Application of amido guanidine compound in resisting tumors and treating myocardial injury caused by adriamycin

Abstract

The invention provides an application of an acylamino guanidine compound in resisting tumors and treating myocardial injury caused by adriamycin. Various pharmacological experiments prove that the NHE1 inhibitor (N-(4-guanidine butyl)-4-hydroxy-3-methoxybenzamide) involved in the invention inhibits oxidative stress and myocardial cell apoptosis caused by adriamycin by reducing the activity of NHE1, and finally relieves myocardial damage caused by adriamycin. In addition, the NHE1 inhibitor provided by the invention has an inhibition effect on human or mouse breast cancer tumor cells. Meanwhile, the NHE1 inhibitor can cooperate with adriamycin to achieve the anti-tumor effect, and the anti-tumor effect of adriamycin is enhanced by promoting the oxidative stress reaction caused by tumor cells and promoting the tumor cell apoptosis level.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and relates to amidoguanidine compounds. Specifically, it relates to the application of amidoguanidine compounds in anti-tumor and the treatment of doxorubicin-induced myocardial injury. Background Art

[0002] Doxorubicin (Dox) is an effective chemotherapeutic drug commonly used to treat various hematological and solid tumors and other malignancies in adults and children. Due to the strong cardiotoxicity of Dox, it can cause dose-dependent cardiomyopathy. Therefore, many cancer patients treated with Dox will develop cardiomyopathy of varying severities, and even lead to death. Clinically, the cumulative dose of Dox is tentatively set at 450 - 600 mg / m 2 . Currently, Dexrazoxane is the only cardiac protectant approved by the FDA to reduce Dox-induced cardiac injury and myocardial dysfunction. It is reported that Dexrazoxane will significantly reduce the response rate of Dox chemotherapy in patients with advanced and metastatic breast cancer, but it will affect the anti-tumor efficacy of Dox. Moreover, Dexrazoxane has carcinogenic potential and may cause acute myeloid leukemia and myelodysplastic syndromes, so its clinical application is limited. Therefore, there is an urgent need to develop safer and more effective therapeutic drugs to overcome Dox-induced myocardial toxicity. Currently, the mechanism of Dox-induced myocardial toxicity is not yet clear, including oxidative stress, mitochondrial dysfunction, metabolic disorders, autophagy disorders, ferroptosis, and apoptosis, etc. Among them, Dox inhibits oxidative phosphorylation and ATP production in H9c2 cells, which can lead to mitochondrial damage and cardiomyocyte apoptosis. Therefore, mitochondrial dysfunction can lead to mitochondrial respiratory inhibition and the production of reactive oxygen species (ROS), all of which are considered to be the key to the pathogenesis of Dox-induced cardiomyopathy. In addition, due to the imbalance between oxidation and antioxidant systems in the body caused by Dox, it can further induce the occurrence of cardiomyocyte apoptosis.

[0003] Na + / H + exchanger (Na + / H + exchanger, NHE) is a transmembrane protein widely present in mammalian cells and is crucial for maintaining normal physiological functions of cells, including regulating intracellular pH value and cell volume, maintaining intracellular Na + and Ca 2+Stability, etc. NHE includes multiple subunits (NHE1 - NHE10). Among them, NHE1 is mainly expressed in cardiac tissues, and its activation is closely related to various pathological processes such as myocardial mitochondrial damage, calcium overload, oxidative stress, fibrosis, inflammation, apoptosis, etc., and is involved in the occurrence and development of arrhythmia, myocardial ischemia - reperfusion injury, cardiac hypertrophy, heart failure, and diabetes. It is reported that NHE1 inhibitors can inhibit the + / H + exchange, avoid excessive Na + from entering the cell, and thus inhibit the Na + / Ca 2+ exchange, prevent the excessive increase of intracellular Ca 2+ level from causing cell contraction and necrosis, thereby having a good protective effect on myocardial ischemia - reperfusion injury. Previous studies have shown that the increase in NHE1 activity is closely related to oxidative stress - related reactions and plays a key role in the occurrence and development of pathological cardiac hypertrophy and heart failure. Therefore, selective inhibition of NHE1 can reduce Dox - induced cardiotoxicity by inhibiting oxidative stress, inflammation, and apoptosis. Therefore, targeted inhibition of NHE1 activity has important value in the treatment of Dox - induced cardiotoxicity. Currently, it is reported that NHE1 inhibitors are divided into selective inhibitors and non - selective inhibitors. Among them, Cariporide, Eniporide, and Zoniporide have all entered the clinical research stage, but the clinical studies of Cariporide and Eniporide have not obtained the expected results. Therefore, finding new structural types of NHE - 1 inhibitors has become a work of great significance. Previous studies synthesized multiple amidinoguanidine compounds, and some of these compounds can significantly inhibit NHE1 activity and have a significant protective effect on Dox - induced myocardial injury. However, the specific mechanism of the protective effect of such amidinoguanidine compounds on Dox - induced myocardial injury is not clear, and whether such amidinoguanidine compounds have other synergistic effects with Dox also needs to be further explored. All of the above are major problems that need to be solved urgently in this field. SUMMARY OF THE INVENTION

[0004] In view of the deficiencies of the prior art, the present invention provides the application of amidinoguanidine compounds in anti - tumor and the treatment of myocardial injury caused by doxorubicin. Based on previous research, taking N - (4 - guanidinobutyl) - 4 - hydroxy - 3 - methoxybenzamide, the NHE1 inhibitor with the strongest protective effect on Dox - induced myocardial injury screened in the previous stage, as the research object, the specific mechanism of its protective effect on Dox - induced myocardial injury and its effect on tumor cells were detected. Through a variety of pharmacological experiments, it was proved that the NHE1 inhibitor involved in the present invention (the structure is shown in Figure 1)It can relieve the cardiomyocyte cytotoxicity induced by Dox by inhibiting oxidative stress, inhibiting apoptosis, and protecting mitochondrial damage. Meanwhile, it has antitumor effects, can synergize with Dox, and enhance the antitumor effect of Dox.

[0005] In the first aspect of the present invention, there is provided the use of an amidoguanidine compound in the preparation of an antitumor drug, characterized in that the amidoguanidine compound is an NHE1 inhibitor.

[0006] Further, the amidoguanidine compound is N-(4-guanidinobutyl)-4-hydroxy-3-methoxybenzamide, and its structural formula is:

[0007]

[0008] Further, the tumor is a solid tumor or a hematological tumor.

[0009] Still further, the tumor is breast cancer.

[0010] In the second aspect of the present invention, there is provided a combined drug for antitumor use, characterized in that the combined drug is the combination of doxorubicin (Dox) and an amidoguanidine compound, and the amidoguanidine compound is an NHE1 inhibitor.

[0011] Further, the amidoguanidine compound is N-(4-guanidinobutyl)-4-hydroxy-3-methoxybenzamide, and its structural formula is:

[0012]

[0013] Further, the drug further comprises a pharmaceutically acceptable carrier or excipient.

[0014] Further, the tumor is a solid tumor or a hematological tumor.

[0015] Further, the tumors include acute leukemia (lymphocytic and granulocytic), malignant lymphoma, breast cancer, bronchogenic carcinoma (undifferentiated small cell and non-small cell), ovarian cancer, soft tissue sarcoma, osteosarcoma, rhabdomyosarcoma, Ewing's sarcoma, blastoma, neuroblastoma, bladder cancer, thyroid cancer, prostate cancer, head and neck squamous cell carcinoma, testicular cancer, gastric cancer, liver cancer, etc.

[0016] Still further, the tumor is breast cancer.

