Use of necrostatin-1 in preparation of drugs for preventing and / or treating acute aristolochic acid nephropathy

Abstract

The present application relates to the application of Necrostatin-1 in the preparation of a drug for preventing and / or treating acute aristolochic acid nephropathy. The typical pathological change of acute aristolochic acid nephropathy is the necrosis of renal tubular epithelial cells, and Necrostatin-1 can target this change to prevent and / or treat acute aristolochic acid nephropathy. Necrostatin-1 can significantly inhibit the kinase activity of RIPK1, block the endogenous and exogenous apoptosis pathways dependent on RIPK1, delay the apoptosis and GSDME-dependent pyroptosis of renal tubular epithelial cells, effectively improve the damage of renal tubular epithelial cells, and achieve the purpose of treating acute aristolochic acid nephropathy. The present application can provide a new strategy for the treatment of acute aristolochic acid nephropathy. Necrostatin-1 can be widely used in the preparation of drugs for treating acute aristolochic acid nephropathy.

Description

Technical Field

[0001] This invention relates to the fields of cell biology and medicine, specifically to novel medicinal uses and applications of specific small molecule chemical inhibitors in acute aristolochic acid nephropathy. Background Technology

[0002] Aristolochic acid (AA) is a collective term for a class of nitrophenanthrene carboxylic acids, a class of compounds that are abundant and widely used in traditional Chinese medicine pharmacopoeias. AA has significant nephrotoxicity, and 70% of AAN patients will develop end-stage renal disease (ESRD). [1] Its pathogenic mechanism is not yet clear, and there is no obvious and effective treatment strategy for aristolochic acid nephropathy (AAN).

[0003] AAN (anti-inflammatory kidney disease) refers to any form of kidney disease caused by consuming foods or medications containing amino acids (AA). The cause of AAN is direct exposure to AA. Epidemiological analysis shows that AAN is mainly distributed in Asia and the Balkans. [2] In Asia, traditional Chinese medicine is widely used, especially herbs rich in amino acids (AA), including plants from the genera Aristolochia and Asarum. These herbs are widely used as medicinal materials or in prepared Chinese medicines due to their analgesic, anti-infective, and anti-inflammatory effects. [3] In Asia, AAN is mainly caused by improper consumption of traditional Chinese medicine containing amino acids. [4-5] In the Balkans, AAN, also known as Balkan nephropathy (BEN), is caused by the pollution of soil and crops by plants of the Aristolochia genus, resulting in long-term chronic exposure to an environmental toxin in the population. To date, approximately 100,000 people are at risk of developing BEN, with 25,000 already diagnosed. [6] .

[0004] AAN can be classified into acute AAN and chronic AAN based on the mode of exposure. Acute AAN is caused by a single high dose of amino acids (AA), and occasionally occurs with low doses. The lesions primarily occur in the proximal tubules, especially in the cortical and corticomedullary junction. Symptoms include brush border shedding of tubular epithelial cells (TECs), cloudy and swollen cells, and necrotic shedding forming epithelial casts. Extensive "naked membrane" of the tubular basement membrane is observed, with lesions appearing in patchy or diffuse distribution. Chronic AAN is generally caused by low doses of AA. The lesions mainly occur in the superficial renal cortex and corticomedullary junction. Symptoms include flattened, cloudy, and swollen TECs, tubular destruction, and interstitial fibrosis without cellular infiltration. The lesions are focally distributed. [7] AAN can progress from early acute kidney injury (AKI) to chronic kidney disease (CKD) and even ESRD.

[0005] Early manifestations of acute kidney injury (AKI) in atrial angina are primarily characterized by proximal tubular epithelial atrophy and damage to proximal tubular epithelial cells (PTEC). [8-9] Studying the death mechanism of PTEC from the perspective of programmed cell death and exploring targeted therapies against PTEC death has long been a research hotspot in AAN.

[10] Programmed cell death is a complex system, including not only the common apoptosis but also pyroptosis, programmed necrosis, and ferroptosis. These death mechanisms complement and transform each other, collectively forming a complex programmed cell death system. To date, research on the programmed cell death mechanisms induced by apoptosis (AA) in PTEC has mainly focused on autophagy and apoptosis. Man et al.'s research showed that AA can induce type II programmed cell death, autophagy, in PTEC. Autophagy promotes epithelial-mesenchymal transition (EMT), potentially mediating AA-induced renal fibrosis, and inhibiting autophagy can significantly alleviate the EMT process.

