Application of MIF inhibitor in preparation of medicine for preventing and / or treating peritoneal ultrafiltration failure or peritoneal fibrosis

Abstract

The invention provides application of an MIF inhibitor in preparation of a medicine for preventing and / or treating peritoneal ultrafiltration failure or peritoneal fibrosis. The MIF inhibitor is used for protecting peritoneal mesothelial cells and peritoneal macrophages induced by high-concentration glucose peritoneal dialysis fluid from being damaged, so that peritoneal fibrosis is relieved, the time that a patient can accept peritoneal dialysis is prolonged, and the arrival of peritoneal ultrafiltration failure is delayed.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to the application of MIF inhibitors in the preparation of drugs for the prevention and / or treatment of peritoneal ultrafiltration failure or peritoneal fibrosis. Background Technology

[0002] Peritoneal dialysis (PD) is one of the main renal replacement therapies for patients with end-stage renal disease (ESRD) and uremia. In PD, a hypertonic solution is used to remove excess water from the body through the peritoneum (PM). Currently, the most commonly used osmotic agent in PD solutions is a 1.5%–4.25% glucose solution, such as Dianeal from Baxter (Deerfield, IL, USA). As dialysis time increases, low-concentration glucose dialysate quickly becomes insufficient to remove enough water, requiring the use of high-concentration glucose dialysate. However, with the increased frequency of high-concentration glucose dialysate use, dialysis efficiency typically declines after two years of dialysis, making it impossible for patients to continue relying solely on peritoneal dialysis and necessitating conversion to hemodialysis. Statistics show that the incidence of ultrafiltration failure increases with the duration of PD, and ultrafiltration failure is the main reason why peritoneal dialysis patients are forced to discontinue peritoneal dialysis. Preventing and slowing down peritoneal fibrosis is the fundamental way to avoid ultrafiltration failure in peritoneal dialysis patients and is the key to ensuring their long-term dialysis success and extending their lifespan.

[0003] The peritoneum consists of three layers: the mesothelial layer, the basal layer, and the submesothelial supporting tissue. The submesothelial supporting tissue is rich in microvessels, and mesothelial cells and submesothelial microvessels are the most important cellular and structural basis for peritoneal dialysis. Morphologically, peritoneal fibrosis is characterized by a reduction in mesothelial cells, with dead mesothelial cells being replaced by myofibroblasts or fibroblasts, inflammatory cell infiltration, and a large accumulation of extracellular matrix accompanied by an increased number of blood vessels. Therefore, mesothelial cell death and peritoneal inflammation are the root causes of peritoneal fibrosis. The current mainstream view is that the factors leading to mesothelial cell death and peritoneal inflammation are high glucose concentrations themselves, as well as the abnormal cell metabolism and free radicals induced by high glucose concentrations. Therefore, in recent years, many scientific and clinical studies have focused on finding alternative dialysates to high-concentration glucose solutions, but unfortunately, none have achieved the effect of preventing peritoneal fibrosis. Summary of the Invention

[0004] The purpose of this invention is to provide the use of MIF inhibitors in the preparation of medicaments for the prevention and / or treatment of peritoneal ultrafiltration failure or peritoneal fibrosis.

[0005] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0006] This invention provides the use of MIF inhibitors in the preparation of medicaments for the prevention and / or treatment of peritoneal ultrafiltration failure or peritoneal fibrosis.

[0007] Because MIFs are closely associated with the progression of many inflammatory diseases, much research has focused on finding MIF-directed therapies. The application of biologics (such as anti-MIF antibodies) as novel therapeutic agents is one direction of development. This approach has been used in numerous studies. A clinical trial of an anti-MIF antibody has been reported, but no further results have been revealed. Another approach is to develop small molecules that bind to MIFs to interfere with their function. Due to its unique structure, MIFs also possess enzymatic activity, containing two catalytic centers: one is a thiol oxidoreductase activity, and the other is a tautomerase activity, the latter containing D-dopachrome tautomerase and phenylpyruvate tautomerase. It has been reported that the catalytically active sites of MIFs can also be considered pro-inflammatory sites; mutations in these active sites significantly reduce the pro-inflammatory activity of MIFs. These active sites are Pro-1, Lys-32, Ile-64, Tyr-95, and Asn-97. Currently, several small molecule inhibitors of peritoneal ultrafiltration (MIF) have been reported. Their mechanisms of inhibiting MIF enzyme activity can be broadly categorized into five types: direct binding to the active site; allosteric inhibition; covalent modification of the Pro1 residue at the active site; dissociation of the dimer induced by the compound leading to the destruction of the active site; and stabilizing the MIF monomer to prevent the formation of the active trimer. However, to date, there are no reports of MIF inhibitors being used for the prevention and / or treatment of peritoneal ultrafiltration failure or peritoneal fibrosis.

[0008] According to some embodiments, the MIF inhibitor is selected from MIF tautomerase inhibitors and / or MIF oxidoreductase inhibitors.