[0017] In the third aspect of the present invention, there is provided the use of the combination of an amidoguanidine compound and doxorubicin (Dox) in the preparation of an antitumor drug, characterized in that the amidoguanidine compound is an NHE1 inhibitor.

[0018] Further, the amidoguanidine compound is N-(4-guanidinobutyl)-4-hydroxy-3-methoxybenzamide, and its structural formula is:

[0019]

[0020] Further, the tumor is a solid tumor or a hematological tumor.

[0021] Further, the tumors include acute leukemia (lymphocytic and granulocytic), malignant lymphoma, breast cancer, bronchogenic carcinoma (undifferentiated small cell and non-small cell), ovarian cancer, soft tissue sarcoma, osteosarcoma, rhabdomyosarcoma, Ewing's sarcoma, blastoma, neuroblastoma, bladder cancer, thyroid cancer, prostate cancer, squamous cell carcinoma of the head and neck, testicular cancer, gastric cancer, liver cancer, etc.

[0022] Even further, the tumor is breast cancer.

[0023] In the fifth aspect of the present invention, there is provided the use of an amidoguanidine compound in the preparation of a drug for reducing the increase in lactate dehydrogenase (LDH) in cardiomyocytes caused by doxorubicin, characterized in that the amidoguanidine compound is an NHE1 inhibitor.

[0024] Further, the amidoguanidine compound is N-(4-guanidinobutyl)-4-hydroxy-3-methoxybenzamide, and its structural formula is:

[0025]

[0026] In the sixth aspect of the present invention, there is provided the use of an amidoguanidine compound in the preparation of a drug for inhibiting doxorubicin-induced cardiomyocyte hypertrophy, characterized in that the amidoguanidine compound is an NHE1 inhibitor.

[0027] Further, the amidoguanidine compound is N-(4-guanidinobutyl)-4-hydroxy-3-methoxybenzamide, and its structural formula is:

[0028]

[0029] In the seventh aspect of the present invention, there is provided the use of an amidoguanidine compound in the preparation of a drug for inhibiting the decline in mitochondrial viability of cardiomyocytes caused by doxorubicin, characterized in that the amidoguanidine compound is an NHE1 inhibitor.

[0030] Further, the amidoguanidine compound is N-(4-guanidinobutyl)-4-hydroxy-3-methoxybenzamide, and its structural formula is:

[0031]

[0032] In the eighth aspect of the present invention, there is provided the use of an amidoguanidine compound in the preparation of a drug for inhibiting the oxidative stress response of cardiomyocytes caused by doxorubicin, wherein the amidoguanidine compound is an NHE1 inhibitor.

[0033] Further, the inhibition of the oxidative stress response of cardiomyocytes caused by doxorubicin is specifically manifested as restoring the decrease in the content of superoxide dismutase (SOD), the increase in the content of malondialdehyde (MDA), or the increase in reactive oxygen species (ROS) in cardiomyocytes caused by doxorubicin.

[0034] Further, the amidoguanidine compound is N-(4-guanidinobutyl)-4-hydroxy-3-methoxybenzamide, and its structural formula is:

[0035]

[0036] In the ninth aspect of the present invention, there is provided the use of an amidoguanidine compound in the preparation of a drug for reducing the apoptosis of cardiomyocytes caused by doxorubicin, wherein the amidoguanidine compound is an NHE1 inhibitor.

[0037] Further, the amidoguanidine compound is N-(4-guanidinobutyl)-4-hydroxy-3-methoxybenzamide, and its structural formula is:

[0038]

[0039] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described hereinafter (such as in the examples) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be repeated one by one here.

[0040] Compared with the prior art, the present invention has the following advantages and progressiveness:

[0041] Based on previous research, the present invention takes N-(4-guanidinobutyl)-4-hydroxy-3-methoxybenzamide, the NHE1 inhibitor with the strongest protective effect on doxorubicin-induced myocardial injury screened in the previous stage, as the research object, and detects the specific mechanism of its protective effect on doxorubicin-induced myocardial injury and its effect on tumor cells.

[0042] The NHE1 inhibitor involved in the present invention (the structure is shown in Figure 1)By reducing NHE1 activity, inhibiting doxorubicin-induced oxidative stress and cardiomyocyte apoptosis, and ultimately alleviating doxorubicin-induced myocardial injury. The present invention has demonstrated the inhibitory effect of this NHE1 inhibitor on NHE1 activity and protein expression through various pharmacological methods; by detecting intracellular LDH, SOD, MDA, ROS and other indicators, the inhibitory effect of this NHE1 inhibitor on doxorubicin-induced oxidative stress in cardiomyocytes was further detected, and it was confirmed by detecting mitochondrial membrane potential that this inhibitor has a protective effect on doxorubicin-induced mitochondrial damage in cardiomyocytes. Finally, the effect of this NHE1 inhibitor on doxorubicin-induced cardiomyocyte apoptosis was detected by detecting apoptosis indicators (Bcl-2, Bax, Caspase3 and TUNEL staining).

[0043] In addition, the NHE1 inhibitor of the present invention has an inhibitory effect on human or mouse breast cancer tumor cells. At the same time, the NHE1 inhibitor of the present invention can synergistically achieve an anti-tumor effect with doxorubicin and enhance the anti-tumor effect of doxorubicin. Specifically, the NHE1 inhibitor of the present invention can promote the oxidative stress response caused by breast cancer cells, thereby enhancing the anti-cancer activity of Dox; the NHE1 inhibitor of the present invention can promote the apoptosis level of breast cancer cells, thereby enhancing the anti-cancer activity of Dox; at the same time, the present invention further verified through cell experiments and in vivo experiments that the NHE1 inhibitor of the present invention can significantly synergistically promote the anti-tumor effect of doxorubicin. Brief Description of the Drawings

[0044] The drawings forming a part of the present invention are used to provide a further understanding of the present invention. The schematic embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention.

[0045] Figure 1 Shows the amidoguanidine structure as an NHE1 inhibitor involved in the embodiments of the present invention.

[0046] Figure 2 Shows the effect of the NHE1 inhibitor in the examples of the present invention on the survival rate of H9c2 cardiomyocytes. This figure shows that the NHE1 inhibitor has no toxicity to H9c2 cardiomyocytes.

[0047] Figure 3 Shows the effect of the NHE1 inhibitor in the examples of the present invention on the survival rate of MCF-7 human breast cancer cells. This figure shows that the NHE1 inhibitor has an inhibitory effect on MCF-7 human breast cancer cells.

[0048] Figure 4 Shows the effect of the NHE1 inhibitor in the examples of the present invention on the survival rate of 4T1 mouse breast cancer cells. This figure shows that the NHE1 inhibitor has an inhibitory effect on 4T1 mouse breast cancer cells.

[0049] Figure 5 It shows the effect of the NHE1 inhibitor combined with doxorubicin on the survival rate of MCF-7 human breast cancer cells in the examples of the present invention. This figure indicates that the NHE1 inhibitor has no inhibitory effect on the anticancer activity of doxorubicin.

[0050] Figure 6 It shows the effect of the NHE1 inhibitor combined with doxorubicin on the survival rate of 4T1 mouse breast cancer cells in the examples of the present invention. This figure indicates that the NHE1 inhibitor has no inhibitory effect on the anticancer activity of doxorubicin.

[0051] Figure 7 It shows the effect of the NHE1 inhibitor on the cytotoxicity of doxorubicin-induced H9c2 cardiomyocytes in the examples of the present invention. This figure indicates that the addition of the NHE1 inhibitor can alleviate the decrease in the survival rate of cardiomyocytes caused by doxorubicin.

