CN117304264B - Pentapeptide RP5 with intestinal barrier protection function and application thereof - Google Patents
Pentapeptide RP5 with intestinal barrier protection function and application thereof Download PDFInfo
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- CN117304264B CN117304264B CN202311587342.3A CN202311587342A CN117304264B CN 117304264 B CN117304264 B CN 117304264B CN 202311587342 A CN202311587342 A CN 202311587342A CN 117304264 B CN117304264 B CN 117304264B
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- pentapeptide
- enzymolysis
- intestinal barrier
- protein
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Classifications
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- C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
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- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/17—Amino acids, peptides or proteins
- A23L33/18—Peptides; Protein hydrolysates
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- A—HUMAN NECESSITIES
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
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- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
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Abstract
The invention discloses a pentapeptide RP5 with an intestinal barrier protection function and application thereof, and belongs to the technical field of biology. The amino acid sequence of the pentapeptide RP5 is RPRGP, the pentapeptide RP5 is extracted from sea cucumber enzymatic hydrolysate, has potential interaction with the tight junction protein 1 (ZO-1), can remarkably reduce inflammatory reaction of a colonitis model mouse, increase intestinal barrier function protein expression, protect and relieve intestinal barrier damage of the mouse caused by modeling drugs, and can be effectively used in chronic colonitis relieving or treating drugs.
Description
Technical Field
The invention relates to a small molecular peptide and application thereof, in particular to a pentapeptide RP5 with an intestinal barrier protection function and application thereof in chronic colitis relieving or treating medicines, and belongs to the technical field of biology.
Background
Inflammatory bowel disease is a chronic, recurrent, inflammatory bowel disease, mainly including ulcerative colitis and crohn's disease, and its pathological manifestations include intestinal barrier damage, crypt structure destruction, basal plasma cell augmentation, lymphocyte aggregation, and lamina propria fibrosis, etc. Studies have shown that long-term inflammatory bowel disease increases the risk of colon cancer.
Impairment of intestinal barrier function is both a pathological manifestation of inflammatory bowel disease and considered one of the pathogenesis of inflammatory bowel disease. Therefore, protecting/restoring the intestinal barrier function is a new idea for treating inflammatory bowel disease. The mechanical barrier is an important component of the intestinal barrier, and under the normal state, cells are tightly connected and sealed in gaps between adjacent intestinal epithelial cells under the action of a tight junction protein (ZO), so that substances such as bacteria, antigens and the like in intestinal cavities are prevented from entering the intestinal mucosa lamina propria to activate immune cells in the lamina propria, and abnormal immune reaction of the mucosa is avoided. Among them, claudin 1 (ZO-1) is an important claudin closely related to intestinal barrier function. At present, partial treatment medicines for inflammatory bowel diseases exist, but have certain side effects; while there are still few screening and excavation of natural active substances with the functions of protecting the intestinal barrier and relieving inflammatory bowel disease. The active peptide from food-borne proteolysis has the advantages of high specificity, small toxic and side effects, good curative effect, larger available medicine dosage and the like, and is a good strategy for protecting intestinal barriers and relieving inflammatory bowel diseases.
At present, active peptides with intestinal barrier protection and intestinal inflammation relief functions are prepared by using animal, plant and marine organism proteins as raw materials through an enzymolysis mode. Different enzymolysis solutions are obtained from protein raw materials of different sources in different enzymolysis processes, and the enzymolysis solutions are a series of active peptide sets with different structures, various sequences and different functions. The active peptide with intestinal barrier protection and intestinal inflammation relief functions has very large difference in amino acid composition and structure, no fixed or unified amino acid composition, and the components of proteolytic products in food are quite complex, so that the separation and extraction difficulty is greatly increased.
