CN116726028B

CN116726028B - Use of fucose in IgA nephropathy treatment

Info

Publication number: CN116726028B
Application number: CN202310250111.7A
Authority: CN
Inventors: 李亚峰; 卿剑波; 李昌群; 宋文柱; 户学利; 支文强; 韩慧敏; 秦智琦; 龚豪
Original assignee: Beijing Haodingrui Biotechnology Co ltd
Current assignee: Beijing Haodingrui Biotechnology Co ltd
Priority date: 2022-04-26
Filing date: 2023-03-13
Publication date: 2023-12-19
Anticipated expiration: 2043-03-13
Also published as: CN116726028A

Abstract

The application relates to the technical field of disease treatment, in particular to application of fucose in IgA nephropathy treatment and a corresponding pharmaceutical composition for treating nephropathy.

Description

Use of fucose in IgA nephropathy treatment

Technical Field

The application belongs to the field of disease treatment, and particularly relates to an application of fucose in IgA nephropathy treatment.

Background

IgA nephropathy is an autoimmune disease driven by the common disturbances of the environmental, genetic and immune, especially the adaptive and innate immune systems, and is the most common primary glomerular disease worldwide characterized by mesangial region immune complex deposition, mesangial cell proliferation and mesangial matrix expansion, glomerulosclerosis, tubular interstitial fibrosis and haematuria, proteinuria, etc. The incidence rate of IgA nephropathy in China is in an ascending trend, so that IgA nephropathy becomes an important cause of end-stage nephropathy at present, but no specific treatment means exist yet. The current medicines for treating IgA nephropathy mainly comprise a renin-aldosterone system blocker, an immunosuppressant and a monoclonal antibody medicine, and the medicines are effective, but still can not prevent the diseases from progressing to end-stage renal disease, and the long-term survival rate of patients is not obviously improved due to the limited curative effect and certain side effect of the medicines. Therefore, the development of novel drugs is of great importance for the treatment of IgA nephropathy.

Fucose, which is found in a large amount in seaweed and gums and in polysaccharides of some bacteria and also in intestinal glycoproteins, is a main constituent structure of fucose, and has been widely used in various fields such as functional foods, health products, cosmetics, biomedical materials, plant growth stimulants, etc. all over the world at present, fucose mediates host microbial symbiosis, inhibits virulence of pathogens and pathogenic bacteria, and improves systemic infection and inflammation in the center of the intestinal tract. The fucose can also effectively remove helicobacter pylori, condition intestinal flora, promote tumor rehabilitation, and play a unique role in promoting chronic wound healing, moisturizing skin, promoting crop growth and other fields.

The prior art describes therapeutic uses of fucoidan in chronic kidney disease and diabetic kidney disease, but there is no disclosure of application to IgA kidney disease, and there is no disclosure of application of fucoidan in IgA kidney disease treatment.

In view of this, the present application is presented.

Disclosure of Invention

In order to solve the technical problems, the application finds that immune factors related to the pathogenesis of IgA nephropathy are mainly focused on the rise of macrophages and the reduction of T cells, and fucose can regulate the expression of key genes causing the immune phenomenon of IgA nephropathy; on the other hand, fucose itself has various immunological activities, and can regulate symbiotic relationship between host and intestinal flora, reduce complement C3 deposition on renal tubules, infiltrate immune cells, and inhibit macrophage activation; the above process is also closely related to the pathogenesis of IgA nephropathy; in addition, fucose is a powerful antioxidant, and can reduce tissue injury caused by inflammation and protect renal function. Based on this, the present application proposes that fucose can be used for the treatment of IgA nephropathy.

It is therefore a first object of the present application to provide the use of fucose in the treatment of kidney disease (especially IgA kidney disease) and its use in the manufacture of a medicament for the treatment of IgA kidney disease;

a second object of the present application is to provide the use of fucose for the manufacture of a medicament for the treatment of IgA nephropathy;

a third object of the present application is to provide a pharmaceutical composition for the treatment of kidney disease, in particular IgA kidney disease.

In particular, the present application first provides the use of fucose for the manufacture of a medicament for preventing and/or treating a disease or disorder, or for reducing the risk of a disease or disorder.

Further, the disease is kidney disease;

further, the kidney disease is IgA kidney disease.

The present application also provides a pharmaceutical composition comprising the active ingredient fucose.

Further, the pharmaceutical composition further comprises optionally a pharmaceutically acceptable excipient, carrier and/or diluent.

Further, the pharmaceutical composition is used for preventing and/or treating kidney diseases;

further, the kidney disease includes IgA kidney disease.

The present application also provides a method of preventing or treating a disease, disorder or syndrome, comprising administering to a subject in need thereof an effective amount of fucose or a pharmaceutical composition as described above.

Further, the medicament or the pharmaceutical composition described above is administered to the subject subcutaneously, intravenously, orally, rectally or intraperitoneally.

The present application also provides a method of inhibiting a cell or tissue disorder in vitro, the method comprising administering to the cell or tissue an effective amount of fucose or a pharmaceutical composition as described above, the disorder being an IgA nephropathy disorder.

Further, the cells are kidney cells and the tissue is kidney tissue;

further preferably, the cells and tissues are ex vivo.

The application also provides an application of the fucose in preparing an animal model of kidney diseases, in particular to an application in an animal model of IgA kidney diseases.

The present application also provides the use of an agent for detecting macrophage elevation and T cell depletion for the preparation of an agent for assessing the occurrence of IgA nephropathy; preferably, the T cells are cd4+ T cells, more preferably iTreg cells.

