CN116942707A - Application of enterobacteria in preparation of medicine or biological agent for preventing or treating nephrotic syndrome - Google Patents
Application of enterobacteria in preparation of medicine or biological agent for preventing or treating nephrotic syndrome Download PDFInfo
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- CN116942707A CN116942707A CN202210392727.3A CN202210392727A CN116942707A CN 116942707 A CN116942707 A CN 116942707A CN 202210392727 A CN202210392727 A CN 202210392727A CN 116942707 A CN116942707 A CN 116942707A
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- nephrotic syndrome
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Abstract
The invention belongs to the technical field of clinical medical treatment, and discloses application of enterobacteria in medicines or biological preparations for preventing or treating nephrotic syndrome. The invention finds that specific intestinal bacterial species are closely related to the occurrence and development of immune-mediated nephrotic syndrome, abnormal changes in the intestinal tract of these bacteria can induce the occurrence of immune-mediated nephrotic syndrome and can exacerbate the disease progression after the occurrence of nephrosis. The 21 bacteria provided by the invention have reduced relative content in intestinal tracts of 12 immune-mediated nephrotic syndrome patients and are closely related to morbidity. The relative expression of 9 other bacteria including AKK bacteria in intestinal tract of patient with immune mediated nephrotic syndrome is obviously inversely related to the clinical bad indexes of immune mediated nephrotic syndrome. The effect of treating or assisting in treating the immune-mediated nephrotic syndrome is achieved by supplementing a patient suffering from the immune-mediated nephrotic syndrome with any one or more combinations of these 22 genera.
Description
Technical Field
The invention relates to the technical field of clinical medical treatment, in particular to a combined application of a group of intestinal bacteria or independent application of the intestinal bacteria, and application of the intestinal bacteria in preparing medicines or health-care products for preventing and/or treating nephrotic syndrome. Can be used for preventing and/or treating nephrotic syndrome by oral administration/intestinal tract implantation, etc., and can be used as medicine or health product.
Background
Immune-mediated nephrotic syndrome is a special glomerular disease mediated by immunity and is the most common cause of idiopathic syndrome in non-diabetic adults worldwide. Immune-mediated nephrotic syndrome is mainly divided into two types, namely idiopathic immune-mediated nephrotic syndrome and secondary immune-mediated nephrotic syndrome, wherein idiopathic immune-mediated nephrotic syndrome is mainly kidney-restricted and accounts for about 80% of cases; secondary immune-mediated nephrotic syndrome is associated with other systemic diseases or exposures, accounting for approximately 20%. As a kidney-specific, autoimmune glomerular disease, idiopathic immune-mediated nephrotic syndrome has several central pathological features, mainly involving massive immune complex deposition of glomerular basement membrane, glomerular shrinkage and tubular interstitial injury, and thus is often accompanied by increased urinary proteins and by glomerular injury. Furthermore, the occurrence of inflammation and dysbacteriosis of the intestinal tract are also important pathological manifestations in the course of disease onset.
In recent years, the correlation between intestinal bacteria and hosts is gradually brought into the field of view of people, and the imbalance of intestinal homeostasis is closely related to the occurrence and development of various diseases, such as obesity, diabetes, cardiovascular diseases, nephropathy and the like, so that the intestinal bacteria and hosts are a current research hot spot for intestinal brain axis, intestinal mandrel axis, intestinal bone axis, intestinal kidney axis and the like. There are a number of clinical evidence that accompanies dysbacteriosis in the intestinal tract in chronic kidney disease. The theory of the intestinal and renal axes states that bi-directional communication between intestinal bacteria and chronic kidney disease affects the homeostasis of intestinal bacteria, and deregulated intestinal bacteria produce nephrotoxin metabolites that exacerbate kidney injury. Considering the great role of intestinal bacteria and their metabolites in the host, some new therapies are gradually coming into the field of view of people, for example, through probiotics and prebiotics, fecal transplantation, etc., to correct the disorder and improve the disease symptoms. Wherein fecal transplantation (fecal microbiota transplantation, FMT) is the injection of fecal microorganisms from a healthy donor into the intestinal tract of a recipient to remodel the intestinal microbial composition of the recipient and provide health benefits. FMT has been used successfully to treat recurrent clostridium difficile infection. The colonization of intestinal bacteria or the increase of specific intestinal bacteria supply provides a new therapeutic direction for disease treatment.
Disclosure of Invention
The technical problem solved by the present invention is to provide a group of applications of intestinal flora capable of improving nephrotic syndrome, including individual applications and free combination applications of these intestinal flora. The main purpose is to prevent the occurrence of nephrotic syndrome in healthy people, and for patients already suffering from nephrotic syndrome, the application or additional supplementation of the flora can play a role in improving the progression of nephrotic syndrome, including reducing urine proteins, blood urea, creatinine, low-density lipoprotein cholesterol, triglyceride and the like, and improving kidney pathological damage.
According to the invention, 16s rRNA intestinal flora sequencing and analysis are carried out on 40 clinical immune-mediated nephrotic syndrome patients and 41 healthy person controls (with matched ages and sexes) corresponding to the patients, and through a multiple linear regression equation, the down regulation of 12 bacteria is found to be obviously related to the occurrence of the immune-mediated nephrotic syndrome, so that the bacteria are possibly provided with a protective effect on the occurrence and the development of the immune-mediated nephrotic syndrome. The other 9 bacteria are significantly related to the clinical index of immune-mediated nephrotic syndrome. The feces of the patient are transplanted into the intestinal tract of the sterile mice, which proves that the feces can induce kidney injury of the mice, increase urea nitrogen in blood, generate urine protein and be accompanied with the phenomena of intestinal canal tight junction protein injury and the like. Thus, the addition of these populations to patients with immune-mediated nephrotic syndrome provides us with a means to prevent and treat immune-mediated nephrotic syndrome, and also improves prognosis. In addition, using the abundance of these gut flora in the gut, a random forest classifier can be constructed for judging the occurrence of immune-mediated nephrotic syndrome and prognosis of immune-mediated nephrotic syndrome treatment.
