Application of a gut microbiota combination in the preparation of a drug for treating SLE

Abstract

The present invention belongs to the technical field of biomedicine, and discloses the application of an intestinal microbiota combination in the preparation of a medicament for treating Systemic lupus erythematosus (SLE). The intestinal microbiota combination of the present invention comprises Ligilactobacillus murinus and / or Faecalibaculum rodentium. After the intestinal microbiota transplantation of the intestinal microbiota combination in lupus mice, the intestinal inflammatory response of the lupus mice can be improved, and the expression levels of autoantibodies and inflammatory factors can be effectively reduced, significantly improving the disease progression of the lupus mice, providing a new idea and method for the treatment of SLE diseases.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and specifically relates to the application of a gut microbiota combination in the preparation of a drug for treating Systemic lupus erythematosus (SLE). Background Art

[0002] Systemic lupus erythematosus (SLE) is a chronic autoimmune disease that affects multiple organs and systems throughout the body. In this disease, the immune system mistakenly attacks the body's own tissues, resulting in inflammation and damage. Basophils are a type of typical innate immune cell. Although their proportion in peripheral blood mononuclear cells (PBMCs) is less than 1%, their powerful functions have been increasingly emphasized. A series of recent studies have found that basophils play an important role in autoimmune diseases. Autophagy is a lysosome-dependent cellular degradation pathway that maintains the balance of body metabolism and the stability of the internal environment by degrading macromolecules and damaged organelles within the cell.

[0003] Gut microbiota refers to a group of microorganisms that live in the human gut, including various bacteria, fungi, etc. Gut microbiota has important metabolic and regulatory functions, which can help the human body break down food, synthesize vitamins, enzymes and other substances. In addition, gut microbiota can also promote the absorption of nutrients and the balance of water, regulate energy and fat metabolism, and maintain the balance of the body's internal environment. Recent studies have also shown that there is a close connection between gut microbiota and many human diseases, including intestinal diseases, metabolic diseases, neurological diseases and cancers. Therefore, gut microbiota has become a new direction for the research and treatment of various diseases. Summary of the Invention

[0004] The purpose of the present invention is to overcome the deficiencies of the prior art and provide the application of a gut microbiota combination in the preparation of a drug for treating SLE.

[0005] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0006] In the first aspect, the present invention provides a gut microbiota combination for preventing and / or treating SLE, including Ligilactobacillus murinus and / or Faecalibaculum rodentium.

[0007] By constructing a lupus mouse model with autophagy deficiency in basophils (Baso), it was found that autophagy deficiency in basophils could improve the disease progression of lupus mice. The feces of lupus mice were collected for metagenomic sequencing, and it was found that the expression of Ligilactobacillus murinus and Faecalibaculum rodentium was significantly increased in the group with autophagy deficiency in basophils, and was negatively correlated with the expression of autoantibodies and inflammatory factors. Subsequently, intestinal microbiota transplantation of Ligilactobacillus murinus and Faecalibaculum rodentium was performed in lupus mice. The results showed that intestinal microbiota transplantation of Ligilactobacillus murinus and Faecalibaculum rodentium could improve the intestinal inflammatory response in lupus mice and effectively reduce the expression of autoantibodies and inflammatory factors, and the treatment effect of the group with intestinal microbiota transplantation of Ligilactobacillus murinus combined with Faecalibaculum rodentium was significantly better than that of the group with single intestinal microbiota transplantation.

[0008] In a second aspect, the present invention provides a drug for preventing and / or treating SLE, comprising the intestinal microbiota combination.

[0009] As a preferred embodiment of the drug according to the present invention, it further comprises a pharmaceutically acceptable carrier or excipient.

[0010] In a third aspect, the present invention applies the intestinal microbiota combination in the preparation of a drug for preventing and / or treating SLE.

[0011] As a preferred embodiment of the application according to the present invention, the drug is administered by gavage or intestinal microbiota transplantation.

[0012] As a preferred embodiment of the application according to the present invention, the functions of the drug are at least one of the following:

[0013] Ⅰ. Improve the intestinal inflammatory response;

[0014] Ⅱ. Reduce the expression of autoantibodies and inflammatory factors.

