouse for about 4cm, slowly withdrawing the stomach tube, injecting about 100 mu L of solution while injecting, and completely withdrawing the stomach tube, wherein the anus is free from liquid outflow;

3. intestinal sampling

Killing the mouse by dislocation of cervical vertebra, longitudinally cutting open the abdominal cavity along the abdominal midline, dissociating colon and distal ileum, and taking the whole colorectal from the anus to the tail end of the cecum; observing the general morphology of the colon of each group of mice, measuring the colorectal length (the confluent end of the small intestine and the cecum to the anus); dissecting the colon and rectum in longitudinal rows, washing the colon and rectum with precooled sterile normal saline, observing the condition of the inner wall of the intestinal tract and taking a picture; cutting the intestinal tract along the long axis in two parts, soaking part of lesion tissues in 4% paraformaldehyde at 4 ℃ overnight, embedding paraffin, slicing for H.E. staining for pathological examination, and freezing and storing the rest liquid nitrogen for subsequent RNA and protein extraction;

4. intestinal tissue lymphocyte isolation

1) The method comprises the following steps of (1) quickly disinfecting a mouse after the mouse is killed, opening the abdominal cavity layer by layer, taking down the colon as soon as possible, placing the colon in precooled PBS + 2% FBS liquid, excising mesentery tissues, cutting open an intestinal canal along a longitudinal axis, carefully removing residual excrement and rinsing;

2) transferring the intestinal tissue into a new centrifuge tube, cutting into pieces with sterile scissors, adding 2-3mL of digestive juice, and horizontally shaking at 230rpm/37 ℃ for 30 min;

3) centrifuging at 400g/4 ℃ for 5min, discarding the supernatant, adding a proper amount of precooled PBS and 2% FBS for resuspending tissues, and filtering through a 70-micron sterile cell filter to remove impurities;

4) centrifuging at 400g/4 ℃ for 5min, resuspending the tissue with 2.4mL of RMPI 1640 medium + 2% FBS, mixing with 1.6mL of 100% Percoll to prepare 40% Percoll, slowly adding the 40% Percoll to the upper layer of the 70% Percoll separating medium, and then slowly adding 30% Percoll to the surface of the 40% Percoll to form a 30%/40%/70% Percoll separating system, taking care not to damage the liquid plane;

5) centrifuging at 2000 rpm/room temperature for 20min, and setting the speed increasing and decreasing gradient as 1;

6) collecting 40%/70% Percoll interlayer cells, washing twice by using a large amount of PBS, and then suspending in 1mL of PBS + 2% FBS and placing at 4 ℃ for later use;

5. cell surface, nuclear staining and flow cytometry analysis

1) Counting intestinal immune cells of the mice and calculating the cell activity, wherein the cell activity is not lower than 90%;

2) transferring the cells into a flow tube, centrifuging at 400g/4 ℃ for 5min, and resuspending in 500 μ L PBS;

3) adding appropriate amount of surface staining antibody into each tube according to the instruction, and incubating at 4 deg.C in dark for 30 min;

4) adding 2mL PBS to wash the cells, and centrifuging for 5min at 400g/4 ℃;

5) repeating the step 4;

6) adding 1mL of fixing/membrane-breaking liquid, and fixing for 30min at room temperature;

7) adding 2mL of 1 Xmembrane-breaking solution to wash cells, and centrifuging at 400g/4 ℃ for 5 min;

8) repeating the step 7;

9) adding appropriate amount of nuclear staining antibody into each tube according to the instruction requirement, and incubating for 30-40min at room temperature in the dark;

10) adding 2mL of 1 Xmembrane-breaking solution to wash cells, and centrifuging at 400g/4 ℃ for 5 min;

11) repeating the step 7;

12) adding 300 mu L PBS for resuspension and preparing for flow cytometry detection;

13) data analysis was performed using Flowjo X.