In order to solve the technical problems of the invention, the invention provides the following technical scheme:
the technical scheme of the invention provides application of intestinal flora in preparing a medicament or a biological preparation for preventing or treating nephrotic syndrome, which is characterized in that the intestinal flora comprises Bacteroides, faecalibacterium, agathobacter, dialister, roseburia, parabacteroides, alistipes, lachnospira, paraprevotella, butyricicoccus, barnesiella, parasutterella, sutterella, lachnoclostridium, fusobacterium, collinsella, bifidobacterium, akkermansia and flavonifractor, lactobacillus, streptococcus, phascolarctobacterium, and the intestinal flora comprises one or a combination of a plurality of intestinal flora.
Compared with healthy people, the intestinal bacteria of bacteria, agalactia, dialist, roseburia, paralacteroides, alistipes, lachnospira, paraprex, butyciclicoccus, barnesiella, parasutella, sutterella, lachnoclostricium, fusobacterium, collinella, bifidobacteria, akkermannia, flavanifactor, lactobacilli, streptococci, and Phascobacterium are significantly changed in the intestinal tract of the immune-mediated nephrotic syndrome patient, and participate in the occurrence and disease progression of the immune-mediated nephrotic syndrome.
The reduction of the intestinal bacteria Faecaliberium, agathobacterium, dialister, roseburia, parabacterides, alistipes, lachnospira, parapreverella, butyricicoccus, barnesiella, sutterella, bacteroides can significantly increase the incidence of immune-mediated nephrotic syndrome.
The intestinal bacteria Collinella and Bifidobacterium are inversely related to diastolic pressure (DBP) and to systolic pressure (SBP), suggesting that the reduction of these two intestinal bacteria may lead to an increase in blood pressure in patients with immune-mediated kidney disease syndrome.
The relative abundance of the enterobacteria Fusobacterium is inversely related to the low density lipoprotein cholesterol, while the reduction of the enterobacteria in the intestinal tract is positively related to the abnormal lipid metabolism of the patients suffering from the immune-mediated nephrotic syndrome by the enterobacteria Parabacterial, akkermansia, flavofacitor, bacterioides, alistines, roseburia, lactobacillus and streptococci.
The relative abundance of enterobacteria paralytic and Akkermansia in the gut is inversely related to IgG deposition on glomeruli in kidney tissue biopsies; phascoloarcobacterium is inversely related to the concentration of IgA in the peripheral blood of the patient; the reduction of these intestinal bacteria in the intestinal tract is associated with immune status in patients with immune-mediated nephrotic syndrome, i.e. the reduction of these bacteria may lead to an over-immune activation of the body.
The reduction of Lactobacillus in the intestinal tract of patients with nephrotic syndrome has the relative abundance inversely related to the number of leukocytes and neutrophils in peripheral blood, which suggests that the reduction of the relative abundance of Lactobacillus in the intestinal tract may increase the number of neutrophils and leukocytes in peripheral blood of patients with the marked correlation of the intestinal Lactobacillus with the basic immune status of patients with immune-mediated nephrotic syndrome.
The occurrence of the nephrotic syndrome of the mice can be induced by transplanting the fecal bacteria of the patient with the immune-mediated nephrotic syndrome into the sterile mice, and the kidney injury and the kidney inflammation state are aggravated; the human faecal bacteria comprise the following 21 bacteria: bacteria, agalactia, dialister, roseburia, parabacterides, alistipes, lachnospira, parapreviella, butyriciococcus, barnesiella, parasutterella, sutterella, lachnoclavidium, fusobacterium, collinella, bifidobacterium, akkermannia, flavonifacter, lactobacilli, streptococcus.
The oral administration of Lactobacillus curvatus, bifidobacterium and Acremonium (AKK bacteria) significantly improves the urine protein of the doxorubicin-induced nephrotic syndrome by inducing the formation of a model of the nephrotic syndrome in rats by the tail vein injection of doxorubicin.
The biological agent comprises a live bacteria preparation, an inactivated bacteria preparation and a live bacteria culture extract preparation.
The nephrotic syndrome comprises nephrotic syndrome caused by immunity, nephrotic syndrome caused by nephrotoxic drugs and IgA nephritis nephrotic syndrome.
The immune-mediated nephrotic syndrome refers to the clinical diagnosis of the immune-mediated nephrotic syndrome, and the kidney pathology detection of a patient shows that IgG or IgA or complement C3 immune complex is deposited in glomerulus; a group of syndromes with clinical characteristics of massive proteinuria, hypoproteinemia, edema and hyperlipidemia, and nephrotic syndromes caused by various autoimmune diseases.
Detailed description of the invention:
in a first aspect, the invention provides a population associated with the onset of immune-mediated nephrotic syndrome comprising the following 12 intestinal microorganisms, which are significantly reduced in both faeces and intestinal tract of an immune-mediated nephrotic syndrome patient compared to the normal population: faecalibacterium, agathobacterium, dialister, roseburia, parabacteroides, alistipes, lachnospira, paraprevitetella, butyricicoccus, barnesiella, sutterella, bactroides. In the present invention, the immune-mediated nephrotic syndrome refers to a patient diagnosed with the immune-mediated nephrotic syndrome by clinical pathological and biochemical indexes.
