CN114306296A - Application of disulfiram in preparation of medicine for treating membranous nephropathy - Google Patents
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Abstract
The invention discloses an application of disulfiram in preparing a medicine for treating membranous nephropathy, wherein the disulfiram plays a kidney protection effect by inhibiting glomerular podocyte apoptosis. The invention proves the therapeutic effect of disulfiram in membranous nephropathy through an in vitro complement-mediated podocyte injury model and a membranous nephropathy animal model, possibly further expands the clinical application range of disulfiram, innovates the therapeutic method and thought of membranous nephropathy, and is expected to generate wide social and economic benefits.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to application of disulfiram in preparation of a medicine for treating membranous nephropathy.
Background
Membranous Nephropathy (MN) is a kidney-specific autoimmune disease, the most common cause of nephrotic syndrome in non-diabetic patients, and also a common cause of end-stage renal disease. In recent years, the prevalence of MN increases year by year at home and abroad, and the MN has a trend of youthfulness, thereby causing heavy social and economic burden. The core mechanism of MN kidney tissue injury is that glomerular podocyte antigens are combined with autoantibodies thereof to form a sub-epithelial immune complex, so that a complement system is activated, a complement activation fragment and a sub-soluble C5b-9 are generated, and the podocyte and glomerular filtration barrier is damaged. The patients show a large amount of typical manifestations of nephrotic syndrome such as proteinuria, hypoproteinemia and edema. Currently, MN treatment approaches are still very limited and controversial, and both the safety and effectiveness of immunosuppressive treatment regimens, such as hormone/alkylating agent, calcineurin inhibitor, and recently reported CD20 mab, are still more limited. The discovery of new therapeutic drugs is a prominent problem to be solved urgently in clinical diagnosis and treatment of MN.
Apoptosis (pyroptosis) is a newly discovered mode of programmed necrotic cell death. Pathogen-related molecular patterns, danger-related molecular patterns and change of cellular homeostasis, which are generated by cells under the action of stimulation factors such as exogenous infection and injury, can activate pattern recognition receptors, so that classical inflammatory corpuscle assembly and activation are caused, Caspase-1 in the inflammatory corpuscles is activated after being cut, and then protein gasdermin D (GSDMD) is cut and subjected to apoptosis execution, and the cut and activated GSDMD N segment is combined with phosphoinositide and cardiolipin of a cell membrane and oligomerizes to form a pore channel, so that the intracellular and extracellular osmotic pressure difference is destroyed, and the cells are swollen and ruptured, which is the main occurrence process of cell apoptosis. By utilizing a human immortalized podocyte injury model mediated by complement C3a/C5a cultured in vitro, a membranous nephropathy (passive Heymann nephritis) rat model and an MN patient queue, the inventor has proved in previous research work that glomerular podocyte apoptosis is one of important mechanisms of MN kidney tissue injury. This result suggests that inhibition of cellular apoptosis is likely to constitute a breakthrough for MN treatment. However, none of the currently reported cell apoptosis inhibitors with kidney protective effects have been approved for clinical use. The research evidence of the kidney protection effect is weak and all the results stay in vitro experiments or in vivo animal experiments. The safe and effective scorch inhibitor found in the existing clinical drugs is applied to the treatment of kidney diseases, and is the key for carrying out rapid clinical transformation on the earlier research results of the inventor and innovating MN therapy.
Disulfiram (formula: C10H20N2S4, CAS number: 97-77-8) is an ancient, safe, inexpensive clinical Drug approved by The U.S. Food and Drug Administration, FDA, for The clinical treatment of alcohol addiction as early as 1951. Recent research shows that disulfiram can exert anti-focal death effect by covalently modifying Cys191/Cys192 sites of human/mouse focal death executive protein GSDMD so as to inhibit the membrane perforation process after activation. Given that podocyte burn-out constitutes an important mechanism of MN renal tissue injury, this result suggests to the inventors that disulfiram, an anti-burn drug, is likely to have a renoprotective effect in MN. However, the prior art does not disclose or suggest the use and mechanism of disulfiram in the preparation of medicaments for the treatment of MN.
Disclosure of Invention
The invention aims to provide a new application of disulfiram, in particular to an application in preparing a medicament for treating membranous nephropathy.
The technical scheme of the invention is as follows: an application and a mechanism of disulfiram in preparing a medicine for treating membranous nephropathy.
The disulfiram is applied to preparing the medicine for treating membranous nephropathy by inhibiting glomerular podocyte scorching.
