Application of enteric microorganism composition in preparation of medicine for treating SLE (systemic lupus erythematosus)

Abstract

The invention belongs to the technical field of biological medicines, and discloses an application of an enteric microorganism composition in preparation of a medicine for treating systemic lupus erythematosus (SLE). In particular, the present invention relates to an enteric microbial composition comprising Lactobacillus murinus (Lactobacillus murinus) and / or Fecalibacterium rodentium (Fecalibacterium rodentium), and to a method for preparing the same. After the enteric microorganism combination is subjected to enteric microorganism transplantation of the lupus mice, the intestinal inflammatory response of the lupus mice can be improved, the expression level of autoantibodies and inflammatory factors of the lupus mice can be effectively reduced, the disease progress of the lupus mice can be remarkably improved, and a new thought and method are provided for treatment of the SLE disease.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to an application of an intestinal microbial combination in preparing 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, causing inflammation and damage. Basophils (Baso) are a typical innate immune cell. Although they account for less than 1% of peripheral blood mononuclear cells (PBMCs), their powerful functions are increasingly valued. A series of studies in recent years have found that the role of basophils in autoimmune diseases cannot be underestimated. Autophagy is a lysosome-dependent cell degradation pathway that maintains the balance of body metabolism and the stability of the internal environment by degrading intracellular macromolecules and damaged organelles.

[0003] Intestinal microbes refer to a group of microorganisms that live in the human intestine, including various bacteria and fungi. Intestinal microbes have important metabolic and regulatory functions, which can help the human body break down food, synthesize vitamins and enzymes, etc. In addition, intestinal microbes can also promote the absorption of nutrients and water balance, regulate energy and fat metabolism, maintain the balance of the body's internal environment, etc. Studies in recent years have also shown that there is a close connection between intestinal microbes and many human diseases, including intestinal diseases, metabolic diseases, nervous system diseases and cancer. Therefore, intestinal microbes have become a new direction for the study and treatment of many diseases. Summary of the invention

[0004] The purpose of the present invention is to overcome the shortcomings of the prior art and provide an application of an intestinal microbial 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 a first aspect, the present invention provides an intestinal microbial combination for preventing and / or treating SLE, comprising Ligilactobacillus murinus and / or Faecalibaculum rodentium.

[0007] The present invention constructs a lupus mouse model with basophil (Basophils, Baso) autophagy defect, and finds that basophil autophagy defect can 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 Lactobacillus murinus and Faecalibaculum rodentium was significantly increased in the basophil autophagy defect group, and was negatively correlated with the expression of autoantibodies and inflammatory factors. Subsequently, intestinal microbial transplantation of lupus mice Ligilactobacillus murinus and Faecalibaculum rodentium was performed. The results showed that intestinal microbial transplantation of Ligilactobacillus murinus and Faecalibaculum rodentium can improve the intestinal inflammatory response of lupus mice, and effectively reduce the expression of autoantibodies and inflammatory factors, and the intestinal microbial transplantation group of Ligilactobacillus murinus combined with Faecalibaculum rodentium has a significantly better therapeutic effect than the transplantation group of a single intestinal microorganism.

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

[0009] As a preferred embodiment of the drug of the present invention, it also includes a pharmaceutically acceptable carrier or excipient.

[0010] In a third aspect, the present invention provides a health product for preventing and / or treating SLE, comprising the intestinal microorganism combination.

[0011] As a preferred embodiment of the health care product of the present invention, it also includes a carrier or auxiliary material acceptable to food.

[0012] As a further preferred embodiment of the medicine or health product of the present invention, the effective concentration of Lactobacillus murinus and / or Faecalibaculum rodentium is 10 7 ~10 9 CFU / 200μL.

[0013] In a fourth aspect, the present invention uses the intestinal microbial combination in the preparation of a drug for preventing and / or treating SLE.

[0014] As a preferred embodiment of the application of the present invention, the drug is administered by intragastric administration or intestinal microorganism transplantation.

[0015] As a preferred embodiment of the application of the present invention, the function of the drug is at least one of the following:

[0016] Ⅰ. Improve intestinal inflammatory response;