[0009] According to some further embodiments, the MIF inhibitor is one or more selected from ISO-1, (±)-CPSI-1306, ISO-66, Jorgensen-3g, Jorgensen-3h, Dziedzic-3bb, 4-IPP, isothiocyanate, K664-1, iguratimod, AV411 or AV1013.

[0010] ISO-1 inhibits mif isomerase activity in a dose-dependent manner. It binds to the substrate p-hydroxyphenylpyruvate at the same position, with an IC50 of approximately 7 μM.

[0011] CPSI-1306, a small molecule inhibitor of isoxazoline scaffold MIF tautomerase, lacks the characteristic phenolic function, which is advantageous for in vivo application. Phenolic functions are generally considered non-pharmacological due to their tendency to undergo phase II bioconjugation, leading to rapid inactivation and excretion in vivo. In a mouse model of multiple sclerosis, mice orally administered this inhibitor showed milder symptoms compared to untreated mice. Subsequently, another isoxazoline small molecule inhibitor, ISO-66, was reported in 2014. In MIF variant enzyme assays, ISO-66 had an IC50 of 1.5 μM. In mouse models of colon cancer or melanoma, long-term administration of ISO-66 showed no cytotoxicity and significantly reduced tumor burden.

[0012] In 2010, 1,2,3-triazole derivatives were reported as MIF inhibitors. The most potent compounds, Jorgensen-3g and Jorgensen-3h, exhibited MIF isomerase activity of approximately 1 μm and an IC50 value for MIF-cd74 binding. Subsequently, in 2015, improvements were made by synthesizing several optimized biaryltriazoles. This provided potent compounds with phenolic hydroxyl groups that bind to the MIF tautomerase active site. However, some compounds exhibited limited water solubility. The activity of these compounds was further enhanced by adding a fluorine atom near the phenolic hydroxyl group to strengthen the hydrogen bond interaction with the MIF residue Asn-97. This yielded the most potent compound, Dziedzic-3bb, with a Ki value of 0.057 μ and solubility within the normal range for oral administration.

[0013] The specific reactivity of proline at the active site of MIFs offers an opportunity to develop covalent inhibitors. In 2008, a phenylpyrimidine compound, 4-IPP, was reported to inactivate the catalytic function of MIFs through dehalogenation and the formation of a covalent bond between the C-4 of the pyrimidine and the n-terminal nitrogen of Pro-1 at the miftautoerase active site. This compound also interferes with the biological function of MIFs, and has been reported to irreversibly inhibit the migration and anchorage-independent growth of lung adenocarcinoma cells. Later studies showed that 4-IPP inhibits the growth of thyroid cancer cells by inducing apoptosis and mitotic cell death. In 2009, isothiocyanates were discovered as irreversible inhibitors of MIF variant enzymes. The isothiocyanate BITC exhibits covalent modification of the Pro-1 residue at the MIF active site. This drastically alters the tertiary structure of MIFs, leading to the loss of their tautomerase activity and inhibition of MIF binding to CD74.

[0014] Therefore, these compounds not only inhibit MIF enzyme activity but also show some inhibitory effects on MIF-induced biological functions. Our results have confirmed that the MIF inhibitor ISO-1 can protect against damage to peritoneal mesothelial cells and peritoneal macrophages induced by high-concentration glucose peritoneal dialysis fluid. Therefore, we suggest that the above-mentioned MIF inhibitors can all protect against damage to peritoneal mesothelial cells and peritoneal macrophages, especially the long-term use of the non-cytotoxic ISO-66, which can significantly protect against peritoneal ultrafiltration failure and peritoneal fibrosis in mice undergoing peritoneal dialysis.

[0015] According to some embodiments, the concentration of the MIF inhibitor is 10–1000 μM, further, the concentration of the MIF inhibitor is 20–800 μM, further, the concentration of the MIF inhibitor is 30–500 μM, further, the concentration of the MIF inhibitor is 40–400 μM, further, the concentration of the MIF inhibitor is 50–400 μM, further, the concentration of the MIF inhibitor is 60–400 μM, and further, the concentration of the MIF inhibitor is 100–400 μM.

[0016] According to some embodiments, the MIF inhibitor can be added to peritoneal dialysis fluid for use, or the MIF inhibitor can be prepared into a formulation with pharmaceutical excipients for use.

