Application of Lg-like domain protein and compound thereof in preparation of medicine for relieving irritable bowel syndrome

Abstract

The invention relates to the field of biological medicine, in particular to application of Lg-like domain protein and a compound thereof in preparation of a medicine for relieving irritable bowel syndrome. The Lg structural domain protein Llp-495, the Lg sample structural domain protein Llp-495 and the coptis chinensis extract composition provided by the invention can obviously reduce the content of tryptase in intestinal tissues, and inhibit the expression of mast cell surface membrane protein 1, mast cell protease 4-like protein, mast cell protease 9, carboxypeptidase, chymotrypsin and the like, so that the activation of mast cells is inhibited, and the intestinal tract is protected. The viscera hypersensitivity of the irritable bowel syndrome rats is relieved. Besides, the Lg structural domain protein Llp-495, the Lg-like structural domain protein Llp-495 and a coptis chinensis extract composition can improve intestinal flora disorder of the irritable bowel syndrome rats, so that generation of pro-inflammatory substances such as LPS in intestinal tracts is reduced.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to the use of Lg-like domain proteins and compounds thereof in the preparation of drugs for alleviating irritable bowel syndrome. Background Art

[0002] Irritable bowel syndrome (IBS) is a common gastrointestinal functional disorder characterized by persistent or intermittent abdominal pain and bloating, often accompanied by changes in bowel habits and abnormal stool characteristics, but without organic lesions. Patients often experience symptoms such as abdominal discomfort or abdominal pain, abnormal bowel movements, etc. The pain is mostly located in the lower abdomen and can also be migratory, with uncertain onset and duration, and often relieved after passing gas or defecation. Diarrhea often occurs in the morning or after meals, without blood in the stool. Constipation is often accompanied by a feeling of incomplete defecation, and some patients experience alternating diarrhea and constipation. Irritable bowel syndrome has a great impact on individuals and society, seriously affecting patients' work, study and life, and reducing their quality of life, and requires active treatment.

[0003] At present, irritable bowel syndrome is generally treated with drugs to relieve the pain of patients, such as antihistamines, corticosteroids, etc., which can reduce the sensitivity of the nervous system and relieve pain and discomfort. However, drug treatment may cause some adverse reactions, such as dry mouth, constipation, dizziness, drowsiness, weight gain, etc. Therefore, it is urgent to find a safe and long-term treatment method to improve the quality of life of patients with irritable bowel syndrome. Summary of the invention

[0004] The purpose of the present invention is to provide an application of an Lg-like domain protein and a compound thereof in the preparation of a drug for relieving irritable bowel syndrome, so as to solve the problems existing in the above-mentioned prior art. The Lg-like domain protein and a compound thereof provided by the present invention can effectively relieve visceral hypersensitivity, especially irritable bowel syndrome.

[0005] Lg domain proteins are a class of proteins containing an Lg domain. The Lg domain is a domain composed of about 90 amino acids, has a highly conserved tertiary structure, and can bind to a variety of ligands, such as carbohydrates, lipids, peptides, ions, etc. Lg domain proteins are involved in a variety of biological processes, such as cell adhesion, signal transduction, immune regulation, neural development, etc. The present invention provides an Lg domain protein that can significantly relieve the symptoms of irritable bowel syndrome. Unlike the existing use of probiotics to relieve irritable bowel syndrome, previous probiotic preparations such as Lactobacillus plantarum are mostly live bacteria or inactivated bacteria and can only be taken orally. The purified Lg domain protein Llp-495 provided by the present invention can be taken orally or by injection, and is easy to use clinically.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides an application of an Lg-like domain protein Llp-495 in preparing a drug for alleviating irritable bowel syndrome. The amino acid sequence of the Lg-like domain protein Llp-495 is shown in SEQ ID NO.1.

[0008] Preferably, the nucleotide sequence of the gene encoding the Lg-like domain protein Llp-495 is shown as SEQ ID NO.2.

