CN109718234A - L-Trp is alleviating the application in intestinal inflammation and barrier function disorder - Google Patents
L-Trp is alleviating the application in intestinal inflammation and barrier function disorder Download PDFInfo
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Abstract
The invention discloses L-Trps to alleviate the application in mouse intestinal inflammatory reaction and barrier function disorder.Be shown experimentally that: L-Trp can be relieved the body weight loss of the mouse of DSS induction, the death rate increases, colon shortens, Disease Activity Index rises and intestinal lesion；Not only improve tight junction protein structural damage caused by DSS, reduce the infiltration of neutrophil leucocyte, macrophage and the T cell of DSS induction to colonic lamina propria and submucosa, and it is relieved the decline of inflammatory cytokine IL-6, TNF-α, the raising of the mRNA level in-site of IL-17a, G-CSF, MIP-1a and CXCL-2 and the TGF-β 2 of DSS induction and the mRNA level in-site of TGF-β 3.The above result shows that: L-Trp can reduce the expression of inflammatory factor, improve gut barrier disorder, can be used as the effective Nutrition Strategies for preventing and treating IBD.
Description
Technical field
The invention belongs to field of biotechnology, and in particular to L-Trp is disorderly in alleviation intestinal inflammation and barrier function
Application in unrest, in particular to L-Trp are in mouse intestinal inflammatory reaction and the barrier function for alleviating dextran sulfate sodium induction
Application in disorder.
Background technique
Inflammatory bowel disease (inflammatory bowel disease, IBD) can be divided into ulcerative colitis
Two kinds of (Ulcerative Colitis) and Crohn disease (Crohn ' s Disease).IBD, which is that a kind of chronic progressivity is immune, to be situated between
The disease led, it is characterised in that repeated for intestinal inflammatory seriously affects intestinal health and quality of life.IBD has become
A kind of global health problem.Immune response of the generation of IBD by heredity, environment, enteric microorganism and body etc. is more
Kind factor influences.IBD is typically considered under nature-nurture factor collective effect, and the immunity function of body gets muddled and draws
The intestinal inflammatory risen.The inflammatory reaction of enteron aisle is mostly due to gut epithelium barrier breakdown, and pathogen enters interior environment in enteric cavity, in turn
Cause neutrophil leucocyte and macrophages infiltration, generate and discharges a variety of inflammatory cytokines and chemotactic factor (CF).Glucan sulfuric acid
Sodium (dextran sodium sulfate, DSS) be capable of inducing mouse colitis (Johansson ME et al.2014,
63:281-91.；Johansson ME et al.2010,5:e12238.；Cooper HS et al.1993,69:238-
49.) inflammatory cytokine (such as TNF-α, IL-17, IL-6, IFN-γ, IL-4 and IL-10), chemotactic factor (CF) and blood vessel, are improved
The expression and secretion of the factor are generated, and participates in the inflammatory reaction of T cell mediation, diarrhea, weight loss, colon occurs and shortens, is hidden
Nest loses and hematochezia (Perse M, Cerar A.2012,2012:718617.).
There are microorganisms abundant in enteric cavity.Nutriment in daily ration can be with the digestion in enteric cavity after entering enteron aisle
Enzyme, microorganism contact and interact, and may directly or indirectly influence the intestinal barrier function of host, and then influence IBD's
Generation, development and prognosis.Some researches show that IBD can influence enteral nutrition substance, such as vitamin, minerals and amino acid
Deng be absorbed and utilized.
L-Trp (L-tryptophan, Trp) is the essential amino acid of mammal.In addition to synthesizing body protein,
Tryptophan is also the precursor for synthesizing serotonin and melanin；In vivo when niacinamide deficiency, tryptophan may participate in synthesis
A part of niacinamide.Tryptophan can also in liver cell tryptophan 2,3- dioxygenase (tryptophan 2,3-
Dioxygenase), or in leucocyte or other intracellular indoleamine 2s, 3 dioxygenases (indoleamine 2,
3dioxygenase, IDO) it is degraded under effect, while a variety of catabolites are generated, is referred to as kynurenin.
Summary of the invention
It is an object of the present invention to provide the new applications of L-Trp.