[0017] Compared with the prior art, the present invention has the following advantages:

[0018] This invention protects peritoneal mesothelial cells and peritoneal macrophages from damage induced by high-concentration glucose peritoneal dialysis fluid using MIF inhibitors, thereby alleviating peritoneal fibrosis, prolonging the time patients can undergo peritoneal dialysis, and delaying the onset of peritoneal ultrafiltration failure. Attached Figure Description

[0019] Figure 1 This is a graph showing the detection results from Example 1;

[0020] Figure 2 This is a graph showing the detection results from Example 2;

[0021] Figure 3 This is a graph showing the detection results in Example 3;

[0022] Figure 4 This is a diagram of the detection results in Example 4;

[0023] Figure 5 This is a diagram showing the detection results in Example 5;

[0024] Figure 6 This is a graph showing the detection results in Example 6;

[0025] Figure 7This is a graph showing the detection results in Example 7;

[0026] Figure 8 This is a graph showing the detection results in Example 8;

[0027] Figure 9 This is a graph showing the detection results in Example 9;

[0028] Figure 10 This is a graph showing the detection results in Example 10. Detailed Implementation

[0029] The present invention will be further described below with reference to embodiments. However, the present invention is not limited to the following embodiments. The implementation conditions used in the embodiments can be further adjusted according to different requirements of specific use, and the implementation conditions not specified are conventional conditions in this industry. The technical features involved in the various embodiments of the present invention can be combined with each other as long as they do not conflict with each other. In the specific embodiments of the present invention, the raw materials used can all be obtained commercially.

[0030] Example 1:

[0031] 1. Transcriptomic and proteomic data from a previous high-glucose-induced peritoneal mesothelial cell injury model showed increased MIF expression. Therefore, we collected peritoneal dialysis supernatant from overnight peritoneal dialysis patients, using the supernatant from the second peritoneal lavage after catheter placement as a control group, and measured MIF content. We used ELISA to detect MIF content in peritoneal dialysis supernatant. The kit was purchased from Wuhan Elabscience Biotechnology Co., Ltd. (CAT#E-EL-H6170). Results Figure 1 As shown in A, from Figure 1 As can be seen, the content of MIF in the supernatant of peritoneal dialysis fluid of peritoneal dialysis patients is significantly increased, and it increases with the increase of glucose concentration in the dialysis fluid.

[0032] 2. Human peritoneal mesothelial cell line HMrSV5 was seeded into DMEM high-glucose medium (hyclone, Cat No: SH30022.01) containing 10% (v / v) FBS and 1% penicillin / streptomycin. The cells were cultured at 37°C in a 5% CO2 incubator, and cell growth was observed. The medium was changed every 2 days. After the cells reached 90% confluence, they were passaged and digested with 0.25% trypsin at 37°C for 3 min. After digestion, the cells were collected, centrifuged at 1000 rpm at room temperature for 5 min, resuspended in complete medium, and counted. Cells were then seeded into 6-well plates at 700,000 cells / well. Mesothelial cells adhered for 16-24 h, followed by 12 h of starvation, and then treated with peritoneal dialysis fluid containing different concentrations of glucose (Glu 83mM, 136mM, 236mM) for 6, 12, and 24 hours. The culture supernatant of peritoneal mesothelial cells treated as described above was collected. The results showed that the secretion of mesothelial cells (MIFs) increased with prolonged high-glucose treatment time, and further increased with increasing glucose concentration. Figure 1 B), the method for detecting MIF content is the same as described in section 1 above. Simultaneously, the activity of MIF dopa tautomerase in high-concentration glucose-induced peritoneal mesothelial cell culture medium was detected, and its activity was found to be significantly increased (B). Figure 1 C), where the detection principle of MIF dopa tautomerase activity is as follows: The D-dopachrome methyl esterase activity of MIF can, under suitable conditions, turn the orange D,L-dopachrome methyl ester solution colorless and convert it into a 2,3-indolediic acid derivative. A decrease in absorbance can be detected at a wavelength of 475 nm. The greater the increase in MIF enzyme activity, the greater the decrease in absorbance. When MIF enzyme activity is inhibited, the decrease in absorbance is less. Detection method: According to the method described in the literature, at room temperature, 30 μL of orange-red L-dopachrome methyl ester solution was reacted with 60 μL of recombinant human MIF purified protein (360 nM) dissolved in 50 mM potassium phosphate buffer (containing 50 mM KH2PO4, 50 mM K2HPO4*3H2O, 0.5 mM EDTA, pH = 6) in a 96-well plate. The solution immediately changed from orange to colorless. Since L-dopachrome methyl ester is unstable at room temperature, it must be prepared fresh before use. It can be prepared by reacting 6 mM L-3,4-dihydroxyphenylalanine methyl ester and 12 mM INaO4 in equal volumes at room temperature for 3-5 minutes. After adding recombinant human MIF protein, the reaction was carried out at room temperature for 20-25 minutes. The absorbance value was detected at 475 nm using a full-wavelength microplate reader, with measurements taken every 10 seconds for a total of 400 seconds.

