CN110538172B - Application of ferroptosis inhibitor in preparation of medicine for treating auranofin hepatotoxicity - Google Patents

Abstract

The invention discloses application of an iron death inhibitor in preparing a medicament for treating auranofin hepatotoxicity, wherein the iron death inhibitor is Ferrostatin-1. The invention provides evidence of Ferrostatin-1 in inhibiting iron death process caused by auranofin, provides theoretical basis for treating auranofin liver injury with iron death as target, and especially provides basis for combined administration of auranofin and iron death inhibitor in clinical process.

Description

Application of ferroptosis inhibitor in preparation of medicine for treating auranofin hepatotoxicity

technical field

The invention relates to the application of Ferrostatin-1, an inhibitor of ferroptosis, in the treatment of auranofin hepatotoxicity.

Background technique

Ferroptosis is an iron-dependent cell death pathway, which is different from other death pathways such as apoptosis, necrosis, and autophagy. Ferroptosis manifests as comprehensive changes in multiple indicators such as the level of lipid peroxidation and the increased expression of the marker gene Ptgs2, which can be specifically inhibited by iron ion chelators.

Ferrostatin-1 is currently recognized as a ferroptosis inhibitor. As a compound containing N-cyclohexyl, Ferrostatin-1 has a high affinity with the cell membrane phospholipid bilayer, and can effectively remove cell membrane lipid peroxidation. In vitro experiments, Ferrostatin-1 acts on cancer cells to inhibit the accumulation of Erastin-induced cytoplasmic and lipid ROS, thereby inhibiting ferroptosis. In Huntington's disease model, Ferrostatin-1 inhibits neuronal ferroptosis induced by glutamate toxicity; in hereditary hemochromatosis, Ferrostatin-1 inhibits hepatic ferroptosis induced by iron overload.

Invention of Application No. 201710919871.7 "Application of Ferroptosis Inhibitors in the Preparation of Drugs to Prevent Myocardial Ischemia-Reperfusion Injury" and Invention of Application No. 201710137097.4 "Application of Ferroptosis Inhibitors in the Preparation of Drugs Inhibiting Cardiotoxicity Caused by Doxorubicin" , the invention of Patent No. 201610307748.5 "Application of Ferroptosis Inhibitors in the Preparation of Drugs for Treating Iron Overload Diseases" discloses the corresponding effect of Ferrostatin-1.

In addition, Ferrostatin-1 cannot inhibit extracellular signal-regulated kinase (ERK) phosphorylation or iron sequestration, indicating that Ferrostatin-1 inhibits ferroptosis not by regulating MEK/ERK pathway, cellular iron level or inhibiting protein synthesis. Similar to reducing agents such as tocopherol, Ferrostatin-1 is unstable and easily oxidized to stable 2,2-diphenyl-1-picrylhydrazine (DPPH).

Auranofin (Auranofin) is a kind of oral gold preparation specially developed by Smith Kline & French for the treatment of rheumatoid arthritis. Auranofin is slightly soluble in water and easily soluble in liposomes. It was approved by the US Food and Drug Administration (FDA) in 1985 for the treatment of rheumatoid arthritis. After oral administration of auranofin, 15% to 25% of the drug can be detected in plasma, mainly bound to albumin. The plasma half-life of the drug is 15-25 days, and it is almost completely excreted after 55-80 days. 85% of auranofin is excreted through feces, only 15% is excreted through urine, and only 0.4% of the administered dose is accumulated in the kidney. The incidence of adverse reactions of auranofin is as high as 30% to 50%, and most of them occur within 3 months after taking the medicine, mainly manifested as liver toxicity and hematopoietic inhibition. Up to now, Auranofin has gradually withdrawn from the first-line clinical use.

The cause of Auranofin (Auranofin) liver toxicity is still unclear, and there is no relevant report on the correlation between ferroptosis and Auranofin toxicity in vivo. It is also unknown whether high-dose injection of Auranofin will cause Ferroptosis; that is, at present There are no reports of an association between auranofin and hemochromatosis or ferroptosis. At present, there is no drug that can effectively treat/alleviate the toxicity of auranofin (Auranofin), and clinically, drug withdrawal for auranofin toxicity is often done.

