Use of trapidil for the preparation of a medicament for the prevention and / or treatment of doxorubicin-induced organ toxicity

Abstract

The application provides application of Trapidil in preparation of a drug for preventing and / or treating doxorubicin-induced organ toxicity, and belongs to the technical field of biological medicines.The application discloses that Trapidil plays an organ protection role by activating a cAMP / PKA / CREB signal axis and driving two parallel downstream effect channels: on the one hand, up-regulating an NRF2-GPX3 positive feedback loop to inhibit doxorubicin-induced myocardial cell pyroptosis and glomerular podocyte pyroptosis; and on the other hand, activating an NRF2 / HO-1 channel to inhibit myocardial cell apoptosis and glomerular podocyte apoptosis.In-vivo and in-vitro experiments prove that Trapidil can improve doxorubicin-induced cardiac dysfunction and kidney function damage, reduce histopathological damage, reduce the level of oxidative stress, inhibit the pyroptosis and apoptosis of myocardial cells and glomerular podocytes, and does not affect the antitumor effect of doxorubicin.The application provides a new drug strategy for doxorubicin chemotherapy-induced multi-organ toxicity.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically the application of tropidil in the preparation of drugs for the prevention and / or treatment of doxorubicin-induced organ toxicity. Background Technology

[0002] Doxorubicin (DOX) is a broad-spectrum and highly effective anthracycline antitumor antibiotic widely used in the clinical treatment of various malignant tumors, including breast cancer, leukemia, and lymphoma. However, its clinical application is limited by its severe and dose-dependent organ toxicity. Cardiotoxicity is the most common and fatal, manifesting as arrhythmias, cardiomyopathy, and even congestive heart failure. Its pathological mechanisms involve calcium homeostasis imbalance, mitochondrial damage, and reactive oxygen species accumulation, inducing various programmed cell death processes in cardiomyocytes, including apoptosis and pyroptosis. Furthermore, doxorubicin can induce nephrotoxicity, with glomerular podocyte apoptosis being the core pathological process, clinically manifesting as proteinuria, decreased renal function, and even end-stage renal disease. In addition, doxorubicin can cause damage to organs such as the liver, spleen, and lungs, further exacerbating the systemic toxicity burden in chemotherapy patients.

[0003] The only clinically approved drug for preventing anthracycline cardiotoxicity is dexrazoxane, but its indications are strictly limited to metastatic breast cancer patients who have received a cumulative dose of doxorubicin and need to continue treatment. However, dexrazoxane has several drawbacks: it may interfere with the antitumor efficacy of doxorubicin, studies suggest it may increase the risk of secondary tumors, and it may exacerbate side effects such as chemotherapy-induced myelosuppression. Currently, there are no specific drugs to prevent or treat doxorubicin nephrotoxicity; only symptomatic and supportive treatment can be provided clinically. Other drugs, such as angiotensin-converting enzyme inhibitors / angiotensin II receptor blockers (e.g., losartan has shown some renal protective effects in animal studies), beta-blockers, and statins, have insufficient or controversial evidence regarding their clinical prevention and treatment of doxorubicin cardiotoxicity and nephrotoxicity. Therefore, there is an urgent clinical need for a novel drug that can effectively prevent or treat doxorubicin cardiotoxicity, nephrotoxicity, and multi-organ toxicity without affecting antitumor efficacy.

[0004] Trapidil (TRA) is a clinically marketed non-selective phosphodiesterase inhibitor, traditionally used as a vasodilator and antiplatelet drug. Currently, there are no reports on the use of trapidil for the prevention or treatment of doxorubicin-induced organ toxicity (especially cardiotoxicity and nephrotoxicity). Therefore, developing a drug that can effectively protect multiple organs such as the heart and kidneys without affecting the antitumor efficacy of doxorubicin is an important technological direction in this field. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to provide the use of tropidil in the preparation of drugs for the prevention and / or treatment of doxorubicin-induced organ toxicity. Tropidil can effectively improve doxorubicin-induced cardiac dysfunction and renal damage, reduce histopathological changes, reduce oxidative stress and cell death, and does not affect the antitumor efficacy of doxorubicin.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides the use of tropidil in the preparation of drugs for the prevention and / or treatment of doxorubicin-induced organ toxicity.

[0007] Preferably, the organ toxicity is cardiotoxicity.

[0008] Preferably, the organ toxicity is nephrotoxicity.

[0009] More preferably, the drug exerts a cardioprotective effect by inhibiting myocardial cell pyroptosis and / or apoptosis.

[0010] More preferably, the drug inhibits cardiomyocyte pyroptosis by activating the cAMP / PKA / CREB signaling axis, upregulating the NRF2-GPX3 positive feedback loop.

[0011] More preferably, the drug inhibits cardiomyocyte apoptosis by activating the NRF2 / HO-1 signaling pathway.

[0012] More preferably, the drug inhibits pyroptosis of glomerular podocytes by activating the cAMP / PKA / CREB signaling axis, upregulating the NRF2-GPX3 positive feedback loop.

[0013] More preferably, the drug inhibits glomerular podocyte apoptosis by activating the NRF2 / HO-1 signaling pathway.

[0014] Preferably, the organ toxicity also includes hepatotoxicity, spleen toxicity, and / or pulmonary toxicity.

[0015] Preferably, the drug is an oral preparation or an injectable preparation.

[0016] More preferably, the oral formulation is a tablet, capsule, or suspension.

[0017] Preferably, the drug further includes a pharmaceutically acceptable carrier.

[0018] Compared with the prior art, the present invention has the following advantages: (1) This invention reveals for the first time the dual protective effect of tropidil in the prevention and treatment of doxorubicin-induced cardiotoxicity and nephrotoxicity, with significant efficacy and mechanism innovation. In vivo experiments show that tropidil (25-50 mg / kg / day) gavage treatment can significantly improve cardiac function, including significant increases in left ventricular ejection fraction (LVEF), left ventricular fractional shortening (LVFS), stroke volume (SV), and heart rate (HR), and significant decreases in left ventricular end-systolic diameter (LVIDs) and left ventricular end-diastolic diameter (LVIDd); it can significantly reduce serum myocardial injury markers, including creatine kinase isoenzyme (CK-MB) and cardiac troponin T (cTnT) levels, and alleviate myocardial tissue pathological damage and collagen deposition; at the same time, it can significantly reduce mouse urinary protein (UPRO), serum creatinine (Scr), and blood urea nitrogen (BUN) levels, alleviate kidney tissue pathological damage, and also have significant pathological improvement effects on major organs such as liver, spleen, and lungs. The above results indicate that tropidil provides comprehensive protection against doxorubicin-induced multi-organ damage at multiple levels, including organs, tissues, and cells. Further validation in a 4T1 tumor-bearing mouse model showed that tropidil effectively improved doxorubicin-induced cardiac function decline (left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS) were significantly better than in the doxorubicin monotherapy group), demonstrating its cardioprotective efficacy in the presence of tumors.

[0019] (2) This invention further reveals the unified molecular mechanism of tropidil's cardio-renal protection. At the phenotypic level, tropidil can simultaneously inhibit doxorubicin-induced pyroptosis and apoptosis in cardiomyocytes and glomerular podocytes. At the molecular level, tropidil exerts its protective effect by upregulating NRF2 and its downstream target genes GPX3 and HO-1: the NRF2-GPX3 positive feedback loop is responsible for inhibiting pyroptosis, and the NRF2 / HO-1 pathway is responsible for inhibiting apoptosis. At the upstream signaling level, tropidil activates the cAMP / PKA / CREB signaling axis, thereby driving the above two parallel downstream effector pathways. Mechanism verification experiments confirmed that GPX3 siRNA, NRF2 inhibitor ML385, PKA inhibitor H89 (cardiomyocyte), or PKA siRNA knockdown of PKA (kidney) can significantly weaken the protective effect of tropidil, fully verifying the necessity of the signaling axis "cAMP / PKA / CREB → NRF2 → (GPX3 / HO-1)". This mechanism of "a single upstream hub driving two parallel effect pathways" provides solid molecular evidence for the unique broad-spectrum protective effect of tropidil.