[0033] 3. A mouse peritoneal fibrosis model was established by intraperitoneal injection of hypertonic peritoneal dialysis fluid (4.25% PD). Twelve C57WT mice were divided into two groups: a control group (n=6) receiving daily intraperitoneal injections of 0.9% normal saline (NS) at a volume equal to 120 ml / kg of body weight in sterile saline; and a model group (n=6) receiving daily intraperitoneal injections of 4.25% peritoneal dialysis fluid at a volume equal to 120 ml / kg of body weight. This continued for six weeks. At the start of the experiment, there was no significant difference in body weight between the groups. From day one, mouse body weight was monitored and recorded daily. The remaining mice exhibited normal eating and activity levels, good mental state, and increased body weight compared to the initial weight. At the end of the experiment, body weight was found to be lower in the model group than in the normal control group. However, statistical analysis revealed no significant differences.

[0034] Animal sample collection, specimen processing, and preservation

[0035] In week 6 of the experiment, a peritoneal balance test was performed to monitor peritoneal function and collect tissue samples from mice. The peritoneal balance test was conducted 2 hours after mice stopped peritoneal dialysis for 48 hours. Mice were anesthetized by intramuscular injection of 1% sodium pentobarbital at a dose of 0.07 ml / 10 g.

[0036] After anesthetizing the mice, they were fixed in a supine position on the operating table. The abdomen was routinely disinfected, and a sterile surgical drape was applied. The operator wore a sterile surgical gown, mask, and gloves. A small incision was then made along the linea alba using sterile forceps. Each mouse received an intraperitoneal injection of 3 mL of 4.25% peritoneal dialysis fluid. A timer was used to control the flow, and the abdomen was gently agitated during dialysis. After 1 hour of retention in the peritoneum, 100 μL of dialysis fluid was aspirated into a clean 600 μL EP tube and placed on ice. Simultaneously, 100 μL of blood was drawn from the tail vein and placed into an EP tube, which was then labeled. After 2 hours of retention in the peritoneum, the dialysis fluid was aspirated from the peritoneum using a sterile syringe until all fluid was removed. The volume of peritoneal dialysis fluid in the syringe was recorded and transferred to a 5 mL centrifuge tube, which was then labeled. At the same time, 100 μL of blood was drawn from the tail vein. Next, use a scalpel to open the abdominal cavity along the linea alba, bluntly separating the fascia and muscles layer by layer, avoiding damage to small blood vessels. Wipe away any blood seeping from ruptured small blood vessels to prevent blood from flowing into the abdominal cavity and affecting the calculation of the ultrafiltration volume. Ultrafiltration volume: Gently soak a sterile gauze pad with the fluid from the abdominal cavity and calculate the net increase in volume of the gauze. Ultrafiltration volume (UF) = Volume of dialysate in the syringe + Net increase in volume of gauze - 3 ml.

[0037] Sampling: The abdominal wall was fully exposed. Parietal peritoneum from both sides of the abdominal incision was harvested, washed multiple times in pre-cooled PBS, cut into small pieces, and fixed separately in 4% PFA (PFA volume more than 10 times the tissue volume), embedded in OCT and immediately placed in liquid nitrogen, and then placed in 1.5ml EP tubes in liquid nitrogen for protein and RNA detection. The appearance of the mesentery and other visceral peritoneum was observed. A portion of the visceral peritoneum was aliquoted into EP tubes, labeled, and immediately flash-frozen in liquid nitrogen. The samples in liquid nitrogen were then quickly frozen at -80°C. Tubes containing tissue and 4% PFA were stored at 4°C.

[0038] Separating peritoneal dialysis fluid: Place 600 μL centrifuge tubes containing peritoneal dialysis fluid samples after 1 h and 2 h of peritoneal retention in a 4°C centrifuge at 3500 rpm for 10 min. After centrifugation, carefully transfer the supernatant to a new 600 μL EP tube and immediately perform parameter testing or temporarily store at -20°C.

[0039] Separating cells from peritoneal dialysis fluid: Place the recovered peritoneal dialysis fluid on ice, centrifuge at 3500 rpm and 4 degrees Celsius for 10 minutes, transfer the peritoneal dialysis fluid supernatant, and remove any red blood cells from the sediment at the bottom of the tube. Finally, wash the cells three times with pre-cooled PBS, centrifuge again at low temperature, discard the supernatant, recover the cells, and add an appropriate amount of protein extraction solution according to the cell volume to extract proteins.

[0040] The parietal peritoneum was paraffin-embedded and sectioned, and the tissue was fixed in OCT for the preparation of frozen sections.

[0041] Immunohistochemical results confirmed a significant increase in MIF expression in peritoneal fibrosis tissue. Figure 1 D).