The present application also provides a use of fucose for modulating C3AR1, CYBB, CTSS, IGTB2, FCER1G, TYROBP, CD, RAC2, HCK, IFI30, IL10RA, NCF2, TLR2 and/or HCLS1 gene expression in cells, tissues or patients of IgA nephropathy in vivo or in vitro.

The application also relates to the use of fucose for the preparation of a medicament for modulating expression of C3AR1, CYBB, CTSS, IGTB2, FCER1G, TYROBP, CD, RAC2, HCK, IFI30, IL10RA, NCF2, TLR2 and/or HCLS1 genes in cells, tissues or patients of IgA nephropathy.

The present application also relates to a method of modulating C3AR1, CYBB, CTSS, IGTB2, FCER1G, TYROBP, CD, RAC2, HCK, IFI30, IL10RA, NCF2, TLR2 and/or HCLS1 gene expression in cells, tissues or patients of IgA nephropathy in vivo or in vitro, characterized in that it is administered fucose.

The application also relates to the use of the C3AR1, CYBB, CTSS, IGTB, FCER1G, TYROBP, CD, RAC2, HCK, IFI30, IL10RA, NCF2, TLR2 and/or HCLS1 genes as an indicator of IgA nephropathy.

The present application also relates to the use of an agent for detecting C3AR1, CYBB, CTSS, IGTB, FCER1G, TYROBP, CD, RAC2, HCK, IFI30, IL10RA, NCF2, TLR2 and/or HCLS1 genes in the manufacture of a product for diagnosing a disease or disorder, said disease or disorder being IgA nephropathy.

Further, the subject includes human or non-human animals (including mammals), such as humans, non-human primates (apes, gibbons, gorillas, chimpanzees, orangutans, macaques), domestic animals (dogs and cats), farm animals (poultry such as chickens and ducks, horses, cows, goats, sheep, pigs), and laboratory animals (mice, rats, rabbits, guinea pigs). Human subjects include fetal, neonatal, infant, adolescent and adult subjects. Further, the subject also includes an animal disease model.

The beneficial technical effect of this application:

the application proposes that fucose has therapeutic application to IgA nephropathy for the first time;

the present application illustrates for the first time the underlying mechanism of fucose in the treatment of IgA nephropathy;

the present application also establishes a series of genes potentially indicative of IgA nephropathy by letter analysis.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the description of the embodiments or the prior art will be briefly described, and it is obvious that the drawings in the description below are some embodiments of the present invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.

In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings that are needed in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present application, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1, data merge and remove batch effect results;

FIG. 2, results of a single sample gene set enrichment analysis;

FIG. 3 shows the result of immunoinfiltration clustering of IgA nephropathy;

FIG. 4 shows the results of differential expression analysis of IgA nephropathy genes;

FIG. 5, HUB gene screening results;

FIG. 6, results of gene function enrichment analysis;

FIG. 7 is a graph showing the trend of gene expression;

FIG. 8, model construction results;

fig. 9, change in serum creatinine concentration after 8 weeks of gastric lavage;

FIG. 10, blood urea nitrogen concentration change after 8 weeks of gastric lavage;

FIG. 11, change in urine protein concentration 24h after 8 weeks of gastric lavage;

FIG. 12 shows PAS staining results of kidney pathological sections of IgA nephropathy mice after administration.

Detailed Description

The following description of the embodiments of the present application will be made apparent and fully in view of the accompanying drawings, in which some, but not all embodiments of the invention are shown. All other embodiments, which can be made by one of ordinary skill in the art without undue burden from the present disclosure, are within the scope of the present disclosure.

Partial term definition

Unless defined otherwise hereinafter, all technical and scientific terms used in the detailed description of the present application are intended to be the same as commonly understood by one of ordinary skill in the art. While the following terms are believed to be well understood by those skilled in the art, the following definitions are set forth to better explain the present application.

As used in this application, the terms "comprising," "including," "having," "containing," or "involving" are inclusive or open-ended and do not exclude additional unrecited elements or method steps. The term "consisting of …" is considered to be a preferred embodiment of the term "comprising". If a certain group is defined below to contain at least a certain number of embodiments, this should also be understood to disclose a group that preferably consists of only these embodiments. The indefinite or definite article "a" or "an" when used in reference to a singular noun includes a plural of that noun.

The terms "about", "substantially" in this application refer to a range of accuracy that one of ordinary skill in the art would understand yet still guarantee the technical effect of the features in question. The term generally means a deviation of + -10%, preferably + -5%, from the indicated value. Furthermore, the terms first, second, third, (a), (b), (c), and the like in the description and in the claims, are used for distinguishing between similar elements and not necessarily for describing a sequential or chronological order. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments described herein are capable of operation in other sequences than described or illustrated herein.

Use of fucose according to the present application for the manufacture of a medicament for the prevention and/or treatment of a disease or disorder, or for reducing the risk of a disease or disorder.

In some embodiments, the disease described herein is kidney disease.

As used herein, "kidney disease" refers to damage to kidney structure and function from a variety of causes, and may include, but is not limited to, primary glomerular diseases such as acute nephritis, chronic nephritis, latent nephritis, nephrotic syndrome, etc., secondary glomerular diseases such as lupus nephritis, purpura nephritis, hepatitis b virus associated nephritis, igA nephropathy, etc., hereditary kidney diseases such as polycystic kidney, thin basement membrane kidney disease, etc., interstitial nephritis such as acute interstitial nephritis, chronic interstitial nephritis, etc.