In a second aspect, the invention provides intestinal flora, including Lachnoclostrichum and Fusobacteria, that is significantly associated with reduced renal function. In the present invention, the decrease in renal function means a significant increase in urea nitrogen in blood, creatinine in blood, albumin in urine, and the like.
In a third aspect, the invention provides an intestinal flora associated with blood pressure in a patient suffering from immune-mediated kidney disease syndrome, comprising Collinella and Bifidobacterium. Wherein the blood pressure comprises the clinical common indicators systolic pressure (SBP) and diastolic pressure (DBP).
In a fourth aspect, the invention provides an intestinal flora associated with dyslipidemia in a patient suffering from immune-mediated kidney disease syndrome, comprising Parabacterides, akkermansia, flavofactor, bactroides, alistines, roseburia, lactobacillus, and streptococcus. The dyslipidemia referred to in the present invention refers to the clinical indication of elevated triglyceride and low density lipoprotein cholesterol and reduced high density lipoprotein in the peripheral blood of the patient.
In a fifth aspect, the invention provides intestinal flora associated with immune overactivation in patients with immune-mediated kidney disease syndrome, including Parabacterides, lactobacillus and Akkermansia, wherein immune overactivation in the invention is a significant increase in IgA content, neutrophil count and total number of leukocytes in peripheral blood of patients as demonstrated by kidney tissue biopsy.
In a sixth aspect, the invention provides any one of the following uses of the intestinal flora associated with the onset and progression of immune-mediated nephrotic syndrome, the intestinal flora associated with reduced renal function, the intestinal flora associated with abnormally elevated blood pressure, the intestinal flora associated with dyslipidemia, the intestinal flora associated with excessive activation of the immune system:
1) Biomarkers for the diagnosis and prognosis of immune-mediated nephrotic syndrome;
2) Is used for evaluating kidney function, dyslipidemia and immune overactivation of patients with immune mediated nephrotic syndrome, predicting abnormal blood pressure and evaluating prognosis;
3) Preparation of a reagent, kit or system for preventing and predicting the occurrence and progression of immune mediated nephrotic syndrome;
4)
in a seventh aspect, the present invention provides a biological agent focus application of the intestinal flora related to the onset and progression of the immune-mediated nephrotic syndrome, the intestinal flora related to reduced renal function, the intestinal flora related to abnormally high blood pressure, the intestinal flora related to dyslipidemia, the intestinal flora related to excessive activation of the immune system for preventing or treating the immune-mediated nephrotic syndrome;
preferably, the biological agents include, but are not limited to, live bacteria agents, inactivated bacteria agents, live bacteria culture extract agents, and the like.
In an eighth aspect, the invention provides primers (e.g. 16srDNA detection primers) and/or probes for detecting the intestinal flora associated with the onset and progression of the immune-mediated nephrotic syndrome, the intestinal flora associated with reduced renal function, the intestinal flora associated with abnormally high blood pressure, the intestinal flora associated with dyslipidemia, the intestinal flora associated with excessive activation of the immune system.
The beneficial effects of the invention are as follows:
1) The invention discovers intestinal flora closely related to the onset and progress of immune-mediated nephrotic syndrome for the first time, and the effect is related to the participation of the flora in intestinal mucosa protection and uremic toxin substance metabolism;
2) The invention discovers the specific intestinal flora related to dyslipidemia, abnormal blood pressure, overimmune activation and reduced renal function of patients with immune-mediated nephrotic syndrome for the first time, and the test of the aseptic mice transplanted by the fecal bacteria proves the prediction results found clinically.
3) The invention provides a biomarker for discovering and prognosis judging of patients suffering from immune-mediated nephrotic syndrome through an intestinal flora marker. The sample collection is convenient and quick, the organism injury can not be caused, and the basis is provided for clinically judging the disease progress.
4) The present invention provides a biological method that can be used to prevent and treat immune-mediated nephrotic syndrome by increasing the enteral feeding of specific flora.
Drawings
FIG. 1 case selection chart
FIG. 2 shows the relative abundance of intestinal flora in patients with model kidney disease and healthy control population obtained by generalized linear model analysis.
FIG. 3 Szechwan rank correlation (Spearman's rank correlation) analysis found that the intestinal flora correlated with clinical signs, mainly those of particular genus that were negatively correlated with the signs of immune-mediated nephrotic syndrome
FIG. 4A flow chart of the operation of transplanting faecal bacteria
FIG. 5 shows a microbial viability assay
FIG. 6. Fecal transplantation affects renal Biochemical index
FIG. 7. Fecal transplantation affects local inflammation of the kidneys
FIG. 8. Fecal transplantation affects systemic inflammation
FIG. 9. Faecal fungus transplantation affects colon and kidney pathology
FIG. 10 oral administration of Lactobacillus acidophilus, bifidobacterium and Acremonium (AKK) significantly improved the renal function of doxorubicin-induced nephrotic syndrome in rats
Detailed Description
The object of the present invention is to provide a method for the prevention, treatment or adjuvant treatment of immune-mediated nephrotic syndrome by supplementing and increasing specific intestinal flora. Meanwhile, the relative abundance of the bacteria in the intestinal tract of the human body also provides a group of effective markers for diagnosis and prognosis judgment of the immune-mediated nephrotic syndrome. The invention will be further illustrated with reference to specific examples. The following examples are illustrative only and are not to be construed as limiting the invention.