[0020] (3) This invention demonstrates that tropidil does not affect the antitumor efficacy of doxorubicin. In vitro MTT assays confirmed that tropidil does not affect the half-maximal inhibitory concentration (IC50) of doxorubicin against various tumor cells (mouse breast cancer cells 4T1, human breast cancer cells MCF-7, human liver cancer cells HepG2, and human non-small cell lung cancer cells A549); in the 4T1 tumor-bearing mouse model, there was no significant difference in tumor growth rate and final tumor weight between the tropidil and doxorubicin combination therapy group and the doxorubicin monotherapy group.

[0021] Since tropidil is a marketed drug with sufficient safety data, it can be quickly translated into clinical practice, providing chemotherapy patients with a safe, effective, and cost-controllable new strategy for the prevention and treatment of multi-organ toxicity. It solves the clinical problem that existing preventive drugs (such as dexrazoxane) may affect the anti-tumor efficacy and increase the risk of secondary tumors when preventing doxorubicin cardiotoxicity, and lack effective intervention for doxorubicin-induced nephrotoxicity. Attached Figure Description

[0022] Figure 1 The effects of tropidil on cardiac function, structure, and myocardial injury markers in doxorubicin-induced model mice; A is a schematic diagram of the animal experimental protocol; B is a representative echocardiogram; CH represent the left ventricular ejection fraction (LVEF), left ventricular fractional shortening (LVFS), stroke volume (SV), heart rate (HR), left ventricular end-systolic diameter (LVIDs), and left ventricular end-diastolic diameter (LVIDd) of each group of mice, respectively. n =4); IJ represents the levels of creatine kinase isoenzyme (CK-MB) and cardiac troponin T (cTnT). n =4).

[0023] Figure 2 To improve the pathological damage of myocardial tissue in doxorubicin model mice using tropidil; A is a hematoxylin-eosin (HE) staining image of mouse myocardial tissue (scale bar 50µm); B is a Masson staining image of mice in each group (scale bar 50µm); C is a quantitative statistical analysis of perivascular collagen volume fraction (CVF) in mouse myocardial tissue.

[0024] Figure 3 The study aimed to reduce oxidative stress levels in myocardial tissue of doxorubicin-induced mice and in neonatal myocardial cells (NMCMs) induced by doxorubicin using tropidil. A represents the quantitative analysis of glutathione (GSH) in mouse myocardial tissue; B represents the quantitative analysis of catalase (CAT) in mouse myocardial tissue; C represents representative ROS fluorescent staining images in NMCMs (scale bar 20 µm); and D represents the quantitative analysis of ROS levels.

[0025] Figure 4Trapidil inhibits pyroptosis in the myocardial tissue of a mouse model of doxorubicin; A is a transmission electron microscopy observation of the ultrastructure of cardiomyocytes (mitochondrial morphology, cristae structure, intracellular material) (scale bar, 2µm); B is a representative Western blot image of NLRP3, Cleaved-CASP1, Cleaved-IL-1β, Cleaved-IL-18, and GSDMD-N in mouse myocardium; C and D are quantitative analyses of protein levels in B. n =4).

[0026] Figure 5 The effect of tropidil on the expression of GPX3 and NRF2 proteins and mRNA in the myocardial tissue of doxorubicin model mice; A is a representative Western blot plot of GPX3 and NRF2; B and C are the quantitative analysis of protein levels in A. n =4); DE represent the mRNA expression levels of GPX3 and NRF2, respectively. n =4).

[0027] Figure 6 Trapidil inhibits doxorubicin-induced pyroptosis in mouse cardiomyocytes by regulating the NRF2-GPX3 positive feedback loop; AG represents the GPX3 siRNA intervention experiment; A is a Western blot representation of GPX3, NRF2, NLRP3, and Cleaved-CASP1 in NMCMs; BE represents the quantitative analysis of protein levels in A. n =4); F is a representative fluorescent staining image of ROS (scale bar 20µm); G is the quantitative analysis of ROS levels; the groups are: Control, DOX, DOX+TRA and DOX+TRA+GPX3siRNA (tropidil combined with GPX3 siRNA treatment group); HL is the NRF2 inhibitor ML385 intervention experiment; H is a representative Western blot image of GPX3 and NRF2 in NMCMs; IJ are the quantitative analysis of protein levels in H ( n =4); K is a representative ROS fluorescence staining image (scale bar 20µm); L is the quantitative analysis of ROS level; the groups are: Control, DOX, DOX+TRA, Control+ML385 (NRF2 inhibitor ML385 group) and DOX+TRA+ML385 (tropidil combined with NRF2 inhibitor ML385 treatment group).

[0028] Figure 7To demonstrate the activation of the cAMP / PKA / CREB signaling axis by tropidil in doxorubicin-treated primary cardiomyocytes, and the blocking of its downstream NRF2-GPX3 regulation by the PKA inhibitor H89; AH serves as validation of the in vitro and in vivo cAMP / PKA / CREB signaling axis activation: A shows representative Western blot plots of cAMP, p-PKA, and p-CREB in mouse myocardial tissue; BD represents the quantitative analysis of protein levels in A. n =4); E is a representative Western blot plot of cAMP, p-PKA, and p-CREB in NMCMs; FH is a quantitative analysis of protein levels in E ( n =4); IL is the H89 blockade experiment of PKA inhibitor: I is a representative Western blot of p-CREB, GPX3, and NRF2 in NMCMs; J and L are the quantitative analysis of protein levels in I ( n =4); the groups were: Control, DOX, DOX+TRA, H89 intervention group (PKA inhibitor H89 group) and DOX+TRA+H89 (tropidil combined with PKA inhibitor H89 treatment group).

[0029] Figure 8 The effect of tropidil on apoptosis of myocardial cells in doxorubicin-induced mouse model; A is a representative Western blot plot of Bcl2, BAX, and Cleaved-CASP3 in myocardial tissue; B and D are quantitative analyses of protein levels in A, respectively. n =4); E is a representative image of TUNEL staining, red fluorescence represents TUNEL-positive apoptotic cells, blue (DAPI) represents cell nuclei, scale bar 50μm; F is a quantitative analysis of the percentage of TUNEL-positive cells.

[0030] Figure 9 Trapidil inhibits doxorubicin-induced apoptosis in mouse cardiomyocytes by regulating the NRF2 / HO-1 pathway; AC represents the expression of NRF2 and HO-1 proteins in mouse myocardial tissue; A is a representative Western blot image of NRF2 and HO-1 in mouse myocardial tissue; BC represents the quantitative analysis of protein levels in A. n =4); DK represents the NRF2 inhibitor (ML385) intervention experiment; D represents the Western blot representation of NRF2, HO-1, Bcl2, BAX, and Cleaved-CASP3 in NMCMs; EI represents the quantitative analysis of protein levels in D ( n=4); J represents representative TUNEL staining images, red fluorescence represents TUNEL-positive apoptotic cells, blue (DAPI) represents cell nuclei, scale bar 50μm; K represents quantitative analysis of the percentage of TUNEL-positive cells; the groups are: Control, DOX, DOX+TRA, Control+ML385 (NRF2 inhibitor ML385 group) and DOX+TRA+ML385 (tropidil combined with NRF2 inhibitor ML385 treatment group).

