Application of protease-related active preparations in the treatment of inflammatory diseases with hyperresponsive immune responses

Abstract

The present invention discloses the application of protease-related active preparations in treating inflammatory diseases with hyperactive immune responses, and belongs to the field of biomedical technology. In order to provide a method for effectively improving allergic intestinal inflammation, the present invention provides the application of cysteine ​​protease inhibitors in the preparation of drugs for treating allergic intestinal inflammation, and provides new ideas for treating and preventing allergic enteritis.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to the application of protease-related active preparations in treating inflammatory diseases with hyperimmune response. Background Art

[0002] In recent years, the incidence of inflammatory bowel disease (IBD) has increased year by year worldwide, including two types of diseases: Crohn's disease and ulcerative colitis, both of which are accompanied by abdominal pain, blood in the stool and weight loss. The causes of IBD are closely related to environmental changes, family genetic genes and disordered flora. Epidemiological surveys show that after people migrate from areas with low IBD incidence to areas with high incidence, the overall incidence rate increases significantly, which may be related to local dietary habits. In addition, the prevalence of tobacco and irrational drug use in recent years have seriously damaged the homeostasis of the body's immune system, which may be related to the increase in the incidence of IBD. With the development of genomics, 163 potential gene loci associated with the onset of IBD have been discovered, including IL-10 receptor, IL-23 receptor, Nod2 and CARD9. Nod2 is an important cell receptor that regulates intestinal flora. It can induce the synthesis and secretion of antimicrobial peptides such as α-defensins, and directly participate in regulating the microbial composition of the intestine. In addition, Nod2 mutations lead to decreased expression levels of IL-10, and clinical patients show chronic inflammatory responses mediated by IL-12 and IFN-γ. Intestinal flora is a key microorganism that maintains intestinal immune homeostasis. In IBD patients, the diversity of their intestinal flora is low, and the proportion of Escherichia coli and Bacteroides genus increases significantly. In summary, the causes of IBD are complex and diverse, but they are all caused by an imbalance in the immune system, which causes excessive immune responses and leads to inflammatory responses and pathological changes in intestinal tissues. It is necessary to find drugs with significant therapeutic and preventive effects. Summary of the invention

[0003] The purpose of the present invention is to provide a method for effectively improving allergic intestinal inflammation.

[0004] The present invention provides an application of a cysteine ​​protease inhibitor in preparing a medicine for treating allergic intestinal inflammation.

[0005] The present invention provides an application of a cysteine ​​protease inhibitor in preparing a drug for auxiliary treatment of allergic intestinal inflammation.

[0006] It is further defined that the dosage form of the drug is any one of tablets, capsules, granules, powders, and liquid preparations.

[0007] Further defined, the amino acid sequence of the cysteine ​​protease inhibitor is shown as SEQ ID NO.10.

[0008] Further defined, allergic intestinal inflammation is induced by OVA (chicken ovalbumin).

[0009] The invention provides a method for obtaining a cysteine ​​protease inhibitor. The gene sequence of the cysteine ​​protease inhibitor is obtained and inserted into a Pet-28a vector, which is transformed into a competent state. After being screened with kanamycin, a monoclonal strain is selected, and a plasmid is extracted and transformed into BL21 (DE3). Recombinant protein is obtained through expanded culture and IPTG induction.

[0010] Beneficial effects: In the OVA-induced intestinal inflammation model, the mortality rate of mice in the recombinant cysteine ​​protease inhibitor treatment group was reduced on the 20th day; the weight loss rate of mice in the recombinant cysteine ​​protease inhibitor treatment group was improved on the 20th day. The level of OVA-specific IgE antibodies in the serum of mice in the recombinant cysteine ​​protease inhibitor treatment group was significantly reduced. The tissue damage and pathological changes of the intestine of mice in the recombinant cysteine ​​protease inhibitor treatment group were improved. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 The SDS-page images of the lysate proteins of the recombinant OVA-expressing bacteria, 1: empty vector control; 2: OVA-expressing bacteria;

[0012] Figure 2 Western Bolt images of lysate proteins of recombinant OVA-expressing bacteria, 1: empty vector control; 2: OVA-expressing bacteria;

[0013] Figure 3 Figure 1 shows the identification of OVA protein expression by E. coli in mice. 1: Feces of mice infected with empty vector E. coli; 2: Feces of mice infected with OVA-expressing bacteria; 3: Small intestinal lavage fluid of mice infected with OVA-expressing bacteria; 4: Colonic lavage fluid of mice infected with OVA-expressing bacteria;

[0014] Figure 4 The PCR results of colon lavage fluid of mice infected with E. coli at different time periods are shown in Figure 1: negative control; 2-5: colon lavage fluid of empty vector E. coli infected on days 3, 5, 7, and 9; 6-9: colon lavage fluid of OVA-expressing bacteria infected on days 3, 5, 7, and 9;

