CN115501225A - Application of CK-666 in inhibiting iron death and preparation of medicine thereof - Google Patents

Abstract

The invention provides application of CK-666 in inhibiting iron death and preparation of a medicament thereof. The research shows that an Arp2/3 compound inhibitor CK-666 can effectively inhibit iron death induced by various modes, and the cell iron death inhibition effect is enhanced along with the increase of the concentration of CK-666; and CK-666 also has good oxidation resistance, can prevent the oxidative burst of liposome, and shows that CK-666 can inhibit the death of iron by reducing the peroxidation of cell lipid. Meanwhile, CK-666 can obviously reduce the blood creatinine and urea nitrogen level of the mice subjected to ischemia reperfusion, can obviously relieve renal tissue injury when being used for the mice subjected to ischemia reperfusion, has better effectiveness, and provides a new choice for the research and development of related drugs of high-efficiency iron death inhibitors.

Description

Application of CK-666 in inhibiting iron death and preparation of medicine thereof

Technical Field

The invention belongs to the technical field of biological medicines. More particularly, it relates to the application of CK-666 in inhibiting iron death and its medicine preparation.

Background

Iron death was a novel cell-regulated death mode proposed in 2012 and was caused by iron ion-dependent lipid peroxidation accumulation. Accumulation of iron ions, synthesis of polyunsaturated fatty acid-containing lipids and disturbance of the cellular redox system are key to the induction of iron death. Iron ions oxidize polyunsaturated fatty acid-containing lipids through fenton reaction or lipid oxidase, and intracellular redox pathways, particularly Glutathione Peroxidase 4 (Glutathione Peroxidase 4, gpx4), prevent cellular iron death by reducing lipid peroxidation. At present, more and more evidences show that iron death is involved in the occurrence of various diseases, is related to diseases caused by oxidative stress related injuries of various tissues and organs, has complex mechanisms, such as neurodegenerative diseases, ischemia-reperfusion injuries, acute liver and kidney injuries and the like, and can also be induced by various compounds, such as erastin or clinical medicine sorafenib and the like, and can induce cell-triggered iron death. Therefore, clinically, not only can the iron death related diseases be effectively relieved, but also the side effects of the medicines can be reduced by inhibiting iron death, and the development of the efficient iron death inhibitor related medicines can bring unprecedented opportunities for treating the iron death related diseases.

However, currently known iron death inhibitors are mainly used for in vitro experiments of cells, but the stability and the effectiveness of the iron death inhibitors in animal bodies are insufficient, typical iron death inhibitors such as Ferrostatin-1 (Fer-1) can inhibit the HT-1080 cell iron death induced by RSL3 or Erastin, but the metabolism of the Fer-1 in the body is unstable, and the inhibition effect is poor; although the iron death inhibitor Liproxstatin-1 (Lip-1) has stronger stability, the safety and the effectiveness of the iron death inhibitor are far from the clinical application standard, so the screening and the development of the iron death inhibitor become one of the hot spots and the difficulties in the iron death research field.

CK-666 is an inhibitor of the Arp2/3 complex, an actin-related protein, and it is useful for studying Arp 2/3-mediated structural changes of cells by binding to the Arp2/3 complex and stabilizing the inactive state thereof. The Arp2/3 complex is a protein containing 7 subunits, and can nucleate microfilaments, inhibit the formation of platy pseudopodes, and influence migration, endocytosis and the like of cells. CK-666 is currently mainly studied in lower organisms such as cells, yeast and insects in vitro, and few relevant studies on CK-666 in mammals such as mice have been reported. The action mechanism and effectiveness of CK-666 in mice are not clear, and no research shows that CK-666 is related to iron death mechanism at present. Therefore, the development of more iron death inhibitors and medicines thereof with better effect and better safety has great significance for treating related diseases caused by iron death.

Disclosure of Invention

The invention aims to overcome the defects and shortcomings of the problems and provides application of CK-666 in inhibiting iron death and preparation of medicines thereof.

The invention aims to provide a new application of CK-666 in inhibiting iron death and preparing medicines for inhibiting cell iron death.

The above purpose of the invention is realized by the following technical scheme:

the research of the invention shows that CK-666 can effectively inhibit iron death induced by various modes, and CK-666 also has good oxidation resistance, can prevent the oxidative rupture of liposome, and shows that CK-666 can inhibit iron death by reducing cell lipid peroxidation. Meanwhile, CK-666 can obviously reduce the blood creatinine and urea nitrogen level of the mice subjected to ischemia reperfusion, and can obviously relieve the renal tissue injury when being used for the mice subjected to ischemia reperfusion, so that the method has better effectiveness.

