Composition containing unsaturated dicarboxylic acid monomethyl ester and its use in the preparation of a medicament for preventing and treating renal fibrosis

Abstract

The present application relates to the technical field of biological medicine, and more particularly to a composition and application thereof in preparation of an anti-fibrosis drug. 2+ The combination has a significant prevention and treatment effect on kidney fibrosis and has obvious synergistic effect, thereby providing a new selection for clinical treatment of kidney fibrosis.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to compositions containing unsaturated dicarboxylic acid monomethyl esters and their application in the preparation of drugs for the prevention and treatment of renal fibrosis. Background Technology

[0002] Renal fibrosis, a key pathological process in the progression of various chronic kidney diseases (CKD) to end-stage renal disease, is characterized by abnormal proliferation of renal interstitial cells and excessive deposition of extracellular matrix, ultimately leading to irreversible loss of renal function and seriously threatening human health. The pathogenesis of renal fibrosis is complex, with oxidative stress playing a crucial role. During the development of CKD, the body is in a state of oxidative stress imbalance, with mitochondrial pathways and NADPH oxidase pathways promoting the excessive generation of reactive oxygen species (ROS). Excessive ROS not only damages cell structure and function, triggering lipid peroxidation and the accumulation of advanced glycation end products (AGEs), leading to severe tissue damage, but also participates in various pathological processes such as renal anemia, accelerating the process of renal fibrosis. When the pro-oxidative and anti-oxidative systems are imbalanced, leading to damage to glomerular and tubular epithelial cells, inflammatory and fibrotic pathways such as nuclear factor kappa-B (NF-κB) and inflammasomes are activated, thereby triggering renal fibrosis. Currently, clinical treatment options for renal fibrosis are limited. Existing drugs are mostly multi-target and multi-route medications, such as pirfenidone and nintedanib. However, due to side effects, these drugs are difficult to widely use in patients with renal fibrosis, making the exploration of new treatment strategies urgent. Therefore, finding highly effective and safe drugs for the prevention and treatment of renal fibrosis has become a pressing research need. Summary of the Invention

[0003] In view of this, the present invention provides the use of a composition containing an unsaturated dicarboxylic acid monomethyl ester in the preparation of a medicament for the prevention and treatment of renal fibrosis. The composition comprises a dicarboxylic acid monomethyl ester and a metal ion. The present invention has found that the combined use of the unsaturated dicarboxylic acid monomethyl ester and the metal ion has a significant therapeutic effect on renal fibrosis.

[0004] This invention provides a composition for preventing and treating renal fibrosis, comprising component A and component B; Component A is an unsaturated dicarboxylic acid monomethyl ester; Component B is a metal ion; the metal ion includes Zn. 2+ Al 3+ Mn 2+ Mg 2 + at least one of them.

[0005] Furthermore, the molar ratio of component A to component B is (1~3):(1~5), preferably (1~2.25):(1~4.5), specifically 1:1, 1:2, 1:3, 1:4.5, 1.5:2 or 2.25:2.

[0006] Specifically, the dicarboxylic acid monomethyl ester is preferably an unsaturated dicarboxylic acid monomethyl ester, more preferably at least one of maleic acid monomethyl ester, fumarate monomethyl ester, and itaconic acid monomethyl ester, and even more preferably maleic acid monomethyl ester.

[0007] In this invention, the metal ions include Zn. 2+ Al 3+ Mn 2+ Mg 2+ At least one of them, preferably Mn 2+ This invention does not have specific limitations on the specific source of the metal ions; any soluble metal salt commonly used in the art is acceptable, such as chloride or sulfate salts of the aforementioned metal ions. Chloride salts, such as zinc chloride, aluminum chloride, manganese chloride, and magnesium chloride, which are easily soluble in water, are preferred to facilitate the formation of a stable system with component A during the formulation process. In a specific embodiment of this invention, the metal ion is Mn. 2+ Specifically, it is in the form of manganese chloride.

