Use of ido2 activators in the manufacture of a medicament for reducing cisplatin-induced acute kidney injury

Abstract

The application discloses an application of an IDO2 activator in preparation of a medicine for relieving cisplatin-induced acute kidney injury, and in particular finds that ginsenoside Rb2 plays an anti-ferroptosis role by activating an IDO2 protein level, and further relieves cisplatin-related acute kidney injury, and can become an effective clinical medicine for preventing and treating AKI.

Description

Technical Field

[0001] This invention belongs to the pharmaceutical field, specifically relating to the application of an IDO2 activator in the preparation of a drug for alleviating cisplatin-induced acute kidney injury, particularly the application of ginsenoside Rb2 in the preparation of a drug for the prevention and / or treatment of cisplatin-induced acute kidney injury. Background Technology

[0002] Cisplatin is one of the most effective chemotherapy drugs in clinical practice and is widely used to treat a variety of malignant diseases, including those affecting the respiratory, digestive, urinary, and reproductive systems. However, severe adverse reactions such as nephrotoxicity, ototoxicity, and emetic effects limit its clinical use. Acute kidney injury (AKI) caused by nephrotoxicity is the most significant limiting factor. Clinically, the prevention and treatment of cisplatin-induced AKI mainly rely on supportive measures such as hydration and correction of electrolyte imbalances; targeted therapies that can specifically halt disease progression and promote kidney repair are currently lacking. While the efficacy of cisplatin is directly proportional to the dosage, its nephrotoxicity also exhibits a dose-dependent effect. Cisplatin-induced nephrotoxicity involves multiple mechanisms, including renal cell necrosis, apoptosis, ferroptosis, a large number of inflammatory cytokines, cytotoxicity, and oxidative stress. Reducing cisplatin nephrotoxicity, better utilizing its anti-tumor effects, and improving the quality of chemotherapy for patients are urgent problems to be solved. Therefore, developing new and effective drugs that can improve cisplatin-induced AKI has significant clinical implications.

[0003] In recent years, ferroptosis, as a novel cell death mechanism driven by iron-dependent lipid peroxidation, has been shown to play a more upstream and central role than apoptosis and necrosis in cisplatin-induced renal tubular epithelial cell injury. However, existing intervention strategies for cisplatin-induced kidney injury mainly focus on anti-apoptosis and conventional antioxidant pathways, which are insufficient to effectively block ferroptosis. Currently, some ginsenoside monomers (such as ginsenosides Rg1, Rg2, and Rg5) have been reported to have a certain ameliorative effect on cisplatin-induced kidney injury. However, the known mechanisms of action of these saponins are mainly limited to conventional pathways such as anti-apoptosis, anti-inflammation, or free radical scavenging, and none of them involve the ferroptosis pathway. Furthermore, their targets are vague, require high doses, and lack specificity, failing to fundamentally address the core pathological link of cisplatin-triggered ferroptosis.

[0004] Indoleamine 2,3-dioxygenase 2 (IDO2) is an enzyme that catalyzes the first step in the catabolism of tryptophan, an essential amino acid, in the kynurenine pathway. This pathway is crucial for the regulation of tryptophan metabolism and has implications for immune regulation. However, whether IDO2 is involved in cisplatin-induced acute kidney injury, and in particular whether IDO2 can regulate ferroptosis in renal tubular epithelial cells, remains unreported in the literature.

[0005] Ginsenoside Rb2, a major saponin extracted from ginseng (CAS number 11021-13-9), possesses various pharmacological activities including antioxidant, anti-inflammatory, and anti-tumor effects. Existing research indicates that ginsenoside Rb2 plays a role in metabolic diseases and neuroprotection. However, to date, no published literature or patents, domestic or international, report that ginsenoside Rb2 can exert anti-ferroptosis effects by activating IDO2 protein levels, nor does it offer any technological inspiration for using ginsenoside Rb2 through this specific mechanism to alleviate cisplatin-induced acute kidney injury. Summary of the Invention

[0006] Purpose of the invention: The technical problem to be solved by the present invention is to address the shortcomings of the prior art by providing the application of IDO2 activator in the preparation of drugs for alleviating cisplatin-induced acute kidney injury, particularly the application of ginsenoside Rb2 in the preparation of drugs for the prevention and / or treatment of cisplatin-induced acute kidney injury.

