Application of response marker for treating ulcerative colitis by coprophilous fungus transplantation

Abstract

The invention discloses an application of a response marker for treating ulcerative colitis by coprophilous fungi transplantation, and belongs to the technical field of biology, as a further optimization scheme of the invention, the coprophilous fungi comprise Streptococcus, Faecalibacteria, Weissella, Lactococcus and Rombout, and the application of the response marker for treating ulcerative colitis by coprophilous fungi transplantation has the advantages that the application of the response marker for treating ulcerative colitis by coprophilous fungi transplantation is realized, and the application of the response marker for treating ulcerative colitis by coprophilous fungi transplantation is realized. As a further optimization scheme of the invention, the coprophilous bacteria further comprise Streptococcus pluviae, Bifidobacterium longum, Lactococcus garviae, Weissella parasites, Lactococcus fermentum and Lactococcus lactis, and the Lactococcus pluviae, the Lactococcus pluviae, the Lactococcus pluviae, the Lactococcus pluviae, the Lactococcus pluviae, the Lactococcus pluviae, the Lactococcus pluviae, the Lactococcus pluviae, the Lactococcus pluviae, the Lactococcus pluviae, the Lactococcus pluviae, the Lactococcus pluviae, the Lactococcus pluviae, the Lactococcus pluviae, the Lactococcus pluviae, the Lactococcus pluviae, the Lactococcus pluviae and the In the invention, the flora change before and after coprophilous fungus transplantation treatment is researched on a patient with ulcerative colitis, a series of specific coprophilous fungus varieties are identified, and the effect of the strains in UC treatment is not confirmed in the previous research. Through quantitative analysis of existence and abundance of the specific strains, response conditions of UC patients to FMT treatment are predicted.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and specifically to the application of fecal microbiota transplantation in the treatment of ulcerative colitis response markers. Background Art

[0002] Ulcerative colitis (UC) is an inflammatory bowel disease caused by multiple factors, among which intestinal bacterial disturbance is considered to be one of the main pathogenic factors. The incidence of UC is generally on the rise worldwide, and the severity of the disease is also increasing.

[0003] The intestinal flora mainly exists in the intestines of the body. Under physiological conditions, the intestinal flora is non-pathogenic and coexists mutually with the host. The intestinal flora can not only provide enzymes to help the human body break down carbohydrates, fats and proteins and produce SCFAs, but also play a key role in the synthesis of important metabolites such as folic acid, vitamin K and vitamin B12. In addition, the intestinal flora also plays an important role in immune regulation, maintaining the structural integrity of the intestinal mucosal barrier and resisting the colonization of pathogenic microorganisms. Although FMT treatment has been successful in some cases, its specific mechanism for the treatment of UC is still unclear, which limits the optimization and improvement of the treatment method. For this reason, the application of fecal microbiota transplantation response markers for the treatment of ulcerative colitis is proposed. Summary of the invention

[0004] The present invention aims to solve at least one of the technical problems existing in the prior art. To this end, one object of the present invention is to propose the application of fecal microbiota transplantation to treat ulcerative colitis response markers.

[0005] To achieve the above object, the present invention provides the following technical solutions:

[0006] Application of fecal microbiota transplantation to treat ulcerative colitis response markers.

[0007] As a further optimization scheme of the present invention, fecal bacteria include the genera Streptococcu, Faecalibacterium, Weissella, Lactococcus, and Romboutsia.

[0008] As a further optimization scheme of the present invention, fecal bacteria also include Streptococcus pneumoniae, Bifidobacterium longum, Lactococcus garvieae, Weissella paramesenteroides, Lactobacillus fermentum and Lactococcus lactis.

[0009] Compared with the prior art, the present invention has the following beneficial effects:

[0010] In the present invention, the changes in the microbiota of patients with ulcerative colitis before and after fecal microbiota transplantation were studied, and a series of specific fecal microbiota species were identified, which have not been confirmed in previous studies to play a role in the treatment of UC. These species include Streptococcus, Bifidobacterium longum, Lactococcus garvieae, Weissella paramesenteroides, Lactobacillus fermentum, and Lactococcus lactis. By quantitatively analyzing the presence and abundance of these specific species, the response of UC patients to FMT treatment was predicted.

