Application of cladosporium globiforme and / or fermentation product of cladosporium globiforme in preparation of medicine for preventing and / or treating enteritis

Abstract

The invention relates to application of cladosporium globiforme and / or a fermentation product of the cladosporium globiforme in preparation of a medicine for preventing and / or treating enteritis, and belongs to the technical field of microorganisms. The invention provides application of cladosporium globiforme and / or a fermentation product of the cladosporium globiforme in preparation of a medicine for preventing and / or treating enteritis. In combination with in-vivo and in-vitro researches, the cladosporium globiforme participates in regulating the AMP metabolic process depending on a complete genetic loop of the cladosporium globiforme, and the cladosporium globiforme enhances the barrier integrity of epithelial cells and promotes the proliferation and differentiation of the epithelial cells so as to play an anti-inflammatory role and reduce proinflammatory cytokines and chemotactic factors; the method for increasing docking protein and goblet cell expression protein belongs to intestinal protective fungi.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, and in particular to application of Cladosporium cocci and / or its fermentation products in preparing medicines for preventing and / or treating enteritis. Background Art

[0002] C.sphaerospermum (Cladosporium Sphaerospermum) is a widespread saprophytic fungus found in both natural and artificial environments. It is primarily airborne and is present in both indoor and outdoor air. As a salt-tolerant microorganism, it thrives in areas with high salinity. Studies have shown that C. sphaerospermum can survive and reproduce in highly radioactive areas and reduce radiation levels (Averesch NJH, Shunk GK, Kern C. Cultivation of the Dematiaceous Fungus Cladosporium sphaerospermum Aboard the International Space Station and Effects of Ionizing Radiation. Front Microbiol. 2022 Jul 5; 13: 877625. doi: 10.3389 / fmicb.2022.877625. PMID: 35865919; PMCID: PMC9294542.); industrial waste emissions such as aromatic hydrocarbons, ketones and certain organic acids can also be degraded by C. sphaerospermum, making C. sphaerospermum a potential model for studying natural biofiltration mechanisms; C. sphaerospermum can also produce gibberellins, a plant growth hormone necessary for plant growth and development (Hamayun, M., Afzal Khan, S., Ahmad, N. et al. Cladosporium sphaerospermum as a new plant growth-promoting endophyte from the roots of Glycine max (L.) Merr.. World J Microbiol Biotechnol 25, 627–632 (2009). https: / / doi.org / 10.1007 / s11274-009-9982-9.), and therefore may also become a substitute for chemical fertilizers; in addition, studies have shown that extracts of C. sphaerospermum can effectively inhibit bacterial quorum sensing (QS)-related virulence factors and biofilm formation and may be used in combination with existing antimicrobial drugs to enhance the therapeutic effect of drug-resistant bacteria (Liu DR, Yan QX, Zou ZB, Xie CL, Yang XW, Jia AQ.Cladosporium sphaerospermum extractinhibits quorum sensing associated virulence factors of Serratiamarcescens.Biofilm.2023 Aug 1;6:100146.doi:10.1016 / j.bioflm.2023.100146.PMID:37560185;PMCID:PMC10406620.). .

[0003] The human digestive system is composed of a wide variety of microorganisms, including bacteria, archaea, viruses, and fungi, among which the so-called intestinal fungal community constitutes an important part of the healthy microbial community. Although it only accounts for a small part of the microbiome (0.01% to 1%), the fungal population in the human intestine has a considerable impact on various physiological and pathological processes. At present, there are few studies on C. sphaerospermum in the human digestive system. It is now known that through the annotation of a large number of intestinal fungal species genomes, it is found that in addition to common fungi such as Candida, C. sphaerospermum is also found to appear in feces under specific conditions or in specific populations, but the activity is not high.

[0004] So far, C. sphaerospermum has only been found in human feces. However, as a fungus, what role it plays in the intestinal microecological environment and what functional metabolic pathways it participates in have not been reported. Summary of the invention

[0005] The purpose of the present invention is to overcome the shortcomings of the prior art and provide the use of Cladosporium cocci and / or its fermentation products in the preparation of drugs for preventing and / or treating enteritis.