The present invention provides L-Trps in following A1)-A10) in it is any in application:
A1 the application in the product of prevention and or treatment inflammation enteropathy and/or colitis) is prepared；
A2) prevention and or treatment inflammation enteropathy and/or colitis；
A3) intestinal inflammation and/or the barrier function disorder of dextran sulfate sodium induction are alleviated in preparation；
A4) alleviate intestinal inflammation and/or the barrier function disorder of dextran sulfate sodium induction；
A5 gut integrity) is maintained；
A6 the tight junction protein structure of intestinal epithelial cell) is maintained；
A7) lower intestinal inflammatory factor IL-6 and/or TNF-α and/or IL-17a and/or G-CSF and/or MIP-1a and/
Or the mRNA expression of CXCL-2；
A8 the mRNA expression of intestinal inflammatory factor TGF-β 2 and/or TGF-β 3) is raised；
A9 infiltration of the immunocyte of dextran sulfate sodium induction to colonic lamina propria and submucosa) is reduced；
A10 the growth performance of animal individual) is improved.
In above-mentioned application, it is tight that the tight junction protein structure for maintaining intestinal epithelial cell is embodied in raising epithelial cell
The expression of close connection albumen ZO-1 and/or occludin and/or claudin-1.
In above-mentioned application, the intestinal inflammation and/or barrier function disorder body for alleviating dextran sulfate sodium induction
Any one of present following B1)-B5):
B1 the weight of the animal individual of dextran sulfate sodium induction) is improved；
B2 the survival rate of the animal individual of dextran sulfate sodium induction) is improved；
B3 the Disease Activity Index of the animal individual of dextran sulfate sodium induction) is reduced；
B4 the colon lengths of the animal individual of dextran sulfate sodium induction) are improved；
B5) alleviate the intestinal bleeding of the animal individual of dextran sulfate sodium induction.
In above-mentioned application, the immunocyte for reducing dextran sulfate sodium induction is to colonic lamina propria and submucosa
Infiltration, which is embodied in, reduces colonic mucosa immunocyte quantity；
The colonic mucosa immunocyte is specially neutrophil leucocyte and/or macrophage and/or T cell.
In above-mentioned application, it is described improve animal individual growth performance be embodied in increase animal individual amount of drinking water and/or
Feed intake.
It is a further object to provide a kind of products；
The product have following C1)-C8) and in any function:
C1) prevention and or treatment inflammation enteropathy and/or colitis；
C2) alleviate intestinal inflammation and/or the barrier function disorder of dextran sulfate sodium induction；
C3 gut integrity) is maintained；
C4 the tight junction protein structure of intestinal epithelial cell) is maintained；
C5) lower intestinal inflammatory factor IL-6 and/or TNF-α and/or IL-17a and/or G-CSF and/or MIP-1a and/
Or the mRNA expression of CXCL-2；
C6 the mRNA expression of intestinal inflammatory factor TGF-β 2 and/or TGF-β 3) is raised；
C7 infiltration of the immunocyte of dextran sulfate sodium induction to colonic lamina propria and submucosa) is reduced；
C8 the growth performance of animal individual) is improved.
The active constituent of product provided by the invention is L-Trp.
In the said goods, it is tight that the tight junction protein structure for maintaining intestinal epithelial cell is embodied in raising epithelial cell
The expression of close connection albumen ZO-1 and/or occludin and/or claudin-1.
In the said goods, the intestinal inflammation and/or barrier function disorder body for alleviating dextran sulfate sodium induction
Any one of present following B1)-B5):
B1 the weight of the animal individual of dextran sulfate sodium induction) is improved；
B2 the survival rate of the animal individual of dextran sulfate sodium induction) is improved；
B3 the Disease Activity Index of the animal individual of dextran sulfate sodium induction) is reduced；
B4 the colon lengths of the animal individual of dextran sulfate sodium induction) are improved；
B5) alleviate the intestinal bleeding of the animal individual of dextran sulfate sodium induction.
In the said goods, the immunocyte for reducing dextran sulfate sodium induction is to colonic lamina propria and submucosa
Infiltration, which is embodied in, reduces colonic mucosa immunocyte quantity；
The colonic mucosa immunocyte is specially neutrophil leucocyte and/or macrophage and/or T cell.
In the said goods, it is described improve animal individual growth performance be embodied in increase animal individual amount of drinking water and/or
Feed intake.
In above-mentioned application or product, the animal is mammal, and the mammal can be people or mouse.
