Application of lupeol in preparation of medicine for preventing or treating renal injury caused by cis-platinum

Abstract

The invention belongs to the technical field of biology, and particularly relates to application of lupeol to preparation of a medicine for preventing or treating renal injury caused by cis-platinum. The invention discovers that the natural compound lupeol has a potential protection effect on the renal injury caused by the cis-platinum for the first time, and in-vivo and in-vitro models prove that the lupeol remarkably relieves renal dysfunction, tissue pathological injury and rising of renal injury markers caused by the cis-platinum, inhibits cell apoptosis and promotes proliferation of renal tubular cells. A transcriptome sequencing result shows that lupeol enhances cell survival and proliferation by activating a PI3K / Akt signal channel, regulates an Nrf2 channel to resist oxidative stress, inhibits nuclear translocation of NF [kappa] B, reduces pro-inflammatory cytokines and inhibits inflammatory response. Results show that lupeol may become a choice for relieving renal toxicity of cis-platinum.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to the application of lupeol in the preparation of drugs for the prevention or treatment of cisplatin-induced kidney damage. Background Technology

[0002] Acute kidney injury (AKI) is a clinical syndrome characterized by a rapid decline in renal function, manifested as a sudden decrease in glomerular filtration rate (GFR), accumulation of nitrogenous products, and reduced urine output. AKI can be caused by a variety of pathological conditions, including severe infection and hypovolemia (40%-60%), urinary tract obstruction (15%-25%), or nephrotoxic damage caused by nonsteroidal anti-inflammatory drugs (NSAIDs), nephrotoxic antibiotics, or chemotherapy drugs (20%-40%). Globally, the incidence of AKI is showing a significant upward trend, and the mortality rate remains high, with approximately 13,300,000 new cases and about 170,000 deaths annually (among hospitalized patients, the incidence rate in adults is approximately 21.6%, and the mortality rate is approximately 23.9%, while the incidence rate in children is as high as 33.7%, and the mortality rate is approximately 13.8%). Up to 50% of patients in the intensive care unit (ICU) develop acute kidney injury (AKI), and up to 8% of these cases progress to chronic kidney disease (CKD), resulting in excessive consumption of medical resources and a huge socioeconomic burden.

[0003] Cisplatin, as a first-line platinum-based anti-tumor drug, is widely used to treat solid tumors such as bladder cancer, lung cancer, ovarian cancer, and head and neck tumors. In the body, cisplatin is primarily metabolized in the liver and kidneys, with over 90% excreted through the kidneys. Therefore, nephrotoxicity is one of the most common adverse reactions of cisplatin. Currently, approximately 20%–30% of cancer patients receiving cisplatin treatment worldwide experience acute kidney injury annually, and about 500,000 patients are forced to adjust their treatment regimens due to kidney injury. Of these, approximately 17% progress to chronic kidney disease, significantly increasing the risk of death. According to recent studies on the mechanisms of cisplatin nephrotoxicity, cisplatin primarily mediates acute kidney injury through local accumulation, disruption of DNA damage and repair, oxidative stress, activation of programmed cell death, activation of the inflammatory cascade, endoplasmic reticulum stress, and mitochondrial damage. Summary of the Invention

[0004] In view of this, the present invention provides the use of lupeol in the preparation of medicaments for the prevention or treatment of cisplatin-induced kidney damage, in order to solve the above-mentioned problems.

[0005] To achieve the above solution, the technical solution of the present invention is as follows: This invention provides the use of lupeol in the preparation of medicaments for the prevention or treatment of kidney damage.

[0006] Optionally, the kidney injury is cisplatin-induced kidney injury.

[0007] Optionally, the lupeol can be administered via intraperitoneal injection, subcutaneous injection, or oral administration.

[0008] Optionally, the lupeol can be administered orally.

[0009] Optionally, the drug may also include a pharmaceutically acceptable carrier.

[0010] Optionally, the pharmaceutically acceptable carrier includes at least one of a diluent, binder, surfactant, lubricant, filler, and disintegrant.

[0011] Optionally, the dosage form of the drug includes granules, powders, tablets, capsules, syrups, suppositories, injections, and elixirs.

[0012] The present invention also provides a pharmaceutical composition comprising cisplatin and lupeol.

[0013] The present invention also provides a pharmaceutical product comprising the pharmaceutical composition described above.

[0014] Optionally, the medicine may also include a pharmaceutically acceptable carrier.

[0015] This invention offers the following beneficial effects: For the first time, this invention discovers that the natural compound lupeol has a potential protective effect against cisplatin-induced kidney injury. Through in vivo and in vitro models, it was confirmed that lupeol significantly alleviated cisplatin-induced renal dysfunction, histopathological damage, and elevated levels of kidney injury markers, inhibited apoptosis, and promoted the proliferation of renal tubular cells. Transcriptome sequencing results showed that lupeol enhanced cell survival and proliferation by activating the PI3K / Akt signaling pathway, while simultaneously regulating the Nrf2 pathway to combat oxidative stress, inhibiting NFκB nuclear translocation, and reducing pro-inflammatory cytokines, thereby suppressing the inflammatory response. These results suggest that lupeol may be a potential option for alleviating cisplatin-induced nephrotoxicity. Attached Figure Description