[0006] To achieve the above object, the technical solution adopted by the present invention is:

[0007] In a first aspect, the present invention provides the use of Cladosporium sphaerocephalum and / or its fermentation products in the preparation of a medicament for preventing and / or treating enteritis.

[0008] The Cladosporium sphaerocephalum and / or its fermentation product of the present invention enhances the integrity of the epithelial cell barrier and promotes the proliferation / differentiation of epithelial cells, thereby exerting an anti-inflammatory effect, and belongs to intestinal protective fungi.

[0009] Furthermore, the fermentation product contains adenosine phosphate.

[0010] The present invention detects adenine nucleotide (AMP) in the culture supernatant of Cladosporium sphaerocephalum at fixed time points within a continuous time period, and AMP is detected at 72 hours of culture but not at the baseline (0 hours), indicating that Cladosporium sphaerocephalum can synthesize and secrete AMP, and AMP may be a differential output metabolite of Cladosporium sphaerocephalum that acts on downstream hosts, and is a key active substance in the prevention of intestinal inflammation by Cladosporium sphaerocephalum.

[0011] Furthermore, the medicine is a medicine for treating intestinal damage.

[0012] Furthermore, the drug is a drug for repairing the intestinal epithelial barrier.

[0013] Furthermore, the drug is a drug that promotes proliferation and / or differentiation of intestinal epithelial cells.

[0014] Furthermore, the drug is a drug that reduces pro-inflammatory cytokines and / or chemokines.

[0015] In a specific embodiment of the present invention, the proinflammatory cytokines include at least one of IL-1α, IL-1β, IL-1f9, IL-2ra, IL-6, IL-17a and IL-1β. The present invention uses the above-mentioned proinflammatory cytokines as examples for detection and verification.

[0016] In a specific embodiment of the present invention, the chemokine includes at least one of Ccl2, Ccl8, Ccl9, Ccl11, Cxcl1, Cxcl2, Cxcl3, Cxcl5, Cxcl9, Cxcl10, and Cxcl13. The present invention uses the above-mentioned chemokines as examples for detection and verification.

[0017] Furthermore, the drug is a drug that increases the docking protein and / or goblet cell expression protein.

[0018] In a specific embodiment of the present invention, the docking protein includes Claudin1 and / or E-Cadherin; the goblet cell expressed protein includes Mucin2; the present invention uses the above-mentioned docking protein and goblet cell expressed protein as examples for detection and verification.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] The present invention combines in vivo and in vitro studies to illustrate that Cladosporium cocci relies on its complete genetic circuit to participate in regulating the AMP metabolic process. Cladosporium cocci enhances the integrity of the epithelial cell barrier and promotes epithelial cell proliferation and differentiation, thereby exerting an anti-inflammatory effect, reducing proinflammatory cytokines and chemokines, and increasing docking proteins and goblet cell expression proteins. It is an intestinal protective fungus, which is a major discovery in the study of intestinal inflammation. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 To sequence and annotate C. sphaerospermum. A is the experimental process; B is the whole genome sequencing analysis, annotating the genes related to AMP metabolism; C is the AMP metabolic pathway, and the genes marked in red are those in C. sphaerospermum; D is the ion flow diagram of AMP; E is the ion flow diagram of IMP.

[0022] Figure 2 The effects of oral administration of C. sphaerospermum on the body weight and intestinal tissue pathology of the enteritis mouse model. A is the drug administration and oral administration process; B is the weight of the mouse; C is the length of the mouse intestine; and D is the H&E staining result of the mouse intestinal tissue.

[0023] Figure 3 Transcriptome sequencing was used to detect the gene expression levels of pro-inflammatory cytokines and chemokines in mouse intestinal tissues.

[0024] Figure 4 The expression levels of AMP and IMP in the serum of enteritis mouse model.

[0025] Figure 5 Transcriptome sequencing was used to detect the transcriptional levels of genes related to cell junctions, tight junctions and Wnt signaling pathways. A is Gene Ontology (GO) pathway analysis; B is Gene Set Enrichment Analysis (GSEA).