[11] Xie et al.'s research indicates that...

[12] Relaxin can alleviate AA-induced apoptosis in PTEC by activating the PI3K / Akt pathway, which is a typical autophagy-inhibiting pathway, suggesting that AA-induced autophagy in PTEC may mediate apoptosis. A review of existing literature indicates that apoptosis is the main cell death process in AAN pathology. AA can induce oxidative stress in cells, leading to calcium... 2+ A rapid increase in concentration subsequently induces endoplasmic reticulum and mitochondria stress, leading to the release of cytochrome C (Cyt C), caspase activation, and apoptosis-mediating pathways including the PI3K / Akt pathway.

[12] MAPK pathway

[13] and AMPK pathway

[14] These cellular responses proceed in an orderly manner, ultimately leading to apoptosis. However, Zeng et al. investigated the relationship between autophagy and apoptosis in an acute AAN model. Their results showed that apoptosis was not the primary damage caused by acute AAN in PTEC. Autophagy could inhibit AA-induced apoptosis through the mitochondrial pathway, but it promoted other non-apoptotic programmed cell death pathways.

[15] The specific pathways of non-apoptotic programmed cell death induced by apoptosis (AA) and their regulatory mechanisms remain to be studied.

[0006] Pyroptosis is a lytic inflammatory programmed cell death process that can induce a severe inflammatory response in the body. The induction mechanisms of pyroptosis are divided into classical and non-classical pathways. In the classical pathway, pyroptosis depends on caspase-1. When cells are exposed to pathogen-associated molecular patterns (PAMPs) or danger-associated molecular patterns (DAMPs), pattern recognition receptors (PRRs) recognize them and activate the NF-κB pathway, inducing the expression of various NOD-like receptor proteins, as well as pro-IL-1β and pro-IL-18. NOD-like receptors rapidly recognize DAMPs and assemble with the adaptor protein ASC, recruiting pro-caspase-1. Pro-caspase-1 undergoes autocleavage to produce activated caspase-1. Caspase-1 cleaves pro-IL-1β and pro-IL-18, promoting their maturation and mediating secretion; it also induces the cleavage of Gasdermin D (GSDMD), exposing GSDMD-NT and relieving the autoinhibitory state of GSDMD. GSDMD-NT can bind to phosphorylated phosphatidylinositol (p-PI) or cardiolipin, and oligomerize on the membrane to form 16-polymer channels with an inner diameter of 10-14 nm. These channels promote cell rupture and release IL-1β and IL-18 directly into the extracellular space. In the non-classical pathway, pyroptosis is mediated by Gasdermin E (GSDME). If caspase-3 is activated and GSDME is present in the cell, caspase-3 cleaves GSDME, exposing GSDME-NT, which functions identically to GSDMD-NT, causing the cell to rapidly transition from apoptosis to pyroptosis, or directly proceed to pyroptosis. [16-17] AA can induce apoptosis in PTEC, indicating that AA can induce the activation of caspase-3. Given the presence of GSDME in normal kidney cells, there is sufficient reason to believe that AA can activate caspase-3 in PTEC, cleave GSDME, and induce pyroptosis. However, whether AA induces pyroptosis in PTEC and the molecular mechanism of this induction have not yet been investigated.

[0007] During acute cell injury, there is an interconversion between different programmed cell death modes.

[18] To explore the regulatory targets of GSDME through PCD interactions, we screened a series of PCD-specific inhibitors. Inhibitor treatment results showed that the receptor-interacting protein kinase 1 (RIPK1) inhibitor Necrostatin-1 provided the greatest protective effect on cells. RIPK1 is a key regulator of inflammation and cell death, and can regulate inflammation through pathways such as NF-κB.

[19] It can also induce programmed necrosis through the RIPK3 / MLKL pathway.

[20] It has become a potential therapeutic target for a variety of diseases.

