Application of aromatic aldehyde compound in preparation of product for relieving or treating infectious colitis induced by drug-resistant escherichia coli

Abstract

The invention relates to the technical field of biological medicines, in particular to application of aromatic aldehyde compounds in preparation of products for relieving or treating infectious colitis induced by drug-resistant escherichia coli. It is found that the aromatic aldehyde compound can effectively relieve or treat infectious colitis induced by drug-resistant Escherichia coli, a reference is provided for deep research on application of the aromatic aldehyde compound to preparation of drugs for treating infectious colitis, and the aromatic aldehyde compound has very important practical significance and application value in treatment of inflammatory bowel diseases.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to application of aromatic aldehyde compounds in preparing products for alleviating or treating infectious colitis induced by drug-resistant Escherichia coli. Background Art

[0002] Infectious colitis is a disease caused by bacteria, viruses or parasites that can affect individuals of all ages, with an increased incidence with age. It is important to note that infectious colitis is different from chronic colitis (such as ulcerative colitis and Crohn's disease) and may be related to genetics or immune system abnormalities. Most infectious colitis is caused by bacteria, with foodborne bacteria being the main cause. Multidrug-resistant (MDR) Gram-negative bacteria, such as Escherichia coli and Salmonella, are the most common microorganisms causing infectious colitis, and their unique outer membrane lipopolysaccharide (LPS) plays an important role in inducing colitis. Recent studies have shown that infectious colitis can lead to rapid development in the colon, accompanied by bleeding or hemolytic uremic syndrome, causing high morbidity and mortality worldwide. Treatment of infectious colitis is usually antibiotic therapy, which may also lead to the development of antibiotic resistance.

[0003] Plant extracts (e.g., phenols, flavonoids, alkaloids, quinones, etc.) have attracted extensive attention in recent years. Plant extracts have become the subject of much research attention due to their potential antimicrobial and anti-inflammatory properties, as well as low cytotoxicity and reduced potential for the development of bacterial resistance. Vanillin (also known as vanillin), an active ingredient found in vanilla, has long been recognized to have health benefits. It not only has important flavoring functions, but is also commonly used as an additive in food products such as biscuits, chocolate, milk tea, ice cream, candy, chewing gum, and cakes. In addition, vanillin has many beneficial effects on human health due to its strong antimutagenic, anti-metastatic, and antidepressant activities. It is non-toxic and can be rapidly absorbed across the blood-brain barrier even when used at high concentrations; therefore, it has great promise in the treatment of neurological diseases. However, there are no studies on whether vanillin has an effect on infectious colitis. Summary of the invention

[0004] The present invention proves through in vivo experiments that aromatic aldehyde compounds can effectively alleviate or treat infectious colitis induced by drug-resistant Escherichia coli. Based on this, the following technical solution is proposed.

[0005] Firstly, the present invention provides the use of aromatic aldehyde compounds in preparing products; the products are used for alleviating or treating infectious colitis induced by drug-resistant Escherichia coli.

[0006] Preferably, the aromatic aldehyde compound is vanillin.

[0007] Preferably, the product is a medicine, a food, a food additive, a feed or a feed additive.

[0008] Preferably, the drug contains aromatic aldehyde compounds as active ingredients.

[0009] Preferably, the medicine comprises aromatic aldehyde compounds and other pharmaceutically acceptable excipients.

[0010] In the specific implementation process, other pharmaceutically acceptable excipients include fillers, excipients, lubricants, wetting agents, diluents and the like.

[0011] In the specific implementation process, the dosage form of the drug is a solid preparation or a liquid preparation.

[0012] Preferably, the solid preparation is a powder, granule, capsule or tablet, and the liquid preparation is an oral solution or injection.

[0013] In a specific implementation process, the drug-resistant Escherichia coli is Escherichia coli resistant to amikacin, benzocillin, chloramphenicol, clindamycin, doxycycline, erythromycin, gentamicin, lincomycin, penicillin, streptomycin, sulfamethoxazole or vancomycin.

[0014] Preferably, the drug-resistant Escherichia coli is Escherichia coli CVCC195.

