Application of ephedrine in the preparation of drugs for the prevention or treatment of peritoneal fibrosis

Abstract

This invention discloses the application of ephedrine in the preparation of drugs for the prevention or treatment of peritoneal fibrosis, relating to the field of biomedical technology. The key technical point is: a pharmaceutical preparation for the prevention or treatment of peritoneal fibrosis, the active ingredient of which is ephedrine. This pharmaceutical preparation can achieve preventive and therapeutic effects on peritoneal fibrosis by inhibiting the mesothelial metastatic process (MMT) in peritoneal mesothelial cells, downregulating the expression of fibrosis-related proteins, and improving peritoneal ultrafiltration and solute transport functions, thus providing a new drug option for the treatment of end-stage renal disease patients undergoing peritoneal dialysis.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and more specifically, to the use of ephedrine in the preparation of drugs for the prevention or treatment of peritoneal fibrosis. Background Technology

[0002] Chronic kidney disease (CKD) has a global prevalence of approximately 10%, making it one of the major chronic diseases threatening human health. Peritoneal dialysis (PD), as one of the main renal replacement therapies for end-stage renal disease (ESRD), is widely used clinically due to its ease of operation, cost-effectiveness, and ability to effectively protect residual renal function. However, with prolonged PD treatment, progressive pathological changes often occur in the peritoneal tissue, leading to a gradual decline in peritoneal function and ultimately ultrafiltration failure. This severely impacts treatment outcomes and significantly increases hospitalization and mortality rates.

[0003] Currently, there are still no effective drugs for treating peritoneal structural damage and functional decline caused by long-term peritoneal dialysis. Studies have shown that high concentrations of glucose in peritoneal dialysis fluid are a key factor causing peritoneal damage. A high-glucose environment can induce mesothelial-to-mesenchymal transition (MMT) in peritoneal mesothelial cells, a process considered a core mechanism of peritoneal structural and functional impairment. During MMT, peritoneal mesothelial cells lose epithelial polarity, tight junctions between cells are disrupted, and they gradually transform into mesenchymal cells with a myofibroblast phenotype, exhibiting high migration and invasive capabilities. Simultaneously, the expression of mesothelial cell markers such as E-cadherin significantly decreases, while the expression of mesenchymal cell markers such as vimentin, α-smooth muscle actin, and fibronectin significantly increases.

[0004] Baimaside is a flavonoid compound isolated from plants such as Apocynum venetum. Current research suggests that flavonoids play a regulatory role in various physiological and pathological processes. For example, in the nervous system, these compounds can improve mood function by regulating monoamine neurotransmitter levels or inhibiting monoamine oxidase activity; its aglycone, quercetin, has been shown to promote neuroplasticity and exert antidepressant effects by activating the BDNF / TrkB pathway. In the skin, flavonoids exhibit significant antioxidant and anti-inflammatory activities, mitigating photoaging through mechanisms such as free radical scavenging and COX-2 inhibition. Metabolically, some flavonoid components can improve hepatic lipid metabolism and insulin resistance through the AMPK / PGC-1α and PPARα-related autophagy pathways.

[0005] Although ephedrine has shown potential effects in neuroprotection, anti-skin aging, and metabolic regulation, its function in other physiological or pathological processes remains unclear. In particular, its role in the mesothelial process of peritoneal mesothelial cells (MMT) and the development of peritoneal fibrosis is currently a blank area of ​​research. Summary of the Invention

[0006] To address the shortcomings of existing technologies, the purpose of this invention is to provide the application of ephedrine in the preparation of drugs for the prevention or treatment of peritoneal fibrosis. As the first disclosed application of ephedrine, this invention provides an innovative drug target for the prevention and treatment of peritoneal fibrosis.

[0007] The above-mentioned technical objective of the present invention is achieved through the following technical solution: Firstly, it provides the application of ephedrine in the preparation of drugs for the prevention or treatment of peritoneal fibrosis.

[0008] Preferably, the application is achieved by inhibiting the epithelial-mesenchymal transition process of peritoneal mesothelial cells.

[0009] Preferably, the application downregulates the expression of fibrosis-related proteins, wherein the fibrosis-related proteins are at least one of fibronectin, vimentin, and collagen I.

