Application of Tnfaip2 as target spot in preparation of medicine for preventing and treating candida albicans fungal dermatosis

Abstract

The invention relates to application of Tnfaip2 as a target spot in preparation of a medicine for preventing and treating candida albicans fungal dermatosis, and belongs to the technical field of biological medicine. The invention discloses the application of Tnfaip2 as a target gene in the aspect of treating candida albicans dermatosis for the first time, and particularly, the Tnfaip2 realizes the treatment effect by mediating the polarization of macrophages to the M1 direction and enhancing the sterilization capability of the macrophages. At present, although Tnfaip2 is applied to cancer treatment, Tnfaip2 gene mediating polarization of macrophages to M1 type and participation in research on inflammatory skin diseases is still in the starting stage, and particularly, the Tnfaip2 gene promoting the sterilization ability of the macrophages and the molecular mechanism acting on Candida albicans skin disease treatment are not clearly elaborated in existing literatures and reports. The research clarifies a molecular mechanism that the Tnfaip2 gene mediates macrophages to be polarized to the M1 type through an NF kappa B signal channel, and a more targeted treatment strategy can be derived according to a Tnfaip2 gene target spot, so that the symptoms of a patient are effectively relieved, and the life quality of the patient is promoted.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical technology, and particularly to the application of Tnfaip2 as a target in the preparation of drugs for preventing and treating fungal skin diseases caused by Candida albicans. In particular, it relates to the application of Tnfaip2 as a target in the development or screening or preparation of drugs for preventing and treating Candida albicans skin diseases. Background Art

[0002] Candida albicans is a common opportunistic fungal infection disease, mostly superficial infections of the oral cavity, vagina or skin, and the symptoms include leukoplakia, erythema, itching and irritation symptoms. Conditions that contribute to Candida albicans skin infection include: hot and humid climate, poor hygiene habits, diabetes, HIV infection or the use of immunosuppressants, etc. In 2023, according to the newly revised Merck Manual of Diagnosis and Therapy, the main symptoms and signs of cutaneous mucous membrane candidiasis are as follows: The infection in the folded area is manifested as itching, well-defined erythema of various sizes and shapes, which may be less noticeable in patients with darker skin color, and there may be satellite lesions such as papules and pustules around the primary patch. Perianal candidiasis is accompanied by white maceration and itching. Vulvovaginal candidiasis causes itching and discharge. Currently, the commonly used treatment methods include topical or oral antifungal drugs, etc. However, although these methods can control inflammation and fungal infection to a certain extent, there are many drawbacks. For example, long-term use of topical antifungal drugs may cause adverse reactions such as local skin allergy and burning sensation; oral antifungal drugs may cause systemic side effects such as liver and kidney function damage and gastrointestinal discomfort, and some patients are prone to drug resistance, which greatly reduces the treatment effect.

[0003] Innate immunity is the first line of defense for the body to resist infection, mainly through the pathogen recognition receptors (PRRs: TLR, C-type lectin receptors, CLRs, the galectin family proteins) of innate immune cells to recognize pathogen-associated molecular patterns (PAMPs). Innate immune cells include phagocytes, NK cells, basophils and eosinophils, and other epithelial or endothelial cell components (keratinocytes, respiratory epithelial cells, etc.). The pathogenesis of cutaneous candidiasis involves the central role of macrophages in the innate immune response. Some studies have shown that the main mechanism for the host to resist Candida albicans infection is the recognition, phagocytosis and killing by macrophages at the infection site (Hu Shaohua, Ge Jiaqi, Han Qi. A Candida albicans strain expressing GFP / mCherry: construction and application in the research on macrophage - C. albicans interaction. Acta Microbiologica Sinica, 2023, 63(11): 4208-4217). In the field of immunotherapy for fungal skin diseases, there are also significant deficiencies in the existing technologies. Current immunotherapy for fungal skin diseases mostly relies on generally activating innate immune cells, lacking precise targeting and being unable to specifically enhance the recognition, phagocytosis and killing efficiency of macrophages against Candida albicans. Macrophage polarization is a dynamic process, and it is difficult for existing technologies to ensure that macrophages always maintain a polarized state that is beneficial to combating Candida albicans infection, resulting in the difficulty of lasting immunotherapy effects. Moreover, the existing immunotherapy technologies have a relatively narrow scope of application. For special populations with immunodeficiency such as diabetes, HIV infection or those undergoing immunosuppressive therapy, conventional immunotherapy methods often have poor effects and cannot effectively compensate for their own immune deficiencies and enhance their ability to resist Candida albicans infection. In addition, during the immunotherapy process of existing technologies, it is easy to trigger an excessive immune response, leading to the massive release of inflammatory factors, which in turn causes damage to the normal tissues of the body and aggravates the pain of patients.