[0009] The present invention provides an application of an Lg-like domain protein Llp-495 and a coptis chinensis extract in preparing a drug for alleviating irritable bowel syndrome. The amino acid sequence of the Lg-like domain protein Llp-495 is shown in SEQ ID NO.1.

[0010] Preferably, the nucleotide sequence of the gene encoding the Lg-like domain protein Llp-495 is shown as SEQ ID NO.2.

[0011] Preferably, the preparation method of the Coptis chinensis extract comprises the following steps:

[0012] After mixing the coptis root with the ethanol solution, performing a first ultrasonic alcohol extraction to obtain a first alcohol extract and a first alcohol extraction residue;

[0013] The first alcohol extraction residue is mixed with an ethanol solution, and subjected to a second ultrasonic alcohol extraction to obtain a second alcohol extract;

[0014] The first alcohol extract and the second alcohol extract are combined and concentrated to obtain the Coptis chinensis extract.

[0015] The present invention provides a drug for relieving irritable bowel syndrome. The active ingredient of the drug comprises Lg-like domain protein Llp-495. The amino acid sequence of the Lg-like domain protein Llp-495 is shown in SEQ ID NO.1.

[0016] Preferably, the nucleotide sequence of the gene encoding the Lg-like domain protein Llp-495 is shown as SEQ ID NO.2.

[0017] Preferably, the active ingredient of the medicine also includes Coptis chinensis extract.

[0018] Preferably, the preparation method of the Coptis chinensis extract comprises the following steps:

[0019] After mixing the coptis root with the ethanol solution, performing a first ultrasonic alcohol extraction to obtain a first alcohol extract and a first alcohol extraction residue;

[0020] The first alcohol extraction residue is mixed with an ethanol solution, and subjected to a second ultrasonic alcohol extraction to obtain a second alcohol extract;

[0021] The first alcohol extract and the second alcohol extract are combined and concentrated to obtain the Coptis chinensis extract.

[0022] Preferably, the drug further comprises pharmaceutically acceptable excipients.

[0023] The present invention discloses the following technical effects:

[0024] The Lg domain protein Llp-495 of Lactobacillus plantarum and the Lg-like domain protein Llp-495 and the composition of the Rhizoma Coptidis extract provided by the present invention can significantly reduce the tryptase content in the intestinal tissue, inhibit the expression of genes such as mast cell surface membrane protein 1 (Mcemp1), mast cell protease 4-like protein (Mcpt4l1), mast cell protease 9 (Mcpt9), carboxypeptidase (Cpa3) and chymotrypsin (Cam1), thereby inhibiting the activation of mast cells and alleviating the visceral hypersensitivity of rats with irritable bowel syndrome. In addition, the Lg domain protein Llp-495 and the Lg-like domain protein Llp-495 and the composition of the Rhizoma Coptidis extract can reduce the generation of pro-inflammatory substances such as LPS in the intestine by improving the intestinal flora disorder of rats with irritable bowel syndrome. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0026] Figure 1 It is the recombinant expression of Lg domain protein Llp-495;

[0027] Figure 2 Characteristic myoelectric potentials ((a) and (b)) and fecal morphology (c) of the abdomen of IBS rats at different expansion volumes;

[0028] Figure 3 The effects of Lg-like domain protein Llp-495 and the combination of Lg-like domain protein Llp-495 and Coptis chinensis extract on colonic mast cells in IBS rats; wherein, (a) is mast cell staining in the colon, with a magnification of 200 times; (b) is mast cell count; (c) is tryptase content in colon tissue;

[0029] Figure 4 The effect of Lg-like domain protein Llp-495 on the expression of colonic mast cell membrane proteins in IBS rats;

[0030] Figure 5It is the species composition of rat intestinal flora at the genus level; among them, Protein is Llp-495;

[0031] Figure 6 The species differences of rat intestinal flora at the genus level; among them, Protein is Llp-495;