The present invention chooses 40 eight week old C57BL/6 male mices and is randomly divided into 4 groups: control group, DSS group, Trp group and
Trp+DSS group.Chmice acute colitis mould is established with 2% dextran sulfate sodium (dextran sodium sulfate, DSS)
Type, record mouse amount of drinking water, feed intake and weight during test.It is living by body weight loss, the death rate, colon lengths, disease
Sex index, Intestinal Morphology, tight junction protein and Immune Indexes evaluation L-Trp are to the DSS mouse colitis induced
Relaxation effect.Result of study shows: L-Trp can be relieved the body weight loss of the mouse of DSS induction, the death rate increases,
Colon shortens, Disease Activity Index rises and intestinal lesion；Not only improve tight junction protein ZO-1 caused by DSS,
Occludin and claudin-1 structural damage, neutrophil leucocyte, macrophage and the T cell for reducing DSS induction are solid to colon
There is the infiltration of layer and submucosa, and is relieved the inflammatory cytokine IL-6 of DSS induction, TNF-α, IL-17a, G-
The decline of the mRNA level in-site of the raising of the mRNA level in-site of CSF, MIP-1a and CXCL-2 and TGF-β 2 and TGF-β 3.The above knot
Fruit shows: L-Trp can reduce the expression of inflammatory factor, improve gut barrier disorder, can be used as and prevents and treats IBD
Effective Nutrition Strategies.
Detailed description of the invention
Fig. 1 is control group and the daily amount of drinking water of Trp group mouse (Figure 1A), feed intake (Figure 1B) and daily gain (Fig. 1 C).Number
Value is expressed as average value ± S.E.M.；N=6 cage, 3-4 mouse of every cage.* being expressed as Trp group and control group relatively has conspicuousness
Difference (P < 0.05).
Fig. 2 is the acute inflammatory reaction that DSS induction is alleviated in tryptophan addition.It is added to off-test (1-10 days) from DSS
Control group, DSS group, percentage (Fig. 2A), the death rate (Fig. 2 B), mouse of Trp group and the daily body weight loss of Trp+DSS group mouse
Colon lengths (Fig. 2 C) and Disease Activity Index (Fig. 2 D).Wherein each group mouse Colon length is measured in the last day of test.
Numerical value is expressed as average value ± S.E.M., and every group of n=4-10 mouse.* is expressed as DSS group, Trp group or Trp+DSS in fig. 2
Group mouse has significant difference (P < 0.05) compared with control group mice.* is expressed as DSS group and Trp+DSS group ratio in fig. 2b
Relatively there is significant difference (P < 0.05).4 groups of Multiple range test in Fig. 2 C and 2D, *, which is expressed as different two groups of symbol, relatively to be had
Significant difference (P < 0.05).
Fig. 3 is that tryptophan addition reduces colon caused by DSS is handled and the damage of jejunum Intestinal Tract Morphology.Control group, DSS
Group, Trp group and Trp+DSS group mouse Colon (Fig. 3 A) and small intestine (Fig. 3 C and Fig. 3 D) histopathology slide (H&E dyeing, DSS
Treated the tenth day) and fecal occult blood situation analysis (treated the 4th day by DSS).Scale bar=200 micron (Fig. 3 A and
Fig. 3 D) or 1000 microns (Fig. 3 C).
Fig. 4 is the damage that intestinal barrier integrity caused by DSS is handled is alleviated in tryptophan addition.Control group, DSS group, Trp
Group and being immunized for Trp+DSS group mouse Colon OCT embedded section tight junction protein ZO-1, claudin-1 and occludin are glimmering
Light analysis.3 repetitions of each processing, are as a result expressed as representative picture.Scale bar=200 micron.
Fig. 5 is the infiltration that colon immunization cell caused by DSS is handled is alleviated in tryptophan addition.Control group, DSS group, Trp group
With Trp+DSS group mouse Colon OCT embedded section F4/80+、CD11b+、Gr1+、CD4+、CD8+And CD31+Immunocyte is immunized
Fluorescence analysis.Red staining is expressed as F4/80+, CD11b+, Gr1+, CD4+, CD8a+ or CD31+Immunocyte；Blue dye
Color table is shown as nucleus (being dyed with Hoechst 33258).3 repetitions of each processing, are as a result expressed as representative picture.Ratio
Ruler=200 micron.
Fig. 6 is the variation that the colitis factor caused by DSS is handled is alleviated in tryptophan addition.Control group, DSS group, Trp group
With Trp+DSS group mouse Colon IL-6, TNF α, IL-17a, G-CSF, MIP-1 α, CXCL-2, TGF-β 2 and TGF-β 3 mRNA
Expression analysis.Numerical value is expressed as average value ± S.E.M., and every group of n=4-10 mouse.4 groups of Multiple range test, * or * * are indicated
There is significant difference (P < 0.05) for different two groups of symbol.