Arranz A et al in article Vasoactive intestinal peptide as a healing mediator in Crohn's disease report that the active peptide GPA obtained from fish skin gelatin enzymatic hydrolysate has the effects of repairing intestinal epithelial barrier injury and reducing IL-6, IL-12 and active oxygen content in a DSS induced colitis mouse model; ila Joshi et al in article A Meretrix meretrix visceral mass derived peptide inhibits lipopolysaccharide-stimulated responses in RAW264.7 cells and adult zebrafish model reported that the active peptide GQCC obtained from clam viscera enzymatic hydrolysate has an effect of inhibiting inflammatory factor gene expression in LPS-induced zebra fish model; chang-Bum Ahn et al in article Antioxidant and anti-inflammatory peptide fraction from salmon byproduct protein hydrolysates by peptic hydrolysis reported that the active peptide PAY obtained from salmon by-product proteolytic liquid has a relieving effect on LPS-induced inflammatory cell model, and has inhibition rates of 63.80% and 45.33% on NO and PGE2, respectively. Ziyan Wang et al in article Targeted screening of an anti-inflammatory polypeptide from Rhopilema esculentum Kishinouye cnidoblasts and elucidation of its mechanism in alleviating ulcerative colitis based on an analysis of the gut microbiota and metabolites report that the active peptide PKKVV obtained from jellyfish proteolytic liquid has the effect of repairing intestinal epithelial barrier damage, targeting ZO-1 in a DSS induced colitis mouse model.
Sea cucumber belongs to echinoderm, is an important ocean food and drug, and various sea cucumber species are being developed in the fields of food and pharmacy due to the rich functional composition. Sea cucumber is a natural food and medical functional material with biological activity, and is rich in protein (40.7% -63.3%). The preparation and biological activity of sea cucumber polypeptides have been focused, and it has been found and evaluated that the biological activity of sea cucumber polypeptides includes antioxidant, antidiabetic, ACE inhibiting, immunomodulating, anticancer, antifatigue, anti-aging, neuroprotection, trace metal sequestration and the like. Jing Mao et al in article Sea Cucumber Peptide Alleviates Ulcerative Colitis Induced by Dextran Sulfate Sodium by Alleviating Gut Microbiota Imbalance and Regulating miR-155/SOCS1 Axis in Mice report that sea cucumber proteolytic liquid mediates intestinal epithelial barrier through miR-155/SOCS1 Axis in DSS induced colitis mouse model, has the function of repairing intestinal barrier damage, but no further identification analysis is carried out on functional components and structures thereof in sea cucumber proteolytic liquid.
Disclosure of Invention
The invention aims to provide the pentapeptide RP5 which is screened from sea cucumber enzymatic hydrolysate, has an intestinal barrier protection function and can be applied to chronic colitis relieving or treating medicines.
In order to achieve the above object, the present invention adopts the following technical scheme:
a pentapeptide RP5 with intestinal barrier protection function, wherein the amino acid sequence of the pentapeptide RP5 is RPRGP, and the pentapeptide RP5 is shown in a sequence table SEQ ID NO: 3.
The application of the pentapeptide RP5 with the intestinal barrier protection function in medicines for relieving or treating chronic colitis.
When the pentapeptide RP5 with the intestinal barrier protection function is applied to chronic colitis relieving or treating drugs, the pentapeptide RP5 is obtained by solid phase synthesis, and the solid phase synthesis method specifically comprises the following steps: adopting Fmoc solid phase synthesis strategy, taking Fmoc protected amino acid as raw material, selecting polystyrene resin as solid phase carrier, and extending peptide chain from C end to N end according to the sequence of arginine-proline-arginine-glycine-proline to obtain pentapeptide RP5 by solid phase synthesis; or, the pentapeptide RP5 is obtained by enzymolysis of sea cucumbers, and the method for enzymolysis of the sea cucumbers is specifically as follows: (1) Taking sea cucumbers, adding compound enzyme according to an enzyme bottom ratio of 0.5% for enzymolysis, wherein the enzymolysis temperature is 55 ℃, the pH value is 8, and the enzymolysis time is 6 hours, so as to obtain an enzymolysis product, wherein the compound enzyme is formed by mixing alkaline protease and neutral protease according to a mass ratio of 1:1; (2) After the enzymolysis is finished, filtering an enzymolysis product by using eight layers of gauze, and removing residues to obtain clarified polypeptide enzymolysis liquid; (3) And freeze-drying the polypeptide enzymolysis liquid to obtain a sea cucumber peptide sample, wherein the sea cucumber peptide sample contains more pentapeptide RP5.