[0032] Figure 7 This is the effect of Lg-like domain protein Llp-495 on the intestinal LPS content in IBS rats. DETAILED DESCRIPTION

[0033] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0034] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0035] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0036] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0037] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0038] Example 1 Preparation of Lactobacillus plantarum Lg domain protein Llp-495

[0039] The experimental process is as follows Figure 1As shown, the genome of Lactobacillus plantarum AR495 (CGMCC No.14004, recorded in “CN108467843B A Lactobacillus plantarum for improving osteoporosis”) was used as a template, and colony PCR was performed using primers F1 / R1 (its specific sequence is shown in Table 1). The C-terminus of the Lg domain protein Llp-495 was fused with a His-tag label, and the purity was tested by agarose gel electrophoresis and then recovered to obtain the Lg domain protein Llp-495 gene. The nucleotide sequence of the Lg domain protein Llp-495 gene is shown in SEQ ID NO.2, and the amino acid sequence of the Lg domain protein Llp-495 encoded by it is shown in SEQ ID NO.1.

[0040] Table 1 Primer sequence list of Lg domain protein Llp-495

[0041]

[0042] The amino acid sequence of Lg domain protein Llp-495 is: MKKLMIAVTGLAMVATLGAAVPVTGITANAATKKTTATKNVAPSLKVNAIYNNTTQITGTATKGAKITVKSTANAKKNLGTATASKTTGKYTVKLAKTLKAQSNVYVYATNPTTKAYFYRVIRVQTAK TTASSTKKTTNKTTTTKKVATKSTAATSSSKKTTKSFKVNAPAGTWKSNTTNKYSQILTFSAKNGFNQTLYKNGKQVKKLVSYASYSMTAKTPTFWKVSYKSGSTTKTLYLRYTATNKFTVVNAKNQIVKVKVGNAPAAVWTFTKK, SEQ ID NO.1.

[0043] The nucleotide sequence of the Lg domain protein Llp-495 gene is: ATGAAGAAATTGATGATTGCTGTAACTGGTTTAGCCATGGTGGCAACGCTGGGAGCAGCGGTACCCGTTACAGGGATTACAGCGAATGCGGCGACTAAGAAGACAACGGCTACGAAGAATGTCGCACCAAGTTTGAAGGTCAATGCAATTTACAATAATACGACGCAAATTACAGGTACGGCAACCAAGGGCGCTAAGATCACCGTAAAAAGTACGGCCAACGCCAAGAAAAATCTTGGAACTGCAACGGCCTCTAAAACGACGGGAAAGTACACGGTTAAATTAGCCAAGACATTGAAGGCGCAAAGCAATGTTTATGTGTACGCCACGAATCCAACGACTAAGGCTTATTTCTACCGCGTCATCCGTGTTCAGACTGCCAAAACTACTGCCTCTTCAACTAAGAAGACGACTAACAAGACAACCACGACTAAGAAAGTGGCAACTAAATCGACGGCGGCCACTTCGTCATCCAAGAAGACGACTAAATCGTTTAAAGTCAACGCGCCAGCGGGTACTTGGAAGTCGAATACGACTAACAAGTACTCCCAAATCTTAACGTTTAGCGCTAAGAATGGATTTAATCAGACGCTTTATAAGAATGGCAAACAAGTTAAAAAGCTGGTCTCGTATGCGAGTTATAGCATGACTGCCAAGACGCCAACGTTCTGGAAAGTCAGCTATAAGTCAGGCTCGACTACTAAGACCCTATATTTACGCTATACAGCAACTAATAAGTTTACGGTCGTTAATGCTAAAAATCAGATCGTTAAAGTTAAGGTTGGCAATGCCCCAGCAGCTGTTTGGACGTTTACCAAGAAGTGA, SEQ ID NO.2.