[0008] This invention has discovered that unsaturated dicarboxylic acid monomethyl esters (such as monomethyl maleate, monomethyl fumarate, and monomethyl itaconic acid) alone or in combination with metal ions (such as Zn) 2+ Al 3+ Mn 2+ Mg 2+ It has a good preventive and therapeutic effect on renal fibrosis, and the effect is more significant when used in combination. Among them, monomethyl maleate and Mn 2+ Combined prevention and control measures yield the best results.

[0009] In some embodiments of the present invention, the metal ion in the composition of the present invention is Mn. 2+ The unsaturated dicarboxylic acid monomethyl ester is maleic acid monomethyl ester, and the maleic acid monomethyl ester and Mn 2+ The molar ratio is (1~2.25):(1~4.5), specifically 1:1, 1:2, 1:3, 1:4.5, 1.5:2 or 2.25:2.

[0010] The present invention also provides the use of component A as described above or the above composition in the preparation of a medicament for the prevention and treatment of renal fibrosis.

[0011] In the above applications, component A or the composition has at least one of the following effects: regulating the level of inflammatory factors, reducing the level of oxidative stress, improving renal function, and improving the degree of renal fibrosis.

[0012] Specifically, the inflammation-related factors include at least one of TNF-α, IL-6, IL-1β and IL-11.

[0013] The indicators of oxidative stress include at least one of NRF2, NQO1, HO-1, and SOD2.

[0014] The renal function indicators include at least one of BUN and Scr.

[0015] The renal fibrosis markers include at least one of TGF-β, α-SMA, and COL1A1.

[0016] In this invention, the renal fibrosis refers to renal fibrosis in humans or animals. The animals include mice, rats, guinea pigs, rabbits, apes, monkeys, chimpanzees, cattle, sheep, pigs, horses, dogs, or cats.

[0017] The present invention also provides a medicament for the prevention and treatment of renal fibrosis, comprising component A as described above or the composition described in the present invention.

[0018] The drug described in this invention may also include other drugs for the prevention and treatment of renal fibrosis, which may be commonly used Western or traditional Chinese medicines in the field for the prevention and treatment of renal fibrosis. Specifically, the Western medicines include, but are not limited to, pirfenidone.

[0019] The pharmaceutical products described in this invention also include pharmaceutically acceptable excipients. This invention does not impose any special restrictions on the types of excipients; any type that complies with relevant regulations and standards and does not affect the activity and function of the composition is acceptable. Specific selection can be made according to the product dosage form requirements. The excipients include, but are not limited to, fillers, binders, disintegrants, lubricants, suspending agents, matrix, preservatives, flavoring agents, sweeteners, etc.

[0020] The compositions of this invention can be formulated into pharmaceutically acceptable dosage forms, including but not limited to granules, tablets, capsules, pellets, oral liquids, syrups, suspensions, lozenges, orally disintegrating tablets, ointments, pastes, patches, and sprays, using conventional pharmaceutical manufacturing methods.

[0021] The present invention also provides a method for preventing and treating renal fibrosis, comprising: administering component A as described above, the composition as described above, or the drug as described above.

[0022] To improve patient compliance, components A and B can be administered separately or simultaneously, depending on the clinical treatment plan. When administered separately, the time interval between the administration of components A and B should not be too long; therefore, components A and B can be formulated as independent preparations. If administered simultaneously, components A and B can be formulated as a compound preparation, meaning that components A and B exist in the same preparation, or they can be formulated as single preparations and then mixed.

[0023] In the applications, drugs, and methods described above in this invention, the effective concentrations of components A and B are 3.25 to 15 mM. Specifically, the effective concentrations can be 3.25 mM, 7.5 mM, 15 mM, or any value between any two values ​​or any small range.