[0011] The fecal bacteria species in the present invention affect the therapeutic effect of UC through immune regulation, barrier function enhancement, and the influence of metabolites in the response after FMT treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 It is a genus level indicator species statistical chart of the present invention; wherein, Figure A: T test of the response group before treatment vs. the response group after treatment; Figure B: Wilcox rank sum test of the response group before treatment vs. the response group after treatment; Figure C: T test of the non-response group after treatment vs. the response group after treatment; Figure D: Wilcox rank sum test of the non-response group after treatment vs. the response group after treatment.

[0013] Figure 2 It is a species level indicator species statistical chart of the present invention; wherein, Figure A: T test of the response group before treatment vs. the response group after treatment; Figure B: Wilcox rank sum test of the response group before treatment vs. the response group after treatment; Figure C: T test of the non-response group after treatment vs. the response group after treatment; Figure D: Wilcox rank sum test of the non-response group after treatment vs. the response group after treatment.

[0014] Figure 3 This is a mouse DAI score chart of the present invention; wherein, the horizontal axis is the experimental time (DAYS); the vertical axis is the disease activity score (DAI SCORES); the upper left corner is the name of each group marked with different colors.

[0015] Figure 4 The figures are graphs showing the degree of shortening of the colon of each group of mice of the present invention; wherein, Figure A: a general picture of the colon of each group of mice; Figure B: a statistical comparison of the colon length of each group of mice, the abscissa is the name of each group, and the ordinate is the average colon length of each group of mice at the end point of the experiment.

[0016] Figure 5It is a diagram of pathological changes in the mouse colon of the present invention; wherein, Figure A: HE staining of the colon of each group of mice, the horizontal axis is the name of each group, and the vertical axis is the magnification; Figure B: pathological scores of each group of mice, the horizontal axis is the name of each group, and the vertical axis is the pathological score.

[0017] Figure 6 It is the expression diagram of barrier protein in mouse colon tissue of the present invention; wherein, Figure A: barrier protein blot; Figures B, C, D, E: grayscale analysis of protein blot.

[0018] Figure 7 This is an immunohistochemical image of barrier proteins in mouse colon tissue of the present invention. DETAILED DESCRIPTION

[0019] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only 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.

[0020] Embodiment 1

[0021] 69 UC patients who received FMT and regularly collected stool samples were selected for 16S sequencing. Among the 69 patients, 42 responded to FMT treatment and 27 did not respond. Key bacterial communities were found at the genus and species levels.

[0022] T-test and Wilcox rank-sum test were used to calculate and compare the relative abundance changes of bacterial species in the response group before and after treatment and in the non-response group vs. the response group after treatment. The following differences were found at the genus level:

[0023] As attached Figure 1 As shown, compared with before treatment, the bacterial abundance of the response group Streptococcu (Rs_UC_Post vs. Rs_UC_Pre = 3.84% vs. 1.44%, P = 0.0028), Faecalibacterium (Rs_UC_Post vs. Rs_UC_Pre = 7.59% vs. 2.90%, P = 0.0084), Weissella (Rs_UC_Post vs. Rs_UC_Pre = 2.87% vs. 2.67%, P = 0.0453), and Lactococcus (Rs_UC_Post vs. Rs_UC_Pre = 1.93% vs. 0.46%, P = 0.0026) genera increased significantly after treatment.

[0024] In the patients after treatment, species analysis showed that compared with the non-responder group, the responder group had a higher abundance of bacteria in the Weissella genera (No_UC_Post vs. Rs_UC_Post = 0.82% vs. 2.87%, P = 0.0110), Romboutsia genera (No_UC_Post vs. Rs_UC_Post = 1.25% vs. 2.67%, P = 0.0431), and Lactococcus genera (No_UC_Post vs. Rs_UC_Post = 0.65% vs. 1.93%, P = 0.0056).