[0044] The pET30α vector was connected to the Lg domain protein Llp-495 gene to construct a recombinant expression vector pET30α-Llp-495. pET30α-Llp-495 was transferred into the Escherichia coli BL21 (DE3) strain and coated on LB solid medium for screening. The LB solid medium contained Kan resistance (100 μg / mL) to obtain a recombinant expression strain. After the recombinant expression strain was fermented in a 5L fermenter, the bacterial cells were collected, the recombinant strain cells were ultrasonically broken, and the protein was purified by a nickel column. After desalting and renaturation, the protease expression was detected by polyacrylamide gel electrophoresis, and the recombinant protein was successfully obtained.

[0045] The nucleotide sequence of Lg domain protein Llp-495 is 825 bp long, and its molecular weight is about 29 KDa ( Figure 1 ). The protein was sequenced and found to be an Lg-like domain protein consisting of 274 amino acids (SEQ ID NO.1) with an average molecular weight of 29340 Da.

[0046] Example 2 Establishment of rat irritable bowel syndrome (IBS) model and experimental grouping

[0047] 1. Establishment of rat visceral hypersensitivity model:

[0048] Acetic acid enema plus restraint: Female Wistar rats (180 g, 5-6 weeks old) Female Wistar rats were acclimatized for 1 week without any treatment.

[0049] The rats were fasted for 24 hours before the experiment, but not water. After intraperitoneal injection of 10% chloral hydrate (300 mg / kg), a PC tube connected to a 5mL syringe was inserted through the anus (8 cm from the anus), and 1 mL of 40mL / L acetic acid was poured into the colon. The rats' tails were raised for 30 seconds, and the anus was pressed by hand. Then the colon was flushed with 1 mL of PBS solution (0.01 mol / L) and the rats were returned to the cage to eat and drink freely.

[0050] From the second day on, restraint stress was performed every day. Female Wistar rats were restrained with masking tape to restrict the movement of the upper body and forelimbs, and their heads were restricted, but their free activities were not restricted. After 2 hours, the female Wistar rats were placed in the breeding cage. All stress training was performed at the same time of the day (between 10 am and 12 am) to minimize the influence of circadian rhythm and movement rhythm.

[0051] Female Wistar rats were randomly divided into 5 groups after adaptive feeding for one week, with 8 rats in each group. The body weight was taken into consideration when grouping to ensure that there was no significant difference in body weight among the rats before the experiment (150-156 g / rat). The 5 groups were control group (Control), model group (IBS), Lg-like domain protein Llp-495 intervention group (Llp-495), Coptis chinensis extract group (CRE) and Lg-like domain protein Llp-495 + Coptis chinensis extract compound group (Llp-495+CRE); among them, control group (Control group): on the first day, rats were gavaged with 1 mL of normal saline instead of acetic acid, and from the 8th day to the 15th day, rats were gavaged with 1 mL of normal saline; model group (IBS group): on the After acetic acid enema on day 1, restraint stress was applied every day. From day 8 to day 15, rats were gavaged with 1 mL of normal saline once a day. Lg-like domain protein Llp-495 intervention group (Llp-495): After acetic acid enema on day 1, restraint stress was applied every day. From day 8 to day 15, rats were gavaged with 1 mL of the recombinant protein solution obtained in Example 1 (the concentration of the recombinant protein in the solution was 1 mg / mL) once a day. Coptis chinensis extract group (CRE): After acetic acid enema on day 1, restraint stress was applied every day. From day 8 to day 15, rats were gavaged with 1 mL of Coptis chinensis extract (10 g / mL, wherein the preparation method of the Coptis chinensis extract is as follows: 500 g of crushed Coptis chinensis was accurately weighed in a round-bottom flask, and 1 L of Soak in 95% (volume percentage) ethanol solution at 60°C for 2h, and treat with ultrasound at 40Khz for 30min; install the round-bottom flask containing the drug residue on a Soxhlet extraction device, heat and reflux at 80°C for 2h, collect the first alcohol extract and the first alcohol extraction residue; add 1L The second extraction was carried out with 95% (volume percentage) ethanol (immersion in ethanol solution at 60°C for 2h, ultrasonic treatment at 40Khz for 30min; the round-bottom flask containing the drug residue was equipped with a Soxhlet extraction device, heated at 80°C for reflux for 2h, and the second extract was collected), the two alcohol extracts were combined and filtered under reduced pressure, and concentrated to about 50mL by rotary evaporation; the drug solution was freeze-dried and configured to a 10g / mL solvent for standby use), and the mixture was gavaged once a day; Lg-like domain protein Llp-495+Coptis extract composite group (Llp-495+CRE): after acetic acid enema on the first day, restraint stress was performed every day, and from the 8th day to the 15th day, rats were gavaged with 1mL of a solution containing the recombinant protein obtained in Example 1 and Coptis extract (the concentration of the recombinant protein in the solution was 1mg / mL, and the concentration of the Coptis extract was 10g / mL), and the mixture was gavaged once a day.