[0024] This invention provides a composition and its use in a medicament for the prevention and treatment of renal fibrosis, the composition comprising monomethyl maleate and Mn 2+ This invention is the first to discover that Mn 2+ When combined with monomethyl maleate, it can reduce oxidative damage, regulate inflammatory response and improve renal fibrosis, playing a significant role in the treatment of renal fibrosis and thus providing a new option for the clinical treatment of renal fibrosis. Attached Figure Description

[0026] Figure 1A This represents the rate of change in body weight of mice after unilateral ureteral obstruction surgery via gavage 7 days later; Figure 1B This indicates the serum BUN concentration in mice in Example 1; Figure 1C This indicates the serum Scr concentration in mice in Example 1; Figure 1D The kidneys of mice in each group in Example 1 are shown. TGF-β mRNA expression levels; Figure 1E The kidneys of mice in each group in Example 1 are shown. α-SMA mRNA expression levels; Figure 1F The kidneys of mice in each group in Example 1 are shown. SOD2 mRNA expression level Figure 1G The kidneys of mice in each group in Example 1 are shown. NQO-1 mRNA expression levels; Figure 1H The kidneys of mice in each group in Example 1 are shown. IL-6 mRNA expression levels; Figure 1I This refers to the kidneys of mice in each group in Example 1. TNF-α mRNA expression levels; Figure 1J The kidneys of mice in each group in Example 1 are shown. IL-11 mRNA expression levels; Figure 2A This indicates H&E and Masson staining of mouse kidneys in Example 1; Figure 2B This indicates the quantification of the renal fibrosis area in mice in Example 1; Figure 2C This indicates the renal tubular injury score in Example 1; Figure 3A This represents the rate of change in body weight of mice after unilateral ureteral obstruction surgery via gavage 7 days later; Figure 3B This indicates the serum BUN concentration in mice in Example 2; Figure 3C This indicates the serum Scr concentration in mice in Example 2; Figure 3D The kidneys of mice in each group in Example 2 represent... TGF-β Level of expression; Figure 3E The kidneys of mice in each group in Example 2 represent... α-SMA mRNA expression levels; Figure 3F The kidneys of mice in each group in Example 2 represent... SOD2 mRNA expression levels; Figure 3G The kidneys of mice in each group in Example 2 represent... NQO-1 mRNA expression levels; Figure 3H The kidneys of mice in each group in Example 2 represent... IL-6 mRNA expression levels; Figure 3I The kidneys of mice in each group in Example 2 represent... IL-11 mRNA expression levels; Figure 4A This represents the rate of change in body weight of mice after unilateral ureteral obstruction surgery via gavage 7 days later; Figure 4B This indicates the serum BUN concentration in mice in Example 3; Figure 4C This indicates the serum Scr concentration in mice in Example 3; Figure 4D This represents the kidneys of mice in each group in Example 3. TGF-β mRNA expression levels; Figure 4E The kidneys of mice in each group in Example 3 represent... α-SMA mRNA expression levels; Figure 4F This represents the kidneys of mice in each group in Example 3. COL1A1 mRNA expression level Figure 4G This represents the kidneys of mice in each group in Example 3. SOD2 mRNA expression levels; Figure 4HThis represents the kidneys of mice in each group in Example 3. NQO-1 mRNA expression levels; Figure 4I The kidneys of mice in each group in Example 3 represent... IL-6 mRNA expression levels; Figure 4J The kidneys of mice in each group in Example 3 represent... IL-11 mRNA expression levels; Figure 5A This represents the rate of change in body weight of mice after unilateral ureteral obstruction surgery via gavage 7 days later; Figure 5B This indicates the serum BUN concentration in mice in Example 4; Figure 5C This indicates the serum Scr concentration in mice in Example 4; Figure 5D The kidneys of mice in each group in Example 4 represent... TGF-β mRNA expression levels; Figure 5E The kidneys of mice in each group in Example 4 represent... α-SMA mRNA expression levels; Figure 5F The kidneys of mice in each group in Example 4 represent... COL1A1 mRNA expression level Figure 5G The kidneys of mice in each group in Example 4 represent... SOD2 mRNA expression levels; Figure 5H The kidneys of mice in each group in Example 4 represent... HO-1 mRNA expression levels; Figure 5I The kidneys of mice in each group in Example 4 represent... NQO-1 mRNA expression levels; Figure 5J The kidneys of mice in each group in Example 4 represent... IL-6 mRNA expression levels; Figure 5K The kidneys of mice in each group in Example 4 represent... TNF-α mRNA expression levels; Figure 5L The kidneys of mice in each group in Example 4 represent... IL-11 mRNA expression levels; Figure 6A This indicates that in Example 4, pirfenidone and monomethyl maleate were combined with Mn 2+ H&E and Masson staining of mouse kidneys after drug administration; Figure 6B This indicates that in Example 4, pirfenidone and monomethyl maleate were combined with Mn2+ Quantification of renal fibrosis area in mice after drug administration; Figure 6C This indicates that in Example 4, pirfenidone and monomethyl maleate were combined with Mn 2+ Renal tubular injury score after drug administration. Detailed Implementation

