Application of nitrate in preparation of product for preventing drug-induced liver injury

Abstract

The invention relates to the technical field of medicines, in particular to application of nitrate in preparation of a product for preventing drug-induced liver injury. The invention finds that nitrate plays a role in protecting acute liver injury caused by methotrexate by inhibiting liver cell apoptosis and promoting liver cell proliferation and repair; in addition, nitrate can activate a Wnt / beta-catenin signal channel by up-regulating FZD6 to play a role in liver protection. Therefore, the nitrate can be used as a preventive drug for the drug-induced liver injury and has the advantage of high safety.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical technology, and particularly to the application of nitrate in the preparation of products for preventing drug-induced liver injury. Background Art

[0002] Methotrexate (MTX) is widely used clinically. It is currently the first-line drug for the treatment of rheumatoid arthritis. At the same time, as an anti-folate anti-tumor drug, it can be used to treat malignant tumors such as head and neck cancer, acute leukemia, malignant lymphoma, breast cancer, etc. However, its hepatotoxicity limits its clinical application. Studies have shown that among patients with rheumatoid arthritis who take methotrexate for a long time, about 70% of the patients have recurrent elevated serum transaminases. Therefore, there is an urgent need for a product with high safety and a protective effect on methotrexate-induced drug-induced liver injury. Summary of the Invention

[0003] To solve the above problems, the present invention provides the application of nitrate in the preparation of products for preventing drug-induced liver injury. The present invention discovers that nitrate plays a protective role in acute liver injury caused by methotrexate by inhibiting hepatocyte apoptosis and promoting hepatocyte proliferation and repair, and has high safety and can be used as a prophylactic drug for drug-induced liver injury.

[0004] To achieve the above object, the present invention provides the following technical solutions:

[0005] The present invention provides the application of nitrate in the preparation of products for preventing drug-induced liver injury, and the drug includes methotrexate.

[0006] Preferably, the nitrate includes sodium nitrate.

[0007] Preferably, the product includes a product for inhibiting hepatocyte apoptosis and / or promoting hepatocyte proliferation and repair.

[0008] Preferably, the inhibition of hepatocyte apoptosis includes promoting the expression of anti-apoptotic genes and / or inhibiting the expression of pro-apoptotic genes; the anti-apoptotic genes include Bcl2 and / or Bcl-xl; the pro-apoptotic gene includes Bax.

[0009] Preferably, the promotion of hepatocyte proliferation and repair includes promoting the expression of hepatocyte growth factor; the hepatocyte growth factor includes Hgf.

[0010] Preferably, the product includes a product for activating the Wnt / β-catenin signaling pathway.

[0011] Preferably, the activation of the Wnt / β-catenin signaling pathway includes upregulating the expression of FZD6 protein.

[0012] Preferably, the product includes reducing the activity of one or more of alanine aminotransferase, aspartate aminotransferase, lactate dehydrogenase, and alkaline phosphatase.

[0013] Preferably, the product includes products that reduce the degree of hepatocyte edema and necrosis and / or reduce lipid accumulation.

[0014] Preferably, the product includes drugs.

[0015] Beneficial effects:

[0016] The present invention provides the application of nitrate in the preparation of products for preventing drug-induced liver injury, and the drug includes methotrexate. The present invention finds that nitrate plays a protective role in acute liver injury caused by methotrexate by inhibiting hepatocyte apoptosis and promoting hepatocyte proliferation and repair; in addition, nitrate can play a hepatoprotective role by upregulating FZD6 and activating the Wnt / β-catenin signaling pathway. It can be seen that nitrate can be used as a prophylactic drug for drug-induced liver injury and has the advantage of high safety. Description of the drawings

[0017] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required in the embodiments.

[0018] Figure 1 It is the detection results of serum biochemical indexes and liver tissue pathology of mice in different groups in Example 1; among them, A to D are the detection results of the activities of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), and lactate dehydrogenase (LDH) in serum in turn; E is the HE staining result, the scale in the lower right corner of the first row of pictures is 300μm, and the scale in the lower right corner of the second row of pictures is 60μm;

[0019] Figure 2 It is the staining results of mice in different groups in Example 1; among them, A is the staining results of TUNEL (green fluorescence), Ki67 (red fluorescence), and Dapi (blue fluorescence), and the scale in the lower right corner is 100μm; B is the statistical chart of TUNEL expression level; C is the statistical chart of Ki67 expression level;

[0020] Figure 3 It is the relative mRNA expression levels of apoptosis-related factors Bcl2, Bclxl, Bax, and hepatocyte growth factor Hgf in the livers of mice in different groups in Example 1;