[0015] Preferably, the product alleviates or treats infectious colitis induced by drug-resistant Escherichia coli through at least one of the following approaches: (1) Improve colon epithelial morphology and mucosal structure; (2) Regulate inflammation-related factors; (3) Alleviate oxidative stress; (4) Changes in the intestinal α-diversity index; (5) Improve the distribution of intestinal microbiota.

[0016] Preferably, the inflammation-related factors include at least one of IL-6, IL-1β, TNF-α, p38, pp38, p65, pp65, MYD88 or IL-10.

[0017] Aromatic aldehyde compounds can reduce the expression of inflammation-related factors IL-6, IL-1β, TNF-α, p38, pp38, p65, pp65, and MYD88 in mice, and increase the expression of anti-inflammatory factor IL-10.

[0018] In addition, the present invention provides the use of aromatic aldehyde compounds in the preparation of antibacterial products; the antibacterial products are used to inhibit at least one of Escherichia coli, Salmonella typhimurium, and Staphylococcus aureus.

[0019] Preferably, the product is a medicine, a feed additive, a disinfectant or a bactericide.

[0020] Preferably, during use, the dosage of the aromatic aldehyde compound is 15 mg / kg or more, more preferably 30 mg / kg or more, and even more preferably 60 mg / kg or more.

[0021] Compared with the prior art, the present invention has the following beneficial effects: The present invention discovered that aromatic aldehyde compounds can effectively alleviate or treat infectious colitis induced by drug-resistant Escherichia coli, providing a reference for in-depth research on the application of aromatic aldehyde compounds in the preparation of drugs for treating infectious colitis, and has very important practical significance and application value for the treatment of inflammatory bowel disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 These are the results of the cytotoxicity test of vanillin on mouse macrophages (RAW264.7).

[0023] Figure 2 This is the detection result of IL-1β.

[0024] Figure 3 This is the test result of IL-6.

[0025] Figure 4 This is the test result of IL-10.

[0026] Figure 5 This is the result of TNF-α detection.

[0027] Figure 6 This is the detection result of pp65.

[0028] Figure 7 This is the test result of MYD88.

[0029] Figure 8 This is the detection result of p38.

[0030] Fig. 9 This is the detection result of p65.

[0031] Fig.10 This is the detection result of pp38.

[0032] Fig.11 This is the result of mouse colon tissue pathology scoring.

[0033] Fig.12This is a histopathological picture of mouse colon.

[0034] Fig.13 This is a graph of the weight changes of mice.

[0035] Fig.14 This is a diagram of the changes in mouse intestinal microorganisms at the phylum level.

[0036] Fig.15 This is a diagram of the changes in mouse intestinal microorganisms at the genus level.

[0037] Fig.16 This is a diagram of the changes in mouse intestinal microorganisms at the species level. DETAILED DESCRIPTION

[0038] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be described clearly and completely below. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0039] In the examples provided in this specification, if no specific techniques or conditions are specified, the techniques or conditions described in the literature in this field or the product instructions are used. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased through regular channels.

[0040] All data in the following examples were analyzed using GraphPad Prism 9 statistical software, and the results were expressed as mean ± standard deviation. The differences between the groups were evaluated using the least significant test. p <0.05 was considered statistically significant.

[0041] Example 1 In this example, the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of vanillin were determined by microtiter broth dilution method. Escherichia coli, Salmonella typhimurium and Staphylococcus aureus were cultured until they reached the logarithmic growth phase. 5 CFU / mL, added to a 96-well plate (90 μL / well). Vanillin dissolved in sterile phosphate buffered saline (PBS) was diluted 2-fold and added to each well (10 μL / well). PBS was used as a negative control (CK). The cells were then incubated at 37°C for 16-18 h until visible turbidity was observed in the negative control. The MIC value of vanillin against the tested strains was determined as the lowest concentration that completely inhibited bacterial growth. The MBC value was calculated as the vanillin concentration at which the initial inoculum was reduced by 99.9%. All experiments were repeated 3 times.

[0042] The test results are shown in Table 1.