Specific embodiment
Experimental method used in following embodiments is conventional method unless otherwise specified.
The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
Quantitative test in following embodiments, is respectively provided with three repeated experiments, and results are averaged.
Tight junction protein antibody ZO-1, claudin-1 and occludin purchase in following embodiments is certainly
Invitrogen company (Chinese Shanghai).Internal reference antibody GAPDH is bought from Santa Cruz company.Immune factor antibody Gr1,
CD11b, CD4, CD8a and CD31 purchase are bought from BD company, F4/80 from Biolegend company.Cy3- coupling-sheep blood serum anti-rabbit
IgG and TRIzon is bought from CWBIO biotech firm (BeiJing, China).Other reagents are purchased from Sigma- except non-specifically indicating
Aldrich (Chinese Shanghai).
From dimension company, tonneau China, China, mouse is raised for eight week old C57BL/6 male mices purchase in following embodiments
In the laboratory animal room of China Agricultural University's specific pathogen free.Mouse receives 12-h/12-h light/dark cycle illumination, freely
Feeding and drinking-water, from Fukang Biology Science Co., Ltd, Beijing China, mouse daily ration amino acid is dense for feeding conventional mouse daily ration purchase
Degree is as shown in table 1.All animal experiments are all ratified by local Institutional Animal protection and using the committee.
Table 1, mouse daily ration amino acid concentration
| Amino acid | Concentration g/kg daily ration |
| Asp | 20.82±0.92 |
| Glu | 43.33±1.65 |
| Ser | 11.94±0.48 |
| His | 4.97±0.23 |
| Gly | 10.14±0.47 |
| Thr | 7.43±0.35 |
| Arg | 13.24±0.70 |
| Ala | 11.18±0.37 |
| Tyr | 4.00±0.20 |
| Val | 11.30±0.38 |
| Phe | 10.80±0.29 |
| Ile | 9.63±0.34 |
| Leu | 18.02±0.54 |
| Lys | 15.81±0.44 |
| H-Pro | 1.22±0.04 |
| Pro | 21.09±2.28 |
| Cystine | 0.38±0.00 |
| Trp | 1.92±0.04 |
| Met | 5.96±0.14 |
Embodiment 1, L-Trp are disorderly in the mouse intestinal inflammatory reaction and barrier function for alleviating dextran sulfate sodium induction
Application in unrest
One, experimental method
1, experimental group and processing
40 eight week old C57BL/6 male mices are randomly divided into 4 groups by this test: control group, DSS group, Trp group and Trp+
DSS group, every group of 10 mouse.Experiment process process is divided into laundering period (7 days), pretreatment phase (7 days), DSS induction acute colonic
Scorching phase (7 days) and convalescence (3 days), the processing method of different times each group is as follows:
Control group (Control): the laundering period 7 days, mouse normally fed water and conventional mouse daily ration, recorded amount of drinking water daily
The intake of tryptophan is accurately calculated with feed intake；The pretreatment phase 7 days, the processing method and record index of mouse were the same as adaptation
Phase records the intake of amount of drinking water, feed intake, daily gain and tryptophan daily；DSS is induced the acute colitis phase 7 days, mouse
Processing method is the same as the phase that pre-processes；Convalescence 3 days, mouse processing method was according to the pretreatment phase.
DSS group: the laundering period 7 days, mouse normally fed water and conventional mouse daily ration, record daily amount of drinking water and feed intake with
Accurately calculate the intake of tryptophan；The pretreatment phase 7 days, processing method and the record index same laundering period of mouse recorded daily
Amount of drinking water, feed intake, the intake of daily gain and tryptophan；DSS is induced the acute colitis phase 7 days, and mouse is in addition to according to pre-
Process phase is fed outside water, and it is that (from MP Biomedicals company, molecular weight is 2%DSS for purchase that mass fraction is also added in water
36,000-50,000) acute colitis is induced；Convalescence 3 days, mouse processing method (observed intestinal mucosa according to the pretreatment phase
Restore).
Trp group: the laundering period 7 days, mouse also added tryptophan in addition to normally feeding water and conventional mouse daily ration in water
(0.1mg/g bw) records amount of drinking water and feed intake daily to accurately calculate the intake of tryptophan；The pretreatment phase 7 days, mouse
Processing method and the record index same laundering period, every day entry amount of drinking water, feed intake, the intake of daily gain and tryptophan；
DSS is induced the acute colitis phase 7 days, and mouse processing method is the same as the phase that pre-processes；Convalescence 3 days, mouse processing method was according to pre- place
The reason phase.