[21] The effects of RIPK1 on GSDME-mediated pyroptosis have never been reported in the literature. The protective effect of Necrostatin-1 prompted us to investigate the regulatory role of RIPK1 in GSDME. We detected RIPK1 expression in cells and tissues, and the results showed that with AA exposure, RIPK1 kinase activity in cells increased in a dose- and time-dependent manner, and phosphorylation levels also increased significantly in animal models. Necrostatin-1 treatment significantly reduced RIPK1 kinase activity and greatly inhibited GSDME-mediated pyroptosis. This suggests that RIPK1 may play a key regulatory role in GSDME-mediated pyroptosis. Given the conclusion in the literature that GSDME is cleaved by caspase-3, we focused on the RIPK1-dependent apoptosis pathway based on the link between RIPK1 and caspase-3. RIPK1 has both scaffold protein activity and kinase activity. Its kinase form can form a Ripoptosome complex including FADD and caspase-8, thereby activating caspase-3.

[22] Necrostatin-1s have been shown to inhibit the activity of proteins involved in the RIPK1-dependent apoptosis pathway. Necrostatin-1s can alleviate GSDME-mediated pyroptosis by inhibiting the RIPK1-dependent apoptosis pathway, thereby increasing cell viability and reducing LDH release. In summary, we can conclude that RIPK1 is a key regulator of GSDME-mediated pyroptosis, and Necrostatin-1 may alleviate GSDME-mediated pyroptosis and protect cells from necrosis by inhibiting the RIPK1-dependent apoptosis pathway.

[0008] References

[0009] [1]Jadot, I., Decleves, AE, Nortier,J., Caron, N. An integrated view of aristolochic acid nephropathy: Update of the literature. Int. J. Mol.Sci., 2017, 18(2): E297

[0010] [2]Debelle, FD, Vanherweghem, JL,Nortier, JL Aristolochicacid nephropathy: a worldwide problem. Kidney Int., 2008, 74(2): 158-69

[0011] [3]Zhang, H. M., Zhao, X. H., Sun, Z. H.,Li, G. C., Liu, G. C., Sun,L. R., Hou, J. Q., Zhou, W. Recognition of the toxicity of aristolochic acid.J. Clin. Pharm. Ther.,2019, 44(2): 157-162

[0012] [4]Ng, A. W. T., Poon, S. L., Huang, M.N., Lim, J. Q., Boot, A., Yu,W., Suzuki, Y., Thangaraju, S., Ng, C. C. Y., Tan, P. Aristolochic acids andtheir derivatives are widely implicated in livercancers in Taiwan andthroughout Asia. Sci. Transl. Med.,2017, 9(412): pii: eaan6446

[0013] [5]Chen, C. H., Dickman, K. G., Moriya,M., Zavadil, J., Sidorenko, V.S., Edwards, K. L., Gnatenko, D. V., Wu, L., Turesky, R. J., Wu, X. R., Pu,Y. S., Grollman, A. P. Aristolochicacid-associated urothelial cancer inTaiwan. Proc. Natl. Acad. Sci. U. S. A.,2012, 109(21): 8241-6

[0014] [6]Stiborova, M., Arlt, V. M., Schmeiser,H. H. Balkan endemicnephropathy: an update on its aetiology. Arch. Toxicol.,2016, 90(11): 2595-2615

[0015] [7]Vanherweghem, J. L., Depierreux, M.,Tielemans, C., Abramowicz, D.,Dratwa, M., Jadoul, M., Richard, C., Vandervelde, D., Verbeelen, D.,Vanhaelen-Fastre, R., et al. Rapidlyprogressive interstitial renal fibrosisin young women: association with slimming regimen including Chinese herbs.Lancet,1993, 341(8842): 387-91

[0016] [8]Matsui, K., Kamijo-Ikemorif, A.,Sugaya, T., Yasuda, T., Kimura, K.Renal liver-type fatty acid binding protein (L-FABP) attenuates acute kidneyinjury in aristolochic acid nephrotoxicity.Am. J. Pathol.,2011, 178(3): 1021-32

[0017] [9]Nortier, J. L., Deschodt-Lanckman, M.M., Simon, S., Thielemans, N.O., de Prez, E. G., Depierreux, M. F., Tielemans, C. L., Richard, C.,Lauwerys, R. R., Bernard, A. M., Vanherweghem, J. L.Proximal tubular injuryin Chinese herbs nephropathy: monitoring by neutral endopeptidase enzymuria.Kidney Int.,1997, 51(1): 288-93

[0018]

[10] Ramos, A. M., Gonzalez-Guerrero, C.,Sanz, A., Sanchez-Nino, M.D., Rodriguez-Osorio, L., Martin-Cleary, C., Fernandez-Fernandez, B., Ruiz-Ortega, M., Ortiz, A. Designing drugs that combatkidney damage. Expert OpinDrug Discov,2015, 10(5): 541-56

