Use of curcumin in preparation of medicine for treating renal injury and enhancing chemotherapy and immunotherapy of tumor

Abstract

The application discloses application of curcumin in preparation of a medicine for treating kidney injury and enhancing tumor chemotherapy immunotherapy, and belongs to the technical field of biological medicine. 15 H 24 O2, and a chemical structural formula is further provided.The kidney injury is acute kidney injury or chronic kidney injury caused by the acute kidney injury.The application provides a medicine composition for treating kidney injury and a sensitizer for tumor chemotherapy immunotherapy, and both of them contain an effective dose of curcumin.It is found that the curcumin has the effect of effectively reducing kidney injury and enhancing tumor chemotherapy immunotherapy, can be used for kidney function protection and tumor treatment, and has low toxic and side effects, so that the curcumin has wide clinical application prospects as a kidney injury treatment and tumor sensitizer.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of turmeric dione in the preparation of drugs for treating kidney damage and sensitizing tumor chemotherapy and immunotherapy. Background Technology

[0002] In recent years, the application of immune checkpoint inhibitors (ICIs) has significantly changed the treatment landscape for various solid tumors. However, single-treatment strategies face challenges such as limited response rates, primary resistance, and acquired resistance (Immunity. 2026, 59, 29-33). Traditional chemotherapy drugs such as cisplatin not only have a clear tumor burden reduction effect but can also enhance the response to immunotherapy by promoting tumor antigen release and reshaping the tumor immune microenvironment. Therefore, chemotherapy combined with immunotherapy has become an important strategy for improving the efficacy of cancer treatment (N Engl J Med. 2022; 387, 217-226). However, while chemotherapy-immunotherapy combination therapy improves efficacy, it also increases the risk of treatment-related toxicities. The kidneys are simultaneously affected by the direct toxicity of chemotherapy drugs and the immune inflammatory response, resulting in acute kidney injury (AKI) with complex damage characteristics. This not only affects patients' renal function and quality of life but may also lead to dose reduction, interruption, or termination of anti-tumor therapy, weakening the overall treatment benefit. Therefore, chemotherapy-immunotherapy combination therapy-related kidney injury has become a significant issue restricting the safety and sustained benefit of cancer treatment.

[0003] Currently, there is a lack of specific prevention and treatment strategies for kidney injury associated with combination cancer therapy. Clinically, treatment mainly relies on hydration, diuresis, dose adjustment, treatment pauses, and glucocorticoid intervention. However, these intervention strategies can affect the continuity of anti-tumor therapy and the effectiveness of tumor control, and increase the risk of infection and metabolic disorders. Therefore, mitigating kidney injury without weakening the anti-tumor effect is an urgent problem to be solved in the safety management of combination cancer therapy. However, the specific mechanism of acute kidney injury (AKI) associated with chemotherapy combined with immunotherapy is not yet clear, and there is no specific treatment strategy that can prevent or reverse the pathological progression of kidney injury. Therefore, elucidating the key molecular mechanisms of combination therapy-related AKI and screening small molecule intervention strategies with renal protective potential without weakening the anti-tumor effect based on these mechanisms is of great value.

[0004] Curcuma dione is a natural sesquiterpene compound derived from Curcuma aromatica. Existing research indicates that curcuma dione possesses broad-spectrum organ-protective and antitumor activities. Regarding organ injury, curcuma dione can alleviate D-galactosamine / lipopolysaccharide-induced acute liver injury, improve cerebral ischemia-reperfusion injury, and alleviate doxorubicin or isoproterenol-induced myocardial injury. Furthermore, it can reduce pulmonary fibrosis, liver fibrosis, and renal interstitial fibrosis caused by unilateral ureteral obstruction, and improve sepsis-related acute lung injury. In cancer treatment, curcuma dione can inhibit the growth of tumor cells such as breast cancer, uterine leiomyosarcoma, colorectal cancer, and non-small cell lung cancer. Overall, curcuma dione possesses potential for organ protection, anti-fibrosis, and antitumor sensitization; however, its application in chemotherapy-combined immunotherapy-related acute kidney injury and its simultaneous enhancement of antitumor efficacy still lacks systematic research. Summary of the Invention

[0005] To overcome the above-mentioned shortcomings of the existing technology, the purpose of this invention is to provide the application of turmeric dione in the preparation of drugs for treating kidney damage and sensitizing tumor chemotherapy and immunotherapy.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] In a first aspect, the present invention provides the use of curcuminone in the preparation of a medicament for treating kidney injury and sensitizing tumor chemotherapy and immunotherapy; wherein the molecular formula of curcuminone is C 15 H 24 O2, chemical structural formula is .

[0008] Preferably, the kidney injury is acute kidney injury or chronic kidney injury resulting from acute kidney injury.

[0009] Preferably, the acute kidney injury is acute kidney injury associated with chemotherapy combined with immunotherapy.