[0031] Figure 10 Results of tropidil improving renal function in doxorubicin model mice; A: urinary protein (UPRO) level in mice; B: blood urea nitrogen (BUN) level in mice; C: serum creatinine (Scr) level in mice; (n=4).

[0032] Figure 11 To improve the pathological damage of renal tissue in doxorubicin model mice using tropidil; A is a hematoxylin-eosin (HE) staining image of mice (scale bar 50µm); B is a Masson staining image of mice (scale bar 50µm); C is a quantitative statistical analysis of perivascular collagen volume fraction (CVF) in the renal tissue of mice.

[0033] Figure 12 To improve doxorubicin-induced oxidative stress levels in glomerular podocytes using tropidil; A shows a representative ROS fluorescent staining image (scale bar 20µm); B shows quantitative analysis of ROS levels.

[0034] Figure 13 Trapidil inhibits the expression of pyroptosis-related proteins in the kidney tissue of doxorubicin model mice; A is a Western blot representation of NLRP3, Cleaved-CASP1, Cleaved-IL-1β, Cleaved-IL-18, and GSDMD-N in mouse kidney tissue; BF are quantitative analyses of the protein levels in A. n =4).

[0035] Figure 14 The effect of tropidil on the expression of GPX3 and NRF2 proteins in the kidney tissue of doxorubicin model mice is shown in Figure A. Western blot diagram of GPX3 and NRF2 in mouse kidney tissue is shown in Figure B. Quantitative analysis of protein levels in A is shown in Figure C.

[0036] Figure 15Tropidil inhibits doxorubicin-induced pyroptosis in glomerular podocytes by regulating the NRF2-GPX3 positive feedback loop; AE represents the GPX3 siRNA intervention experiment; A is a Western blot representation of GPX3, NRF2, NLRP3, and Cleaved-CASP1 in glomerular podocytes; BE represents the quantitative analysis of protein levels in A. n =4); the groups were: Control, DOX, DOX+TRA and DOX+TRA+GPX3 siRNA (tropidil combined with GPX3 siRNA treatment group); FH is the NRF2 inhibitor (ML385) intervention experiment: F is a representative Western blot of GPX3 and NRF2 in glomerular podocytes; GH are the quantitative analysis of protein levels in F (n=4); the groups were: Control, DOX, DOX+TRA, Control+ML385 (NRF2 inhibitor ML385 group) and DOX+TRA+ML385 (tropidil combined with NRF2 inhibitor ML385 treatment group).

[0037] Figure 16 To activate the cAMP / PKA / CREB signaling axis in doxorubicin-treated glomerular podocytes, and to block the regulation of downstream NRF2-GPX3 by PKA siRNA knockdown of PKA; AG shows the in vitro activation verification of the cAMP / PKA / CREB signaling axis; A is a representative Western blot image of cAMP, p-PKA, and p-CREB in mouse podocytes; BD represents the quantitative analysis of protein levels in A. n =4); E is a representative Western blot of NRF2 and GPX3 in renal podocytes; FG is the quantitative analysis of protein levels in E (n=4); HL is the PKA siRNA knockdown PKA blocking experiment; H is a representative Western blot of p-PKA, p-CREB, GPX3, and NRF2 in glomerular podocytes; IL is the quantitative analysis of protein levels in H (n=4). n =4); the groups were: Control, DOX, DOX+TRA, PKA siRNA (normal cells + PKA siRNA) and DOX+TRA+PKA siRNA (tropidil combined with PKA knockdown treatment group).

[0038] Figure 17 The effect of tropidil on the expression of apoptosis proteins in renal tissue cells of doxorubicin model mice; A is a representative Western blot plot of WT-1, BCL2, BAX, and Cleaved-CASP3 protein expression in renal tissue; B and C are quantitative analyses of protein levels in A. n =4).

[0039] Figure 18 Tropidil inhibits doxorubicin-induced glomerular podocyte apoptosis by regulating the NRF2 / HO-1 pathway; AB shows the expression of HO-1 in mouse kidney tissue: A is a representative Western blot image of HO-1 in mouse kidney tissue; B is the quantitative analysis of protein levels in A. n =4); CH represents the NRF2 inhibitor ML385 intervention experiment; C is a representative Western blot of NRF2, HO-1, Bcl2, BAX, and Cleaved-CASP3 in glomerular podocytes; DH represents the quantitative analysis of protein levels in C ( n =4); the groups were: Control, DOX, DOX+TRA, Control+ML385 (NRF2 inhibitor ML385 group) and DOX+TRA+ML385 (tropidil combined with NRF2 inhibitor ML385 treatment group).

[0040] Figure 19 Trapidil does not affect the antitumor activity of doxorubicin against tumor cells; A represents the cell viability curves of 4T1, MCF-7, HepG2, and A549 cells after treatment with different concentrations of doxorubicin for 24 hours, determined by the MTT assay, to determine the IC50 of each cell line. 50 value( n =3); B is the administration of DOX (IC) to each tumor cell. 50 Cell viability after treatment with DOX (concentration) or DOX combined with tropidil (50 μM) for 24 h ( n =3); grouped as Control (blank control), DOX (doxorubicin IC50) 50 ), DOX+TRA (IC 50 DOX + 50 μM TRA). Statistical markers are shown in the attached figure.

[0041] Figure 20 To demonstrate that tropidil did not affect the antitumor efficacy of doxorubicin in tumor-bearing mouse models and still improved cardiac function; AC represents cardiac function assessment: A is a representative echocardiogram image of the mouse heart; B is a quantitative analysis of left ventricular ejection fraction (LVEF) (n=5); C is a quantitative analysis of left ventricular fractional shortening (LVFS) (n=5); DF represents antitumor efficacy assessment: D is the mouse tumor growth curve; E is a representative tumor image of each group of mice; F is the mouse tumor weight ( n =6); Groups were as follows: PBS: control group, DOX: doxorubicin model group, DOX+TRA: tropidil treatment group; p <0.01, compared with the PBS group; # p <0.05, compared with the DOX group.

[0042] Figure 21 The effects of tropidil on the pathological characteristics of liver, spleen, and lung tissues and liver and kidney function in doxorubicin model mice; A is a representative HE-stained image of mouse liver, spleen, and lung tissues (scale bar 50µm); B shows the serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels in mice. n =4).

[0043] In the accompanying drawings of this invention, unless otherwise specified, the abbreviations used have the following meanings: Control: Normal control group; DOX: Doxorubicin model group; DOX+TRA: Trapidil treatment group (specific dosage see the methods of each embodiment); DOX+TRA-L: Low-dose trapidil treatment group (25 mg / kg / day, see the kidney-related drawings); DOX+TRA-H: High-dose trapidil treatment group (50 mg / kg / day, see the kidney-related drawings); DOX+LOS: Losartan positive control group (animal experiments or cell experiments, see the kidney-related drawings); Data are expressed as mean ± SD; * p <0.05,** p <0.01, *** p <0.001 indicates a comparison with the Control group; # p <0.05, ## p <0.01, ### p <0.001 indicates a comparison with the DOX group; & p <0.05, && p <0.01 indicates a comparison with the DOX+TRA group (if applicable); other special groups appearing in each figure (such as GPX3 siRNA, ML385, H89, etc.) will be explained separately in the corresponding figure caption. Detailed Implementation

[0044] This invention provides the use of trapidil in the preparation of a drug for the prevention and / or treatment of doxorubicin-induced organ toxicity. Trapidil (TRA), chemically known as 5-methyl-7-diethylamino-1,2,4-triazole[1,5-α]pyrimidine, is commercially available. Trapidil is a clinically marketed drug, with a routine adult clinical dose of 150-300 mg / day (divided into oral doses, i.e., 50-100 mg three times daily). To evaluate the protective effect of trapidil against doxorubicin-induced organ toxicity, this invention, referring to the above-mentioned clinical dose, converts the routine clinical dose to an equivalent mouse dose (approximately 25-55 mg / kg / day) based on body surface area conversion principles. The drug can be formulated into oral preparations (e.g., tablets, capsules, suspensions) or injections, containing a therapeutically effective amount of trapidil and a pharmaceutically acceptable carrier. In clinical application, physicians can individualize the above-mentioned routine dose according to the patient's specific condition.