[0052] Figure 8 It shows the effect of the NHE1 inhibitor on the intracellular pH in the examples of the present invention. This figure indicates that the NHE1 inhibitor reduces NHE1 activity and the inhibitory effect is significantly higher than that of the typical NHE1 inhibitor cariporide.

[0053] Figure 9 It shows the change in the expression level of NHE1 protein by the NHE1 inhibitor in the examples of the present invention. This figure indicates that the NHE1 inhibitor reduces the expression of NHE1 protein.

[0054] Figure 10 It shows the effect of the NHE1 inhibitor on the activity of lactate dehydrogenase in H9c2 cardiomyocytes in vivo in the examples of the present invention. This figure indicates that the addition of the NHE1 inhibitor can reduce the increase in LDH caused by doxorubicin.

[0055] Figure 11 It shows the effect of the NHE1 inhibitor on the surface area of H9c2 cardiomyocytes in the examples of the present invention. This figure indicates that the addition of the NHE1 inhibitor can inhibit cardiomyocyte hypertrophy caused by doxorubicin.

[0056] Figure 12 It shows the effect of the NHE1 inhibitor on the mitochondrial viability of H9c2 cardiomyocytes in the examples of the present invention. This figure indicates that the NHE1 inhibitor reduces the decrease in mitochondrial viability of cells caused by doxorubicin.

[0057] Figure 13 It shows the effect of the NHE1 inhibitor on the content of superoxide dismutase in H9c2 cardiomyocytes in vivo in the examples of the present invention. This figure indicates that the addition of the NHE1 inhibitor can restore the decrease in SOD content caused by doxorubicin.

[0058] Figure 14Shows the effect of the NHE1 inhibitor in the examples of the present invention on the malondialdehyde content in H9c2 cardiomyocytes in vivo. This figure shows that the addition of the NHE1 inhibitor can inhibit the increase in MDA content caused by doxorubicin.

[0059] Figure 15 Shows the effect of the NHE1 inhibitor in the examples of the present invention on the reactive oxygen species in H9c2 cardiomyocytes. This figure shows that the addition of the NHE1 inhibitor can inhibit the increase in ROS caused by doxorubicin.

[0060] Figure 16 Shows the effect of the NHE1 inhibitor in the examples of the present invention on the apoptosis level of H9c2 cardiomyocytes. This figure shows that the NHE1 inhibitor reduces the apoptosis caused by doxorubicin.

[0061] Figure 17 Shows the change in the expression level of Bcl-2 by the NHE1 inhibitor in the examples of the present invention. This figure shows that the NHE1 inhibitor increases the expression of Bcl-2 protein while the NHE1 agonist reverses this effect.

[0062] Figure 18 Shows the change in the expression level of Bax by the NHE1 inhibitor in the examples of the present invention. This figure shows that the NHE1 inhibitor reduces the expression of Bax protein while the NHE1 agonist reverses this effect.

[0063] Figure 19 Shows the change in the expression level of cleaved-Caspase-3 by the NHE1 inhibitor in the examples of the present invention. This figure shows that the NHE1 inhibitor reduces the expression of cleaved-Caspase-3 protein while the NHE1 agonist reverses this effect.

[0064] Figure 20 Shows the effect of the NHE1 inhibitor in the examples of the present invention on the reactive oxygen species in 4T1 murine breast cancer cells. This figure shows that the NHE1 inhibitor has a promoting effect on the oxidative stress produced by tumor cells.

[0065] Figure 21 Shows the effect of the NHE1 inhibitor in the examples of the present invention on the apoptosis level of 4T1 murine breast cancer cells. This figure shows that the NHE1 inhibitor can promote the apoptosis level of 4T1 murine breast cancer cells.

[0066] Figure 22 Shows the effect of the NHE1 inhibitor in the examples of the present invention on the body weight of tumor-bearing mice and the tumors in vivo in 4T1 breast cancer cells. This figure shows that the NHE1 inhibitor can significantly promote the anti-tumor effect of doxorubicin.

[0067] Figure 23Shows the effect of the NHE1 inhibitor on tumors in 4T1 breast cancer cells in vivo in the examples of the present invention. This figure shows that the NHE1 inhibitor can significantly enhance the anti-tumor effect of doxorubicin. Detailed implementation mode

[0068] The present invention provides the application of amidoguanidine compounds in anti-tumor and in treating myocardial injury caused by doxorubicin. The following is a specific description of the present invention in combination with examples to facilitate the further understanding of the present invention by those skilled in the art. However, the examples described below are only a part of the examples of the present invention and should not be regarded as any form of limitation to the present invention. It should be noted that the adjustments and improvements made by those of ordinary skill in the art based on the concept of the present invention should be regarded as the protection scope of the present invention. For the specific technical operation steps and operators not specified in the examples, they are all carried out according to the general technical conditions described in the literature in this field or the relevant product specifications.

[0069] Example 1: Cell lines and cell culture

[0070] The cells used in this example are H9c2 rat cardiomyocytes, MCF-7 human breast cancer cells and 4T1 mouse breast cancer cells. H9c2 rat cardiomyocytes are cultured in DMEM medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin solution; MCF-7 cells and 4T1 cells are cultured in 1640 medium containing 10% FBS and 1% penicillin-streptomycin. All cells are cultured at 37 °C and 5% CO2.

[0071] Example 2: Effect of NHE1 inhibitor on the survival rate of H9c2 rat cardiomyocytes

[0072] The specific grouping and drug administration are as follows: H9c2 rat cardiomyocytes were divided into the following eight groups, namely the control group (CON), the NHE1 inhibitor 0.05 μM group (0.05 μM), the NHE1 inhibitor 0.1 μM group (0.1 μM), the NHE1 inhibitor 0.5 μM group (0.5 μM), the NHE1 inhibitor 1 μM group (1 μM), the NHE1 inhibitor 5 μM group (5 μM), the NHE1 inhibitor 10 μM group (10 μM), and the NHE1 inhibitor 20 μM group (20 μM). Except for the cells in the CON group which were added with blank medium, the remaining groups were respectively treated with different concentrations of NHE1 inhibitor (0.05 μM, 0.1 μM, 0.5 μM, 1 μM, 5 μM, and 10 μM). The cells in each of the above groups were seeded in 96-well plates, 5000 cells per well. After treatment with drugs according to different groups for 24 h, the cells were washed with PBS, then added with MTT reagent at a final concentration of 0.5 mg / ml, incubated at 37 °C for 3 h, the medium was aspirated, and 150 μl of DMSO was added. The absorbance values of each well at a wavelength of 572 nm were detected using a microplate reader. The cell survival rate of each group was calculated as follows:

[0073]

[0074] Figure 2 It shows the effect of NHE1 inhibitor (0.05 - 20 μM) on the survival rate of H9c2 cardiomyocytes. In this range, there was no significant difference in the cell survival rate compared with the CON group, indicating that this inhibitor has no toxic effect on H9c2 cardiomyocytes within the concentration range of 0.05 - 20 μM.