[0004] Tnfaip2 (TNF alpha induced protein 2 Gene), also known as tumor necrosis factor alpha-induced protein 2, is upregulated upon TNFα stimulation. Studies have found that Tnfaip2 plays multiple roles in various physiological and pathological conditions. The representative functions of Tnfaip2 include triggering inflammatory responses, promoting angiogenesis, promoting cell proliferation, adhesion, migration, and inducing the formation of tunneling nanotubes. Currently, although there are studies on Tnfaip2 in cancer treatment, the research on the regulation of macrophage polarization mediated by Tnfaip2 is less, especially there is no relevant report on the application of Tnfaip2 as a target in the development, screening, or preparation of drugs for the prevention and treatment of Candida albicans skin diseases. Existing treatment methods cannot precisely target and regulate the process of macrophage polarization, making it difficult to efficiently enhance the body's own immune defense ability against Candida albicans. However, this invention is expected to overcome the above deficiencies in the treatment of Candida albicans skin diseases by studying Tnfaip2 and develop a safer and more effective treatment strategy. Summary of the Invention

[0005] In view of the above problems, the present invention provides the application of the Tnfaip2 gene as a target in the development, screening, or preparation of drugs for the prevention and treatment of Candida albicans skin diseases.

[0006] To achieve the above invention purposes, the present invention provides the following technical solutions.

[0007] The present invention discloses the application of a regulator targeting the Tnfaip2 gene in the preparation of drugs for the treatment of Candida albicans skin diseases.

[0008] Further, the regulator targeting the Tnfaip2 gene is to promote the expression of Tnfaip2.

[0009] Further, the regulator includes α-(1,6)(1,2)-mannoprotein.

[0010] The present invention also discloses the application of a reagent for detecting the expression level of Tnfaip2 in the preparation of a product for predicting the efficacy of drugs for the treatment of Candida albicans fungal skin diseases.

[0011] Further, the drugs for the treatment of Candida albicans fungal skin diseases include α-(1,6)(1,2)-mannoprotein.

[0012] Further, the product contains specific primers for amplifying the Tnfaip2 gene and probes hybridizing with the nucleotide sequence of the Tnfaip2 gene.

[0013] Further, it is characterized in that the specific primer sequences for amplifying the Tnfaip2 gene are shown in SEQ ID NO.1 and SEQ ID NO.2.

[0014] The present invention also discloses an immunomodulatory reagent, which is characterized by comprising a reagent for regulating the Tnfaip2 gene.

[0015] The present invention also discloses a reagent for regulating macrophage M1 polarization, which is characterized by comprising a reagent for regulating the Tnfaip2 gene.

[0016] Furthermore, according to the reagent described in any one of the above, it is characterized in that the reagent for regulating the Tnfaip2 gene comprises a reagent for promoting Tnfaip2 expression or interfering nucleic acid targeting the Tnfaip2 gene; the reagent for promoting Tnfaip2 expression includes α-(1,6)(1,2)-mannan protein; the interfering nucleic acid is small interfering RNA (siRNA), and its sequences are shown in SEQ ID NO:3~4.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows.

[0018] The present invention reveals the detailed molecular mechanism of the upregulation of the TNFAIP2 gene through the NFκB pathway after Candida albicans infects macrophages. By constructing a gene knockdown model, it is found that the deletion of Tnfaip2 significantly inhibits the migration ability of macrophages, reduces the secretion of inflammatory factors such as IL-6 and TNF-α, and hinders the M1 polarization process. Experiments confirm that after knocking down the Tnfaip2 gene with small interfering RNA, the bactericidal activity of macrophages against Candida albicans decreases. Stimulation with α-(1,6)(1,2)-mannan protein can significantly upregulate Tnfaip2 expression by activating the NFκB pathway, enhance the M1 polarization phenotype (CD86 + ) and improve the bactericidal function. Further studies show that overexpression of Tnfaip2 can promote the migration defect and bactericidal activity of knockdown cells. The present invention clarifies for the first time the protective role of Tnfaip2 in Candida albicans infection, and its expression level is positively correlated with macrophage function, providing a new molecular target and immunomodulatory strategy for the treatment of Candida albicans-related skin diseases.

[0019] Patent CN109021083A discloses a Candida albicans cell wall mannan protein that induces macrophage polarization and enhances its immune function. By purifying a cell wall polysaccharide α-(1,6)(1,2)-mannan protein from Candida albicans, it has been found through research that this protein can stimulate macrophages to activate in the M1 direction. However, the content related to Tnfaip2 and α-(1,6)(1,2)-mannan protein has not been disclosed. Through a large number of experiments, the present invention has demonstrated that the key gene regulating macrophage M1 polarization is the Tnfaip2 gene, and it has also been clarified that knocking down the Tnfaip2 gene using small interfering RNA can reduce the bactericidal activity of macrophages and inhibit macrophage polarization in the M1 direction. In addition, it has been found that α-(1,6)(1,2)-mannan protein can promote the overexpression of the Tnfaip2 gene. This series of discoveries has laid a solid foundation for the successful implementation of the "targeted immunomodulation" strategy in the prevention and treatment of Candida albicans skin diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 For the transcriptome sequencing results, it is found that a large number of differentially expressed genes in raw264.7 cells treated with α-(1,6)(1,2)-mannan protein are enriched in the NFκB pathway and the expression of the Tnfaip2 gene is upregulated. A is the heat map of differentially expressed genes, B is the KEGG pathway analysis of differentially expressed genes, and C is the volcano plot of differentially expressed genes.

[0021] Figure 2 For the QPCR and WB results of raw264.7 cells. A shows the changes in the mRNA levels of NFKB2, NFKBIA, iNOS, Arg-1, and IL-6. B shows the changes in the mRNA level of Tnfaip2 in raw264.7 cells. C shows the changes in the proteins related to Tnfaip2 and the NFκB pathway in raw264.7 cells and the gray value analysis of the WB results.

