Application of Paquinimod in the preparation of drugs for treating secondary lymphedema

Abstract

The present invention is applicable to the field of biomedical technology, and provides the use of Paquinimod in the preparation of a drug for treating secondary lymphedema. The present invention uses Paquinimod, a small molecule inhibitor of S100A8 / A9 dimer, to treat lymphedema mice. The results show that Paquinimod can significantly inhibit the secretion of inflammatory factors in model mice, effectively alleviate edema, and inhibit the activation of the S100A8 / A9-TLR4-CCL2 signaling pathway. Therefore, the present invention uses Paquinimod, an inhibitor of the S100A8 / A9-TLR4 signaling pathway, in the preparation of a drug for treating secondary lymphedema (SL), which opens up new ideas and methods for the treatment of SL and effectively solves the problem of the lack of effective treatment methods for SL.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and in particular relates to the application of Paquinimod in preparing a drug for treating secondary lymphedema. Background Art

[0002] Secondary lymphedema (SL) is a chronic tissue edema caused by impaired lymphatic drainage due to external injury. It is common in patients with gynecological tumors and patients after radiotherapy. For example, about 30% of women will develop lymphedema after receiving breast cancer treatment. The clinical characteristics of lymphedema include progressive swelling, skin fibrosis, chronic inflammation and fat accumulation, which seriously affect the quality of life of patients. Due to the complex pathogenesis of lymphedema and the difficulty in early diagnosis, there is a lack of effective prevention or treatment methods and drugs in clinical practice. Existing treatments such as liposuction and physical therapy often cannot fundamentally solve the cause of lymphedema and may be accompanied by certain side effects.

[0003] S100A8 and S100A9 (denoted as S100A8 / A9), also known as calmodulin, are members of the S100 protein family and are mainly expressed in myeloid cells, especially neutrophils. S100A8 / A9 plays an important role in regulating inflammatory responses. After activation, S100A8 / A9 heterodimers are secreted. These heterodimers can bind to Toll-like receptor 4 (TLR4) as damage-associated molecular patterns (DAMPs), activate proinflammatory cytokines and chemokines, and thus amplify the inflammatory response. Studies have confirmed that elevated levels of S100A8 / A9 are associated with diseases such as rheumatoid arthritis, systemic lupus erythematosus, and psoriasis, and have been identified as biomarkers for COVID-19. Existing studies have not directly linked S100A8 / A9 to SL, leading to the neglect of its potential targets.

[0004] Paquinimod is a small molecule immunomodulatory compound that can prevent S100A8 / A9 from binding to TLR4, thereby inhibiting TLR4-mediated natural immunity, reducing the secretion of proinflammatory factors, and regulating immune responses. It was originally developed for the treatment of systemic lupus erythematosus and has been reported to reduce the viral load of SARS-Cov-2 infected mice and is used in pneumonia research. Based on the above background, the present invention proposes the use of Paquinimod in the preparation of a drug for the treatment of secondary lymphedema. Summary of the invention

[0005] The object of the present invention is to provide the use of Paquinimod in the preparation of a medicament for treating secondary lymphedema, aiming to solve the problems raised in the above-mentioned background technology.

[0006] To achieve the above object, the present invention provides the following technical solutions:

[0007] Use of Paquinimod in the preparation of a drug for treating secondary lymphedema.

[0008] Furthermore, the Paquinimod inhibits the activation of the S100A8 / A9-TLR4-CCL2 signaling pathway, reduces the secretion of inflammatory factors in the model mice, and alleviates the edema symptoms in the model mice.