Example 1 screening of intestinal flora associated with immune-mediated nephrotic syndrome
1. Sample collection
All clinical studies were conducted with subjects informed and voluntary, and the study protocol was approved by the ethics committee of the university of medical science, beijing, co. From 5 months 2019 to 10 months 2019, 81 subjects from beijing co-hospital and beijing university third hospital were co-enrolled, including 40 immune-mediated nephrotic syndrome patients and 41 corresponding healthy controls. All IMN patients meeting the conditions of the study were accurately diagnosed as meeting the criteria of proteinuria, hyperlipidemia, edema, hypoalbuminemia, immunofluorescence detection of primary immunoglobulin 4 (IgG 4) deposition in glomeruli and platelet-activating protein 2R1 autoantibodies (PLA 2R 1) positivity. Patient exclusion criteria were as follows (1) patients with complex disease, acute and chronic infections; (2) patients with chronic inflammatory bowel disease and celiac disease; (3) Patients who received antibiotics, immunosuppressants and functional foods (probiotics) for three months. The healthy control has the characteristics that (1) the healthy human has normal kidney function and no kidney disease, celiac disease and other complications; (2) Patients who never received antibiotics, immunosuppressants, and functional foods (probiotics) within three months. All participants were between 18-70 years of age. The two groups are matched in age and gender. The case selection flow chart and baseline levels of biochemical indicators of the patient are shown in fig. 1 and table 1. All participants' fresh faeces were immediately placed in sterile test tubes and stored at-80 ℃.
Table 1. Basic clinical indicators of immunocompromised nephrotic syndrome patients and normal control populations.
2.16S rRNA sequencing
Sequencing of sample 16S rRNA was performed by Beijing Nodezafion Bioinformation technologies Inc., the following steps:
2.1 extraction of genomic DNA
The genomic DNA of the sample was extracted by CTAB or SDS methods, and the procedures were carried out according to instructions.
1) Sample: 1000ul of CTAB lysate was aspirated into a 2.0ml EP tube, 20ul of lysozyme was added, the appropriate amount of sample was added to the lysate, and the mixture was stirred in a 65℃water bath (for fecal samples, water bath time was 2 hours) and inverted and mixed several times during this time to allow the sample to be thoroughly lysed.
2) 950ul of the supernatant was centrifuged and phenol (pH 8.0) was added in an equal volume to the supernatant: chloroform: isoamyl alcohol (25:24:1), mixed upside down, and centrifuged at 12000rpm for 10min.
3) Taking the supernatant, adding an equal volume of chloroform: isoamyl alcohol (24:1), the mixture was inverted and centrifuged at 12000rpm for 10min.
4) The supernatant was pipetted into a 1.5mL centrifuge tube, 3/4 of the supernatant volume of isopropanol was added, and the mixture was shaken up and down and precipitated at-20 ℃.
5) Centrifuge at 12000rpm for 10 minutes, pour out the liquid, take care not to pour out the pellet. The remaining small amount of liquid can be collected again by centrifugation by washing 2 times with 1ml of 75% ethanol and then sucked out with a gun head.
6) The ultra clean bench is blow dried or air dried at room temperature (the DNA sample is not too dry or otherwise difficult to dissolve).
7) mu.L of ddH2O was added to dissolve the DNA sample and incubated at 55-60℃for 10min to aid solubilization, if necessary.
8) RNase A1 ul was added to digest RNA and left at 37℃for 15min.
2.2 determination of purity and concentration of DNA samples
The purity and concentration of the DNA were checked by agarose gel electrophoresis, an appropriate amount of sample DNA was taken in a centrifuge tube, and the sample was diluted to 1 ng/. Mu.l with sterile water.
Detecting parameters: genomic DNA-gel concentration: 1%; voltage: 100v; electrophoresis time: for 40min; sample loading amount: 5 mu L
2.3PCR amplification
The diluted genomic DNA was used as a template, and specific primers with Barcode were used according to the selection of the sequencing region, new England Biolabs companyHigh-Fidelity PCR Master Mix with GC Buffer and High-efficiency High-fidelity enzyme to perform PCR, thereby ensuring the amplification efficiency and accuracy.
2.3.1 primer design
Primer corresponding region: 16S V3-V4 region primer (341F and 806R): identifying bacterial diversity;
according to the designated sequencing region, a specific primer with barcode is synthesized, and the primer sequence is as follows:
341F：5'-CCTAYGGGRBGCASCAG-3'；
806R:5’-GGACTACNNGGGTATCTAAT-3’
2.3.2PCR amplification
1) The reaction system was prepared as shown in Table 2.
TABLE 2PCR reaction System
2) Amplification of
PCR amplification was performed using a model Applied Biosystems 2720 PCR apparatus, the amplification procedure being: 95℃for 5min, (94℃for 1min,57℃for 45s,72℃for 1 min). Times.35 cycles, 72℃for 10min,16℃for 5min.
2.4 mixing and purification of PCR products
The PCR products were electrophoretically detected using 2% concentration agarose gel; equal amount of mixing is carried out according to the concentration of the PCR product, after the mixture is fully and evenly mixed, 2% agarose gel electrophoresis is used for detecting the PCR product, and a gel recovery kit provided by Qiagen company is used for recovering the target strip.
Detecting parameters: PCR product-gel concentration: 2%; voltage: 80v; electrophoresis time: for 40min; sample loading amount: 3 mu L
2.5 library construction and on-machine sequencing
UsingThe DNA PCR-Free Sample Preparation Kit library construction kit is used for constructing a library, the constructed library is quantified by Qubit and Q-PCR, and after the library is qualified, novaSeq6000 is used for sequencing on the machine.