[0015] Compared with the prior art, the beneficial effects of the present invention are:

[0016] The present invention discovers that Ligilactobacillus murinus and Faecalibaculum rodentium are significantly increased in expression in the group with autophagy deficiency of basophils, and are negatively correlated with the expression of autoantibodies and inflammatory factors; the intestinal microbiota transplantation of Ligilactobacillus murinus and Faecalibaculum rodentium can improve the intestinal inflammatory response in lupus mice and effectively reduce the expression of autoantibodies and inflammatory factors, and the treatment effect of the intestinal microbiota transplantation group combining Ligilactobacillus murinus and Faecalibaculum rodentium is significantly better than that of the transplantation group of a single intestinal microbiota. After the intestinal microbiota transplantation with the intestinal microbiota combination (Ligilactobacillus murinus combined with Faecalibaculum rodentium) of the present invention, the disease progression of lupus mice can be significantly improved, providing a new idea and method for the treatment of SLE disease. Brief Description of the Drawings

[0017] Figure 1 Detection results of plasma autoantibodies and inflammatory factors in lupus mice with autophagy deficiency of basophils; in the figure, A is the detection result of anti-nuclear autoantibodies in the plasma of lupus mice, B is the detection result of anti-dsDNA autoantibodies in the plasma of lupus mice, C is the detection result of TNF-α inflammatory factor in the plasma of lupus mice, D is the detection result of IFN-γ inflammatory factor in the plasma of lupus mice, E is the detection result of IL-1β inflammatory factor in the plasma of lupus mice, F is the detection result of IL-2 inflammatory factor in the plasma of lupus mice, G is the detection result of IL-4 inflammatory factor in the plasma of lupus mice, H is the detection result of IL-6 inflammatory factor in the plasma of lupus mice, I is the detection result of IL-13 inflammatory factor in the plasma of lupus mice, J is the detection result of IL-17 inflammatory factor in the plasma of lupus mice; P<0.05 indicates significant difference, marked with *.

[0018] Figure 2 Results of fecal metagenomic sequencing of lupus mice with autophagy deficiency of basophils; in the figure, A is the phylogenetic tree analysis generated by LEfSe, B is the bar chart analysis generated by LEfSe, red represents the intestinal microbiota enriched in the Atg5KO group, and green represents the intestinal microbiota enriched in the control group.

[0019] Figure 3Analysis of the correlation between gut microbiota, autoantibodies, and inflammatory factors in lupus mice with basophil autophagy deficiency; in the figure, A is the RDA analysis of gut microbiota and autoantibodies, B is the RDA analysis of gut microbiota and inflammatory factors. The length of the arrow represents the magnitude of the correlation between the disease severity index and the sample distribution. The longer the line, the greater the correlation. The angle between the arrow and the sorting axis represents the correlation, with an acute angle indicating a positive correlation and an obtuse angle indicating a negative correlation; C is the heatmap analysis of gut microbiota, autoantibodies, and inflammatory factors. The R value is represented by different colors, with red indicating a positive correlation and blue indicating a negative correlation. The darker the color, the stronger the positive / negative correlation; P<0.05 indicates a significant difference, marked with an asterisk.

[0020] Figure 4 Results of H&E staining of intestinal pathology in lupus mice with gut microbiota transplantation.

[0021] Figure 5 Results of detection of plasma autoantibodies and inflammatory factors in lupus mice with gut microbiota transplantation; in the figure, A is the result of detecting anti-nuclear autoantibodies in the plasma of lupus mice, B is the result of detecting anti-dsDNA autoantibodies in the plasma of lupus mice, C is the result of detecting TNF-α inflammatory factors in the plasma of lupus mice, D is the result of detecting IFN-γ inflammatory factors in the plasma of lupus mice, and E is the result of detecting IL-17 inflammatory factors in the plasma of lupus mice; P<0.05 indicates a significant difference, marked with an asterisk. Detailed implementation manners

[0022] To better illustrate the purpose, technical solution, and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments. Those skilled in the art should understand that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0023] Unless otherwise specified, the experimental methods used in the examples are all conventional methods; the materials, reagents, etc. used, unless otherwise specified, can all be obtained from commercial channels.

[0024] Example 1: Detection of autoantibodies and inflammatory factors in lupus mice with basophil autophagy deficiency

[0025] Autophagy gene Atg5 knockout mice (Atg5 flox / flox Cre + / - , Atg5 - / - ) and autophagy gene Atg5 non-knockout control mice (Atg5 flox / flox Cre - / - , Atg5 + / +) provided by Shanghai Model Organisms Center, Inc. The basophil autophagy-deficient mouse model was constructed with reference to the Chinese invention patent document CN 118120702B (A method for constructing a basophil autophagy-deficient mouse and its application).