[0042] from Figure 1 It is evident that high-glucose peritoneal dialysis fluid leads to increased production of mesothelial cell fibroblasts (MIFs), accompanied by enhanced activity of MIF dopa tautomerase. Figure 1 A showed that in the supernatant of peritoneal dialysis fluid from patients who underwent overnight peritoneal dialysis, the ELISA results indicated an increased level of MIF, which increased with increasing glucose concentration in the peritoneal dialysis fluid (*P<0.05, **p<0.01). Figure 1 B shows that in peritoneal mesothelial cell injury models induced by different concentrations of glucose in peritoneal dialysis fluid, the secretion of MIF in cell culture supernatant increased, with the most significant increase at 24 hours (*P<0.05, **p<0.01 vs NG). Figure 1 C shows that the activity of dopa tautomerase in peritoneal mesothelial cell culture supernatant treated with high glucose for different time periods was detected. The detection showed that the activity of dopa tautomerase in MIF was increased, and was significantly upregulated from 6 hours. N=3, *p<0.05, ****p<0.0001 vs NG. Figure 1 D shows a paraffin section of a wild-type mouse peritoneal fibrosis model. Immunohistochemical results indicate increased expression of MIF in the peritoneal tissue of peritoneal fibrosis. The brown areas in the figure and the red asterisks indicate positive signals. Scale bar = 50 μM.

[0043] Example 2:

[0044] Ten-week-old male MIF wild-type and MIF- / - (KO) mice were each divided into two groups: a normal saline (NS) control group and a 4.25% glucose dialysis group (n = 6 / genotype). Samples were collected 6 weeks after intraperitoneal injection. The modeling and sample collection methods were the same as in Part 3 of Example 1. Wild-type MIF mice developed ultrafiltration failure after 6 weeks of dialysis. Figure 2 A), while ultrafiltration in MIF KO mice was essentially normal. The level of LDH in the dialysate 2 hours after MIF knockout was significantly reduced ( Figure 2 B), LDH detection was performed using the Nanjing Jiancheng Bioengineering Institute's lactate dehydrogenase (LDH) kit (CAT#A020-2-2). The specific detection method was the same as the kit instructions. Masson staining was used to observe the morphology and thickness of the mouse peritoneum to assess the degree of fibrosis. Compared to the saline group, the high-glucose dialysis group in the control group showed a large amount of subcutaneous collagen fiber deposition in the interstitial tissues, with more and denser blue collagen fibers, indicating a successful establishment of the mouse peritoneal fibrosis model. In contrast, the MIF KO dialysis group showed only a small amount of blue collagen fiber accumulation (…). Figure 2 CD), where blue represents collagen fibers and red represents muscle fibers, with a scale bar of 50 μm. Western blot results showed that caspase 3 activation and GSDME cleavage could also be detected in exfoliated cells of the peritoneal dialysis fluid from wild-type control mice, but this phenomenon was inhibited after MIF KO. Figure 2 E). N=6. *p<0.05,**p<0.01,***p<0.001vs WT Nacl. #p<0.05,##p<0.01vs MIF- / -PD group. MIF knockout reduced peritoneal fibrosis and the number of blood vessels and lymphatic vessels in mice. Immunohistochemical staining for changes in fibrosis markers showed that Fibronectin expression in the model group was significantly reduced compared to the wild-type model group after MIF knockout. Figure 2 F). To assess the number of blood vessels in the peritoneal tissue of a MIF knockout mouse model of peritoneal fibrosis induced by high-glucose dialysis fluid, we used immunohistochemical staining to detect the vascular endothelial marker proteins CD31 and lymphatic endothelial receptor 1 (LYVE-1), such as... Figure 2As shown in GH, in the simple high glucose model group, the number of CD31 and LYVE-1 positive cells significantly increased in the thickened peritoneal tissue and exhibited a vascular distribution in the peritoneal mesocortex. MIF knockout significantly inhibited the upregulation of CD31 and LYVE-1 (Scale bar = 50 μm). These data indicate that MIF knockout can alleviate peritoneal fibrosis and ultrafiltration failure in mice induced by high glucose dialysate, and that MIF-mediated pyroptosis plays an important role in the progression of peritoneal fibrosis and ultrafiltration failure induced by high glucose dialysate.

[0045] Example 3:

[0046] A peritoneal mesothelial cell line with specific MIF knockout was prepared using CRISPR-Cas9 technology. The MIF knockout cell line was purchased from Ubigene Biotech (https: / / www.ubigene.com / ). Western blotting was used to verify the MIF knockout efficiency (n=3, **P<0.01 vs Control). Figure 3 As shown in Figure A, MIF expression was significantly reduced, indicating that the MIF knockout cells were successfully constructed. Figure 3 B is a standard optical micrograph, with the arrow indicating pyrode bubbles, and the scale bar is 50 μm. Figure 3 C represents crystal violet staining of cells; a deeper blue indicates more surviving cells, and vice versa. Both ordinary light microscopy and crystal violet staining results confirmed that MIF knockout cells significantly reduced high glucose-induced peritoneal mesothelial cell death. Figure 3 Western blot results also confirmed that after MIF knockout, the degree of cleavage of Cleaved-Caspase 3 and GSDME was significantly reduced (BC). Figure 3 D), n=3, ****P<0.0001. This suggests that MIF knockout of peritoneal mesothelial cells can significantly protect against high glucose-induced peritoneal mesothelial cell damage.