[0010] Preferably, the kidney injury is acute kidney injury caused by cisplatin (Cisp), chronic kidney injury caused by repeated administration of cisplatin (Cisp3), or combined kidney injury induced by cisplatin combined with PD-1 monoclonal antibody (aPD1C).

[0011] Preferably, the turmeric dione is achieved by downregulating one or more of the following kidney injury-related indicators: serum creatinine (Cr), blood urea nitrogen (BUN), alanine aminotransferase (ALT), and aspartate aminotransferase (AST).

[0012] Preferably, the curcuminone is achieved by downregulating one or more of the following: the levels of kidney injury markers KIM-1 and NGAL, the levels of inflammation-related proteins NLRP3, C-GSDMD, C-Caspase-1, IL-1, the tumor proliferation marker Ki67, and the levels of drug resistance-related proteins NLRP3 and PD-L1.

[0013] Preferably, the drug uses an effective dose of turmeric as the active ingredient, or may also include pharmaceutically acceptable excipients; the concentration of turmeric is 0.01-200 mg / kg.

[0014] More preferably, the concentration of the curcuminone is 50 mg / kg.

[0015] Preferably, the dosage form of the drug is at least one of the following: injection, tablet, pill, capsule, or nanoformulation.

[0016] A second aspect of the invention provides a pharmaceutical composition for treating kidney injury, comprising an effective dose of turmeric dione, wherein turmeric dione has the molecular formula C 15 H 24 O2, chemical structural formula is The concentration of the turmeric dione is 0.01-200 mg / kg; the kidney injury is acute kidney injury related to chemotherapy combined with immunotherapy.

[0017] Preferably, the concentration of turmeric dione is 50 mg / kg.

[0018] A third aspect of the present invention provides a sensitizer for tumor chemotherapy immunotherapy, comprising an effective dose of curcuminidine, wherein the molecular formula of curcuminidine is C2. 15 H 24 O2, chemical structural formula is The concentration of the turmeric dione is 0.01-200 mg / kg, and the tumor chemotherapy and immunotherapy is cisplatin combined with PD-1 monoclonal antibody for tumor treatment.

[0019] Preferably, the concentration of turmeric dione is 50 mg / kg.

[0020] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0021] This invention, through experiments, first discovered that turmeric dione has the effect of effectively reducing kidney damage and enhancing the sensitization of tumor chemotherapy and immunotherapy. It can be used for kidney function protection and tumor sensitization, with low toxicity and side effects. Its mechanism of action is related to turmeric dione reducing inflammatory damage. As a kidney damage treatment and tumor sensitizer, it has broad clinical application prospects. Attached Figure Description

[0022] Figure 1 The images show hematoxylin-eosin (HE) staining and changes in liver and kidney function indicators in mice with a single cisplatin-induced acute kidney injury model after administration of turmeric dione in the examples. A: HE staining image and tubular injury score; B: serum creatinine, blood urea nitrogen, AST, and ALT levels.

[0023] Figure 2 The images shown are HE staining images and changes in liver and kidney function indicators in mice after administration of turmeric dione in a mouse model of kidney injury induced by repeated cisplatin administration in the examples; A: HE staining image and tubular injury score map; B: serum creatinine, blood urea nitrogen, AST and ALT levels.

[0024] Figure 3 The images show HE staining and changes in liver and kidney function indicators in mice with a cisplatin-PD-1 monoclonal antibody-induced kidney injury model after administration of turmeric dione in the examples; A: HE staining and tubular injury score; B: serum creatinine, blood urea nitrogen, AST, and ALT levels.

[0025] Figure 4 The images shown are: immunohistochemical staining of KIM1 and NGAL levels and a statistical graph of relative protein levels after administration of turmeric dione in a mouse model of acute kidney injury induced by a single cisplatin dose (A); and Western blotting of KIM1 and NGAL levels and a statistical graph of relative protein levels (B).

[0026] Figure 5 The images shown are: immunohistochemical staining of KIM1 and NGAL levels and a statistical graph of relative protein levels after administration of turmeric dione in a mouse model of renal injury induced by repeated cisplatin administration (A); and Western blotting of KIM1 and NGAL levels and a statistical graph of relative protein levels (B).

[0027] Figure 6 The images shown are: (A) Immunohistochemical staining of KIM1 and NGAL levels and relative protein level statistics in a mouse kidney injury model induced by cisplatin combined with PD-1 monoclonal antibody; and (B) Western blotting images of KIM1 and NGAL levels and relative protein level statistics in the same model.

[0028] Figure 7 The images show Western blotting plots and relative protein level statistics of NLRP3, C-GSDMD, C-Caspase-1, and IL-1 in a mouse kidney injury model induced by a single cisplatin dose in the examples.