[0025] The above analysis showed that patients who responded to FMT treatment showed a clear enrichment trend of specific bacterial communities after treatment.

[0026] Embodiment 2

[0027] Regarding the changes in bacterial species at the species level after FMT treatment, such as Figure 2 As shown, at the species level, the abundance of Streptococcus pneumoniae (Rs_UC_Post vs. Rs_UC_Pre = 0.88% vs. 0.18%, P = 0.0155), Bifidobacterium longum (Rs_UC_Post vs. Rs_UC_Pre = 3.57% vs. 1.06%, P = 0.0091), and Lactococcus garvieae (Rs_UC_Post vs. Rs_UC_Pre = 1.16% vs. 0.37%, P = 0.0040) in the response group after treatment was significantly higher than that before treatment.

[0028] After FMT treatment, compared with the non-response group, the abundance of Weissella paramesenteroides (No_UC_Post vs. Rs_UC_Post = 0.78% vs. 2.62%, P = 0.0149), Lactobacillus fermentum (No_UC_Post vs. Rs_UC_Post = 0.13% vs. 3.70%, P = 0.0369), Lactococcus lactis (No_UC_Post vs. Rs_UC_Post = 0.10% vs. 0.64%, P = 0.02), and Lactococcus garvieae (No_UC_Post vs. Rs_UC_Post = 0.53% vs. 1.16%, P = 0.0224) increased in the responder group.

[0029] Based on the above microbial analysis results, it can be seen that Streptococcus pneumoniae, Bifidobacterium longum, Lactococcus garvieae, Weissella paramesenteroides, Lactobacillus fermentum and Lactococcus lactis are related to the efficacy of FMT in the treatment of UC.

[0030] Embodiment 3

[0031] FMT and single bacteria gavage treatment improved the DAI score of enteritis mice:

[0032] As attached Figure 3 As shown, after 3 days of DSS induction, except for the Control group, all other groups drank 2.5% DSS solution, and the DAI scores increased. Among them, the DSS group given PBS by gavage had the highest and fastest increase in DAI score, and the weight loss and diarrhea and bloody stool observed by naked eyes during the experiment were the most serious.

[0033] The DAI scores of mice in the DSS+FMT group, DSS+L.lactis group, DSS+W.paramesenteroides group, DSS+L.fermentum group, and DSS+L.garvieae group increased slowly. Among them, the DSS+FMT group and DSS+L.garvieae group had the best effect on mice.

[0034] At the end of the experiment on the 7th day, the DAI scores of the DSS group were significantly higher than those of the Control group (P < 0.0001), and the DAI scores of the DSS+FMT group and the DSS+L.garvieae group were significantly lower than those of the DSS group (P = 0.0018 and P = 0.0050). The DSS+L.lactis, DSS+W.paramesenteroides and DSS+L.fermentum groups showed a downward trend compared with the DSS group, but there was no statistical difference (P values ​​were 0.4031, 0.1339, and 0.2681, respectively).

[0035] Embodiment 4

[0036] FMT and single bacteria gavage treatment improved the degree of colon shortening in enteritis mice:

[0037] As attached Figure 4 As shown, at the end of the seventh day of the experiment, compared with the Control group, the feces in the colon of the DSS group were unformed, with dark red blood in the stool, the intestinal wall became thinner and brittle, and the colon was significantly shortened (6.06±0.34 vs. 11.83±0.19, P<0.0001).

[0038] The colon conditions of mice in the DSS+FMT group, DSS+L.lactis group, DSS+W.paramesenteroides group, DSS+L.fermentum group, and DSS+L.garvieae group recovered compared with those in the DSS group. The feces were granular, and some intestinal walls were thickened and stiff. The average colon lengths were (8.07±1.09) cm, (7.55±0.82) cm, (7.90±0.90) cm, (7.27±1.2 5)cm, (7.04±0.72)cm, among which the DSS+FMT group, DSS+L.lactis group, DSS+W.paramesenteroides group and DSS+L.garvieae group had significant differences compared with the DSS group (P values ​​were 0.0053, 0.001, 0.0030 and 0.0065, respectively), and there was no statistical difference in the degree of colon shortening between the DSS+L.fermentum group and the DSS group (P=0.0643).