[0047] Example 4:

[0048] To further investigate the impact of MIF knockout on the production of inflammatory factors, we further examined the levels of inflammatory factors TNF-α and IL-6 in the supernatant of high glucose-induced peritoneal mesothelial cell knockout MIF cell culture. We used ELISA to detect the levels of TNF-α and IL-6 in peritoneal dialysis fluid. The kits were purchased from Wuhan Elabscience Biotechnology Co., Ltd. (TNF-α cat#E-EL-H0109; IL-6 cat#E-EL-H6156), and the specific detection method was the same as the kit instructions. Figure 4AB showed that the release of inflammatory factors IL6 and TNFα in cell culture supernatant was increased, and the knockout of MIF in peritoneal mesothelial cells could significantly reduce the production of high glucose-induced inflammatory factors TNF-α and IL-6. Among them, N=3, **P<0.01, ****P<0.0001.

[0049] Example 5:

[0050] Peritoneal mesothelial cells from both control and MIF knockout groups were stimulated with high-concentration glucose (236 mM) for 12 hours. RNA was collected for transcriptomic sequencing. Venn diagrams were plotted to compare upregulated genes in the control high-glucose group with downregulated genes in the MIF knockout + high-glucose stimulation group. The results showed that the MIF knockout high-glucose group significantly downregulated 888 genes upregulated in the high-glucose control group. Figure 5 A). Further GO enrichment analysis of genes jointly regulated by both revealed multiple gene pathways related to angiogenesis and P38MAPK signaling cascade activation. Figure 5 B). Further KEGG pathway enrichment of genes jointly regulated by both revealed enrichment in Ribosome, TNFα signaling pathway, MAPK, PI3K-AKT and other signaling pathways. Figure 5 C). Further, a heatmap was created to display genes involved in the TNFα signaling pathway, angiogenesis, and MAPK signaling pathway. Figure 5 DF).

[0051] Example 6:

[0052] Since peritoneal macrophages also play an important role in the development and progression of peritoneal fibrosis, we further investigated the production of mesenchymal fibrosis (MIF) in the culture supernatant of peritoneal macrophages damaged by high-glucose peritoneal dialysis. We first performed primary culture of mouse peritoneal macrophages.

[0053] (1) Preparation: 25-30g C57-WT mice of the same sex were injected intraperitoneally with 1ml of 4% broth for 4 consecutive days.

[0054] Prepare the following equipment: autoclaved instruments (2 surgical scissors, 4 ophthalmic forceps), 1mL sterile syringe, 5mL sterile syringe, cell culture medium prepared with Gibco 1640 medium containing 2% FBS and 10% FBS, PBS, red blood cell lysis buffer, and 75% alcohol.

[0055] (2) Operation steps

[0056] ① Euthanize mice by cervical dislocation and soak them in 75% alcohol for 5 minutes. ② Make a small incision in the abdomen and inject 5 ml of 2% FBS 1640 medium into the incision using a 5 mL syringe (with a 1 mL syringe needle). Massage the abdomen for 8-15 minutes. ③ Aspirate the medium from the abdominal cavity and add it to a 15 mL centrifuge tube. Centrifuge at 1000 rpm for 10 minutes at 4°C. Remove the supernatant and collect the cell pellet. ④ If the pellet contains a large number of red blood cells, lyse them with red blood cell lysis buffer at room temperature for 1 minute. Stop the lysis with cold PBS and centrifuge again. ⑤ Resuspend the cell pellet in 10% FBS 1640 medium, count the cells, and seed them into six-well plates. Mix the cells well and place them in a cell culture incubator. ⑥ Replace with fresh 10% FBS 1640 medium after 2 hours. Drug stimulation can be given after 6 hours.

[0057] (3) Primary mouse peritoneal macrophages were stimulated with 83mM, 139mM, and 236mM glucose for 6h, 12h, and 24h. Cell morphology changes were observed, and cell culture supernatants were harvested. The content of MIF in the peritoneal macrophage culture supernatant was detected using ELISA. The kit was purchased from Wuhan Elabscience Biotechnology Co., Ltd. (Cat#E-EL-M0771). The results showed that high-glucose peritoneal dialysis fluid could also induce the production of MIF in peritoneal macrophages. Figure 6 In different high-glucose-induced macrophage injury models, the secretion of MIF in cell culture supernatant increased, with the most significant increase at 24 hours. *P<0.05 vs NG. N=3.

[0058] Example 7:

[0059] Primary cultures of peritoneal macrophages from MIF knockout mice were performed to determine the response of MIF knockout to high glucose-induced TNF-α and IL-6 production in peritoneal macrophages. First, primary cultures of peritoneal macrophages from male wild-type and MIF knockout mice (weighing 22-25g) were conducted, following the same procedures as in Example 6. After stimulation with 83mM, 139mM, and 236mM glucose for 24 hours, cell morphology changes were observed, and cell culture supernatants were harvested. The levels of TNF-α and IL-6 in peritoneal dialysis fluid were detected using ELISA kits purchased from Wuhan Elabscience Biotechnology Co., Ltd. (TNF-α cat#E-EL-M1210; IL-6 cat#E-EL-M0044). The specific detection methods were as per the kit instructions. Experimental results are as follows: Figure 7As shown in Figures AB, the results indicated that MIF knockout significantly reduced the production of TNF-α and IL-6 in peritoneal macrophages induced by high glucose. N = 3, ****P < 0.0001.