[0043] Table 1 Antibacterial activity of vanillin

[0044] a indicates resistance to amikacin, benzocillin, chloramphenicol, clindamycin, doxycycline, erythromycin, gentamicin, lincomycin, penicillin, streptomycin, sulfamethoxazole and vancomycin. b indicates resistance to lincomycin, amoxicillin, amikacin, ampicillin, oxacillin, erythromycin, sulfamethoxazole, neomycin, azithromycin, kanamycin, gentamicin and penicillin.

[0045] Example 2 In this example, the CCK-8 method was used to detect the effect of aromatic aldehyde compound-vanillin on cell viability. Mouse macrophage RAW264.7 cells were cultured in DMEM medium containing 10% fetal bovine serum in a humidified incubator at 37°C and 5% carbon dioxide. RAW264.7 cells (2.5×10 4 Cells were inoculated with 10 μL of CK8 solution (0.04 mg / mL) in 96-well plates and incubated for 24 h. Subsequently, cells were treated with different concentrations of vanillin (ranging from 0.04 mg / mL to 5 mg / mL) for another 24 h. After that, each well was supplemented with 10 μL of CCK8 solution. After incubation for 1 h, the absorbance (OD) was measured at a wavelength of 450 nm using a microplate reader.

[0046] Test results such as Figure 1 As shown in the figure, the cell viability of vanillin was 82% and 92% at concentrations of 0.04 and 0.08 mg / mL, respectively. In addition, the cell viability of vanillin in the concentration range of 0.16-0.63 mg / mL exceeded 100%, indicating that vanillin had no cytotoxicity to mouse macrophages.

[0047] Example 3 In this example, the aromatic aldehyde compound vanillin was used to treat colitis in mice induced by Escherichia coli CVCC195 to observe its effectiveness. The mice were randomly divided into six groups (8 mice / group): control group (oral gavage), Escherichia coli CVCC195 infection group, Escherichia coli CVCC195 + 11.8 mg / kg polymyxin B (PMB) group, Escherichia coli CVCC195 + 60 mg / kg vanillin group, Escherichia coli CVCC195 + 30 mg / kg vanillin group and Escherichia coli CVCC195 + 15 mg / kg vanillin group. Each group of mice was orally gavaged with Escherichia coli CVCC195 every day (1 time / day, 200 μL each time, 1×10 8CFU / mL) for 20 consecutive days. Vanillin or PMB was gavaged daily (once a day) for 10 consecutive days. After 10 days of vanillin treatment, mice were anesthetized with isoflurane, euthanized, and samples were collected.

[0048] 1. Take mouse serum for testing, use ELISA kit to detect the expression levels of TNF-α, IL-6, IL-1β and IL-10, and then use an enzyme reader to measure the absorbance at a wavelength of 520 nm.

[0049] The results of IL-1β detection are as follows Figure 2 As shown, compared with the control group, Escherichia coli CVCC195 significantly increased the protein level of IL-1β, and compared with the Escherichia coli CVCC195 treatment group, the aromatic aldehyde compound-vanillin intervention significantly reduced the protein level of IL-1β.

[0050] The results of IL-6 detection are as follows Figure 3 As shown, compared with the control group, Escherichia coli CVCC195 increased the protein level of IL-6, and compared with the Escherichia coli CVCC195 treatment group, the aromatic aldehyde compound-vanillin intervention significantly reduced the protein level of IL-6.

[0051] The results of IL-10 detection are as follows Figure 4 As shown, compared with the control group, Escherichia coli CVCC195 reduced the protein level of IL-10, and compared with the CVCC195 treatment group, the aromatic aldehyde compound-vanillin intervention significantly increased the protein level of IL-10.

[0052] The results of TNF-α detection are as follows Figure 5 As shown, compared with the control group, Escherichia coli CVCC195 increased the TNF-α protein level, and compared with the CVCC195 treatment group, the aromatic aldehyde compound-vanillin intervention significantly reduced the TNF-α protein level.