[0075] Example 3: Effect of NHE1 inhibitor on the survival rate of breast cancer cells

[0076] The specific grouping and drug administration are as follows: MCF-7 human breast cancer cells and 4T1 mouse breast cancer cells were each divided into the following four groups, namely the control group (CON), the NHE1 inhibitor 0.05 μM group (0.05 μM), the NHE1 inhibitor 1 μM group (1 μM), and the NHE1 inhibitor 10 μM group (10 μM). Except for the cells in the CON group which were added with blank medium, the remaining groups were respectively treated with different concentrations of NHE1 inhibitor (0.05 μM, 1 μM, and 10 μM). The cells in each of the above groups were seeded in 96-well plates, 5000 cells per well. After treatment with drugs according to different groups for 24 h, the cells were washed with PBS, then added with MTT reagent at a final concentration of 0.5 mg / ml, incubated at 37 °C for 3 h, the medium was aspirated, and 150 μl of DMSO was added. The absorbance values of each well at a wavelength of 572 nm were detected using a microplate reader. The cell survival rate of each group was calculated as follows:

[0077]

[0078] As Figure 3 and Figure 4 shown. After adding the NHE1 inhibitor, as the concentration increased, the survival rates of both MCF-7 cells and 4T1 cells decreased. Compared with the CON group, when the concentration of the NHE1 inhibitor reached 10 μM, the survival rate of MCF-7 tumor cells was approximately 70% (P < 0.001), and the survival rate of 4T1 cells was approximately 80% (P < 0.001). This indicates that NHE1 inhibition has a certain inhibitory effect on MCF-7 and 4T1 tumor cells.

[0079] Example 4: Effect of NHE1 Inhibitor Combined with Dox on the Survival Rate of Breast Cancer Cells

[0080] The specific grouping and drug administration are as follows: MCF-7 human breast cancer cells and 4T1 mouse breast cancer cells are each divided into the following five groups, namely the control group (CON), the Dox group (Dox), the Dox + 0.05 μM inhibitor group (0.05 μM), the Dox + 1 μM inhibitor group (1 μM), and the Dox + 10 μM (10 μM) inhibitor group. Cells in the CON group were added with blank medium, cells in the Dox group were added with 2 μM Dox, and cells in each inhibitor group were added with 2 μM Dox and different concentrations of the NHE1 inhibitor (0.05 μM, 1 μM, and 10 μM respectively) for treatment. Cells in the above groups were all seeded in 96-well plates, 5000 cells / well. After treatment with drugs according to different groups for 24 h, the cells were washed with PBS, MTT reagent with a final concentration of 0.5 mg / ml was added, and incubated at 37 °C for 3 h. The medium was aspirated, and 150 μl of DMSO was added. The absorbance value of each well at a wavelength of 572 nm was detected using an enzyme-linked immunosorbent assay (ELISA) reader. The survival rate of each group of cells was calculated as follows:

[0081]

[0082] In this example, the effect of this inhibitor combined with Dox on the survival rate of tumor cells was further detected, as Figure 5 shown. The survival rate of MCF-7 tumor cells in the group treated with Dox alone was approximately 80% (P < 0.001). After adding the NHE1 inhibitor, as the concentration increased, the survival rate of MCF-7 cells decreased (P < 0.01). When the concentration of the NHE1 inhibitor reached 10 μM, the survival rate of MCF-7 tumor cells was approximately 70%. As Figure 6As shown, compared with the CON group, the survival rate of 4T1 tumor cells in the Dox group alone was approximately 70% (P < 0.001). After adding the NHE1 inhibitor, as the concentration increased, the survival rate of 4T1 cells decreased (P < 0.05). When the concentration of the NHE1 inhibitor reached 1 μM and 10 μM, the survival rates of 4T1 tumor cells were approximately 65% and 60% (P < 0.05). The above results prove that this inhibitor can synergistically enhance the anti-tumor effect of Dox.

[0083] Example 5: Effect of NHE1 inhibitor on the reduction of survival rate of H9c2 rat cardiomyocytes caused by Dox

[0084] The specific grouping and drug administration are as follows: H9c2 rat cardiomyocytes were divided into the following seven groups, namely the control group (CON), the Dox group (Dox), the Dox + 0.05 μM inhibitor group (0.05 μM), the Dox + 0.1 μM inhibitor group (0.1 μM), the Dox + 0.5 μM inhibitor group (0.5 μM), the Dox + 1 μM inhibitor group (1 μM), and the Dox + 10 μM inhibitor group (10 μM). Cells in the CON group were added with blank medium, cells in the Dox group were added with 2 μM Dox, and cells in each inhibitor group were respectively added with 2 μM Dox and different concentrations of NHE1 inhibitor (0.05 μM, 0.1 μM, 0.5 μM, 1 μM, and 10 μM) for treatment. Cells in the above groups were all inoculated in 96-well plates, 5000 cells / well. After treatment with drugs according to different groupings for 24 h, the cells were washed with PBS, MTT reagent with a final concentration of 0.5 mg / ml was added, and incubated at 37 °C for 3 h. The medium was aspirated, and 150 μl of DMSO was added. The absorbance values of each well at a wavelength of 572 nm were detected using an enzyme-linked immunosorbent assay (ELISA) reader. The calculation of the survival rate of each group of cells is as follows:

[0085]

[0086] As Figure 7 shown, when treating H9c2 cardiomyocytes with Dox, simultaneously adding the NHE1 inhibitor (0.05 - 10 μM) could significantly increase the cell survival rate (P < 0.001). When the concentration of the NHE1 inhibitor was 1 μM, the cell survival rate was the highest. Therefore, the NHE1 inhibitor can alleviate the decrease in the survival rate of H9c2 cardiomyocytes caused by Dox.

[0087] Example 6: Detection of NHE1 activity

[0088] After 24 h of inoculating each group of H9c2 rat cardiomyocytes into 96-well plates, the grouping and treatment were as follows: The cells were divided into the following six groups, namely the acidification group (Model), Dox group (Dox), Dox + 0.05 μM inhibitor group (0.05 μM), Dox + 1 μM inhibitor group (1 μM), Dox + 10 μM inhibitor group (10 μM), and Dox + 10 μM cariporide group (Cariporide). Cells in the Model group were added with blank medium, cells in the Dox group were added with 2 μM Dox, cells in the Cariporide group were added with 2 μM Dox and 10 μM Cariporide, and cells in each inhibitor group were respectively added with 2 μM Dox and different concentrations of the novel NHE1 inhibitor (0.05 μM, 1 μM, and 10 μM respectively) for treatment. After incubating each group of cells with Krebs solution (135 mM sodium chloride, 5.9 mM potassium chloride, 1.5 mM calcium chloride, 1.2 mM magnesium chloride, 11.6 mM HEPES, 11.5 mM glucose, adjusted to pH 7.4 with KOH, stored at 4 °C) for 30 min, 1 μM BCECF-AM fluorescent probe was added to each well and incubated for 30 min, then washed away, and the cells were washed with Krebs solution. Subsequently, except for the standard curve group, the remaining groups were acidified with NH4Cl for 30 min, washed away, and then washed twice with sodium-free Krebs solution (135 mM choline chloride, 5.9 mM potassium chloride, 1.5 mM calcium chloride, 1.2 mM magnesium chloride, 11.6 mM HEPES, 11.5 mM glucose, adjusted to pH 7.4 with KOH, stored at 4 °C). Finally, the fluorescence values (FIR 始 ) of each group of cells were measured by a fluorescence microplate reader at an emission wavelength of 480 nm and 440 nm and an excitation wavelength of 530 nm. Subsequently, Krebs solution was added, and the fluorescence values (FIR 末 ) were continuously measured at the above wavelengths. The calculation method of FIR was as follows: FIR = FIR 480nm / FIR 440nm ×100%. For the preparation of the pHi standard curve, cells in five wells of the 96-well plate that had been inoculated and not treated were taken, incubated with a high-potassium solution containing nigericin, and the pH values were 6.5, 6.8, 7.1, 7.4, and 7.7 respectively. The FIR values of the cells in each well were measured according to the above method. Subsequently, the regression equation was obtained by using the pH value of each well of cells and the corresponding FIR, and the pHi standard curve was made. Finally, according to the standard pHi curve, the pHi values corresponding to FIR 始 and FIR 末 were obtained, which were pHi 始 and pHi 末 . The activity of NHE1 was expressed as ΔpHi = (pHi 末 - pHi 始 ).