[0022] Figure 3 For the positive control group (AC), the blank control group (NC), macrophages treated with α-(1,6)(1,2)-mannan protein (MP), inhibition of the NFκB pathway (PDTC), and inhibition of the expression of the Tnfaip2 gene (si-Tnfaip2), the results of detecting the ROS secretion of macrophages are shown.

[0023] Figure 4 For the blank control group (NC), macrophages treated with α-(1,6)(1,2)-mannan protein (MP), and inhibition of the expression of the Tnfaip2 gene (si-Tnfaip2), the results of the scratch experiment to detect macrophage migration are shown.

[0024] Figure 5 For the results of flow cytometry detecting macrophage surface markers. A is the flow cytometry result, and B is the statistical chart of the flow cytometry result.

[0025] Figure 6 To establish a Tnfaip2 gene knockdown mouse model using C57BL / 6 and the QPCR results of skin tissues. A is the flowchart for establishing mice with Tnfaip2 gene knockdown and control blank small interfering mice by subcutaneous injection, B is the QPCR result of the effect of intradermal injection of MP concentration gradient on the Tnfaip2 gene, and C is the QPCR result of the Tnfaip2 gene expression in the PBS group, MP group, Chol-2’OMe-siTnfaip2, and Chol-2’OMe–siNC groups.

[0026] Figure 7 A is the flowchart for mouse model establishment, and B and C are representative pictures of the mouse model establishment results.

[0027] Figure 8 Representative pictures of the HE staining results of mouse model tissues, pathological score, and statistical results of epidermal thickness. A is the HE staining picture, B is the tissue pathological score, and C is the epidermal thickness result.

[0028] Figure 9 Representative pictures of the immunohistochemical results of mouse model tissues. A is the representative immunohistochemical picture, and B and C are the statistical results of the number of CD86 + and CD163 + positive cells. Detailed implementation manners

[0029] The following uses specific examples to further describe the present invention in detail. However, this should not be understood that the scope of the above-mentioned subject matter of the present invention is limited to the following examples. All technologies implemented based on the content of the present invention belong to the scope of the present invention.

[0030] Unless otherwise specified, the reagents and materials used in the present invention are all commercially available.

[0031] In the attached drawings, NC represents the blank control group, MP represents the Tnfaip2 agonist group, i.e., the group of macrophages treated with α-(1,6)(1,2)-mannan protein, PDTC represents the group of macrophages pretreated with the NFκB pathway inhibitor and then stimulated with MP, and si-Tnfaip2 represents the group of macrophages stimulated with MP after knocking down the Tnfaip2 gene with small interfering RNA. The experimental methods in the following examples are all conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples can be obtained through commercial channels unless otherwise specified. In the attached drawings of the present invention, NS represents no statistical significance, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0032] Prepare α-(1,6)(1,2)-mannoprotein according to the content disclosed in Patent CN109021083A for subsequent experiments.

[0033] Example 1. Results obtained by performing transcriptome sequencing on macrophage Raw264.7 cells treated with α-(1,6)(1,2)-mannoprotein ( Figure 1 ). The heatmap shows obvious expression differences between the blank control group (NC) and the α-(1,6)(1,2)-mannoprotein treatment group (MP) ( Figure 1 A). KEGG analysis shows that most of the differential genes are clustered in the NFκB pathway ( Figure 1 B). The volcano plot shows the differentially expressed genes (DEGs) of the blank group and macrophages treated with α-(1,6)(1,2)-mannoprotein. Red indicates significantly upregulated genes (Tnfaip2), and blue indicates significantly downregulated genes. After macrophages are treated with α-(1,6)(1,2)-mannoprotein, the expression of the Tnfaip2 gene increases significantly ( Figure 1 C).

[0034] Example 2. Verify the relationship between macrophages and Tnfaip2.

[0035] In subsequent experiments, 4 experimental groups are set up, namely: Blank control group (NC); Tnfaip2 agonist group (MP): Treat macrophages with α-(1,6)(1,2)-mannoprotein.

[0036] NFκB pathway inhibitor group (PDTC): Pretreat cells with PDTC to inhibit the NFκB pathway before adding α-(1,6)(1,2)-mannoprotein; Tnfaip2 gene knockdown group (si-Tnfaip2): Knock down the expression of the Tnfaip2 gene using RNA interference technology and then treat the cells with α-(1,6)(1,2)-mannoprotein.