In some specific embodiments, the kidney disease described herein is IgA kidney disease.

As used herein, "IgA nephropathy" is an autoimmune disease that is driven by a combination of environmental, genetic and immune disorders, particularly disorders of the adaptive and innate immune systems, and is the most common primary glomerular disease characterized by mesangial region immune complex co-deposition, mesangial cell proliferation and mesangial matrix expansion, glomerulosclerosis, tubular interstitial fibrosis and hematuria, proteinuria, and the like.

The "fucose" described in the present application, english name: fucose, chinese name: 6-deoxy-L-galactose, is one of monosaccharides and hexoses that are present in a large amount in intestinal glycoproteins.

In this application, the terms "disease" or "disorder" are used interchangeably and generally refer to any deviation of a subject from a normal state, such as any change in the state of the body or certain organs, that impedes or disrupts performance of the function, and/or that causes symptoms such as discomfort, dysfunction, pain, or even death in a person suffering from or in contact with the disease. The disease or disorder may also be referred to as a disorder (disorder), an malaise (ailment), a malady (ailment), a disorder (disorder), a disease (hickness), an illness (illness), a physical malaise (compatibility), an index of disorder, or an affection.

In this application, the term "preventing and/or treating" includes not only preventing and/or treating a disease, but also generally includes preventing the onset of a disease, slowing or reversing the progression of a disease, preventing or slowing the onset of one or more symptoms associated with a disease, reducing and/or alleviating one or more symptoms associated with a disease, reducing the severity and/or duration of a disease and/or any symptoms associated therewith and/or preventing further increases in the severity of a disease and/or any symptoms associated therewith, preventing, reducing or reversing any physiological damage caused by a disease, and any pharmacological effects that would normally be beneficial to a patient being treated. The RNA inhibitors or pharmaceutical compositions of the present application form viable therapeutic agents without the need to achieve complete cure or eradication of any symptoms or manifestations of the disease. As recognized in the relevant art, drugs used as therapeutic agents may reduce the severity of a given disease state, but need not eliminate every manifestation of the disease to be considered useful therapeutic agents. Similarly, a prophylactically administered treatment constitutes a viable prophylactic agent and need not be completely effective in preventing the onset of the condition. It may be sufficient to simply reduce the impact of the disease in the subject (e.g., by reducing the number or severity of its symptoms, or by increasing the effectiveness of another treatment, or by producing another beneficial effect), or to reduce the likelihood of disease occurrence or exacerbation.

The application also relates to a pharmaceutical composition comprising the active ingredient fucose for use in the prevention and/or treatment of kidney diseases.

In some embodiments, the kidney disease described herein is IgA kidney disease.

In this application, the term "subject" generally refers to a human or non-human animal (including mammals) in need of diagnosis, prognosis, amelioration, prevention and/or treatment of a disease, such as humans, non-human primates (apes, gibbons, gorillas, chimpanzees, gorillas, macaques), domestic animals (dogs and cats), farm animals (poultry such as chickens and ducks, horses, cattle, goats, sheep, pigs), and laboratory animals (mice, rats, rabbits, guinea pigs). Human subjects include fetal, neonatal, infant, adolescent and adult subjects. The subject includes an animal disease model. In some embodiments, the subject of the present application is particularly a kidney disease subject, and in other specific embodiments, the subject of the present application is particularly an IgA kidney disease subject.

In some specific embodiments, the subject described herein is a human or a mouse.

In some embodiments, the pharmaceutical compositions described herein further comprise optionally a pharmaceutically acceptable excipient, carrier, and/or diluent.

In this application, the term "pharmaceutically acceptable" generally refers to one or more non-toxic substances that do not interfere with the effectiveness of the biological activity of the active ingredient. Such formulations may generally contain salts, excipients, buffers, preservatives, compatible carriers and optionally other therapeutic agents. Such pharmaceutically acceptable formulations may also generally comprise compatible solid or liquid fillers, diluents or encapsulating materials suitable for administration to humans. For pharmaceutical use, the salts should be pharmaceutically acceptable salts, but non-pharmaceutically acceptable salts may be conveniently used to prepare pharmaceutically acceptable salts and are not excluded from the scope of the present application. Such pharmacologically and pharmaceutically acceptable salts include, but are not limited to, salts prepared from the following acids: hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, maleic acid, acetic acid, salicylic acid, citric acid, boric acid, formic acid, malonic acid, succinic acid, and the like. Pharmaceutically acceptable salts may also be prepared as alkali metal salts or alkaline earth metal salts, such as sodium, potassium or calcium salts. The term "acceptable excipients, carriers, and/or diluents" refers to excipients, carriers, and/or diluents that are pharmacologically and/or physiologically compatible with the subject and active agent, are well known in the art (see, e.g., remington's pharmaceutical sciences. Mediated by Gennaro AR,19th ed.Pennsylvania:Mack Publishing Company,1995), and include, but are not limited to, pH adjusters, surfactants, adjuvants, and ionic strength enhancers, and the like. For example, pH modifiers include, but are not limited to, phosphate buffers; surfactants include, but are not limited to, cationic, anionic or nonionic surfactants, such as Tween-80; ionic strength enhancers include, but are not limited to, sodium chloride.

The present application also relates to a method of preventing or treating a disease, disorder or syndrome, comprising administering to a subject in need thereof an effective amount of fucose or a pharmaceutical composition as described above.