[0026] Figure 6 The results of immunohistochemical staining of the junction protein Claudin1 (CLDN1), E-Cadherin (CDH1) and goblet cell expressed protein Mucin2 (MUC2). A is Claudin1 (CLDN1); B is Mucin2 (MUC2); C is E-Cadherin (CDH1).

[0027] Figure 7 C. sphaerospermum promotes epithelial cell proliferation. A is a photo of the scratch test of the intestinal epithelial cell line HT29; B is a quantitative graph of the scratch test of the intestinal epithelial cell line HT29; C is an intestinal organoid isolated from humans. DETAILED DESCRIPTION

[0028] To better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with specific examples. Other materials, reagents, etc. used in the examples, unless otherwise specified, can be obtained from commercial sources.

[0029] The product name of Cladosporium sphaerospermum used in the examples of the present invention is: Cladosporium sphaerospermum, the product number is BMZ146644, the brand is Mingzhoubio, and the manufacturer is Ningbo Mingzhou Biotechnology Co., Ltd.

[0030] Example 1 C. sphaerospermum has a complete genetic circuit to regulate AMP metabolism

[0031] 1. Experimental Methods

[0032] 1. If Figure 1 As shown in A, whole genome sequencing and transcriptome sequencing of C. sphaerospermum were performed: C. sphaerospermum was cultured in vitro using a specific culture medium. The culture medium components and culture conditions referred to the information provided by the China Industrial Microbiology Collection Center (CICC). The culture medium was yeast nitrogen basal medium (Yeast Nitrogen base, YNB, https: / / www.yeasen.com / products / detail / 4236) supplemented with 5% (w / v) glucose. The culture medium was sterilized at 121°C for 15 min, and no agar was added to the liquid culture medium. C. sphaerospermum was inoculated in the liquid culture medium, cultured at 30°C, with a shaker speed of 180 rpm for 72 h, and the cells were collected by centrifugation for whole genome sequencing and transcriptome sequencing, and the genomic information and active transcripts were annotated.

[0033] 2. Detection of adenine nucleotide (AMP) and inosine nucleotide (IMP) contents in the culture supernatant of C. sphaerospermum: In the in vitro culture experiment, the culture supernatant of C. sphaerospermum at fixed time points within a continuous period of time was collected, and the contents of AMP and IMP in the culture supernatant were detected by HPLC / MS technology.

[0034] 2. Experimental Results

[0035] 1. If Figure 1 B and Figure 1 (C) The complete genetic circuit of AMP and IMP metabolism in the purine recycling pathway was discovered by annotating the whole genome and active transcripts of C. sphaerospermum.

[0036] 2. Purine permease is a type of transporter protein in the process of purine secretion. It plays a role on the cell membrane and is responsible for the transmembrane transport of purine substances. In the present invention, this type of transporter protein is annotated at the DNA level and is actively transcribed at the RNA level.

[0037] 3. If Figure 1 D and Figure 1 As shown in E, the AMP and IMP contents in the culture supernatant of C. sphaerospermum were detected at fixed time points within a continuous period of time. The results showed that AMP was detected at 72 hours of culture, but not at baseline (0 hours), while the abundance of IMP did not differ significantly between different time points.

[0038] The above results indicate that C. sphaerospermum can synthesize and secrete AMP, and AMP may be a differential output metabolite of C. sphaerospermum to act on downstream hosts.

[0039] Example 2C. sphaerospermum relieves intestinal inflammation

[0040] 1. Experimental Methods

[0041] 1. If Figure 2 As shown in A, the mice in the control group were given PBS, and the mice in the experimental group were given C. sphaerospermum bacterial solution, 200 μL / mouse / time, and the bacterial content in the C. sphaerospermum bacterial solution was 10 6 The bacterial solution also contained C. sphaerospermum fermentation products and was given by gavage every other day for 4 weeks.

[0042] 2. Construction of mouse enteritis model: Dextran sulfate sodium (DSS) at a concentration of 2% (w / v) was used to induce enteritis in mice by adding it into drinking water for 6 days. During the administration of DSS, no PBS or C. sphaerospermum was administered by gavage.