[0060] Example 8:

[0061] MIF expression is widespread, found in immune cells such as monocytes / macrophages, B cells, and T cells, as well as some non-immune cells such as endothelial cells, epithelial cells, and endocrine cells. We have already demonstrated that MIF expression is high in peritoneal mesothelial cells and macrophages, and that high glucose can induce MIF production in both cell types. Therefore, we further cultured primary peritoneal mesothelial cells from MIF knockout mice and used a pancreatic enzymatic peritoneal digestion method.

[0062] Preparation of materials: Prepare primary cell culture medium, DMEM F12 medium with 5% FBS, 0.125% trypsin-0.01% EDTA digestion solution, and phosphate-buffered saline (PBS) in advance. Preheat the above liquids in a 37°C water bath for 30 minutes. Prepare one pair of scissors and two curved forceps by autoclaving. Autoclave and dry pipette tips. Prepare 1ml, 5ml, and 20ml sterile syringes, 15ml and 50ml sterile centrifuge tubes, multiple sterile six-well plates, and laboratory paper. Place all prepared materials in a laminar flow hood and sterilize with UV light for 30 minutes. Coat the six-well plates with gelatin and place them in a cell culture incubator. Additionally, prepare 1L of 75% alcohol and place it in the transfer window for later use. b. Sacrifice the target genotype mice by cervical dislocation. Immediately place the mice in a small dish containing 75% alcohol and immerse them for 5 minutes, turning them intermittently during immersion to ensure thorough immersion. After immersion, drain the alcohol from the mice and place them supine in a laminar flow hood lined with sterile paper towels. c. Exposing the abdominal wall: Quickly pass scissors and forceps through an alcohol lamp, allow them to cool slightly at room temperature, then make a small incision along the linea alba of the mouse's abdomen in the lower abdomen, peeling off only the skin without opening the abdominal wall. d. Irrigating the peritoneal cavity: Take a 5ml syringe and a 1ml syringe, and replace the original needle of the 5ml syringe with the needle of the 1ml syringe to create a modified syringe. Then, gently lift the abdominal wall above the skin opening with forceps, and inject 5ml of cold PBS into the peritoneal cavity using the modified syringe. Gently and thoroughly shake the mouse's body, and aspirate the peritoneal irrigating fluid with the syringe. Repeat the irrigation twice, 2 minutes each time. e. Digesting the abdominal wall: Inject 5ml of preheated pancreatic enzyme digestion solution into the peritoneal cavity using the modified syringe, and digest at room temperature for 15 minutes. During digestion, gently shake the mouse's body to ensure complete digestion. Maintain aseptic technique, keep the mouse warm, and keep the abdominal wall moist during the procedure. f. Collecting cells: The peritoneal digestion time needs to be controlled, otherwise a large number of fibroblasts will detach. After digestion, massage the mouse abdomen again to suspend the peritoneal cells. Use a sterile syringe to collect the digestive fluid from the peritoneal cavity; the pinkish digestive fluid will turn pale yellow. Remove the needle and transfer the digestive fluid to DMEM F12 medium containing 5% FBS to terminate digestion. To collect more cells, inject DMEM F12 medium containing 5% FBS again, thoroughly flush the peritoneal cavity, and then collect the cells back into the same centrifuge tube. Centrifuge the digestive fluid at 4°C, 1000 rpm for 10 min. g. Red blood cell removal: If red blood cells were introduced during the experiment, they need to be removed using red blood cell lysis buffer. Add 1 ml of red blood cell lysis buffer to the cell clumps and mix thoroughly by pipetting and aspirating. Incubate at room temperature for 3 min, then add more than twice the volume of PBS to terminate lysis. Centrifuge at 1000 rpm, 4°C for 5 min. After centrifugation, wash the cells twice with pre-cooled sterile PBS, and finally centrifuge at 4°C, 1000 rpm for 10 min to obtain the cell pellet.h. Cell Culture: Remove the gelatin-coated six-well plates, discard the gelatin using a pipette, and wash twice with sterile PBS. Resuspend the cells in preheated primary culture medium, count them using a Newton-Bauer counting chamber, and aliquot them into six-well plates, adjusting the cell density to approximately 1 x 10^5 cells / ml. Add the dedicated culture medium to 2 ml. Observe cell morphology under a microscope and culture at 37°C with 5% CO2. Change the culture medium every 3 days, without repeatedly removing the cells. Peritoneal mesothelial cells adhere to the plate in about three to five days, and after 10-14 days, the primary cells grow rapidly, exhibiting typical cobblestone growth. B. Peritoneal mesothelial cells from successfully cultured MIF knockout and control mice were administered high-glucose peritoneal dialysis solution at 236 mM. NaCl with the same osmotic pressure was used as an osmotic control. The cell culture supernatant was collected. The levels of VEGF-A and VEGF-C were detected using an ELISA kit purchased from Wuhan Elabscience Biotechnology Co., Ltd. VEGF-A: Cat#E-EL-M1292; VEGF-C: Cat#E-EL-M1230. Results showed that MIF knockout significantly reduced high glucose-induced production of VEGF-A and VEGF-C in peritoneal mesothelial cells. Figure 8 AB)N=3, **P<0.01, ****P<0.0001.