Contents of the invention

The technical problem to be solved by the present invention is to provide the application of Ferrostatin-1, an inhibitor of ferroptosis, in the preparation of drugs for the treatment of auranofin hepatotoxicity, so as to provide a basis for new drug research and development and innovative therapy.

In order to solve the above technical problems, the present invention provides an application of a ferroptosis inhibitor in the preparation of a drug for treating/relieving auranofin hepatotoxicity, and the ferroptosis inhibitor is Ferrostatin-1.

Although ferroptosis is the pathogenic mechanism of many liver injury diseases, there are many forms of liver toxicity caused by drugs, such as anti-tumor chemotherapy drugs, anti-tuberculosis drugs, antipyretic and analgesic drugs, immunosuppressants, hypoglycemic and lipid-lowering drugs The hepatotoxicity caused by antibacterial, antifungal and antiviral drugs is different; therefore, the known Ferrostatin-1 inhibits ferroptosis of liver cells caused by iron overload and cannot provide technical inspiration for the present invention.

In the course of the invention, the inventors found that high doses of auranofin can lead to ferroptosis; the inventors found that in the Huh7 liver cancer cell line, Ferrostatin-1 can significantly inhibit the ferroptosis caused by auranofin. It was confirmed by the mouse model that Ferrostatin-1 can significantly inhibit the ferroptosis and liver damage caused by auranofin. The results of this study provide a theoretical basis for Ferrostatin-1 to alleviate the side effects of auranofin, especially for the combination of auranofin and ferroptosis inhibitors in the clinical process.

During the invention process, it was found that the intraperitoneal administration of Ferrostatin-1 was ineffective against the liver toxicity caused by the above-mentioned anti-tumor chemotherapy drugs, anti-tuberculosis drugs, antipyretic analgesics, immunosuppressants, etc. therapeutic effect.

Ferrostatin-1, a ferroptosis inhibitor, is used in the treatment of auranofin toxicity, and its usage and dosage are as follows: daily intramuscular injection or intravenous infusion of ferrostatin-1 (1 mg/kg body weight), 3 weeks as a course of treatment.

Auranofin is a drug for the clinical treatment of rheumatoid arthritis, but there are side effects such as liver damage, which limits the current clinical use of auranofin. The present invention uses experiments to prove that ferrostatin-1 can effectively prevent the hepatotoxic side effects of auranofin (Figure 2).

In summary, the present invention provides an application of a ferroptosis inhibitor in the treatment of auranofin hepatotoxicity, specifically, the ferroptosis inhibitor Ferrostatin-1 can be used to prepare a drug for treating/relieving auranofin hepatotoxicity, Or a supplement in combination with auranofin to prevent ferroptosis hepatotoxicity. The beneficial effects of the present invention are mainly reflected in: the present invention provides the evidence that Ferrostatin-1 is suppressing the ferroptosis process that auranofin causes, and provides a theoretical basis for the treatment of auranofin liver injury with ferroptosis as the target, especially It provides the basis for the combination of auranofin and ferroptosis inhibitors in the clinical process.

Description of drawings

The specific implementation manners of the present invention will be described in further detail below in conjunction with the accompanying drawings.

Fig. 1 is that Ferrostatin-1 suppresses the ferroptosis that auranofin causes in vitro;

A: blank treatment (Con), auranofin (2.5 μM) plus dimethyl sulfoxide (DMSO), auranofin (2.5 μM) plus Ferrostatin-1 (2 μM), auranofin (2.5 μM) plus apoptosis Inhibitor Z-VAD-FMK (10 μg/mL), auranofin (2.5 μM) plus necrosis inhibitor Necrostatin-1 (10 μg/mL) co-treated mouse liver primary cells for 24 hours, and the rescue effect on cell viability.