[0016] Figure 1The results of the assay for lupeol in alleviating cisplatin-induced acute kidney injury in mice are shown in the figures. A represents the mouse body weight change curve; B represents the renal function evaluation results of each group; C represents the H&E and PAS staining images of mouse kidneys; D represents the serum Kim-1 and NGAL levels detected by ELISA, and the relative mRNA expression levels of the kidney injury markers Kim-1 and NGAL in mouse kidneys; E represents the representative immunofluorescence staining image of Kim-1 in kidney tissue; and F represents the representative immunohistochemical staining image of NGAL in kidney tissue. Data are expressed as mean ± standard error (mean ± SEM) (n = 6); *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 compared to the cisplatin group; Weight represents body weight, Time represents time, and Day represents days. Figure 2 The results of experiments related to lupeol's ability to mitigate cisplatin-induced cytotoxicity in HK-2 cells are presented. A represents the viability of HK-2 cells after treatment with cisplatin and increasing concentrations of lupeol, as detected by the CCK-8 assay. B represents the dose-response curve and EC50 value of lupeol in cisplatin-treated HK-2 cells. C represents the relative expression levels of Kim-1 and NGAL in HK-2 cells, detected by RT-qPCR. D represents transmission electron microscopy (TEM) images of HK-2 cells. Data are expressed as mean ± standard error (mean ± SEM) (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 compared to the cisplatin group. Cell Viability represents cell viability, Concentration represents concentration, Relative represents relative value, and Level represents level. Figure 3The results of experiments demonstrating the inhibition of apoptosis and promotion of proliferation of HK-2 cells by lupeol are as follows: A represents the apoptosis status of HK-2 cells after Annexin V-FITC / PI staining by flow cytometry; B represents the expression levels of apoptosis-related proteins Bax, Bcl-2, and cleaved Caspase-3 obtained by Western blotting; C represents the proliferation capacity of HK-2 cells under different treatment conditions as determined by EdU cell proliferation assay; and D represents the relative mRNA expression level of Ki67 obtained by RT-qPCR. Data are expressed as mean ± standard error (mean ± SEM) (n = 3); **P < 0.01; ****P < 0.0001 compared with the cisplatin group; Apoptitic Cells represent apoptotic cells. Figure 4 The figure shows the effect of lupeol treatment following cisplatin exposure on the transcriptome sequencing of HK-2 cells. A is a bar chart of differentially expressed genes, B is a volcano plot, C is a hierarchical clustering heatmap, D is the GO functional enrichment analysis result, E is the DO (disease ontology) enrichment analysis result, and F is the KEGG signaling pathway enrichment analysis result. The definition criteria for differentially expressed genes are: fold change > 1.5 and adjusted P < 0.05. Number represents the number of genes, Differential statistics represents the differential statistics, and volcano represents the volcano pattern. Figure 5 Figure 1 shows the results of experiments related to lupeol activation of the PI3K / Akt signaling pathway. A represents the immunofluorescence staining of PI3K expression, B represents the immunofluorescence staining of phosphorylated Akt (p-Akt) expression, and C represents the protein expression levels of PI3K, Akt, and p-Akt detected by Western blotting. Data are expressed as mean ± standard error (mean ± SEM) (n = 3); **P < 0.01, ****P < 0.0001 compared to the cisplatin group. Figure 6Figure 1 shows the results of an assay related to lupeol's reduction of cisplatin-induced oxidative stress in HK-2 cells. Figure A shows the immunofluorescence analysis results of Nrf2 expression and nuclear translocation; Figure B shows the protein expression levels of Nrf2 and its downstream antioxidant enzymes HO-1 and NQO1, analyzed by Western blotting; Figure C shows the intracellular reactive oxygen species (ROS) levels detected by DCFH-DA fluorescence assay; Figure D shows the ROS fluorescence intensity statistics; Figure E shows the levels of malondialdehyde (MDA) and glutathione (GSH) in HK-2 cells. Data are expressed as mean ± standard error (mean ± SEM) (n = 3); *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 compared to the cisplatin group; Relative fluorescent intensity indicates relative fluorescence intensity. Figure 7 Figure 1 shows the results of experiments related to the inhibition of inflammatory response by lupeol. Figure A shows the immunofluorescence analysis results of NFκB expression and nuclear translocation; Figure B shows the levels of inflammatory cytokines in the serum of cisplatin-induced AKI mice detected by ELISA; Figure C shows the relative mRNA expression levels of inflammatory cytokines in HK-2 cells detected by RT-qPCR. Data are expressed as mean ± standard error (mean ± SEM) (n = 3); ***P < 0.001, ****P < 0.0001 compared with the cisplatin group. Detailed Implementation

[0017] The present invention will be further illustrated by specific examples below. However, it should be noted that the specific material ratios, process conditions and results described in the embodiments of the present invention are only for illustrating the present invention and cannot be used to limit the scope of protection of the present invention. All equivalent changes or modifications made in accordance with the spirit and essence of the present invention should be covered within the scope of protection of the present invention.

[0018] The present invention will be described in detail below through specific examples and embodiments. It should also be understood that the following embodiments are only for specific illustration of the present invention and should not be construed as limiting the scope of protection of the present invention. Any non-essential improvements and adjustments made by those skilled in the art based on the above description of the present invention are within the scope of protection of the present invention. The specific process parameters, etc., in the following examples are merely examples within a suitable range; that is, those skilled in the art can make appropriate selections within the appropriate range based on the description herein, and are not intended to be limited to the specific values ​​in the examples below.

[0019] 1. Reagents and Materials Lupeol (HY-N0790), Cisplatin (HY-17394), and RIPA lysis buffer were all purchased from MedChemExpress (New Jersey, USA).

[0020] Reduced protein loading buffer (5X) was purchased from Suzhou New Saimai Biotechnology Co., Ltd. (Suzhou, China).