[0053] 2. 0.02 g of mouse colon tissue was lysed and centrifuged at 12,000 × g for 5 min to obtain the supernatant. Subsequently, 30 μg of protein was analyzed by SDS-PAGE and transferred to a PVDF membrane for immunoblotting. After blocking with a solution containing 5% skim milk powder for 1 h, the membrane was washed with a buffer containing Tween 20 and incubated with MYD88, p65, pp65, pp38, and p38 primary antibodies (diluted 1:1000) at 4°C overnight. After washing three times with TBST, goat anti-rabbit IgG H&L antibody containing AlexaFluor® dye was incubated at room temperature for 1 h. Finally, western blot analysis was performed using the BioRad Chemical Gel System.

[0054] The results of pp65 detection are as follows Figure 6As shown, compared with the control group, Escherichia coli CVCC195 significantly increased the expression level of pp65, and compared with the Escherichia coli CVCC195 treatment group, the aromatic aldehyde compound-vanillin intervention significantly reduced the protein level of pp65.

[0055] The test results of MYD88 are as follows Figure 7 As shown, compared with the control group, Escherichia coli CVCC195 significantly increased the expression level of MYD88, and compared with the Escherichia coli CVCC195 treatment group, the aromatic aldehyde compound-vanillin intervention significantly reduced the protein level of MYD88.

[0056] The results of p38 detection are as follows Figure 8 As shown, compared with the control group, Escherichia coli CVCC195 significantly increased the expression level of p38, and compared with the Escherichia coli CVCC195 treatment group, the aromatic aldehyde compound-vanillin intervention significantly reduced the protein level of p38.

[0057] The results of p65 detection are as follows Fig. 9 As shown, compared with the control group, Escherichia coli CVCC195 increased the expression level of p65, and compared with the Escherichia coli CVCC195 treatment group, the aromatic aldehyde compound-vanillin intervention significantly reduced the protein level of p65.

[0058] The results of pp38 detection are as follows Fig.10 As shown, compared with the control group, Escherichia coli CVCC195 significantly increased the expression level of pp38, and compared with the CVCC195 treatment group, the intervention of aromatic aldehyde compound-vanillin significantly reduced the protein level of pp38.

[0059] 3. Mouse colon tissue (3-4 mm) was taken and fixed with 4% paraformaldehyde. Subsequently, the tissue was dehydrated, embedded in paraffin blocks, sliced ​​to 5 μm using a microtome, and mounted on a staining evaluation slide above the biopolymer label. The slide was placed on a hot plate at 60°C for 2 h and stained with 1% Mayer's H&E aqueous solution. Finally, the samples were observed under a microscope. The depth of chicken lesions was scored according to the degree of intestinal inflammation: 0: no inflammatory reaction; 1: superficial inflammation and mild mucosal hyperplasia; 2: mild ulceration, the lesion extent enters the submucosa; 3: mucosa and submucosa sometimes form moderate hyperplasia, sometimes form a transdural membrane; 4: mucosa and submucosa often have obvious transdural hyperplasia, accompanied by moderate to obvious goblet cell loss and crypt loss.

[0060] The rating results are as follows Fig.11As shown, according to the scoring criteria, E. coli CVCC195 significantly increased the pathological score compared with the blank control group. Compared with the E. coli CVCC195 treatment group, the aromatic aldehyde compound-vanillin intervention significantly reduced the colon inflammation score.

[0061] Colon tissue pathology of mice Fig.12 As shown in the results, the colon of the E. coli CVCC195 treated group showed obvious histological changes and acute damage, including epithelial cell necrosis, intestinal mucosal shedding, a certain degree of intestinal gland exposure, and a slight increase in mucosal fibrous tissue. Vanillin can significantly reduce the pathological changes caused by E. coli, and the integrity and arrangement of the intestinal mucosal epithelial cells remain intact.

[0062] 4. Daily detection of mouse weight changes The weight changes of mice Fig.13 As shown, compared with the control group, the mice in the Escherichia coli CVCC195 challenge group lost weight; compared with the Escherichia coli CVCC195 treated group, 60 mg / kg vanillin could significantly increase the body weight of mice with infectious colitis.

[0063] In summary, compared with the E. coli treated group, mice treated with vanillin significantly alleviated the E. coli-induced changes in colon epithelial morphology and improved mucosal structure. Through cytotoxicity tests, it was determined that at a certain dose, vanillin had no toxic effect on cells and did not affect the liver and kidney organs of mice, indicating good safety.