[0045] In this invention, the organ toxicity is preferably cardiotoxicity. Experimental results of this invention show that when the organ toxicity is cardiotoxicity, the left ventricular ejection fraction (LVEF), left ventricular fractional shortening (LVFS), stroke volume (SV), heart rate (HR), left ventricular end-systolic diameter (LVIDs), and left ventricular end-diastolic diameter (LVIDd) in the tropidil-treated group mice significantly increased compared to the model group; serum CK-MB and cTnT levels significantly decreased; myocardial tissue pathological damage and collagen deposition were significantly reduced; TUNEL-positive cell rate decreased; and the expression of NLRP3, Cleaved-CASP1, Cleaved-IL-1β, Cleaved-IL-18, GSDMD-N, BAX, and Cleaved-CASP3 was downregulated while BCL2 expression was upregulated. In in vitro mechanism verification, primary cardiomyocytes from C57BL / 6 neonatal mice were isolated and treated with 1 μmol / L doxorubicin and 50 μmol / L tropidil for 24 hours. The results showed that tropidil significantly reduced intracellular reactive oxygen species (ROS) levels, decreased the rate of TUNEL-positive cells, and inhibited the expression of pyroptosis-related proteins (NLRP3, Cleaved-CASP1, GSDMD-N) and pro-apoptotic proteins (BAX, Cleaved-CASP3), while upregulating the expression of the anti-apoptotic protein BCL2. These results indicate that tropidil can simultaneously inhibit both pyroptosis and apoptosis, two programmed cell death pathways, in the heart.

[0046] Building upon this foundation, this invention further elucidates the molecular targets by which tropidil exerts its dual protective effects. Detection revealed that the protein and mRNA expression levels of NRF2 and its downstream target genes GPX3 and HO-1 in myocardial tissue significantly increased in the tropidil-treated group. Knockdown of GPX3 using GPX3 siRNA significantly weakened the upregulation of NRF2 by tropidil, while the expression of pyroptosis-related proteins (Cleaved-CASP1 and NLRP3) increased, and ROS levels rose again, indicating a positive feedback regulatory relationship between GPX3 and NRF2, and that this loop is crucial for the anti-pyroptosis effect of tropidil. After using the NRF2 inhibitor ML385, the upregulation of GPX3 and HO-1 by tropidil was inhibited, and its anti-pyroptosis and anti-apoptosis effects were significantly reversed (pyroptosis protein levels increased, BAX and Cleaved-CASP3 levels increased, BCL2 decreased, and TUNEL positivity rate increased again). The above results confirm that NRF2 is a core node connecting two downstream effector pathways: tramidil inhibits cardiomyocyte pyroptosis by upregulating the NRF2-GPX3 positive feedback loop, and at the same time inhibits cardiomyocyte apoptosis by activating the NRF2 / HO-1 signaling pathway.

[0047] After identifying key downstream targets, this invention further traced the upstream signaling mechanism of tropidil's activation of NRF2. Detection revealed that the expression of cAMP, p-PKA, and p-CREB proteins was significantly upregulated in the myocardial tissue and primary cardiomyocytes of mice treated with tropidil. Pretreatment with the PKA-specific inhibitor H89 significantly weakened the upregulation of p-CREB, NRF2, and GPX3 by tropidil, demonstrating that the cAMP / PKA / CREB signaling axis is the core upstream hub for tropidil's activation of NRF2 and its downstream pathways. In summary, tropidil comprehensively alleviates doxorubicin-induced cardiotoxicity by activating the cAMP / PKA / CREB signaling axis, driving the NRF2-GPX3 positive feedback loop (anti-pyroptosis) and the NRF2 / HO-1 pathway (anti-apoptosis).

[0048] In this invention, the organ toxicity is preferably nephrotoxicity. Experimental results of this invention show that when the organ toxicity is nephrotoxicity, the levels of urinary protein (UPRO), serum creatinine (Scr), and blood urea nitrogen (BUN) in mice treated with tropidil are significantly lower than those in the model group. Kidney tissue pathological damage (glomerular sclerosis, podocyte injury, and vacuolar degeneration of renal tubular epithelial cells) is significantly reduced. The expression of NLRP3, Cleaved-CASP1, Cleaved-IL-1β, Cleaved-IL-18, GSDMD-N, Cleaved-CASP3, and BAX is downregulated, while the expression of WT-1 and BCL2 is upregulated. Its protective effect is comparable to or better than that of the losartan control group. In in vitro mechanism verification, MPC-5 mouse glomerular podocytes were cultured and treated with 2 μmol / L doxorubicin and 50 μmol / L tropidil for 24 hours. The results showed that tropidil significantly reduced intracellular reactive oxygen species levels, inhibited the expression of pyroptosis-related proteins (NLRP3, Cleaved-CASP1, Cleaved-IL-1β, Cleaved-IL-18, GSDMD-N) and pro-apoptotic proteins (BAX, Cleaved-CASP3), while upregulating the expression of podocyte marker protein WT-1 and anti-apoptotic protein BCL2. These results indicate that tropidil can simultaneously inhibit pyroptosis and apoptosis in kidney podocytes.

[0049] Building upon this foundation, this invention further elucidates the molecular targets by which tropidil exerts its dual protective effect in the kidney. Detection revealed that the expression levels of NRF2 and its downstream target genes GPX3 and HO-1 in kidney tissue significantly increased in the tropidil-treated group. Knockdown of GPX3 using GPX3 siRNA significantly weakened the upregulation of NRF2 by tropidil, while pyroptosis protein expression increased, indicating that the NRF2-GPX3 positive feedback loop also plays a crucial anti-pyroptosis role in podocytes. After using the NRF2 inhibitor ML385, the upregulation of both GPX3 and HO-1 by tropidil was inhibited, and both anti-pyroptosis and anti-apoptosis effects were reversed, confirming that NRF2 is also a core node connecting the two effector pathways in the kidney.

[0050] After identifying key downstream targets, this invention further traced the upstream signaling mechanism by which tropidil activates NRF2 in the kidney. Detection revealed that the expression of cAMP, p-PKA, and p-CREB proteins was significantly upregulated in the kidney tissue and podocytes of mice treated with tropidil. Knockdown of PKA by transfecting with PKA-specific siRNA significantly weakened the upregulation of p-CREB, NRF2, and GPX3 by tropidil, demonstrating that the cAMP / PKA / CREB signaling axis is also a core upstream hub for tropidil's activation of NRF2 and its downstream pathways in the kidney. In summary, tropidil comprehensively alleviates doxorubicin-induced nephrotoxicity by activating the cAMP / PKA / CREB signaling axis, driving the NRF2-GPX3 positive feedback loop (anti-pyroptosis) and the NRF2 / HO-1 pathway (anti-apoptosis).