The invention has the advantages that: the intestinal barrier protection pentapeptide RP5 is extracted from sea cucumber enzymatic hydrolysate, has potential interaction with the tight junction protein 1 (ZO-1), can obviously reduce inflammatory response of a colonitis model mouse, increase intestinal barrier function protein expression, protect and relieve intestinal barrier damage of the mouse caused by modeling drugs, and can be effectively used in chronic colonitis relieving or treating drugs.
Drawings
FIG. 1 is a graph showing changes in the Disease Activity Index (DAI) of mice;
FIG. 2 is a graph showing the weight gain of mice;
FIG. 3 is a graph of colon length statistics for mice;
FIG. 4 is a graph showing the content of inflammatory factors in mouse serum, wherein A is a graph showing the content of TNF-alpha in mouse serum, B is a graph showing the content of IL-1β in mouse serum, C is a graph showing the content of IL-10 in mouse serum, and D is a graph showing the content of IL-22 in mouse serum;
FIG. 5 is a diagram showing transcription of inflammatory factor gene in colon tissue of mouse, wherein A is a diagram showing transcription of gene tnf-alpha, B is a diagram showing transcription of gene il-1β, C is a diagram showing transcription of gene il-10, and D is a diagram showing transcription of gene il-22;
FIG. 6 is a graph showing the expression of intestinal barrier function protein in colon tissue of mice.
Detailed Description
The invention is described in detail below with reference to the drawings and the specific embodiments.
1. Preparation of sea cucumber peptide samples
Mixing alkaline protease and neutral protease according to a mass ratio of 1:1 to obtain the complex enzyme.
Taking sea cucumber, adding compound enzyme according to an enzyme-to-enzyme ratio of 0.5% for enzymolysis, wherein the enzymolysis temperature is 55 ℃, the pH value is 8, and the enzymolysis time is 6 hours.
And after the enzymolysis is finished, filtering an enzymolysis product by using eight layers of gauze, and removing residues to obtain a clarified polypeptide enzymolysis liquid.
Lyophilizing the polypeptide enzymolysis liquid to obtain sea cucumber peptide sample.
2. Obtaining polypeptide sequences in sea cucumber peptide samples
And carrying out mass spectrometry on the sea cucumber peptide sample obtained by the method by adopting LC-MS/MS, and analyzing a mass spectrometry determination result by adopting mass spectrometry analysis software to obtain a plurality of polypeptide sequences.
The LC-MS/MS measurement conditions were:
(1) Liquid phase method: the column was C18,3 μm,250mm×75 μm (Eksigent), phase A was water, 0.1% formic acid; phase B is acetonitrile, 0.1% formic acid, the flow rate is 300nL/min, the sample injection volume is 4 mu L, the chromatographic gradient is 60min, and the specific elution gradient is: 0-48min, and the phase A is uniformly reduced from 95% to 60%;48-55min, uniformly reducing the phase A from 60% to 30%, and uniformly reducing the phase A from 30% to 0 in 55-56 min; and (5) maintaining the phase A at 0% for 56-60 min.
(2) Mass spectrometry method: orbitrap Exploris 480 (thermosusher), positive ion detection mode, first order resolution of 120000, AGC set to 300, scan range of 200-1600m/z. MIPS mode is peptide, valence state 1-5 is selected, secondary resolution is 15000, and separation window is 1.6m/z.
3. Screening active oligopeptide with abundance of more than 0.001% and amino acid number of less than or equal to 6
From the several polypeptide sequences obtained above, 33 active oligopeptides with abundance >0.001% and amino acid number less than or equal to 6 were finally screened, and the screening results are shown in table 1.
TABLE 1 active oligopeptide sequences with abundance >0.001% and amino acid number less than or equal to 6 in sea cucumber peptide samples
4. Screening active oligopeptide with strong binding capacity with ZO-1 protein and less than or equal to 6 amino acids
Polypeptide sequences with the amino acid number less than or equal to 6 are selected from a plurality of polypeptide sequences obtained in the prior art, molecular docking is carried out on the polypeptide sequences with the amino acid number less than or equal to 6 and the ZO-1 protein respectively by using Discovery Studio software, the 2D structure of the polypeptide is converted into a 3D structure by energy minimization before docking, and the polypeptide sequences with strong binding capacity with the ZO-1 protein are obtained by screening.