Further, the glomerular podocyte is an in vitro cultured complement C3a/C5a mediated immortalized human glomerular podocyte and a glomerular podocyte of a membranous nephropathy rat model.
Further, preparing a serum-free culture solution of the complement C3a/C5 a-mediated in vitro cultured immortalized human glomerular podocytes, wherein the concentration of the C3a/C5a in the serum-free culture solution is 50-500 nM; the concentration of disulfiram in serum-free medium is 100-500 nM.
Furthermore, the disulfiram is added into 0.5 percent of sodium carboxymethylcellulose to prepare suspension, and the membranous nephropathy is taken for 2 times per day according to the dosage of 10-100 mg/kg.
Further, the membranous nephropathy is passive Heymann nephritis.
Furthermore, the apoptosis refers to a programmed necrotic death mode of cells and a signal path for regulating the occurrence of the programmed necrotic death mode, and comprises NLRP3, ASC, Caspase-1, IL-1 beta, IL-18 and GSDMD.
Furthermore, disulfiram exerts anti-pyrophoric kidney protective effects by inhibiting renal tissue pyrophoric signal pathways and membrane perforation after activation of the glomerular podocyte executive protein GSDMD.
Further, the medicine for treating membranous nephropathy comprises a pharmaceutical composition in a pharmaceutically acceptable carrier.
Advantageous effects
(1) The invention respectively utilizes an immortalized human glomerular podocyte injury model and a membranous nephropathy (passive Heymann nephritis) rat model mediated by complement C3a/C5a cultured in vitro to prove that disulfiram can reduce the podocyte injury mediated by complement and the renal tissue injury of the membranous nephropathy (passive Heymann nephritis) rat model, thereby disclosing the potential application value of the disulfiram in preparing the medicine for treating the membranous nephropathy.
(2) The invention further clarifies the mechanism of the therapeutic effect of disulfiram on membranous nephropathy by respectively utilizing an immortalized human glomerular podocyte injury model and a rat model of membranous nephropathy (passive Heymann nephritis) mediated by complement C3a/C5a cultured in vitro. Namely, disulfiram can play a kidney protection role in membranous nephropathy through inhibiting glomerular podocyte apoptosis in vitro and in vivo.
Drawings
FIG. 1 shows the Lactate Dehydrogenase (LDH) release levels from each group of podocytes;
FIG. 2 shows the results of Western immunoblotting (Western-Blot) of each group of podocytes NLRP3, ASC, Caspase-1, IL-18, GSDMD, and internal reference GAPDH;
FIG. 3 shows the immunofluorescent staining results of each group of podocytes GSDMDM, ZO-1, cell nuclei (DAPI) and merged cells (Merge);
wherein, NC: a normal control group; c3 a: c3a stimulation group; and (3) DSF: disulfiram treatment group; c3a + DSF: c3a stimulation group + disulfiram treatment group; c5 a: c5a stimulation group; c5a + DSF: c5a stimulation group + disulfiram treatment group; data are described as mean ± standard deviation; using anova with the t-test for least significant difference (e.g., data variance alignment)/Welch's approximate anova with Dunnett's T3 test (e.g., data variance alignment) to infer differences between groups in panel a and compare them pairwise; **: p is less than 0.01.
FIG. 4A shows groupsDetecting results of 24h urine protein (24h-UPro) 1 day, 5 days, 8 days and 15 days after modeling of the rat; FIG. 4B shows the results of serum Albumin (ALB) assays at 1, 5, 8, and 15 days after modeling for each group of rats; FIGS. 4C-D are the results of the ultrastructural drawing and the measurement of the width of the podocyte foot process under the glomerular electron microscope of each group of rats,
showing sub-epithelial electron density deposition; "meshed" indicates podocyte foot process fusion. FIG. 4E shows the real-time fluorescent quantitative PCR detection results of glomerular NLRP3, ASC, Caspase-1, IL-18, and GSDMD of each group of rats; FIGS. 4F-G show the results of immunohistochemical staining and semi-quantitative analysis of groups of rat kidney tissue NLRP3, ASC, Caspase-1, IL-18, GSDMDM (N); FIG. 4H shows the results of Western immunoblotting (Western-Blot) of rat kidney tissues NLRP3, ASC, Caspase-1p20, IL-1. beta. mass form, IL-18, GSDMDM (N), and internal reference GAPDH in each group. FIG. 4I shows the results of immunofluorescent staining of rat kidney tissues GSDMDM (N), Synaptopodin, ZO-1 and pooled (Merge) in each group.