[0037] The si-Tnfaip2 sequence is (Sense 5′-GCACCUGCACCUAGUGAAATT-3′, antisense 5′-UUUCACUAGGUGCAGGUGCTT- 3′) 2. Primers for Arg-1, IL-6, Tnfaip2, NFKB2, and NFKBIA were prepared according to the published literature for qPCR reactions. Arg-1 (forward: 5′-ACATTGGCTTGCGAGACGTA-3′, reverse: 5′-ATCACCTTGCCAATCCCCAG-3′); IL-6 (forward: 5′-TCCATCCAGTTGCCTTCT-3′, reverse: 5′-TAAGCCTCCGACTTGTGA-3′); Tnfaip2 (forward: 5′-AGGAGGAGTCTGCGAAGAAGA-3′, reverse: 5′-GGCAGTGGACCATCTAACTCG-3′); NFKB2 (forward: 5′-GGCCGGAAGACCTATCCTACT-3′, reverse: 5′-CTACAGACACAGCGCACACT-3′); NFKBIA (forward: 5′-TGAAGGACGAGGAGTACGAGC-3′, reverse: 5′-TTCGTGGATGATTGCCAAGTG-3′). The reaction system and conditions were set according to the qPCR kit instructions, and the relative expression levels of the target genes were detected by a fluorescence quantitative PCR instrument ( Figure 2 A). Judging from the results, MP promoted the expression of Tnfaip2 ( Figure 2 B), indicating that this protein is an agonist of Tnfaip2. With the assistance of MP, the expression of Tnfaip2 was up-regulated, which in turn promoted the expression of pro-inflammatory genes such as IL-6 and promoted the polarization of macrophages to M1. After knocking down the Tnfaip2 gene (si-Tnfaip2 group), there were significant differences compared with the Tnfaip2 agonist group (MP group) and the NC group, and the expression of pro-inflammatory genes was down-regulated, indicating that the deletion of the Tnfaip2 gene affected the expression of these genes, thereby inhibiting the polarization of macrophages to M1 and affecting the immune function and inflammatory response of macrophages. The change in the expression level of the Tnfaip2 gene greatly affected the process of macrophage M1 polarization. Since macrophages play a key immune role in defending against dermatophytosis caused by Candida albicans, the difference in Tnfaip2 expression plays an important role in the pathogenesis and immune defense of dermatophytosis caused by Candida albicans by affecting macrophage function, and MP assists in regulating the expression of Tnfaip2 in this process.

[0038] Detection of WB level changes and its statistical chart were performed in the raw264.7 cell line ( Figure 2C), presenting the bands of proteins such as Tnfaip2, p-p65, p65, and Iκbα, as well as GAPDH as an internal reference. The expression levels of proteins such as Tnfaip2, p-p65, Iκbα, and p65 relative to the internal reference GAPDH were quantified respectively. It can be seen that there were significant changes in the expression levels of Tnfaip2, p-p65, and Iκbα proteins treated with the Tnfaip2 agonist compared with the NC group. Among them, the expression levels of Tnfaip2 and p-p65 increased, while the expression level of Iκbα decreased ( Figure 2 C). There were also differences to varying degrees between the PDTC treatment group and the si-Tnfaip2 treatment group and the NC group, indicating that these treatment factors had a regulatory effect on protein expression. These results suggested that the treatment with the Tnfaip2 agonist affected the NFκB signaling pathway by regulating the expression of proteins such as Tnfaip2, p-p65, p65, and Iκbα. After knocking down the Tnfaip2 gene (si-Tnfaip2 group), the expression level of the Tnfaip2 protein decreased, the phosphorylation level of p-p65 decreased, and the expression level of the Iκbα protein increased relatively, indicating that the activation of the NF-κB signaling pathway was inhibited.

[0039] The Raw264.7 cells in the NC, MP, PDTC, and si-Tnfaip2 groups were treated accordingly, ROS probes were added, stained according to the instructions, and the fluorescence intensity of ROS in the cells was detected using a fluorescence microscope or a flow cytometer to reflect the intracellular reactive oxygen species level ( Figure 3 ). AC was the positive control provided with the reactive oxygen species detection kit. It could be visually seen that the fluorescence in the NC group was weak; the green fluorescence in the Tnfaip2 agonist group (MP group) was significantly stronger than that in the NC group, indicating that the treatment with the Tnfaip2 agonist (MP) significantly increased the intracellular ROS level; the fluorescence intensities in the PDTC group and the si-Tnfaip2 group also increased, but not as significantly as in the Tnfaip2 agonist group, suggesting that the promotion effect of these two treatments on the intracellular ROS level was not as good as that of the Tnfaip2 agonist ( Figure 3 A). The abscissa was different treatment groups, and the ordinate was Mean density of ROS fluorescence (reflecting the ROS level). The experimental results showed that the treatment with the Tnfaip2 agonist (MP) could significantly increase the intracellular ROS level in Raw264.7 cells, while the effects of PDTC and si-Tnfaip2 treatments on the intracellular ROS level were not as significant as that of the Tnfaip2 agonist under the experimental conditions. This was of great significance for studying the oxidative stress response of cells and the action mechanisms of related treatment factors. Figure 3B). The average fluorescence density of ROS in the si-Tnfaip2 group was much lower than that in the Tnfaip2 agonist MP group, further indicating that after knocking down the Tnfaip2 gene, the production of ROS in macrophages could not increase significantly like that in the Tnfaip2 agonist MP treatment group, suggesting that the Tnfaip2 gene has an impact on the process of MP-induced ROS production in macrophages. Upregulation of Tnfaip2 gene expression promotes ROS secretion and macrophage bactericidal activity.