[0010] Preferably, the application improves peritoneal ultrafiltration or solute transport function.

[0011] In a second aspect, a pharmaceutical composition for the prevention or treatment of peritoneal fibrosis is provided, comprising ephedrine as an active ingredient and a pharmaceutically acceptable carrier or excipient.

[0012] Preferably, the pharmaceutical composition is administered by gavage.

[0013] Preferably, the pharmaceutical composition is used to prevent and treat peritoneal fibrosis associated with peritoneal dialysis.

[0014] Compared with the prior art, the present invention has the following beneficial effects: 1. This invention demonstrates that ephedrine can significantly alleviate the pathological progression of peritoneal fibrosis by inhibiting excessive extracellular matrix deposition and peritoneal thickening, thereby reversing fibrotic damage. Experiments show that in a mouse model induced by high-glucose dialysis fluid, peritoneal thickness significantly decreased after ephedrine intervention, and Masson staining results clearly indicated a reduction in the degree of fibrosis. This can address the tissue remodeling problem caused by long-term exposure to a high-glucose environment in peritoneal dialysis patients, providing a basis for clinical prevention and treatment. 2. This invention demonstrates that ephedrine effectively inhibits the MMT process and prevents peritoneal mesothelial cells from transforming into myofibroblasts by regulating key signaling pathways (such as TGF-β). Experiments have confirmed that ephedrine reverses TGF-β-induced upregulation of fibronectin (FN) in a concentration-dependent manner, thereby maintaining cell epithelial properties and blocking the fibrosis process at the molecular level, exhibiting advantages such as high specificity and low side effects. 3. This invention demonstrates that ephedrine can significantly reduce the abnormally high expression of fibrosis marker proteins such as fibronectin (FN), collagen I, and vimentin, thereby reducing extracellular matrix accumulation. Immunohistochemical results showed that under ephedrine intervention, the positive signals of FN and Collagen I in mouse peritoneal tissue were significantly weakened, indicating that it alleviates fibrosis by regulating protein expression, helps delay peritoneal function decline, and reduces the risk of ultrafiltration failure. 4. This invention demonstrates that ephedrine can effectively restore the ultrafiltration capacity and solute transport efficiency of the peritoneum, thereby ensuring the long-term effectiveness of peritoneal dialysis. Mouse experiments show that ultrafiltration volume and glucose reabsorption are significantly improved after ephedrine intervention, which is directly related to the efficient utilization of dialysate and waste removal, thus solving the problem of treatment failure caused by peritoneal dysfunction and improving the quality of life of patients. Attached Figure Description

[0015] The accompanying drawings, which are included to provide a further understanding of embodiments of the invention and form part of this application, do not constitute a limitation thereof. In the drawings: Figure 1 These are Masson staining images and statistical analysis diagrams of peritoneal tissues from mice in different treatment groups in Example 1 of the present invention; wherein, A is a representative image of Masson staining of peritoneal tissues from each group of mice, and B is a quantitative statistical analysis diagram of the Masson staining results; Figure 2 This is a graph showing the detection results of peritoneal ultrafiltration and glucose reabsorption in mice of different treatment groups in Example 2 of the present invention; wherein, A is the statistical result of peritoneal ultrafiltration in each group of mice, and B is the statistical result of peritoneal glucose reabsorption in each group of mice. Figure 3 These are immunohistochemical staining and quantitative images of FN and Collagen I protein in peritoneal tissue of mice in different treatment groups in Example 3 of the present invention; wherein, A is the immunohistochemical positive staining image of FN and Collagen I protein in peritoneum of mice in each group, and B and C are quantitative statistical analysis images of the immunohistochemical positive staining results of FN and Collagen I protein, respectively. Figure 4These are the detection results of fibrosis-related proteins in human epithelial cells (MET-5A) from different treatment groups in Example 4 of the present invention; wherein, Figure A is the Western Blot detection result of FN protein, and Figure B is the gray value quantitative analysis of FN protein expression. Detailed Implementation

[0016] To make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the embodiments and accompanying drawings. The illustrative embodiments and descriptions of the present invention are only used to explain the present invention and are not intended to limit the present invention.