In the present application, the term "administration" generally refers to the introduction of the pharmaceutical formulation of the present application into the body of a subject by any route of introduction or delivery. Any method known to those skilled in the art for contacting cells, organs or tissues with the drug may be used. The administration may include, but is not limited to, intravenous, intra-arterial, intranasal, intra-abdominal, intramuscular, subcutaneous transdermal, or oral. The daily dose may be divided into one, two or more doses of a suitable form to be administered at one, two or more times during a certain period of time. In some embodiments, the medicament or the pharmaceutical composition described herein is administered to the subject subcutaneously, intravenously, orally, or intraperitoneally.

In this application, the term "effective amount" or "effective dose" generally refers to an amount sufficient to achieve, or at least partially achieve, a desired effect. A "therapeutically effective amount" or "therapeutically effective dose" of a drug or therapeutic agent is generally any amount of drug that, when used alone or in combination with another therapeutic agent, promotes regression of the disease (as evidenced by a decrease in severity of symptoms of the disease, an increase in the frequency and duration of disease asymptomatic periods, or prevention of damage or disability due to the disease). "prophylactically effective amount" or "prophylactically effective dose" of a drug generally refers to an amount of a drug that inhibits the progression or recurrence of a disease when administered alone or in combination with another therapeutic agent to a subject at risk of disease progression or recurrence. The ability of a therapeutic or prophylactic agent to promote regression of a disease or inhibit the progression or recurrence of a disease can be assessed using a variety of methods known to those of skill in the art, such as in a human subject during a clinical trial, in an animal model system to predict efficacy in humans, or by assaying the activity of the agent in an in vitro assay. In certain embodiments, an "effective amount" refers to an amount of fucose that produces a desired pharmacological, therapeutic, or prophylactic result.

In some embodiments, the subject described herein is a subject having kidney disease, preferably an IgA kidney disease subject.

The application also provides applications of fucose in preparing animal models of kidney diseases, in particular IgA kidney disease. It will be appreciated that upon determining that fucose is useful for treating kidney disease, one skilled in the art can prepare or assist in preparing animal models of kidney disease accordingly.

The application also relates to the use of fucose in screening for inhibitors of kidney disease; preferably, the kidney disease is IgA kidney disease. It will be appreciated that upon determining that fucose is useful in the treatment of kidney disease, one skilled in the art will be able to screen accordingly for other corresponding kidney disease inhibitor compounds.

The present application relates to the use of fucose in vitro to improve immune cell infiltration, inhibit macrophage activation, and/or reduce tissue damage caused by inflammation; the cells are IgA nephropathy cells and the tissue is micro IgA nephropathy tissue.

Furthermore, the present application establishes that immune characteristics of IgA nephropathy are manifested by an increase in macrophages and a decrease in CD4+ T cells, and thus, the present application may relate to an assessment for the occurrence of nephropathy based on macrophage increase and T cell decrease, in some embodiments, the T cells are CD4+ T cells, preferably iTreg cells.

The present application establishes that immune factors associated with the onset of IgA nephropathy are primarily focused on elevation of macrophages and reduction of T cells, while fucose can regulate expression of key genes responsible for this immune phenomenon of IgA nephropathy, including C3AR1, CYBB, CTSS, IGTB2, FCER1G, TYROBP, CD53, RAC2, HCK, IFI30, IL10RA, NCF2, TLR2, and HCLS1.

The present application thus also relates to the use of fucose for modulating C3AR1, CYBB, CTSS, IGTB2, FCER1G, TYROBP, CD, RAC2, HCK, IFI30, IL10RA, NCF2, TLR2 and/or HCLS1 gene expression in cells, tissues or patients of IgA nephropathy.

The present application also relates to methods of modulating C3AR1, CYBB, CTSS, IGTB2, FCER1G, TYROBP, CD, RAC2, HCK, IFI30, IL10RA, NCF2, TLR2 and/or HCLS1 gene expression in cells, tissues or patients of IgA nephropathy in vivo or in vitro by administering fucose thereto.

The present application is further described by the accompanying drawings and the following examples, which are provided merely to illustrate particular embodiments of the present application and are not to be construed as limiting the scope of the present application in any way.

Example 1 data collection and processing

The keyword "IgA nephropathy" was used to search the GEO database for IgA gene expression profile, and for accuracy and completeness all transcriptome sequencing data of IgA glomeruli were obtained from the GEO database, and 5 datasets (including 4 microarray datasets and 1 RNA seq dataset) were downloaded, the information of which is shown in Table 1. Then, gene name conversion and log2 log conversion were performed to perform gene expression profile analysis. In addition, for a combination of multiple data sets, the present embodiment first uses the R software package "inslicomering" to merge the data sets, then uses the "combat" function to remove the batch effect, and finally gets the "datal" after removing the batch effect.

Table 1The information of 5datasets obtained from GEO database

The results showed that the 4 gene chip datasets were pooled into a new matrix "datal" using the inlicomering software package, which included 100 IgA nephropathy patients and 70 healthy controls. A box plot, density plot and UAMP plot were generated to show the difference between the samples before and after removal of the batch effect (fig. 1). It can be observed that the sample distribution of each dataset before the batch effect is removed varies greatly, indicating the presence of the batch effect. After the batch effect is removed, the data distribution of each data set tends to be consistent.