[0007] This application is the first to discover a significant downregulation of IDO2 protein levels in the kidney tissue of mice with cisplatin-induced acute kidney injury, and reveals for the first time that the downregulation of IDO2 is a key upstream event triggering ferroptosis in renal tubular epithelial cells. Based on this novel discovery, this invention proposes that activating IDO2 function with exogenous compounds can stabilize GPX4 protein in a non-tryptophan metabolism-dependent manner, thereby specifically blocking ferroptosis and improving cisplatin-induced acute kidney injury.

[0008] To address the aforementioned technical problems, this invention discloses the application of an IDO2 activator in the preparation of a drug for alleviating cisplatin-induced acute kidney injury.

[0009] In one embodiment, the IDO2 activator is ginsenoside Rb2.

[0010] Specifically, ginsenoside Rb2 exerts its anti-ferroptosis effect by activating IDO2 protein levels, thereby alleviating cisplatin-induced acute kidney injury. More importantly, this mechanism of action of ginsenoside Rb2 is completely different from that of known ginsenosides Rg1, Rg2, and Rg5. While known saponins mainly exert their effects through broad-spectrum anti-apoptosis or conventional antioxidant pathways, this invention establishes a novel signaling axis of "IDO2 activation—GPX4 stabilization—lipid peroxidation inhibition—ferroptosis blocking," representing an unprecedented and highly specific mode of action.

[0011] Ginsenoside Rb2 reduces the expression of KIM-1 and NGAL in mice with cisplatin-induced acute kidney injury.

[0012] Ginsenoside Rb2 inhibits lipid peroxidation and improves cellular oxidative stress.

[0013] This application also proposes the use of ginsenoside Rb2 in the preparation of drugs for activating the activity of IDO2 protein.

[0014] Furthermore, this application proposes the use of ginsenoside Rb2 as the sole active ingredient in the preparation of a drug for alleviating cisplatin-induced acute kidney injury.

[0015] Specifically, ginsenoside Rb2 upregulates IDO2 protein levels and GPX4 protein levels in kidney tissue; it also reduces cisplatin-induced ferroptosis in renal tubular epithelial cells.

[0016] This invention explores the use of ginsenoside Rb2 as an IDO2 activator in alleviating symptoms associated with cisplatin-induced acute kidney injury (AKI) by applying it to cisplatin-induced acute kidney injury (AKI) in mouse and in vitro cell models. Results showed that intervention with ginsenoside Rb2 in both cisplatin-induced mouse and in vitro cell models significantly improved renal structure and function in AKI and inhibited tubular cell ferroptosis. This invention provides an effective clinical candidate drug for the prevention and treatment of AKI.

[0017] Compared with the prior art, the present invention has the following significant beneficial effects and unexpected technical effects:

[0018] (1) Target novelty: This invention is the first to discover that IDO2 is a key functional target for regulating ferroptosis in cisplatin-induced acute kidney injury. All known ginsenosides (such as Rg1, Rg2, and Rg5) used for cisplatin-induced nephroprotection in the prior art do not involve IDO2 protein or ferroptosis pathway.

[0019] (2) Unique Mechanism: This invention confirms that ginsenoside Rb2 exerts its anti-ferroptosis effect by specifically activating IDO2 protein levels. This is fundamentally different from the known mechanisms by which saponins exert their effects through anti-apoptosis (such as the Caspase pathway) or conventional antioxidant pathways (such as ROS scavenging). This invention is the first to use ferroptosis as the core intervention target and has identified the IDO2-GPX4 specific signaling axis.

[0020] (3) Non-obviousness: Although Rg1, Rg2, Rg5 and Rb2 belong to the same class of ginsenosides, the pharmacological activities and intracellular targets of different saponin monomers vary greatly. The existing technology only suggests that some saponins have conventional nephroprotective effects, but does not provide any technical guidance to those skilled in the art to try to use Rb2 to activate IDO2 to inhibit ferroptosis. Attached Figure Description

[0021] Figure 1 Ginsenoside Rb2 showed that it significantly improved kidney injury and kidney function in cisplatin-induced acute kidney injury;

[0022] Figure 2 The study showed that ginsenoside Rb2 downregulated the levels of NGAL and KIM-1 proteins in kidney tissue in a cisplatin-induced acute kidney injury model.