[0017] The specific operating steps of the Western blot, Masson staining, immunohistochemical experiments, and statistical analysis methods involved in this invention are as follows: (1) Western blot: Total protein was extracted from tissues or cells using a RIPA lysis buffer (Beyotime) and a protease inhibitor mixture cocktail (Merck). Protein concentration was determined by the BCA method and normalized. Equal volumes of protein samples were subjected to SDS-PAGE electrophoresis (7% or 10% polyacrylamide gel concentration), followed by wet transfer to a PVDF membrane at a constant current of 300 mA for 2 hours. The membrane was blocked with 5% BSA at room temperature for 1 hour, then incubated overnight at 4°C with primary antibody (FN antibody purchased from Abcam, Gapdh internal control antibody purchased from Huabio). After three washes with TBST, the corresponding secondary antibody was added and incubated at room temperature for 1 hour, followed by another wash. ECL chemiluminescence was used for color development, and images were acquired using a ChemiD MP imaging system. The gray values ​​of the bands were analyzed using ImageJ software, and the relative expression level was expressed as the ratio of the target protein's gray value to the Gapdh gray value.

[0018] (2) Masson staining: Mouse peritoneal tissue was fixed in 4% paraformaldehyde for 24–48 hours, followed by sequential dehydration with graded ethanol, clearing with xylene, paraffin embedding, and sectioning. After dewaxing to water, sections were stained as follows: washed with running water and distilled water; hematoxylin staining of the nuclei for 5–10 minutes, followed by blue staining with running water; staining with Masson's compound stain for 5–10 minutes; rinsing with 2% glacial acetic acid aqueous solution; differentiation with 1% phosphomolybdic acid for 3–5 minutes; direct counterstaining with aniline blue or light green for 5 minutes; rinsing with 0.2% glacial acetic acid; dehydration with graded ethanol, clearing with xylene, and mounting with neutral resin. Microscopic observation and image acquisition were performed to assess the degree of tissue fibrosis.

[0019] (3) Immunohistochemistry: After fixation, dehydration, embedding, sectioning, and dewaxing, tissues were subjected to microwave thermal retrieval using citrate buffer (pH 6.0) (medium heat for 10 minutes). After cooling, the tissues were blocked with goat serum for 1 hour, and primary antibodies (FN and Collagen I antibodies, both purchased from Abcam) were added. The tissues were incubated overnight at 4°C. After washing with PBS, DAB staining was performed, and the nuclei were counterstained with hematoxylin. The tissues were then dehydrated, cleared, and mounted. The distribution and intensity of positive signals were observed under a microscope, and image analysis was performed.

[0020] (4) Statistical methods All experimental data are expressed as mean ± standard error (mean ± SEM). One-way ANOVA was used for comparisons among multiple groups. P < 0.05 was considered statistically significant. P < 0.05 was marked as *, P < 0.01 as **, and P < 0.001 as ****.

[0021] Example 1: Ephedrine improves peritoneal fibrosis induced by high-glucose dialysis fluid in mice.

[0022] Patients undergoing long-term peritoneal dialysis often experience persistent and progressive peritoneal fibrosis due to frequent and continuous exposure to high-concentration glucose dialysate (typically ranging from 2.5% to 4.25%), coupled with the continuous accumulation and long-term buildup of advanced glycation end products (AGEs) in the body. This pathological process is characterized by abnormally excessive deposition and accumulation of extracellular matrix (ECM) components in the peritoneal mesothelial subepithelial space, accompanied by significant excessive angiogenesis and proliferation. These structural changes ultimately lead to the gradual loss and complete failure of peritoneal ultrafiltration function. Statistics show that ultrafiltration failure caused by this mechanism accounts for approximately 40% to 60% of all peritoneal dialysis failures.