[0089] As Figure 8 shown, compared with the Model group, the NHE1 activity of cells in the Dox group was significantly increased (P<0.01); while both 1 μM and 10 μM inhibitors could significantly reduce the increase in NHE1 activity caused by Dox (P<0.001 or P<0.01), and the 0.05 μM inhibitor had no effect on the increase in NHE1 activity caused by Dox. Cariporide is a typical NHE1 inhibitor at present. Cariporide could significantly reduce the increase in NHE1 activity caused by Dox (P<0.01), but its inhibitory effect was significantly lower than that of the 1 μM inhibitor group.

[0090] Example 7: Detection of NHE1 protein level

[0091] (1) Extraction of total cellular proteins

[0092] The cell grouping and treatment were as follows: Cells were inoculated and cultured for 24 h, and divided into a control group (CON), a Dox group (Dox), a 1 μM inhibitor group (1 μM), and a 10 μM inhibitor group (10 μM). Cells in the CON group were added with blank medium; cells in the Dox group were added with 2 μM Dox; cells in different concentration inhibitor groups were respectively added with 2 μM Dox and different concentrations of inhibitors (1 μM and 10 μM respectively). After 24 h of drug administration, cells were washed three times with PBS. After aspiration, cell lysate was added according to the cell density. Cells were scraped off on ice with a cell scraper, and the liquid was aspirated into a sorted centrifuge tube, mixed well and placed at -80 °C for 30 min. Subsequently, the centrifuge was pre-cooled at 4 °C, and the rotation speed was set at 12000 rpm for 10 min. Finally, the supernatant was aspirated into a new centrifuge tube. Except for taking a small amount for protein concentration measurement by BCA method, 5×loading buffer was added according to the volume, and it was boiled in a constant temperature water bath at 100 °C for 10 min, and stored at -20 °C after cooling to room temperature for standby.

[0093] (2) Determination of protein concentration

[0094] This experiment was carried out according to the instructions of the BCA kit (Shanghai Hongye Biotechnology Co., Ltd., product number GK10009). According to the number of samples, the BSA working solution was prepared at a volume ratio of A solution:B solution = 50:1. 25 μl of each of protein standards 1 - 8 (corresponding to 2000 μg / ml, 1500 μg / ml, 1000 μg / ml, 750 μg / ml, 500 μg / ml, 250 μg / ml, 125 μg / ml and 0 μg / ml respectively) were added to the 96-well plate in turn. An appropriate amount of the proteins extracted from each group above was added to the wells, and an appropriate amount of PBS was added to make up to 25 μL. The working solution was added and incubated at 37 °C for 30 min. The absorbance was measured at 562 nm with an enzyme-linked immunosorbent assay (ELISA) reader, and the BSA standard curve was plotted.

[0095] (3) Western blot

[0096] Electrophorese the above-extracted proteins using a 10% SDS-PAGE gel. Uniformly add the denatured proteins to the gel lanes and add protein marker for localization. Subsequently, set the electrophoresis program voltage to a constant 90 V for operation, observe the proteins running to the boundary between the stacking gel and the separating gel, and adjust the voltage to 120 V for about 60 min. Then cut a PVDF membrane of appropriate size and soak it in methanol for 4 min. After rinsing with water, place it in the transfer buffer for use. Take out the gel, cut off the stacking gel, and then cut a suitable size and place it on a sponge pad with filter paper. Subsequently, place the PVDF membrane, remove the air bubbles. Then cover it with filter paper. Clamp it and put it into the transfer tank, add the transfer buffer, set the program current to 200 mA, and run at a constant current of 4 °C for 2 h to transfer the proteins and the marker to the PVDF membrane. Block with 5% non-fat milk for about 2 h, and then wash three times with TBST. Incubate with the primary antibodies against caspase3, Bcl-2, Bax, NHE1, GAPDH, and β-actin at 4 °C overnight. The next day, wash three times with TBST, incubate with the secondary antibody at room temperature for 90 min, then wash three times with TBST, develop the color with a chemiluminescent kit, and use Image J to statistically analyze the gray value of the bands.

[0097] As Figure 9 shown, the expression level of NHE1 protein in the Dox group was about 1.6 times that in the CON group (P < 0.05). The expression levels of NHE1 protein in cells treated with 1 μM and 10 μM inhibitors were significantly decreased compared with the Dox group (P < 0.05). Therefore, NHE1 inhibitors can significantly reduce the increase in NHE1 protein expression level caused by Dox, but there is no concentration dependence.

[0098] Example 8: Determination of lactate dehydrogenase (LDH) level

[0099] Grouping and treatment of H9c2 rat cardiomyocytes are as follows: The cells were divided into the following five groups: control group (CON), Dox group (Dox), 1 μM inhibitor group (1 μM), 10 μM inhibitor group (10 μM), and LiCl group (LiCl). Cells in the CON group were added with blank medium; cells in the Dox group were added with 2 μM Dox; cells in different concentration inhibitor groups were added with 2 μM Dox and different concentrations of inhibitors (1 μM and 10 μM respectively), and cells in the LiCl group were added with 2 μM Dox, 1 μM inhibitor and 1 mM LiCl. After 24 hours of drug administration, the culture medium was aspirated for detection. Take centrifuge tubes, add samples in sequence and mix well according to the operation table of the kit (Nanjing Jiancheng Bioengineering Institute, product number A020-1), and incubate at 37 °C for 15 min. Subsequently, 2,4-dinitrophenylhydrazine was added and incubated at 37 °C for 15 min. Then 0.4 mol / L NaOH solution was added, and after mixing, it was left at room temperature for 3 minutes. 100 μL / well of the supernatant was added to each well, and the OD value of each well was read at a wavelength of 440 nm. Calculate the intracellular LDH activity (U / L) according to the formula.

[0100]

[0101] As Figure 10 shown, the LDH activity of H9c2 cardiomyocytes in each group was detected using a kit. The LDH activity in the supernatant of cells in the Dox group was significantly higher than that of cells in the CON group (P < 0.001). After treatment with NHE1 inhibitors, compared with the Dox group, the LDH activities of cells in the 1 μM and 10 μM inhibitor groups were significantly decreased (P < 0.001); and the NHE1 agonist LiCl could significantly reverse the above effects of NHE1 inhibitors (P < 0.001). The above results indicate that NHE1 inhibitors protect cardiomyocytes by inhibiting NHE1 and reducing the increase in LDH caused by Dox.

[0102] Example 9: Detection of cell size by Action-Tracker Green staining

[0103] H9c2 rat cardiomyocytes were seeded in confocal dishes and divided into a control group (CON), a Dox group (Dox), a 1 μM inhibitor group (1 μM), a 10 μM inhibitor group (10 μM), and a LiCl group (LiCl). Cells in the CON group were added with blank medium; cells in the Dox group were added with 2 μM Dox; cells in the different concentration inhibitor groups were added with 2 μM Dox and different concentrations of inhibitors (1 μM and 10 μM respectively), and cells in the LiCl group were added with 2 μM Dox, 1 μM inhibitor, and 1 mM LiCl. After 24 h of drug treatment, the H9c2 rat cardiomyocytes were washed twice with PBS, then fixed with paraformaldehyde for 10 min. After sucking off the fixing solution, the cells were washed 3 times with PBS containing 0.1% Triton X-100. Subsequently, Action-TrackerGreen was diluted 1:100 with PBS containing 5% BCA and 0.1% Triton X-100, and the cells were incubated for 60 min. Then the cells were washed 3 times with PBS containing 0.1% Triton X-100. Finally, the cells were counterstained with DAPI, washed 3 times with PBS containing 0.1% Triton X-100, observed under a fluorescence microscope (excitation wavelength / emission wavelength were 495 nm / 518 nm respectively), and the cell area was analyzed using Image J.