[0051] This invention further verifies that tropidil does not affect the antitumor efficacy of doxorubicin. In vitro MTT assays showed that, in the presence of tropidil (50 μmol / L), the half-maximal inhibitory concentration (IC50) of doxorubicin against 4T1 breast cancer cells, MCF-7 breast cancer cells, HEPG2 liver cancer cells, and A549 lung cancer cells was not significantly different from that of the doxorubicin monotherapy group. In an in vivo tumor-bearing mouse model, the tumor volume and weight of the doxorubicin (5 mg / kg, every 4 days) combined with tropidil (25 mg / kg / day) treatment group were not significantly different from those of the doxorubicin monotherapy group. Furthermore, tropidil not only did not aggravate the pathological damage to major organs such as the liver, spleen, and lungs induced by doxorubicin in mice, but also significantly improved it. Therefore, tropidil, while preventing and treating doxorubicin-induced cardiotoxicity and / or nephrotoxicity, does not affect the antitumor efficacy of doxorubicin, demonstrating good tissue tolerability and drug safety.

[0052] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0053] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods. Unless otherwise specified, the experimental materials used in the following embodiments are commercially available products.

[0054] Example 1: Protective effect of tropidil against doxorubicin-induced cardiotoxicity in mice. 1. Experimental Methods Six- to eight-week-old male C57BL / 6 mice were randomly divided into three groups: a normal control group (Control), an doxorubicin model group (DOX), and a tropidil treatment group (DOX+TRA), with ten mice in each group. Chronic cardiotoxicity models were established in the doxorubicin model group and the tropidil treatment group by intraperitoneal injection of doxorubicin (5 mg / kg, once every four days for a total of four injections, with a cumulative dose of 20 mg / kg). In the tropidil treatment group, tropidil (25 mg / kg) was administered by gavage daily for 14 consecutive days, starting one day before the first doxorubicin injection. Figure 1 In the control group (A), an equal volume of normal saline was administered. One day after the last administration, echocardiography was used to measure left ventricular ejection fraction (LVEF), left ventricular fractional shortening (LVFS), stroke volume (SV), heart rate (HR), left ventricular end-systolic diameter (LVIDs), and left ventricular end-diastolic diameter (LVIDd). Serum samples were collected to detect myocardial injury markers creatine kinase isoenzyme (CK-MB) and cardiac troponin T (cTnT). Myocardial tissue was collected for HE staining and Masson staining to observe pathological changes.

[0055] 2. Experimental Results like Figure 1 BJ and Figure 2 The results from the AC study showed that, compared with the normal control group, the cardiac function indicators (LVEF, LVFS, SV, HR) of mice in the doxorubicin model group were significantly reduced. p <0.01, cardiac structural indicators (LVIDs, LVIDd) significantly increased ( p <0.05, serum CK-MB and cTnT levels were significantly elevated ( p <0.01, myocardial tissue showed obvious myofibril disorder, inflammatory cell infiltration, and collagen deposition. Compared with the doxorubicin model group, the above cardiac function indicators in the tropidil treatment group significantly recovered ( p <0.01, cardiac structural indicators significantly improved ( p <0.01, serum myocardial injury marker levels were significantly reduced ( p (<0.01), myocardial tissue pathological damage and collagen deposition were significantly reduced. These results indicate that tropidil can effectively improve doxorubicin-induced cardiac dysfunction, reduce myocardial tissue pathological damage, and has a significant protective effect against this cardiotoxicity.

[0056] Example 2: Verification of the mechanism by which tropidil inhibits doxorubicin-induced pyroptosis of cardiomyocytes by activating the NRF2-GPX3 positive feedback loop. 1. Experimental Methods (1) Animal tissue detection: The myocardial tissue of mice in Example 1 was taken and the levels of GSH and CAT were detected by biochemical method; the ultrastructure of myocardium was observed by transmission electron microscopy; the expression of NLRP3, Cleaved-CASP1, Cleaved-IL-1β, Cleaved-IL-18, GSDMD-N, NRF2 and GPX3 proteins were detected by Western blot; and the mRNA expression levels of GPX3 and NRF2 were detected by RT-qPCR.

[0057] (2) Primary cardiomyocyte culture and treatment: Primary cardiomyocytes (NMCMs) from C57BL / 6 mice were isolated and divided into the following groups: normal control group, doxorubicin group (1 μmol / L), tropidil group (1 μmol / L DOX + 50 μmol / L TRA), GPX3 siRNA interference group (DOX + TRA + GPX3 siRNA), NRF2 inhibitor group (DOX + TRA + ML385, ML385 concentration 10 μmol / L), and tropidil combined with H89 treatment group (DOX + TRA + H89, H89 concentration 10 μmol / L). After 24 hours of treatment, the ROS level of cells in each group was detected (DCFH-DA probe), and the expression of NRF2, GPX3, p-CREB, NLRP3, Cleaved-CASP1, GSDMD- and other proteins was detected by Western blot.

[0058] 2. Experimental Results (1) Tropidil reduces oxidative stress and inhibits myocardial cell pyroptosis. To verify the effects of tropidil on oxidative stress and pyroptosis, oxidative stress-related indicators were first examined. For example... Figure 3 As shown in Figure AB, the levels of glutathione (GSH) and catalase (CAT) in the myocardial tissue of doxorubicin model mice were significantly lower than those in the normal control group. p <0.01%, the above antioxidant indicators significantly rebounded after treatment with tropidil ( p <0.01). Meanwhile, such as Figure 3 As shown in the CD, compared with the normal control group, the ROS fluorescence intensity in the doxorubicin group NMCMs was significantly increased ( p <0.01); compared with the doxorubicin group, the tropidil group had a significantly lower ROS level ( p <0.01). The above results indicate that tropidil can effectively alleviate doxorubicin-induced oxidative stress.

[0059] Building upon this, the occurrence of pyroptosis was further investigated at the morphological and molecular levels. Transmission electron microscopy observations ( Figure 4In Figure A), the ultrastructure of cardiomyocytes in the normal control group mice was normal, with regular mitochondrial morphology and abundant, dense cristae. In the doxorubicin model group, cardiomyocytes showed obvious morphological changes typical of pyroptosis, including mitochondrial swelling, disordered and broken cristae, and loss of intracellular material. Tropidil treatment significantly reduced the aforementioned mitochondrial damage. Figure 4 As shown in Figure BG, compared with the normal control group, the expression of pyroptosis-related proteins (NLRP3, Cleaved-CASP1, Cleaved-IL-1β, Cleaved-IL-18, and GSDMD-N) in the myocardial tissue of mice in the doxorubicin model group was significantly increased. p <0.01); Compared with the doxorubicin model group, the expression of the above proteins was significantly downregulated in the tropidil treatment group ( p <0.05, p <0.01). The above results indicate that tropidil can effectively inhibit doxorubicin-induced pyroptosis of cardiomyocytes.

[0060] (2) Tropidil mediates the above-mentioned protective effect through the NRF2-GPX3 positive feedback loop. After confirming the oxidative stress and pyroptosis phenotypes, further investigation of molecular pathways was conducted. For example... Figure 5 As shown in the middle AE, compared with the normal control group, the protein and mRNA expression levels of NRF2 and GPX3 in the myocardial tissue of mice in the doxorubicin model group were significantly reduced. p <0.01); Compared with the doxorubicin model group, the expression of NRF2 and GPX3 in the tropidil treatment group was significantly restored ( p <0.01).