[0040] Add serum-free medium to the upper chamber of the Transwell chamber and serum-containing medium to the lower chamber ( Figure 4 ). After digesting and counting the Raw264.7 cells treated with the NC, MP, PDTC, and si-Tnfaip2 groups, inoculate them into the upper chamber ( Figure 4 A). After culturing for 24 h, take out the chamber, wipe off the non-migrated cells in the upper chamber, stain and count the migrated cells in the lower chamber, and analyze the cell migration ability ( Figure 4 B). In the NC group, the number of cells migrated to the lower chamber was small; in the Tnfaip2 agonist group, the number of cells in the lower chamber of the MP group increased significantly, and the blue-stained cells were dense, indicating that the treatment with the Tnfaip2 agonist MP significantly promoted the migration of Raw264.7 cells; the number of cells in the lower chamber of the si-Tnfaip2 group was small and similar to that in the NC group, suggesting that after knocking down the Tnfaip2 gene, the migration ability of the cells was significantly inhibited and close to the level of the untreated NC group. The number of migrated cells in the Tnfaip2 agonist MP group was significantly higher than that in the NC group, further confirming that the treatment with the Tnfaip2 agonist MP could greatly enhance the cell migration ability; while there was no significant difference between the si-Tnfaip2 group and the NC group, and the number of migrated cells was much lower than that in the Tnfaip2 agonist MP group, indicating again that knocking down the Tnfaip2 gene would inhibit the promoting effect of the Tnfaip2 agonist on cell migration. Generally speaking, the experimental results show that upregulation of Tnfaip2 expression significantly promotes the migration of Raw264.7 cells, while knocking down the Tnfaip2 gene weakens this promoting effect.

[0041] Use flow cytometry to detect cell surface markers ( Figure 5 ). Collect the cells of the blank control group, the Tnfaip2 agonist group, and the Tnfaip2 knockdown group, stain them with anti-CD86 antibody and anti-CD206 antibody, then incubate at 4 °C for 60 min and wash the cells 3 times with PBS, and then resuspend the cells with PBS to prepare a solution with a cell concentration of 1×10 5 cells / mL. Use a flow cytometer for flow cytometry analysis and process the data using FlowJo software.

[0042] The figure shows the expression of two surface markers, CD86 and CD206, in cells of four groups: NC (control group), MP (Tnfaip2 agonist group), PDTC (NFκB pathway inhibitor group), and si-Tnfaip2 (treatment group with knocked-down Tnfaip2 gene). Each figure is divided into four quadrants (Q1-Q4), and cell populations in different quadrants represent different marker expression characteristics. The numbers in the figure indicate the proportion of cells in each quadrant ( Figure 5 A).

[0043] Blank control group: Most cells are concentrated in the Q4 quadrant, indicating that cells in this group have low expression of CD206 and low expression of CD86.

[0044] Tnfaip2 agonist group: There are obvious changes in cell distribution, and the proportion of cells in the Q3 quadrant increases significantly, meaning that after treatment with α-(1,6)(1,2)-mannoprotein, cells have high expression of CD86 and low expression of CD206.

[0045] PDTC group: The cell distribution changes significantly compared with that of the Tnfaip2 agonist group, and the number of cells in the Q3 quadrant decreases significantly.

[0046] si-Tnfaip2 group: The cell distribution is between that of the blank control group and the Tnfaip2 agonist group, and there are a certain number of cells in both the Q3 and Q4 quadrants, indicating that after knocking down the Tnfaip2 gene, the expression of cell surface markers has changed, but the effect is not as good as that of Tnfaip2 agonist treatment.

[0047] The proportion of CD86 + macrophages in different groups was counted, visually showing the differences between groups.

[0048] Blank control group: The proportion of CD86 + macrophages is very low.

[0049] Tnfaip2 agonist group: The proportion of CD86 + macrophages increases significantly, showing a significant difference compared with the NC group.

[0050] PDTC group: CD86 + is significantly lower than that of the Tnfaip2 agonist group si-Tnfaip2 group: CD86 + macrophages are significantly lower than those of the Tnfaip2 agonist group.

[0051] Overall, this set of figures shows that treatment with Tnfaip2 agonist can significantly promote the expression of CD86 in macrophages, and knocking down the Tnfaip2 gene weakens the effect of MP. The M1 polarization of macrophages is closely related to the upregulation of Tnfaip2.

[0052] Example 3. Experiment on the correlation between Tnfaip2 agonist (MP) and Candida albicans dermatosis.

[0053] Described are the results of skin tissue QPCR after the establishment of a mouse gene knockdown model and drug treatment. Facilitate the study of the role of the Tnfaip2 gene in the process of Candida albicans infection and the impact of differences in Tnfaip2 expression levels on the disease condition ( Figure 6 A-C).

[0054] The specific operations are as follows.

[0055] 1. Preparation of Candida albicans: The Candida albicans strain (ATCC90028) used in this study was purchased from Beijing Na Biotechnology Co., Ltd.

[0056] Candida albicans was cultured in YDP medium. After taking it out of the storage environment, it was inoculated into YDP medium and placed in an incubator at 37 °C, and cultured with shaking at a speed of 160 rpm / min for 16-18 h. After the culture was completed, Candida albicans was collected by centrifugation under the condition of 2500 g, and configured to 1×10 9 CFU / mL for subsequent in vitro and in vivo infection experiments, and the injection volume was 100 μL.