[0063] Example 9:

[0064] MIF is a pluripotent molecule widely involved in physiological and pathological processes such as cellular inflammation, cell proliferation and differentiation, cell survival and apoptosis. Since we detected a significant increase in dopa tautomerase activity of MIF in the supernatant of culture medium from high glucose-induced peritoneal mesothelial cell injury, we first investigated the effect of MIF inhibitors on high glucose-induced peritoneal mesothelial cell injury. Using the MIF inhibitor ISO-1 (purchased from MedChenExpress (Cat#HY-16692)), we pretreated peritoneal mesothelial cells with 150 μM and 300 μM MIF inhibitor for 30 minutes, followed by simultaneous treatment with 236 mM glucose for 24 hours. Cell death was then assessed. Figure 9 AB results showed that both ordinary light microscopy and crystal violet staining indicated that the MIF enzyme inhibitor ISO-1 could alleviate peritoneal mesothelial cell damage caused by high-glucose peritoneal dialysis fluid. Figure 9 A is a standard optical micrograph, with the arrow indicating pyrode bubbles, and the scale bar is 50 μm. Figure 9 B shows the cells stained with crystal violet. The darker the blue, the more surviving cells there are, and vice versa.

[0065] Example 10:

[0066] To clarify the effect of MIF inhibitors on the release of inflammatory factors from peritoneal macrophages, and to investigate the effects of different doses of MIF inhibitors on this release, we first conducted primary culture of wild-type mouse peritoneal macrophages, following the same procedures as in Example 6. Primary mouse peritoneal macrophages were pretreated with 100 μM and 300 μM MIF inhibitors for 30 minutes, followed by simultaneous treatment with 236 mM glucose for 24 hours. The cell culture supernatant was then collected for analysis. We used ELISA to detect the levels of TNF-α and IL-6 in the peritoneal dialysis fluid. The kits were purchased from Wuhan Elabscience Biotechnology Co., Ltd. (TNF-α cat#E-EL-M1210; IL-6 cat#E-EL-M0044), and the specific detection methods were the same as those in the kit instructions. The results showed that the production of TNFα and IL-6 decreased significantly with increasing dose of the MIF inhibitor. The MIF inhibitor ISO-1 significantly reduced the production of TNFα and IL-6 in peritoneal macrophages induced by high glucose. N=3. *P<0.05,**P<0.01,***P<0.001,****P<0.0001.

[0067] The present invention has been described in detail above, with the aim of enabling those skilled in the art to understand and implement the invention. However, this description should not be construed as limiting the scope of protection of the invention. All equivalent changes or modifications made in accordance with the spirit and essence of the invention should be included within the scope of protection of the invention.

Claims

1. The use of an MIF inhibitor in the preparation of a medicament for the prevention and / or treatment of peritoneal ultrafiltration failure or peritoneal fibrosis.

2. The application according to claim 1, characterized in that: The MIF inhibitor is selected from MIF tautomerase inhibitors and / or MIF oxidoreductase inhibitors.

3. The application according to claim 1, characterized in that: The MIF inhibitor is selected from one or more of ISO-1, (±)-CPSI-1306, ISO-66, Jorgensen-3g, Jorgensen-3h, Dziedzic-3bb, 4-IPP, isothiocyanate, K664-1, iguratimod, AV411 or AV1013.

4. The application according to claim 1, characterized in that: The concentration of the MIF inhibitor is 10–1000 μM.

5. The application according to claim 4, characterized in that: The concentration of the MIF inhibitor is 20–800 μM.

6. The application according to claim 5, characterized in that: The concentration of the MIF inhibitor is 30–500 μM.

7. The application according to claim 6, characterized in that: The concentration of the MIF inhibitor is 40–400 μM.

8. The application according to claim 7, characterized in that: The concentration of the MIF inhibitor is 50–400 μM.

9. The application according to claim 8, characterized in that: The concentration of the MIF inhibitor is 60–400 μM.

10. The application according to claim 1, characterized in that: The MIF inhibitor can be added to peritoneal dialysis fluid for use, or the MIF inhibitor can be prepared into a formulation with pharmaceutical excipients for use.

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