[0019]

[11] Man, Y. L., Rui, H. L., Chen, Y. P.,Wang, G. Q., Sun, L. J.,Cheng, H. Aristolochic acid-induced autophagy promotes epithelial-to-myofibroblast transition in human renal proximal tubuleepithelial cells.Evid. Based Complement. Alternat. Med.,2017, 2017: 9596256

[0020]

[12] Xie, X. C., Zhao, N., Xu, Q. H., Yang,X., Xia, W. K., Chen, Q.,Wang, M., Fei, X. Relaxin attenuates aristolochic acid induced human tubularepithelial cell apoptosis in vitro by activation ofthe PI3K / Akt signalingpathway. Apoptosis,2017, 22(6): 769-776

[0021]

[13] Romanov, V., Whyard, T. C., Waltzer, W.C., Grollman, A. P.,Rosenquist, T. Aristolochic acid-induced apoptosis and G2 cell cycle arrestdepends on ROS generation and MAP kinases activation. Arch.Toxicol.,2015, 89(1): 47-56

[0022]

[14] Kwak, D. H., Lee, S. Aristolochic AcidI Causes Testis Toxicity byInhibiting Akt and ERK1 / 2 Phosphorylation. Chem. Res. Toxicol.,2016, 29(1):117-24

[0023]

[15] Zeng, Y., Li, S., Wu, J., Chen, W.,Sun, H., Peng, W., Yu, X.,Yang, X. Autophagy inhibitors promoted aristolochic acid I induced renaltubular epithelial cell apoptosis via mitochondrialpathway but alleviatednonapoptotic cell death in mouse acute aritolochic acid nephropathy model.Apoptosis,2014, 19(8): 1215-24

[0024]

[16] Wang, Y., Gao, W., Shi, X., Ding, J.,Liu, W., He, H., Wang, K.,Shao, F. Chemotherapy drugs induce pyroptosis through caspase-3 cleavage of agasdermin. Nature,2017, 547(7661): 99-103

[0025]

[17] Rogers, C., Fernandes-Alnemri, T.,Mayes, L., Alnemri, D.,Cingolani, G., Alnemri, E. S. Cleavage of DFNA5 by caspase-3 during apoptosismediates progression to secondarynecrotic / pyroptotic cell death. Nat Commun,2017, 8: 14128

[0026]

[18] Bedoui, S., Herold, M., Strasser, A.Emerging connectivity ofprogrammed cell death pathways and its physiological implications. Naturereviews. Molecular cell biology,2020, 21(11): 678-695

[0027]

[19] Kondylis, V., Kumari, S., Vlantis, K.,Pasparakis, M. Theinterplay of IKK, NF-κB and RIPK1 signaling in the regulation of cell death,tissue homeostasis and inflammation. Immunol.Rev.,2017, 277(1): 113-127

[0028]

[20] Delanghe, T., Dondelinger, Y.,Bertrand, M. RIPK1 kinase-dependentdeath: A symphony of phosphorylation events. Trends Cell Biol.,2020, 30(3):189-200

[0029]

[21] Degterev, A., Ofengeim, D., Yuan, J.Targeting RIPK1 for the treatment of human diseases. Proc. Natl. Acad. Sci. USA, 2019, 116(20):9714-9722

[0030]

[22] Mifflin, L., Ofengeim, D., Yuan, J. Receptor-interacting proteinkinase 1 (RIPK1) as a therapeutic target. Nature reviews. Drug discovery, 2020, 19(8): 553-571 Summary of the Invention

[0031] The purpose of this invention is to provide the use of Necrostatin-1 in the preparation of drugs for the prevention and / or treatment of acute aristolochic acid nephropathy, and to provide an effective drug for the prevention or treatment of acute aristolochic acid nephropathy, thus providing a new option for the treatment of acute aristolochic acid nephropathy.