3. Data analysis
3.1 Sequencing data processing
Splitting each sample data from the next machine data according to the Barcode sequence and the PCR amplification primer sequence, cutting off the Barcode and the primer sequence, and then splicing reads of each sample by using FLASH (V1.2.7, http:// ccb.jhu.edu/software/FLASH /), wherein the obtained spliced sequence is the original Tags data (Raw Tags); the Raw Tags obtained by splicing need to be subjected to strict filtering treatment to obtain high-quality Tags data (Clean Tags), and refer to the Tags quality control flow of Qiame (V1.9.1http:// qiime.org/scripts/split_lists_fastq.html). The Tags obtained after the above processing need to be subjected to processing of removing the chimeric sequences, the Tags sequences are compared with a species annotation database through (https:// github. Com/torognes/vsearch /), the chimeric sequences are detected, and finally the chimeric sequences are removed, so that final Effective data (Effective Tags) are obtained.
3.2 OTU clustering and species annotation
All Effective Tags of all samples are clustered by using Upsse software (Upsse v7.0.1001, http:// www.drive5.com/Uparse /), sequences are clustered into OTUs (Operational Taxonomic Units) by default with 97% consistency (Identity), meanwhile, a representative sequence of OTUs is selected, and a sequence with the highest occurrence frequency in the OTUs is selected as the representative sequence of the OTUs according to the algorithm principle. Species annotation was performed on OTUs sequences, species annotation analysis (threshold was set to 0.8-1) was performed with the Mothur method with the ssarrna database of SILVA132 (http:// www.arb-SILVA. De /), taxonomic information was obtained and at each level of classification: the community composition of each sample was counted by kingdom, phylum, class, order, family, genus, species. Quick multiple sequence alignment was performed using MUSCLE (Version 3.8.31, http:// www.drive5.com/MUSCLE /) software to obtain the phylogenetic relationship of all OTUs representing sequences. And finally, carrying out homogenization treatment on each sample data.
4 data analysis
4.1. Generalized Linear Model (GLM) analysis is performed by GLM software analysis packages in the R language (https:// www.r-project. Org /).
4.2.Spearman rank relevance analysis is performed by means of a relevant software analysis package in the R language (https:// www.r-project. Org /).
5 results
5.1. After control of possible confounding factors (such as age, sex and BMI), GLMs were used to identify the differential genus between immune-mediated nephrotic syndrome and healthy control group, with negative correlation coefficients representing negative correlation with immune-mediated nephrotic syndrome as protective species. The specific genera are listed in Table 3, and the abundance of each genus between the two groups is shown in FIG. 2.
TABLE 3 analysis of generalized Linear model (generalized linear model) to obtain intestinal flora bacteria with significant negative correlation to the onset of immune-mediated nephrotic syndrome
5.2. Through correlation analysis of the relative abundance of each genus in the intestinal tract of the patient with immune-mediated nephrotic syndrome and clinical indexes, the relative abundance of Lachnoclostrichum is found to be in negative correlation with urine protein of the patient with immune-mediated nephrotic syndrome, and Fusobacterium is found to be in significant negative correlation with urea nitrogen in blood. Collinella is inversely related to diastolic pressure (SBP), collinella is inversely related to Bifidobacterium and systolic pressure DBP. Fusobacteria are inversely related to LDL-C. And HDL-C, parabacterides, akkermansia, flavofactors, bactoides, alistipes, roseburia, lactobacillus and Streptococcus are positively correlated with each other for the protection of human body. The relative abundance of the two species of bacteria is inversely related to the deposition of IgG on glomeruli in kidney tissue biopsies by parkacteroides and Akkermansia. Phascoloarcobacterium is inversely related to the concentration of IgA in the peripheral blood of a patient. The relative abundance of Lactobacillus is inversely related to the content of white blood cells WBC and neutrophils in the peripheral blood. The specific results are shown in FIG. 3.
EXAMPLE 2 patient sample faecal fungus transplantation
1. Fecal microorganism viability detection
Using Invitrogen TM LIVE/DEAD TM BacLight TM Bacterial Viability Kit the microbial activity in the feces was examined.
(1) Patient faeces of a certain mass were weighed, ground in a volume of sterile physiological saline according to a ratio of 1:10 using a 40 μm sieve, centrifuged for 3min at 800g of grinding fluid, the dregs removed and the supernatant transferred to a fresh 1.5ml EP tube.
(2) The bacterial culture was concentrated by centrifugation at 10000 Xg and allowed to stand for 10-15min.
(3) The supernatant was removed and resuspended in 2ml of 0.85% NaCl buffer.
(4) To each of two 50ml centrifuge tubes, 1ml of this suspension was added, and the centrifuge tubes contained 20ml of 0.85% NaCl buffer (live bacteria control tube) or 20ml of 70% isopropanol (dead bacteria control tube).
(5) Incubate for 1 hour at room temperature, mix every 15 minutes.
(6) Centrifuge at 10000 Xg for 10-15min.
(7) Resuspended in 20ml of 0.85% NaCl or appropriate buffer and centrifuged as in step 1.6.
(8) The suspension was resuspended in 10ml of 0.85% NaCl or appropriate buffer, respectively, in different tubes.
(9) The absorbance (OD 670) of 3 ml of the bacterial suspension at 670nm was determined.