6. Enzyme-Linked Immunosorbent Assay (Enzyme Linked Immunosorbent Assay, ELISA)

1) The standard substance in the kit is diluted by sample diluent according to the instruction, and the standard substance with corresponding concentration is prepared;

2) washing with washing solution for 2 times, adding 100 μ L/well sample diluent, adding standard (each concentration standard has a secondary well) and 50 μ L/well sample, and adding 50 μ L/well calibrator diluent into blank well; adding an enzyme-labeled reagent at a concentration of 50 mu L/hole, covering the cover, and incubating at room temperature for 2 h;

3) discarding the liquid, washing with washing solution for 6 times, adding 100 μ L/well of horseradish peroxidase, and incubating at room temperature for 1 h;

4) discarding the liquid, washing with washing solution for 6 times, adding 100 μ L/hole of color development solution, incubating at room temperature in dark place for 30min, and gradually changing the color to blue;

5) adding 100 μ L of stop solution into the wells, measuring absorbance of each well at 450nm with enzyme-labeling instrument within 15 min;

6) and (3) drawing a standard curve by taking the OD value as a vertical coordinate and the concentration of the standard substance as a horizontal coordinate, finding out the concentration of the sample on the standard curve according to the OD value of the sample, and multiplying the concentration by the dilution factor to obtain the concentration of the sample.

7. Real-time fluorescent quantitative PCR

7.1 mouse colorectal tissue RNA extraction

1) Preparation in the early stage of the experiment: soaking sterile scissors, tweezers and homogenate steel balls in 0.1% DEPC aqueous solution overnight, and sterilizing with high pressure steam for 15 min;

2) tissue homogenization: taking out about 200mg of each mouse colorectal tissue frozen in liquid nitrogen, putting the mouse colorectal tissues into a glass grinder filled with a proper amount of liquid nitrogen in advance, fully grinding the mouse colorectal tissues into powder, and continuously adding the liquid nitrogen to keep the temperature low in the process; transferring the powder tissue into a 2.0mL EP centrifuge tube, adding 1mL Trizol reagent, placing into a homogenizer to homogenize at 120hz for 1min, taking out and standing at room temperature for 5 min;

3) phase separation: adding 200 μ L of trichloroethanol into each tube, vortex oscillating for 15s, standing at room temperature for 5min, and centrifuging at 12000rpm/4 deg.C for 10 min;

4) and (3) RNA precipitation: transferring 400 μ L of upper water phase to a new 1.5ml RNase-free EP centrifuge tube, adding equal volume of isopropanol, fully inverting and mixing uniformly, standing at room temperature for 15min, and centrifuging at 12000rpm/4 ℃ for 10 min;

5) washing RNA: the supernatant was removed and the bottom RNA pellet of the EP tube was observed. Adding 1ml 75% ethanol diluted with precooled DEPC water into the tube, slightly inverting the washed precipitate, and centrifuging at 12000rpm/4 ℃ for 10 min;

6) removing supernatant, air drying the precipitate at room temperature, and adding appropriate amount of DEPC water to fully dissolve the precipitate when the precipitate becomes transparent slightly.

7.2 Total RNA concentration and purity determination

Detecting the concentration and purity of the extracted total RNA according to the NanoDrop operating instruction; according to OD₂₆₀/OD₂₈₀The ratio of (A) to (B) can estimate the purity of RNA, and 1.8-2.0 is an ideal numerical range; measuring the concentration of the total RNA with qualified quality for subsequent experiments, and storing the total RNA in a refrigerator at the temperature of minus 80 ℃ for a long time;

7.3RNA reverse transcription

7.3.1.1 removal of residual genomic DNA

Preparing a reaction system according to the specification of a reverse transcription kit, and lightly blowing, beating and uniformly mixing; incubating at 42 deg.C for 2 min;

7.3.1.2RNA reverse transcription

Preparing a reaction system according to the specification of a reverse transcription kit, and lightly blowing, beating and uniformly mixing;

the reaction system is used for finishing reverse transcription on a PCR instrument at 37 ℃ (15min) and 85 ℃ (5 min);

7.4 real-time quantitative fluorescent PCR

The Real-Time fluorescent quantitative PCR uses an Applied Biosystems 7500Real-Time PCR System, a SYBR Green I method is used for relative quantitative analysis, and each sample is provided with three auxiliary holes; according to the specification of the reverse transcription kit, a reaction system is prepared as follows:

the reaction System is uniformly mixed and then placed in a special 96-well plate (suitable for Applied Biosystems 7500Real-Time PCR System) and then placed in a machine; the amplification conditions were set as follows:

the specificity of amplification can be determined by whether the dissolution curve of the product is a single peak or not, and the relative expression level of the target gene mRNA in the sample can be calculated according to the Ct value (Cycle threshold, which is the number of cycles that the fluorescence signal in each reaction tube reaches a set threshold) of the target gene and the Ct value of the housekeeping gene GAPDH. Differential gene expression data analysis was performed using the classical Δ Δ Ct method, Δ Δ Ct ═ Ct (Ct experimental sample-Ct experimental reference) - (Ct control sample-Ct control reference). Fold difference in gene expression of 2^-ΔΔCt。