[0021] Primary antibodies used for immunofluorescence (IF), immunohistochemistry (IHC), and Western blot included: GAPDH (390035, Zhengneng Biotech, Chengdu, China), Cleaved-Caspase 3 (341034, Zhengneng Biotech, Chengdu, China), Caspase 3 (R23315, Zhengneng Biotech, Chengdu, China), Bax (R22708, Zhengneng Biotech, Chengdu, China), Nrf2 (R380773, Zhengneng Biotech, Chengdu, China), PI3K (R22768, Zhengneng Biotech, Chengdu, China), Bcl2 (15071; Cell Signaling Technology, Massachusetts, USA), phospho-Akt (4060; Cell Signaling Technology, Massachusetts, USA), Akt (4691; Cell Signaling Technology, Massachusetts, USA), Kim-1 (A24493, Abbkine, Wuhan, China), NGAL (A2092, Abbkine, Wuhan, China), HO-1 (10701-1-AP, Proteintech, Wuhan, China), NQO1 (T56710, Abbimat, Shanghai, China).

[0022] 2. Animal experiments Male specific pathogen-free (SPF) C57BL / 6J mice (6 weeks old, weighing 16 - 18 g) were purchased from the Experimental Animal Center of Chongqing Medical University (License No.: SCXK [Yu] 2022 - 0010). The mice were housed in a 12 h light / 12 h dark cycle environment with sufficient food and water available for free intake. All animal experiments were strictly conducted in accordance with the procedures approved by the Animal Ethics Committee of Children's Hospital of Chongqing Medical University (CHCMU-IACUC20230309006).

[0023] After one week of adaptive feeding, 30 mice were randomly divided into the following five groups: CIS+H-Lup (abbreviated as H-Lup, high-dose lupeol group, 40mg / kg, n=6): Lupeol was administered orally once daily via gavage at a dose of 40mg / kg; CIS+M-Lup (abbreviated as M-Lup, medium-dose lupeol group, 20 mg / kg, n=6): Lupeol was administered orally once daily by gavage at a dose of 20 mg / kg; CIS+L-Lup (abbreviated as L-Lup, low-dose lupeol group, 10 mg / kg, n=6): Lupeol was administered orally once daily by gavage at a dose of 10 mg / kg; CIS (cisplatin group, 20 mg / kg, n=6): PBS buffer was used instead of lupeol, at a dose of 180 μL; CON (control group, n=6): PBS buffer was used instead of lupeol, at a dose of 180 μL; The above-mentioned drugs were administered by gavage for 7 consecutive days. On the 4th day, except for the CON group, all other groups received a single intraperitoneal injection of cisplatin (dose of 20 mg / kg). The experiment was completed 72 hours after the cisplatin injection. During this process, the body weight of mice in each group was monitored, and the average value was used as the test result for the corresponding group. The results are as follows: Figure 1 As shown in A; 72 hours after injection, the mice were sacrificed, and their hearts, livers, spleens, lungs, kidneys and serum were collected. The renal function biochemical indicators in the serum (specifically serum creatinine (SCr) content and blood urea nitrogen (BUN) content) were tested. The renal function biochemical indicators were measured by the Department of Clinical Laboratory of Children's Hospital Affiliated to Chongqing Medical University. The results are shown in 1B. Left kidneys from mice in each of the above groups were collected and fixed in 4% paraformaldehyde solution for 24 hours. After programmed dehydration, the specimens were embedded in paraffin and cut into 4 µm thick sections. After dewaxing and rehydration, the sections were stained with H&E and PAS according to the manufacturer's instructions. Subsequently, they were dehydrated using gradient ethanol solutions, mounted, and observed under an optical microscope. The results are as follows: Figure 1 As shown in C; Serum was collected from each group of mice, and the levels of kidney injury markers Kim-1 and NGAL were detected. The procedure was strictly performed according to the ELISA kit instructions (Shanghai Senxiong Biotechnology). The absorbance at 450 nm was measured using a microplate reader. Kim-1 concentration (pg / ml) was calculated using the formula: Kim-1 concentration (pg / ml) = 269.13 × OD value − 15.977. NGAL concentration (ng / ml) was calculated using the formula: 17.2524 × OD value − 0.00979. The results are as follows: Figure 1As shown in D; the relative mRNA expression levels of the kidney injury markers Kim-1 and NGAL in mouse kidneys were detected. The specific steps were as follows: Total RNA was extracted from kidney tissue and reverse transcribed into cDNA according to the instructions of the qPCR reverse transcription kit (MCE, New Jersey, USA); the cDNA was mixed with primers, SYBR GreenqPCR Master Mix (MCE, New Jersey, USA), and enzyme-free water, and amplified using the Bio-Rad CFX Connect™ real-time quantitative PCR system. The primer sequences for the target gene and GAPDH are shown in Table 1. After amplification using the Bio-Rad CFX Connect™ real-time quantitative PCR system, a table of CT values ​​was generated. The relative gene expression level was determined by calculating 2−ΔΔCt using the CT values. The results are shown in Table 1. Figure 1 As shown in D; Representative immunofluorescence staining and NGAL immunohistochemical staining of Kim-1 in kidney tissue were performed. The specific steps were as follows: Paraffin sections of mouse kidneys from each group were dewaxed and rehydrated, then placed in 0.01 mmol / L citrate buffer for antigen retrieval via a boiling water bath; endogenous peroxidase was blocked using 3% hydrogen peroxide, followed by blocking with 0.5% BSA for 1 h at room temperature, then adding primary antibody (0.5% BSA, 1:200 dilution) and incubating overnight at 4°C; washing three times with PBS buffer (10 min each time), then adding the corresponding secondary antibody (PBS buffer, 1:200 dilution) and incubating, followed by washing again with PBS buffer; immunofluorescence staining was performed using a DAPI-containing anti-fluorescence quenching mounting medium, and imaging was performed under a fluorescence microscope. The results are shown below. Figure 1 As shown in E; immunohistochemical staining was performed using the DAB staining method followed by nuclear staining. After dehydration and mounting, the slides were observed under a light microscope. The results are as follows. Figure 1 As shown in F.