[0032] The technical solution of the present invention is as follows:

[0033] The use of Necrostatin-1 in the preparation of drugs for the prevention and / or treatment of acute aristolochic acid nephropathy, the use including at least one of the following aspects:

[0034] 1) Application in the preparation of drugs for preventing and treating renal tubular epithelial cell damage caused by aristolochic acid;

[0035] 2) Application in the preparation of drugs that inhibit aristolochic acid-induced increase in RIPK1 kinase activity;

[0036] 3) Application in the preparation of drugs that inhibit the activation of the aristolochic acid-induced endogenous apoptosis pathway;

[0037] 4) Application in the preparation of drugs that inhibit the activation of aristolochic acid-induced exogenous apoptosis pathway;

[0038] 5) Application in the preparation of drugs for preventing and treating apoptosis of renal tubular epithelial cells induced by aristolochic acid;

[0039] 6) Application in the preparation of drugs for preventing and treating GSDME-mediated pyroptosis of renal tubular epithelial cells induced by aristolochic acid;

[0040] 7) Application in the preparation of drugs for preventing and treating kidney inflammation caused by aristolochic acid.

[0041] A drug for the prevention and / or treatment of acute aristolochic acid nephropathy, the active ingredient of which is / contains Necrostatin-1 and its similar structural component Necrostatin-1 stable (Necrostatin-1s). The structural formulas of both are as follows:

[0042]

[0043] The drugs mentioned above, with Necrostatin-1 as the main active ingredient, for the prevention and treatment of acute aristolochic acid nephropathy, can be introduced into the body through inhalation, intravenous injection, intramuscular injection, oral administration, or by preparations that are mixed or encapsulated with other chemical substances.

[0044] Drugs with Necrostatin-1 as the active ingredient may, when necessary, incorporate one or more pharmaceutically acceptable carriers. These carriers include diluents, excipients, fillers, binders, wetting agents, disintegrants, absorption enhancers, surfactants, adsorbents, lubricants, etc., commonly used in the pharmaceutical field. The drug can be administered via, but is not limited to, oral, intravenous, or intramuscular injection, and its dosage form can be formulated as injections, tablets, powders, granules, capsules, oral liquids, and other forms. All of the above dosage forms can be prepared using conventional methods in the pharmaceutical field.

[0045] The advantages of this invention are as follows: The typical pathological change in acute aristolochic acid nephropathy is renal tubular epithelial cell necrosis, and Necrostatin-1 can target this change to treat acute aristolochic acid nephropathy. Necrostatin-1 can significantly inhibit the kinase activity of RIPK1, block RIPK1-dependent intrinsic and extrinsic apoptosis pathways, delay renal tubular epithelial cell apoptosis and GSDME-dependent pyroptosis, effectively improve renal tubular epithelial cell damage, and thus achieve the goal of treating acute aristolochic acid nephropathy. This invention can provide a new strategy for the treatment of acute aristolochic acid nephropathy. Necrostatin-1 can play a wide range of roles in the preparation of drugs for the treatment of acute aristolochic acid nephropathy. Attached Figure Description

[0046] Figure 1 Aristolochic acid I (AAI) induces damage to renal tubular epithelial cells.

[0047] Figure 2 AAI induces apoptosis in renal tubular epithelial cells.

[0048] Figure 3 AAI induces pyroptosis in renal tubular epithelial cells.

[0049] Figure 4The effect of AAI on RIPK1 kinase activity.

[0050] Figure 5 Necrostatin-1 (Nec) inhibits aristolochic acid-induced cell damage.

[0051] Figure 6 Nec inhibits the activity of cell damage-related marker proteins.

[0052] Figure 7 To quantify the RIPK1 kinase activity induced by aristolochic acid inhibited by Nec.

[0053] Figure 8 To quantify the pathway by which Nec inhibits aristolochic acid-induced extrinsic apoptosis.

[0054] Figure 9 To quantify the Nec pathway that inhibits aristolochic acid-induced endogenous apoptosis.

[0055] Figure 10 To quantify the inhibition of aristolochic acid-induced apoptosis by Nec.

[0056] Figure 11 To quantify the GSDME-mediated pyroptosis induced by Nec inhibition of aristolochic acid. Detailed Implementation

[0057] The present invention will be described below through specific experimental implementation methods. The present invention includes, but is not limited to, these methods.

[0058] Unless otherwise specified, the experimental methods described in the following examples are conventional methods. Unless otherwise specified, all reagents and materials are commercially available.

[0059] Example 1: Pharmacodynamic test of Necrostatin-1 protecting cells from acute aristolochic acid nephropathy.