Example 2 Single sample Gene set enrichment analysis (ssGSEA)

Since ciberport and other methods do not obtain sufficient T cell subtype results, this example uses ImmuneCellAI, a tool to estimate the abundance of 24 immune cells from a gene expression dataset, consisting of 18T cell subtypes and 6 other immune cells, for single sample gene set enrichment analysis of the combined chip data and RNA-seq data.

The abundance of 24 immune cells in datal and dataset GSE141295 was calculated using ImmuCell AI, including 10 layer1 cells (DC (dendritic cells), B cell (B cells), monocyte (monocytes), macrotage (macrophages), NK cell (natural killer cells), neutrophil (neutrophils), CD4+T cell (CD4+T cells), CD8+T cell (CD8+T cells), NKT (natural killer T cells), tgd (γδT cells) and 14 layer2 cells, which comprises 14T cell subtypes (cd4+ naive T cells), tr1 (regulatory T cell 1), nTreg (natural regulatory T cells), nTreg (inducible regulatory T cells), th1 (helper T cell 1), th2 (helper T cell 2), th17 (helper T cell 17), tfh (T follicular helper cells), cd8+ naive T cells (cd8+ naive T cells), tc (cytotoxic T cells), tex (depleting T cells), MAIT (mucosa-associated constant T cells), tcm (central memory T cells), tem (effector memory T cells)).

Referring specifically to fig. 2, fig. 2A shows the abundance of 10 layer1 immune cells in data1 and dataset GSE 141295. In the IgA nephropathy samples of data1, DCs (dendritic cells), macrotage (macrophages), cd8+ T cells (cd8+ T cells), tex (depleting T cells), MAIT (mucosa-associated constant T cells) and Tem (effector memory T cells) were increased, whereas cd4+ T cells (cd4+ T cells), neutrophil (neutrophils), iTreg (inducible regulatory T cells), nTreg (natural regulatory T cells), tcm (central memory T cells), th2 (helper T cells 2), th17 (helper T cells 17) were decreased (fig. 2B and C). Meanwhile, macrotage (macrophages) and NKT (natural killer T cells) were increased, B cell (B cell), cd4+ T cell (cd4+ T cell), tr1 (regulatory T cell 1), nTreg (natural regulatory T cell), nTreg (inducible regulatory T cell), th1 (helper T cell 1) and Tfh (T follicle helper cell) were decreased in IgA nephropathy samples of the dataset GSE141295 (fig. 2D and E). According to the ssGSEA results of data1 and dataset GSE141295, immune characteristics of IgA nephropathy are manifested by an increase in macrophages and a decrease in cd4+ T cells, especially in iTreg cells.

EXAMPLE 3 immune infiltration clustering of IgA nephropathy

While GEO databases do not have sufficient clinical information for these samples, cluster analysis can help find a subset of diseases. Thus, this example uses "ConensusClusterPlus" to perform cluster analysis, a class discovery tool with confidence assessment and item tracking, uses aggregated PAM clusters with 1-Pearson correlation distances, and resamples 80% of the samples 10 replicates. The optimal cluster number is determined using an empirical cumulative distribution function map. Differences in the identified immune cell clusters are compared to distinguish their immune characteristics.

Based on the abundance of 24 immune cells obtained from ssGSEA, 100 IgA nephropathy samples in data1 were subjected to cluster analysis, which indicated that: when the K value=2, the decrease trend of CDF delta is the slowest (fig. 3A), while the area under the CDF curve increases with the increase of the K value (fig. 3B), this embodiment needs to keep the decrease of CDF delta as slow as possible while keeping the area under the CDF curve as large as possible, and finally, to achieve the highest average uniformity within the group, the number of clusters selected is k=2 (fig. 3C). 100 IgA nephropathy samples were divided into two clusters (FIG. 3D), 53 samples in cluster 1 and 47 samples in cluster 2. Then, this example compares the abundance of immune cells in groups 1 and 2, with DC, macrophages and NK cells in group 2 being significantly higher than in group 1, while macrophages and NK cells in group 2 were not different from the control (fig. 3E). Thus, in IgA nephropathy samples, cluster 2 is considered to represent severe inflammation, while cluster 1 represents mild inflammation.

Example 4 differential expression analysis of IgA nephropathy Gene

In this example, igA nephropathy and control Differential Expression Genes (DEGs) in the pooled data were screened using "LIMMA" in R software, differential analysis was performed on dataset GSE141295 using "DESeq2 (Version 1.32.0)", and the results of both parts were pooled to obtain two common DEGs up-and down-regulated in the sequencing data using P <0.05 as the standard for screening the DEGs, and we named the reconstructed expression matrix of these common DEGs as "data2".

3754 DEGs were identified in total between IgA nephropathy in data1 and control group according to P <0.05, up-regulated genes 1553 and down-regulated genes 2203. 2818 up-regulated genes and 7097 down-regulated genes were obtained from dataset GSE1412950 (fig. 4A), 1104 common DEGs were screened out in combination with data1 and data set GSE141295 DEGs, 637 up-regulated genes and 467 down-regulated genes (fig. 4B and C), and these 1104 common DEGs were selected to reconstruct the expression matrix, generating data2 for further analysis.