[0024] Depend on Figure 1 It was found that after intraperitoneal injection of cisplatin, the body weight of mice in all intervention groups decreased significantly compared with the control group. With the extension of cisplatin injection time, the medium and high dose lupeol groups could significantly reduce the cisplatin-induced decrease in mouse body weight. Figure 1 A). Lupeol can alleviate the increase in serum creatinine (SCr) and blood urea nitrogen (BUN) induced by cisplatin. Figure 1 B). HE staining results showed that after cisplatin intervention, the kidneys exhibited disordered glomerular structure, renal tubular dilation, tubular epithelial cell swelling, nuclear pyknosis and fragmentation, brush border loss, extensive formation of protein casts within the tubules, and interstitial inflammatory cell infiltration; PAS staining results showed that after cisplatin intervention, the brush border of renal tubular epithelial cells disappeared, the basement membrane was ruptured and irregular, renal interstitial edema and inflammatory cell infiltration were observed, and glycogen deposition was visible in the mesangial area. Figure 1(C) Compared to the cisplatin group, the above-mentioned pathological manifestations in the lupeol intervention group were all alleviated. The results of serum and kidney marker detection in mice showed that lupeol could reduce the cisplatin-induced increase in Kim-1 and NGAL (C). Figure 1 (DF). In summary, lupeol can alleviate cisplatin-induced acute kidney injury in mice.

[0025] 3. Cell Culture and Processing Human renal tubular epithelial cell line HK-2 (Chinese Academy of Sciences Cell Bank) was cultured in DMEM / F12 medium (Baidi Biotech, L310KJ, Shanghai, China) containing 10% fetal bovine serum (FBS; BDBIO, F814-500, Guangzhou, China) and 1% penicillin-streptomycin (NCM Biotech, C100C5, Suzhou, China) at 37°C and 5% CO2. The optimal concentrations of lupeol (40 µM, 50 µM, 60 µM) were determined using a CCK-8 assay; when the cells reached approximately 50% confluence, they were divided into the following five groups: CIS+H-Lup (lupeol, 60 µM): Cells were cultured for 24 h in 8 ml of complete medium containing 60 µM lupeol and 20 µM cisplatin. CIS+M-Lup (lupeol, 50 µM): Culture cells in 8 ml of complete medium containing 50 µM lupeol and 20 µM cisplatin for 24 h. CIS+L-Lup (lupeol, 40 µM): Cells were cultured for 24 h in 8 ml of complete medium containing 40 µM lupeol and 20 µM cisplatin. CIS (cisplatin, 20 µM): Cells were cultured in 8 ml of complete medium containing 20 µM cisplatin for 24 h. CON (control group): Cells were cultured in 8 ml of complete culture medium for 24 h; HK-2 cells were collected after 24 hours to test their viability. The specific steps were as follows: Logarithmic growth phase HK-2 cells were evenly seeded into 96-well plates and treated with cisplatin (20 μM, final concentration) and different concentrations of lupeol (0, 1, 5, 10, 15, 30, 45, 60, 75 μM, all final concentrations) for 24 hours. Then, 10% CCK-8 reagent (NCM Biotech, C6005, Suzhou, China) was added, and the plates were incubated at 37℃ and 5% CO2 for 2 hours. The absorbance was measured at 450 nm using a microplate reader. Cell viability (%) was calculated using the formula: Cell viability (%) = Sample OD value / Average OD value at 0 μM × 100. The results are shown below. Figure 2 As shown in A; Will Figure 2 Data A was transformed and calculated using GraphPad Prism v10.2.1 to plot the corresponding curve, resulting in... Figure 2 B, and calculated EC50 using GraphPad Prism v10.2.1, the results are as follows. Figure 2 As shown in B.

[0026] 4. Real-time quantitative reverse transcription-polymerase chain reaction (RT-qPCR) Total RNA was extracted from kidney tissue and HK-2 cells. Following the instructions of the qPCR reverse transcription kit (MCE, New Jersey, USA), the extracted total RNA was reverse transcribed into cDNA. The cDNA was mixed with primers, SYBR Green qPCR Master Mix (MCE, New Jersey, USA), and enzyme-free water, and amplified using the Bio-Rad CFX Connect™ real-time quantitative PCR system. The primer sequences for the target gene and GAPDH are shown in Table 1. The relative gene expression level was determined by calculating 2−ΔΔCt using the Bio-Rad CFX Connect™ real-time quantitative PCR system (a table of CT values ​​will be generated after amplification). The results are expressed as 2−ΔΔCt (normalized to GAPDH). Figure 2 C, as shown in 3D.

[0027]

[0028] 5. Transmission electron microscopy (TEM) HK-2 cells were collected and fixed with 2.5% glutaraldehyde solution; subsequently, they were post-fixed with 1% osmium tetroxide and embedded in epoxy resin; 70 nm ultrathin sections were carefully prepared using an ultramicrotome, stained with uranium acetate and lead citrate, and observed under a transmission electron microscope (HITACHI, HT7700, Tokyo, Japan). The results are as follows. Figure 2 As shown in D.