[0061] To verify the regulatory relationship between NRF2 and GPX3 and the necessity of this loop in the anti-pyroptosis effect of tropidil, GPX3 siRNA knockdown and NRF2 inhibitor ML385 intervention experiments were conducted. Figure 6 As shown in the AG, after GPX3 was knocked down by transfecting GPX3 siRNA, the upregulation effect of tropidil on NRF2 was significantly weakened, while the expression of Cleaved-CASP1 and NLRP3 rebounded, and the ROS level increased again. p The value <0.01 indicates a positive feedback regulatory relationship between GPX3 and NRF2, and this loop is crucial for the antipyrolysis effect of tropidil.

[0062] like Figure 6 As shown in HL, treatment with the NRF2 inhibitor ML385 significantly inhibited the upregulation of GPX3 by tropidil. p <0.01, further confirming that NRF2 is an upstream regulator of GPX3; at the same time, the scavenging effect of tropidil on ROS was also significantly reversed ( p<0.01).

[0063] (3) The cAMP / PKA / CREB signal axis is the upstream hub of the loop activated by Tropical. After confirming the NRF2-GPX3 positive feedback loop, the upstream signal mechanism of tropidil activation of NRF2 was further investigated. For example... Figure 7 As shown in Figure AH, compared with the doxorubicin model group, the expression of cAMP, p-PKA, and p-CREB proteins in the myocardial tissue and NMCMs of mice treated with tropidil was significantly upregulated. p The value <0.01 indicates that tropidil can activate the cAMP / PKA / CREB signaling pathway both in vivo and in vitro. Further reverse verification was performed using the PKA-specific inhibitor H89, such as... Figure 7 As shown in the IL, H89 pretreatment significantly weakened the upregulation effect of tropidil on p-CREB, NRF2, and GPX3. p <0.01), proving that the cAMP / PKA / CREB signal axis is the key upstream hub for the upregulation of the NRF2-GPX3 loop by tropidil.

[0064] The above results indicate that tropidil inhibits NLRP3 inflammasome-mediated cardiomyocyte pyroptosis by activating the cAMP / PKA / CREB signaling axis, upregulating the NRF2-GPX3 positive feedback loop, and reducing oxidative stress.

[0065] Example 3: Tropidil inhibits doxorubicin-induced cardiomyocyte apoptosis by activating the NRF2 / HO-1 signaling pathway. 1. Experimental Methods (1) Animal tissue detection: mouse myocardial tissue from Example 1 was taken and Western blot was used to detect the expression of NRF2, HO-1, BAX, BCL2 and Cleaved-CASP3 proteins; TUNEL staining was used to detect the apoptosis rate of myocardial tissue cells.

[0066] (2) Primary cardiomyocyte experiment: The cells were divided into four groups: normal control group (Control), doxorubicin group (DOX, 1 μmol / L), tropidil group (DOX+TRA, 1 μmol / L DOX+50 μmol / L TRA), and NRF2 inhibitor group (DOX+TRA+ML385, ML385 concentration 10 μmol / L). After 24 hours of treatment, the expression of NRF2, HO-1, BAX, BCL2, and Cleaved-CASP3 proteins was detected by Western blot; the apoptosis rate was detected by TUNEL staining.

[0067] 2. Experimental Results (1) Tropidil inhibits doxorubicin-induced cardiomyocyte apoptosis The aforementioned results have confirmed that tropidil can effectively alleviate doxorubicin-induced oxidative stress and inhibit pyroptosis. Figure 3 , Figure 4 Based on this, the effect of tropidil on cell apoptosis was further investigated.

[0068] like Figure 8 As shown in the AF, compared with the normal control group, the expression of the anti-apoptotic protein BCL2 in the myocardial tissue of mice in the doxorubicin model group was significantly reduced ( p <0.01), and the expression of pro-apoptotic proteins BAX and Cleaved-CASP3 was significantly increased ( p <0.01), the percentage of TUNEL-positive cells increased significantly ( p <0.01); Compared with the doxorubicin model group, BCL2 expression was significantly upregulated in the tropidil treatment group ( p <0.01), BAX and Cleaved-CASP3 expression were significantly downregulated ( p <0.01), the TUNEL-positive cell rate was significantly reduced ( p <0.01).

[0069] (2) Tropidil inhibits apoptosis by activating the NRF2 / HO-1 pathway. To investigate the molecular mechanism of tropidil's anti-apoptotic effect, the expression of NRF2 and its downstream target gene HO-1 was examined. Figure 9 As shown in the AC diagram, compared with the normal control group, the expression of NRF2 and HO-1 proteins in the myocardial tissue of mice in the doxorubicin model group was significantly reduced ( p <0.01); Compared with the doxorubicin model group, the expression of NRF2 and HO-1 was significantly upregulated in the tropidil treatment group ( p <0.01).

[0070] The aforementioned results have confirmed that the scavenging effect of tropidil on ROS was significantly reversed after the use of the NRF2 inhibitor ML385. Figure 6 The presence of KL (in the middle lipoprotein) suggests that NRF2-mediated antioxidant effects play an important role in the protective effect of tropidil. To verify whether the anti-apoptotic effect of tropidil depends on the NRF2 / HO-1 pathway, intervention was performed using the NRF2 inhibitor ML385. Figure 9 As shown in the middle DK, compared with the tropidil group, HO-1 expression was significantly suppressed after ML385 treatment ( p <0.05), BAX and Cleaved-CASP3 expression rebounded ( p <0.01), BCL2 expression decreased ( p <0.05%, the TUNEL-positive cell rate increased again ( p <0.01).

[0071] The above results indicate that tropidil inhibits oxidative stress by activating the NRF2 / HO-1 signaling pathway, thereby blocking the mitochondrial-dependent cardiomyocyte apoptosis pathway.

[0072] Example 4: Protective effect of tropidil against nephrotoxicity in doxorubicin model mice 1. Experimental Methods Six- to eight-week-old male C57BL / 6 mice were randomly divided into four groups: a normal control group (Control), an doxorubicin model group (DOX), a positive control group (DOX+LOS, doxorubicin + losartan 10 mg / kg / day), a low-dose tropidil group (DOX+TRA-L, doxorubicin + tropidil 25 mg / kg / day), and a high-dose tropidil group (DOX+TRA-H, doxorubicin + tropidil 50 mg / kg / day), with ten mice in each group. A nephropathy model was established by tail vein injection of doxorubicin (10 mg / kg). Each treatment group received the corresponding drug via gavage daily for four weeks, starting the day after doxorubicin injection. The normal control group received an equal volume of physiological saline. Two weeks after doxorubicin injection, urine samples were collected to determine urinary protein (UPRO) levels to confirm model success. After the last administration, serum creatinine (Scr) and blood urea nitrogen (BUN) levels were measured; kidney tissue was collected for HE staining and Masson staining to observe pathological changes.

[0073] 2. Experimental Results like Figure 10 AC and Figure 11 As shown in the AC diagram, compared with the normal control group, the levels of UPRO, BUN, and Scr in the doxorubicin model group mice were significantly increased ( p <0.01%, significant glomerular sclerosis, vacuolar degeneration of renal tubular epithelial cells, and collagen deposition were observed in the kidney tissue. Compared with the doxorubicin model group, serum UPRO, BUN, and Scr levels were significantly decreased in both low- and high-dose tramidil groups. p The levels of troponin (<0.01) significantly reduced renal tissue pathological damage and collagen deposition, with the high-dose group showing better results than the low-dose group. The positive control group (losartan) also effectively improved the above indicators, but there was no statistically significant difference compared to either the high-dose or low-dose tropidil groups. These results indicate that tropidil can effectively improve doxorubicin-induced renal function damage and has a significant protective effect against nephrotoxicity.