B: blank treatment (Con), auranofin (2.5 μM) plus dimethyl sulfoxide (DMSO), auranofin (2.5 μM) plus Ferrostatin-1 (2 μM), auranofin (2.5 μM) plus apoptosis Inhibitor Z-VAD-FMK (10 μg/mL), auranofin (2.5 μM) plus necrosis inhibitor Necrostatin-1 (10 μg/mL) co-treated mouse liver primary cells for 12 hours, the effect on cell lipid peroxidation Inhibitory effect;

C: blank treatment (Con), auranofin (2.5 μM) plus dimethyl sulfoxide (DMSO), auranofin (2.5 μM) plus Ferrostatin-1 (2 μM), auranofin (2.5 μM) plus apoptosis Inhibitor Z-VAD-FMK (10 μg/mL), auranofin (2.5 μM) plus necrosis inhibitor Necrostatin-1 (10 μg/mL) co-treated mouse liver primary cells for 12 hours, inhibited the expression of Ptgs2 mRNA in cells Effect;

Figure 2 shows that intraperitoneal administration of Ferrostatin-1 can inhibit ferroptosis and improve liver damage in hemochromatosis mice;

A: Ferrostatin-1 (Ferr-1, 1mg/kg body weight per day) was injected intraperitoneally for 6 weeks, and the lethal curves of male and female Hfe ^-/- mice;

Explanation: In the right picture, AF+Ferr1 and control are completely overlapped, indicating that the two have the same effect;

B: Ferrostatin-1 (Ferr-1, every day 1mg/kg body weight) intraperitoneal injection 3 weeks, male Hfe ^-/- mouse liver malondialdehyde (MDA) content;

C: Ferrostatin-1 (Ferr-1, 1mg/kg body weight per day) was injected intraperitoneally for 3 weeks, and the expression level of Ptgs2 mRNA in the liver of male Hfe ^-/- mice;

D: Ferrostatin-1 (Ferr-1, 1 mg/kg body weight per day) was injected intraperitoneally for 3 weeks, and the liver of male Hfe ^-/- mice was stained with Sirius red.

AF stands for Auranofin.

Detailed ways

The present invention is further described below in conjunction with specific embodiment, but protection scope of the present invention is not limited thereto:

Example 1:

1. Materials and methods:

1.1 Preparation of experimental raw materials:

Compound monomer Auranofin was purchased from MedChemExpress Company, and Ferrostatin-1, Z-VAD-FMK and Necrostatin-1 were purchased from SIGMA Company, all of which were dissolved in sterile dimethyl sulfoxide (DMSO) and prepared to the desired concentration.

1.2 Huh7 liver cancer cell line:

Huh7 liver cancer cell line culture conditions: DMEM high-glucose medium (GIBCO) containing 10% FBS (GIBCO), 37° C., 5% CO2 saturated humidity incubator. After the cells adhered to the wall, add DMSO, auranofin (2.5 μM, final concentration, the same below) + DMSO, auranofin (2.5 μM) + apoptosis inhibitor (10 μg/mL), auranofin Fen (2.5 μM) + necrosis inhibitor (10 μg/mL), auranofin (2.5 μM) + Ferrostatin-1 (2 μM). Cell viability was measured by MTT 24 hours after treatment (see 1.3). After 12 hours of treatment, the cells were collected for Real-time PCR (see 1.4) and determination of membrane lipid peroxidation (see 1.5).

The apoptosis inhibitor is Z-VAD-FMK; the necrosis inhibitor is Necrostatin-1.

1.3 Cell viability assay (MTT):

The Huh7 cells treated with drugs for 24 hours were detected by MTT colorimetric method, and the MTT kit was purchased from Biyuntian.

1.4 RNA extraction and Real-time PCR:

Trizol (Life Technologies) method was used to extract RNA from cells and tissues, and specific operations were performed according to the instructions. The RNA purity (OD260/OD280≈1.9-2.1) and RNA concentration (ng/μl) were detected on the Nanodrop1000 Spectrophotometer, and the RNA concentration was adjusted to 1 μg/μl.