[0029] Depend on Figure 2 It was found that lupeol significantly improved CIS-induced cell viability at concentrations of 45 μM, 60 μM, and 75 μM. Figure 2 A); The calculated EC50 for lupeol in alleviating cisplatin-induced kidney injury was 48.13 μM. Based on the EC50, a lupeol intervention concentration gradient of 40 μM, 50 μM, and 60 μM was established. Figure 2 B); Consistent with in vivo results, lupeol reduced cisplatin-induced increases in Kim-1 and NGAL ( Figure 2C); Transmission electron microscopy of HK2 cells showed that, compared with the control group, the cisplatin-treated group had swollen mitochondria and lost mitochondrial cristae, while lupeol intervention could alleviate this pathological change ( Figure 2 D).

[0030] 6. Flow cytometry detection of apoptosis HK-2 cells were collected after treatment with cisplatin (20 μM) and lupeol (40, 50, 60 μM) for 24 h. Following the kit instructions, cells were stained using the Annexin V-FITC / PI staining kit (BD Biosciences, USA). Cell apoptosis was then analyzed by flow cytometry (BD FACSCalibur, USA). Results are shown below. Figure 3 As shown in Figure A.

[0031] 7. Western Blot HK-2 cells were homogenized with RIPA buffer and then sonicated for lysis. The cells were centrifuged at 12,000 rpm for 20 min at 4°C, and the supernatant was collected. Protein concentration was determined using the BCA method, and loading buffer was added. The cells were then denatured in a 100°C metal bath. SDS-PAGE gel electrophoresis was performed at 80 V / 120 V. Subsequently, proteins were transferred to the membrane at a constant current of 400 mA in an ice bath and blocked with rapid blocking buffer (specifically, incubation with Neo-Synephrine rapid blocking buffer on a shaker at room temperature for 10 min). Primary antibody (specifically...) was added... Figure 3 B: Cleaved-Caspase 3, Caspase 3, Bax, Bcl2, GAPDH; Figure 5 C: PI3K, p-Akt, Akt, GAPDH; Figure 6 B: Nrf2, HO-1, NQO1, GAPDH) are diluted according to the corresponding antibody instructions, with the amount used to completely cover the band. Incubate overnight at 4°C on a shaker. Wash the membrane three times with TBST (10 min each time), then add the corresponding secondary antibody (specifically, goat anti-mouse / anti-rabbit secondary antibody, diluted 1:10000, enough to completely cover the band). Wash the membrane three more times with TBST, then add ECL substrate (ECL substrate is prepared by mixing ECL chemiluminescence solution A and solution B at a volume ratio of 1:1, with the amount used to completely cover the band). Perform chemiluminescence detection and save the image. The results are as follows: Figure 3 As shown in B, 5C and 6B.

[0032] 8. EdU cell proliferation assay Cell proliferation activity was assessed using an EdU assay kit (Beyotime Biotechnology, C0071S, Shanghai, China). HK-2 cells were seeded onto cell slides in 24-well plates and treated with cisplatin and lupeol for 24 h. Cells were then co-incubated with 10 µM EdU (final concentration) at 37°C and 5% CO2 for 2 h. Subsequently, the cells were fixed and permeabilized, and incubated with Alexa Fluor 488-labeled azide at room temperature in the dark for 30 min. After nuclear staining and mounting, cell proliferation activity was analyzed using a fluorescence microscope. The results are shown below. Figure 3 As shown in C.

[0033] Depend on Figure 3 It can be seen that cisplatin alone significantly induced apoptosis in HK2 cells, while lupeol co-treatment significantly reduced the apoptosis rate, and the degree of reduction was dose-dependent. Figure 3 A). Western blot results showed that cisplatin intervention significantly upregulated the expression levels of apoptosis-related proteins Bax and Cleaved-Caspase 3, and downregulated the expression of the anti-apoptotic protein Bcl2, while lupeol intervention significantly reversed this trend. Figure 3 B). EdU cell proliferation assay results showed that cisplatin treatment significantly inhibited HK2 cell proliferation, while lupin co-treatment significantly restored the proliferative capacity of HK2 cells after cisplatin damage. Figure 3 C). RT-qPCR results showed that the mRNA expression level of the proliferative gene Ki67 increased compensatorily after cisplatin-only intervention, while lupeol co-treatment further increased Ki67 mRNA expression, exhibiting a stronger proliferative effect. Figure 3 D).

[0034] 9. RNA sequencing (RNA-Seq) analysis HK-2 cells were treated with cisplatin (20 µM) and cisplatin (20 µM) combined with lupeol (50 µM) for 24 h, respectively. Total RNA was extracted using TRIzol reagent (Invitrogen) and sent to a biotechnology company for transcriptome sequencing. Gene expression levels were quantified using featureCounts and differential expression analysis was performed using DESeq2 software. The significance threshold was set at fold change > 1.5 and corrected P-value < 0.05. Enriched biological processes and signaling pathways were explored through GO, DO, and KEGG pathway analysis. All data analysis was performed on the Meiji Biotechnology Cloud Platform. The results are as follows: Figure 4 As shown.

[0035] Depend on Figure 4It can be seen that by performing transcriptome sequencing on HK2 cells in the cisplatin group and the lupeol co-treatment group to screen for differentially expressed genes, a total of 1869 differentially expressed genes were screened, of which 1099 were upregulated genes and 770 were downregulated genes. Figure 4 (AC). GO enrichment analysis of these 1869 differentially expressed genes showed that, in terms of biological processes, they were enriched in stress and inflammation responses, cell death regulation, and metabolic disorders. In terms of molecular function, they were mainly associated with oxidoreductase activity, cytokine binding, calcium ion binding, and protein binding. In terms of cellular components, they were enriched in subcellular localizations such as membrane structures, plasma membranes, and intercellular junctions. DO enrichment analysis of these 1869 differentially expressed genes showed significant enrichment in kidney-related diseases, especially inflammatory diseases. KEGG enrichment analysis of these 1869 differentially expressed genes showed that they were mainly enriched in the PI3K-Akt signaling pathway, the FoxO signaling pathway, and the Ras signaling pathway.