[0039] It can be seen that FMT fecal suspension and oral gavage of each group of single bacteria have a certain protective effect on DSS-damaged colon, among which FMT fecal suspension and Lactococcus garvieae, Weissella parames enteroides and Lactococcus lactis have better effects.

[0040] Embodiment 5

[0041] FMT and single-bacteria oral gavage therapy improve colon pathological damage in enteritis mice:

[0042] As attached Figure 5 As shown, the colon epithelium of the Control group was intact, the crypt glands were neatly arranged, the goblet cells were numerous and densely distributed, there was no obvious neutrophil infiltration in the lamina propria, and the pathological score was 0, which was a healthy colon pathological phenotype.

[0043] In the DSS group, colon epithelial damage, crypt structure destruction and disordered arrangement were observed, neutrophils proliferated massively and infiltrated into the submucosal layer of the intestinal wall. The pathological score was 12±2.24 points, which was significantly different from that in the Control group (P<0.0001).

[0044] The colon pathological damage of mice in the DSS+FMT group, DSS+L.lactis group, and DSS+L.garvieae group was alleviated to varying degrees, such as preserving most of the normally arranged crypts, reducing the degree of inflammatory infiltration, and reducing the range of crypt damage. The pathological scores of these three groups were 8.33±3.44, 8.17±3.66, and 7.57±4.16, respectively, which were statistically different from those in the DSS group (P values ​​were 0.04, 0.04, and 0.03, respectively). The pathological scores of the DSS+W.paramesenteroides group and DSS+L.fermentum group were 9.67±2.73 and 11.17±1.60, which were not statistically different from those in the DSS group (P values ​​were 0.12 and 0.46, respectively).

[0045] As attached Figure 6 As shown in the results, Lactococcus garvieae has the best effect in improving the DAI score of the DSS enteritis mouse model, reducing the degree of colon shortening and colon pathological damage, restoring the diversity of intestinal flora, and inhibiting the growth of pathogens. At the molecular level, the expression levels of colon epithelial barrier proteins in mice were detected by extracting mouse colon proteins and performing Western blotting. It was found that compared with the control group mice, the expression of Claudin-1, ZO-1, Occludin and MUC-2 proteins in the colon of the DSS group mice was reduced (P=0.0093, P=0.0001, P=0.0003 and P=0.0004), and the expression of MUC-2 protein in the colon of the DSS+L.garvieae group mice increased (P=0.017). The expression levels of barrier proteins in other groups showed an increasing trend after treatment.

[0046] As attached Figure 7 As shown in the figure, this experiment further detected the expression position and relative expression of barrier proteins by immunohistochemistry, and found that the colon epithelium of mice in the DSS group was damaged, the crypt structure was destroyed and the arrangement was disordered, the expression of barrier proteins was reduced, and ZO1 had obvious positional structural changes. After treatment, the colon crypt structure of some mice in the FMT group and DSS+L.garvieae group was restored, and the expression of barrier proteins was also enhanced, among which the change of MUC2 protein was more obvious.

[0047] Parts not involved in the present invention are the same as the prior art or can be implemented by the prior art. Although the embodiments of the present invention have been shown and described, it is understood by those skilled in the art that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and spirit of the present invention, and the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. Application of fecal microbiota transplantation response markers in the treatment of ulcerative colitis.

2. The use of fecal microbiota transplantation to treat ulcerative colitis response markers according to claim 1, characterized in that: Fecal bacteria include the genera Streptococcu, Faecalibacterium, Weissella, Lactococcus, and Romboutsia.

3. The use of fecal microbiota transplantation to treat ulcerative colitis response markers according to claim 1, characterized in that: Fecal bacteria also include Streptococcus pneumoniae, Bifidobacterium longum, Lactococcusgarvieae, Weissella paramesenteroides, Lactobacillus fermentum and Lactococcuslactis.

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