[0060] 1 Experimental Methods

[0061] 1.1 Cell Culture and AAI Treatment

[0062] The cell line used was the immortalized human proximal renal tubular epithelial cell line HK2, purchased from the American Type Culture Center (ATCC). The culture medium was DMEM / F12 supplemented with 10% fetal bovine serum. Cells were cultured in a CO2 incubator at 37°C with a CO2 concentration of 5%. AAI concentrations of 10, 20, 30, 40, and 50 μg / mL were prepared using the culture medium. When HK2 cells reached 90% confluence in six-well plates, 2 mL of AAI solution was added to each well. After 48 h of exposure, AAI induced significant damage to HK2 cells.

[0063] 1.2 Survival rate

[0064] This invention uses the Cell Counting Kit-8 (CCK-8) reagent kit to determine cell viability. HK2 cells were seeded into 96-well plates, 1 × 10⁶ cells per well. 4 Cells were cultured for 24 h, and then 10, 20, 30, 40, and 50 μg / mL AAI solutions were added to the culture medium, and the cells were cultured for 24 or 48 h. The CCK-8 reagent was diluted 20-fold with culture medium. 200 μL was added to each well, and the cells were cultured for 2–4 h. The absorbance of each well was read at 450 nm using a microplate reader. The survival rate of the control group was normalized to 100%, and the cell viability of each group was calculated.

[0065] 1.3 ATP content

[0066] This invention uses an enhanced ATP assay kit to determine ATP content. Logarithmically growing HK2 cells were seeded in 12-well plates, 5 × 10⁶ cells per well. 5 Cells were cultured for 24 h, and then 10, 20, 30, 40, and 50 μg / mL AAI solutions were added to the culture medium, and the cells were cultured for 24 or 48 h. The culture medium was removed, and 200 μL of lysis buffer was added to fully lyse the cells. The lysis buffer was collected, centrifuged at 12000×g for 5 min, and the supernatant was collected. ATP detection working solution was added, and the chemiluminescence intensity of each group was detected using a microplate reader. The ATP content of the control group was normalized to 100%.

[0067] 1.4 LDH Measurement

[0068] This invention uses an LDH cytotoxicity kit to detect LDH content in the supernatant. HK2 cells were treated as described above, and the supernatant was collected and centrifuged at 1500×g for 5 min. 120 μL of the supernatant was added to a 96-well plate, followed by 60 μL of LDH detection reagent. The plates were incubated in the dark for 30 min, and the absorbance of each well was read at 490 nm using a microplate reader. The survival rate of the control group was normalized to 1.

[0069] 1.5 TUNEL Experiment

[0070] Paraffin sections of kidney tissue from mouse models were dewaxed, digested with proteinase K working solution, rehydrated with equilibration buffer, incubated with TdT enzyme solution in the dark, washed, and stained with DAPI. Images were taken using a Leica DM4000B fluorescence microscope.

[0071] 1.6 Flow cytometry detection of apoptosis

[0072] This invention uses the Annexin V-FITC apoptosis detection kit to determine the proportion of apoptotic cells. Cells were processed and collected according to the procedure described in 1.2, centrifuged, resuspended in 200 μL of Annexin V-FITC binding buffer, and incubated at 25°C for 10 min with 5 μL of Annexin V-FITC solution. Then, 5 μL of PI staining solution was added, and apoptotic cells were immediately detected by flow cytometry.

[0073] 1.7 Western Blot Experiment

[0074] Cells were processed and collected according to the aforementioned procedure. Cells or tissues were lysed on ice, and whole protein was extracted. 1 / 4 volume of 5× loading buffer was added, and the mixture was thoroughly mixed and heated in a metal water bath at 95°C for 10-15 min. The sample was loaded onto a gel plate, and proteins were separated using polyacrylamide gel electrophoresis at a stacking gel voltage of 80 V and a separating gel voltage of 120 V. A sandwich structure was then constructed to transfer the proteins onto a 0.22 μm PVDF membrane. The membrane was blocked with 5% skim milk powder for 1 h, followed by incubation with 1:1000 primary and secondary antibodies. The band intensity was then developed using a chemiluminescent reagent in a gel imaging system.

[0075] 1.8 Statistical Methods

[0076] The results presented in this study are representative of at least three independent replicate experiments. Immunofluorescence and Western blotting results were quantified using ImageJ or Gelproanalyzer software, and the results are presented as mean ± standard deviation. After one-way heteroscopy, the minimum significance method was used to calculate the p-value between groups. A p-value < 0.05 was considered statistically significant.