(10) The concentration of the bacterial liquid (living bacteria and inactivated bacteria) is regulated to be 1 multiplied by 10 8 bacteria/mL (OD 670 about 0.03) then diluted 1:100 in double distilled water to a final 1X 10 6 bacteria/mL.
(11) 5 bacterial liquids were prepared in different proportions in a 1.5ml EP tube according to Table 4. The volumes of the 5 sample tubes were all 500. Mu.l.
TABLE 4 bacterial liquid ratio map
(12) To 5 sample tubes (500. Mu.l) were added 1.5. Mu.l of each mixed dye, and to single-stained tubes (1 ml) were added 0.3. Mu.l of each dye, and each tube was pipetted up and down several times and thoroughly mixed.
(13) Incubate for 15min at room temperature in the dark.
(14) And (5) detecting on-line in a streaming mode.
2 preparation of sterile mice
Antibiotic interference was used to simulate a sterile environment. 50mg/kg of vancomycin, 100mg/kg of metronidazole, 50mg/kg of cefoxitin, 50mg/kg of gentamicin and 100mg/kg of neomycin are prepared. Each mouse was dosed at 200ul, 2 times a day for 7 days. After the end of the antibiotic administration for 24 hours, the patient was subjected to a fecal transplantation experiment.
3 faecal fungus transplantation
(1) Recipient mouse origin
18-20g of C57BL/6 male mice were taken as recipient mice and randomly divided into 5 groups.
Group1, complete control (without any treatment) (n=10);
group2 antibiotic + sterile saline (n=10);
group3 antibiotic + faecal bacteria transplantation healthy Group faeces (n=10);
group4 antibiotic + faecal bacteria transplantation immune mediated kidney disease syndrome patient faeces (n=10);
before the beginning of the transplantation, the recipient mice and the transplanted material were examined for the content of microorganisms in the feces.
(2) Faecal fungus transplanting
Dissolving the fecal transplantation material in sterile physiological saline at a concentration of 100mg/ml, grinding to homogenize the fecal transplantation material, filtering with a 40 μm sieve, removing small particles, centrifuging for 3min at 800g, collecting supernatant, and adding sodium bicarbonate with a final concentration of 1.2% into the fecal suspension. 200 μl of the extract was taken orally by stomach irrigation. And 6 times of total transplantation. And collecting a related biological sample, and detecting a related index. The detailed fecal fungus transplanting flow is shown in figure 4.
EXAMPLE 3 Effect of oral administration of Lactobacillus curvatus, bifidobacterium and Acremonium (AKK) on renal function in Adriamycin-induced nephrotic syndrome rats construction and treatment of Adriamycin-induced nephrotic syndrome rat models
(1) Rat source and grouping
180-200g of SD rats were taken, of which 22 received a single tail vein doxorubicin injection (8 mg/kg, dissolved in sterilized normal saline) to induce doxorubicin nephritis, and of which 8 tail veins were injected with the same dose of normal saline as a normal control group. After 1 week, all rats were harvested and the ratio of urine protein to urine creatinine was measured as a quantitative indicator of urine protein. Of these, 22 rats received doxorubicin tail vein, which had urine protein values 10 times higher than the normal control group, were considered successful in molding, and were admitted to the next step of group administration. The rats successfully modeled were divided into 2 groups, one group being a model control group, were treated with water of the same volume as the stomach (n=8), the second group (n=8), and were given with lactobacillus, bifidobacterium, and AKK for 4 weeks immediately after grouping.
(2) Gastric lavage live bacteria dosage
Bifidobacterium longum bifidobacterium, viable bacteria 10 8 CFU/day, after dilution with 2mL distilled water, the stomach was irrigated for 4 weeks.
Lactobacillus acidophilus Lactobacillus acidophilus, live bacteria, 10 4 CFU/day, after dilution with 2mL distilled water, the stomach was irrigated for 4 weeks.
Akkermansia muciniphila (Acremonium AKK) live bacteria 10 4 CFU/day, after dilution with 2mL distilled water, the stomach was irrigated for 4 weeks.
After 4 weeks of gastric lavage, each group of mice was collected for 24 hours and tested for urine protein levels and urea nitrogen and creatinine in the blood.
Results 4 results
Before fecal bacteria is transplanted, the microbial activity in fecal samples is detected in order to ensure the efficiency of the transplantation. Using Invitrogen TM LIVE/DEAD TM BacLight TM Bacterial Viability Kit microbial viability in feces was detected by flow cytometry (FIG. 5A) and analyzed using FITC-A, percp-Cy5.5-A channels. The results show that the activity of the two groups of microorganisms is about 90%, and the subsequent transplantation experiments can be performed. Feces were collected, feces suspension was prepared, LB plate test was performed, culture was performed for 48 hours, and colony formation was observed. The results prove that the effect of antibiotic intervention and the effect of fecal transplantation are both possible. (FIG. 5B).