[0036] 10. Immunofluorescence and Western Blot Detection To verify whether transcriptome sequencing results regulate cisplatin-induced HK2 cell translation, this invention performed immunofluorescence detection on proteins related to the PI3K / Akt signaling pathway in each group of cells. The specific steps of immunofluorescence detection were as follows: HK-2 cells were seeded on cell spreaders in 24-well plates and treated with cisplatin (20 µM) and cisplatin (20 µM) combined with lupeol (50 µM) for 24 h. The culture medium was discarded, and the cells were gently washed twice with pre-warmed PBS to remove residual serum. Subsequently, 4% paraformaldehyde was added for fixation at room temperature for 20 min. After fixation, the cells were washed three times with PBS for 10 min each time. The cells were then punched with 0.3% Triton X-100 for 5 min, followed by washing three times with PBS for 10 min each time. Blocking with 0.5% BSA at room temperature for 60 min was performed. The blocking solution was then discarded, and primary antibodies (PI3K and p-Akt, diluted to an appropriate concentration with 0.5% BSA) were directly added dropwise. Dilute 1:200 (enough to completely cover the cell culture slide), incubate overnight at 4°C in a humidified chamber, and rinse three times with PBS for 10 minutes each time after incubation. Add fluorescently labeled secondary antibody (goat anti-rabbit secondary antibody, enough to completely cover the cell culture slide), incubate at room temperature in the dark for 1 hour, and then rinse three times with PBS for 10 minutes each time. Remove the cell culture slide from the culture dish, mount it on a glass slide with a DAPI-containing anti-fluorescence quenching mounting medium, and observe and acquire images using a fluorescence microscope at the corresponding excitation wavelength. The results are as follows: Figure 5 As shown in A and 5B; Western blotting was then performed, following the steps outlined above, and the results are as follows: Figure 5 As shown in C.

[0037] Depend on Figure 5 Immunofluorescence assay results showed that, compared with the control group, the expression of PI3K and p-Akt in HK2 cells was significantly reduced after cisplatin-only intervention, while the addition of lupeol co-treatment improved the reduced levels of PI3K and p-Akt. Figure 5 (A, B) Western blot results also corroborated this conclusion. Western blot results further showed that with increasing lupeol intervention dose, the activation level of the PI3K / Akt pathway also increased. Figure 5 C).

[0038] 11. Detection of protein expression of Nrf2 pathway downstream antioxidant enzymes HO-1 and NQO1 by cellular immunofluorescence, Western blotting, and other methods. Immunofluorescence assays were performed to test the Nrf2 protein expression levels in HK2 cells from each group. The specific steps were as follows: HK-2 cells were seeded onto cell spreaders in 24-well plates and treated with cisplatin (20 µM) and cisplatin (20 µM) combined with lupeol (50 µM) for 24 h. Discard the culture medium and gently wash the cells twice with pre-warmed PBS to remove residual serum. Then, fix with 4% paraformaldehyde at room temperature for 20 min. After fixation, wash three times with PBS for 10 min each time. Puncture cells with 0.3% Triton X-100 for 5 min, then wash three times with PBS for 10 min each time. Block with 0.5% BSA at room temperature for 60 min. Discard the blocking solution and directly add primary antibody (Nrf2, diluted 1:200 with 0.5% BSA, just enough to completely cover the slide). Incubate overnight at 4°C in a humidified chamber. After incubation, wash three times with PBS for 10 min each time. Add secondary antibody labeled with fluorescent dye (goat anti-rabbit secondary antibody, just enough to completely cover the slide), incubate at room temperature in the dark for 1 h, then wash three times with PBS for 10 min each time. Remove the cell slides from the culture dish and mount them on a glass slide with a DAPI-containing anti-fluorescence quenching mounting medium. Observe and acquire images using a fluorescence microscope at the corresponding excitation wavelength. The results are as follows: Figure 6 As shown in Figure A.

[0039] Western blotting was performed to test the expression levels of Nrf2 protein and the downstream antioxidant enzymes HO-1 and NQO1 in HK2 cells of each group. The specific steps were as described above, and the results are as follows. Figure 6 As shown in B.

[0040] 12. ROS detection Intracellular reactive oxygen species (ROS) levels were assessed using a ROS detection kit (Solarbio, CA1410, Beijing, China). The specific steps were as follows: HK-2 cells that had undergone different interventions were incubated with 10 µM DCFH-DA (final concentration) at 37°C for 30 min; fluorescence intensity was observed under a fluorescence microscope, and the results are as follows: Figure 6 As shown in Figure D, the fluorescence intensity was measured using a fluorescence microplate reader (excitation wavelength: 485 nm; emission wavelength: 535 nm), and statistical analysis was performed based on the fluorescence intensity measured by the microplate reader, resulting in the following graph: Figure 6 As shown in C.