[0043] 3. Mouse body weight detection: After administering DSS to mice, weigh them once a day. After 6 days, allow them to drink normal water and continue to detect their body weight for 2 to 3 days.

[0044] 4. Collect the mouse intestines to measure the intestinal length and perform H&E staining on the intestinal tissues.

[0045] 5. RT-qPCR was used to detect the gene expression levels of proinflammatory cytokines and chemokines in mouse intestinal tissues.

[0046] Proinflammatory cytokines include interleukin-1α (IL-1α), interleukin-1β (IL-1β), interleukin 1 family member 9 (IL-1f9), interleukin 2 receptor alpha chain (IL-2ra), interleukin-6 (IL-6), interleukin-17a (IL-17a), and tumor necrosis factor-α (TNF-α).

[0047] Chemokines include monocyte chemoattractant protein 1 (Ccl2), monocyte chemoattractant protein 2 (Ccl8), macrophage inflammatory protein 1γ (Ccl9), eosinophil chemoattractant protein (Ccl11), growth-related gene α (Cxcl1), growth-related gene β (Cxcl2), growth-related gene γ (Cxcl3), epithelial neutrophil-activating protein 78 (Cxcl5), monocyte chemoattractant protein-1-induced protein (Cxcl9), interferon-γ-induced protein 10 (Cxcl10) and B lymphocyte-attracting chemokine 1 (Cxcl13).

[0048] 6. Detection of AMP content in mouse serum.

[0049] 7. RT-qPCR was used to detect the transcription levels of genes related to cell junctions, tight junctions (such as Claudin1 (CLDN1) and E-Cadherin (CDH1)) and Wnt signaling pathways; immunohistochemistry was used to stain the junction proteins Claudin1 (CLDN1), E-Cadherin (CDH1) and goblet cell-expressed protein Mucin2 (MUC2).

[0050] Junction proteins include tight junction protein 1: Claudin1 (CLDN1) and cadherin E: E-Cadherin (CDH1). Goblet cell expressed proteins include mucin 2: Mucin2 (MUC2).

[0051] 8. Scratch healing experiment: HT-29 cells were seeded on 6-well cell culture plates at 200,000 cells per well and cultured under standard conditions (37°C, 5% CO2) until a confluent cell monolayer was formed (about 48 hours). A 200 μL pipette tip was used to prepare precise and reproducible scratches in each well. After scratching, the culture medium in each well was aspirated and gently rinsed with PBS to remove detached cells and debris, and then 2000 μL of control culture medium or culture medium containing 500 μM AMP or 7.5% (v / v) C. sphaerospermum culture supernatant was added. Immediately after scratching, an initial image was taken at 20× using a microscope, and images were taken every 24 hours within 72 hours of culture. The relative scratch closure rate (%) was calculated by measuring the change in the distance between the edges of the scratch at the preset points over time using ImageJ.

[0052] 9. Organoid culture: Primary intestinal crypts were isolated from human subjects and cultured in vitro. During the culture process, organoids were seeded in matrix gel containing more than 50% (w / v) Matrigel and cultured using organoid growth medium (human). When the organoids reached confluence, the matrix blocks containing organoids were collected in cold PBS and vigorously resuspended to dissociate the matrix. The organoids were centrifuged at 500×g for 5min. Subsequently, they were washed at least twice with cold basal medium (Advanced DMEM:F12, 1xGlutaMAX, 10mM HEPES), and then the organoids were seeded into Matrigel matrix domes. After the matrix solidified, the domes were covered with the above growth medium. 2000μL of control medium or medium containing 500μM AMP or 7.5% (v / v) C.sphaerospermum culture supernatant was added. Initial images were taken using a microscope at 20× magnification immediately after scratching, and images were taken every 24h for 10 days in culture.

[0053] 2. Experimental Results

[0054] 1. Mouse weight detection: Compared with the control group mice (administered PBS), the experimental group mice (administered C. sphaerospermum) showed significantly less weight loss after oral administration of DSS, indicating that intestinal inflammation in the experimental group mice was significantly reduced ( Figure 2 B).