[0077] 2 Results

[0078] 2.1 AAI-induced renal tubular epithelial cell damage

[0079] like Figure 1 As shown in Figure A, with increasing AAI dosage, HK2 cell damage significantly worsened, with cell tentacles shrinking and cell morphology becoming rounder. Figure 1 B showed that HK2 cell survival gradually decreased with increasing dose, and when the AAI concentration was greater than 20 μg / mL, the survival rate was significantly lower than that of the control group. AAI exposure at 48 h resulted in significantly greater damage than at 24 h. ATP survival and LDH extracellular release results suggested the same conclusions (see [link to study]. Figure 1 C and Figure 1 D), AAI can significantly induce damage in HK2 cells.

[0080] 2.2 AAI induces apoptosis in renal tubular epithelial cells

[0081] The results of the TUNEL experiment are as follows: Figure 2 As shown in Figure A, in the AAI-treated group, HK2 cell nuclei were FITC-positive, while those in the control group were FITC-negative, suggesting that AAI exposure can induce DNA breaks in HK2 cells. Flow cytometry results are as follows. Figure 2 As shown in Figure B, compared with the control group, the number of late apoptotic or necrotic cells stained with FITC+ / PI+ was significantly increased, with the proportion rising from 3% to about 17%; the proportion of early apoptotic cells rose from about 1% to about 20%, indicating that AAI induced significant apoptosis. Figure 2 The results showed that the apoptosis-executing protein caspase-3, the endogenous pathway marker protein caspase-9, and the exogenous pathway marker protein caspase-8 were all significantly cleaved, suggesting that AAI induces apoptosis in HK2 cells through endogenous and exogenous apoptosis pathways.

[0082] 2.3 AAI induces pyroptosis in renal tubular epithelial cells

[0083] like Figure 3 As shown, with increasing AAI exposure time and dose, the pyroptosis executive protein GSDME underwent significant cleavage, with a significant decrease in GSDME-FL content and a significant increase in GSDME-NT content (see [link to relevant documentation]). Figure 3 A and Figure 3 B) ， The quantitative results are shown below. Figure 3 C and Figure 3 D indicates that AAI induces GSDME-mediated pyroptosis in HK2 cells.

[0084] 2.4 Effects of AAI on RIPK1 kinase activity

[0085] Figure 4 Figure A shows the changes in RIPK1 kinase activity in the kidney tissue of AAI-exposed mice. Quantitative graph is shown below. Figure 4 B. Figure 4 C and Figure 4 E represents the effect of AAI dose gradient and exposure time on RIPK1 kinase activity, respectively. Quantitative plots are shown below. Figure 4 D and Figure 4 F. The results showed that AAI exposure significantly increased RIPK1 kinase activity in proximal renal tubular epithelial cells.

[0086] 2.5 Nec inhibits AAI-induced cell damage

[0087] Figure 5Figure A shows the effects of Nec and its homologous small molecule Nec-1s on AAI-induced cell damage. The results indicate that Nec and Nec-1s significantly blocked AAI-induced cell damage. Pretreatment of cells with Nec and Nec-1s significantly alleviated AAI-induced cell rounding and necrosis, and significantly improved cell morphology. Survival results ( Figure 5 (B) showed that, compared with the AAI group, Nec and Nec-1s pretreatment significantly improved HK2 cell survival. Compared with the AAI group, Nec and Nec-1s pretreatment significantly reduced LDH extracellular secretion. Figure 5 C). Figure 5 The results suggest that Nec can inhibit AAI-induced cell damage.

[0088] 2.6 Nec inhibits the activity of cell damage-related marker proteins

[0089] Figure 6 The figure shows the effects of Nec treatment on RIPK1 kinase activity, endogenous and exogenous apoptosis pathways, and the activity of apoptosis and pyroptosis marker proteins. As can be seen from the figure, AAI significantly induced an increase in RIPK1 kinase activity, and also significantly increased levels of cleaved caspase-8, cleaved caspase-9, cleaved caspase-3, and GSDME-NT. Nec pretreatment suppressed the levels of pathway-related marker proteins to control levels, suggesting that Nec can block GSDME-mediated pyroptosis by inhibiting the RIPK1-dependent endogenous and exogenous apoptosis pathways and thus play a protective role for cells.