[0041] 13. Determination of MDA and GSH According to the kit instructions, the levels of malondialdehyde (MDA) and glutathione (GSH) in HK-2 cells were measured using the MDA and GSH detection kits (Solarbio, BC0020 / BC1175, Beijing, China). The absorbance at 532 nm, 600 nm (for MDA), and 412 nm (for GSH) was measured using a microplate reader. The MDA content was calculated using the formula (nmol / 10⁻⁶). 4 The malondialdehyde (MDA) content is calculated as follows: (cell) = 32.258 × [(A532 assay − A532 blank) − (A600 assay − A600 blank)] / total cell count (in tens of thousands). The sample concentration x is calculated using the formula derived from the standard curve of each experiment. Then, the GSH content (ug / 10) is calculated using the formula... 6 cell) = x / number of cells (in 10) 6 The content of glutathione (GSH) was calculated using a calculator, and the results are as follows: Figure 6 As shown in E.

[0042] Depend on Figure 6 The results of the immunofluorescence assay showed that, compared with the control group, the expression of Nrf2 protein in HK2 cells treated with cisplatin alone was reduced, while the lupeol co-treatment group significantly improved the reduction of Nrf2 protein expression. Simultaneously, Nrf2 protein translocation from the cytoplasm to the nucleus was observed. Combined with the Nrf2 protein expression results in Western blotting, this indicates that lupeol activates the Nrf2 pathway. Figure 6 A, B). Protein expression analysis of the downstream antioxidant enzymes HO-1 and NQO1 in the Nrf2 pathway showed that the lupin alcohol co-treatment group significantly upregulated the levels of these proteins. Figure 6B). The results of ROS, MDA, and GSH level detection in each group of cells showed that cisplatin treatment significantly increased ROS fluorescence intensity, increased MDA in the cell supernatant, and decreased GSH. Lupeol could improve the cisplatin-induced increase in ROS and MDA and decrease in GSH. Figure 6 CE).

[0043] 14. Detection of cellular inflammatory factor expression The nuclear translocation of NFκB pathway was verified by immunofluorescence assay. The specific steps were as follows: HK-2 cells were seeded onto cell spreaders in 24-well plates and treated with cisplatin (20 µM) and cisplatin (20 µM) combined with lupeol (50 µM) for 24 h. The culture medium was discarded, and the cells were gently washed twice with pre-warmed PBS to remove residual serum. Subsequently, 4% paraformaldehyde was added for fixation at room temperature for 20 min. After fixation, the cells were washed three times with PBS for 10 min each time. The cells were then punched with 0.3% Triton X-100 for 5 min, followed by three more washes with PBS for 10 min each time. Blocking with 0.5% BSA at room temperature for 60 min was then performed. The blocking solution was then discarded, and primary antibody (Nrf2, diluted to an appropriate concentration with 0.5% BSA) was directly added dropwise. Dilute 1:200 (enough to completely cover the cell culture slide), incubate overnight at 4°C in a humidified chamber, and rinse three times with PBS for 10 minutes each time after incubation. Add fluorescently labeled secondary antibody (goat anti-rabbit secondary antibody, enough to completely cover the cell culture slide), incubate at room temperature in the dark for 1 hour, and then rinse three times with PBS for 10 minutes each time. Remove the cell culture slide from the culture dish, mount it on a glass slide with a DAPI-containing anti-fluorescence quenching mounting medium, and observe and acquire images using a fluorescence microscope at the corresponding excitation wavelength. The results are as follows: Figure 7 As shown in A; The levels of inflammatory factors in the serum of mice with cisplatin-induced acute kidney injury were detected using ELISA. The specific steps were as follows: serum was collected from each group of mice, and the levels of inflammatory factors were detected. The operation was strictly performed according to the ELISA kit instructions (Shanghai Senxiong Biotechnology). The absorbance value at 450 nm was measured using a microplate reader. The concentration of IL-1α was calculated using the formula: IL-1α concentration (pg / ml) = 89.676 × OD value − 7.0518; the concentration of IL-6 was calculated using the formula: IL-6 concentration (pg / ml) = 75.564 × OD value − 4.8061; and the concentration of TNF-α was calculated using the formula: TNF-α concentration (pg / ml) = 353.92 × OD value − 1.3913. The results are as follows: Figure 7 As shown in B; The mRNA expression of relevant inflammatory cytokine genes in cisplatin-induced HK2 cells was detected by RT-qPCR, following the steps described above. The results are as follows: Figure 7 As shown in C.

[0044] Depend on Figure 7 As can be seen from the results of the cellular immunofluorescence assay, compared with the control group, the cisplatin-only intervention group showed significant NFκB nuclear translocation, while lupeol intervention could inhibit this nuclear translocation phenomenon. Figure 7 A), ELISA results showed that the serum inflammatory factor levels in mice with cisplatin-induced acute kidney injury ( Figure 7 B), RT-qPCR results showed that there was a significant inflammatory response in cisplatin-mediated acute kidney injury, and lupeol intervention could significantly improve this inflammatory response.

[0045] In summary, this invention, through in vitro and in vivo experiments, found that lupeol, by regulating the PI3K / Akt / Nrf2 signaling axis, enhances the expression of the downstream anti-apoptotic protein Bcl2, significantly reduces the expression of apoptosis-related proteins Bax and Cleaved-Caspase 3. EdU staining and elevated Ki67 mRNA levels indicate that lupeol not only inhibits apoptosis but also actively drives damaged HK2 cells into the cell cycle for regeneration and repair. On the other hand, it initiates antioxidant responses, promotes the expression of antioxidant enzymes, inhibits inflammatory pathways, and suppresses the release of pro-inflammatory factors, thereby improving cisplatin-induced kidney damage.