[0052] 2. Effects of Lg-like domain protein Llp-495 and the combination of Lg-like domain protein Llp-495 and Coptis chinensis extract on colon sensitivity in IBS rats

[0053] Rats with different treatments were fasted but not watered for 12 h before the experiment. After that, the rats were anesthetized and an 8F catheter with a balloon was inserted through the anus (the end of the balloon was inserted 7 cm into the anus and fixed to the base of the rat's tail 1 cm outside the anus). The rats were placed in a homemade transparent plastic barrel cage and could only move forward and backward. The experiment began after the rats were stabilized for 30 min and fully adapted. Each rat was given balloon dilation 3 times with a capacity of 0.2 mL, 0.3 mL, 0.4 mL and 0.5 mL, respectively. Each dilation lasted for 5 min with an interval of 30 s.

[0054] Rats were equipped with two sets of silver needle electrodes, three in each set, implanted in the external oblique muscle, 2 cm above the inguinal ligament. The catheter and electrodes were exposed on the back of the rat's neck and fixed to the rat's tail. The rat's electromyographic activity was recorded by colorectal distention (CRD) connected to an electromyograph, which was recorded as the abdominal withdrawal reflex (AWR). Rats were placed in a transparent plastic barrel cage for several days before the experiment to minimize recording artifacts.

[0055] Colonic sensitivity can be quantified by monitoring the electromyographic signal after colon distension in rats. Figure 2 As shown in the figure, the IBS rats were successfully modeled (visceral hypersensitivity and reduced pain threshold). At 0.3-0.5 mL expansion volume, the sensitivity of the IBS group was significantly higher than that of the control group. Lg-like domain protein Llp-495 and Lg-like domain protein Llp-495+CRE intervention alleviated the sensitivity of colon expansion and increased the pain threshold. Compared with the control group, the feces of the model group were generally mushy stools with unclear edges and high water content; Lg-like domain protein Llp-495 and Lg-like domain protein Llp-495+CRE intervention can significantly improve the feces morphology, and the feces morphology is sausage-shaped.

[0056] 3. Effects of Lg-like domain protein Llp-4955 and the combination of Lg-like domain protein Llp-495 and Coptis chinensis extract on colonic mast cells in IBS rats