[0064] 5. Detection of changes in intestinal microorganisms using the 16S rDNA method Changes in intestinal microbiota at the phylum level in mice Fig.14 As shown, compared with the control group, Firmicutes and Bacteroides were the dominant bacteria in the mice in the Escherichia coli CVCC195 challenge group; compared with the Escherichia coli CVCC195 treatment group, the dominant bacteria in the mice in the vanillin intervention group were Firmicutes and Bacteroidetes, vanillin increased the abundance of Bacteroidetes and reduced the abundance of Firmicutes; colistin B significantly reduced the abundance of Firmicutes.

[0065] Changes in intestinal microbiota at the genus level in mice Fig.15 As shown, vanillin reduced the Ligilactobacillus , Lachnospira Lachnospiracea The abundance of _NK4A136_group increased the genus Prevotella Prevotellaceae _UCG-001, another mycobacterium Alistipes and Bacteroides Bacteroides The abundance of Prevotella and Ruminococcus was increased by colistin B, while the abundance of Lachnospira was decreased.

[0066] Changes in intestinal microbiota at the species level in mice Fig.16 As shown, vanillin reduced the Lachnospiraceae_bacterium _COE1, Lachnospiraceae_bacterium _A4 and Clostridium Clostridiales_bacterium The abundance of _CIEAF_020 increased the abundance of Bacteroidetes Bacteroides caecimuris Parabacteroides gordonii Bacteroides goldstein II abundance; in the group treated with colistin B, the abundance of intestinal flora of mice was the lowest, and colistin B reduced the abundance of Lachnospiraceae Lachnospiraceae _bacterium_COE1 and Clostridium Clostridium _sp_Culture - 27 abundance.

[0067] Therefore, the aromatic aldehyde compound vanillin can be used to alleviate or treat infectious colitis induced by drug-resistant Escherichia coli. The development of drugs for the treatment of colitis based on the aromatic aldehyde compound vanillin can help (1) treat intestinal inflammation caused by Escherichia coli; (2) provide a cheap, safe and effective drug for the treatment of infectious colitis.

[0068] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. Application of aromatic aldehyde compounds in the preparation of products; the products are used to alleviate or treat infectious colitis induced by drug-resistant Escherichia coli.

2. The use according to claim 1, characterized in that: The product is a medicine, food, food additive, feed or feed additive.

3. The use according to claim 2, characterized in that: The medicine uses aromatic aldehyde compounds as active ingredients.

4. The use according to claim 3, characterized in that: The medicine comprises aromatic aldehyde compounds and other pharmaceutically acceptable auxiliary materials.

5. The use according to claim 2, characterized in that: The dosage form of the drug is a solid preparation or a liquid preparation; preferably, the solid preparation is a powder, granules, capsule or tablet, and the liquid preparation is an oral solution or an injection.

6. The use according to claim 1, characterized in that: The drug-resistant Escherichia coli is Escherichia coli resistant to amikacin, benzocillin, chloramphenicol, clindamycin, doxycycline, erythromycin, gentamicin, lincomycin, penicillin, streptomycin, sulfamethoxazole or vancomycin.

7. The use according to claim 6, characterized in that: The drug-resistant Escherichia coli is Escherichia coli CVCC195.

8. The use according to claim 1, characterized in that: The product alleviates or treats infectious colitis induced by drug-resistant Escherichia coli through at least one of the following pathways: (1) Improve colon epithelial morphology and mucosal structure; (2) Regulate inflammation-related factors; (3) Alleviate oxidative stress; (4) Changes in the intestinal α-diversity index; (5) Improve the distribution of intestinal microbiota.

9. The use according to claim 8, characterized in that: The inflammation-related factors include at least one of IL-6, IL-1β, TNF-α, p38, pp38, p65, pp65, MYD88 or IL-10.

10. Use of aromatic aldehyde compounds in the preparation of antibacterial products; the antibacterial products are used to inhibit at least one of Escherichia coli, Salmonella typhimurium, and Staphylococcus aureus.

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