Therefore, the invention provides the application of CK-666 in inhibiting iron death, preparing iron death inhibitors, being used as iron death inhibitors, preparing medicines for inhibiting cell iron death and preparing medicines for treating diseases caused by iron death.

CK-666 employed in the present invention is a cell permeability inhibitor of the actin-related protein Arp2/3 complex, and has the chemical formulaIs C ₁₈ H ₁₇ FN ₂ O, structural formula is shown as the following formula (I):

preferably, CK-666 is used as an inhibitor of iron death in the preparation of a medicament for treating diseases caused by iron death.

In particular, CK-666 provided by the invention can be used for or used for preparing a medicament for treating diseases caused by iron death and related diseases caused by iron death as an iron death inhibitor, wherein the diseases comprise but are not limited to renal/hepatic/cardiac ischemia reperfusion, neurodegenerative diseases or acute liver and kidney injury.

Preferably, the CK-666 is used for inhibiting iron death of cells and/or tissues and organs.

Preferably, the cell is a fibrosarcoma cell, an embryonic lung fibroblast, and/or a mouse embryonic fibroblast.

Preferably, the medicament contains a pharmaceutically acceptable carrier or excipient.

The invention has the following beneficial effects:

the invention provides application of CK-666 in inhibiting cell iron death and medicine preparation thereof, and researches show that an Arp2/3 compound inhibitor CK-666 can effectively inhibit iron death induced by various modes, can obviously save iron death induced by RSL3 and Erastin and Pfa1 cell iron death caused by Gpx4 gene knockout, and has better cell iron death inhibition effect along with the increase of CK-666 concentration; and CK-666 also has good oxidation resistance, can prevent the oxidative rupture of liposome, and shows that CK-666 can inhibit the death of iron by reducing the peroxidation of cell lipid.

Meanwhile, CK-666 can obviously reduce the blood creatinine and urea nitrogen level of an ischemia reperfusion mouse, can obviously relieve kidney tissue injury when being used for the ischemia reperfusion mouse, and can be used for preparing a medicament for treating diseases caused by iron death. The CK-666 provided by the invention can be used as an iron death inhibitor, has better effect than Lip-1, has better oxidation resistance and safety, and provides a new choice for the research and development of high-efficiency iron death inhibitor related medicines.

Drawings

FIG. 1 is a graph showing the results of detection of iron-dead cells induced in different ways (a. RSL3 induces iron death of HT1080 cells; b. RSL3 induces iron death inhibition rate at different CK-666 concentrations; c. Erastin induces HT1080 cells; d. RSL3 induces iron death of 293T cells; e. Pfa1 cell iron death caused by Gpx4 gene knockout);

FIG. 2 is a graph showing the results of the antioxidant capacity (a) and the inhibition of liposome oxidative burst (b) in vitro;

FIG. 3 is a graph showing the results of renal ischemia reperfusion injury (a. Creatinine level in mouse serum; b. Urea nitrogen level in mouse serum; c.H & E staining of tubular region).

Detailed Description

The invention is further described with reference to the drawings and specific examples, which are not intended to limit the invention in any way. The reagents, methods and apparatus employed in the present invention are conventional in the art, except as otherwise indicated.

Unless otherwise indicated, reagents and materials used in the following examples are commercially available.

The material sources used in the following examples were: human fibrosarcoma cell HT1080 and human embryonic lung fibroblast 293T were purchased from China center for type species Collection (CTCC); immortalized mouse embryo fibroblast capable of inducing GPX4 knockout cell pfa1 was given to professor of cistanche deserticola of river-south university; male 8-12 week old C57 mice were purchased from Beijing Huafukang Biotech, inc.

Example 1 Effect of CK-666 on cellular iron death

In this example, two cell iron death inducers, namely glutathione peroxidase 4 (GPX 4) inhibitors RSL3 and Erastin, were used to induce cell iron death, and then CK-666 inhibitors at different concentrations were added for treatment, while no CK-666 inhibitor was added as a control group (ctl group), to examine cell survival.