[0021] Figure 4 It is the top 30 KEGG bubble chart of the upregulated pathway in the nitrate treatment group compared with the methotrexate modeling group;

[0022] Figure 5Results of GSEA analysis of the Wnt signaling pathway in the methotrexate modeling group and the nitrate treatment group;

[0023] Figure 6 Heatmap of genes related to the Wnt pathway in the methotrexate modeling group and the nitrate treatment group;

[0024] Figure 7 Relative mRNA expression levels of Fzd6, Ccnd1, and Axin2 in mice of each group in Example 1;

[0025] Figure 8 Expression results of proteins of Fzd6, p-GSK3β, GSK3β, β-catenin, c-Myc, Cyclin D1, and β-actin in mice of each group in Example 1;

[0026] Figure 9 Results of the cck8 experiment and mRNA levels of apoptosis-related factors in cells of each group in Example 2; among them, A is the results of the cck8 experiment of each group of cells; B-D are the relative mRNA expression levels of apoptosis-related factors Bcl2, Bclxl, and Bax in AML12 cells;

[0027] Figure 10 Ki67 staining conditions and statistical results in Example 2; the scale bar in the lower right corner is 80 μm for all;

[0028] Figure 11 Relative mRNA expression levels of genes related to the Wnt / β-catenin signaling pathway, Fzd6, Myc, Axin2, and Ccnd1, in Example 2;

[0029] Figure 12 Schematic diagram of the mechanism of nitrate in preventing drug-induced liver injury;

[0030] Among them, * indicates P < 0.05, ** indicates P < 0.01, and *** indicates P < 0.001. Detailed implementation manners

[0031] The present invention provides the application of nitrate in the preparation of a product for preventing drug-induced liver injury, and the drug includes methotrexate.

[0032] As an implementation manner, the nitrate can be sodium nitrate.

[0033] As an implementation manner, the product includes a product that inhibits hepatocyte apoptosis and / or promotes hepatocyte proliferation and repair.

[0034] As an implementation manner, the inhibition of hepatocyte apoptosis includes promoting the expression of anti-apoptotic genes and / or inhibiting the expression of pro-apoptotic genes; the anti-apoptotic genes include Bcl2 and / or Bcl-xl; the pro-apoptotic gene may be Bax.

[0035] As an implementation manner, the promotion of hepatocyte proliferation and repair includes promoting the expression of hepatocyte growth factor; the hepatocyte growth factor may be Hgf.

[0036] As an implementation manner, the product includes a product that activates the Wnt / β-catenin signaling pathway.

[0037] As an implementation manner, the activation of the Wnt / β-catenin signaling pathway may be to up-regulate the expression of FZD6 protein.

[0038] As an implementation manner, the product includes reducing the activity of one or more of alanine aminotransferase, aspartate aminotransferase, lactate dehydrogenase, and alkaline phosphatase.

[0039] As an implementation manner, the product includes a product that reduces the degree of hepatocyte edema and necrosis and / or reduces lipid accumulation.

[0040] As an implementation manner, the product may be a drug.

[0041] Through in-vivo experiments on mice and cell experiments, the present invention found that nitrate plays a protective role against acute liver injury caused by methotrexate by inhibiting hepatocyte apoptosis and promoting hepatocyte proliferation and repair; in addition, nitrate can play a hepatoprotective role by up-regulating FZD6 and activating the Wnt / β-catenin signaling pathway (see the schematic diagram of the mechanism in Figure 12 ). It can be seen that nitrate can be used as a prophylactic drug for drug-induced liver injury, and the use of nitrate has no adverse effects on mice, having the advantage of high safety.

[0042] To further illustrate the present invention, the application of nitrate provided by the present invention in the preparation of a product for preventing drug-induced liver injury will be described in detail below in conjunction with the drawings and embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0043] The detection methods used in the following examples are as follows:

[0044] 1. Serum biochemical indexes: The freshly collected serum is placed on ice and detected for the activities of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), and lactate dehydrogenase (LDH) on a full-automatic biochemical analyzer ( RxL ) within 6 hours.

[0045] 2. HE staining, RT-qPCR, WB: Detection was performed according to the method described in the reference [Dietary Inorganic Nitrate Protects Hepatic Ischemia-Reperfusion Injury Through NRF2-Mediated Antioxidative Stress doi: 10.3389 / fphar.2021.634115]. The primer sequences used for RT-qPCR are shown in Table 1.