[0029] Figure 8The images show Western blotting plots and relative protein level statistics of NLRP3, C-GSDMD, C-Caspase-1, and IL-1 in a mouse kidney injury model induced by repeated cisplatin administration, as well as a statistical graph of their relative protein levels.

[0030] Figure 9 The images show Western blotting plots and relative protein level statistics of NLRP3, C-GSDMD, C-Caspase-1, and IL-1 in a mouse kidney injury model induced by cisplatin combined with PD-1 monoclonal antibody, as well as the mouse model of inflammatory proteins NLRP3, C-GSDMD, C-Caspase-1, and IL-1 after administration of turmeric dione in the examples.

[0031] Figure 10 To illustrate the synergistic sensitizing effect of turmeric dione in cisplatin monotherapy for tumor treatment in the examples; A: Dynamic growth curve of mouse tumor volume over time; B: Quantitative statistical analysis of tumor weight in each group of mice at the experimental endpoint; C: Kaplan-Meier survival curves of mice in each group; D: HE staining image of tumor tissue, immunohistochemical staining image of Ki67 level, and statistical graph of relative protein level.

[0032] Figure 11 The following are examples of the synergistic sensitizing effect of turmeric dione in cisplatin combined with PD-1 monoclonal antibody antitumor therapy: A: Dynamic growth curve of mouse tumor volume over time; B: Quantitative statistical analysis of tumor weight in each group of mice at the experimental endpoint; C: Kaplan-Meier survival curves of mice in each group; D: HE staining image of tumor tissue, immunohistochemical staining image of Ki67 level, and statistical graph of relative protein level.

[0033] Figure 12 The images shown are: (A) Immunohistochemical staining images and relative protein level statistics of NLRP3 and PD-L1 levels in cisplatin monotherapy for tumors; and (B) Immunohistochemical staining images and relative protein level statistics of NLRP3 and PD-L1 levels in cisplatin combined with PD-1 monoclonal antibody therapy for tumors. Detailed Implementation

[0034] To more fully understand and demonstrate the technical solutions, objectives, and advantages of the present invention, the technical effects produced by the present invention will be further described in detail and completely below with reference to the accompanying drawings and specific embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. It should be noted that other embodiments obtained by those skilled in the art without departing from the concept of the present invention are all within the protection scope of the present invention.

[0035] The technical solution of the present invention will be described in more detail below with reference to embodiments:

[0036] Experimental Example 1: Curcuma dione can significantly alleviate cisplatin-induced acute kidney injury in vivo.

[0037] 1. Experimental Methods

[0038] 1.1 Animal Grouping

[0039] Male C57BL / 6 mice aged 6-8 weeks were randomly divided into the following 4 groups (n=6-8 per group) after 7 days of acclimatization in an SPF-grade animal facility: control group (physiological saline), model group (cisplatin 20 mg / kg), low-dose treatment group (cisplatin 20 mg / kg + turmeric dione 50 mg / kg), and high-dose treatment group (cisplatin 20 mg / kg + turmeric dione 100 mg / kg). Turmeric dione was freshly prepared with DMSO. All injections were administered by volume calculated based on body weight, according to the above grouping scheme.

[0040] 1.2 Model Establishment (Cisplatin-Induced Acute Kidney Injury)

[0041] Mice were given a single intraperitoneal injection of cisplatin (20 mg / kg).

[0042] 1.3 Dosing regimen

[0043] The turmeric dione treatment group received their first intraperitoneal injection of turmeric dione (50 or 100 mg / kg, dissolved in normal saline with DMSO aid) on day 1 after cisplatin administration, once daily until the end of the experiment.

[0044] 1.4 Sample Collection

[0045] Mice in each group were sacrificed 72 hours after cisplatin administration, and bilateral kidney tissue was collected. Some kidney tissue was immediately fixed in 4% paraformaldehyde for pathological analysis; the rest was cryopreserved at -80°C for subsequent molecular biological assays.

[0046] 1.5 Histological and Pathological Analysis

[0047] Kidney tissue was fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned to a thickness of 4 micrometers. Sections were stained with hematoxylin and eosin (HE) according to standard operating procedures. Under an optical microscope, renal tubular injury was assessed based on the severity and extent of tubular epithelial cell necrosis, vacuolar degeneration, cast formation, and luminal dilation (0 = no injury; 1 = less than 10% tubular damage; 2 = 10%-25% tubular damage; 3 = 26%-50% tubular damage; 4 = 50%-75% tubular damage; 5 = more than 75% tubular damage). At least five non-overlapping high-power fields (400x magnification) were randomly selected from each section for scoring, and the average score for each sample was calculated.

[0048] 1.6 Liver and Kidney Function Analysis

[0049] Serum samples and kidney tissue were collected 72 hours later. The levels of serum creatinine, blood urea nitrogen, AST and ALT in the animal model serum were measured according to the instructions of the serum creatinine, blood urea nitrogen, AST and ALT kit (purchased from Nanjing Jiancheng Biotechnology Institute). The creatinine determination results are shown in Table 1.