After 2.0 μg of RNA was treated with DNase (Promega), reverse transcription was performed with M-MLV reverse transcriptase (Promega) and Oligo(dT)18primer (Takara Bio Inc.). Realtime PCR was carried out in CFX96 Real-Time System (Bio-Rad), using iQ SYBR Green Supermix (Bio-Rad) as the reagent, and the reaction system was 10 μl, including 5 μl of SYBGREEN, 1 μl of cDNA, 2 μl of primers, and 2 μl of ddH ₂ O. The Real-Time program is pre-denaturation at 95°C for 3 minutes, 40 cycles (each cycle is 95°C for 10 seconds, fluorescence is detected during 60°C for 15 seconds), and the melting curve is detected. The primer sequences are as follows:

Mouse Actb (β-actin):

forward:AAATCGTGCGTGACATCAAGA

reverse: GCCATCTCCTGCTCGAAGTC

mouse Ptgs2:

forward: CTGCGCCTTTTCAAGGATGG

reverse: GGGGATACACCTCTCCCACCA.

1.5 Determination of cell membrane lipid peroxidation level:

The Huh7 cells treated with drugs for 12 hours were digested with trypsin to form a single cell suspension. C11-BODIPY (10 μM) was incubated at room temperature in the dark for 30 minutes, washed 3 times with PBS, and detected by flow cytometry. C11-BODIPY was purchased from Invitrogen.

Mouse liver tissue was homogenized with PBS, and the content of lipid peroxidation product malondialdehyde (MDA) was detected by colorimetry. MAD kit was purchased from Biyuntian.

1.6 Experimental animals:

Hfe-knockout (Hfe-/-) mice are an animal model of classical hemochromatosis, and their phenotype is similar to that of HFE-HH patients, showing an abnormal decrease in hepcidin expression and systemic iron overload. Hfe-/- mice were free from symptoms and associations of liver injury and ferroptosis.

Hfe ^-/- mice were raised in SPF environment until 8 weeks old, and were randomly divided into three groups according to body weight, with 8 males and 8 males in each group. According to the experimental design, the three groups were given daily intraperitoneal injection of normal saline, intraperitoneal injection of auranofin (25 mg/kg body weight), and intraperitoneal injection of auranofin (25 mg/kg body weight)+Ferrostatin-1 (1 mg/kg body weight). At 6 weeks, the lethal curve of mice was recorded.

Hfe ^-/- mice were raised in SPF environment until 8 weeks old, and were randomly divided into three groups according to body weight, with 6 males in each group. According to the experimental design, the three groups were given daily intraperitoneal injection of normal saline, intraperitoneal injection of auranofin (25 mg/kg body weight), and intraperitoneal injection of auranofin (25 mg/kg body weight)+Ferrostatin-1 (1 mg/kg body weight). 3 weeks. After intraperitoneal anesthesia with 5% chloral hydrate, collect blood from the heart, separate serum, and collect liver, spleen and kidney tissues (preserved in liquid nitrogen), and detect liver lipid peroxidation level (same as 1.5), fibrosis index (see 1.7) and liver Ptgs2 Expression amount (method is the same as 1.4).

1.7 Detection of liver damage indicators:

Liver fibrosis is commonly used clinically to measure the level of liver damage. Hepatic fibrosis was performed by Sirius red staining on liver paraffin sections. The red-stained area was collagen, a product of liver fibrosis. Sirius red dye was purchased from Sigma, and the staining method was in accordance with the instructions.

1.8 Statistical methods

The statistics used were analyzed by R software, and the experimental data were expressed as Mean ± SEM. Tukey's test (ANOVA) was used for comparison between groups in cell and animal experiments, and Student's t-test was used for comparison between two groups. P<0.05 was considered statistically significant, and different letters indicate P<0.05.