The experimental results are as follows:

1. the tryptophan enema is given to obviously relieve the enteritis of mice

The general situation of the intervention group (2.5% DSS + Trp group) mice is obviously better than that of the model group mice, and the symptom model groups (2.5% DSS + H) such as hematochezia and weight reduction₂O) mice were more severe and persisted longer; compared with the model group mice, the weight reduction degree of the intervention group mice is obviously reduced, the disease activity index is obviously reduced, and the colorectal shortening is also obviously less (figure 2. A-B); H.E. section staining shows that the intestinal tracts of the mice in the model group have mucosal erosion, inflammatory cell enrichment, glandular fossa morphological change and mucosal muscularis fracture; the integrity of the intestinal tract of the mice in the intervention group is better, and the serious damage of the intestinal tract such as mucosal muscularis rupture does not appear (figure 2. C); the control mice had no evidence of intestinal inflammation.

2. The tryptophan enema is given, so that the proportion of proinflammatory cells in intestinal tracts of enteritis mice can be obviously reduced, and the proportion of the proinflammatory cells in the intestinal tracts of the enteritis mice is increased:

after immune cells are extracted from intestinal tissues of each group of mice, the local immune pattern change of the intestinal tract is analyzed by using flow cytometry, and the intervention group is found to have no significant change in the ratio of Th1/Treg cells (figure 3.A) and significantly increased ratio of Th2 cells (figure 3.B) in a comparison model group; the proportion of Th17/Treg cells that play a key role in the mouse DSS enteritis model was significantly reduced (fig. 3. C); at the same time, the number of M-MDSC in the inflammation-suppressing cells was significantly increased in the cells derived from the myeloid line (FIG. 4.A), while the number of G-MDSC was not significantly different (FIG. 4. B); the proportion of non-M1-type macrophages was also significantly reduced compared to the model group (fig. 4. C-D).

The in vivo experiment proves that the tryptophan enema intervention effectively improves the clinical symptoms of the test animal such as weight loss, diarrhea, hematochezia and other colonitis, reduces the pathological damage of colon tissues and inhibits the infiltration of inflammatory cells of the test mouse to the colon tissues; the result proves that the compound has good anti-inflammatory immunosuppressive activity and higher safety, and has good application prospect in the aspect of preparing enema medicines for inflammatory bowel diseases used clinically.

Drawings

FIG. 1 shows a specific process scheme for mouse DSS modeling and tryptophan enema intervention, wherein,

control: control, DSS + H2O: model group, DSS + Trp: tryptophan enema preparation group, H₂O: and (3) water.

Fig. 2. tryptophan enema significantly eases the degree of intestinal inflammation in mice, wherein,

A. tryptophan enema intervention can obviously slow down the weight loss of the model mouse. B. The tryptophan enema intervention can obviously slow down the shortening of the intestinal tract of a model mouse, the left graph is a general schematic diagram of the intestinal tract length of the mouse, and the right graph is a statistical analysis result of each group of data and a staining result of an H.E. section of an intestinal tract tissue; data are expressed as mean. + -. standard error, H₂O+H₂O: blank control, DSS + 1% Trp: model group, DSS + H₂O: dry run, P < 0.05, P < 0.001.

FIG. 3 Tryptophan enema intervention significantly reduced the intestinal Th17/Treg ratio and increased the Th2 ratio in enteritis mice, wherein,

the ratio of various types of T cells in the local intestinal tract is changed, the data are expressed by the mean value +/-standard error, and Trp: model group, DSS: dry pre-group, P < 0.05.

FIG. 4 Tryptophan enema intervention significantly increased the ratio of M-MDSC to non-M1 type macrophages in intestinal tract of enteritis mice, wherein,

the proportion of various local medullary line cells in the intestinal tract is changed, the data are expressed by the average value +/-standard error, and Trp: model group, DSS: dry pre-group, P < 0.05.