Trp+DSS group: the laundering period 7 days, mouse also added tryptophan in addition to normally feeding water and conventional mouse daily ration in water
(0.1mg/g bw) records amount of drinking water and feed intake daily to accurately calculate the intake of tryptophan；The pretreatment phase 7 days, mouse
Processing method and the record index same laundering period, every day entry amount of drinking water, feed intake, the intake of daily gain and tryptophan；
DSS is induced the acute colitis phase 7 days, and other than adding tryptophan in water, the DSS that also addition mass fraction is 2% is lured mouse
Lead acute colitis；Convalescence 3 days, mouse processing method was according to pretreatment phase (recovery of observation intestinal mucosa).
2, growth performance and Disease Activity Index (disease activity index, DAI)
Weight, feed intake and the amount of drinking water of mouse are recorded in experimentation, and observe pellet morphology and hematochezia situation, and adopt
Disease Activity Index is measured with blind, Disease Activity Index (DAI) is point according to body weight loss, pellet morphology and hematochezia situation
Number calculates, specifically can be according to document " Lu F, Fernandes SM, Davis AE, 3rd.The role of the
complement and contact systems in the dextran sulfate sodium-induced colitis
model:the effect of C1inhibitor in inflammatory bowel disease.American
journal of physiology Gastrointestinal and liver physiology.2010；298:G878-
Method in 83. " calculates Disease Activity Index (DAI).The score of each parameter calculated summary are as follows: relative to test first day
Body weight loss scoring (0:<1%, 1:1-5%, 2:5-10%, 3:10-15%, 4:>15%)；Pellet morphology scoring (0: it is normal,
2: it is loose, 4: watery stools)；The scoring of hematochezia situation (0: negative, 1: weakly positive is occulted blood, and 2: the positive is occulted blood, and 3: strong positive is occulted blood,
4: dominant hematochezia).Wherein, testing cassete (Nanjing of China builds up biotech firm) is occulted blood according to operation manual measurement excrement using fecaluria
The degree occulted blood.
3, caecum length and shoot formation
The 10th day after addition DSS, to the anesthetic of mouse injection high dose, (anesthetic is that ketamine, plug are drawn piperazine and life
Reason salt water is uniformly mixed so as to obtain, and the final concentration of ketamine and Sai La piperazine is respectively 100mg/kg BW and 8mg/kg BW), after anesthesia,
Abdominal cavity is opened, the colon between ileocecus and proximal rectum is separated；The length of colon is measured, small intestine, heart, spleen, kidney are separated
Dirty and liver, and weigh weight.According to following formula: relative organ's index=(absolute organ's index × 100%)/mouse is killed
The weight on the same day calculates relative organ's index.
4, histochemistry's parameter
The 10th day after addition DSS, after taking 1.5-2cm colon and jejunum to be fixed for 24 hours with 4% paraformaldehyde, it is stored in 70%
Alcoholic solution in.Then by tissue dewatering, paraffin embedding, slice (5 μm), and dyed with h and E
(hematoxylin and eosin, H&E).Specific experiment step are as follows: (1) be dehydrated.Successively use the alcohol immersion group of increasing concen-trations
Knit block: 85% alcohol (30min), 95% alcohol (30min), 100% alcohol (1h), 100% alcohol (1h)；(2) transparent.With two
Toluene soak tissue block is until tissue block is fully transparent；(3) waxdip.Transparent tissue block is placed in the paraffin dissolved,
It is put into wax-dissolving box heat preservation 2h, until paraffin is completely immersed in tissue block；(4) it embeds.Paraffin-embedded tissue block is poured into embedded box, to
Paraffin is cooled and solidified into blocks；(5) it is sliced, opens up piece and roasting piece.Wax stone is fixed on slicer, 5 μm of thin slice is cut into.It cuts
Thin slice is put into 42 DEG C of water and is attached on glass slide after flattening, puts in 37 DEG C of insulating boxs and dries overnight.Then in 60 DEG C of roasting piece 1h；
(6) dewaxing, rehydration.The paraffin in slice is sloughed with dimethylbenzene, then is successively immersed in the alcohol of descending concentrations: 100% alcohol
(5min), 95% alcohol (5min), 70% alcohol (5min), 50% alcohol (5min), are finally putting into distilled water；(7) H&E contaminates
Color.Slice is put into hematoxylin aqueous solution and dyes 2min, is rinsed 2 times, each 2min with tap water, observation is thin under the microscope
Karyon is dyed to blue；Slice is immersed in 1% hydrochloric acid differentiation 5s and takes off extra coloring agent, rinses slice 2 times with tap water,
Each 2min；Slice is then placed on eosin stains agent 2min, is rinsed 2 times with tap water, each 2min；(8) it is dehydrated.Successively use
Following reagent impregnates slice: 70% alcohol (5s), 95% alcohol (1min), 100% alcohol (5min), dimethylbenzene (10min)；
(9) mounting.By the transparent upper natural gum of slice drop, covered sealing is observed after drying.