[0104] As Figure 11 shown, compared with the CON group, after stimulating H9c2 cardiomyocytes with Dox, the cell surface area increased significantly (P < 0.001). The cell surface area in the 1 μM NHE1 inhibitor group was significantly reduced compared with the Dox group (P < 0.001), indicating that this inhibitor could significantly inhibit Dox-induced H9c2 cell hypertrophy. The NHE1 agonist Licl increased the cell surface area (P < 0.001). The above results suggest that the NHE1 inhibitor alleviates Dox-induced H9c2 cardiomyocyte hypertrophy by inhibiting NHE1 activity.

[0105] Example 10: Detection of mitochondrial membrane potential in cells by Mito-Tracker Red CMXRos staining

[0106] H9c2 rat cardiomyocytes were seeded in confocal dishes. After 24 h of culture, the cells were divided into a control group (CON), a Dox group (Dox), a 1 μM inhibitor group (1 μM), a 10 μM inhibitor group (10 μM), and a LiCl group (LiCl). Cells in the CON group were added with blank medium; cells in the Dox group were added with 2 μM Dox; cells in the different concentration inhibitor groups were added with 2 μM Dox and different concentrations of inhibitors (1 μM and 10 μM respectively), and cells in the LiCl group were added with 2 μM Dox, 1 μM inhibitor and 1 mM LiCl. After 24 h of drug treatment, the medium was aspirated and the cells were rinsed once with PBS. Subsequently, Mito-Tracker Red CMXRos was diluted with blank medium at a ratio of 1:2000, and the cells were incubated for 30 min. Then the cells were washed 3 times with PBS. Paraformaldehyde and Triton X-100 were added for fixation and permeabilization for 30 min, and then the cells were washed 3 times with PBS. Finally, DAPI was added for counterstaining, and the cells were washed 3 times with PBS. The cells were observed under a fluorescence microscope (excitation wavelength / emission wavelength were 579 nm / 599 nm respectively), and the fluorescence intensity was statistically analyzed using Image J.

[0107] The results are as Figure 12 shown. Compared with the CON group, the red fluorescence intensity of H9c2 cardiomyocytes treated with Dox was significantly decreased (P < 0.001). The red fluorescence intensity in the 1 μM and 10 μM inhibitor groups was significantly increased compared with the Dox group (P < 0.001). However, the LiCl group reversed the above effects and decreased the red fluorescence intensity (P < 0.001). It can be seen that Dox can reduce the activity of cell mitochondria through mitochondrial membrane potential, thereby inducing cell apoptosis, and the novel NHE1 inhibitor can alleviate the above situation.

[0108] Example 11: Detection of SOD level by WST-1 method

[0109] The grouping and treatment of H9c2 rat cardiomyocytes were as follows: The cells were divided into the following five groups: a control group (CON), a Dox group (Dox), a Dox + 1 μM inhibitor group (1 μM), a Dox + 10 μM inhibitor group (10 μM), and a Dox + 1 μM inhibitor + LiCl group (LiCl). Cells in the CON group were added with blank medium; cells in the Dox group were added with 2 μM Dox; cells in the different concentration inhibitor groups were added with 2 μM Dox and different concentrations of inhibitors (1 μM and 10 μM respectively), and cells in the LiCl group were added with 2 μM Dox, 1 μM inhibitor and 1 mM LiCl. After 24 h of drug administration, the medium was aspirated, and after washing with PBS, an appropriate amount of lysis buffer was added. The cells were scraped off with a cell scraper and aspirated into a 1.5 mL Eppendorf tube. After mixing, the samples were placed at -80 °C. Another small amount of protein sample was taken to measure the protein concentration.

[0110] Take a centrifuge tube, add samples in sequence and mix well according to the kit operation manual (Nanjing Jiancheng Bioengineering Institute, catalog number A001-3), incubate at 37°C for 20 min, and read the OD value of each well at a wavelength of 450 nm. Calculate the intracellular SOD content (U / mg prot) according to the formula.

[0111]

[0112] SOD activity = SOD inhibition rate ÷ 50% × dilution factor of the reaction system ÷ protein concentration of the sample to be tested

[0113] The SOD, MDA, and ROS levels of H9c2 cardiomyocytes in each group were detected using a kit, and the results were as Figure 13 shown. After treatment with Dox, the SOD activity of H9c2 cardiomyocytes decreased significantly compared with that of the CON group (P<0.001). Compared with the Dox group, 1 μM and 10 μM inhibitors significantly increased the SOD activity of Dox group cells (P<0.001). Compared with the 1 μM inhibitor group, LiCl significantly decreased the SOD level (P<0.01).

[0114] Example 12: Detection of MDA level by the TBA method

[0115] The grouping and treatment of H9c2 rat cardiomyocytes were as follows: The cells were divided into the following five groups: control group (CON), Dox group (Dox), 1 μM inhibitor group (1 μM), 10 μM inhibitor group (10 μM), and LiCl group (LiCl). Cells in the CON group were added with blank medium; cells in the Dox group were added with 2 μM Dox; cells in different concentration inhibitor groups were added with 2 μM Dox and different concentrations of inhibitors (1 μM and 10 μM respectively), and cells in the LiCl group were added with 2 μM Dox, 1 μM inhibitor, and 1 mM LiCl. After 24 h of drug administration, the medium was aspirated, and after washing with PBS, an appropriate amount of lysis buffer was added. The cells were scraped off with a cell scraper and aspirated into a 1.5 mL Eppendorf tube, mixed well, and placed at -80°C. Another small amount of protein sample was taken to measure the protein concentration. Take a centrifuge tube and make small holes in the tube cap. Add samples in sequence and mix well according to the kit operation manual (Nanjing Jiancheng Bioengineering Institute, catalog number A003-2), heat in a water bath at 95°C for 40 min, cool to room temperature in running water, centrifuge at 4000 rpm / min for 10 min, take the supernatant and add it to a 96-well plate, with 100 μL / well of the supernatant added to each well. Read the OD value of each well at a wavelength of 532 nm. Calculate the intracellular MDA content (nmol / mg ptot) according to the formula.

[0116]

[0117] The results were as Figure 14As shown, after treatment with Dox, the MDA content in H9c2 cardiomyocytes increased significantly (P<0.001). Compared with the Dox group, 1 μM and 10 μM inhibitors significantly reduced the MDA level in the Dox group cells (P<0.05). Compared with the 1 μM inhibitor group, LiCl significantly increased MDA (P<0.05). Therefore, NHE1 inhibitors protect cardiomyocytes by inhibiting NHE1 and reducing the oxidative stress response induced by Dox.

[0118] Example 13: Detection of Reactive Oxygen Species (ROS) using DCFH-DA fluorescent probe

[0119] H9c2 cells were seeded in confocal dishes. After 24 h of culture, they were divided into a control group (CON), a Dox group (Dox), a 1 μM inhibitor group (1 μM), and a LiCl group (LiCl). Cells in the CON group were added with blank medium; cells in the Dox group were added with 2 μM Dox; different concentration inhibitor groups were respectively added with 2 μM Dox and 1 μM inhibitor, and cells in the LiCl group were added with 2 μM Dox, 1 μM inhibitor, and 1 mM LiCl. After 24 h of drug treatment, H9c2 cells were washed twice with PBS, and then fixed with paraformaldehyde. After aspirating the fixing solution, they were washed 3 times with PBS. Subsequently, they were incubated with 10 μM DCFH-DA fluorescent probe at 37 °C for 30 min and washed 3 times with PBS. Finally, they were observed under a fluorescence microscope (excitation wavelength / emission wavelength were 502 nm / 530 nm respectively), and the fluorescence intensity was statistically analyzed using Image J.