To observe the effect of fecal transplantation on kidney function and lipid metabolism in mice, blood and urine of mice were collected after the completion of fecal transplantation for the detection of relevant biochemical indicators (fig. 6). For both TG and CHO indices in blood, there was found to be a trend of increasing in the FMT-IMN group compared to the FMT-Healthy group, but no significant difference. For LDL in blood, a significant increase in LDL in blood was found in the FMT-IMN group (P < 0.05) compared to the FMT-Healthy group. Blood creatinine, blood urea nitrogen, was used to reflect the ability of the kidneys to excrete metabolic waste, and it was found that blood urea nitrogen levels were significantly elevated (P < 0.01) in the FMT-IMN group compared to the FMT-healthcare group. Endotoxin lipopolysaccharide LPS is the main component of the cell wall of gram negative bacteria, and intestinal mucosa is damaged to make LPS enter blood from colon tissue to cause systemic inflammation. The blood LPS levels were measured using ELISA and found to be significantly elevated in the FMT-IMN group compared to the FMT-Healthy group (P < 0.001). Alb/Cre in urine samples is a gold standard reflecting renal function, and the results indicate that the FMT-IMN group urine protein is significantly elevated (P <0.01.Vs. FMT-health group). Neutrophil gelatinase-associated lipocalin NGAL and NAG (N-acetyl- β -D-glucosidase) are markers reflecting kidney inflammation and kidney injury, respectively, and the results show elevated NGAL, NAG levels in urine from FMT-IMN group, with significant differences in NAG (P <0.05, vs. In addition, we also record the change of the weight of mice in the transplanting process, and can find that compared with normal mice, the weight of the mice is reduced by the administration of antibiotics, the weight of the mice is gradually increased after the fecal bacteria is transplanted, and the weights of the FMT-health group and the FMT-IMN group mice are higher than those of the FMT-0.9% group, so that the intestinal flora of the mice can be prompted to have the functions of increasing the nutrition of organisms and providing energy.
To see if fecal transplantation can cause a local inflammatory phenotype in the kidney, kidney tissue inflammatory factor expression will be detected from mRNA levels (fig. 7). In earlier studies, we performed transcriptome sequencing of the kidney of mice after fecal transplantation. By enriching the differential genes of the FMT-Healthy group and the FMT-IMN group mice, the differential genes were found to be mainly enriched in the following pathways: cytokine receptor interaction pathways, TNF signaling pathways, chemokine signaling pathways, and NOD-like receptor signaling pathways are all associated with inflammation and bacterial invasion. In particular, cytokine receptor interaction pathways are mainly enriched for the following genes: ccl12, cxcl2, ccl7, ccl5, IL9r, ccl20; the TNF signaling pathway is mainly enriched in Gm5431, ccl12, cxcl2, ccl20, ccl5; the chemokine signal pathway is mainly enriched in Ccl12, cxcl2, ccl7, adcy2, ccl5, ccl20; the NOD-like receptor signaling pathway is mainly enriched in Mefv, cxcl2, ccl12, ccl5. The expression quantity of the genes in each group is analyzed, so that the expression quantity of each inflammatory factor of the kidney of the FMT-IMN group mice is obviously higher than that of the FMT-health group mice, and the expression quantity of each inflammatory factor of the kidney of the FMT-IMN group mice has obvious difference.
To verify the effect of fecal transplantation on systemic inflammation, the amount of inflammatory factors in serum of fecal transplanted mice was tested using a multi-factor test kit, and a total of 13 inflammatory factors in serum were tested, including CXCL1, tgfβ, IL18, IL23, CCL22, IL10, IL12p70, IL6, tnfα, GCSF, CCL17, IL12p40, IL1 β. The concentration of 8 inflammatory factors was too low to be detected, and the content of the remaining 5 inflammatory factors among groups is shown in FIG. 8. The results showed that the serum inflammatory factor content of the FMT-IMN group mice all tended to increase, but there was no significant difference compared to the FMT-Healthy group mice.
To further observe the effect of fecal transplantation on the gut, kidney, we used immunohistochemistry and HE staining to observe histopathological conditions. The tissue structure of the intestinal tract and the kidney is observed through HE staining, and the expression of inflammatory factors and inflammatory cell infiltration of the intestinal tract and the kidney are observed through immunohistochemistry. The results showed that compared with FMT-Healthy group, FMT-IMN group mice had increased kidney macrophage infiltration, and simultaneously increased expression of IL17 and NGAL, and by HE staining, it was found that FMT-IMN group mice had increased kidney glomeruli and increased pathological damage to the tubular vessels (FIG. 9). Also in colon tissue, inflammatory cell infiltration was increased in FMT-IMN group mice and inflammatory cytokine expression was increased. By HE staining, it was found that the intestinal tissue structural integrity of the FMT-IMN group mice was disrupted compared to the FMT-healthcare group mice (fig. 9).
To verify the effect of the flora on nephrotic syndrome urine protein, we used doxorubicin induction to establish a nephrotic syndrome mouse model, and after urine protein appears in the mice, the transplanted intestinal flora observed the effect on urine protein and urea nitrogen and creatinine in the blood. The results indicate that the intestinal flora reduced the urine protein level caused by nephrotic syndrome and reduced the urea nitrogen and creatinine levels in the blood compared to the complete control group (fig. 10).
From the above, it can be seen that specific genera associated with the onset of immune-mediated nephrotic syndrome are found in the examples, and that some of them are significantly associated with important features of the disease. Implantation of the patient's faeces into the intestinal tract of a sterile mouse can significantly exacerbate the dysbacteriosis of the mouse and lead to an increased degree of systemic inflammation in the mouse with concomitant impairment of renal function. Therefore, the dysregulated flora can indeed cause or aggravate kidney injury, so that the flora is taken as a target or a treatment means to increase the colonization or supplementation of the kidney beneficial bacteria, and the dysregulated flora can be used as a brand-new means for preventing, treating or assisting in treating the immune-mediated nephrotic syndrome.