[0026] The mice were grouped as follows:

[0027] The Control group was MRL / lpr mice without any intervention; Atg5 - / - The Baso adoptive transfer group was MRL / lpr mice with basophil autophagy deficiency; Atg5 + / + The Baso adoptive transfer group was control MRL / lpr mice with the autophagy gene Atg5 not knocked out that had received adoptive transfer of basophils; at the same time, MRL / MpJ mice were set as the normal control group for MRL / lpr lupus mice.

[0028] (1) Detection of anti-nuclear autoantibodies in the plasma of lupus mice

[0029] The ELISA kit was used to detect anti-nuclear antibody in the plasma of mice:

[0030] Thaw the cryopreserved plasma on ice. Open the Mouse anti-nuclear antibody ELISA Kit (AlphaDiagnostic, USA). Dilute the samples 1:150 with the prepared 1×Working Sample Diluent and Low NSB Sample Diluent. Using the 96-well plate in the anti-nuclear antibody kit, add 100 μL of the standard, samples, and negative control to the pre-determined wells respectively, and seal with sealing film. Incubate at room temperature for 60 min. Invert the plate to remove the liquid in the wells. Use a multi-channel pipette to add 200 μL of 1×Working Wash Solution to each well, gently tap the plate by hand to wash, then invert the plate to remove the liquid. Invert the 96-well plate on a tissue paper and tap it several times forcefully to ensure the removal of the liquid in the wells. Repeat the washing process 3 more times in the same way. Add 100 μL of Anti-Mouse Ig HRP to the wells containing the standard, samples, and negative control respectively, then seal it with sealing film and incubate at room temperature for 30 min. Invert the plate to remove the liquid in the wells. Add 200 μL of 1×Working Wash Solution to each well and gently tap the plate to wash, then invert the plate to remove the liquid. Invert the 96-well plate on a tissue paper and tap it several times forcefully to ensure the removal of the liquid in the wells. Repeat the washing process 4 more times in the same way. Add 100 μL of TMB Substrate to each well and incubate in the dark for 15 min. At this time, the liquid in the wells will start to turn blue. Add 100 μL of Stop Solution to each well and pipette up and down to mix evenly. Measure the OD value at 450 nm and the OD value at 630 nm with an ELISA reader to normalize the well background. Finally, calculate the net OD value (OD 450 -OD 630 ) for each sample, establish a standard curve, and calculate the titer of anti-nuclear autoantibody for each sample based on this curve.

[0031] (2) Detection of anti-dsDNA autoantibody in the plasma of lupus mice

[0032] Detect anti-dsDNA antibody in the plasma of mice using an ELISA kit:

[0033] Thaw the cryopreserved plasma on ice. Open the Mouse anti-dsDNA (Alpha Diagnostic, USA) kit and dilute the samples 1:3000 with the prepared 1×Working Sample Diluent and Low NSB Sample Diluent. Using the 96-well plate in the dsDNA kit, add 100 μL of the standard, samples, and negative control to the designated wells respectively, seal with sealing film, and incubate at room temperature for 60 min. Invert the plate to remove the liquid in the wells. Use a multi-channel pipette to add 200 μL of 1×Working Wash Solution to each well, gently tap the plate by hand to wash, then invert the plate to remove the liquid, and invert the 96-well plate on a paper towel and tap it several times forcefully to ensure the removal of the liquid in the wells. Repeat the washing process 3 more times in the same way. To the wells with the standard, samples, and negative control, add 100 μL of Anti-Mouse Ig HRP each, then seal it with sealing film and incubate at room temperature for 30 min. Invert the plate to remove the liquid in the wells, add 200 μL of 1×Working Wash Solution to each well and gently tap the plate to wash, then invert the plate to remove the liquid, and invert the 96-well plate on a paper towel and tap it several times forcefully to ensure the removal of the liquid in the wells. Repeat the washing process 4 more times in the same way. Add 100 μL of TMB Substrate to each well and incubate in the dark for 15 min. At this time, the liquid in the wells will start to turn blue. Add 100 μL of Stop Solution to each well and pipette to mix evenly. Use an ELISA reader to detect the OD value at 450 nm and the OD value at 630 nm to normalize the well background. Finally, calculate the net OD value (OD 450 -OD 630 ) for each sample, establish a standard curve, and calculate the titer of anti-dsDNA autoantibody for each sample accordingly.