2. Results

2.1 Ferrostatin-1 inhibits auranofin-induced ferroptosis in Huh7 liver cancer cells

Huh7 cells were cultured in vitro, and DMSO, auranofin (2.5 μM, final concentration, the same below)+DMSO, auranofin (2.5 μM)+apoptosis inhibitor Z-VAD-FMK (10 μg/mL ), Auranofin (2.5 μM) + necrosis inhibitor Necrostatin-1 (10 μg/mL), auranofin (2.5 μM) + Ferrostatin-1 (2 μM). Cell viability was detected after 24 hours. After 12 hours, ferroptosis indicators were detected, including cell membrane lipid peroxidation and Ptgs2 mRNA levels. Three replicate wells were set up for each treatment, and the experiment was repeated three times.

As shown in Figure 1, the experimental results found that compared with Control, auranofin + DMSO stimulation can significantly kill cells (Figure 1A); up-regulate ferroptosis indicators, including cell membrane lipid peroxidation level (Figure 1B) and Ptgs2 mRNA expression (Figure 1C); indicating that auranofin at the cellular level in vitro can trigger ferroptosis. Compared with auranofin + DMSO, auranofin + Ferrostatin-1 treatment can significantly rescue cell viability (Figure 1A), and inhibit the up-regulation of auranofin on lipid peroxidation (Figure 1B) and Ptgs2 expression (Figure 1C) ; while auranofin+apoptosis inhibitor Z-VAD-FMK and auranofin+necrosis inhibitor Necrostatin-1 were more effective than auranofin+DMSO on cell viability (Fig. 1A), lipid peroxidation (Fig. 1B) and Ptgs2 expression levels had no effect (Fig. 1C). This shows that the cell death mode caused by auranofin is ferroptosis, rather than apoptosis or necrosis. Ferrostatin-1 can inhibit the ferroptosis induced by auranofin in vitro.

2.2 Ferrostatin-1 inhibits the toxicity of auranofin

8-week-old Hfe ^-/- mice were randomly divided into three groups (8 males and females in each group), and were given daily intraperitoneal injection of normal saline, intraperitoneal injection of auranofin (25 mg/kg body weight), and intraperitoneal injection of auranofin (25 mg/kg body weight). /kg body weight)+Ferrostatin-1 (1 mg/kg body weight). After 6 weeks of treatment, the lethal curve of the mice was recorded; after 3 weeks of treatment, the content of MDA in the liver, the level of Ptgs2 mRNA, and liver fibrosis were detected.

The results in Figure 2A show that, regardless of male mice or female mice, all 100% of the mice in the auranofin treatment group died within 5 weeks after injection, while the combined treatment of Ferrostatin-1+auranofin significantly delayed and reduced the death of mice (FIG. 2A). In terms of physiological and biochemical indicators of ferroptosis, after 3 weeks of injection of auranofin, the content of MDA in the liver of mice (Figure 2B), the level of Ptgs2 mRNA (Figure 2C), and the level of liver fibrosis (Figure 2D) were significantly increased compared with those in the normal saline group (control), It shows that in vivo treatment of auranofin can lead to ferroptosis. Compared with Auranofin alone treatment group, combined treatment of Auranofin + Ferrostatin-1 can significantly restore liver MDA content (Figure 2B), Ptgs2 mRNA level (Figure 2C), liver fibrosis level (Figure 2D), indicating that Ferrostatin- 1 In the body, it can inhibit the ferroptosis caused by auranofin and improve the liver damage caused by ferroptosis.

Based on this, inhibition of ferroptosis by Ferrostatin-1 is a therapeutic approach for the treatment of auranofin hepatotoxicity. Therefore, targeting ferroptosis and inhibiting auranofin-induced ferroptosis through ferroptosis inhibitors is of great significance for the treatment of auranofin hepatotoxicity and the safe use of auranofin.

Finally, it should be noted that the above examples are only some specific embodiments of the present invention. Obviously, the present invention is not limited to the above embodiments, and many variations are possible. All deformations that can be directly derived or associated by those skilled in the art from the content disclosed in the present invention should be considered as the protection scope of the present invention.

sequence listing

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Claims (1)

1. Application of a ferroptosis inhibitor in the preparation of a medicament for treating auranofin hepatotoxicity, the ferroptosis inhibitor is Ferrostatin-1, and Ferrostatin-1 can obviously inhibit ferroptosis and liver damage caused by auranofin.

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