[0015] Figure 5 Schematic diagram of establishing the OT-II mouse enteritis model and the related inflammation evaluation system;

[0016] Figure 6 The result graph is the number of mice surviving in each group;

[0017] Figure 7 The figure is the result of the change of body weight of each mouse;

[0018] Figure 8 This is the HE staining result of mouse small intestine tissue;

[0019] Fig. 9 This is the HE staining result of mouse colon tissue;

[0020] Fig.10 Schematic diagram of inflammation evaluation of intestinal tissue;

[0021] Fig.11 Schematic diagram of the treatment of OVA-IBD mice with recombinant cysteine ​​protease inhibitors;

[0022] Fig.12 This is the survival rate result graph of mice on day 20;

[0023] Fig.13 This is a graph showing the weight loss of mice on the 20th day;

[0024] Fig.14 The figure is the concentration result of OVA-specific antibody IgE in the serum of mice on the 20th day;

[0025] Fig.15 Schematic diagram of inflammation evaluation of mouse intestinal tissue. DETAILED DESCRIPTION

[0026] In order to more intuitively and clearly illustrate the purpose, technical solutions and advantages of the present invention, the present invention is further described in detail below according to the accompanying drawings. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0027] The specific implementation of the present invention is described in detail below in conjunction with specific embodiments.

[0028] Example 1. Method for constructing a chronic intestinal inflammation model

[0029] 1. A method for preparing an OVA (chicken ovalbumin) expressing bacterium, comprising the following steps:

[0030] Obtain the gene sequence of OVA and predict the signal peptide of the protein. Design primers that remove the signal peptide and have BamH I and HindIII restriction sites. The specific primer sequences are shown in Table 1. Amplify the target gene from the cDNA of chicken oviduct tissue. Connect the target gene to the Pet-32a vector and transform it into BL21 (DE3) Escherichia coli. Pick a single clone colony for expansion culture. The Pet-32a vector can be induced by T7 RNA polymerase in Escherichia coli to express the recombinant protein, such as Figure 1 and Figure 2 shown.

[0031] 2. Construction of OVA-induced chronic intestinal inflammation model, including the following steps:

[0032] Step 1) Intestinal flora colonization detection. 1×10 7 cfu / 200 μL of OVA recombinant E. coli were gavaged into mice, and the feces, small intestinal lavage fluid and colonic lavage fluid of the mice were collected after 3, 5, 7 and 9 days. PCR amplification of the intestinal lavage fluid was performed to identify the presence of recombinant OVA expressing bacteria and control bacteria. The specific primers are shown in Table 1. The results are shown in Figure 4 As shown. Then, the BSA kit was used to quantify it, and 50 μg of protein in the 9th day feces was separated by polyacrylamide gel electrophoresis, and then the protein was transferred to the PVDF membrane using western bolt technology. The PVDF membrane was blocked with 5% skim milk powder for 2 hours; then the membrane was incubated with diluted anti-OVA antibody at 37°C for 1 hour, and the membrane was washed three times with TBST; the membrane was incubated with diluted HRP-labeled anti-rabbit IgG antibody at 37°C for 1 hour, and the membrane was washed three times with TBST; the specific OVA protein band was detected using ECL substrate for color development, and the results are shown as follows Figure 3 shown.

[0033] Step 2) OVA was first injected intraperitoneally into OTII mice on days -14 and -7; piroxicam (50 μg / g body weight) was orally administered on day -3; and 1×10 7 cfu / 200 μL of OVA-expressing bacteria, and the mice were killed on day 21. Figure 5 shown.

[0034] Step 3) Evaluation of intestinal inflammation. First, the daily survival of each group of mice was counted and the survival rate of the mice was calculated, such as Figure 6 Then the mice were weighed, as shown in Figure 7 The fecal status and anal prolapse of the mice were observed, and the DAI score was calculated according to Table 1. On the last 21 days, the small intestine and colon tissues of the mice were collected, fixed with 4% paraformaldehyde for 24 hours, dehydrated and embedded in paraffin, sliced ​​and HE stained, and the pathological changes of intestinal tissues were observed. The scoring criteria are shown in Table 2, including: 0 (normal), 1 (mild lesions), 2 (moderate lesions), 3 (severe lesions) and 4 (extremely severe lesions). The results are shown in Figure 8 , 9 , as shown in 10.