Human fibrosarcoma cells HT1080 and human embryonic lung fibroblasts 293T were seeded at 4000/100. Mu.L/well in 96-well plate cell culture plates, respectively, and after 12-24 hours of adherent cell growth, the cells were added to 100. Mu.L of medium containing RSL3 (0, 4nM, 8nM, 16nM, 31nM, 125nM, 500nM, 2000 nM) or erastin (0, 0.15. Mu.M, 0.3. Mu.M, 0.6. Mu.M, 1.25. Mu.M, 2.5. Mu.M, 5. Mu.M, 10. Mu.M) at the corresponding concentrations and CK-666 inhibitor (0, 6. Mu.M, 12.5. Mu.M, 25. Mu.M, 50. Mu.M, 100. Mu.M) at different concentrations, respectively. Each drug concentration was set with 3 multiple wells. After 24h of treatment, nucleic acid dyes Hoechst and Propidium Iodide (PI) were added to stain for 15 min, and Hoechst was able to penetrate the cell membrane and stain the nuclei of all cells. PI cannot penetrate the intact cell membrane and only stains dead cells. Hoechst and PI therefore stain total and dead cells differently, respectively. Then, photographing is carried out through a high-throughput confocal imaging system, and the cell death rate is counted.

Since Pfa1 cell iron death was also caused by the knockout of the Gpx4 gene, pfa1 cell iron death after the knockout of the Gpx4 gene was also measured, and the cells were prepared as described above, and then 100. Mu.L of a medium containing 1. Mu.M of 4-OHT and 100. Mu.M of CK-666 was added, and after 72 hours of treatment, the cell activity was measured by the same method.

As shown in FIG. 1, in HT1080 cells, the cell survival rate was also reduced with the increase of the concentrations of the iron death inducers RSL3 (FIG. 1 a) and Erastin (FIG. 1 c), the iron death induced by RSL3 and Erastin was significantly saved after CK-666 was added, and the iron death induced by RSL3 was more inhibited with the increase of CK-666 concentration (FIG. 1 b). At the same time, RSL 3-induced iron death was also significantly inhibited by CK-666 on 293T cells (FIG. 1 d). In addition, iron death of Pfa1 cells caused by Gpx4 gene knock-out was also significantly inhibited by CK-666 (FIG. 1 e). These results indicate that CK-666 is effective in inhibiting iron death induced by a variety of cellular processes.

Example 2 in vitro antioxidant Capacity of CK-666

1. DPPH free radical assay

DPPH free radical has single electron, its alcoholic solution is purple, and has strong absorption at 515 nm. In the presence of an antioxidant, DPPH free radicals are eliminated, the solution becomes lighter in color, the absorbance at 515nm decreases, and the change in absorbance within a certain range is proportional to the degree of radical elimination. Therefore, the change of the absorbance can be detected by the microplate reader to reflect the scavenging of the free radicals. The in-vitro oxidation resistance of CK-666 was tested by using DPPH assay kit, and the specific procedures were performed according to the method provided on the kit, wherein vitamin C (3. Mu.M, 12.5. Mu.M, 50. Mu.M) with different concentrations was used as positive control, and iron death inhibitors Fer-1 and Lip-1 were used for treatment.

2. Effect of CK-666 on Liposome oxidative cleavage

The terbium ions are wrapped by phospholipid molecules to form liposome, and the liposome is prepared from Fe ²⁺ And H ₂ O ₂ Under the action of the liposome, the liposome gradually undergoes oxidative rupture and releases terbium ions. By utilizing the characteristic that terbium ions can react with dipicolinic acid (DPA) to emit fluorescence, the rupture condition of the liposome can be reflected by detecting the change of the fluorescence value through a microplate reader.

2mg of soybean phospholipid was dissolved in 500. Mu.L of chloroform, and dried with nitrogen in a small round-bottomed flask to form a uniform plasma membrane at the bottom of the flask. 1mL buffer TL (20 mM HEPES pH =7.4, 0.1M sodium chloride, 50mM sodium citrate, 15mM terbium chloride) was added to resuspend the plasma membrane from the previous step, and the liposome was prepared from mini-extruder as described in the specification.

And centrifugally filtering and cleaning the mixture by a 100KD cut-off ultrafiltration tube to remove the unencapsulated terbium ions in the liquid. After the final centrifugation, 500. Mu.L of buffer L (20mM HEPES pH =7.4, 0.1M sodium chloride) was used for resuspension. mu.L liposome was mixed with 10. Mu.M H ₂ O ₂ 、50μM FeSO ₄ 1 μ M Lip-1 or 100 μ M CK-666, buffer L to a total volume of 100 μ L. The fluorescence signal was recorded every 60s with a microplate reader set for 270nm excitation and 490nm emission, and after 3 hours 0.1% Triton X-100 was added to disrupt all liposomes while detecting the fluorescence signal 10 times. All the obtained fluorescence intensity signal changes were counted.