[0023] Figure 3 The results showed that in a cisplatin-induced acute kidney injury model, ginsenoside Rb2 upregulated IDO2 protein levels and GPX4 protein levels in kidney tissue; and significantly reduced cisplatin-induced ferroptosis in renal tubular epithelial cells.

[0024] Figure 4 The results showed that in an in vitro renal tubular epithelial cell model, ginsenoside Rb2 exerted an anti-ferroptosis effect by activating IDO2. Detailed Implementation

[0025] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, and the advantages of the present invention in the above and / or other aspects will become clearer.

[0026] The materials and methods used in the following embodiments are as follows:

[0027] (a) Materials and reagents:

[0028] Ginsenoside Rb2 was purchased from Shanghai Yuanye Biotechnology Co., Ltd.; IDO2, GPX4, 4-HNE, KIM1, and NGAL antibodies were purchased from Abcam; Cleaved caspase3 and Cleaved PARP were purchased from CST; GAPDH and Tubulin antibodies were purchased from Proteintech; and the PI / Hoechst kit was purchased from Solarbio.

[0029] (II) Cell Culture and Treatment:

[0030] Human renal tubular epithelial cells (HK-2) were cultured in DMEM / F12 medium containing 10% fetal bovine serum, 1% penicillin, and streptomycin at 37°C, 5% carbon dioxide, and 95% air. To investigate the role of ginsenoside Rb2 in cisplatin-induced renal tubular epithelial cell damage, HK-2 cells were cultured in DMEM / F12 medium without fetal bovine serum when they reached 80% confluence. Pretreatment with ginsenoside Rb2 (dissolved in DMSO) for 1 h or the same treatment with DMSO was performed, followed by the addition of cisplatin. Cells were then incubated for another 24 h, and finally, Western blot analysis was performed.

[0031] (III) Cisplatin-induced acute kidney injury mouse model and experimental grouping:

[0032] Mice were randomly divided into two groups (n=5 per group): a model group (Cisplatin) and a ginsenoside Rb2 treatment group (Cisplatin + Rb2). All mice underwent a single intraperitoneal injection of cisplatin (20 mg / kg) to establish an acute kidney injury model. The Rb2 treatment group received ginsenoside Rb2 (20 mg / kg, dissolved in physiological saline) via gavage for 3 days prior to cisplatin injection, and continued this treatment daily on the day of and after cisplatin modeling. The model group served as a control, receiving an equal volume of physiological saline via gavage. Seventy-two hours after modeling, all mice were sacrificed, and blood and kidney tissue samples were collected. All animal experimental procedures complied with the Chinese Regulations on the Management and Use of Laboratory Animals.

[0033] (iv) Renal tubular injury and collagen fiber deposition score:

[0034] The degree of tubular damage was observed in the PAS pathological staining of the kidney, and the tubular damage was assessed according to the semi-quantitative damage scoring criteria. Normal renal tubular tissue was scored as 0 points, renal tubular damage less than 30% was scored as 1 point, renal tubular damage between 30% and 60% was scored as 2 points, and renal tubular damage greater than 60% was scored as 3 points.

[0035] (v) Western blot of proteins:

[0036] Total protein was extracted from kidney tissue using RIPA lysis buffer. Protein concentration was determined using the BCA method. 20 μg of protein was loaded onto a PVDF membrane and subjected to 10% polyacrylamide gel electrophoresis (SDS-PAGE). The membrane was wet-transferred to a PVDF membrane using a rapid transfer buffer at 400 mA for 40 min. The membrane was blocked with 5% skim milk at room temperature for 2 h. After TBST elution, primary antibody was added, and the membrane was incubated overnight at 4°C. The membrane was washed three times with TBST for 10 min each time, followed by incubation with the corresponding secondary antibody at room temperature for 1 h, and then washed three times with TBST for 10 min each time. Antigen-antibody complexes were visualized using enhanced chemiluminescence (ECL) on X-ray film in a dark room. Protein quantification was performed by analyzing the grayscale values ​​of the target bands, with the relative expression level of the target protein expressed as the grayscale value of the target band / GAPDH grayscale value. Western blot results were analyzed using ImageJ software.