[0009] Compared with the prior art, the present invention has the following beneficial effects:

[0010] The present invention firstly uses the public data set GSE4333 to obtain the transcriptome data of the mouse tail tissue of lymphedema mice, performs differentially expressed gene analysis, and finds that S100A8 / A9 is significantly upregulated in the tissue of lymphedema mice, and is confirmed as a key network node through PPI network construction. Subsequently, by constructing a lymphedema mouse model, it is confirmed that S100A8 / A9 and its receptor TLR4 are significantly upregulated in edema tissue, and an in vitro experiment is conducted through an LPS (Lipopolysaccharide)-induced LEC cell model to further verify that S100A8 / A9 is a potential target for the treatment of SL. Based on the above findings, Paquinimod, a small molecule inhibitor of S100A8 / A9 dimers, was used to treat lymphedema mice. The results showed that Paquinimod could significantly inhibit the secretion of inflammatory factors in model mice, effectively alleviate edema, and inhibit the activation of the S100A8 / A9-TLR4-CCL2 signaling pathway. Therefore, the present invention applies Paquinimod, an inhibitor of the S100A8 / A9-TLR4 signaling pathway, to the preparation of a drug for treating secondary lymphedema (SL), which opens up new ideas and methods for the treatment of SL and effectively solves the problem of lack of effective treatment methods for SL. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 The results of cluster analysis of transcriptional activity in tail tissue of mice with lymphedema.

[0012] Figure 2 The PPI network construction and functional enrichment analysis results of differentially expressed genes in the tail of lymphedema mice; (a) is the PPI network construction result, and (b) is the functional enrichment analysis result.

[0013] Figure 3 The results of S100A8 and S100A9 gene expression and protein expression in the tail tissue of lymphedema mice; (a) is the gene expression result; (b) is the protein expression result.

[0014] Figure 4The results of gene expression and protein expression of TLR4, S100A8 and S100A9 in LEC cells induced by LPS; (a) is the gene expression result; (b) is the protein expression result.

[0015] Figure 5 Cluster analysis results of LPS-induced transcriptional activity in LEC cells.

[0016] Figure 6 The results of LPS-induced up-regulation of cytokine expression in LEC cells; (a) is the relative mRNA expression level of inflammatory factors IL-8, IL-6, IL-1α, CXCL3 and CCL2 in LEC induced by LPS; (b) is the ELISA experiment to detect the change in the concentration of IL-6 in the culture supernatant of LEC cells after LPS induction; (c) is the ELISA experiment to detect the change in the concentration of IL-8 in the culture supernatant of LEC cells after LPS induction; (d) is the ELISA experiment to detect the change in the concentration of CCL2 in the culture supernatant of LEC cells after LPS induction.

[0017] Figure 7 The results of Paquinimod inhibiting the expression of S100A8 and inflammatory factors in LEC cells induced by LPS; (a) Paquinimod inhibits the expression of IL-1α, an inflammatory factor in LEC cells induced by LPS, at the mRNA level; (b) Paquinimod inhibits the expression of IL-6, an inflammatory factor in LEC cells induced by LPS, at the mRNA level; (c) Paquinimod inhibits the expression of CCL2, an inflammatory factor in LEC cells induced by LPS, at the mRNA level; (d) Paquinimod inhibits (e) Paquinimod inhibits the expression of LPS-induced inflammatory factor CXCL3 in LEC cells at the mRNA level; (f) Paquinimod inhibits the secretion of IL-6 in the supernatant of LEC cells induced by LPS; (g) Paquinimod inhibits the secretion of IL-8 in the supernatant of LEC cells induced by LPS; (h) Paquinimod inhibits the secretion of CCL2 in the supernatant of LEC cells induced by LPS.

[0018] Figure 8 The figures are specific results of Paquinimod treatment of mice with tail lymphedema; (a) is the observation result of the tail circumference of each group of mice; (b) is the change chart of the tail volume of each group of mice within 28 days.