[0023] In this embodiment, healthy male C57BL / 6J mice (with an initial weight controlled between 25 and 27 grams) were selected as experimental subjects and randomly assigned to three different experimental groups: a blank control group (CTR group), a peritoneal fibrosis model group (PDS group), and a ginsenoside drug intervention group (PDS+BAI group). Mice in the model group and the drug intervention group received a daily intraperitoneal injection of 4.25% glucose dialysate at a dose of 100 mg / kg body weight. This modeling process was continuous for a full 6 weeks. The blank control group mice received an equal volume of sterile saline intraperitoneally during the same time period. In the drug intervention group, in addition to receiving the same high-glucose dialysate injection as the model group, they were also treated with a 0.1 mg / L ginsenoside solution administered via gavage at a dose of 100 mg / kg body weight. At the end of the experimental period, peritoneal tissue samples were collected from mice in all groups and subjected to specialized staining and histopathological analysis using Masson's trichrome staining method.

[0024] Experimental results are as follows Figure 1 As shown in Figure A, when compared with the blank control group, a significant abnormal thickening of the peritoneal tissue in the model group mice can be clearly observed. This morphological change strongly confirms that an animal model of peritoneal fibrosis was successfully established through injection of high-glucose dialysis fluid. Figure 1 As shown in Figure B, after treatment with ephedrine solution, the peritoneum thickness of mice in the drug intervention group showed significant shrinkage and reduction. This comparative result clearly indicates that ephedrine can effectively inhibit and reverse the pathological process of peritoneal fibrosis induced by high-concentration glucose dialysis fluid.

[0025] Based on the above results, it can be concluded that, at the animal model experimental level, ephedrine can indeed significantly alleviate and improve peritoneal fibrosis lesions caused by long-term peritoneal dialysis, demonstrating its potential therapeutic value.

[0026] Example 2: Study on the effect of ephedrine on peritoneal ultrafiltration function and solute transport function. Peritoneal ultrafiltration and solute transport are the physiological basis for maintaining the efficacy of peritoneal dialysis. Dysfunction of these functions can directly affect the treatment process and even lead to treatment failure.

[0027] The animal grouping and modeling methods in this embodiment are consistent with those in Example 1. After a 6-week experimental period, the experimental animals were intraperitoneally injected with 4.25% glucose dialysate at a dose of 100 mg per kilogram of body weight. Two hours after injection, residual ascites fluid was collected from the peritoneal cavity. The ultrafiltration volume of the peritoneum was calculated by subtracting the volume of injected fluid from the volume of recovered fluid. Simultaneously, the glucose concentration in the ascites fluid and the original dialysate was measured using a glucose assay kit, and the glucose reabsorption volume was calculated based on this.

[0028] like Figure 2 As shown in Part A, the ultrafiltration rate of the model group mice decreased significantly; while... Figure 2 As shown in Part B, glucose reabsorption was also significantly reduced, suggesting that peritoneal function in the model group mice was significantly impaired. After intervention with ephedrine, both of the above key indicators showed a significant improvement trend, indicating that ephedrine has a clear positive effect in protecting peritoneal transport function.

[0029] Conclusion: Ephedrine can effectively improve peritoneal ultrafiltration dysfunction and solute transport abnormalities in mice induced by high-glucose dialysis fluid.

[0030] Example 3: Ephedrine significantly downregulated the expression levels of fibrosis-related proteins.

[0031] In the pathological process of peritoneal fibrosis, the abnormally high expression of various fibrosis-related proteins is a key molecular biological feature. Among them, the most representative proteins include fibronectin (FN) and collagen I, which are usually regarded as the core indicators for assessing the degree of fibrosis.

[0032] Based on the animal model grouping in Example 1, peritoneal tissue samples were collected from each group and analyzed qualitatively and quantitatively using immunohistochemistry to examine the expression changes and distribution of FN and Collagen I proteins. The immunohistochemical results clearly showed: Figure 3 As shown, compared with the normal control group, the positive expression signals of FN and Collagen I in the peritoneal tissue of the model group animals were significantly enhanced, showing extensive and dense staining areas. In the treatment group treated with ephedrine, the positive signal intensity of these two proteins was significantly weakened, and their expression range and staining depth were significantly reduced.

[0033] In conclusion, ephedrine can effectively inhibit the overexpression of fibrosis-related proteins such as FN and Collagen I, thereby reducing abnormal fibrous deposition and structural remodeling in peritoneal tissue at the molecular level, and has a clear positive effect on alleviating and delaying the process of peritoneal fibrosis.