[0017] Ⅱ. Reduce the expression of autoantibodies and inflammatory factors.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] The present invention finds that the expression of Lactobacillus murinus and Faecalibaculum rodentium is significantly increased in the basophil autophagy defect group, and is negatively correlated with the expression of autoantibodies and inflammatory factors; the intestinal microbial transplantation of Ligilactobacillus murinus and Faecalibaculum rodentium can improve the intestinal inflammatory response of lupus mice, and effectively reduce the expression of autoantibodies and inflammatory factors, and the intestinal microbial transplantation group of Ligilactobacillus murinus combined with Faecalibaculum rodentium has a significantly better therapeutic effect than the transplantation group of a single intestinal microorganism. After the intestinal microbial combination (Ligilactobacillus murinus combined with Faecalibaculum rodentium) of the present invention is transplanted with intestinal microorganisms, the disease progression of lupus mice can be significantly improved, providing a new idea and method for the treatment of SLE diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The results of plasma autoantibodies and inflammatory factors detection in lupus mice with basophil autophagy defect; in the figure, A is the detection result of anti-nuclear autoantibodies in lupus mice plasma, B is the detection result of anti-dsDNA autoantibodies in lupus mice plasma, C is the detection result of TNF-α inflammatory factor in lupus mice plasma, D is the detection result of IFN-γ inflammatory factor in lupus mice plasma, E is the detection result of IL-1β inflammatory factor in lupus mice plasma, F is the detection result of IL-2 inflammatory factor in lupus mice plasma, G is the detection result of IL-4 inflammatory factor in lupus mice plasma, H is the detection result of IL-6 inflammatory factor in lupus mice plasma, I is the detection result of IL-13 inflammatory factor in lupus mice plasma, and J is the detection result of IL-17 inflammatory factor in lupus mice plasma; P<0.05 indicates significant difference, marked with *.

[0021] Figure 2The fecal metagenomic sequencing results of lupus mice with defective basophil autophagy; in the figure, A is the evolutionary graph analysis generated by LEfSe, and B is the bar chart analysis generated by LEfSe. Red indicates intestinal microorganisms enriched in the Atg5KO group, and green indicates intestinal microorganisms enriched in the control group.

[0022] Figure 3 This is the correlation analysis between intestinal microorganisms and autoantibodies and inflammatory factors in lupus mice with basophil autophagy deficiency; in the figure, A is the RDA analysis of intestinal microorganisms and autoantibodies, B is the RDA analysis of intestinal microorganisms and inflammatory factors, the length of the arrow indicates 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 indicates the correlation, the acute angle indicates positive correlation, and the obtuse angle indicates negative correlation; C is the heat map analysis of intestinal microorganisms, autoantibodies and inflammatory factors, the R value is represented by different colors, red indicates positive correlation, blue indicates negative correlation, and the darker the color, the stronger the positive / negative correlation; P<0.05 indicates significant difference, marked with *.

[0023] Figure 4 H&E staining results of intestinal pathology in lupus mice transplanted with intestinal microorganisms.

[0024] Figure 5 These are the results of plasma autoantibody and inflammatory factor detection in lupus mice with intestinal microbiota transplantation; in the figure, A is the result of anti-nuclear autoantibody detection in lupus mouse plasma, B is the result of anti-dsDNA autoantibody detection in lupus mouse plasma, C is the result of TNF-α inflammatory factor detection in lupus mouse plasma, D is the result of IFN-γ inflammatory factor detection in lupus mouse plasma, and E is the result of IL-17 inflammatory factor detection in lupus mouse plasma; P<0.05 indicates significant difference, marked with *. DETAILED DESCRIPTION

[0025] 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 embodiments. It should be understood by those skilled in the art that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0026] Unless otherwise specified, the experimental methods used in the examples are all conventional methods; the materials, reagents, etc. used, unless otherwise specified, can be obtained from commercial channels.

[0027] Example 1: Detection of autoantibodies and inflammatory factors in basophil autophagy-deficient lupus mice

[0028] Autophagy gene Atg5 knockout mice flox / flox Cre + / - ,Atg5- / - ) and autophagy gene Atg5 non-knockout control mice (Atg5 flox / flox Cre - / - ,Atg5 + / + ), provided by Shanghai Model Organisms Technology Co., Ltd. The basophil autophagy-deficient mouse model was constructed, specifically referring to the Chinese invention patent document CN 118120702B (a method for constructing a basophil autophagy-deficient mouse and its application).

[0029] The mice were divided into the following groups:

[0030] The control group consisted of MRL / lpr mice without any intervention; Atg5 - / - Baso adoptive transfer group is MRL / lpr mice with defective basophil autophagy; Atg5 + / + The Baso adoptive transfer group consisted of control MRL / lpr mice in which the autophagy gene Atg5 of basophils was not knocked out; at the same time, MRL / MpJ mice were set as the normal control group of MRL / lpr lupus mice.