[0050] Table 1

[0051]

[0052] Creatinine content (μmol / L) = [(A2-K*A1)-(Blank A2-K*Blank A1)] / [(Standard A2-K*Standard A1)-(Blank A2-K*Blank)]*Standard concentration (442μmol / L). Urea nitrogen test results are shown in Table 2.

[0053] Table 2

[0054]

[0055] Urea nitrogen content (mmol / L) = (measured OD value - blank measured value) / (standard OD value - blank OD value) * standard concentration (10 mmol / L) * sample dilution factor before testing. AST measurement is shown in Table 3, and ALT measurement is shown in Table 4.

[0056] Table 3

[0057]

[0058] Table 4

[0059]

[0060] 2. Experimental Results

[0061] 2.1 Curcumaone alleviates renal tubular injury in a cisplatin-induced acute kidney injury model

[0062] HE staining results ( Figure 1 A) The results showed that the renal tubular structure in the control group was normal, with no obvious pathological changes. The Cisp model group showed significant acute renal tubular injury, manifested as extensive brush border shedding of epithelial cells, cell necrosis, and cast formation. The degree of renal tubular injury in the turmeric dione treatment group was significantly reduced, specifically manifested as a decrease in necrotic cells and casts, and better maintenance of tubular structural integrity.

[0063] 2.2 Curcuma zedoaria reduced serum creatinine, blood urea nitrogen, AST, and ALT levels in a cisplatin-induced acute kidney injury model.

[0064] In the Cisp model group, serum creatinine and blood urea nitrogen levels were significantly elevated, and renal function deteriorated. Curcuma dione effectively reduced serum creatinine and blood urea nitrogen levels in the model group. Figure 1 B). The above experimental results further confirm the hepatoprotective and renal protective effects of turmeric dione in acute kidney injury.

[0065] Experimental Example 2: Curcuma dione can significantly alleviate renal injury induced by repeated cisplatin administration (Cisp3) in vivo.

[0066] 1. Experimental Methods

[0067] Male C57BL / 6 mice aged 6-8 weeks were acclimatized for 3 days in an SPF-grade animal facility and then randomly divided into the following 4 groups (n=6-8 per group): control group (physiological saline), model group (cisplatin 20 mg / kg, every 5 days), low-dose treatment group (cisplatin 20 mg / kg + turmeric dione 50 mg / kg), and high-dose treatment group (cisplatin 20 mg / kg + turmeric dione 100 mg / kg). All injections were administered by body weight. Turmeric dione or control solvent was administered according to the above grouping scheme. Serum samples and kidney tissue were collected 12 days later. The serum creatinine, blood urea nitrogen, AST, and ALT levels in the animal model serum were measured according to the instructions of the kits (purchased from Nanjing Jiancheng Bioengineering Institute), using the same methods as above.

[0068] 2. Experimental Results

[0069] 2.1 Curcumone alleviates renal tubular injury in a cisplatin-repeated renal injury model

[0070] HE staining results ( Figure 2 A) The results showed that the renal tubular structure in the control group was normal, with no obvious pathological changes. The Cisp3 model group exhibited significant acute tubular injury, characterized by extensive brush border shedding of epithelial cells, cell necrosis, and cast formation. The degree of tubular injury was significantly reduced in the turmeric-dione treatment group. Specifically, the number of necrotic cells and casts decreased, and the integrity of the tubular structure was better maintained.

[0071] 2.2 Curcuma zedoamine reduced serum creatinine, blood urea nitrogen, AST, and ALT levels in a cisplatin-induced renal injury model.

[0072] In the Cisp3 model group, serum creatinine and blood urea nitrogen levels were significantly elevated, and renal function deteriorated. Curcuma dione effectively reduced serum creatinine and blood urea nitrogen levels in the model group. Figure 2 B). The above experimental results further confirm the hepatoprotective and renal protective effects of turmeric dione in cisplatin-induced kidney injury after repeated administration.

[0073] Experimental Example 3: Curcuma dione can significantly alleviate kidney injury induced by cisplatin combined with PD-1 monoclonal antibody administration (aPD1C) in vivo.

[0074] 1. Experimental Methods

[0075] Male C57BL / 6 mice aged 6-8 weeks were acclimatized for 3 days in an SPF-grade animal facility and then randomly divided into the following 4 groups (n=6-8 per group): control group (physiological saline), model group 1 (cisplatin 20 mg / kg, every 5 days), model group 2 (cisplatin 20 mg / kg, every 5 days + PD-1 monoclonal antibody 160 μg / mice, every 5 days), and treatment group (cisplatin 20 mg / kg, every 5 days + PD-1 monoclonal antibody 160 μg / mice, every 5 days + turmeric dione 100 mg / kg). All injections were administered by body weight. Turmeric dione or control solvent was administered according to the above grouping scheme. Serum samples and kidney tissue were collected 12 days later. The serum creatinine, blood urea nitrogen, AST, and ALT levels in the animal models were measured according to the instructions of the kits (purchased from Nanjing Jiancheng Biotechnology Research Institute), using the same methods as above.