[0037] (vi) Immunofluorescence staining of cells:

[0038] Human renal tubular epithelial cells (HK-2) were seeded into 24-well culture plates containing sterile cell spreaders. When HK-2 cells reached 80% confluence, the medium was replaced with DMEM / F12 medium without fetal bovine serum (FBS). After 12 hours, the cells were pretreated with Rb2 for 1 hour, followed by cisplatin treatment for 12 hours. After intervention, the medium was aspirated, and the cells were gently washed three times with pre-cooled PBS. Subsequently, the cells were fixed with 4% paraformaldehyde at room temperature for 15 minutes, followed by three more washes with PBS. To increase cell membrane permeability, the cells were permeabilized with PBS containing 0.1% Triton X-100 at room temperature for 10 minutes, followed by three washes with PBS. Immediately afterward, nonspecific antigens were blocked with 3% bovine serum albumin (BSA) at room temperature for 1 hour. After blocking, the BSA was aspirated, and the primary antibody working solution diluted with 3% BSA (4-HNE: 1:200) was added directly, and the cells were incubated overnight in a humidified chamber at 4°C. The following day, after rewarming, the primary antibody was recovered and thoroughly washed three times with PBS to remove unbound antibody. Then, fluorescently labeled secondary antibody working solution (1:100) was added, and the mixture was incubated at room temperature in the dark for 1 hour. After incubation, the slides were washed three times with PBS in the dark to remove residual secondary antibody. Finally, the slides were mounted using a DAPI-containing anti-fluorescence quenching mounting medium, and observed and images were acquired under an inverted microscope.

[0039] (vii) PI / Hoechst staining:

[0040] Human renal tubular epithelial cells (HK-2) were seeded into 24-well culture plates containing sterile cell slides. When the HK-2 cells reached 80% confluence, the medium was replaced with DMEM / F12 medium without fetal bovine serum. After 12 hours, the cells were pretreated with Rb2 for 1 hour, followed by cisplatin treatment for 12 hours. After the intervention, the medium was aspirated, and the cells were gently washed three times with pre-cooled PBS. The cells were then incubated with PI / Hoechst staining solution (1:200) at 4°C for 30 minutes, washed thoroughly with PBS three times, mounted with anti-fluorescence quenching mounting medium, and observed and imaged under an inverted microscope.

[0041] (viii) Statistical Analysis:

[0042] Data are expressed as mean ± SEM. A t-test was used to compare data between two groups. A p-value < 0.05 was considered statistically significant.

[0043] Example 1: Ginsenoside Rb2 improves kidney injury and renal function in a cisplatin-induced acute kidney injury model.

[0044] To evaluate the role of ginsenoside Rb2 in protecting against cisplatin-induced acute kidney injury, we measured renal-related biochemical indicators in mouse serum. Seventy-two hours after intraperitoneal injection of cisplatin to induce the acute kidney injury model, serum creatinine and blood urea nitrogen levels were significantly elevated, and renal pathological damage, such as tubular dilation and necrosis, was also severe. However, after treatment with ginsenoside Rb2, the corresponding renal damage and renal function indicators significantly decreased. Figure 1 (A, 1B, 1C). Furthermore, the renal tubular injury score also indicated that ginsenoside Rb2 treatment could improve cisplatin-induced renal pathological damage. Therefore, ginsenoside Rb2 can not only improve renal function but also alleviate renal pathological damage. These results suggest that ginsenoside Rb2 can protect against cisplatin-induced acute kidney injury, and it itself has not shown significant toxic side effects.

[0045] To further demonstrate the role of ginsenoside Rb2 in protecting against cisplatin-induced acute kidney injury, we tested early-stage specific biomarkers for acute kidney injury, NGAL and KIM-1. Figure 2 Western blot results showed that KIM-1 and NGAL were highly expressed in the kidneys of mice with cisplatin-induced acute kidney injury. After treatment with ginsenoside Rb2, their expression levels decreased significantly, indicating that ginsenoside Rb2 can improve cisplatin-induced acute kidney injury.

[0046] Example 2: Ginsenoside Rb2 upregulates IDO2 protein levels in kidney tissue and inhibits ferroptosis.