[0035] Table 1: List of specific primers

[0036]

[0037] Table 2: DAI scores of disease models

[0038]

[0039] Example 2. Preparation of a cysteine ​​protease inhibitor derived from Trichinella spiralis

[0040] 1. A method for preparing a cysteine ​​protease inhibitor derived from Trichinella spiralis, comprising the following steps:

[0041] Obtain the gene sequence of cysteine ​​protease inhibitor (GeneBank accession number: EU263325.1; protein sequence accession number: ABY60755.1),

[0042] WN10 gene sequence: (SEQ ID NO.9)

[0043]

[0044] WN10 amino acid sequence: (SEQ ID NO.10)

[0045] SIQKMSFMHCIFVVLFFAVGEAQILGETTHYGRNDPVMLRNAHEALFSSDLKQESGVFHKLLELEESSTMGILTTMKVVMQDTDCPVSFALLSYYDVLVNCQGEGRRKHCTMEYTHRNPSKATVSKCFEEVEEPLIIPQRVKMIGGRAVYIDSNADVEEQMQMLGETTHYGRNDPVMLPKAREALFSSDSKEQSGVLHKLVELEESSTMGILTTMKVVIQDTECRVSSAYSSYYDVLHYCHGKGPRKHCTLEYRHRTPSTATVSECFEEVEEPLIVPQRVQRVNGRTIYLDSSDDVEEQVVSQRSQMLGGTTKYTDSNVHIKEEVKQAIFESDKKKSSGTYLLLDKIVEGFNMGISSRFQVLVKETECGIKEKAFNSYEDVYKNCSGSGDSKVCSVEYKYFDPTKSTVEC;

[0046] WN10 gene sequence without signal peptide: (SEQ ID NO.11)

[0047]

[0048] Specific primers for removing signal peptides were designed. The cDNA of the third-day adult Trichinella spiralis was specifically amplified to obtain the target sequence and inserted into the Pet-28a vector and transformed into competent cells. After kanamycin screening, monoclonal strains were selected, cultured and sequenced. The correct plasmid was then transformed into BL21 (DE3). Recombinant protein was obtained by culture and IPTG induction. The recombinant protein was separated by nickel column affinity chromatography using an AKTA protein purifier to obtain a soluble single recombinant protein. After protein quantitative calculation using the BCA kit, the recombinant cysteine ​​protease inhibitor was packaged and stored at -80 ℃.

[0049] Effect data: The intervention effect of recombinant cysteine ​​protease inhibitor on mouse enteritis:

[0050] In the OVA-induced intestinal inflammation model, 50 μg of recombinant cysteine ​​protease inhibitor was injected into mice by intraperitoneal injection on days 2, 9, and 16. The mice were killed on day 20 and intestinal inflammation was evaluated. The experimental process is as follows Fig.11 shown.

[0051] 1) Fig.12 The mice in the groups treated with the indicated recombinant cysteine ​​protease inhibitors had reduced mortality on day 20; Fig.13 The results showed that the weight loss rate of mice in the recombinant cysteine ​​protease inhibitor treatment group was improved on the 20th day.

[0052] 2) Collect serum and detect the IgE content in serum. Coat 100 μL of 5 μg / mL OVA protein on a 96-well ELISA plate and incubate overnight at 4°C; wash three times with PBST, add diluted serum samples, and incubate at 37°C for one hour; wash three times with PBST, add HRP-labeled anti-mouse IgG antibody dilution, and incubate at 37°C for one hour; wash three times with PBST, add TMB color development substrate to react for 10 minutes, add stop solution to terminate the reaction, and detect the absorbance at 450nm. Calculate the IgE concentration in serum based on the IgE standard curve. The results are as follows: Fig.14 As shown, the level of OVA-specific IgE antibodies in the serum of mice in the recombinant cysteine ​​protease inhibitor treatment group was significantly reduced.

[0053] 3) Intestinal tissue was collected, fixed with 4% paraformaldehyde for 24 hours, dehydrated and embedded in paraffin, sliced ​​using a microtome, dewaxed, and stained with hematoxylin-eosin. Pathological changes of intestinal tissue were observed under a microscope. The scoring criteria included: 0 (normal), 1 (mild lesions), 2 (moderate lesions), 3 (severe lesions) and 4 (extremely severe lesions). The results were as follows: Fig.15 As shown, the tissue damage and pathological changes in the intestine of mice treated with recombinant cysteine ​​protease inhibitors were improved.

[0054] The above are only preferred embodiments of the present invention. It should be pointed out that, for those skilled in the art, several modifications and improvements can be made without departing from the concept of the present invention. These should also be regarded as the protection scope of the present invention. These will not affect the effect of the implementation of the present invention and the practicality of the patent.

Claims

1. The use of WN10 in the preparation of a drug for treating allergic intestinal inflammation, characterized in that: The amino acid sequence of WN10 is shown in SEQ ID NO.

10.

2. The use of WN10 in the preparation of a drug for the auxiliary treatment of allergic intestinal inflammation, characterized in that: The amino acid sequence of WN10 is shown in SEQ ID NO.

10.

3. The use according to claim 1, characterized in that: The dosage form of the drug is any one of tablets, capsules, granules, powders and liquid preparations.

Images