[0034] Example 4: Experimental study on the inhibition of TGF-β-induced human skin cell fibrosis by ephedrine Transforming growth factor-β (TGF-β) is a core and key cytokine that induces epithelial-mesenchymal transition (EMT) in mesothelial cells, driving cell fibrosis during this process. This study investigated the inhibitory effect of ephedrine on TGF-β-induced fibrosis, using the human epithelial cell line MET-5A as the experimental model. MET-5A cells were uniformly seeded in 12-well cell culture plates and divided into the following treatment groups according to the experimental design: a blank control group without treatment, a fibrosis model group stimulated only with TGF-β (10 ng / mL), a co-treatment group receiving 5 μM ephedrine in addition to TGF-β stimulation, and a co-treatment group receiving 10 μM ephedrine in addition to TGF-β stimulation. Cells were starved in serum-free medium for 24 hours before the experiment to synchronize the cell cycle, and then subjected to the corresponding treatments for 48 hours. After treatment, cells from each group were collected, and the expression levels of the fibrosis marker fibronectin (FN) were detected by Western blot.

[0035] Experimental results are as follows Figure 4 As shown in the figure, the model group data showed that, compared with the blank control group, TGF-β treatment significantly upregulated the expression level of the pro-fibrotic protein FN, which indicates that the cells successfully transformed into a fibrotic phenotype. In the ephedrine co-treatment group, ephedrine showed a significant intervention effect, effectively reversing the above-mentioned protein expression changes induced by TGF-β in a dose-dependent manner; that is, as the ephedrine concentration increased (5 μM to 10 μM), the upregulation of FN was inhibited.

[0036] Experimental conclusion: Ephedrine can effectively inhibit the TGF-β-induced fibrosis process in human epithelial cells at the cellular level. The potential molecular mechanism of this inhibitory effect may be closely related to the ability of ephedrine to inhibit FN expression, thereby antagonizing the epithelial-mesenchymal transition process.

[0037] This invention, through systematic in vivo animal experiments and in vitro cell experiments, confirms that ephedrine can effectively intervene in the occurrence and development of peritoneal fibrosis. Its core mechanism of action lies in ephedrine's ability to significantly inhibit the key process of epithelial-mesenchymal transition (MMT) in peritoneal mesothelial cells, while simultaneously downregulating the abnormally high expression of various fibrosis-related proteins, including fibronectin (FN), vimentin, and type I collagen I. Based on these beneficial cellular and molecular-level regulations, ephedrine further improves the physiological function of the peritoneum, specifically by enhancing peritoneal ultrafiltration function and restoring normal solute transport function. Therefore, the experimental evidence from this invention fully demonstrates that ephedrine can effectively prevent and treat peritoneal fibrosis. This invention is the first to publicly disclose a novel application of ephedrine in the prevention and treatment of peritoneal fibrosis, successfully filling a gap in existing technology in this area. It opens up new drug targets and provides innovative treatment options for the clinical prevention and treatment of peritoneal dialysis-related complications, possessing significant scientific value and important clinical application prospects.

[0038] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above description is only a specific embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. Application of ephedrine in the preparation of drugs for the prevention or treatment of peritoneal fibrosis.

2. The application according to claim 1, characterized in that, The application is achieved by inhibiting the epithelial-mesenchymal transition process of peritoneal mesothelial cells.

3. The application according to claim 1, characterized in that, The application downregulates the expression of fibrosis-related proteins, which are at least one of fibronectin, vimentin, and collagen I.

4. The application according to claim 1, characterized in that, The application improves peritoneal ultrafiltration or solute transport.

5. A pharmaceutical composition for the prevention or treatment of peritoneal fibrosis, characterized in that, It contains ephedrine as the active ingredient, as well as pharmaceutically acceptable carriers or excipients.

6. The pharmaceutical composition according to claim 5, characterized in that, The pharmaceutical composition is administered by gavage.

7. The pharmaceutical composition according to claim 5, characterized in that, The pharmaceutical composition is used to prevent and treat peritoneal fibrosis associated with peritoneal dialysis.

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