[0034] (3) Detection of inflammatory factors in the plasma of lupus mice

[0035] The plasma samples of lupus mice were sent to Guangzhou Juyan Biotechnology Co., Ltd. for use MAP liquid-phase protein chip kit (Millipore, Billerica, MA, USA) was used to detect the levels of plasma inflammatory cytokines, including TNF-α, IFN-γ, IL-1β, IL-2, IL-4, IL-6, IL-13, and IL-17.

[0036] The experimental results are shown in Figure 1 , Atg5 - / - The Baso adoptive transfer group compared with the Control group and Atg5 + / +The expression of autoantibodies and inflammatory factors in the Baso adoptive transfer group was significantly reduced. The above results indicate that Baso autophagy deficiency reduces the production of autoantibodies and inflammatory factors in lupus mice.

[0037] Example 2: Analysis of the correlation between gut microbiota, inflammatory factors and autoantibodies in lupus mice with basophil autophagy deficiency

[0038] (1) Metagenomic sequencing of gut microbiota in lupus mice with basophil autophagy deficiency

[0039] Fecal samples from the lupus mice with basophil autophagy deficiency in Example 1 were sent to Wuhan Maiwei Metabolism Biotechnology Co., Ltd. for metagenomic sequencing.

[0040] (2) Analysis of the correlation between gut microbiota, plasma autoantibodies and inflammatory factors in lupus mice with basophil autophagy deficiency

[0041] Results of metagenomic sequencing Figure 2 , the gut microbiota in the basophil autophagy deficiency group was mainly enriched in Ligilactobacillus murinus and Faecalibaculum rodentium, while the Control group (basophils with autophagy activation and without Atg5 knockout) was mainly enriched in Parvibacter caecicola, Bacteroides acidifaciens, etc. Correlation analysis showed that Ligilactobacillus murinus and Faecalibaculum rodentium were negatively correlated with autoantibodies and inflammatory factors.

[0042] Example 3: Detection of disease progression in lupus mice with gut microbiota transplantation

[0043] To evaluate the effect of intestinal microorganisms on disease progression in lupus mice, MRL / lpr lupus mice were divided into: Control group (lupus control without any intervention), Antibiotic group (antibiotics to eliminate intestinal microorganisms), F. rodentium-FMT group (antibiotics to eliminate intestinal microorganisms and then transplant a single Faecalibaculum rodentium), L. murinus-FMT group (antibiotics to eliminate intestinal microorganisms and then transplant a single Ligilactobacillus murinus), L. murinus + F. rodentium-FMT group (antibiotics to eliminate intestinal microorganisms and then transplant Ligilactobacillus murinus and Faecalibaculum rodentium). At the same time, MRL / MpJ mice were set as the normal control group of MRL / lpr lupus mice.

[0044] Among them, the Antibiotic group was treated with antibiotics to remove the intestinal microorganisms of mice. The intervention was carried out at 8 weeks of age. The method was as follows: broad-spectrum antibiotics were added to the drinking water of mice, with the following formula: Ampicillin (0.2g / L), Metronidazole (0.2g / L), Neomycin (0.2g / L), Vancomycin (0.1g / L), and then mixed thoroughly and placed in the IVC cage to replace the drinking water of mice. The drinking water was changed every 3 days for 2 weeks.

[0045] The F. rodentium-FMT group, L. murinus-FMT group, and L. murinus + F. rodentium-FMT group were pretreated with compound antibiotics before intestinal flora transplantation and intervened at 8 weeks of age with the same method as the Antibiotic group. In the F. rodentium-FMT group, Faecalibaculum rodentium was purchased from Shangcheng Beina Chuanglian Biotechnology Co., Ltd. (strain number: BNCC363015) and was used as 10 8 In the L.murinus-FMT group, Ligilactobacillus murinus was purchased from Beina Chuanglian Biotechnology Co., Ltd. (strain number: BNCC194688) at 10 8Gavage at 10 8 CFU / 200 μL. In the L. murinus + F. rodentium - FMT group, the strains Faecalibaculum rodentium (strain number: BNCC363015) and Ligilactobacillus murinus (strain number: BNCC194688) were purchased from Beina Chuanglian Biotechnology Co., Ltd. in the mall. Equal amounts of the two strains were mixed and gavaged at a total of 10 8 CFU / 200 μL. 8 Gavage at 10 8 CFU / 200 μL. All mice subjected to intestinal microbiota transplantation were gavaged three times a week for four weeks, after which the mice were sacrificed for sample collection.