Appendix: chinese and English reference table for strain names
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Claims (13)
1. Use of a population of intestinal bacteria comprising Bacteroides, faecalibacterium, agathobacter, dialister, roseburia, parabacteroides, alistipes, lachnospira, paraprevotella, butyricicoccus, barnesiella, parasutterella, sutterella, lachnoclostridium, fusobacterium, collinsella, bifidobacterium, akkermansia, flavonifractor, lactobacillus, streptococcus, phascolarctobacterium, wherein the population of intestinal bacteria comprises a combination of one or more of the above-mentioned intestinal bacteria, for the preparation of a medicament or a biological agent for the prevention or treatment of nephrotic syndrome.
2. The application of a kind of intestinal flora in preparing a biomarker or a kit for diagnosing and prognosis evaluating immune-mediated nephrotic syndrome is characterized in that the intestinal flora comprises Bacteroides, faecalibacterium, agathobacter, dialister, roseburia, parabacteroides, alistipes, lachnospira, paraprevotella, butyricicoccus, barnesiella, parasutterella, sutterella, lachnoclostridium, fusobacterium, collinsella, bifidobacterium, akkermansia and flavonifractor, lactobacillus, streptococcus, phascolarctobacterium and the intestinal flora comprises one or a combination of more than one intestinal flora.
3. Use according to any one of claims 1-2, characterized in that the intestinal bacteria, agalactia, dialist, roseburia, paralacteoides, alitiges, lachnospira, paraprefotella, butiricus, barnesiella, paraseterella, sutterella, lachnocrostrichum, fusobacteria, collisella, bifidobacteria, akkersia, flavovibacter, lactococcus, phasciences are involved in the occurrence and progression of immune-mediated nephrotic syndrome in the intestinal tract of a patient compared to a healthy person.
4. Use according to any one of claims 1-2, characterized in that the reduction of the enterobacteria faecaliberium, agatholacter, dialister, roseburia, paralacteroides, alistipes, lachnospira, paraprefotella, buticicoccus, barnesiella, sutterella, bacteriodes significantly increases the incidence of immune-mediated nephrotic syndrome.
5. Use according to any one of claims 1-2, characterized in that the enterobacteria collisella are inversely related to diastolic pressure (DBP), collisella are inversely related to bifidobacteria and systolic pressure (SBP), suggesting that a reduction of both enterobacteria is likely to lead to an increase in blood pressure in patients with immune-mediated nephrotic syndrome.
6. Use according to any one of claims 1-2, characterized in that the relative abundance of the intestinal bacteria Fusobacterium is inversely related to low density lipoprotein cholesterol, whereas the reduction of the intestinal bacteria in the intestine is positively related to lipid metabolism abnormalities in patients with immune-mediated renal syndrome, such as the intestinal bacteria paralytic, akkermansia, flavofactor, bacterioides, alistipes, roseburia, lactobacillus and streptococci.
7. Use according to any one of claims 1-2, characterized in that the relative abundance of enterobacteria paralytic and Akkermansia in the gut is inversely related to the deposition of IgG on glomeruli in a kidney tissue biopsy; phascoloarcobacterium is inversely related to the concentration of IgA in the peripheral blood of the patient; the reduction of these intestinal bacteria in the intestinal tract is associated with immune status in patients with immune-mediated nephrotic syndrome, i.e. the reduction of these bacteria may lead to an over-immune activation of the body.
8. Use according to any one of claims 1-2, characterized in that the reduction of the intestinal Lactobacillus in the intestine of a patient suffering from nephrotic syndrome has a relative abundance inversely related to the number of leukocytes and neutrophils in the peripheral blood, suggesting that the intestinal Lactobacillus is significantly related to the basic immune status of the patient suffering from immune-mediated nephrotic syndrome, and that the reduction of the relative abundance of the intestinal Lactobacillus may increase the number of neutrophils and leukocytes in the peripheral blood of the patient.
9. Use according to any one of claims 1-2, characterized in that the occurrence of mouse nephrotic syndrome is induced by implantation of faecal bacteria in immunocompromised nephrotic syndrome patients into sterile mice, exacerbating kidney injury and kidney inflammatory conditions; the human faecal bacteria comprise the following 21 bacteria: bacteria, agalactia, dialister, roseburia, parabacterides, alistipes, lachnospira, parapreviella, butyriciococcus, barnesiella, parasutterella, sutterella, lachnoclavidium, fusobacterium, collinella, bifidobacterium, akkermannia, flavonifacter, lactobacilli, streptococcus.
10. Use according to any one of claims 1-2, characterized in that the formation of a model of the renal syndrome in rats induced by the tail vein injection of doxorubicin, oral administration of lactobacillus curvatus, bifidobacteria and AKK bacteria significantly improves the urinary protein of the nephrotic syndrome induced by doxorubicin.
11. The use according to claim 1, characterized in that the biological agent comprises a live bacterial agent, an inactivated bacterial agent, a live bacterial culture extract agent.
12. The use according to any one of claims 1-2, characterized in that the nephrotic syndrome comprises immune-induced nephrotic syndrome, nephrotoxic drug-induced nephrotic syndrome, igA nephritis nephrotic syndrome.
13. Use according to claim 12, characterized in that said immune-mediated nephrotic syndrome is a clinical diagnosis of immune-mediated nephrotic syndrome, the occurrence of IgG or IgA or complement C3 immune complex deposition in glomeruli in the patient's renal pathology test; a group of syndromes with clinical characteristics of massive proteinuria, hypoproteinemia, edema and hyperlipidemia, including nephrotic syndromes caused by various autoimmune diseases, appear clinically.
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| CN117949648B (en) * | 2024-03-26 | 2024-07-12 | 中国医学科学院北京协和医院 | Marker for detecting ulcerative colitis and application thereof |
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