[0027] This invention provides compositions and their use in the preparation of medicaments for the prevention and treatment of renal fibrosis. Those skilled in the art can refer to the content herein and appropriately modify the process parameters to achieve the desired results. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments, and those skilled in the art will clearly be able to modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0028] The test materials used in this invention are all common commercial products and can be purchased on the market.

[0029] The present invention will be further illustrated below with reference to the embodiments: Example 1 1. Experimental Methods 1.1 Grouping and treatment of experimental animals The experimental mice were SPF-grade male KM mice, aged 6-8 weeks and weighing 20-24 g. They were housed under specific pathogen-free conditions (12 h / 12 ​​h light / dark cycle, 50 ± 5% relative humidity, and 22-26 °C), with free access to safe drinking water and food. All animals underwent 48 h of environmental acclimatization before the start of the experiment.

[0030] Mice were randomly divided into a sham-operated group, a model group, and a drug intervention group, with five mice in each group. Unilateral ureteral obstruction surgery was performed. The specific procedure was as follows: Mice were anesthetized, placed in a supine position, and had their abdomens shaved. The abdomen was disinfected with povidone-iodine. A 0.5 cm straight incision was made on the left side of the midline of the abdomen. The skin, subcutaneous tissue, and peritoneum were dissected layer by layer. The ureter was freed along the ureteropelvic junction and ligated below the left kidney with 4-0 silk suture. The abdominal cavity was then sutured closed. In the sham-operated group, only the left ureter was exposed; no ligation was performed. Mice were weighed and administered the drug via gavage daily post-surgery. On the seventh day, mice were anesthetized, and orbital venous blood was collected. After blood collection, the mice were euthanized by dislocation, and the abdominal cavity was immediately opened. Complete paired kidneys were collected, the renal capsules were removed, and some kidneys were fixed in 4% paraformaldehyde solution and stored at room temperature for histopathological staining. The remaining kidneys were stored at -80 °C for subsequent experiments. After collecting mouse blood, it was left to stand at room temperature for 2-3 hours, and then centrifuged in a low-temperature high-speed centrifuge. The supernatant was then stored at 4 °C for testing of serum biochemical indicators.

[0031] The dosing regimen is as follows: Table 1

[0032] 1.2 Determination of serum urea nitrogen content using the urease method

[0033] (1) Prepare buffer enzyme solution: Prepare according to enzyme stock solution: enzyme diluent = 3:1000; (2) Prepare 100 mmol / L BUN standard stock solution: Prepare by adding 1 mL of primary purified water to 1 vial of BUN standard powder; (3) Prepare 10 mmol / L BUN standard application solution: Dilute 100 mmol / L BUN standard stock solution with primary purified water 10 times.

[0034] Add the sample as follows: Table 2

[0035] BUN concentration (mmol / L) = (Measured OD value - Blank OD value) / (Standard OD value - Blank OD value) × 10

[0036] 1.3 Protein-free endpoint microplate method for detecting serum creatinine levels

[0037] (1) Serum sample preparation: Take 30 μL of serum from each group into a 1.5 mL EP tube, add 270 μL of protein precipitation solution, mix thoroughly, centrifuge at 7500 g for 10 min, and take the supernatant; (2) Preparation of standard working solution: Mix Cr standard (10 mmol / L): Cr standard diluent = 1:99 to obtain 100 μmol / L Cr standard.