[0031] (1) Detection of anti-nuclear autoantibodies in the plasma of lupus mice

[0032] ELISA kit to detect mouse plasma anti-nuclear antibody:

[0033] The frozen plasma was rewarmed on ice, and the Mouse anti-nuclear antibody ELISA Kit (AlphaDiagnostic, USA) was opened. The sample was diluted 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, 100 μL of standard, sample and negative control were added to the predetermined wells, sealed with sealing film, and incubated at room temperature for 60 min. The liquid in the plate was removed by turning it upside down, and 200 μL of 1×Working Wash Solution was added to each well using a spray gun. The plate was gently tapped by hand to wash, and then the plate was turned upside down to remove the liquid. The 96-well plate was turned upside down on a paper towel and tapped several times to ensure that the liquid in the well was removed. The same method was repeated for washing 3 times. To the wells with standard, sample and negative control, 100 μL of Anti-Mouse Ig HRP was added to each well, and then sealed with sealing film and incubated at room temperature for 30 min. Invert the plate to remove the liquid, add 200μL 1×Working Wash Solution to each well and tap the plate gently to rinse, then invert the plate to remove the liquid, invert the 96-well plate on a paper towel and tap it vigorously several times to ensure that the liquid in the wells is removed. Repeat the same washing method 4 times. Add 100μL TMB Substrate to each well and incubate in the dark for 15 minutes. At this time, the liquid in the well will begin to turn blue. Add 100μL Stop Solution to each well and mix well by pipetting. Use an enzyme reader to detect the OD value at 450nm and the OD value at 630nm to normalize the well background. Finally, calculate the net OD value (OD 450 -OD 630 ), a standard curve was established, and the titer of anti-nuclear autoantibodies in each sample was calculated based on it.

[0034] (2) Detection of anti-dsDNA autoantibodies in the plasma of lupus mice

[0035] ELISA kit to detect mouse plasma anti-dsDNA antibody:

[0036] The frozen plasma was rewarmed on ice, and the Mouse anti-dsDNA (Alpha Diagnostic, USA) kit was opened. The sample was diluted 1:3000 with the prepared 1× Working Sample Diluent and Low NSB Sample Diluent. Using the 96-well plate in the dsDNA kit, 100 μL of the standard, sample and negative control were added to the predetermined wells, sealed with sealing film, and incubated at room temperature for 60 min. The liquid in the plate was removed by turning it upside down, and 200 μL of 1× Working Wash Solution was added to each well using a spray gun. The plate was gently tapped by hand to wash, and then the plate was turned upside down to remove the liquid. The 96-well plate was turned upside down on a paper towel and tapped several times to ensure that the liquid in the well was removed. The same method was repeated for washing 3 times. To the wells with the standard, sample and negative control, 100 μL of Anti-Mouse Ig HRP was added to each well, and then sealed with sealing film and incubated at room temperature for 30 min. Invert the plate to remove the liquid, add 200μL 1×Working Wash Solution to each well and tap the plate gently to rinse, then invert the plate to remove the liquid, invert the 96-well plate on a paper towel and tap it vigorously several times to ensure that the liquid in the wells is removed. Repeat the same washing method 4 times. Add 100μL TMB Substrate to each well and incubate in the dark for 15 minutes. At this time, the liquid in the well will begin to turn blue. Add 100μL Stop Solution to each well and mix well by pipetting. Use an enzyme reader to detect the OD value at 450nm and the OD value at 630nm to normalize the well background. Finally, calculate the net OD value (OD 450 -OD 630 ), a standard curve was established, and the titer of anti-dsDNA autoantibodies in each sample was calculated based on it.

[0037] (3) Detection of inflammatory factors in the plasma of lupus mice

[0038] Lupus mouse plasma samples were sent to Guangzhou Juyan Biotechnology Co., Ltd. The MAP liquid 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.

[0039] The experimental results are shown in Figure 1 , Atg5 - / - The Baso adoptive transfer group was significantly higher than that of 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.

[0040] Example 2: Correlation analysis between intestinal microorganisms, inflammatory factors and autoantibodies in lupus mice with basophil autophagy deficiency

[0041] (1) Gut microbial metagenomic sequencing of basophil autophagy-deficient lupus mice

[0042] The feces of the basophil autophagy-deficient lupus mice in Example 1 were sent to Wuhan Maiwei Metabolic Biotechnology Co., Ltd. for metagenomic sequencing.

[0043] (2) Correlation analysis between intestinal microbiota, plasma autoantibodies and inflammatory factors in basophil autophagy-deficient lupus mice

[0044] Metagenomic sequencing results Figure 2 Intestinal microorganisms in the basophil autophagy defective group were mainly enriched in Lactobacillus murinus and Faecalibaculum rodentium, while those in the control group (basophil autophagy activated, basophils without Atg5 knockout) were mainly enriched in Parvibactercaecicola, Bacteroides acidifaciens, etc. Correlation analysis showed that Ligilactobacillus murinus and Faecalibaculum rodentium were negatively correlated with autoantibodies and inflammatory factors.