[0074] Example 5: Tropidil inhibits glomerular podocyte pyroptosis by activating the cAMP / PKA / CREB / NRF2-GPX3 axis. 1. Experimental Methods (1) Animal tissue detection: The kidney tissue of mice in Example 4 was taken and the expression of NLRP3, Cleaved-CASP1, Cleaved-IL-1β, Cleaved-IL-18, GSDMD-N, NRF2, GPX3, p-PKA and p-CREB proteins was detected by Western blot.

[0075] (2) Glomerular podocyte culture and treatment: MPC-5 mouse glomerular podocytes were cultured and divided into the following groups: normal control group (Control), doxorubicin group (DOX, 2 μmol / L), tropidil group (DOX+TRA, 2 μmol / L DOX+50 μmol / L TRA), GPX3 siRNA interference group (DOX+TRA+GPX3 siRNA), NRF2 inhibitor group (DOX+TRA+ML385, ML385 concentration 10 μmol / L), and PKA siRNA group (DOX+TRA+PKA siRNA). After 24 hours of treatment, intracellular ROS levels were detected by DCFH-DA probe; Western blot was used to detect the expression of proteins such as GPX3, NRF2, p-CREB, NLRP3, and Cleaved-CASP1.

[0076] 2. Experimental Results (1) Tropidil reduces oxidative stress and inhibits podocyte pyroptosis. To verify the effects of tropidil on oxidative stress and pyroptosis, oxidative stress-related indicators were first examined. For example... Figure 12 As shown in Figure AB, compared with the normal control group, the intracellular ROS fluorescence intensity of glomerular podocytes in the doxorubicin group was significantly increased ( p <0.01); compared with the doxorubicin group, the tropidil group had a significantly lower ROS level ( p <0.01). The above results indicate that tropidil can effectively alleviate doxorubicin-induced oxidative stress in glomerular podocytes.

[0077] Based on this, the occurrence of pyroptosis and pathway activation were further examined. For example... Figure 13 As shown in the AF, compared with the normal control group, the expression of pyroptosis-related proteins (NLRP3, Cleaved-CASP1, Cleaved-IL-1β, Cleaved-IL-18, GSDMD-N) in the kidney tissue of mice in the doxorubicin model group was significantly increased. p <0.01); Compared with the doxorubicin model group, the expression of the above proteins was significantly downregulated in both the low- and high-dose tropidil groups ( p <0.05, p<0.01), the high-dose group was more effective than the low-dose group; the positive control group also effectively downregulated the expression of the above proteins, but there was no statistically significant difference compared with the high-dose and low-dose tropidil groups. Figure 14 As shown in the AC diagram, compared with the normal control group, the protein expression levels of NRF2 and GPX3 in the kidney tissue of mice in the doxorubicin model group were significantly reduced. p <0.01); Compared with the doxorubicin model group, the expression of NRF2 and GPX3 in both the low- and high-dose tropidil groups significantly increased ( p <0.05, p <0.01), the high-dose group was more effective than the low-dose group.

[0078] (2) Tropidil inhibits pyroptosis of podocytes through the NRF2-GPX3 positive feedback loop. To verify the regulatory relationship between NRF2 and GPX3 and the necessity of this loop in the anti-pyroptosis effect of tropidil, GPX3 siRNA knockdown and NRF2 inhibitor ML385 intervention experiments were conducted. Figure 15 As shown in the middle AE, after GPX3 knockdown by transfection with GPX3 siRNA, the upregulation effect of tropidil on NRF2 was significantly weakened, while the expression of Cleaved-CASP1 and NLRP3 rebounded. p The value <0.01 indicates a positive feedback regulatory relationship between GPX3 and NRF2, and this loop is crucial for the antipyrolysis effect of tropidil. Figure 15 As shown in the middle FH, treatment with the NRF2 inhibitor ML385 significantly inhibited the upregulation of GPX3 by tropidil. p The result of NRF2 <0.01 further confirms that NRF2 is an upstream regulator of GPX3.

[0079] (3) The cAMP / PKA / CREB signal axis is the upstream hub of the loop activated by Tropical. After confirming the NRF2-GPX3 positive feedback loop, the upstream signal mechanism of tropidil activation of NRF2 was further investigated. For example... Figure 16 As shown in the AG, compared with the doxorubicin model group, the expression of cAMP, p-PKA, p-CREB, NRF2, and GPX3 proteins in glomerular podocytes of mice treated with tropidil was significantly upregulated. p <0.01), indicating that tropidil can activate the cAMP / PKA / CREB signaling pathway both in vivo and in vitro. Further reverse validation was performed by constructing a PKA knockdown podocyte model through transfection with PKA-specific siRNA, such as... Figure 16 As shown in HL, PKA knockdown significantly weakened the upregulation effect of tropidil on p-CREB, NRF2, and GPX3. p <0.05,p <0.01), proving that the cAMP / PKA / CREB signal axis is the key upstream hub for the upregulation of the NRF2-GPX3 loop by tropidil.

[0080] The above results indicate that tropidil also reduces oxidative stress and inhibits NLRP3 inflammasome-mediated pyroptosis in glomerular podocytes by activating the cAMP / PKA / CREB signaling axis, upregulating the NRF2-GPX3 positive feedback loop, and inhibiting NLRP3 inflammasome-mediated pyroptosis in glomerular podocytes.

[0081] Example 6: Tropidil inhibits glomerular podocyte apoptosis by activating the NRF2 / HO-1 pathway. 1. Experimental Methods (1) Animal tissue detection: The kidney tissue of mice in Example 4 was taken and the expression of NRF2, HO-1, WT-1, BAX, BCL2 and Cleaved-CASP3 proteins was detected by Western blot.

[0082] (2) Glomerular podocyte experiment: The groups were divided into normal control group (Control), doxorubicin group (DOX, 2 μmol / L), tropidil group (DOX+TRA, 2 μmol / L DOX+50 μmol / L TRA), and NRF2 inhibitor group (DOX+TRA+ML385, ML385 concentration 10 μmol / L). After 24 hours of treatment, the expression of NRF2, HO-1, BAX, BCL2, and Cleaved-CASP3 proteins was detected by Western blot.

[0083] 2. Experimental Results (1) Tropidil inhibits doxorubicin-induced apoptosis of glomerular podocytes. The aforementioned results have confirmed that tropidil can effectively alleviate doxorubicin-induced oxidative stress in podocytes and inhibit pyroptosis. Figure 12 , Figure 13 Based on this, the effect of tropidil on podocyte apoptosis was further investigated.

[0084] like Figure 17 As shown in the middle AE, compared with the normal control group, the expression of glomerular podocyte marker protein WT-1 and anti-apoptotic protein BCL2 in the kidney tissue of mice in the doxorubicin model group was significantly reduced. p <0.01), and the expression of pro-apoptotic proteins BAX and Cleaved-CASP3 was significantly increased ( p <0.01); Compared with the doxorubicin model group, the expression of WT-1 and BCL2 was significantly upregulated in the low- and high-dose tropidil groups ( p <0.05, p <0.01), BAX and Cleaved-CASP3 expression were significantly downregulated (p <0.05, p <0.01). The positive control group also effectively reversed the above-mentioned changes, but there was no statistical difference compared with the high-dose and low-dose tropidil groups.