[0055] 3. Compared with the control group mice (administered PBS), after oral administration, the intestinal length of the experimental group mice (administered C. sphaerospermum) was longer than that of the control group ( Figure 2 C) and showed lesser degree of histopathological damage ( Figure 2 D).

[0056] 4. If Figure 3 As shown, the results of transcriptome sequencing and target gene RT-qPCR detection showed that after oral administration of DSS, the gene expression levels of proinflammatory cytokines and chemokines (including Ccl2, Ccl8, Ccl9, Ccl11, Cxcl1, Cxcl2, Cxcl3, Cxcl5, Cxcl9, Cxcl10, Cxcl13, IL-1α, IL-1β, IL-1f9, IL-2ra, IL-6, IL-17a, IL-1β) in the intestinal tissue of the experimental group mice (given C. sphaerospermum) were significantly lower than those in the control group mice (given PBS).

[0057] 5. If Figure 4 As shown, the AMP level in the serum of mice in the experimental group was increased compared with that in the control group.

[0058] 6. If Figure 5 A and Figure 5 As shown in B, the transcription levels of genes related to cell junctions, tight junctions (such as Claudin1 (CLDN1) and E-Cadherin (CDH1)) and the Wnt signaling pathway were significantly upregulated. Figure 6 A. Figure 6 B and Figure 6 As shown in C, compared with the control group, the expression levels of junction protein Claudin1 (CLDN1), E-Cadherin (CDH1) and goblet cell expressed protein Mucin2 (MUC2) in the intestinal epithelial cells of the experimental group mice were significantly upregulated. After oral administration of C. sphaerospermum, the upregulated genes in the intestinal tissue of mice were all related to the integrity (cell links, tight junctions) and regeneration (Wnt signaling pathway) of the intestinal barrier.

[0059] 7. If Figure 7 A and Figure 7 As shown in B, this is a scratch test of the intestinal epithelial cell line HT29. LGK974 is an inhibitor of the Wnt signaling pathway. It can be seen that when LGK974 is not added, C. sphaerospermum metabolites and AMP can promote cell proliferation. After adding LGK974, there is no promoting effect on cell proliferation, indicating that AMP promotes cell proliferation by inducing Wnt signals. Figure 7 C shows intestinal organoids isolated from humans, where C. sphaerospermum metabolites and AMP promote proliferation.

[0060] The above results indicate that C. sphaerospermum enhances epithelial barrier integrity and promotes epithelial cell proliferation and differentiation, playing a beneficial role in preventing intestinal inflammation, and AMP is the key active substance in C. sphaerospermum in preventing intestinal inflammation.

[0061] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present invention.

Claims

1. Use of Cladosporium cocci and / or its fermentation products in the preparation of drugs for preventing and / or treating enteritis.

2. The use according to claim 1, characterized in that: The fermentation product contains adenosine phosphate.

3. The use according to claim 1, characterized in that: The medicine is a medicine for preventing and / or treating intestinal damage.

4. The use according to claim 3, characterized in that: The drug is a drug for enhancing the intestinal epithelial barrier.

5. The use according to claim 4, characterized in that: The drug is a drug that promotes the proliferation and / or differentiation of intestinal epithelial cells.

6. The use according to claim 1, characterized in that: The drug is a drug that reduces pro-inflammatory cytokines and / or chemokines.

7. The use according to claim 6, characterized in that: The proinflammatory cytokines include at least one of IL-1α, IL-1β, IL-1f9, IL-2ra, IL-6, IL-17a and IL-1β.

8. The use according to claim 6, characterized in that: The chemokine includes at least one of Ccl2, Ccl8, Ccl9, Ccl11, Cxcl1, Cxcl2, Cxcl3, Cxcl5, Cxcl9, Cxcl10, and Cxcl13.

9. The use according to claim 1, characterized in that: The drug is a drug that increases the docking protein and / or goblet cell expression protein.

10. The use according to claim 9, characterized in that: The docking protein includes Claudin1 and / or E-Cadherin; the goblet cell expressed protein includes Mucin2.

Images