Example 5 determination of Critical Gene screening

This example uses R package "WGCNA", and Data2 was used to construct a scaleless co-expression network by weighted gene co-expression network analysis (WGCNA). First, pearson correlation matrix and average linkage analysis were performed on all pairwise combined genes. Then, a weighted adjacency matrix is constructed using a power function a_m= |c_m|β (c_m=pearson correlation between gene m and gene n; a_n=adjacency relationship between gene m and gene n). Beta is a soft threshold parameter that can emphasize strong correlations between genes and penalize weak correlations. After a power of 4 is selected, the adjacency is converted into a Topological Overlap Matrix (TOM) that measures the network connectivity of one gene to all other genes and calculates the corresponding dissimilarity (1-TOM). To classify genes with similar expression profiles into gene modules, average linkage hierarchical clustering was performed according to TOM-based dissimilarity measure, with a minimum size (genome) of the gene dendrogram of 25. To further analyze the modules, we calculated the differences in the module signature genes, selected cut lines for the module dendrogram, and combined some modules. Other parameters include R square-cut=0.85 and deep split=2. The present example then identifies the module with the highest correlation coefficient and screens the HUB gene.

To identify genes significantly associated with IgA nephropathy clusters, 1104 genes in DATA2 were divided into 7 modules using WGCNA based on β=4 (fig. 5A and B). Wherein the MEbrown module correlated most with IgA nephropathy cluster, correlated positively with cluster 2, correlated negatively with cluster 1, and contained 219 genes (fig. 5C). In addition, the present example also calculated the correlation between the module feature vector and the gene expression, resulting in a HUB gene based on the cut-off criteria of |mm| > 0.9. In short, 15 genes with high connectivity in clinically important modules were identified as HUB genes, which were thought to be related to the severity of inflammation of IgA nephropathy, cytoscape 3.8.2 was used to show the correlation between them (fig. 5D).

Example 6 Gene enrichment analysis

This example employed the R software package "clusterifier" (version 3.14.3) for enrichment analysis to obtain the results of the kyoto gene and genome encyclopedia (KEGG) and Gene Ontology (GO) analysis. The minimum gene set was set to 5, the maximum gene set was set to 5000, and p values <0.05 and FDR <0.25 were considered statistically significant.

Enrichment analysis results show that 15 HUB genes are involved in a range of immune processes, such as NK cell mediated cytotoxicity and cell killing pathways in KEGG (fig. 6A), and 15 HUB genes and interleukin 6 (IL-6) and interleukin 10 (IL-10), toll-like receptor signaling pathways, interferon-gamma mediated signaling pathways in the case of GO, macrophage activation are involved in immune responses. (FIG. 6B).

Example 7 Gene expression trend analysis

In this example, the data1 was subjected to a temporal trend analysis of gene expression using a short-time sequential expression mining (STEM) analysis, and genes with the same expression trend were classified into a group from the control group to different clusters, and P value <0.05 was considered as a significant gene cluster.

STEM analysis was used to determine the change in 15 HUB genes from healthy controls to different IgA nephropathy clusters. The results show that: c3AR1, CYBB, CTSS, IGTB2, FCER1G, TYROBP, CD, RAC2, HCK, IFI30, IL10RA, NCF2, TLR2 and HCLS1 present increasing trends in different IgA nephropathy clusters (fig. 7A), C3AR1, CD53, CTSS, CYBB, FCER1G, HCK, IFI, IL10RA, ITGB2, RAC2 and TYROBP in trend 1, NCF2, SAMSN1 and TLR2 in trend 2, and HCLS1 in trend 3 (fig. 7B-D). STEM analysis showed that these 14 genes whose expression tendencies increased from healthy controls to different IgA nephropathy clusters were associated with different immunoinfiltrates with altered IgA nephropathy, which could serve as biomarkers for IgA nephropathy inflammation.

Example 8 construction and evaluation of machine learning model

To better predict and evaluate inflammation of IgA nephropathy, this example used "machine learning" in Shinny application (https:// Shinny. Hiplot. Com. Cn/map learn /) to construct six machine learning models (SvmLinear, svmPoly, neural networks, randomForest, K-NN, naive Bayes) based on HUB genes, the number of cross-validation was set to 10. Finally, the best model is determined based on accuracy and kappa values.

Different machine learning models were constructed by selecting 14 HUB genes that increased with different IgA nephropathy clusters, and model rank sum predictive observations of 6 models were generated according to cross validation times = 10 (fig. 8A and B). Fig. 8C shows the observed and predicted values, and fig. 8D shows the feature importance. In view of accuracy and Kappa values (Table 2), nnet-5-0.1 is the best model for predicting IgA nephropathy inflammation degree based on 14 HUB genes, which is significantly related to the severity of IgA nephropathy glomerular inflammation infiltration, and which is of great value for future prediction of IgA nephropathy glomerular inflammation severity.

Table 2 The accuracy and kappa-value of top 10models

Example 9 identification of potential molecules for the treatment of IgA nephropathy

Inflammatory features of IgA nephropathy can be represented by HUB genes identified by WGCNA, and a database of drug features (DSigDB) in the Enrich r (https:// amp. Pharm. Mssm. Edu/Enrich r /) platform is used to screen drugs that modulate HUB gene expression and to determine candidate molecules for treating IgA nephropathy.

Table 3 lists the first 10 potential molecules predicted from DsigDB, where fucose has the highest odds Ratio (0 dds Ratio) and highest binding Score (Combined Score), and is considered a candidate for treating IgA nephropathy.