[0076] 2. Experimental Results

[0077] 2.1 Curcuma zedoaria alleviates renal tubular injury in a cisplatin-plus-PD-1 monoclonal antibody-induced renal injury model.

[0078] HE staining results ( Figure 3 A) The results showed that the renal tubular structure in the control group was normal, with no obvious pathological changes. The aPD1C model group showed significant acute tubular injury, manifested as extensive brush border shedding of epithelial cells, cell necrosis, and cast formation. The degree of renal tubular injury in the turmeric dione treatment group was significantly reduced, specifically manifested as a decrease in necrotic cells and casts, and better maintenance of tubular structural integrity.

[0079] 2.2 Curcuma zedoaria reduced serum creatinine, blood urea nitrogen, AST, and ALT levels in a cisplatin-plus-PD-1 monoclonal antibody-induced kidney injury model.

[0080] In the aPD1C model group, serum creatinine and blood urea nitrogen levels were significantly elevated, and renal function deteriorated. Curcuma zedoaria effectively reduced serum creatinine and blood urea nitrogen levels in the model group. Figure 3 B). The above experimental results further confirm the hepatoprotective and renal protective effects of turmeric dione in cisplatin-induced kidney injury after repeated administration.

[0081] Experimental Example 4: Effects of Curcumone on KIM1 and NGAL Levels in an In vivo Cisplatin-Induced Acute Kidney Injury Model

[0082] 1. Experimental Methods

[0083] Western Blot Method: Kidney tissue was weighed and protein lysis buffer containing protease inhibitors was added at a mass-to-volume ratio of 10 μL / mg. After thorough grinding, the mixture was transferred to EP tubes and lysed on a shaker at 4°C for 30 min, with the entire process performed on ice. The mixture was then ultracentrifuged at 12,000 rpm for 10 min at 4°C. The supernatant was collected, and the protein concentration was determined using BCA reagent. After electrophoresis, wet transfer, and blocking, the separated proteins were incubated with KIM-1 (a kidney injury molecule) and NGAL antibody, respectively. Each experiment was repeated three times.

[0084] Immunohistochemical staining: Dewaxed sections were subjected to antigen retrieval in citrate buffer (pH 6.0) and incubated overnight at 4°C with anti-KIM1 (1:200) and NGAL (1:200). Signal detection was performed using horseradish peroxidase (HRP)-labeled secondary antibody (1:200 dilution) and 3,3'-diaminobenzidine (DAB) staining.

[0085] 2. Experimental Results

[0086] Western blot and quantitative analysis results are as follows: Figure 4 As shown in Figure A (using β-actin as an internal control), the levels of KIM1 and NGAL were significantly increased in the cisplatin-induced model group. After treatment with curcumin, the protein expression levels of the kidney injury molecules KIM-1 and NGAL were significantly inhibited. The immunohistochemical staining results of KIM-1 and NGAL were consistent with the Western Blot results. Figure 4 B) indicates that turmeric dione has a protective effect against cisplatin-induced acute kidney injury.

[0087] Experimental Example 5: Effects of Curcumone on KIM1 and NGAL Levels in an In Vivo Model of Repeated Cisplatin-Induced Kidney Injury

[0088] 1. Experimental Methods

[0089] Western Blot Method: Kidney tissue was weighed and protein lysis buffer containing protease inhibitors was added at a mass-to-volume ratio of 10 μL / mg. After thorough grinding, the mixture was transferred to EP tubes and lysed on a shaker at 4°C for 30 min, with the entire process performed on ice. The mixture was then ultracentrifuged at 12,000 rpm for 10 min at 4°C. The supernatant was collected, and the protein concentration was determined using BCA reagent. After electrophoresis, wet transfer, and blocking, the separated proteins were incubated with KIM-1 (a cell apoptosis-related protein) and NGAL antibody, respectively. Each experiment was repeated three times.

[0090] 1.2 Immunohistochemical staining: Dewaxed sections were subjected to antigen retrieval in citrate buffer (pH 6.0) and incubated overnight at 4°C with anti-KIM1 (1:200) and NGAL (1:200). Signal detection was performed using horseradish peroxidase (HRP)-labeled secondary antibody (1:200 dilution) and 3,3'-diaminobenzidine (DAB) staining.