[0120] The results were as Figure 15 shown, after treatment with Dox, the ROS content in H9c2 cardiomyocytes increased significantly (P<0.001). Compared with the Dox group, 1 μM and significantly reduced the ROS level in the Dox group cells (P<0.001). Compared with the 1 μM inhibitor group, LiCl significantly increased ROS (P<0.001). Therefore, NHE1 inhibitors protect cardiomyocytes by inhibiting NHE1 and reducing the oxidative stress response induced by Dox.

[0121] Example 14: Detection of cell apoptosis by TUNEL

[0122] H9c2 rat cardiomyocytes were seeded in confocal dishes. After 24 h of culture, the cells were divided into a control group (CON), a Dox group (Dox), a 1 μM inhibitor group (1 μM), a 10 μM inhibitor group (10 μM), and a LiCl group (LiCl). Cells in the CON group were added with blank medium; cells in the Dox group were added with 2 μM Dox; cells in the different concentration inhibitor groups were added with 2 μM Dox and different concentrations of inhibitors (1 μM and 10 μM respectively), and cells in the LiCl group were added with 2 μM Dox, 1 μM inhibitor, and 1 mM LiCl. After 24 h of drug treatment, the H9c2 cells were washed twice with PBS, then fixed with paraformaldehyde for 30 min. After removing the fixing solution, the cells were washed 3 times with PBS. Subsequently, 0.3% Triton X-100 was added and incubated at room temperature for 30 min. After removing it, the cells were washed three times with BSA working solution. The reaction working solution was prepared according to the instructions, added, and incubated in a wet box at 37 °C in the dark for 2 h. Subsequently, the cells were washed 3 times with PBS. Finally, the cells were counterstained with DAPI and washed 3 times with PBS containing. Observation was carried out under a fluorescence microscope (excitation wavelength / emission wavelength were 555 nm / 565 nm respectively), and the number of apoptotic cells and the total number of cells were counted (apoptosis ratio = number of apoptotic cells / number of normal cells).

[0123] The results were as Figure 16 shown. Compared with the CON group, after stimulating H9c2 cardiomyocytes with Dox, the degree of cell apoptosis increased significantly (P < 0.001). The degree of cell apoptosis in the 1 μM and 10 μM NHE1 inhibitor groups was significantly reduced compared with the Dox group (P < 0.001), indicating that this inhibitor could significantly inhibit Dox-induced apoptosis of H9c2 cells. While the NHE1 agonist Licl increased the degree of cell apoptosis (P < 0.001). The above results suggest that the NHE1 inhibitor alleviates Dox-induced cardiomyocyte apoptosis by inhibiting NHE1 activity.

[0124] Example 15: Detection of the expression of related proteins in H9c2 cells by Western blot

[0125] (1) Extraction of total cellular proteins

[0126] The cell grouping and treatment are as follows: The cells were inoculated and cultured for 24 h, and divided into a control group (CON), a Dox group (Dox), a 1 μM inhibitor group (1 μM), a 10 μM inhibitor group (10 μM), and a LiCl group (LiCl). The cells in the CON group were added with blank medium; the cells in the Dox group were added with 2 μM Dox; the cells in the different concentration inhibitor groups were respectively added with 2 μM Dox and different concentrations of inhibitors (1 μM and 10 μM respectively), and the cells in the LiCl group were added with 2 μM Dox, 1 μM inhibitor and 1 mM LiCl. After 24 h of drug administration, PBS was added for washing three times. After sucking it dry, cell lysate was added according to the cell density. The cells were scraped off on ice with a cell scraper, and the liquid was aspirated into a classified centrifuge tube, mixed well and placed at -80 °C for 30 min. Subsequently, the centrifuge was pre-cooled at 4 °C, the rotation speed was set at 12,000 rpm for 10 min. Finally, the supernatant was aspirated into a new centrifuge tube. Except for taking a small amount for protein concentration measurement by BCA method, 5×loading buffer was added according to the volume, and it was boiled in a constant temperature water bath at 100 °C for 10 min. After cooling to room temperature, the protein concentration was measured and stored at -20 °C for standby. The extracted proteins were subjected to conventional SDS-PAGE gel electrophoresis and membrane transfer and blocking. The primary and secondary antibodies of caspase 3, Bcl-2, Bax, NHE1, GAPDH and β-actin were incubated respectively, developed with a hypersensitive luminescence kit, and the gray value of the band was statistically analyzed by Image J.

[0127] The results are as Figures 17 - 19 shown. Compared with the CON group, Dox could significantly reduce the protein expression of Bcl-2 (P<0.001), and increase the protein levels of Bax and cleaved-caspase3 (P<0.01 or P<0.001), indicating that Dox induced cardiomyocyte apoptosis. The 1 μM and 10 μM NHE1 inhibitors could significantly inhibit the decrease in Bcl-2 protein expression and the increase in Bax and cleaved-caspase3 protein expression induced by Dox in H9c2 cardiomyocytes (P<0.05, P<0.01 or P<0.001). LiCl could significantly inhibit the above effects of the novel NHE1 inhibitor (P<0.05 or P<0.001), indicating that the NHE1 inhibitor alleviated Dox-induced H9c2 cell apoptosis by inhibiting NHE1 activity.

[0128] Example 16: Determination of Reactive Oxygen Species (ROS) by DCFH-DA fluorescent probe

[0129] 4T1 mouse breast cancer cells were seeded in confocal dishes. After 24 h of culture, the cells were divided into a control group (CON), a Dox group (Dox), and a Dox + inhibitor group (1 μM). Cells in the CON group were added with blank medium; cells in the Dox group were added with 2 μM Dox; cells in the Dox + inhibitor group (1 μM) were added with 2 μM Dox and 1 μM inhibitor. After 24 h of drug treatment, the 4T1 cells were washed twice with PBS, and then the cells were fixed with paraformaldehyde. After aspirating the fixing solution, the cells were washed 3 times with PBS. Subsequently, the cells were incubated with 10 μM DCFH-DA fluorescent probe at 37 °C for 30 min and then washed 3 times with PBS. Finally, the cells were observed under a fluorescence microscope (excitation wavelength / emission wavelength were 502 nm / 530 nm, respectively).

[0130] The results were as Figure 20 shown. After treatment with Dox, the ROS content in 4T1 mouse breast cancer cells increased significantly, and the ROS level in the combined treatment group was significantly higher than that in the Dox group, indicating that the NHE1 inhibitor promoted the oxidative stress in tumor cells. Therefore, the NHE1 inhibitor could promote the oxidative stress response induced by 4T1 mouse breast cancer cells, thereby enhancing the anti-cancer activity of Dox.

[0131] Example 17: Detection of cell apoptosis by TUNEL

[0132] 4T1 mouse breast cancer cells were seeded in confocal dishes. After 24 h of culture, the cells were divided into a control group (CON), a Dox group (Dox), and a Dox + inhibitor group (1 μM). Cells in the CON group were added with blank medium; cells in the Dox group were added with 2 μM Dox; cells in the Dox + inhibitor group (1 μM) were added with 2 μM Dox and 1 μM inhibitor. After 24 h of drug treatment, the 4T1 cells were washed twice with PBS, and then fixed with paraformaldehyde for 30 min. After aspirating the fixing solution, the cells were washed 3 times with PBS. Subsequently, 0.3% Triton X-100 was added and the cells were incubated at room temperature for 30 min. After aspirating, the cells were washed three times with BSA working solution. The reaction working solution was prepared according to the instructions and added, and then the cells were incubated in a wet box at 37 °C in the dark for 2 h. Subsequently, the cells were washed 3 times with PBS. Finally, the cells were counterstained with DAPI and washed 3 times with PBS containing. The cells were observed under a fluorescence microscope (excitation wavelength / emission wavelength were 555 nm / 565 nm, respectively).