[0046] Table 1 Primer sequences used for RT-qPCR

[0047]

[0048] 3. TUNEL staining: One-step TUNEL Cell Apoptosis Detection Kit (green fluorescence) (manufacturer: Beyotime, product number: C1008).

[0049] 4. Ki67 staining: Performed according to the method described in the reference [Salivary nitrate prevents osteoporosis via regulating bone marrow mesenchymal stem cells proliferation and differentiation doi: 10.1016 / j.jot.2023.12.001].

[0050] 5. CCK8: Cell Counting Kit-8 (CCK-8 kit) (manufacturer: Beyotime, product number: C0038).

[0051] Example 1

[0052] Six to eight-week-old healthy male ICR mice were selected and divided into 4 groups, with 6 mice in each group. The specific grouping and treatment are as follows:

[0053] Modeling group (MTX): After drinking normal water for 5 days, methotrexate (MTX) solution was intraperitoneally injected; the injection amount of methotrexate was 20 mg / kg body weight; the solvent of the methotrexate solution consisted of the following components by volume: 5% dimethyl sulfoxide, 40% polyethylene glycol 300, 5% Tween 80, and 50% normal saline 。

[0054] Normal control group (Ctrl): After drinking normal water for 5 days, the solvent of the methotrexate solution with the same volume as the MTX group was intraperitoneally injected.

[0055] Nitrate control group (Ctrl+Nit): Similar to the Ctrl group, except that ordinary drinking water was replaced with 2 mM sodium nitrate drinking water.

[0056] MTX + Nitrate prevention and treatment group (MTX+Nit): Similar to the MTX group, except that ordinary drinking water was replaced with 2 mM sodium nitrate drinking water.

[0057] Four groups of mice were sacrificed 24 hours after intraperitoneal injection of methotrexate solution or solvent, and samples were taken. The degree of liver injury in mice was observed by detecting serum biochemical indexes and liver tissue pathology analysis. The liver protection effect of nitrate was observed by detecting apoptosis and proliferation levels. The possible liver protection mechanism of nitrate was searched by transcriptome sequencing (RNA-seq). WB and RT-qPCR were used to verify the changes of related pathways. The results are shown in Figures 1 to 8 .

[0058] The results showed that after intraperitoneal injection of methotrexate, the contents of serum alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, and lactate dehydrogenase in mice increased significantly, indicating liver injury. Preventive administration of nitrate drinking water could significantly reduce the levels of serum alanine aminotransferase, aspartate aminotransferase, and lactate dehydrogenase (P < 0.05), and the level of alkaline phosphatase also showed a downward trend ( Figure 1 in A-D).

[0059] The results of HE staining showed that after intraperitoneal injection of methotrexate, hepatocytes around the central vein showed edema and necrosis, and lipid accumulation was visible. Nitrate intervention could reduce the degree of hepatocyte edema and necrosis and reduce lipid accumulation ( Figure 1 in E).

[0060] From Figure 2 and Figure 3 it can be seen that the results of liver TUNEL staining showed that necrotic and apoptotic foci were visible in the liver of the modeling group. After nitrate administration, the number of TUNEL-positive cells decreased significantly. By detecting the expression of liver apoptosis-related genes by RT-qPCR, the results showed that the expression levels of anti-apoptotic genes Bcl2 and Bcl-xl in the modeling group decreased significantly (P < 0.05), and the expression level of pro-apoptotic gene Bax showed an upward trend. After nitrate administration, the expression levels of Bcl2 and Bcl-xl increased significantly, and the expression level of Bax decreased significantly. The above results indicate that nitrate can inhibit hepatocyte apoptosis. The results of liver Ki67 staining showed that compared with the control group, the number of Ki67-positive cells in the liver of the modeling group decreased, and the number of Ki67-positive cells in the liver of the nitrate group increased. The expression of hepatocyte growth factor Hgf also increased after nitrate administration. The above results indicate that nitrate can promote hepatocyte proliferation and repair.

[0061] From Figures 4 to 8As a result, the RNA-seq analysis of liver tissues found that in the pathways downregulated in the model and upregulated by nitrate, the Wnt signaling pathway related to cell proliferation was enriched, which was consistent with the phenotypic results ( Figures 4 to 6 ). The changes in the Wnt pathway in each group were verified at the RNA level and protein level by RT-qPCR and WB, respectively. The results showed that the Wnt pathway was inhibited in the model group and activated after prophylactic administration of nitrate ( Figure 7 and Figure 8 ).