[0047] To further demonstrate the role of ginsenoside Rb2 in protecting against cisplatin-induced acute kidney injury, we examined IDO2 protein levels and ferroptosis-related markers. Western blot results showed that IDO2 expression was significantly upregulated after ginsenoside Rb2 treatment, and the expression of GPX4, a negative regulator of ferroptosis, was also upregulated. Figure 3 A). To further verify the inhibitory effect of IDO2 activation on ferroptosis at the biochemical level, we measured the levels of malondialdehyde (MDA), a lipid peroxidation end product, and reduced glutathione / oxidized glutathione (GSH / GSSG), a key indicator of oxidative stress. The results showed that, compared with the model group, the ginsenoside Rb2 treatment group significantly reduced MDA and increased GSH / GSSG levels in kidney tissue. Figure 3 (B, 3C). These data indicate that IDO2 activation effectively inhibits lipid peroxidation and improves cellular oxidative stress. This suggests that ginsenoside Rb2 can inhibit ferroptosis and improve cisplatin-induced acute kidney injury by activating IDO2.

[0048] Example 3: In an in vitro renal tubular epithelial cell model, ginsenoside Rb2 alleviated cisplatin-induced ferroptosis by activating IDO2.

[0049] Using cultured renal tubular epithelial cells, ginsenoside Rb2 (50 μM) was pretreated for 1 h, followed by treatment with 20 μM cisplatin for another 24 h. Western blot results confirmed that ginsenoside Rb2 activated IDO2 expression and correspondingly regulated downstream cell death signals: the abnormal expression of apoptosis-related proteins (Cleaved Caspase-3, Cleaved PARP) and the ferroptosis key protein GPX4 was significantly reversed. Figure 4 A). Immunofluorescence analysis showed that, compared with the model group, the proportion of PI / Hoechst double-positive cells in the ginsenoside Rb2 treatment group was significantly reduced, and the 4-HNE fluorescence signal of lipid peroxidation damage was also significantly weakened. Figure 4 B). This study demonstrates that ginsenoside Rb2 effectively alleviates cisplatin-induced apoptosis and ferroptosis by activating IDO2, thereby protecting renal tubular epithelial cells.

[0050] In summary, this invention provides the use of ginsenoside Rb2 in the preparation of a drug for alleviating symptoms associated with cisplatin-induced acute kidney injury. The drug is administered by gavage at a dose of 20 mg / kg in mice. It exerts an anti-ferroptosis effect by activating IDO2 protein levels, thereby alleviating cisplatin-related acute kidney injury.

[0051] This invention provides an application idea and method for ginsenoside Rb2 in the preparation of drugs for alleviating cisplatin-related acute kidney injury. Many methods and approaches exist for implementing this technical solution; the above description is merely a preferred embodiment of the invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of this invention, and these improvements and modifications should also be considered within the scope of protection of this invention. All components not explicitly stated in this embodiment can be implemented using existing technologies.

Claims

1. Application of IDO2 activators in the preparation of drugs for alleviating cisplatin-induced acute kidney injury.

2. The application according to claim 1, characterized in that, The IDO2 activator is ginsenoside Rb2.

3. The application according to claim 2, characterized in that, Ginsenoside Rb2 exerts its anti-ferroptosis effect by activating IDO2 protein levels, upregulating GPX4 protein expression, and inhibiting lipid peroxidation, thereby alleviating cisplatin-induced acute kidney injury.

4. The application according to claim 3, characterized in that, Ginsenoside Rb2 reduces the expression of KIM-1 and NGAL in mice with cisplatin-induced acute kidney injury.

5. The application according to claim 3, characterized in that, Ginsenoside Rb2 inhibits lipid peroxidation and improves cellular oxidative stress.

6. Application of ginsenoside Rb2 in the preparation of drugs for activating IDO2 protein activity.

7. Application of ginsenoside Rb2 as the sole active ingredient in the preparation of drugs for alleviating cisplatin-induced acute kidney injury.

8. The application according to claim 7, characterized in that, Ginsenoside Rb2 reduces cisplatin-induced ferroptosis in renal tubular epithelial cells by upregulating IDO2 and GPX4 protein levels in kidney tissue; and its mechanism of action is different from the anti-apoptotic or conventional antioxidant pathways of ginsenosides Rg1, Rg2, and Rg5.

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