[0133] The results were as Figure 21 shown. Compared with the CON group, after stimulating 4T1 cells with Dox, the number of apoptotic cells increased significantly. The 1 μM inhibitor group could significantly promote the number of apoptotic cells in the Dox group, indicating that this inhibitor could significantly promote the apoptosis of 4T1 cells. The results showed that the NHE1 inhibitor could promote the apoptosis level of 4T1 mouse breast cancer cells, thereby enhancing the anti-cancer activity of Dox.

[0134] Example 18: Evaluation of Antitumor Effect in Vivo

[0135] A 4T1 breast tumor model was established by subcutaneously injecting 5×10 5 4T1 cells preserved in RPMI 1640 medium around the fourth pair of mammary glands in BALB / c mice. When the tumor volume increased to approximately 200 mm 3 , the tumor-bearing mice were randomly divided into three groups: Tumor group, Dox group, and NHE1 Inhibitor group. The mice in the Dox group were intravenously injected with Dox (4 mg / kg) via the tail vein every 3 days for approximately 15 days. The mice in the NHE1 Inhibitor group were intravenously injected with Dox (4 mg / kg) and the novel NHE1 inhibitor (5 mg / kg) via the tail vein every 3 days for approximately 15 days. The mice in the tumor group were injected with an equal volume of normal saline. In addition, the body weights of the mice in each group were recorded simultaneously. After the in vivo experiment, the antitumor effect of the novel NHE1 inhibitor was evaluated by continuously measuring the tumor volume (V = length × width 2 × 0.5) for 15 days.

[0136] This example further evaluated the effect of the novel NHE1 inhibitor on the antitumor effect of Dox. First, we detected its antitumor effect by measuring the tumor size. The results were as Figure 22 shown. Both the Dox group and the combination treatment group had obvious antitumor effects (P < 0.001); compared with the Dox group, the antitumor effect of the combination treatment group was significantly enhanced (P < 0.05). During the experiment, the body weights of the tumor-bearing mice in each group decreased. The body weights of the mice after Dox treatment were significantly lower than those of the tumor group (P < 0.05), while the body weights of the mice treated with the combination treatment were significantly higher than those of the doxorubicin group (P < 0.01). This indicates that the novel NHE1 inhibitor can reduce the toxicity induced by Dox. Moreover, both the doxorubicin group and the combination treatment group had the effect of reducing the tumor weight / body weight ratio (P < 0.001), and it was more obvious in the combination treatment group (P < 0.05). The above results show that the novel NHE1 inhibitor can significantly promote the antitumor effect of doxorubicin and reduce the toxicity it induces.

[0137] Example 19: HE Staining

[0138] The section samples were immersed in xylene I for about 15 min, and then replaced with fresh xylene II for about 15 min for dewaxing treatment. During the process, observe to ensure no wax residue. Then, they were successively placed in gradient ethanol for dehydration. The hematoxylin staining solution was dropped with a dropper to completely cover the staining area. After 3 min, it was slightly washed with running water for 1 - 3 s. Then, it was stained with 1% eosin solution for 1 - 3 min. The samples were transferred to 80%, 90%, and 100% ethanol solutions for 3 - 5 s each for dehydration treatment, then changed to xylene I for 3 - 5 s and fresh xylene II for 3 - 5 min, and finally sealed with neutral gum.

[0139] The HE staining results of the tumor tissue sections of each group of mice are as Figure 23 shown. In the doxorubicin group and the combined treatment group, apoptotic cells were separated from normal cells, the cell nuclei were shrunken, and the apoptotic cells were slightly larger. After the combined treatment, there was obvious cell death at the tumor site, indicating that the NHE1 inhibitor of the present invention can significantly promote the anti-tumor effect of doxorubicin.

[0140] As described above, the above are only the preferred embodiments of the present invention and do not impose any limitations on the present invention. For those skilled in the art of the present technology, any modifications and changes made to the above embodiments based on the technical essence of the present invention shall fall within the protection scope of the technical solution of the present invention.

Claims

1. Use of amidoguanidine compounds in the preparation of anti-tumor drugs, characterized in that, The amide guanidine compound is an NHE1 inhibitor; The amide guanidine compound is N-(4-guanidinobutyl)-4-hydroxy-3-methoxybenzamide, and its structural formula is:

2. The application according to claim 1, characterized in that, The tumor is breast cancer.

3. A combined drug for anti-tumor, characterized in that: The combined medication is the combination of doxorubicin and an amide guanidine compound, and the amide guanidine compound is an NHE1 inhibitor; The amide guanidine compound is N-(4-guanidinobutyl)-4-hydroxy-3-methoxybenzamide, and its structural formula is:

4. The drug according to claim 3, characterized in that, The drug further comprises a pharmaceutically acceptable carrier or excipient.

5. The drug according to claim 3, characterized in that, The tumors include acute leukemia, malignant lymphoma, breast cancer, bronchogenic carcinoma, ovarian cancer, soft tissue sarcoma, osteosarcoma, rhabdomyosarcoma, Ewing's sarcoma, blastoma, bladder cancer, thyroid cancer, prostate cancer, squamous cell carcinoma of the head and neck, testicular cancer, gastric cancer, and liver cancer.

6. Use of an amidoguanidine compound in combination with doxorubicin in the preparation of an anti-tumor drug, characterized in that, The amide guanidine compound is an NHE1 inhibitor; The amide guanidine compound is N-(4-guanidinobutyl)-4-hydroxy-3-methoxybenzamide, and its structural formula is:

7. The application according to claim 6, characterized in that The tumors include acute leukemia, malignant lymphoma, breast cancer, bronchogenic carcinoma, ovarian cancer, soft tissue sarcoma, osteosarcoma, rhabdomyosarcoma, Ewing's sarcoma, blastoma, bladder cancer, thyroid cancer, prostate cancer, squamous cell carcinoma of the head and neck, testicular cancer, gastric cancer, and liver cancer.

8. Use of an amidoguanidine compound in the preparation of a drug for reducing the increase in lactic dehydrogenase in cardiomyocytes caused by doxorubicin, inhibiting cardiomyocyte hypertrophy caused by doxorubicin, or inhibiting the decrease in mitochondrial activity of cardiomyocytes caused by doxorubicin, characterized in that, The amide guanidine compound is an NHE1 inhibitor; The amide guanidine compound is N-(4-guanidinobutyl)-4-hydroxy-3-methoxybenzamide, and its structural formula is:

9. Use of an amidoguanidine compound in the preparation of a drug for inhibiting doxorubicin-induced oxidative stress response in cardiomyocytes, characterized in that, The amide guanidine compound is an NHE1 inhibitor; The amide guanidine compound is N-(4-guanidinobutyl)-4-hydroxy-3-methoxybenzamide, and its structural formula is:

10. Use of an amidoguanidine compound in the preparation of a drug for reducing doxorubicin-induced cardiomyocyte apoptosis, characterized in that, The amide guanidine compound is an NHE1 inhibitor; The amide guanidine compound is N-(4-guanidinobutyl)-4-hydroxy-3-methoxybenzamide, and its structural formula is:

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