The 3D structure of protein ZO-1 can be downloaded from the RCSB protein database (PDB ID:4 OEP). The greater the value of-CiE, the greater the ability of the polypeptide to bind ZO-1, and the more likely it is to exhibit intestinal barrier protection, as indicated by the docking score (-CiE). From the polypeptide sequences with the amino acid number less than or equal to 6, 46 active oligopeptides with larger docking scores are screened, and the screening results are shown in Table 2.
TABLE 2 prediction of interaction of sea cucumber peptide components with ZO-1
5. Molecular docking analysis
Of all the active peptides tested, RLSPGA (designated hexapeptide RA6, SEQ ID NO: 1), DLKCPF (designated hexapeptide DF6, SEQ ID NO: 2), RPRGP (designated pentapeptide RP5, SEQ ID NO: 3), PFSRFE (designated hexapeptide PE6, SEQ ID NO: 4), QHRGQ (designated pentapeptide QQ5, SEQ ID NO: 5) and ERGF (designated tetrapeptide EF4, SEQ ID NO: 6) were all greater than 100, -CiE, and since the abundance of RPRGP therein (0.004299957%) was much higher than that of the other 5 active peptides, RPRGP was selected for further molecular docking analysis.
The molecular docking of pentapeptide RP5 with ZO-1 was analyzed as follows:
9H-H bond interactions, 3C-H bond interactions and 4 electrostatic interactions are formed between the pentapeptide RP5 and ZO-1, 28 amino acid residues participate in the interactions between the pentapeptide RP5 and the ZO-1, and the Van der Waals force between the pentapeptide RP5 and the ZO-1 is-14.59 kcal/mol.
6. Evaluation of the in vivo protective intestinal Barrier and inflammation-relieving function of pentapeptide RP5
1. Solid phase synthesis of pentapeptide RP5
Fmoc solid phase synthesis strategy is adopted, fmoc protected amino acid is used as raw material, polystyrene resin is used as solid phase carrier, peptide chain is extended from C end to N end according to the sequence of arginine-proline-arginine-glycine-proline, and pentapeptide RP5 (purity > 90%) is synthesized in solid phase.
2. Animal experiment
At the beginning of the experiment, each mouse was first labeled and weighed, then body weight was weighed every two days, and the mouse body weight, hematochezia, and stool morphology were recorded.
The modeling process of the mouse chronic colitis model is as follows: 7 days 1 cycle, wherein, on 1 day to 5 days, dextran Sodium Sulfate (DSS) is added into drinking water, and the final concentration of DSS is 2.5% (w/v), and on 6 days and 7 days, normal drinking water is obtained; the 3 cycles are repeated.
C57BL/6J mice of 5-6 weeks of age were fed adaptively for one week, randomly divided into 3 groups, each: control group (control), model group (DSS) and RPRGP treatment group (RPRGP).
Mice in the model group and the RPRGP treatment group are subjected to DSS solution modeling, and mice in the control group drink water normally in the whole course; at the same time, the RPRGP treated mice were perfused with 10mg/kg of the aforementioned solid phase synthesized pentapeptide RP5 (dissolved in 200. Mu.L of physiological saline) per day, and the control and model mice were perfused with 200. Mu.L of physiological saline per day. After 3 cycles, it ends. Mice were sacrificed.
3. Sample collection and processing
(1) Serum sample
Whole blood of the mice was collected by taking blood from the eyeballs and placed in a 1.5mL EP tube and centrifuged at 2500rpm in a low temperature centrifuge at 4 ℃ for 5min. Sucking out the upper serum layer with a pipette, sub-packaging, and storing in a refrigerator at-80deg.C for enzyme-linked immunosorbent assay (ELISA).