Wherein, NC: a normal control group; PHN: membranous nephropathy (passive Heymann nephritis) rat model group; PHN + DSF: membranous nephropathy (passive Heymann nephritis) rat model + disulfiram treatment group; IOD: integrating the optical density; area: glomerular area; data are described as mean ± standard deviation; the differences between groups in the A, B, D, E, G graph were inferred and compared pairwise using ANOVA with the least significant difference t test (e.g., uniform data variance)/Welch approximate ANOVA with the Dunnett's T3 test (e.g., non-uniform data variance); *: p is less than 0.05; **: p is less than 0.01.
Detailed Description
Reference will now be made in detail to embodiments of the present invention, examples of which are illustrated in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are illustrative only for the purpose of explaining the present invention, and are not to be construed as limiting the present invention.
The invention adopts the following technical scheme for solving the technical problems:
example 1: disulfiram reduces complement C3a/C5a mediated podocyte injury in vitro by inhibiting apoptosis.
The method comprises the following steps:
(1) cell culture and treatment: human immortalized glomerular podocyte cell line (as donated by professor of Moin A. Saleem, university of British, British) cultured in 10% fetal bovine serum + 1% Insulin-Transferrin-Selenium RPMI 1640 medium at 33 ℃ with 5% CO2Proliferating in the environment, transferring to 37 deg.C when the cell grows to 70-80% full, and adding 5% CO2Differentiating for 7-14 days under the environment. Cells were divided into 4 groups: normal control group (NC), C3a/C5a stimulation group (C3a), disulfiram treatment group (DSF), C3a/C5a stimulation group + disulfiram treatment group (C3a + DSF). The serum-free culture solution is replaced before the cells of each group are treated, and recombinant human complement C3a/C5a is added into a C3a/C5a stimulation group and a C3a/C5a stimulation group and a disulfiram treatment group to the final concentration of 50-500 nM; 100-500nM disulfiram is added to the disulfiram treatment group and the stimulation group of C3a/C5a + disulfiram treatment group, and the disulfiram is added 1 hour before the recombinant human complement C3a/C5a is added, and the incubation is carried out for 1-12 hours. The experiment was repeated 3 times, and 1 duplicate well was set for each experiment.
(2) Sample collection and detection: 1) cellular LDH release was detected using Lactate Dehydrogenase (LDH) cytotoxicity detection kit (Beyotime Biotechnology); 2) detecting the protein levels of NLRP3, ASC, Caspase-1, IL-18 and GSDMDM of the cells by using a Western immunoblotting (Western-Blot) technology; 3) and detecting the expression level of the GSDMD and the co-localization condition of the GSDMD and a cell membrane marker protein ZO-1 by using an immunofluorescence technique.
(3) And (4) observing results: as shown in FIG. 1, C3a/C5a stimulated the group (C3a/C5a) to increase LDH release in cells compared to the normal control group (NC), suggesting that the integrity of the podocyte membrane was destroyed and the cells were damaged. Compared with the C3a/C5a stimulated group (C3a/C5a), the C3a/C5a stimulated group + disulfiram treated group (C3a/C5a + DSF) has significantly reduced LDH release of cells, indicating that disulfiram has a podocyte protection effect. As shown in FIG. 2 and FIG. 3, C3a/C5a stimulated the cell apoptosis signaling pathway proteins NLRP3, ASC, Caspase-1, IL-18, GSDMDM were upregulated, GSDMDM and its N-fragment (GSDMDM (N)) co-localized with ZO-1 increased, suggesting that GSDMDM-N-fragment membrane metastasis increased and that apoptosis was involved in the mechanism of podocyte injury, compared to the control group (C3a/C5 a). Compared with the C3a/C5a stimulation group (C3a/C5a), the C3a/C5a stimulation group + disulfiram treatment group (C3a/C5a + DSF) cells NLRP3, ASC, Caspase-1, IL-18 and GSDMD are down-regulated, and GSDMDM-N fragment membrane metastasis is reduced, so that the disulfiram can inhibit NLRP3-ASC-Caspase-1-IL-18/GSDMD podocyte apoptosis signal pathways and podocyte apoptosis processes. Together, these observations suggest that disulfiram exerts a protective effect during complement-mediated podocyte injury by inhibiting pyroptosis.
Example 2: disulfiram reduces glomerular podocyte injury in a rat model of membranous nephropathy (passive Heymann nephritis) by inhibiting apoptosis.