[0057] 2. Establishment of a gene knockdown mouse model: Chol-2’OMe-siTNFAIP2 is a small interfering RNA of Tnfaip2 with a 2-methoxy-modified base sequence and cholesterol modification at the end, which was synthesized and provided by Hema Biotechnology Co., Ltd., Huzhou, Zhejiang, and used according to the manufacturer's instructions.

[0058] The method for establishing the Chol-2’OMe-siTNFAIP2 mouse model is as follows: Dissolve the Chol-2’OMe-siTNFAIP2 powder in sterile normal saline, inject 5 nmol at a time, inject twice a week, and it takes effect after 4 days for observation ( Figure 6 A).

[0059] The mice were divided into two groups: one group was first subcutaneously injected with Chol-2’OMe-siNC (cholesterol-modified and 2'-methoxy-modified non-targeting control small interfering RNA), and then injected with Chol-2’OMe-siNC again 3 days later. Another 3 days later, Candida albicans was injected. Here, Chol-2’OMe-siNC served as the control group to compare the effects of other treatment groups. The other group was first subcutaneously injected with Chol-2’OMe-siTNFAIP2 (cholesterol-modified and 2'-methoxy-modified small interfering RNA targeting the Tnfaip2 gene), and Chol-2’OMe-siTNFAIP2 was injected again at the same interval of 3 days. Another 3 days later, Candida albicans was also injected (Chol-2’OMe-siTNFAIP2 group). This group was used to explore the effect of knocking down the Tnfaip2 gene on murine Candida albicans dermatosis.

[0060] 3. Dermal injection: C57BL / 6J male mice (8 weeks old, weighing 20 - 25 g) were used. 24 h before injection, the mice were anesthetized and the hair on their backs was shaved. Different concentration gradients of MP (10 μg / mL, 20 μg / mL, 40 μg / mL, 60 μg / mL, 80 μg / mL, 100 μg / mL) were injected respectively. 100 μL of the above liquid was injected into the deep dermis. The mice were sacrificed on the fifth day, and the back tissues were taken for subsequent experiments. Figure 6B). Depilate C57 mice one day in advance, refer to the experimental protocol of the published literature and improve it based on this (Ishikawa Y, Kirikae T, Hirata M, et al. Local skin response in mice induced by a single intradermal injection of bacterial lipopolysaccharide and lipid A. [J]. Infection & Immunity, 1991, 59(6): 1954. DOI: 10.1007 / BF01963949) (Alfituri O A, Mararo E M, Steketee P C, et al. Dermal bacterial LPS-stimulation reduces susceptibility to intradermal Trypanosoma brucei infection [J]. Scientific Reports, 2021, 11(1): 9856). The group injected with PBS was used as a control. After 24 h of injection, the back skin tissue of the mice was extracted. Using the high-purity RNA extraction kit purchased from Beijing TransGen Biotech Co., Ltd. and used according to the instructions, QPCR was used to detect the expression level of Tnfaip2 in the skin tissue to explore the appropriate concentration of MP injection ( Figure 6 B). It was found that there were significant changes in Tnfaip2 in the skin tissue at 80 μg / mL and 100 μg / mL. Therefore, 100 μg / mL of MP was used in subsequent experiments. Mouse tissue QPCR experiments were conducted to explore the role of the Tnfaip2 target ( Figure 6 C). The mice were divided into 4 groups (n = 5 in each group): blank control group (PBS), injected with 100 μg / mL of MP, Tnfaip2 gene knockdown group (Chol-2’OMe-siTNFAIP2), and blank small interfering RNA transfection group (Chol-2’OMe-siNC group). Observe the changes in the mRNA level of the Tnfaip2 gene in the mice models with small interfering RNA knockdown of the Tnfaip2 gene and blank small interfering RNA after 24 h of subcutaneous injection and after the establishment of subcutaneous injection ( Figure 6C). The PBS blank control group showed a baseline level of Tnfaip2 gene mRNA expression. Tnfaip2 gene mRNA levels were significantly lower in the Tnfaip2 knockdown group (Chol-2'OMe-siTNFAIP2), indicating that this knockdown strategy inhibited gene expression. However, Tnfaip2 gene mRNA levels did not differ significantly in the blank small interfering RNA transfection group (Chol-2'OMe-siNC group) compared with the control group, confirming that this group had no interference effect.

[0061] The treatment process of Candida albicans model mice and the skin conditions at different time points are shown ( Figure 7 Thirty 8-week-old C57BL / 6J male mice (weighing 20-25 g) were anesthetized and their back hair was shaved 24 h before infection. They were then divided into six groups (n=5 per group): PBS group, C.albicans+PBS group, C.albicans+MP group, MP+C.albicans group, Chol-2'OMe-siTNFAIP2 group, and Chol-2'OMe-siNC group. Figure 7 A presents the modeling process. Figure 7 BC shows the skin changes of each group from Day 0 to Day 5.

[0062] PBS group: During the observation period, the skin was smooth without redness, swelling or ulcers, indicating that white PBS had no obvious adverse stimulation to the skin and could be used as a control benchmark.

[0063] In the C.albicans+PBS group, the skin at the depilated area was normal on Day 0, and began to turn red on Day 1. The area and severity of the red spots increased over time, and obvious red spots appeared on Day 5, indicating that Candida albicans successfully induced skin inflammation and the inflammation continued to develop.