[0019] Fig. 9The results of Paquinimod targeting the S100A8 / A9-TLR4-CCL2 pathway inhibition; (a) Paquinimod inhibits S100A8 expression at the mRNA level; (b) Paquinimod inhibits S100A9 expression at the mRNA level; (c) Paquinimod inhibits TLR4 expression at the mRNA level; (d) Paquinimod inhibits S100A8 / A9 (the detection result of only S100A8 in the figure is because S100A8 and S100A9 exist in the form of heterodimers) , just detect one of them), TLR4 and CCL2 at the protein level; (e) Paquinimod inhibits the expression of inflammatory factor IL-1α at the mRNA level; (f) Paquinimod inhibits the expression of inflammatory factor IL-6 at the mRNA level; (g) Paquinimod inhibits the expression of inflammatory factor CXCL15 at the mRNA level; (h) Paquinimod inhibits the expression of inflammatory factor CXCL3 at the mRNA level; (i) Paquinimod inhibits the expression of inflammatory factor CCL2 at the mRNA level.

[0020] Fig.10 The results show that Paquinimod targetedly reduces the expression of S100A8 in edematous mouse tail tissue; (a) shows the expression of S100A8 in the edema group (lymphedema); (b) shows the expression of S100A8 in the treatment group (lymphedema+Paquinimod). DETAILED DESCRIPTION

[0021] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0022] The specific implementation of the present invention is described in detail below in conjunction with specific embodiments.

[0023] Embodiment 1, data acquisition and analysis;

[0024] The public dataset GSE4333 was used to obtain transcriptome data of the mouse tail tissue with lymphedema. The specific steps are as follows:

[0025] Data download: Access the GEO database and download the relevant GSE4333 transcriptome data.

[0026] Differentially expressed genes screening: Differential expression analysis was performed using R language, with thresholds set to |logFC| > 1 and adj. p < 0.05, to screen out genes that were significantly upregulated in the tail tissue of mice with lymphedema, where logFC represents the fold change in gene expression and adj. p represents the corrected P value. According to the analysis results, it was found that S100A8 / A9 was significantly upregulated in the tissues of mice with lymphedema (see Figure 1 ).

[0027] Example 2: PPI network construction and functional enrichment analysis;

[0028] After confirming that S100A8 / A9 expression was significantly upregulated in lymphedema mouse tissues, the following steps were performed:

[0029] PPI network construction: The PPI (protein-protein interaction) network of S100A8 / A9 and its related proteins was constructed using the STRING database, key network nodes were identified, and S100A8 / A9 was confirmed to play a key role as a hub gene ( Figure 2 (a).

[0030] GO and KEGG enrichment analysis: Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis were performed on the screened differentially expressed genes, and it was found that hub genes such as S100A8 / A9 were mainly enriched in the immune inflammation regulation pathway ( Figure 2 (b)

[0031] Example 3, mouse model construction and verification;

[0032] Establish a lymphedema mouse model and verify the expression of S100A8 / A9. The specific steps are as follows:

[0033] Model construction: C57BL / 6 mice were selected, and the lymphatic vessels on both sides were cut off by tail circumcision surgery to successfully establish a lymphedema mouse model.

[0034] Expression verification: qPCR and Western Blot (WB) techniques were used to detect the transcription and protein expression of S100A8 / A9 in rat tail edema tissue. The results showed that S100A8 / A9 was significantly upregulated at both the transcription and protein levels ( Figure 3 In a and b, lymphedema refers to lymphedema).

[0035] Example 4: In vitro experiments verify and clarify the regulatory pathway;

[0036] To further validate S100A8 / A9 as a potential target for the treatment of SL, the following in vitro experiments were performed:

[0037] Cell model construction: LPS-induced human lymphatic endothelial cell (LEC) model was used.

[0038] Expression verification: qPCR and WB experiments were used to detect the expression of S100A8 / A9 and receptor TLR4 in LEC cells induced by LPS. The results showed that the expression of S100A8 / A9 and TLR4 was significantly upregulated at the transcriptional and protein levels ( Figure 4 a and b).

[0039] Signaling pathway activation detection: Transcriptome sequencing analysis screened out the inflammatory factors IL-8 (CXCL15), IL-6, IL-1α, CXCL3 and CCL2, which were significantly upregulated ( Figure 5 ). qPCR experiments confirmed that after LPS induction, the above inflammatory factors in LEC cells were significantly upregulated at the transcriptional level ( Figure 6 ELISA results further showed that the expression of IL-6, IL-8 and CCL2 was also significantly upregulated at the serum level ( Figure 6 bd), confirming that LPS can activate the S100A8 / A9-TLR4-CCL2 signaling pathway.