[0091] 2. Experimental Results

[0092] Western blot and quantitative analysis results are as follows: Figure 5 As shown in Figure A (using β-actin as an internal control), the levels of KIM1 and NGAL were significantly increased in the cisplatin-induced model group after repeated dosing. After treatment with curcumin, the protein expression levels of the kidney-damaging molecules KIM-1 and NGAL were significantly inhibited. Immunohistochemical staining results for KIM-1 and NGAL were consistent with Western blotting results. Figure 5 B) indicates that curcumin has a protective effect against renal injury induced by repeated cisplatin administration.

[0093] Experimental Example 6: Effects of Curcumone on KIM1 and NGAL Levels in an In Vivo Model of Renal Injury Induced by Cisplatin Combined with PD-1 Administration

[0094] 1. Experimental Methods

[0095] Western Blot Method: Kidney tissue was weighed and protein lysis buffer containing protease inhibitors was added at a mass-to-volume ratio of 10 μL / mg. After thorough grinding, the mixture was transferred to EP tubes and lysed on a shaker at 4°C for 30 min, with the entire process performed on ice. The mixture was then ultracentrifuged at 12,000 rpm for 10 min at 4°C. The supernatant was collected, and the protein concentration was determined using BCA reagent. After electrophoresis, wet transfer, and blocking, the separated proteins were incubated with KIM-1 (a cell apoptosis-related protein) and NGAL antibody, respectively. Each experiment was repeated three times.

[0096] Immunohistochemical staining: Dewaxed sections were subjected to antigen retrieval in citrate buffer (pH 6.0) and incubated overnight at 4°C with anti-KIM1 (1:200) and NGAL (1:200). Signal detection was performed using horseradish peroxidase (HRP)-labeled secondary antibody (1:200 dilution) and 3,3'-diaminobenzidine (DAB) staining.

[0097] 2. Experimental Results

[0098] Western blot and quantitative analysis results are as follows: Figure 6As shown in Figure A (using β-actin as an internal control), the levels of KIM1 and NGAL were significantly increased in the cisplatin-induced model group after repeated dosing. After treatment with curcumin, the protein expression levels of the kidney-damaging molecules KIM-1 and NGAL were significantly inhibited. Immunohistochemical staining results for KIM-1 and NGAL were consistent with Western blotting results. Figure 6 B) indicates that turmeric dione has a protective effect against kidney injury induced by cisplatin combined with PD-1 monoclonal antibody administration.

[0099] Experimental Example 7: Inhibitory effect of turmeric dione on inflammation-related proteins in in vivo models of kidney injury induced by single cisplatin administration, multiple cisplatin administration, and cisplatin combined with PD-1 monoclonal antibody.

[0100] 1. Experimental Methods

[0101] Western Blot Method: Kidney tissue was weighed and protein lysis buffer containing protease inhibitors and phosphatase inhibitors was added at a mass-to-volume ratio of 10 μL / mg. After thorough grinding, the mixture was transferred to EP tubes and lysed on a shaker at 4°C for 30 min, with the entire process performed on ice. The mixture was then ultracentrifuged at 12,000 rpm for 10 min at 4°C, and the supernatant was collected. Protein concentration was determined using BCA reagent. After electrophoresis, wet transfer, and blocking, the separated proteins were incubated with antibodies against inflammation-related proteins NLRP3, C-GSMDM, C-Caspase-1, and IL-1, respectively, with each group repeated three times.

[0102] 2. Experimental Results

[0103] Western blot and quantitative analysis results are as follows: Figure 7 As shown, turmeric significantly downregulated the expression levels of inflammation-related proteins in a cisplatin-induced acute kidney injury model. In a cisplatin-induced chronic kidney injury model (…),… Figure 8 The same validation was also obtained in kidney injury models induced by cisplatin combined with PD-1 monoclonal antibodies. Figure 9 Curcuma dione can inhibit the expression levels of inflammation-related proteins NLRP3, C-GSMDM, C-Caspase-1, and IL-1.

[0104] Experimental Example 8: Curcuma zedoaria has a sensitizing effect on cisplatin monotherapy (Cisp3) for antitumor treatment.

[0105] 1. Experimental Methods

[0106] 1.1 Tumor-bearing mouse method

[0107] (1) Skin preparation of mice: Male C57BL / 6 mice aged 6-8 weeks were acclimatized in an SPF-grade animal room for 7 days. The hair on the right side of the back of the mice was removed with a shaver to fully expose the skin for subcutaneous inoculation.

[0108] (2) Preparation of cell suspension: Take B16F10 cells in the logarithmic growth phase (70%-80% confluence), digest them with trypsin to prepare a single-cell suspension, centrifuge at 1200 rpm for 5 min, discard the supernatant, add 10 mL of PBS buffer to resuspend the cells, wash, centrifuge at 1200 rpm for 5 min, discard the supernatant, repeat the washing 1-2 times, add 1 mL of PBS buffer to resuspend the cells, and count the cells using a microscope; add PBS buffer according to the counting results to adjust the cell concentration to 5×10⁻⁶. 6 / mL, keep on ice for later use.