(2) Colon specimen
After dissecting the mice, taking out the colon tissue, measuring the length of the colon tissue by a ruler, photographing and recording, then wrapping the colon tissue by tinfoil, quick-freezing the colon tissue by liquid nitrogen, and storing in a refrigerator at-80 ℃ for subsequent tissue protein and RNA extraction.
4. Observing Disease Activity Index (DAI), weight gain and colon length
The DAI of each group of mice was scored against the Disease Activity Index (DAI) scoring criteria (table 3), and the scoring results are shown in fig. 1.
TABLE 3 DAI scoring criteria
From fig. 1, from day 6, the DAI index of the mice in the model group gradually increased, and the overall was about 10; whereas pentapeptide RP5 treatment reduced DAI index in mice with chronic colitis model.
The weight gain of each group of mice is shown in FIG. 2. As can be seen from fig. 2, the weight gain (-1.96±1.60 g) was significantly reduced (p < 0.001) in the mice of the inflammatory model group compared to the control group (weight gain of 2.44±0.93 g); compared with the model group, the weight gain (0.39+/-1.02 g) of the mice in the RPRGP treatment group is obviously improved (p < 0.01). It can be seen that pentapeptide RP5 treatment significantly increased the body weight gain in mice with chronic colitis model.
The statistics of colon length for each group of mice are shown in figure 3. As can be seen from fig. 3, the colon length (4.52±0.48 cm) was significantly reduced (p < 0.001) in the mice of the model group compared to the control group (colon length of 6.32±0.21 cm); compared to the model group, RPRGP treated group mice showed a significant increase in colon length (6.18±1.15 cm) (p < 0.01). It can be seen that pentapeptide RP5 treatment significantly increases the colon length in mice with chronic colitis models.
5. Detecting inflammatory factor content
Diluting a standard substance and a sample to be detected in the kit to corresponding concentrations according to requirements, adding the diluted standard substance and the sample to be detected to the bottom of an ELISA plate containing the coated antibody, incubating for 30min, and washing; then adding an HRP enzyme-labeled antibody to form an antibody-antigen-enzyme-labeled antibody complex, incubating for 30min and washing; after the addition of the color-developing agent and development of color for 10min, the reaction was terminated, and the absorbance value of each well was measured at a wavelength of 450 nm. And drawing a standard curve by using the standard substance, and calculating the inflammatory factor content of each group.
The change of the serum TNF-alpha content of each group of mice is shown in a figure 4A, the change of the serum IL-1 beta content is shown in a figure 4B, the change of the serum IL-10 content is shown in a figure 4C, and the change of the serum IL-22 content is shown in a figure 4D.
As can be seen from fig. 4, the serum TNF- α content (1577.0 pg/mL) was significantly increased (p < 0.001) in the mice of the model group compared to the control group (1384.2 pg/mL serum TNF- α concentration); compared to the model group, the RPRGP treated group mice had significantly reduced serum TNF- α content (1368.0 pg/mL) (p < 0.001). The serum IL-1β content (967.0 pg/mL) was significantly increased (p < 0.001) in the mice of the model group compared to the control group (644.4 pg/mL serum IL-1β concentration); compared with the model group, the serum IL-1β content (827.8 pg/mL) of the mice in the RPRGP treatment group was not significantly reduced (p > 0.05). The serum IL-10 content (1330.4 pg/mL) was significantly increased (p < 0.001) in the mice of the model group compared to the control group (serum IL-10 concentration 1114.5 pg/mL); the serum IL-10 content (1368.0 pg/mL) was not significantly increased (p > 0.05) in the RPRGP treated mice compared to the model group. The serum IL-22 content (1081.1 ng/mL) of mice in the model group was significantly increased (p < 0.001) compared to the control group (serum IL-22 concentration 830.5 ng/mL); compared to the model group, the serum IL-22 content (558.5 ng/mL) was significantly reduced (p < 0.001) in the RPRGP treated group mice.
It can be seen that the pentapeptide RP5 treatment can significantly reduce the contents of inflammatory factors TNF-alpha and IL-22 in the serum of mice in the chronic colitis model, and has no significant effect on the contents of inflammatory factors IL-1 beta and IL-10.