[0057] Colon tissue was taken 8 cm from the anus and stained with toluidine blue. The results were as follows: Figure 3As shown in (a) in the figure. Mast cells are mainly distributed in the small blood vessels in the colon mucosa and submucosa and around the intestinal muscularis nerve plexus. Their cytoplasm is stained purple-red, the nucleus is blue, and the cells are round, spindle-shaped or irregular in shape. Among them, the degranulated cells have ruptured cell membranes, granules gushing out, and the cells are irregular; the non-degranulated cells have intact cell membranes and uniform cytoplasm. As can be seen from the figure, compared with the model group, the number of mast cells did not change significantly in the CRE group, but was significantly reduced in the Llp-495 and Llp-495+CRE groups. Among them, the combined administration of Llp-495+CRE has the best effect in reducing mast cells. Figure 3 The number of mast cells in the physical and chemical staining was statistically analyzed as shown in (b). The colonic mast cell counts in rats in the IBS group were higher than those in the normal control group, and the difference was statistically significant (P<0.05). Statistical data showed that the reduction level of mast cell content after Llp-495+CRE intervention was better than that in the Llp-495 group. The mast cells in the Llp-495+CRE group returned to normal levels, with no significant difference from the control group. To further study the status of mast cells, the tryptase content released in the intestine after mast cell activation and degranulation was measured. The results are shown in Figure 3 As shown in (c), the tryptase content in the colon contents of the control group was 30.61±4.02 ng / g, and the tryptase content in the IBS group was significantly increased to 52.98±2.67 ng / g. Both Lg-like domain protein Llp-495 and Llp-495+CRE intervention reduced the tryptase content, among which the combined administration of Llp-495+CRE had the best effect.

[0058] The expression levels of mast cell-related membrane proteins in colon tissue of rats in different treatment groups (control group (Control), model group (IBS) and Lg-like domain protein Llp-495 intervention group (Llp-495)) were analyzed by q-PCR. The primer sequences used in q-PCR are shown in Table 2. The results are shown in Figure 4 As shown in the data, the transcription levels of mast cell-related membrane proteins and proinflammatory mediators in the colon of IBS rats were significantly upregulated, including mast cell-expressed membrane protein 1 (Mcemp1), mast cell protease 4-like protein (Mcpt4l1), mast cell protease 9 (Mcpt9), carboxypeptidase (Cpa3) and chymotrypsin (Cam1). Intervention with Lg-like domain protein Llp-495 inhibited the transcription levels of mast cell-related genes in the colon of IBS rats, indicating that oral administration of Lg-like domain protein Llp-495 inhibited the activation of mast cells.

[0059] Table 2 Rat mast cell membrane protein specific gene primer sequences

[0060]

[0061] 4. Effects of Lg-like domain protein Llp-495 on intestinal flora and LPS content in the intestine of IBS rats

[0062] Then, the species composition of feces at the genus level of IBS rats in different treatment groups (control group (Control), model group (IBS) and Lg-like domain protein Llp-495 intervention group (Llp-495)) was analyzed. Figure 5 As shown in the figure, Lachnospiraceae is the most abundant species in each group. In the control group, Lachnospiraceae accounts for 30.1%, in the model group, it accounts for 13.5%, and in the protein group, the abundance of this bacterium increases to 18.6%. In addition, Oscillospiraceae, Bacteroides, Lactobacillus, Eubacterium, Limosilactobacillus, Candidayus_Saccharibacteria, Muribaculaceae and Closteridia are also species with high abundance.

[0063] The significance of each bacterial genus was tested by Kruskal-Wallis rank sum analysis of species differences. Figure 6 As shown in the figure, Lachnospiraceae was significantly enriched in the control group (P<0.01), while this strain was significantly downregulated in the model group. After the intervention of Lactobacillus plantarum Lg-like domain protein, this genus was significantly enriched again. The same trend also occurred in unclassified_d__Bacteria. In contrast, Desulfovibrio, Bifidobacterium, Erysipelotrichacea, Ligilactobacillus, and Phascolarctobacterium were significantly enriched in the model group, while their proliferation was inhibited in the control group and protein group. A multi-omics study on IBS reported that the relative abundance of more than 20 Lactobacillus genera was increased in severe IBS-D compared with mild and moderate IBS-D, and the abundance of Phascolarctobacterium was associated with IBS disease.