[0109] (3) Subcutaneous tumor bearing: All procedures were performed in the animal room's clean bench, and all instruments used in the experiment were sterilized at high temperatures. The tumor-bearing sites in mice were disinfected with 75% ethanol. The cell suspension was thoroughly pipetted and mixed. Using a 1 mL syringe, the cell suspension was drawn up and inserted subcutaneously into the mouse with the needle bevel facing upwards. 100 μL of the cell suspension was slowly injected, resulting in each mouse being inoculated with 5 × 10⁶ cells. 5 Identify the cells, then rotate the needle to withdraw it, and press the injection site with a sterile cotton ball for 1-2 minutes.

[0110] (4) Observation and recording: Closely monitor tumor growth after injection. Measure tumor volume every 2 days using calipers, record the longest diameter (A) and shortest diameter (B) of the tumor, and calculate the tumor volume using the formula V = longest diameter × shortest diameter 2 × 0.5. Record the mouse's weight.

[0111] 1.2 Animals and Grouping

[0112] When the tumor volume reaches 80-100 mm 3 Mice were randomly divided into four groups (n=6-8 per group): control group (saline), model group (cisplatin 20 mg / kg every 5 days), low-dose treatment group (cisplatin 20 mg / kg every 5 days + turmeric dione 50 mg / kg once daily), and high-dose treatment group (cisplatin 20 mg / kg every 5 days + turmeric dione 100 mg / kg once daily). All injections were administered by volume calculated based on body weight. Turmeric dione or control solvent was administered according to the above grouping protocol. Tumor size and mouse weight were observed and recorded daily.

[0113] 1.3 Sample Collection

[0114] The average tumor volume in the control group mice reached 2000 mm.3 After blood collection, all mice were euthanized, and tumor tissue was collected. Some of the tumor tissue was immediately fixed in 4% paraformaldehyde for pathological analysis; the rest was cryopreserved at -80°C for subsequent molecular biological assays.

[0115] 1.4 Histological and Pathological Analysis

[0116] After fixation, the tumor tissue was dehydrated, embedded in paraffin, sectioned, and stained with hematoxylin and eosin (HE) to assess the area of ​​tumor necrosis.

[0117] 1.5 Immunohistochemical staining:

[0118] Dewaxed sections were subjected to antigen retrieval using citrate buffer (pH=6.0) and incubated overnight at 4°C with anti-Ki67 (1:200). Signal detection was performed using horseradish peroxidase (HRP)-labeled secondary antibody (1:200 dilution) and 3,3'-diaminobenzidine (DAB) colorimetric method.

[0119] 2. Experimental Results

[0120] This study monitored tumor growth kinetics in each group of tumor-bearing mice. Although Cisco3 alone could inhibit tumor progression to some extent, the anti-tumor effect was significantly amplified after combined intervention with turmeric. Dynamic monitoring showed that the tumor volume growth rate in the combined turmeric group was inhibited, and the growth curve slowed significantly; at the experimental endpoint, the final tumor weight in the combined group was significantly reduced compared to the single-drug group, and the overall tumor burden was effectively controlled. Figure 10 A, B). Kaplan-Meier survival curve analysis further confirmed that the combined application of turmeric dione brought significant survival benefits to tumor-bearing mice, effectively prolonging their median survival (A, B). Figure 10 C). H&E staining results showed that the proportion of necrotic area in tumor tissue significantly increased after combined treatment with turmeric dione. IHC staining analysis of the proliferation marker Ki67 showed that the proportion and abundance of positive cells decreased significantly after combined treatment with turmeric dione, suggesting that tumor cell proliferation was inhibited. Figure 10 D).

[0121] Experimental Example 9: Curcuma dione has a sensitizing effect on antitumor therapy with cisplatin combined with PD-1 monoclonal antibody (aPD1C).

[0122] 1. Experimental Methods

[0123] Subcutaneous tumors were implanted in 6-8 week old male C57BL / 6 mice, and the tumor volume reached 80-100 mm. 3The mice were randomly divided into four groups (n=6-8 per group): control group (saline), model group 1 (cisplatin 20 mg / kg every 5 days), model group 2 (cisplatin 20 mg / kg every 5 days + PD-1 monoclonal antibody 160 μg / mice every 5 days), and treatment group (cisplatin 20 mg / kg every 5 days + PD-1 monoclonal antibody 160 μg / mice every 5 days + turmeric dione 50 mg / kg once daily). All injections were administered by body weight. Turmeric dione or control solvent was administered according to the above grouping scheme. The size of the tumor and the weight of the mice were observed and recorded daily.