[0090] 2.7 Quantitative analysis of Nec's inhibition of AAI-induced RIPK1 kinase activity

[0091] Figure 7 As shown Figure 6 The relative quantification of p-RIPK1 indicates that Nec inhibits RIPK1 kinase activity.

[0092] 2.8 Quantitative analysis of Nec's inhibition of AAI-induced extrinsic apoptosis pathway

[0093] Figure 8 As shown Figure 6 The quantitative analysis of cleaved caspase-8 indicates that Nec inhibits the extrinsic apoptosis pathway.

[0094] 2.9 Quantitative analysis of Nec's inhibition of AAI-induced intrinsic apoptosis pathway

[0095] Figure 9 As shown Figure 6 The quantitative analysis of cleaved caspase-9 indicates that Nec inhibits the intrinsic apoptosis pathway.

[0096] 2.10 Quantitative analysis of Nec's inhibition of AAI-induced apoptosis

[0097] Figure 10 As shown Figure 6 The quantitative analysis of cleaved caspase-3 indicates that Nec inhibits apoptosis.

[0098] 2.11 Quantitative analysis of AAI-induced GSDME-mediated pyroptosis mediated by Nec inhibition

[0099] Figure 11 As shown Figure 6 The quantitative analysis of GSDME-NT indicates that Nec inhibits GSDME-mediated pyroptosis.

[0100] The above results indicate that Nec can significantly inhibit RIPK1 kinase activity, block RIPK1-dependent intrinsic and extrinsic apoptosis pathways, delay renal tubular epithelial cell apoptosis and GSDME-dependent pyroptosis, and effectively improve renal tubular epithelial cell damage. Nec can play a wide range of roles in the preparation of drugs for the treatment of acute aristolochic acid nephropathy.

Claims

1. Use of Necrostatin-1 in the preparation of a medicament for preventing and / or treating acute aristolochic acid nephropathy, characterized in that: Prevent and / or treat the phenotype of GSDME-mediated pyroptosis and pyroptosis-related inflammation in renal tubular epithelial cells.

2. Use of Necrostatin-1 according to claim 1 for the preparation of a medicament for the prevention and / or treatment of acute aristolochic acid nephropathy, characterized in that: The pharmacological mechanism is to block the RIPK1 / caspase-8 / caspase-9 / caspase-3 / GSDME pathway.

3. Use of Necrostatin-1 according to claim 1 or 2 for the preparation of a medicament for the prevention and / or treatment of acute aristolochic acid nephropathy, characterized in that: In the preparation of drugs for preventing and treating aristolochic acid-induced renal tubular epithelial cell damage.

4. Use of Necrostatin-1 according to claim 1 or 2 for the preparation of a medicament for the prevention and / or treatment of acute aristolochic acid nephropathy, characterized in that: In the preparation of drugs for inhibiting aristolochic acid-induced increase in RIPK1 kinase activity.

5. Use of Necrostatin-1 according to claim 1 or 2 for the preparation of a medicament for the prevention and / or treatment of acute aristolochic acid nephropathy, characterized in that: In the preparation of drugs for inhibiting aristolochic acid-induced activation of endogenous apoptosis pathway.

6. Use of Necrostatin-1 according to claim 1 or 2 for the preparation of a medicament for the prevention and / or treatment of acute aristolochic acid nephropathy, characterized in that: In the preparation of drugs for inhibiting aristolochic acid-induced activation of exogenous apoptosis pathway.

7. Use of Necrostatin-1 according to claim 1 or 2 for the preparation of a medicament for the prevention and / or treatment of acute aristolochic acid nephropathy, characterized in that: In the preparation of drugs for preventing and treating aristolochic acid-induced renal tubular epithelial cell apoptosis.

8. Use of Necrostatin-1 according to claim 1 or 2 for the preparation of a medicament for the prevention and / or treatment of acute aristolochic acid nephropathy, characterized in that: In the preparation of drugs for preventing and treating aristolochic acid-induced GSDME-mediated pyroptosis in renal tubular epithelial cells.

9. Use of Necrostatin-1 according to claim 1 or 2 for the preparation of a medicament for the prevention and / or treatment of acute aristolochic acid nephropathy, characterized in that: In the preparation of drugs for preventing and treating aristolochic acid-induced renal inflammation.

Images