6. Real-time fluorescent quantitative PCR detection
(1) Designing primers
The inflammatory factor genes tnf-alpha, il-1 beta, il-10 and il-22 were selected. Real-time fluorescence quantitative PCR (qRT-PCR) Primer design was performed using NCBI website (https:// www.ncbi.nlm.nih.gov /) and Primer 5 software from Premier, canada, and the information of the designed primers is shown in Table 3.
TABLE 4 real-time fluorescent quantitative PCR primer sequences
(2) Total RNA extraction from tissue
Taking out a colon sample from a refrigerator at the temperature of minus 80 ℃, weighing 100mg, putting into a mortar pre-cooled in advance, fully grinding, and continuously adding liquid nitrogen in the grinding process to prevent the sample from being damaged; adding Trizol into the ground tissue, standing and then swirling; total RNA was extracted according to the TransZol Up Plus RNA kit procedure and the concentration and quality of total RNA was determined using Nano Drop 2000 c. RNA was dispensed into RNase-free centrifuge tubes and stored at-80 ℃.
(3) cDNA template synthesis
1 μg of tissue total RNA was taken and a cDNA template was synthesized using the protocol provided by the TransScript All-in-One First-Strand cDNA Synthesis SuperMix for qPCR (One-Step gDNA Removal) kit. The synthesized cDNA can be used as a template for qRT-PCR after being diluted to a certain concentration.
(4) Gene expression detection
The target Gene and the internal reference Gene (beta-actin) are amplified by using a Rotor-Gene 6000 to respectively amplify the cDNA sample, so that the amplification efficiency of the target Gene and the internal reference Gene is similar, and the experimental error is reduced.
Preparation of qRT-PCR system: after properly diluting the cDNA template, 5. Mu.L of the cDNA template was added to the total system, 0.4. Mu.L of the forward primer and the reverse primer were added to the total system, respectively, and 10. Mu.L of 2X TransStartTM Green qPCR SuperMix (containing SYBR Green dye) and 4.2. Mu.L of enzyme-free water were added to the total system, which was 20. Mu.L.
qRT-PCR three-step method: denaturation at 94℃for 10min, amplification reaction for 40 cycles, denaturation at 94℃for 10s, annealing at 50℃for 10s, extension at 72℃for 30s, total extension for 10min.
The gene expression levels of each group were obtained according to the 2- ΔΔct calculation method.
The transcription of gene tnf-alpha is shown in FIG. 5A, the transcription of gene il-1 beta is shown in FIG. 5B, the transcription of gene il-10 is shown in FIG. 5C, and the transcription of gene il-22 is shown in FIG. 5D.
As can be seen from fig. 5, the mice in the model group significantly up-regulated the expression of tnf- α (p < 0.001), il-1β (p < 0.05), il-10 (p < 0.01) and il-22 (p < 0.001) in the colon compared to the control group. Compared to the model group, RPRGP treated mice significantly down-regulated expression of tnf- α (p < 0.001), il-1β (p < 0.05) and il-22 (p < 0.01) in the colon. The expression of il-10 in the colon of mice in the RPRGP treated group was not significantly down-regulated (p > 0.05) compared to the model group.
It can be seen that the DSS modeling process significantly up-regulates the expression of genes tnf- α, il-1β, il-10 and il-22 in the colon of mice. The pentapeptide RP5 treatment has obvious inhibition effect on the transcription of genes tnf-alpha, il-1 beta and il-22 in the colon of a mouse with a chronic colitis model, and has no obvious inhibition effect on the transcription of gene il-10.
6. Western blot detection
(1) Preparation of tissue protein samples
Protein extraction operations were performed on ice. Taking a colon sample out of a refrigerator at the temperature of minus 80 ℃, weighing 100mg, putting into RIPA Lysis Buffer lysate pre-cooled on ice in advance, adding protease inhibitor and phosphatase inhibitor when crushing by a homogenizer, wherein the volume ratio of RIPA Lysis Buffer lysate, protease inhibitor and phosphatase inhibitor is 100:1:1, ensuring that the tissue is sufficiently lysed, then standing on ice for 10min, and then centrifuging for 10min at 13000 Xg in a cryogenic centrifuge, taking the supernatant.