[0046] (1) H&E detection of intestinal pathology in lupus mice with intestinal microbiota transplantation

[0047] Deparaffinization: Place the intestinal tissue slides in xylene I for 20 min and in xylene II for 20 min; place the slides in absolute ethanol I for 5 s, in absolute ethanol II for 5 s → in 95% ethanol I for 5 s, in 95% ethanol II for 5 s → wash with water for 15 s; ③ Place the slides in hematoxylin stain and soak for 2 min → wash with water for 15 s × 2 → soak in 1% hydrochloric acid alcohol for 3 s → wash with water for 15 s → soak in 1% ammonia water for 3 s → wash with water for 15 s; place the slides in eosin stain and soak for 2 min → wash with water for 15 s → absolute ethanol for 2 s → dry by baking, and seal with neutral resin; take pictures.

[0048] (2) Detection of plasma inflammatory factors in lupus mice with intestinal microbiota transplantation

[0049] The detection method for plasma samples of lupus mice with intestinal microbiota transplantation was the same as that in step (3) of Example 1.

[0050] The experimental results are shown in Figures 4 - 5 , in Figure 4 , in the F. rodentium - FMT group and the L. murinus - FMT group, the deposition of intestinal inflammatory cells was improved to varying degrees compared with the Control group and the Antibiotic group, indicating that Faecalibaculum rodentium and Ligilactobacillus murinus improved the disease progression. In the L. murinus + F. rodentium - FMT group compared with the F. rodentium - FMT group and the L. murinus - FMT group, the deposition of intestinal inflammatory cells was more significantly improved, indicating that the combined treatment effect of Faecalibaculum rodentium and Ligilactobacillus murinus was significantly better than single treatment. In Figure 5Among them, the anti-nuclear and anti-dsDNA autoantibodies and inflammatory factors such as TNF-α, IFN-γ, and IL-17 in the F. rodentium-FMT group and the L. murinus-FMT group were decreased to varying degrees compared with the Control group and the Antibiotic group, indicating that Faecalibaculum rodentium and Ligilactobacillus murinus can improve the expression of autoantibodies and inflammatory factors. Compared with the F. rodentium-FMT group and the L. murinus-FMT group, the anti-nuclear and anti-dsDNA autoantibodies and inflammatory factors such as TNF-α, IFN-γ, and IL-17 in the L. murinus+F. rodentium-FMT group were more significantly improved, indicating that the combined treatment effect of Faecalibaculum rodentium and Ligilactobacillus murinus is significantly better than single treatment.

[0051] In summary, compared with the Control group, the intestinal pathology, autoantibodies, and inflammatory factor expression in the Antibiotic group were improved, indicating that the clearance of all intestinal microorganisms by antibiotics can improve the disease progression. The disease progression in the F. rodentium-FMT group and the L. murinus-FMT group was improved to varying degrees compared with the Antibiotic group, indicating that Faecalibaculum rodentium and Ligilactobacillus murinus can improve the disease progression. However, it should be noted that compared with the F. rodentium-FMT group and the L. murinus-FMT group, the disease progression in the L. murinus+F. rodentium-FMT group was more significantly improved, indicating that the combined treatment effect of Faecalibaculum rodentium and Ligilactobacillus murinus is significantly better than single treatment.

[0052] The above results indicate that the combination of Ligilactobacillus murinus and Faecalibaculum rodentium can significantly improve the disease progression in lupus mice, providing a new idea and method for the treatment of SLE.

[0053] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit the protection scope of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. An intestinal microbiota combination for preventing and / or treating systemic lupus erythematosus, characterized in that, including Ligilactobacillus murinus and Faecalibaculum rodentium; the strain number of the Ligilactobacillus murinus is BNCC363015; the strain number of the Faecalibaculum rodentium is BNCC194688.

2. A drug for preventing and / or treating systemic lupus erythematosus, characterized in that, including the intestinal microbiota combination according to claim 1 and a pharmaceutically acceptable carrier or excipient.

3. Use of the intestinal microbiota combination according to claim 1 in the preparation of a drug for preventing and / or treating systemic lupus erythematosus.

4. The application according to claim 3, characterized in that, The drug is administered by gavage or intestinal microbiota transplantation.

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