[0124] 2. Experimental Results

[0125] This study monitored tumor growth kinetics in each group of tumor-bearing mice. The results showed that although aPD1C alone could inhibit malignant tumor progression to some extent, the anti-tumor effect was significantly amplified after combined intervention with turmeric. Dynamic monitoring showed that the tumor volume growth rate in the combined turmeric group was inhibited, and the growth curve slowed significantly; at the experimental endpoint, the final tumor weight in the combined group was significantly reduced compared to the single-drug group, and the overall tumor burden was effectively controlled. Figure 11 A, B). Furthermore, Kaplan-Meier survival curve analysis further confirmed that the combined application of turmeric dione brought significant survival benefits to tumor-bearing mice, effectively prolonging their median survival (A, B). Figure 11 C). H&E staining results showed that the proportion of necrotic area in tumor tissue significantly increased after combined treatment with turmeric dione. IHC staining analysis of the proliferation marker Ki67 showed that the proportion and abundance of positive cells decreased significantly after combined treatment with turmeric dione, suggesting that tumor cell proliferation was inhibited. Figure 11 D).

[0126] Experimental Example 10: Effects of Curcumadione on the levels of drug resistance-related proteins NLRP3 and PD-L1 in tumor treatment

[0127] 1. Experimental Methods

[0128] Immunohistochemical staining: Dewaxed sections were subjected to antigen retrieval with citrate buffer (pH 6.0) and incubated overnight at 4°C with anti-NLRP3 (1:200) and PD-L1 (1:200). Signal detection was performed using horseradish peroxidase (HRP)-labeled secondary antibody (1:200 dilution) and 3,3'-diaminobenzidine (DAB) staining.

[0129] 2. Experimental Results

[0130] In the cisplatin multiple-dose group, the expression levels of NLRP3 and PD-L1 in tumor tissue were significantly increased; after intervention with turmeric dione, the expression of both was significantly reduced. Figure 12 A), similar changes were also observed in the cisplatin plus PD-1 monoclonal antibody treatment group ( Figure 12 B). The above results indicate that curcumone can inhibit the upregulation of NLRP3 inflammatory signaling and PD-L1 expression induced by chemotherapy and chemotherapy combined with immunotherapy, suggesting that it may reduce the adaptive tolerance and immune escape of tumor cells, thereby enhancing the anti-tumor effects of cisplatin monotherapy and cisplatin combined with PD-1 monoclonal antibody.

[0131] The above are merely preferred embodiments of the present invention and are not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. The application of curcuminone in the preparation of drugs for treating kidney injury and sensitizing tumor chemotherapy and immunotherapy, wherein the molecular formula of curcuminone is C 15 H 24 O2, chemical structural formula is .

2. The application according to claim 1, characterized in that, The kidney injury referred to is acute kidney injury or chronic kidney injury resulting from acute kidney injury.

3. The application according to claim 2, characterized in that, The acute kidney injury mentioned refers to acute kidney injury associated with chemotherapy combined with immunotherapy.

4. The application according to claim 1, characterized in that, The kidney injury refers to acute kidney injury caused by cisplatin, chronic kidney injury caused by repeated administration of cisplatin, or combined kidney injury induced by cisplatin combined with PD-1 monoclonal antibody.

5. The application according to any one of claims 1 to 4, characterized in that, The turmeric dione is achieved by downregulating one or more of the following kidney injury-related indicators: serum creatinine, blood urea nitrogen, alanine aminotransferase, and aspartate aminotransferase.

6. The application according to any one of claims 1 to 4, characterized in that, The turmeric dione is achieved by downregulating one or more of the following: the levels of kidney injury markers KIM-1 and NGAL, the levels of inflammation-related proteins NLRP3, C-GSDMD, C-Caspase-1, IL-1, the tumor proliferation marker Ki67, and the levels of drug resistance-related proteins NLRP3 and PD-L1.

7. The application according to claim 1, characterized in that, The drug uses an effective dose of turmeric dione as its active ingredient, wherein the concentration of turmeric dione is 0.01-200 mg / kg; The dosage form of the drug is at least one of the following: injection, tablet, pill, capsule, or nanoformulation.

8. The application according to claim 7, characterized in that, The concentration of the turmeric dione is 50 mg / kg.

9. A pharmaceutical composition for treating kidney injury, characterized in that, It contains an effective dose of turmeric dione, wherein the molecular formula of turmeric dione is C64-C ... 15 H 24 O2, chemical structural formula is The concentration of turmeric dione was 0.01-200 mg / kg; the kidney injury described was acute kidney injury associated with chemotherapy combined with immunotherapy.

10. A sensitizer for tumor chemotherapy and immunotherapy, characterized in that, It contains an effective dose of turmeric dione, wherein the molecular formula of turmeric dione is C64-C ... 15 H 24 O2, chemical structural formula is The concentration of turmeric dione is 0.01-200 mg / kg; the tumor chemotherapy and immunotherapy is cisplatin combined with PD-1 monoclonal antibody therapy for tumors.

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