[0064] Effects of Lg-like domain protein Llp-495 on intestinal LPS content in IBS rats Figure 7As shown in the figure, compared with the normal group, the content of lipopolysaccharide (LPS) in the intestine of rats in the IBS model group was significantly increased. After the intervention of Lg-like domain protein Llp-495, the content of LPS was significantly reduced, and there was no significant difference with the normal group. Lipopolysaccharide (LPS) is a component of the outer wall of the cell wall of Gram-negative bacteria. It is a glycolipid composed of lipids and polysaccharides. As an endotoxin, it participates in host recognition and induces immune response through Toll-like receptors present on the surface of host cell membranes. Intragastric administration of Lg-like domain protein Llp-495 can significantly improve the intestinal flora disorder of IBS rats, inhibit harmful flora, promote the proliferation of beneficial flora, and thus inhibit the production of pro-inflammatory substances in the intestine.

[0065] In summary, the Lg-like domain protein Llp-495 and the composition of Lg-like domain protein Llp-495+Coptis chinensis extract provided by the present invention can effectively alleviate the visceral hypersensitivity of patients with irritable bowel syndrome, by significantly reducing the tryptase content in intestinal tissue, inhibiting the expression of genes such as mast cell surface membrane protein 1 (Mcemp1), mast cell protease 4-like protein (Mcpt4l1), mast cell protease 9 (Mcpt9), carboxypeptidase (Cpa3) and chymotrypsin (Cam1), thereby inhibiting the activation of mast cells and alleviating the visceral hypersensitivity of rats with irritable bowel syndrome. In addition, the Lg-like domain protein Llp-495 and the composition of Lg-like domain protein Llp-495+Coptis chinensis extract can reduce the generation of pro-inflammatory substances such as LPS in the intestine by improving the intestinal flora disorder of rats with irritable bowel syndrome.

[0066] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. Application of Lg-like domain protein Llp-495 in the preparation of drugs for relieving irritable bowel syndrome, It is characterized in that The amino acid sequence of the Lg-like domain protein Llp-495 is shown in SEQ ID NO.

1.

2. The use according to claim 1, It is characterized in that The nucleotide sequence of the gene encoding the Lg-like domain protein Llp-495 is shown in SEQ ID NO.

2.

3. Application of Lg-like domain protein Llp-495 and Coptis chinensis extract in the preparation of drugs for relieving irritable bowel syndrome, It is characterized in that The amino acid sequence of the Lg-like domain protein Llp-495 is shown in SEQ ID NO.

1.

4. The use according to claim 3, It is characterized in that The nucleotide sequence of the gene encoding the Lg-like domain protein Llp-495 is shown in SEQ ID NO.

2.

5. The use according to claim 3, It is characterized in that The preparation method of the Coptidis rhizome extract comprises the following steps: After mixing the coptis root with the ethanol solution, performing a first ultrasonic alcohol extraction to obtain a first alcohol extract and a first alcohol extraction residue; The first alcohol extraction residue is mixed with an ethanol solution, and subjected to a second ultrasonic alcohol extraction to obtain a second alcohol extract; The first alcohol extract and the second alcohol extract are combined and concentrated to obtain the Coptis chinensis extract.

6. A drug for relieving irritable bowel syndrome, It is characterized in that The active ingredient of the drug includes Lg-like domain protein Llp-495; the amino acid sequence of the Lg-like domain protein Llp-495 is shown in SEQ ID NO.

1.

7. The drug according to claim 6, It is characterized in that The nucleotide sequence of the gene encoding the Lg-like domain protein Llp-495 is shown in SEQ ID NO.

2.

8. The drug according to claim 6, It is characterized in that The active ingredients of the medicine also include Coptis chinensis extract.

9. The drug according to claim 8, It is characterized in that The preparation method of the Coptidis rhizome extract comprises the following steps: After mixing the coptis root with the ethanol solution, performing a first ultrasonic alcohol extraction to obtain a first alcohol extract and a first alcohol extraction residue; The first alcohol extraction residue is mixed with an ethanol solution, and subjected to a second ultrasonic alcohol extraction to obtain a second alcohol extract; The first alcohol extract and the second alcohol extract are combined and concentrated to obtain the Coptis chinensis extract.

10. The drug according to claim 6, It is characterized in that The medicine also includes pharmaceutically acceptable excipients.

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