The method comprises the following steps:
(1) establishing and grouping animal models: extracting the kidney proximal tubule brush border Fx1A antigen of SD rat, immunizing New Zealand rabbit to prepare Fx1A antiserum, and identifying the titer of the Fx1A antiserum by enzyme-linked immunosorbent assay and immunofluorescence. Taking SD rat with the weight of 150-. Adding disulfiram into 0.5% sodium carboxymethylcellulose to obtain suspension, and performing intragastric administration at a dose of 50mg/kg for 2 times/day. Dosing was continued the day before modeling PHN rats until the observation endpoint. The study period is 16 days (1 day before modeling and 15 days after modeling), 4 observation time points of 1 day, 5 days, 8 days and 15 days after modeling are set, 24-hour urine and venous blood of each group of rats are collected and serum is separated, each group of rats are killed at an observation end point of 15 days, and kidneys are picked up; the study was divided into three groups: normal control group (NC), membranous nephropathy (passive Heymann nephritis) group (PHN), membranous nephropathy (passive Heymann nephritis) + disulfiram treatment group (PHN + DSF), the number of rats per group included in the statistical analysis was 6. The above animal protocol complies with the "3R" guidelines and is approved by the discussion of the eastern war zone general hospital medical ethical review committee.
(2) Sample collection and detection: 1) urine protein (24h-UPro) and serum Albumin (ALB) levels were measured 1 day, 5 days, 8 days, 15 days 24 hours after modeling for each group of rats using a Bradford protein concentration assay kit (Beyotime Biotechnology) and a fully automated biochemical analyzer (Hitachi, Ltd.); 2) will be 1mm3Fresh rat kidney tissues are fixed, dehydrated, soaked, embedded and polymerized, then sliced into 70nm slices, and the slices are observed under an electron microscope after uranium acetate-lead citrate double staining; 3) detecting the expression level of rat kidney tissues NLRP3, ASC, Caspase-1, IL-1 beta, IL-18 and GSDMDM (N) by using an immunohistochemical technology, calculating the Integral Optical Density (IOD) and the Area of glomerulus (Area) in pictures by using Image-Pro Plus 6.0 software (Media Cybernetics, Inc.), and comparing the signal intensity after calculating the ratio; 3) separating fresh rat renal cortex glomeruli, extracting RNA, carrying out reverse transcription, and detecting the gene expression levels of glomeruli NLRP3, ASC, Caspase-1, IL-1 beta, IL-18 and GSDMD by using a real-time fluorescent quantitative PCR technology; 4) detecting the protein levels of the rat fresh renal cortex NLRP3, ASC, Caspase-1p20, IL-1 beta format, IL-18 and GSDMD by using a Western immunoblotting (Western-Blot) technology; 5) an immunofluorescence technique is used for detecting the expression level of rat glomerulus GSDMD (N) and the co-localization condition of the expression level, podocyte marker protein Synaptopodin and cell membrane marker protein ZO-1.
(3) And (4) observing results: as shown in fig. 4A-D, the urinary protein (24h-Upro) significantly increased at 24 hours from 5 days after modeling in the membranous nephropathy (passive Heymann nephritis) group (PHN) rats, and the serum Albumin (ALB) decreased, as compared to the control group (NC), and the electron microscopy, which is based on the deposition of subcutaneous electron dense matter and diffuse fusion of podocyte foot processes, suggested that the establishment of the membranous nephropathy animal model was successful, and the urinary protein (24h-Upro) significantly decreased at 24 hours from 5 days after modeling in the membranous nephropathy (passive Heymann nephritis) + disulfiram treatment group (PHN + DSF) rats, and the serum Albumin (ALB) significantly increased at 8 days after modeling, as compared to the membranous nephropathy (passive Heymann nephritis) group (PHN). Diffuse fusion of podocyte foot processes is obviously improved under an electron microscope. It is suggested that disulfiram has protective effect on renal tissue injury in rats with membranous nephropathy (passive Heymann nephritis). As shown in FIG. 4E-I, compared with the control group (NC), the glomerular apoptosis signal pathways NLRP3, ASC, Caspase-1, IL-1 beta, IL-18, GSDMD of rats (PHN) are significantly up-regulated on gene expression (mRNA) and protein level, GSDMD (N) has increased co-localization with Synaptodin and ZO-1, i.e., the membrane transfer of glomerular podocyte GSDMD-N fragment is increased, the levels of renal cortex Caspase-1p20 and IL-1 beta format are also increased, suggesting that apoptosis is involved in the mechanism of podocyte injury of the membranous nephropathy (passive Heymann nephritis) rats. Compared with the membranous nephropathy (passive Heymann nephritis) group (PHN) rats, the membranous nephropathy (passive Heymann nephritis) + disulfiram treatment group (PHN + DSF) apoptosis signal channel gene expression (mRNA) and protein level are remarkably reduced, and GSDMDM-N fragment membrane metastasis is reduced, which suggests that disulfiram can inhibit the membranous nephropathy (passive Heymann nephritis) rat glomerular podocyte NLRP3-ASC-Caspase-1-IL-1 beta/IL-18/GSDMDM apoptosis signal channel and the apoptosis process. Together, these observations suggest that disulfiram can exert a nephroprotective effect in a rat model of membranous nephropathy (passive Heymann nephritis) by inhibiting glomerular podocyte apoptosis.