[0038] Add the sample as follows: Table 3

[0039] Serum Cr concentration (μmol / L) = (Measured OD value / Standard OD value) × 100

[0040] 1.4 Real-time quantitative reverse transcription polymerase chain reaction for detecting gene expression levels in kidney tissue

[0041] (1) Extraction of total RNA from mouse kidneys

[0042] 10 mg of kidney tissue sample was thoroughly mixed with 1 mL of Trizol lysis buffer, homogenized using a tissue homogenizer, and allowed to stand at room temperature for 10 min. Then, it was centrifuged at 12000 rpm for 5 min at 4 °C. 700 μL of the supernatant was collected, and 150 μL of chloroform was added. The mixture was thoroughly mixed and allowed to stand at room temperature for 5 min. After standing, it was centrifuged at 12000 rpm for 10 min at 4 °C. 300 μL of the upper layer was collected, and 300 μL of isopropanol was added. The mixture was vortexed thoroughly and then incubated at -20 °C for RNA precipitation for 5 min. After precipitation, it was centrifuged at 12000 rpm for 10 min at 4 °C, the supernatant was discarded, and the sample was washed with pre-chilled 75% ethanol solution. After washing, it was centrifuged at 12000 rpm for 5 min at 4 °C, and the supernatant was discarded. The sample was then placed in a fume hood for clearing. Once the RNA precipitate at the bottom became colorless, 20 μL of DEPC water was added for resuspending to obtain a high-purity total RNA solution. RNA concentration was measured using Nanodrop.

[0043] (2) cDNA preparation

[0044] Calculate the required volume based on the target RNA amount (500 ng) and sample concentration (volume = target amount / concentration).

[0045] Prepare the following mixture in an RNase-free centrifuge tube and gently mix the following components: Table 4

[0046] (3) Real-time quantitative reverse transcription polymerase chain reaction to detect gene expression levels in tissues

[0047] Prepare the following mixture in a PCR tube: Table 5

[0048] The qPCR reaction was performed under the following conditions: Table 6

[0049] (2) The gene primer sequences involved in the experiment are as follows: Table 7 Primer Sequences

[0050] 1.5 H&E staining of kidney tissue sections to assess histomorphological changes

[0051] (1) Sample preparation: Mouse kidney tissue fixed in 4% paraformaldehyde was removed, the tissue was trimmed and flattened in a fume hood, and placed in a dehydration box with the corresponding label.

[0052] (2) Dehydration: Dehydration is carried out in the dehydrator according to the alcohol concentration gradient. The dehydration sequence and time are as follows: 75% alcohol for 4 h, 85% alcohol for 2 h, 90% alcohol for 2 h, 95% alcohol for 1 h, anhydrous ethanol I for 30 min, and anhydrous ethanol II for 30 min.

[0053] (3) Transparency: After dehydration, immerse in xylene I for 6-10 min and xylene II for 6-10 min (the specific time depends on the degree of transparency).

[0054] (4) Wax impregnation: After the glass becomes transparent, immerse in wax I for 1 hour, wax II for 1 hour, and wax III for 1 hour.

[0055] (5) Embedding: The melted wax is placed into the embedding frame, and the tissue is placed into the embedding frame before the wax solidifies. The corresponding label is attached. Cool at -20 °C. After the wax solidifies, the wax block is removed and trimmed.

[0056] (6) Sectioning: Using a paraffin microtome, the wax block is sectioned to a thickness of 3 μm. The sections are floated on a 40 °C warm water spreader to flatten the tissue, and then picked up with a glass slide. The sections are baked in a 60 °C oven until the water is dry and the wax is melted. After baking, they are taken out and stored at room temperature.

[0057] (7) Dewaxing: The sections were placed in xylene I for 20 min, xylene II for 20 min, anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, 75% alcohol for 5 min, and rinsed with water for 2 min.

[0058] (8) Hematoxylin staining: After staining with hematoxylin for 3-5 minutes, differentiate with hydrochloric acid solution, turn blue with ammonia solution, and rinse with water to remove residual liquid.

[0059] (9) Eosin staining: The sections were placed in 85% and 95% alcohol gradients for dehydration, and then stained in eosin staining solution for 5 min.

[0060] (10) Dehydration: The slices were placed in anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, anhydrous ethanol III for 5 min, xylene I for 5 min, and xylene II for 5 min in sequence.

[0061] (11) Mounting: Take out the slide, wipe the surrounding xylene with filter paper, add neutral resin to the slide until it completely covers the sample, cover with a coverslip with tweezers, remove excess neutral resin with qualitative filter paper, air dry naturally, and store at room temperature.