Table3 Suggested top 10small molecules for IgAN

Example 10 evaluation of administration

1. Experimental animals and reagents

The experiment selects 21 IgA nephropathy male mice with the weight of 19 Shi 1g, which is provided by Shanxi province people hospital center laboratory, and the selected IgA nephropathy model mice are MiRNA-23b-3p gene knockout mice (presented by the Kidney disease precision medical innovation center of Beihua university) and are bred for fucose administration experiment after conventional 6 weeks of feeding. Feeding conditions: SPF-class animal house. 21 IgA nephropathy mice are bred in separate cages, 2-5 mice are bred in each cage, the temperature in animal houses is kept at 18-22 ℃, and the relative humidity is kept at 40% -60%. The 12-hour illumination period, the day and night circulation alternation, free water drinking, sufficient food intake, adaptation to the environment, 1 week of feeding, ear nail marking number, weighing and recording.

Experimental medicine and reagent

Fucose preparation: mice in the high dose group took 100 mg/kg/day, so 0.14g fucose was dissolved in 14ml purified water to prepare a one week formulation for the high dose group. The low dose group mice take 50 mg/kg/day of medicine, 0.3325g of fucose is dissolved in 7ml of purified water for preparation, and the packaged mice are put into a refrigerator for refrigeration at 4 ℃ for standby, and corresponding administration doses are timely adjusted according to weekly weighing results of the mice.

10% chloral hydrate solution, namely, dissolving 10g chloral hydrate solid crystals in 100ml physiological saline, fully and uniformly mixing, and preserving in a dark place to obtain the chloral hydrate solid crystals.

2. Experimental method

1) And (3) animal group feeding: 21 SPF-class healthy mice were housed in separate cages, and the mice were randomly divided into three groups, a high-dose fucose-filled group, a low-dose fucose-filled group, and a blank group, each group amounting to 7 IgA nephropathy mice, and after 1 week of acclimation, gastric administration was started, and each group of mice was given a conventional basal feed.

2) Grouping and administration: after 1 week of grouping, 24h urine was collected with a metabolic cage to leave a urine specimen, urine volume and water intake were recorded and 24h urine protein qualitative experiments were detected to confirm that there was no statistical difference between groups. Gastric lavage treatment was started the second week after grouping. The fucose preparation is prepared according to the dosage of 100 mg/kg/day of mice in the high-dosage group and 50 mg/kg/day of mice in the low-dosage group, and the stomach is filled once in a fixed time in noon every day for 8 weeks continuously. According to the weekly weighing result of the mice, the corresponding administration dosage is adjusted in time. Mice are fed with conventional feed during the period of gastric lavage, and the padding is replaced once daily, so that the sufficiency of food and the freedom of drinking water are ensured.

3) Sample collection: the experiment was ended at the end of 8 weeks after gavage (no water forbidden for 12 h), and after weighing, the mice were anesthetized (10% chloral hydrate solution was injected intraperitoneally at 4 ml/kg). After successful anesthesia, the abdominal cavity of the mice was opened to separate and expose the abdominal aorta, and 5ml of abdominal aortic blood was collected by aspiration with a negative pressure blood collection tube. Standing the blood sample for 1 hour, placing the blood sample into a centrifuge for centrifugation for 15 minutes (4 ℃ C., 3000 r/min), separating serum by a pipette, transferring the serum into a freezing tube, and storing the blood sample in an ultralow temperature refrigerator at-80 ℃ until the blood sample is used for measuring serum creatinine and urea nitrogen. Fully exposing the abdominal cavity, shearing the kidneys at the left side and the right side, putting into normal saline for rinsing, wiping with sterile gauze to absorb water, and weighing the weight of the right kidney. The left kidney was cut longitudinally along the kidney gate, half was placed in 4% paraformaldehyde solution to be completely fixed overnight, and the other half was stored in a-80 ℃ freezer and left for use.

4) Preparation of Paraffin sections

Taking out kidney samples fixed by paraformaldehyde solution overnight, and circularly flushing with tap water for 2h;

gradient dehydration of alcohol with different concentrations, namely, dehydration of 75% alcohol, 85% alcohol and 95% alcohol for 15min respectively, dehydration of 100% absolute alcohol for 15min, and repeating for 3 times;

the dimethylbenzene (transparent agent) is completely transparent, wherein the dimethylbenzene I is transparent for 15min, and the dimethylbenzene (II, III) is transparent for 30min respectively; paraffin embedding, namely immersing a kidney specimen in paraffin at 58 ℃, embedding the kidney specimen in paraffin by an embedding machine after the kidney specimen is immersed, and preparing paraffin blocks with the size of 4cm multiplied by 2cm after the paraffin is solidified;

slicing, namely slicing the embedded kidney samples with a slicing machine, wherein the slicing thickness is 3um, cutting each kidney sample into 3 slices, loading the slices, airing, and baking the slices for 30min under a baking machine (58 ℃) for HE dyeing.

5) Index detection

5.1 general State viewing

The general growth state of each group of mice is closely observed in the whole course, including the conditions of spirit, behavior, posture, fur, ingestion, drinking water and defecation.

5.2 Biochemical index determination

After the experiment is finished, taking out the serum sample to be detected from the refrigerator, and respectively detecting two items of 24h urine protein quantification and renal function by using a designated biochemical kit.

5.2.1 24-hour urine protein quantitative determination

Taking a collected mouse urine sample, putting the sample into a centrifuge, setting the rotating speed to 3000r/min, centrifuging for 15 minutes, slowly sucking 1.5ml of supernatant into a new centrifuge tube, and then detecting the protein concentration according to the Bradford method, wherein the detailed steps are as follows:

preparing protein standard stock solution, namely sucking 1mL of protein standard preparation solution by a pipette, transferring into a BSA tube containing 25mg of protein standard substance, shaking thoroughly, dissolving completely to obtain protein standard stock solution (25 mg/mL), and placing into a refrigerator at-20deg.C for standby.