5, quantitative RT-PCR
The 10th day after addition DSS, colonic tissue total serum IgE is prepared according to operation manual TRIzon.It is used according to specification
MRNA is carried out reverse transcription PCR by PrimeScript RT kit (DaLian, China TaKaRa), obtains cDNA.With the cDNA of acquisition
For template, using 7500 sequence of SYBR Premix Ex Taq II kit (DaLian, China TaKaRa) and ABI-Prism
Detection system (Applied Biosystems) carries out real-time quantitative PCR, detects IL-6, TNF-α, IL-17a, G-CSF, MIP-1
The expression quantity of α, CXCL-2, TGF-β 2, TGF-β 3 and β-actin.Primer sequence is as shown in table 2.
Table 2, primer sequence
6, the micro- measurement of immunofluorescence
2cm colon is longitudinally stood, colonic tissue is completely covered with OCT after embedded box, it is spare to be subsequently placed into -80 DEG C of freezings.
After the frost section of colon of OCT embedding, it is being air-dried at room temperature 20min；1h is fixed with acetone, is then closed with 10% lowlenthal serum
30min.Then it is sliced respectively with claudin-1, occludin, ZO-1, F4/80, Gr1, CD11b, CD4, CD8a and CD31
Primary antibodies are incubated overnight in 4 DEG C, are then incubated for 1h with the secondary antibody of Cy3 coupling.After slice is rinsed 3 times with PBS, use
Hoechst 33258 (1mg/mL) contaminates nucleus.Slice is incubated for as negative control using the nonimmune IgG of same concentrations.It is immune
The slice of dyeing is observed at fluorescence microscope (Axio Vert.A1, Zeiss).At least there are three repeat for all tests.
7, data are analyzed
Data are expressed as means ± S.E.M, with the single factor test or two-way ANOVA method of 8.1 software of SAS to data
It is analyzed.The Multiple range test of average is analyzed using Student-Newman-Keuls method between different disposal.P value <
0.05 indicates significant difference.
Two, experimental result
1, tryptophan addition improves the growth performance of mouse
In the pretreatment phase, there was no significant difference (P > 0.05) (Fig. 1 C) for control group and Trp group mouse daily gain, but Trp group
The amount of drinking water and feed intake of mouse are all remarkably higher than control group (P < 0.05) (Figure 1A and Figure 1B), and Trp group mouse tryptophan is taken the photograph
It is higher than control group by 44.81% (2.529mg/ days) to enter amount；2.529mg tryptophan 78.13% is to take in (table 3) by drinking-water.It says
The growth performance of mouse can be improved in light colour propylhomoserin.
The intake (mg/d) of mouse tryptophan in table 3, test
| Processing | Pass through the tryptophan amount of drinking-water intake | Pass through the tryptophan amount of feeding intake |
| Control group | 0 | 5.644±0.163 |
| Trp group | 1.976±0.043 | 6.197±0.114 |
2, body weight loss, dead mouse and the shoot formation variation of DSS induction are alleviated in tryptophan addition
From the point of view of mouse weight, compared with the control group, the weight of the 7-10 days mouse significantly drops after DSS addition in DSS group
Low (P < 0.05)；But except the weight of the 8th day (P < 0.05) outer mouse is without aobvious after compared with the control group, DSS is added in Trp+DSS group
It writes sex differernce (P > 0.05) (Fig. 2A).
From the point of view of mouse survival rate, compared with the control group, DSS addition causes the death of mouse in DSS group, and the death rate is
60%, but compared with the control group, there is no the death of mouse (P < 0.05) (Fig. 2 B) for DSS addition in Trp+DSS group.
From the point of view of shoot formation, compared with the control group, DSS addition significantly improves (P < 0.05) DSS group and Trp+DSS group
The liver shoot formation of mouse, and there was no significant difference between this two groups (P > 0.05), DSS and tryptophan to mouse small intestine, heart,
There was no significant difference for the shoot formation of spleen and kidney (P > 0.05) (table 4).