[0064] In the C.albicans+MP group, MP was injected after pre-injection of Candida albicans. The degree and area of skin redness on Days 1–3 were lighter than those in the C.albicans+PBS group, and the redness remained relatively mild on Day 5, suggesting that MP has an inhibitory effect on established Candida albicans skin inflammation.

[0065] MP+C.albicans group: MP was injected beforehand and then Candida albicans was injected. Although the skin was red on Days 1–5, the overall degree was the mildest and the inflammation developed the slowest, indicating that MP pre-intervention can enhance the skin's ability to resist Candida albicans infection and play a preventive and protective role.

[0066] Chol-2’OMe-siTNFAIP2 group: After knocking down the Tnfaip2 gene, the skin was normal on Day 0, showed flushing on Day 3, and the flushing area expanded and deepened on Day 5. It is speculated that knocking down the Tnfaip2 gene affects the immune or inflammatory regulatory pathway, weakens the skin's resistance to infection, and exacerbates the inflammatory response.

[0067] Chol-2’OMe-siNC group: As a blank control to verify the specificity of the interference operation.

[0068] In summary, MP has both therapeutic and preventive potential for skin inflammation caused by Candida albicans, and the preventive effect of injecting MP first is better; while knocking down the Tnfaip2 gene exacerbates inflammation, further highlighting the positive role of MP intervention.

[0069] Showed the analysis of skin tissues at the modeling site of mice by HE staining ( Figure 8 ), including histological section images ( Figure 8 A) and histological scores ( Figure 8 B), and quantitative analysis of epidermal thickness ( Figure 8 C). The specific example method is as follows: Samples of the skin at the modeling site of mice in different treatment groups were taken on the fifth day, histological sections were made and stained with HE, the tissue morphology was observed through a 5-fold and 10-fold microscope, and the histological scores and epidermal thickness were quantitatively statistically analyzed.

[0070] Analysis of histological section images ( Figure 8 A) PBS group: The skin tissue structure was normal, the epidermal cells were arranged neatly, and there was no obvious infiltration of inflammatory cells, indicating that PBS treatment had no obvious damage to the skin.

[0071] C.albicans + PBS group: The epidermis was significantly thickened, the cell arrangement was disordered, and a large number of inflammatory cells were infiltrated, indicating that Candida albicans infection caused a strong inflammatory response and damaged the skin tissue structure.

[0072] C.albicans + MP group: The degree of epidermal thickening and the infiltration of inflammatory cells were reduced compared with the C.albicans group, indicating that using MP treatment after Candida albicans infection could relieve the inflammatory response and tissue damage to a certain extent.

[0073] MP + C.albicans group: The degree of epidermal thickening and inflammatory cell infiltration was improved compared with the C.albicans group, and the tissue morphology was better than that of the C.albicans + MP group, indicating that pre-treatment with MP had a protective effect on the skin of mice against Candida albicans infection.

[0074] Chol-2’OMe-siNC group: As the control small interfering RNA group, the changes in skin tissues were used to compare the effects of the Chol-2’OMe-siTNFAIP2 group.

[0075] Chol-2’OMe-siTNFAIP2 group: Compared with the Chol-2’OMe-siNC group, the inflammatory response and structural changes in skin tissues were more obvious, and it was used to study the effect of knocking down the Tnfaip2 gene on skin tissues.

[0076] Quantitative analysis.

[0077] Figure 8 B (pathological score): The score of the PBS group was extremely low, indicating that PBS treatment had little damage and inflammation to the skin.

[0078] The score of the C.albicans + PBS group was significantly higher than that of the PBS group, indicating that Candida albicans infection caused severe tissue damage and inflammation.

[0079] The scores of the C.albicans + MP group and the MP + C.albicans group were both lower than that of the C.albicans group, and the score of the MP + C.albicans group was even lower, indicating that MP treatment (regardless of before or after infection) could reduce tissue damage and inflammation, and the effect of pre - using MP was better.

[0080] The score of the Chol-2’OMe-siTNFAIP2 group was higher than that of the Chol-2’OMe-siNC group, indicating that knocking down the Tnfaip2 gene would aggravate skin tissue damage and inflammation.

[0081] Figure 8 C (epidermal thickness): The epidermal thickness of the C.albicans + PBS group was significantly higher than that of the PBS group, confirming that Candida albicans infection led to epidermal thickening.

[0082] MP treatment could reduce the degree of epidermal thickening.

[0083] The epidermal thickness of the Chol-2’OMe-siTNFAIP2 group was higher than that of the Chol-2’OMe-siNC group and was close to that of the C.albicans group, indicating that after knocking down the Tnfaip2 gene, the resistance of skin tissues decreased and the epidermal thickening was aggravated.

[0084] In summary, the experimental results revealed that pretreatment with the Tnfaip2 agonist (MP) activated the bactericidal effect of macrophage M1 polarization in advance, reduced the persistent inflammatory effect of Candida albicans on the skin, and alleviated the skin structure changes caused by its colonization; while knocking down the Tnfaip2 gene would aggravate the inflammatory response and tissue damage.

[0085] showed the expression of CD86 (a surface marker of M1 macrophages) and CD163 (a surface marker of M2 macrophages) in the skin tissues at the modeling site of mice by immunohistochemical quantitative analysis. The specific operation was to sample the modeling sites of mice in different model groups five days after Candida albicans infection and perform immunohistochemical staining ( Figure 9 ).