[0045] Example 3: Disease progression detection in lupus mice with intestinal microbiota transplantation

[0046] 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.

[0047] 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.

[0048] 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 8In the L.murinus+F.rodentium-FMT group, Faecalibaculum rodentium (strain number: BNCC363015) and Ligilactobacillus murinus (strain number: BNCC194688) were purchased from Beina Chuanglian Biotechnology Co., Ltd. in Shangcheng. The two strains were mixed in equal amounts and a total of 10 8 CFU / 200μL was administered orally. All mice transplanted with intestinal flora were administered orally three times a week, and the mice were killed after four weeks to obtain samples.

[0049] (1) H&E detection of intestinal pathology in lupus mice transplanted with intestinal microbiota

[0050] Dewaxing: Place the intestinal tissue slides in xylene I for 20 min and in xylene II for 20 min; Place the slides in anhydrous ethanol I for 5 s and anhydrous ethanol II for 5 s → 95% alcohol I for 5 s and 95% alcohol II for 5 s → Wash with water for 15 s; ③ Place the slides in Subenin staining solution 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 staining solution for 2 min → Wash with water for 15 s → Anhydrous ethanol for 2 s → Bake dry, seal with neutral resin; Take pictures.

[0051] (2) Detection of plasma inflammatory factors in lupus mice transplanted with intestinal microbiota

[0052] The method for detecting the plasma samples of lupus mice transplanted with intestinal microorganisms is the same as step (3) of Example 1.

[0053] The experimental results are shown in Figure 4-5 ,exist Figure 4 In the F.rodentium-FMT group and the L.murinus-FMT group, the intestinal inflammatory cell deposition was improved to varying degrees compared with the Control group and the Antibiotic group, indicating that Faecalibaculum rodentium and Ligilactobacillus murinus have improved the progression of the disease. The L.murinus+F.rodentium-FMT group showed more significant improvement in intestinal inflammatory cell deposition compared with the F.rodentium-FMT group and the L.murinus-FMT group, indicating that the combined treatment of Faecalibaculum rodentium and Ligilactobacillus murinus is significantly better than single treatment. Figure 5Compared with the Control group and Antibiotic group, the anti-nuclear and anti-dsDNA autoantibodies and TNF-α, IFN-γ, and IL-17 inflammatory factors in the F.rodentium-FMT group and the L.murinus-FMT group were reduced to varying degrees, 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 TNF-α, IFN-γ, and IL-17 inflammatory factors in the L.murinus+F.rodentium-FMT group were more significantly improved, indicating that the combined treatment of Faecalibaculum rodentium and Ligilactobacillus murinus is significantly better than that of a single treatment.

[0054] In summary, the Antibiotic group showed improvement in intestinal pathology, autoantibodies, and inflammatory factor expression compared with the Control group, indicating that antibiotics can improve disease progression after eliminating all intestinal microorganisms. The disease progression of 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 improved disease progression. However, it is worth noting that the L.murinus+F.rodentium-FMT group had a more significant improvement in disease progression compared with the F.rodentium-FMT group and the L.murinus-FMT group, indicating that the combined treatment of Faecalibaculum rodentium and Ligilactobacillus murinus is significantly better than single treatment.

[0055] The above results show that Ligilactobacillus murinus combined with Faecalibaculum rodentium can significantly improve the disease progression of lupus mice, providing a new idea and method for the treatment of SLE.

[0056] 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. An intestinal microbial combination for preventing and / or treating SLE, characterized in that: These include Ligilactobacillus murinus and / or Faecalibaculum rodentium.

2. A drug for preventing and / or treating SLE, characterized in that: Comprising the intestinal microbial combination according to claim 1.

3. The drug according to claim 2, characterized in that It also includes pharmaceutically acceptable carriers or excipients.

4. A health product for preventing and / or treating SLE, characterized in that: Comprising the intestinal microbial combination according to claim 1.

5. The health care product according to claim 4, characterized in that: Also included are food-acceptable carriers or excipients.

6. The medicine or health product according to claim 2 or 4, characterized in that: The effective concentration of Lactobacillus murinus and / or Faecalibaculum rodentium is 10 7 ~10 9 CFU / 200μL.

7. Use of the intestinal microbial combination according to claim 1 in the preparation of a medicament for preventing and / or treating SLE.

8. The use according to claim 7, characterized in that: The drug is administered by intragastric administration or intestinal microorganism transplantation.

9. The use according to claim 7, characterized in that: The function of the drug is at least one of the following: Ⅰ. Improve intestinal inflammatory response; Ⅱ. Reduce the expression of autoantibodies and inflammatory factors.

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