Table 4, mouse organs' index (mg/g)
| Small intestine | Heart | Spleen | Kidney | Liver | |
| Control | 46.69±2.76 | 5.91±0.55 | 3.35±0.38 | 11.26±0.33 | 38.14±1.15b |
| DSS | 49.79±1.78 | 5.11±0.38 | 4.64±0.47 | 11.38±0.25 | 45.16±1.44a |
| Trp | 47.34±1.08 | 5.63±0.27 | 3.78±0.15 | 10.47±0.39 | 34.88±0.42b |
| Trp+DSS | 51.43±2.44 | 6.02±0.19 | 4.57±0.35 | 11.15±0.42 | 45.02±1.93a |
3, the activity of intestines problem caused by DSS is alleviated in tryptophan addition
Compared with the control group, DSS addition causes the serious diarrhea of mouse and intestinal bleeding in DSS group, leads to enteron aisle disease
Sick activity dramatically increases (P < 0.05)；Compared with DSS group, the caecum of Trp+DSS group mouse shortens and diarrhea situation is even more slight
(P < 0.05) (Fig. 2 C), the caused intestinal bleeding situation of Trp+DSS group are equally also slighter (Fig. 3 B) than DSS group.From colon shape
State and superstructure analysis shows that, compared with DSS group mouse, Trp+DSS group mouse is soaked because the addition of Trp reduces inflammatory cell
Profit, crypt damage and mucous membrane loss (Fig. 3 A).Similar result is also observed in the morphological analysis picture of jejunum.With DSS group
It compares, Trp+DSS group mouse maintains the villus and crypts integrality (Fig. 3 C and Fig. 3 D) of small intestine because of the addition of tryptophan.Trp+
DSS group mouse reduces the increase (P < 0.05) (Fig. 2 D) of Disease Activity Index caused by DSS due to the addition of tryptophan.
4, tryptophan addition reduces the damage of gut barrier caused by DSS
Compared with the control group, the distribution of DSS group colonic mucosa tight junction protein ZO-1, claudin-1 and occludin
Density reduces and tight connecting device damages, but Trp+DSS group is because the addition of tryptophan effectively maintains colonic mucosa
The integrality (Fig. 4) of tight connecting device and gut barrier.
5, tryptophan addition reduces intestine immunity cellular infiltration caused by DSS
The testing result of immunocyte mark molecule shows: comparing with control group, causes inflammation after DSS addition in DSS group
The infiltration of disease relevant cell improves colon F4/80+And CD11b+Macrophage, Gr1+Neutrophil leucocyte, CD4+And CD8a+T
Cell, CD31+Platelet endothelial cell quantity, the infiltration of immunocyte caused by DSS are predominantly located at lamina propria and submucosa.But
Compared with DSS group, Trp+DSS group mouse Colon mucosa-immune cell quantity significantly reduces (Fig. 5).
6, the expression of the colitis factor caused by tryptophan addition regulation DSS
Cell factor and inflammatory mediator in mouse Colon (IL-6, TNF-α, IL-17a, G-CSF, MIP-1 α, CXCL-2,
TGF-β 2 and TGF-β 3) the testing result of mRNA expression show: compared with the control group, IL-6 in DSS group mouse Colon,
TNF-α, IL-17a, G-CSF and MIP-1 α mRNA expression significantly improve (P < 0.05).But it is added in Trp+DSS group
After tryptophan, inflammatory factor IL-6, TNF-α, IL-17a, G-CSF and MIP-1 α expression be substantially less than DSS group (P <
0.05), and with control group and the no significant difference of Trp group (P > 0.05).Compared with the control group, DSS group and Trp+DSS group
The mRNA expression of CXCL-2 there was no significant difference (P > 0.05).But the expression of the mRNA in Trp group CXCL-2
It significantly reduces (P < 0.05).Compared with the control group, the mRNA expression of TGF-β 2 and TGF-β 3 is significant in DSS group mouse Colon
Property reduce (P < 0.05), but in Trp+DSS group add tryptophan after, the mRNA expression of TGF-β 2 and TGF-β 3 is significantly higher than
DSS group (P<0.05), and there was no significant difference (P>0.05) (Fig. 6) with control group.It may also be seen that: tryptophan is independent
Processing can improve the mRNA level in-site (P < 0.05) of TGF-β 2 in mouse Colon with conspicuousness.