The invention proves the therapeutic effect of disulfiram in membranous nephropathy through a complement-mediated human glomerular podocyte injury model and a membranous nephropathy animal model, and illustrates the application value of disulfiram in preparing a medicine for treating membranous nephropathy. The clinical application range of disulfiram is likely to be further expanded, a treatment method and thought of membranous nephropathy are innovated, and wide social and economic benefits are expected to be generated.
Claims (9)
1. The application of disulfiram in preparing medicine for treating membranous nephropathy.
2. The use according to claim 1, wherein the disulfiram is used for the manufacture of a medicament for the treatment of membranous nephropathy by inhibiting glomerular podocyte apoptosis.
3. The use of claim 2, wherein the glomerular podocytes are complement C3a/C5 a-mediated immortalized human glomerular podocytes cultured in vitro and a glomerular podocyte of a rat model of membranous nephropathy.
4. The use according to claim 3, wherein a serum-free culture of said complement C3a/C5 a-mediated immortalized human glomerular podocytes cultured in vitro is prepared, said C3a/C5a being present in a concentration of 50-500nM in serum-free culture; the concentration of disulfiram in serum-free medium is 100-500 nM.
5. The use according to claim 3, wherein disulfiram is added to 0.5% sodium carboxymethylcellulose to form a suspension, and said membranous nephropathy is administered at 10-100mg/kg 2 times per day.
6. The use according to claim 3, wherein the membranous nephropathy is passive Heymann nephritis.
7. The use of claim 2, wherein said apoptosis is a programmed necrotic death pattern in cells and signaling pathways regulating the occurrence thereof, including NLRP3, ASC, Caspase-1, IL-1 β, IL-18, GSDMD.
8. The use according to claim 7, wherein disulfiram exerts an anti-pyrophoric kidney protective effect by inhibiting the membrane perforation process following activation of the renal tissue pyrophoric signaling pathway and glomerular podocyte pyrophoric executive protein GSDMD.
9. The use according to any one of claims 1 to 8, wherein the medicament for the treatment of membranous nephropathy comprises a pharmaceutical composition in a pharmaceutically acceptable carrier.
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| CN115192563A (en) * | 2022-05-09 | 2022-10-18 | 北京大学第一医院 | Use of C3a/C3aR pathway antagonists for the treatment of primary membranous nephropathy |
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| WO2023049787A1 (en) * | 2021-09-23 | 2023-03-30 | Regeneron Pharmaceuticals, Inc. | Inhibitors of protective loss-of-function genes for the treatment of chronic kidney disease |
| CN116590345A (en) * | 2023-05-06 | 2023-08-15 | 北京中医药大学 | Immortalized mouse podocyte line and preparation method, differentiation method and application thereof |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2023049787A1 (en) * | 2021-09-23 | 2023-03-30 | Regeneron Pharmaceuticals, Inc. | Inhibitors of protective loss-of-function genes for the treatment of chronic kidney disease |
| CN115192563A (en) * | 2022-05-09 | 2022-10-18 | 北京大学第一医院 | Use of C3a/C3aR pathway antagonists for the treatment of primary membranous nephropathy |
| CN115192563B (en) * | 2022-05-09 | 2023-10-13 | 北京大学第一医院 | Use of C3a/C3aR pathway antagonists for the treatment of primary membranous nephropathy |
| CN115844864A (en) * | 2022-12-05 | 2023-03-28 | 复旦大学附属中山医院 | Application of disulfiram in preparation of medicine for preventing and treating aortic aneurysm and aortic dissection |
| CN116590345A (en) * | 2023-05-06 | 2023-08-15 | 北京中医药大学 | Immortalized mouse podocyte line and preparation method, differentiation method and application thereof |
| CN116590345B (en) * | 2023-05-06 | 2024-01-30 | 北京中医药大学 | Immortalized mouse podocyte line and preparation method, differentiation method and application thereof |
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