[0046] After entering the bloodstream, over 90% of cisplatin irreversibly binds to plasma proteins and is distributed throughout the body, particularly in the liver and kidneys. After being freely filtered by the glomeruli, it is concentrated in the renal tubules and actively taken up by organic cation transporters 2 on the apical membrane of renal tubular epithelial cells, slowly releasing active free platinum. The intracellular platinum concentration is much higher than in plasma, resulting in a long-term and sustained cytotoxic effect. Once inside the renal tubular epithelial cells, cisplatin accumulates in the mitochondria, directly damaging mitochondrial DNA and proteins, inhibiting the activity of respiratory chain complexes I-IV, causing electron leakage in the electron transport chain, and generating reactive oxygen species (ROS) bursts. This is consistent with the significant increase in ROS fluorescence intensity in HK2 cells after cisplatin intervention in this invention. Multiple studies have reported that excessive ROS production can directly oxidize Akt or reduce PI3K activity by oxidizing certain amino acid residues (especially tyrosine residues), leading to ineffective PIP3 generation by PI3K and inhibiting the activation of the PI3K / Akt pathway. Furthermore, increased ROS can also oxidize PTEN, an upstream regulator of PI3K / Akt, further enhancing the inhibition of the PI3K / Akt pathway. The PI3K / Akt signaling pathway is widely involved in physiological processes such as cell growth, metabolism, survival, and proliferation. Activation of this pathway produces anti-apoptotic, cell proliferation-promoting, and metabolic-regulating effects to maintain cell survival and function.

[0047] Based on the mechanism of cisplatin nephrotoxicity, oxidative stress plays a crucial role in cisplatin-induced kidney injury. In previous studies, Nrf2, as a master regulator of antioxidant responses, can be activated by the PI3K / Akt pathway, promoting its nuclear translocation and stabilization, initiating antioxidant responses, and increasing the expression of antioxidant enzymes such as HO-1 and NQO1. These antioxidant enzymes, in turn, scavenge excess ROS, alleviating cisplatin-induced oxidative stress and reducing the inhibition of the PI3K / Akt pathway, thereby restoring its anti-apoptotic and cell proliferation-promoting effects. This is also a powerful means to combat cisplatin nephrotoxicity.

[0048] Besides apoptosis and oxidative stress, the inflammatory cascade is a significant factor contributing to the further aggravation of cisplatin-induced kidney injury. In this invention, the serum levels of pro-inflammatory cytokines IL-1α, IL-6, and TNF-α were significantly elevated in mice with cisplatin-induced acute kidney injury. In vitro, cisplatin also promoted the nuclear translocation of NFkB in HK2 cells, and the mRNA levels of inflammatory factors were also significantly increased. This is consistent with previous research results, further demonstrating that the inflammatory response is one of the mechanisms of cisplatin nephrotoxicity. Both in vivo and in vitro, the nuclear translocation of NFkB was inhibited and the expression of inflammatory factors was downregulated after intervention with lupeol. This fully demonstrates that lupeol can alleviate cisplatin-induced kidney injury by reducing the inflammatory response. Multiple studies have shown that Nrf2 activation can feedback-inhibit excessive NF-κB inflammatory responses, and the use of Nrf2 inhibitors can weaken the inhibition of NFkB pathway activity. Therefore, the applicant believes that lupeol exerts direct antioxidant and anti-apoptotic effects by upregulating the PI3K / Akt / Nrf2 signaling pathway, weakening the related pathological effects in the main pathway of cisplatin nephrotoxicity. At the same time, the activated Nrf2 pathway inhibits NFkB activation, inhibits the release of downstream pro-inflammatory factors, breaks the vicious cycle of "damage-inflammation-re-damage", and avoids the further development and deterioration of cisplatin-induced kidney damage.

[0049] This invention explores the potential of lupeol as a novel treatment for cisplatin-induced acute kidney injury (AKI), and its protective effect on the kidneys has been verified both in vitro and in vivo. However, this invention still has certain limitations. The pathophysiological process of acute kidney injury is more complex in humans, and the nephrotoxicity of cisplatin may be influenced by age, sex, and underlying diseases. This invention is primarily based on healthy male mice and a single cell line, and its effects in some pathological conditions remain unclear and require further refinement. Although the involvement of the PI3K / Akt / Nrf2 pathway has been demonstrated, the direct molecular targets of lupeol are not fully elucidated, and further exploration is needed to determine its specific targets. Future research should also utilize inhibitor-based rescue assays to confirm the necessity of these pathways.

[0050] The above embodiments are merely illustrative of the principles and effects of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by those skilled in the art without departing from the spirit and technical concept disclosed in the present invention should still be covered by the claims of the present invention.

Claims

1. Application of lupeol in the preparation of drugs for the prevention or treatment of kidney injury.

2. The application as described in claim 1, characterized in that, The kidney injury was caused by cisplatin.

3. The application as described in claim 2, characterized in that, The lupeol can be administered via intraperitoneal injection, subcutaneous injection, or oral administration.

4. The application as described in claim 3, characterized in that, The administration route of lupeol is selected from oral administration.

5. The application as described in claim 1, characterized in that, The drug also includes a pharmaceutically acceptable carrier.

6. The application as described in claim 5, characterized in that, The pharmaceutically acceptable carriers include at least one of diluents, binders, surfactants, lubricants, fillers, and disintegrants.

7. The application as described in claim 1, characterized in that, The dosage forms of the drug include granules, powders, tablets, capsules, syrups, suppositories, injections, and elixirs.

8. A pharmaceutical composition, characterized in that, The pharmaceutical composition includes cisplatin and lupeol.

9. A medicine, characterized in that, The pharmaceutical product includes the pharmaceutical composition as described in claim 8.

10. The pharmaceutical product as described in claim 9, characterized in that, The medicine also includes a pharmaceutically acceptable carrier.

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