[0062] Example 2

[0063] In vitro experiments were conducted using the normal mouse hepatocyte AML12 cell line for verification. The NO donor NIC (manufacturer: MCE, catalog number: HY-B0341) was used to replace sodium nitrate, and the β-catenin inhibitor MSAB (manufacturer: MCE, catalog number: HY-120697) was used for verification. The grouping and treatment methods were as follows:

[0064] Normal control group (Ctrl): No special treatment after plating AML12 cells;

[0065] Modeling group (MTX): Add methotrexate (MTX) at a final concentration of 250 μM to AML12 cells 24 hours after plating;

[0066] NIC prevention and treatment group (MTX+NIC): Add NIC at a final concentration of 1 μM to AML12 cells 12 hours after plating, and add MTX at a final concentration of 250 μM 12 hours later;

[0067] Inhibitor group (MTX+NIC+MSAB): Add NIC at a final concentration of 20 μM and MSAB at a final concentration of 1 μM to AML12 cells 12 hours after plating, and add MTX at a final concentration of 250 μM 12 hours later.

[0068] After 72 hours of plating, the four groups of AML12 cells were harvested for cck8 experiments, Ki67 staining, and detection of the mRNA levels of apoptosis-related factors and the relative mRNA expression levels of genes related to the Wnt / β-catenin signaling pathway, Fzd6, Myc, Axin2, and Ccnd1. The results are shown in Figures 9 to 11 .

[0069] The CCK8 results showed that methotrexate inhibited cell activity after administration, and prophylactic administration of NIC (NO doner) could partially restore cell activity ( Figure 9 in A).

[0070] The expression levels of apoptosis-related genes were detected by RT-qPCR. In the model group, the expressions of Bcl2 and Bclxl were significantly decreased, while those in the MTX+NIC group were significantly increased. Meanwhile, the expression of Bax in the model group was significantly increased, while that in the MTX+NIC group was significantly decreased (P<0.01)( Figure 9 in Figures B-D).

[0071] The cell proliferation activity was evaluated by Ki67 staining. The quantitative analysis results showed that compared with the control group, the number of Ki67-positive cells in the MTX group was significantly decreased, while that in the MTX+NIC group was significantly increased, and the change was statistically significant (P<0.001)( Figure 10 ).

[0072] The expression levels of molecules related to the Wnt / β-catenin pathway were detected by RT-qPCR, and the results were consistent with those of the in vivo experiment( Figure 11 ).

[0073] When the β-catenin inhibitor MSAB was given simultaneously with NIC, it was found that the beneficial effects of NIC on cell viability, apoptosis, and proliferation disappeared( Figures 9 to 10 ).

[0074] In summary, nitrate plays a protective role against acute liver injury caused by methotrexate by inhibiting hepatocyte apoptosis and promoting hepatocyte proliferation and repair; in addition, nitrate exerts a hepatoprotective effect by upregulating FZD6 and activating the Wnt / β-catenin signaling pathway. It can be seen that nitrate can be used as a prophylactic drug for drug-induced liver injury.

[0075] Although the above embodiments have described the present invention in detail, they are only a part of the embodiments of the present invention, rather than all embodiments. People can also obtain other embodiments according to this embodiment without creative efforts, and these embodiments all fall within the protection scope of the present invention.

Claims

1. Use of nitrate in the preparation of a product for preventing drug-induced liver injury, wherein the drug includes methotrexate.

2. The application according to claim 1, characterized in that The nitrate includes sodium nitrate.

3. The application according to claim 1, wherein The product includes a product for inhibiting hepatocyte apoptosis and / or promoting hepatocyte proliferation and repair.

4. The application according to claim 3, wherein The inhibition of hepatocyte apoptosis includes promoting the expression of anti-apoptotic genes and / or inhibiting the expression of pro-apoptotic genes; the anti-apoptotic genes include Bcl2 and / or Bcl-xl; the pro-apoptotic genes include Bax.

5. The application according to claim 3, wherein The promotion of hepatocyte proliferation and repair includes promoting the expression of hepatocyte growth factor; the hepatocyte growth factor includes Hgf.

6. The application according to claim 1, characterized in that, The product includes a product for activating the Wnt / β-catenin signaling pathway.

7. The application according to claim 6, wherein The activation of the Wnt / β-catenin signaling pathway includes upregulating the expression of FZD6 protein.

8. The application according to claim 1, characterized in that, The product includes reducing the activity of one or more of alanine aminotransferase, aspartate aminotransferase, lactate dehydrogenase and alkaline phosphatase.

9. The application according to claim 1, wherein The product includes a product for reducing the degree of hepatocyte edema and necrosis and / or reducing lipid accumulation.

10. The application according to any one of claims 1 to 9, characterized in that, The product includes a drug.

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