[0086] Immunohistochemical staining images ( Figure 9 A) Images of different treatment groups showed the positive staining distribution of CD86 and CD163, providing an intuitive histological basis for subsequent quantitative analysis.

[0087] Quantitative analysis of positive cells ( Figure 9 B-C) Analysis of CD86 index: PBS group: As a blank control, the number of positive cells of CD86 was low, indicating that the number of M1 macrophages was small under normal conditions and there was no obvious inflammatory reaction.

[0088] C.albicans + PBS group: The number of positive cells of CD86 was significantly higher than that of the PBS group, indicating that Candida albicans infection could induce a large number of macrophages to polarize into M1 type, triggering a strong inflammatory reaction.

[0089] C.albicans + MP group: After treatment with MP after infection, the positive staining of CD86 increased compared with the C.albicans group, indicating that MP could promote the polarization of M1 macrophages to a certain extent after infection and enhance the ability to kill Candida albicans.

[0090] MP + C.albicans group: After pretreatment with MP and then infection with Candida albicans, the number of positive cells of CD86 was higher, indicating that MP pretreatment could activate the M1 polarization of macrophages in advance to eliminate some fungi, enabling the body to activate a large number of macrophages to polarize into M1 type in the early stage of infection, thereby enhancing the ability to resist Candida albicans.

[0091] Chol-2’OMe-siTNFAIP2 group: After knocking down the Tnfaip2 gene, the positive staining of CD86 was lower than that of the Chol-2’OMe-siNC group, indicating that the deletion of the Tnfaip2 gene affected the normal regulation of macrophage M1 polarization, resulting in a decrease in M1 macrophages during infection and making it difficult to effectively control the inflammatory reaction caused by Candida albicans.

[0092] Analysis of CD163 index: The PBS group was the lowest, and the C. albicans + PBS group, C. albicans + MP group, and MP + C. albicans group increased successively, and the differences were statistically significant. It indicates that MP intervention also has an impact on the polarization of M2 macrophages during C. albicans infection. Moreover, the percentage of CD163+ macrophages in the pre-treated MP group (MP + C. albicans) was the highest, and its promoting effect on M2 polarization was more significant.

[0093] There was no significant difference in the number of CD163-positive cells between the Chol-2’OMe-siTNFAIP2 group and the Chol-2’OMe-siNC group, which only showed a tendency of increased M2-polarized macrophages in the Chol-2’OMe-siTNFAIP2 group compared with the Chol-2’OMe-siNC group. This may be because knocking down the Tnfaip2 gene would interfere with the regulation of macrophage M1 polarization, resulting in a slow and persistent inflammatory response caused by fungal infection, prolonging the course of C. albicans skin disease, and polarizing macrophages towards the M2 anti-inflammatory direction.

[0094] Generally speaking, Figure 9 The results show that the intervention of the Tnfaip2 agonist (MP) can activate macrophage M1 polarization in advance by up-regulating the expression of Tnfaip2 to eliminate fungi, reduce subsequent inflammatory responses, and create conditions for M2 polarization repair; while knocking down the Tnfaip2 gene will lead to an imbalance in the regulation of macrophage M1 polarization and exacerbate the inflammatory response. The non-significant difference in the CD163 index indicates that the polarization and repair process of M2 macrophages may be affected by complex factors and further research is needed.

[0095] The above are only the preferred embodiments of the present invention and are not used to limit the patent scope of the present invention. For those skilled in the art, various modifications and changes can be made to the present invention. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. Use of a regulator targeting the Tnfaip2 gene in the preparation of a drug for treating Candida albicans skin disease.

2. The application according to claim 1, wherein The regulator targeting the Tnfaip2 gene promotes the expression of Tnfaip2.

3. The application according to claim 1, characterized in that, The regulator includes α-(1,6)(1,2)-mannoprotein.

4. Use of a reagent for detecting the expression level of Tnfaip2 in the preparation of a product for predicting the efficacy of a drug for treating Candida albicans fungal skin disease.

5. The application according to claim 4, wherein The drug for treating Candida albicans fungal skin disease includes α-(1,6)(1,2)-mannoprotein.

6. The application according to claim 4, characterized in that, The product contains specific primers for amplifying the Tnfaip2 gene and a probe hybridizing with the nucleotide sequence of the Tnfaip2 gene.

7. The application according to claim 6, characterized in that The sequences of the specific primers for amplifying the Tnfaip2 gene are shown as SEQ ID NO.1 and SEQ ID NO.

2.

8. An immunomodulatory reagent, characterized in that, A reagent for regulating the Tnfaip2 gene.

9. A reagent for regulating macrophage M1 polarization, characterized in that, A reagent for regulating the Tnfaip2 gene.

10. The reagent according to any one of claims 6 to 7, characterized in that Among them, the reagent for regulating the Tnfaip2 gene includes a reagent for promoting the expression of Tnfaip2 or interfering nucleic acid targeting the Tnfaip2 gene; the reagent for promoting the expression of Tnfaip2 includes α-(1,6)(1,2)-mannoprotein; the interfering nucleic acid is small interfering RNA (siRNA), and its sequence is as SEQ ID NO:3~4.

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