Detailed Description

Example 1

1. Dextran Sodium Sulfate (DSS) molding and animal grouping

DSS was purchased from midwifery MP biomedicalal and formulated with double distilled water to a 2.5% DSS solution; filtering with 0.22 μm filter to remove impurities and bacteria, and mixing;

2. medicine preparation and enema method

3. intestinal sampling

4. intestinal tissue lymphocyte isolation

5. cell surface, nuclear staining and flow cytometry analysis

4) adding 2mL PBS to wash the cells, and centrifuging for 5min at 400g/4 ℃;

5) repeating the step 4;

8) repeating the step 7;

11) repeating the step 7;

13) data analysis was performed using Flowjo X.

6. Enzyme-Linked Immunosorbent Assay (Enzyme Linked Immunosorbent Assay, ELISA)

7. Real-time fluorescent quantitative PCR

7.1 mouse colorectal tissue RNA extraction

6) removing supernatant, air drying the precipitate at room temperature, and adding appropriate amount of DEPC water to fully dissolve the precipitate when the precipitate becomes transparent slightly. 7.2 Total RNA concentration and purity determination

7.3RNA reverse transcription

7.3.1.1 removal of residual genomic DNA

7.3.1.2RNA reverse transcription

7.4 real-time quantitative fluorescent PCR

Reality of experimental results

The enteritis of the mice can be obviously relieved by the tryptophan enema:

the general situation of the intervention group (2.5% DSS + Trp group) mice is obviously superior to that of the model group mice, and the symptom model group (2.5% DSS + H) with hematochezia and weight reduction₂O) mice were more severe and persisted longer; compared with the model group mice, the weight reduction degree of the intervention group mice is obviously reduced, the disease activity index is obviously reduced, and the colorectal shortening is also obviously less (figure 2. A-B); H.E. section staining shows that the intestinal tracts of the mice in the model group have mucosal erosion, inflammatory cell enrichment, glandular fossa morphological change and mucosal muscularis fracture; the integrity of the intestinal tract of the mice in the intervention group is better, and the serious damage of the intestinal tract such as mucosal muscularis rupture does not appear (figure 2. C); to pairThe control mice had no evidence of intestinal inflammation.

The tryptophan enema is given, so that the proportion of proinflammatory cells in intestinal tracts of enteritis mice can be obviously reduced, and the proportion of the proinflammatory cells in the intestinal tracts of the enteritis mice is increased:

after immune cells are extracted from intestinal tissues of each group of mice, the local immune pattern change of the intestinal tract is analyzed by using flow cytometry, and the intervention group is found to have no significant change in the ratio of Th1/Treg cells (figure 3.A) and significantly increased ratio of Th2 cells (figure 3.B) in a comparison model group; the proportion of Th17/Treg cells that play a key role in the mouse DSS enteritis model was significantly reduced (fig. 3. C); at the same time, the number of M-MDSC in the inflammation-suppressing cells was significantly increased in the cells derived from the myeloid line (FIG. 4.A), while the number of G-MDSC was not significantly different (FIG. 4. B); the proportion of non-M1-type macrophages was also significantly reduced compared to the model group (fig. 4. C-D);

in conclusion, the enema prepared by locally administering tryptophan to the intestinal tract can obviously improve the intestinal inflammation state of DSS-induced enteritis mice and regulate the disordered intestinal immunity pattern, and further, the enema prepared by tryptophan for inhibiting inflammation is used for preparing a medicament for clinically treating inflammatory bowel diseases, so that the enema has a good application prospect.

Claims

1. An anti-inflammatory Tryptophan enema preparation is characterized in that Tryptophan (Trp) is used as an active ingredient, and double distilled water is used for preparing an enema solution, and the solution is filtered, decontaminated and sterilized to prepare the Tryptophan enema preparation.

2. The anti-inflammatory tryptophan enema preparation according to claim 1, wherein the solution for enema is formulated to have a concentration of 1%.

3. The anti-inflammatory tryptophan enema preparation according to claim 1, wherein the prepared solution for enema is filtered by a 0.22 μm filter to remove impurities and bacteria, thereby obtaining the tryptophan enema preparation.

4. The anti-inflammatory tryptophan enema preparation according to claim 1, wherein the tryptophan enema preparation is prepared at the time of use.

5. The anti-inflammatory tryptophan enema preparation according to claim 1, wherein the tryptophan is used for preparing a medicament for inhibiting local inflammation of intestinal tract of inflammatory bowel disease.