The extracted protein was total protein, and protein concentration was measured using BCA kit. The protein was stored in a-80℃refrigerator for further use.
Then carrying out pre-denaturation on protein samples, mixing 30 mug of total amount of each protein sample with 6mL loading buffer to prepare mixed loading liquid, and carrying out metal bath denaturation at 100 ℃ for 10min after full shaking. And (5) after the mixed sample solution is cooled to room temperature, oscillating uniformly again and centrifuging to obtain the sample protein.
(2) Protein separation
(a) Preparation of electrophoresis gel
The preparation method of the electrophoresis gel comprises the following steps:
(i) Cleaning rubber pad on glass plate, sample comb and rubber making frame with detergent, and using ddH 2 Washing for several times, wiping with ethanol, and air drying;
(ii) Placing the two cleaned glass plates on a glue making frame, and horizontally placing the glue making frame on a tabletop;
(iii) Preparing 12% protein isolate gel, specifically taking a clean beaker, adding 6mL of polyacrylamide solution with the concentration of 30wt%, 3.8mL of Tris-HCL buffer with the concentration of 1.5mol/L, 150 mu L of Sodium Dodecyl Sulfate (SDS) solution with the concentration of 10wt%, 150 mu L of ammonium persulfate solution with the concentration of 10wt%, 6 mu L of tetramethyl ethylenediamine (TEMED) and 5mL of deionized water, and uniformly mixing;
(iv) Pouring the 12% protein separation gel between glass plates, immediately covering a layer of double distilled water, and polymerizing the gel after about 30 min;
(v) Preparing 5% concentrated glue, specifically taking a clean beaker, adding 0.85mL of polyacrylamide solution with the concentration of 30wt%, 0.65mL of Tris-HCL buffer solution with the concentration of 1.5mol/L, 50 mu L of SDS solution with the concentration of 10wt%, 50 mu L of ammonium persulfate solution with the concentration of 10wt%, 5 mu L of TEMED and 3.4mL of deionized water, and uniformly mixing;
(vi) Pouring out double distilled water on the protein separating gel, pouring the 5% concentrated gel, and inserting a sample comb.
(b) Protein electrophoresis
The protein isolate was placed in 1 XSDS-PAGE protein buffer. The prepared protein loaded in advance is added into the corresponding hole of each lane, and simultaneously, a high molecular weight protein marker is added. The power supply is switched on, a 120V voltage is firstly used for enabling the protein sample to pass through the concentrated glue, and when the protein marker reaches the junction of the concentrated glue and the separation glue (about 20 min), the voltage is regulated to 70V, so that the protein passes through the separation glue. And when the bromophenol blue indicator in the protein sample reaches the bottom of the gel block, finishing protein separation, and stopping electrophoresis (about 60 min).
(c) Gel electrophoresis protein transfer membrane
Preparing a film transfer liquid: 100mL of 10 Xtransfer Buffer and 150mL of methanol were mixed and water was added to a volume of 1000mL.
Cutting gel strips containing target proteins according to the positions of the proteins and the number of protein samples, cutting out required filter paper and PVDF membrane, soaking the filter paper in electrophoresis liquid for standby, and completely soaking the PVDF membrane in methanol for 15-30s in advance. After the PVDF film becomes semitransparent, the PVDF film is placed in the film transfer solution for 3min of equilibration. And (3) tightly attaching the gel strip and the PVDF film together by adopting a sandwich stacking method, removing bubbles between the PVDF film and the gel strip, then clamping the plate to enable the protein film to be close to the negative electrode, placing the protein film into an electrophoresis tank, adding electrophoresis liquid, adjusting the voltage to 65V, and transferring the film for 90min under an ice bath to obtain the protein film.
(3) Protein immunoassay
(a) Closure
Preparing 5% (W/V) skimmed milk, soaking the protein film in skimmed milk, slowly shaking in a shaking table at normal temperature for 1 hr, and sealing the other unbound protein positions on the protein film.
(b) Incubation of primary antibody
According to the dilution ratio of the primary antibody suggested in the specification, the primary antibody was reasonably diluted with 5% (W/V) skim milk. After the primary antibody was added, the protein film was placed under an environment of 4 ℃ overnight.