[0062] (12) Image acquisition: Observe the morphology of kidney tissue under an optical microscope, acquire multiple field-of-view images at different magnifications, and save the images.

[0063] (13) Tissue damage score: The renal tubular damage was divided into 5 grades according to the degree of damage: normal kidney, <25% damage, 25-50% damage, 50-75% damage and >75% damage. The score was recorded as 0 to 4. The scoring method was blinded.

[0064] 1.6 Masson staining of kidney tissue sections to assess histomorphological changes

[0065] Steps (1)-(9) are the same as H&E staining.

[0066] (10) Collagen staining: Immerse the sections in phosphomolybdic acid solution for mordanting for 5-10 min, then directly transfer them to aniline blue staining solution (or bright green staining solution) for 5-10 min.

[0067] (11) Differentiation and dehydration: Differentiate with 1% glacial acetic acid solution for 30 s, rinse with distilled water, and then put in 95% alcohol for 5 min, anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, anhydrous ethanol III for 5 min, xylene I for 5 min, and xylene II for 5 min in sequence.

[0068] Steps (11)-(12) are the same as H&E staining.

[0069] (13) Calculation of fibrosis area: The fibrosis area was analyzed using ImageJ software based on the Masson staining image results.

[0070] 1.7 Statistical Analysis

[0071] All statistical results are expressed as mean ± standard error (mean ± SEM). Statistical analysis was performed using GraphPad Prism 8.4.2 software. One-way ANOVA was used to compare data among multiple groups, with a confidence interval set at 95%. P < 0.05 was considered statistically significant.

[0072] 2. Experimental Results

[0073] 2.1 Monomethyl maleate can effectively reduce renal fibrosis in mice.

[0074] Figures 1A to 1J The effect of a monomethyl unsaturated dicarboxylic acid ester on renal fibrosis in mice.

[0075] The results showed that, after unilateral ureteral obstruction, drug treatment restored mouse body weight to varying degrees compared with the model group (Figure 1A). Serum BUN and Scr levels in the model group were significantly higher than those in the control group. Different drug treatments all reduced serum BUN and Scr levels, indicating improved renal function in the mice, with monomethyl maleate showing the most significant improvement (Figure 1B and C). RT-qPCR was used to detect mouse renal fibrosis genes. TGF-βand α-SMA Oxidative stress-related genes SOD2 and NQO-1 Inflammation-related genes IL-6 , TNF-α and IL-11 Expression levels were measured, and the results showed that monomethyl maleate had a good effect in reducing renal fibrosis, oxidative stress and inflammation in mice (DJ in Figure 1).

[0076] 2.2 Monomethyl maleate can effectively reduce the degree of morphological damage to mouse kidney tissue.

[0077] Figures 2A-2C Pathological changes in the kidneys of mice after administration of monomethyl maleate.

[0078] H&E staining of kidney tissue sections (Figure 2A) showed that the renal tubules and glomeruli of the control mice were structurally normal. Unilateral ureteral obstruction led to severe pathological changes in the mice's kidneys, including glomerular structural damage, tubular dilation, vacuolation, extensive tubular epithelial detachment and necrosis, and a significantly increased tubular injury score (Figure 2C). In contrast, treatment with monomethyl maleate alleviated the above-mentioned kidney damage. Masson staining (Figure 2A) showed that, compared with the control mice, unilateral ureteral obstruction led to a significantly larger area of ​​renal fibrosis in the mice. Treatment with monomethyl maleate reduced the occurrence of renal fibrosis. Figure 2B ).

[0079] Example 2

[0080] 1. Experimental Methods

[0081] The difference from Example 1 is that the administration method is as follows: Table 8

[0082] 2. Experimental Results

[0083] 2.1 Monomethyl maleate combined with Mn 2+ It can effectively reduce renal fibrosis in mice.

[0084] Figures 3A to 3I The effects of monomethyl maleate combined with four metal ions on renal fibrosis in mice.