3. Results

The in vitro antioxidant capacity result of CK-666 is shown in figure 2a, and shows that CK-666 has certain in vitro antioxidant capacity, the antioxidant activity is superior to that of a 12.5 mu M vitamin positive control, the antioxidant capacity of CK-666 is between that of a known iron death inhibitor Lip-1 and Fer-1, but is significantly superior to that of Lip-1.

The results of the detection of CK-666 inhibiting liposome oxidative burst are shown in FIG. 2b, fe ²⁺ And H ₂ O ₂ Can oxidize and break the liposome, and can prevent the oxidative breakage of the liposome after the CK-666 is added, and the effect is equivalent to that after the iron death inhibitor Lip-1 is adopted.

Taken together, the results suggest that CK-666 can inhibit iron death by reducing cellular lipid peroxidation.

Example 3 Effect of CK-666 on renal ischemia-reperfusion injury

48 male C57BL/6J mice at 8-12 weeks were selected and randomly divided into 4 groups for experiments, which were set as Sham (Sham) (blank control), model (IRI + DMSO) (ischemia reperfusion (positive control)), iron death inhibitor Lip-1 treatment (IRI + Lip-1) (negative control), and CK-666 treatment (IRI + CK-666) (experimental group). Two days before operation, mice are injected with 200 mu LDMSO or 10mg/kg Lip-1 or 30mg/kg CK-666 intravenously and are fasted for 12 hours before operation. During operation, a mouse is anesthetized by using 3% sodium pentobarbital, skin and muscles are cut at a position 0.5cm beside a back vertebra and 0.5cm below a rib, so that a kidney can be seen, renal arteries of two sides of the kidney are carefully separated, the renal arteries of the two sides of the kidney are clamped by an artery clamp rapidly, the artery clamp is loosened after 30min, blood flow is recovered, and an opening is sutured. Mice were sacrificed 24h after which serum and kidneys were taken. The serum is sent to a detection company to detect the content of serum creatinine and urea nitrogen, and is used for evaluating the kidney function. The kidneys were also H & E stained.

The results are shown in fig. 3, where serum creatinine and urea nitrogen levels were significantly increased in mice with ischemia reperfusion injury model (IRI + DMSO) (fig. 3 a-b), and H & E staining also showed significant renal tubular injury such as vacuolar degeneration in the renal tubular region (fig. 3 c), indicating impaired renal function. After the mice subjected to ischemia reperfusion are subjected to the intervention treatment of the iron death inhibitor Lip-1 (IRI + Lip-1), the blood creatinine value of the mice is reduced to some extent, the renal tubular injury condition is relieved to a certain extent, but the change of serum urea nitrogen is not obvious enough. And the CK-666 treatment can obviously reduce the blood creatinine and urea nitrogen level of the mice subjected to ischemia reperfusion, and H & E staining results also show that renal tubular shape disappearance, epithelial cell necrosis and shedding, vacuole-like degeneration and the like are reduced after the CK-666 treatment, which indicates that renal tissue injury is obviously relieved (figure 3 c). These results demonstrate that CK-666 is effective in alleviating ischemia-reperfusion injury of mouse kidney and is superior to the iron death inhibitor Lip-1.

The results show that CK-666 can effectively inhibit cell iron death and mouse iron death related diseases (renal ischemia reperfusion injury), and can be used for preparing anti-iron death medicines.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Claims (10)

1. Use of CK-666 in the inhibition of iron death.
2. 2. The use of claim 1, wherein CK-666 is used in inhibiting iron death in cells and/or tissue organs.
3. 3. Use according to claim 2, wherein the cells are fibrosarcoma cells, embryonic lung fibroblasts and/or mouse embryonic fibroblasts.
4. Application of CK-666 in preparation of iron death inhibitors.
5. The application of CK-666 as an iron death inhibitor is disclosed.
6. Application of CK-666 in preparation of medicines for inhibiting cell iron death.
7. Application of CK-666 in preparing medicine for treating diseases caused by iron death is disclosed.
8. 8. The use according to claim 7, wherein CK-666 is used as an inhibitor of iron death in the preparation of a medicament for the treatment of diseases caused by iron death.
9. 9. The use of claim 7, wherein the disease is renal/hepatic/cardiac ischemia reperfusion, a neurodegenerative disease or acute liver and kidney injury.
10. 10. The use according to claim 6 or 7, wherein the medicament comprises a pharmaceutically acceptable carrier or excipient.

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