[0085] (2) Tropidil inhibits podocyte apoptosis by activating the NRF2 / HO-1 pathway. To investigate the molecular mechanism of tropidil's anti-apoptotic effect, the expression of HO-1, a downstream target gene of NRF2, was examined. Figure 18 As shown in Figure AB, compared with the normal control group, the expression of HO-1 protein in the kidney tissue of mice in the doxorubicin model group was significantly reduced ( p <0.01); Compared with the doxorubicin model group, HO-1 expression was significantly upregulated in the low- and high-dose tropidil groups ( p <0.05, p <0.01), the high-dose group was more effective than the low-dose group.

[0086] To verify whether the anti-apoptotic effect of tropidil also depends on the NRF2 / HO-1 pathway, intervention was performed using the NRF2 inhibitor ML385. Figure 18 As shown in the middle CH, compared with the tropidil group, HO-1 expression was significantly inhibited after ML385 treatment ( p <0.05), BAX and Cleaved-CASP3 expression rebounded ( p <0.05), BCL2 expression decreased ( p <0.05).

[0087] The above results indicate that tropidil reduces oxidative stress and inhibits glomerular podocyte apoptosis by activating the NRF2 / HO-1 signaling pathway.

[0088] Example 7: Trapidil does not affect the antitumor efficacy of doxorubicin. 1. Experimental Methods (1) In vitro experiments: The inhibitory effects of different concentrations of doxorubicin (0-10 μmol / L) on various tumor cells (4T1 breast cancer cells, MCF-7 breast cancer cells, HepG2 liver cancer cells, and A549 lung cancer cells) were detected by the MTT assay, and the IC50 was calculated. 50 In IC 50 At the desired concentration, combined treatment with tropidil (50 μmol / L) for 24 hours was performed to assess its effect on tumor cell viability, and the results were compared with those of doxorubicin monotherapy.

[0089] (2) In vivo experiment: 6-8 week old female BALB / c mice were selected and 4T1 breast cancer cells (1×10⁻⁶) were injected subcutaneously into the right axilla. 6 (Each mouse model bearing a tumor was established by selecting one tumor per mouse). Tumors were collected until they reached approximately 100 mm².3 Mice were randomly assigned to three groups (n=6 per group): a PBS control group (Control), an doxorubicin group (DOX), and an doxorubicin + tropidil group (DOX+TRA). The doxorubicin group received intraperitoneal injections of doxorubicin (5 mg / kg, every 4 days); the doxorubicin + tropidil group received tropidil (25 mg / kg / day) via gavage concurrently with doxorubicin injections; and the PBS control group received an equal volume of physiological saline. Tumor volume was measured every 2-3 days during treatment. Mice were sacrificed after the last administration, and the tumors were harvested and weighed. Simultaneously, left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS) were measured by echocardiography to evaluate the cardioprotective effect of tropidil in tumor-bearing mice.

[0090] 2. Experimental Results (1) Results of in vitro experiments are as follows Figure 19 As shown in Figure AB: The combined treatment of tropidil (50 μmol / L) and doxorubicin (IC50 concentration for each cell type) showed no significant difference in inhibition rate against 4T1, MCF-7, HepG2, and A549 tumor cells compared to the doxorubicin monotherapy group. p >0.05), indicating that tropidil does not affect the in vitro antitumor activity of doxorubicin against a variety of tumor cells.

[0091] (2) Results of in vivo experiments are as follows Figure 20 As shown in the AF diagram: there were no significant differences in tumor growth curves and final tumor weight between the doxorubicin group and the doxorubicin + tropidil group. p >0.05), further confirming that tropidil does not affect the antitumor efficacy of doxorubicin in vivo. Meanwhile, in tumor-bearing mice, the doxorubicin group showed a significant decline in cardiac function (significantly reduced LVEF and LVFS). p <0.01 vs PBS group), while the LVEF and LVFS of mice in the doxorubicin + tropidil group were significantly better than those in the doxorubicin group ( p The value <0.05 indicates that tropidil can effectively protect against doxorubicin-induced cardiotoxicity even in tumor-bearing conditions.

[0092] It should be noted that the tumor-bearing model uses anti-tumor efficacy as the primary endpoint, and the most critical cardiac function test is the LVEF / LVFS index; while the normal model has already conducted a comprehensive and systematic assessment of cardiac and renal function.

[0093] The above results indicate that tropidil, at an effective dose for cardioprotective and renal protection, does not affect the antitumor activity of doxorubicin against tumor cells, demonstrating good safety in combination therapy.

[0094] Example 8: Trapidil improves doxorubicin-induced multi-organ toxicity 1. Experimental Methods Four mice were selected from the normal control group (Control), doxorubicin model group (DOX), and tropidil treatment group (DOX+TRA, 25 mg / kg / day) in Example 1. The mice were sacrificed after the last administration, and liver, spleen, and lung tissues were collected for HE staining to observe histopathological changes. Serum was collected to detect the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) to assess liver function.

[0095] 2. Experimental Results like Figure 21 As shown in Figure A, the liver, spleen, and lung tissues of the normal control group mice showed normal structure. In the doxorubicin model group, the liver tissue of mice exhibited significant hepatocyte vacuolar degeneration, focal necrosis, and inflammatory cell infiltration; the spleen tissue showed red pulp dilation and white pulp atrophy; and the lung tissue showed alveolar septal thickening, capillary congestion, and inflammatory cell infiltration. Compared with the doxorubicin model group, the tropidil treatment group showed significantly reduced pathological damage in the liver, spleen, and lung tissues, with tissue structure tending towards normal.

[0096] like Figure 21 As shown in Figure B, the serum ALT and AST levels in the doxorubicin model group mice were significantly higher than those in the normal control group. p <0.01 indicates that doxorubicin induced significant liver function damage; serum ALT and AST levels in mice treated with tropidil were significantly lower than those in the doxorubicin model group ( p <0.01, p The value of <0.05 indicates that tropidil also has a protective effect against doxorubicin-induced liver function damage.

[0097] The above results indicate that tropidil not only protects against doxorubicin-induced cardiotoxicity and nephrotoxicity, but also significantly improves pathological damage to major organs such as the liver, spleen, and lungs, demonstrating a broad-spectrum protective effect against doxorubicin-induced multi-organ toxicity.

[0098] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Use of tropidil in the preparation of drugs for the prevention and / or treatment of doxorubicin-induced organ toxicity.

2. The application according to claim 1, characterized in that, The organ toxicity mentioned is cardiotoxicity.

3. The application according to claim 1, characterized in that, The organ toxicity is nephrotoxicity.

4. The application according to claim 2, characterized in that, The drug exerts its cardioprotective effect by inhibiting pyroptosis and / or apoptosis of cardiomyocytes.

5. The application according to claim 4, characterized in that, The drug inhibits cardiomyocyte pyroptosis by activating the cAMP / PKA / CREB signaling axis, upregulating the NRF2-GPX3 positive feedback loop.

6. The application according to claim 4, characterized in that, The drug inhibits cardiomyocyte apoptosis by activating the NRF2 / HO-1 signaling pathway.

7. The application according to claim 3, characterized in that, The drug exerts its nephroprotective effect by inhibiting pyroptosis and apoptosis of glomerular podocytes.

8. The application according to claim 7, characterized in that, The drug inhibits pyroptosis of glomerular podocytes by activating the cAMP / PKA / CREB signaling axis, upregulating the NRF2-GPX3 positive feedback loop.

9. The application according to claim 7, characterized in that, The drug inhibits glomerular podocyte apoptosis by activating the NRF2 / HO-1 signaling pathway.

10. The application according to claim 1, characterized in that, The organ toxicity also includes hepatotoxicity, spleen toxicity, and / or lung toxicity.

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