[0085] Based on the screening results of Example 1, monomethyl maleate has a good effect in reducing renal fibrosis. Therefore, monomethyl maleate and Zn were selected. 2+ Al 3+ Mn 2+ and Mg 2+ This study was used to verify whether metal ion adjuvants could further enhance the anti-renal fibrosis effect of monomethyl maleate. After unilateral ureteral obstruction, drug treatment restored mouse body weight to varying degrees compared to the model group. Figure 3A The serum BUN and Scr levels in the model group mice were significantly higher than those in the control group mice. Drug treatment reduced serum BUN and Scr levels in the mice, indicating improved renal function. The combination of monomethyl maleate and Mn... 2+ The effect is most significant ( Figure 3B and Figure 3C RT-qPCR results showed that monomethyl maleate combined with Al 3+ , Mn 2+ Mg 2+ For reducing the gene for renal fibrosis in mice TGF-β and α-SMA Restore oxidative stress-related genes SOD2 and NQO-1 Reduce inflammation-related genes IL-6 and IL-11 Both showed some effect in terms of expression level, with monomethyl maleate combined with Mn showing the best effect. 2+ Optimal results (Figure 3 D-) Figure 3I ).

[0086] Example 3

[0087] 1. Experimental Methods

[0088] The difference from Example 2 is that the administration method is as follows: Table 9

[0089] The experiment involved the following gene primer sequence listing: Table 10

[0090] 2. Experimental Results

[0091] 2.1 Mn 2+ Screening for the optimal ratio of maleic acid monomethyl ester to reduce renal fibrosis in mice

[0092] Figures 4A-4J For different ratios of monomethyl maleate combined with Mn 2+ Effects on renal fibrosis in mice.

[0093] Based on the screening results of Example 2, monomethyl maleate combined with Mn 2+ It has a good effect in reducing renal fibrosis, so the next step was to explore the appropriate ratio of Mn. 2+The combination with monomethyl maleate showed the best effect in preventing and treating renal fibrosis. After unilateral ureteral obstruction, drug treatment restored mouse body weight to varying degrees compared to the model group (Figure 4A). Serum BUN and Scr levels in the model group were significantly higher than in the control group. Drug treatment reduced serum BUN and Scr levels in mice, indicating improved renal function. 2+ The effect was most significant when the ratio of maleic acid monomethyl ester to maleate was 1:2, and it was superior to the positive control drug pirfenidone (Figure 4B and 4C). Figure 4C RT-qPCR results showed different proportions of Mn. 2+ Treatment with monomethyl maleate reduced the renal fibrosis gene in mice TGF-β , α-SMA and COL1A1 Restore oxidative stress-related genes SOD2 and NQO- 1 Reduce inflammation-related genes IL-6 and IL-11 The expression levels of both showed some effect, with Mn being particularly effective. 2+ The optimal ratio of maleate monomethyl ester to pirfenidone was 1:2, which was superior to the positive control drug pirfenidone (Figure 4 D-). Figure 4J ).

[0094] Example 4

[0095] 1. Experimental Methods

[0096] The difference from Example 3 is that the administration method is as follows: Table 11

[0097] The gene primer sequences involved in the experiment are as follows: Table 12

[0098] 2. Experimental Results

[0099] 2.1 Monomethyl maleate combined with Mn 2+ Optimal dose screening

[0100] Figures 5A to 5L Different doses of monomethyl maleate combined with Mn 2+ Effects on renal fibrosis in mice.

[0101] Based on the screening results of Example 2, when Mn 2+ A 1:2 ratio of monomethyl maleate to methyl maleate showed a better effect in reducing renal fibrosis. The following investigation explored the effects of different doses of monomethyl maleate combined with Mn. 2+Comparison of therapeutic effects on renal fibrosis. Following unilateral ureteral obstruction, drug treatment restored mouse body weight to varying degrees compared to the model group (Figure 5A). Serum BUN and Scr levels in the model group were significantly higher than in the control group. Drug treatment reduced serum BUN and Scr levels, indicating improved renal function. The combination of medium-dose monomethyl maleate and Mn... 2+ The effect was most significant and superior to the positive control drug pirfenidone (Figure 5B and Figure 5C (Table 13). RT-qPCR results showed different proportions of Mn. 2+ Treatment with monomethyl maleate reduced the renal fibrosis gene in mice TGF-β , α-SMA and COL1A1 Restore oxidative stress-related genes SOD2 , NQO-1 and HO-1 Reduce inflammation-related genes IL-6 , TNF-α and IL-11 Both showed some effect on expression levels, with the medium dose of monomethyl maleate combined with Mn showing the best effect. 2+ The efficacy was optimal and superior to the positive control drug pirfenidone, with COL1A1 and IL-11 expression levels decreasing by more than 50% compared to the model group (Figure 5 D-). Figure 5L (Tables 13 and 14).