(c) Cleaning
The overnight incubated protein film had bound the primary antibody, the primary antibody was decanted, TBST buffer was added to cover the protein film, and the incubation was slowly shaken on a shaker for 10min, and repeated three times to wash out excess primary antibody that was not bound to the protein film.
(d) Incubation of secondary antibody
The secondary antibody is selected according to the source of the primary antibody. HRP-labeled secondary antibodies were formulated with TBST buffer at a dilution ratio of 1:3000. After adding the secondary antibody, the protein film was placed on a shaker and slowly shaken at room temperature for 1h.
(e) Cleaning and developing
The developing solution A and the developing solution B are prepared for standby according to the volume ratio of 1:1.
The protein film was covered and washed 3 times with TBST buffer. And uniformly covering the prepared developing solution on a protein film, standing for 1min, imaging protein strips by using a BIO-RAD gel imager, and preserving pictures.
(f) Protein band gray scale analysis
Gray scale processing is carried out on the protein imaging pictures by using imageJ software, and gray scale ratio between proteins to be detected (Occludin and ZO-1) and actin is calculated.
The results of processing the protein imaging pictures and the results of calculating the gray scale ratio between the proteins to be tested (Occludin and ZO-1) and actin are shown in fig. 6.
As can be seen from fig. 6, the expression of barrier proteins Occludin and ZO-1 was reduced in the colon of the mice in the model group as compared to the control group; the expression of barrier proteins Occludin and ZO-1 was increased in the colon of the mice in the RPRGP treated group compared to the model group.
It can be seen that the colitis modeling process reduced the expression of barrier proteins Occludin and ZO-1 in the colon of mice, decreasing intestinal barrier function; pentapeptide RP5 treatment can increase the expression of barrier proteins Occludin and ZO-1 in the colon of mice in a model of chronic colitis.
In conclusion, the pentapeptide RP5 (with the sequence of RPRGP) obtained by screening from sea cucumber enzymatic hydrolysate has the intestinal barrier protection function and can be applied to chronic colitis relieving or treating medicines.
It should be noted that the above examples are only examples for clearly illustrating the present invention, and are not limiting to the embodiments of the present invention. Other variations or modifications of the above teachings will be apparent to those of ordinary skill in the art. Not all embodiments are exhaustive. All obvious changes or modifications which are obvious from the technical proposal of the invention are still within the protection scope of the invention.
Claims (4)
1. The pentapeptide RP5 with the intestinal barrier protection function is characterized in that the amino acid sequence of the pentapeptide RP5 is RPRGP, and the sequence table is shown as SEQ ID NO: 3.
2. Use of the pentapeptide RP5 with intestinal barrier protective function of claim 1 in the preparation of a medicament for alleviating or treating chronic colitis.
3. The method for preparing pentapeptide RP5 with intestinal barrier protection function according to claim 1, wherein Fmoc solid phase synthesis strategy is adopted, fmoc protected amino acid is used as raw material, polystyrene resin is used as solid phase carrier, and peptide chain is extended from C end to N end according to the sequence of arginine-proline-arginine-glycine-proline, so as to synthesize pentapeptide RP5 in solid phase.
4. The method for producing pentapeptide RP5 with intestinal barrier protecting function of claim 1 wherein the enzymatic hydrolysis is performed as follows:
(1) Taking sea cucumbers, adding compound enzyme according to an enzyme bottom ratio of 0.5% for enzymolysis, wherein the enzymolysis temperature is 55 ℃, the pH value is 8, and the enzymolysis time is 6 hours, so as to obtain an enzymolysis product, wherein the compound enzyme is formed by mixing alkaline protease and neutral protease according to a mass ratio of 1:1;
(2) After the enzymolysis is finished, filtering an enzymolysis product by using eight layers of gauze, and removing residues to obtain clarified polypeptide enzymolysis liquid;
(3) And freeze-drying the polypeptide enzymolysis liquid to obtain a sea cucumber peptide sample, wherein the sea cucumber peptide sample contains more pentapeptide RP5.
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