Claims (10)
1.L- tryptophan is in following A1)-A10) in it is any in application:
A1 the application in the product of prevention and or treatment inflammation enteropathy and/or colitis) is prepared；
A2) prevention and or treatment inflammation enteropathy and/or colitis；
A3) intestinal inflammation and/or the barrier function disorder of dextran sulfate sodium induction are alleviated in preparation；
A4) alleviate intestinal inflammation and/or the barrier function disorder of dextran sulfate sodium induction；
A5 gut integrity) is maintained；
A6 the tight junction protein structure of intestinal epithelial cell) is maintained；
A7) lower intestinal inflammatory factor IL-6 and/or TNF-α and/or IL-17a and/or G-CSF and/or MIP-1a and/or
The mRNA expression of CXCL-2；
A8 the mRNA expression of intestinal inflammatory factor TGF-β 2 and/or TGF-β 3) is raised；
A9 infiltration of the immunocyte of dextran sulfate sodium induction to colonic lamina propria and submucosa) is reduced；
A10 the growth performance of animal individual) is improved.
2. application according to claim 1, it is characterised in that:
The tight junction protein structure for maintaining intestinal epithelial cell, which is embodied in, improves endothelial cell tight connection albumen ZO-1
And/or the expression of occludin and/or claudin-1.
3. application according to claim 1, it is characterised in that: the intestinal inflammatory for alleviating dextran sulfate sodium induction is anti-
Any one of answer and/or barrier function disorder be embodied in following B1)-B5):
B1 the weight of the animal individual of dextran sulfate sodium induction) is improved；
B2 the survival rate of the animal individual of dextran sulfate sodium induction) is improved；
B3 the Disease Activity Index of the animal individual of dextran sulfate sodium induction) is reduced；
B4 the colon lengths of the animal individual of dextran sulfate sodium induction) are improved；
B5) alleviate the intestinal bleeding of the animal individual of dextran sulfate sodium induction.
4. application according to claim 1, it is characterised in that:
The immunocyte for reducing dextran sulfate sodium induction is embodied in reduction to the infiltration of colonic lamina propria and submucosa
Colonic mucosa immunocyte quantity；
The colonic mucosa immunocyte is specially neutrophil leucocyte and/or macrophage and/or T cell.
5. application according to claim 1, it is characterised in that:
The growth performance for improving animal individual is embodied in the amount of drinking water and/or feed intake for increasing animal individual.
6. a kind of product, active constituent is L-Trp；
The product have following C1)-C8) and in any function:
C1) prevention and or treatment inflammation enteropathy and/or colitis；
C2) alleviate intestinal inflammation and/or the barrier function disorder of dextran sulfate sodium induction；
C3 gut integrity) is maintained；
C4 the tight junction protein structure of intestinal epithelial cell) is maintained；
C5) lower intestinal inflammatory factor IL-6 and/or TNF-α and/or IL-17a and/or G-CSF and/or MIP-1a and/or
The mRNA expression of CXCL-2；
C6 the mRNA expression of intestinal inflammatory factor TGF-β 2 and/or TGF-β 3) is raised；
C7 infiltration of the immunocyte of dextran sulfate sodium induction to colonic lamina propria and submucosa) is reduced；
C8 the growth performance of animal individual) is improved.
7. product according to claim 6, it is characterised in that:
The tight junction protein structure for maintaining intestinal epithelial cell, which is embodied in, improves endothelial cell tight connection albumen ZO-1
And/or the expression of occludin and/or claudin-1.
8. product according to claim 6, it is characterised in that: the intestinal inflammatory for alleviating dextran sulfate sodium induction is anti-
Any one of answer and/or barrier function disorder be embodied in following B1)-B5):
B1 the weight of the animal individual of dextran sulfate sodium induction) is improved；
B2 the survival rate of the animal individual of dextran sulfate sodium induction) is improved；
B3 the Disease Activity Index of the animal individual of dextran sulfate sodium induction) is reduced；
B4 the colon lengths of the animal individual of dextran sulfate sodium induction) are improved；
B5) alleviate the intestinal bleeding of the animal individual of dextran sulfate sodium induction.
9. product according to claim 6, it is characterised in that:
The immunocyte for reducing dextran sulfate sodium induction is embodied in reduction to the infiltration of colonic lamina propria and submucosa
Colonic mucosa immunocyte quantity；
The colonic mucosa immunocyte is specially neutrophil leucocyte and/or macrophage and/or T cell.
10. product according to claim 6, it is characterised in that:
The growth performance for improving animal individual is embodied in the amount of drinking water and/or feed intake for increasing animal individual.
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