[0102] 2.2 Monomethyl maleate combined with Mn 2+ It can effectively reduce the degree of morphological damage to the kidney tissue in mice.

[0103] Figures 6A-6C Pirfenidone and monomethyl maleate combined with Mn 2+ Pathological changes in the kidneys of mice after drug administration.

[0104] H&E staining of kidney tissue sections revealed that the renal tubules and glomeruli of the control group mice were structurally normal. Unilateral ureteral obstruction led to severe pathological changes in the mice's kidneys, including glomerular structural damage, tubular dilation, vacuolation, extensive tubular epithelial detachment and necrosis, and a significantly elevated tubular injury score. In contrast, the use of monomethyl maleate combined with Mn... 2+ Treatment can alleviate the aforementioned kidney damage. Masson staining showed that, compared to control mice, unilateral ureteral obstruction significantly increased the area of ​​renal fibrosis in mice. Treatment with monomethyl maleate combined with Mn... 2+ Treatment can reduce the incidence of renal fibrosis. Figures 6A-6C (Tables 13-14)

[0105] Table 13 Mn 2+ +Effective effect of monomethyl maleate in preventing renal fibrosis

[0106] Table 14 Mn 2+ Comparison of the effects of monomethyl maleate and pirfenidone in preventing and treating renal fibrosis

[0107] The above are merely preferred embodiments of the present invention. It should be noted that, for those skilled in the art, several improvements and structural modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A composition for preventing and treating renal fibrosis, characterized in that, Includes component A and component B; The molar ratio of component A to component B is (1~3):(1~5); Component A is an unsaturated dicarboxylic acid monomethyl ester; Component B is a metal ion; the metal ion includes Zn. 2+ Al 3+ Mn 2+ Mg 2+ At least one of them.

2. The composition according to claim 1, characterized in that, The molar ratio of component A to component B is 1:1, 1:2, 1:3, 1:4.5, 1.5:2 or 2.25:

2.

3. The composition according to claim 1, characterized in that, The unsaturated dicarboxylic acid monomethyl ester is at least one of maleic acid monomethyl ester, fumarate monomethyl ester, and itaconic acid monomethyl ester.

4. The composition according to claim 1, characterized in that, The metal ion is Mn. 2+ .

5. The composition according to any one of claims 1 to 4, characterized in that, The unsaturated dicarboxylic acid monomethyl ester is maleic acid monomethyl ester.

6. The use of component A or the composition according to any one of claims 1 to 5 in the preparation of a medicament for preventing and treating renal fibrosis; wherein component A is component A in the composition according to any one of claims 1 to 5.

7. The application according to claim 6, characterized in that, Component A or the composition is used to regulate the level of inflammatory factors, reduce the level of oxidative stress, and improve renal function and / or the degree of renal fibrosis.

8. The application according to claim 7, characterized in that, The inflammation-related factors include at least one of TNF-α, IL-6, IL-1β, and IL-11; The indicators of oxidative stress include at least one of NRF2, NQO1, HO-1 and SOD2; The renal function indicators include at least one of BUN and Scr; The renal fibrosis markers include at least one of TGF-β, α-SMA, and COL1A1.

9. A drug for preventing and treating renal fibrosis, characterized in that, Includes component A or the composition according to any one of claims 1 to 5.

10. The medicament according to claim 9, characterized in that, It also includes pharmaceutically acceptable excipients; said pharmaceutically acceptable excipients include at least one of fillers, binders, disintegrants, lubricants, suspending agents, preservatives, flavoring agents, and sweeteners.

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