Preparing a protein standard working solution, namely taking 1mL of the protein standard stock solution into a centrifuge tube, adding PBS buffer solution to dilute to 50mL, and preparing the protein standard working solution with the concentration of 0.5mg/mL.

Drawing a standard curve, namely sucking 0,1,2,4,8, 12, 16 and 20ul protein standard working solution by a pipetting gun, sequentially adding the working solution into a 96-well ELISA plate, and then supplementing the working solution by PBS buffer solution to ensure 20ul of working solution per well. New standard protein solutions were obtained at concentrations of 0, 25, 50, 100, 200, 300, 400, 500ug/mL, in order, to form a gradient profile.

Preparing to-be-tested samples, namely adding urine to be tested into the 96-well ELISA plate one by one according to the amount of 20ul of each sample.

And (3) detecting, namely adding 200ul of coomassie brilliant blue solution into each hole, fully and uniformly mixing, standing at room temperature for 5min after shaking, and measuring the absorbance (OD) value of each hole at the wavelength of 595nm by using an enzyme-labeled instrument.

And drawing a coordinate curve, namely drawing a standard coordinate curve by taking the concentration (ug/mL) of a standard protein solution as an X axis and the corresponding absorbance (OD) value as a Y axis.

Calculating the protein concentration (ug/mL) of each urine sample on a standard coordinate curve according to the absorbance (OD) value of the urine sample.

24-hour urine protein (24 hUpro) quantification (mg) =protein concentration of urine sample (ug/mL) ×24-hour urine volume (mL): 1000.

5.2.2 creatinine assay

Creatinine (Scr) content (umol/L) = (assay X2-r X assay X1) one (blank X2-r X blank X1)/(standard X2-r X standard X1) - (blank X2-r X blank X1) X standard concentration (442 u mol/L) dilution factor r= (V-loaded+v enzyme solution a)/(V-loaded+v enzyme solution a+v enzyme solution B) =186/246

5.2.3 Urea Nitrogen determination

And (3) sample adding operation is carried out according to the steps, after color development, 200ul of the sample is absorbed on each tube to 96-well ELISA plates in parallel, and absorbance (OD) values of all the wells are read by an ELISA reader at 640nm wavelength.

Urea Nitrogen (BUN) content (mmol/L) = (detection OD-blank OD)/(standard OD-blank OD) ×standard concentration (10 mmol/L)

5.3 pathological morphological observation of kidney tissue (PAS staining)

(1) Dewaxing to water washing, namely dewaxing by using xylene (I, II and III) respectively for 15min, washing by using alcohol to water of each level, and washing by using 100% alcohol, 95% alcohol and 75% alcohol for 3min and distilled water for 2min respectively.

(2) Placing in 1% periodate solution, standing at room temperature for 5-8min, washing with tap water for 1 time, and soaking with distilled water for 2 times.

(3) The sample was put into a Schiff reagent, stained at room temperature for 15min (note light-shielding), and washed with tap water for 10min.

(4) Hematoxylin staining solution is used for staining for 30s,1% acid alcohol differentiation solution is used for differentiation, and tap water is used for flushing; washing with 1% ammonia water to make the product turn blue.

(5) And (3) dehydrating by alcohol step by step, namely, transparency for dimethylbenzene and sealing by neutral resin.

(6) The results were observed under a mirror and described.

3. Experimental results

1) Kidney function and 24h urine protein concentration variation of three groups of mice before and after gastric lavage

After 8 weeks of gavage, the serum creatinine concentration, the serum urea nitrogen concentration and the 24h urine protein concentration of the IgA nephropathy mice subjected to the gavage by using fucose are obviously reduced compared with the control group, but the low dose group and the high dose group have no obvious difference in related indexes (see figures 9-11), and the results show that the fucose gavage can effectively improve the kidney function and the proteinuria of the IgA nephropathy mice.

2) Pathological changes of kidney

As shown in FIG. 12, the result of PAS staining of kidney pathological section of IgA nephropathy mice after administration shows that mesangial cells of non-administration IgA nephropathy mice are remarkably increased, meanwhile, mesangial matrix expansion of mice under a microscope after administration is lighter, glomerular morphology is normal, and glomerular basement membrane thickening is not obvious. The high-dose or low-dose salt trehalose has obvious protective effect on kidney tissues and cells of IgA nephropathy mice, and can obviously relieve kidney injury of the IgA nephropathy mice.

The foregoing descriptions of specific exemplary embodiments of the present invention are presented for purposes of illustration and description. It is not intended to limit the invention to the precise form disclosed, and obviously many modifications and variations are possible in light of the above teaching. The exemplary embodiments were chosen and described in order to explain the specific principles of the invention and its practical application to thereby enable one skilled in the art to make and utilize the invention in various exemplary embodiments and with various modifications as are suited to the particular use contemplated. It is intended that the scope of the invention be defined by the claims and their equivalents.

Claims

1. Use of fucose as a single active ingredient in the manufacture of a medicament, characterized in that the medicament is for preventing and/or treating a disease or disorder in a subject, or for reducing the risk of a disease or disorder in a subject; the disease or condition is kidney disease; the kidney disease is IgA kidney disease.

2. The use according to claim 1, wherein the subject is a human or non-human animal.