[0040] Example 5, drug treatment experiment;

[0041] Treatment is performed using Paquinimod, a small molecule inhibitor of S100A8 / A9 dimer. The specific steps are as follows:

[0042] Drug treatment: Paquinimod was added to LPS-induced LEC cells to observe its effect on the secretion of inflammatory factors.

[0043] Functional recovery evaluation: qPCR, WB and ELISA experimental results confirmed that Paquinimod can significantly inhibit LPS-induced S100A8 / A9 and TLR4 expression ( Figure 7 e), while significantly inhibiting the secretion of inflammatory factors (such as IL-8, IL-6, IL-1α, CXCL3 and CCL2) ( Figure 7 in ad, fh).

[0044] Example 6, mouse model treatment experiment;

[0045] Experimental groups: control group (Control), edema group (lymphedema), Paquinimod group (Paquinimod), and treatment group (lymphedema+Paquinimod).

[0046] Treatment experiment: The control group was observed for 4 weeks; the edema group was observed for 4 weeks after modeling; the Paquinimod group had no treatment for the first two weeks and received continuous oral administration for the next 2 weeks (4 weeks in total); the treatment group received continuous oral administration of Paquinimod for 2 weeks starting 2 weeks after modeling (4 weeks in total).

[0047] Observation indicators: The circumference of the mouse tail was measured, and qPCR, WB and IHC experiments were performed.

[0048] The results showed that compared with the tail circumference of mice in the edema group, the tail edema symptoms of mice treated with Paquinimod (treatment group) were significantly alleviated, and almost returned to the normal circumference of the control group after 2 weeks of treatment ( Figure 8 In addition, the expression of inflammatory factors IL-1α, IL-6, CXCL15 (the mouse homologue of IL-8), CXCL3 and CCL2 in the treatment group was significantly decreased compared with the edema group ( Fig. 9 zhongei), indicating that Paquinimod can significantly inhibit the inflammatory response of edema mice. And compared with the edema group, the expression of S100A8 / A9, TLR4 and CCL2 at the mRNA and protein levels in the treatment group was significantly reduced ( Fig. 9 Immunohistochemistry (IHC) results ( Fig.10 In a and b), S100A8 was positively expressed around the subcutaneous lymphatic vessels in the edema tissue of the mouse tail. After treatment with Paquinimod, S100A8 expression was significantly reduced, indicating that Paquinimod can effectively inhibit the S100A8 / A9-TLR4-CCL2 signaling pathway.

[0049] in conclusion:

[0050] The present invention utilizes Paquinimod, an inhibitor of the S100A8 / A9-TLR4 signaling pathway, to reduce the secretion of inflammatory factors in model mice by inhibiting the activation of the S100A8 / A9-TLR4-CCL2 signaling pathway, thereby effectively alleviating edema symptoms. Therefore, the present invention applies Paquinimod, an inhibitor of the S100A8 / A9-TLR4 signaling pathway, to the preparation of a drug for treating secondary lymphedema (SL), which opens up new ideas and methods for the treatment of SL and effectively solves the problem of the lack of effective treatment methods for SL.

[0051] The above are only preferred embodiments of the present invention. It should be pointed out that, for those skilled in the art, several modifications and improvements can be made without departing from the concept of the present invention. These should also be regarded as the protection scope of the present invention. These will not affect the effect of the implementation of the present invention and the practicality of the patent.

Claims

1. The use of Paquinimod in the preparation of a drug for treating secondary lymphedema, characterized in that: The Paquinimod inhibits the activation of the S100A8 / A9-TLR4-CCL2 signaling pathway, reduces the secretion of inflammatory factors in the model mice, and alleviates the edema symptoms of the model mice.

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