Application of p62 autophagy to regulation and control of SLC7A11 expression to influence AIC ferroptosis

Abstract

The invention discloses application of p62 autophagy to regulation and control of SLC7A11 expression to influence AIC ferroptosis, and relates to the technical field of medical biology. The invention discloses application of P62 autophagy regulation of SLC7A11 expression to influence AIC ferroptosis, experimental design is carried out from multiple aspects of molecules, cells, animal models and the like, layer-by-layer verification is carried out by adopting advanced technologies such as mass spectrometry, co-immunoprecipitation, light confocal and the like, the effect and molecular mechanism of P62 for directly degrading SLC7A11 through an autophagy approach to induce ferroptosis and promote AIC are verified for the first time, and the application of P62 autophagy regulation of SLC7A11 expression to influence AIC ferroptosis is provided for the first time. A new thought is provided for reducing AIC; based on the above, the invention discloses application of p62 as a drug target for screening drugs for preventing, relieving or / and treating doxorubicin cardiotoxicity. The invention provides an application of regulating expression of SLC7A11 by p62 autophagy to influence AIC ferroptosis, and the application can be used as a reference for development of drugs for inhibiting doxorubicin cardiotoxicity, research of therapeutic targets and the like.

Description

Technical Field

[0001] This invention relates to the field of medical biotechnology, specifically the application of p62 autophagy regulating SLC7A11 expression to affect AIC ferroptosis. Background Technology

[0002] Anthracycline anticancer antibiotics (ACs) are landmark drugs in the development of cancer treatment and have been widely used. Even today, with the emergence of new therapies such as targeted therapy and immunotherapy, they remain first-line drugs for anti-tumor treatment. However, the anthracycline-induced cardiotoxicity (AIC) caused by ACs has also attracted great attention in clinical practice. Terminally differentiated cardiomyocyte death is an important pathological basis for the development of cardiac injury, and ACs can induce various forms of cell death.

[0003] Doxorubicin (DOX) is an anthracycline antibiotic and a commonly used broad-spectrum antitumor drug in clinical practice. It has therapeutic effects on various malignant tumors (such as breast cancer, lymphoma, sarcoma, and certain types of leukemia). Its mechanism of action involves intercalation into human DNA, thereby blocking tumor cell proliferation and inducing apoptosis. However, once DOX was developed and used, it was found to have dose-dependent cardiotoxicity, including various types of arrhythmias, ventricular dilation, and ultimately congestive heart failure. These cardiotoxicities have greatly limited the clinical use of DOX.

[0004] Currently, the main drugs for treating DOX cardiotoxicity include altering the dosage and administration pattern of DOX, liposomal doxorubicin, dextromethorphan and other antioxidants, and drugs for treating heart failure (such as beta-blockers and ACEI / ARB drugs). However, these drugs have difficulty targeting the specific pathogenesis pathways and have limitations in application. For example, studies have shown that dextromethorphan may increase the risk of secondary malignancies in cancer patients. Therefore, in-depth research into the pathogenesis of DOX cardiotoxicity and the search for new therapeutic targets are crucial.

[0005] SQTM1, also known as P62, is located on chromosome 5 and has 8 exons, comprising 440 amino acid residues. It is an important selective autophagy adaptor protein. P62 contains six functional domains: a ubiquitin-associated domain, a Kelch-like epichlorohydrin-associated protein domain, a microtubule-associated protein 1A / 1B light chain 3 domain, a tumor necrosis factor receptor-associated factor 6 domain, Phox and Bem1p domains, and a ZZ-type zinc finger region. These multifunctional domains make it a center for multiple signaling pathways, participating in the maintenance and regulation of basic cellular functions.

[0006] Studies have found that p62 also plays an important role in autophagy-induced ferroptosis (AIC). A study on AIC published in *Toxicology* by the inventors confirmed that abnormal levels of p62 and autophagy promote the occurrence and development of apoptosis, thereby causing myocardial damage. However, the role and mechanism of p62 in AIC ferroptosis remain unclear. Confirming its role and mechanism can provide a basis for the development of cardiotoxic drugs and subsequent research. Summary of the Invention

[0007] The purpose of this invention is to provide an application of p62 autophagy regulating SLC7A11 expression to affect AIC ferroptosis, which can serve as a reference for the development of drugs that inhibit doxorubicin cardiotoxicity and research on therapeutic targets.

[0008] To achieve the above-mentioned technical objectives and effects, the present invention is implemented through the following technical solution:

[0009] The first objective of this invention is to provide the use of p62 as a drug target for screening drugs that prevent, alleviate, or / and treat doxorubicin cardiotoxicity.

[0010] Furthermore, the p62 autophagy-regulated SLC7A11 expression affects AIC ferroptosis and promotes the cardiotoxicity of anthracycline antibiotics.

[0011] A second objective of the present invention is to provide the use of p62 in the preparation of drugs for the prevention, relief, and / or treatment of doxorubicin cardiotoxicity.

[0012] A third objective of this invention is to provide a drug for preventing, alleviating, and / or treating doxorubicin cardiotoxicity, said drug containing a p62 agonist, the p62 gene, or the p62 protein.

[0013] Furthermore, the p62 agonist is a small molecule, antibody, or cooperating nucleic acid that targets and enhances or increases p62 expression.

[0014] Furthermore, the drug also includes a pharmaceutically acceptable carrier.

[0015] The beneficial effects of this invention are as follows:

[0016] This invention discloses the application of p62 autophagy regulating SLC7A11 expression to affect ferroptosis in autoimmune intraepithelial immunodeficiency (AIC). Experiments were designed at multiple levels, including molecular, cellular, and animal models. Advanced technologies such as mass spectrometry, immunoprecipitation, and optical confocal microscopy were used for layer-by-layer verification. This invention is the first to verify that p62 induces ferroptosis and promotes AIC by directly degrading SLC7A11 through autophagy, and the molecular mechanism of this effect is demonstrated, providing a new approach to alleviating AIC.

[0017] The present invention p62 is used as a drug target for screening drugs that prevent, alleviate, or / and treat doxorubicin cardiotoxicity.

[0018] Of course, any product implementing this invention does not necessarily need to achieve all of the advantages described above at the same time. Attached Figure Description

[0019] Figure 1 The results are from the AIC animal model and AIC cell model described in the embodiments of the present invention;

[0020] Figure 2 The protein was expressed at low levels in the AIC animal and AIC cell models described in the embodiments of the present invention.

[0021] Figure 3 The IP described in this embodiment of the invention is used to detect the ubiquitination level of SLC7A11;

[0022] Figure 4 The above is a graph showing the analytical results of the mass spectrometry technique and immunoprecipitation described in the embodiments of the present invention.

[0023] Figure 5 The Western Blot method described in this embodiment of the invention is used to detect the expression of P62 and SLC7A11 in AIC animal and cell models.

[0024] Figure 6 The expression of GPX4, FTH1, and MDA in the cell model described in the embodiments of the present invention;

[0025] Figure 7 The Western Blot method described in this embodiment of the invention is used to detect the expression of P62 and SLC7A11 proteins and mRNAs in an AIC cell model after P62 interference.

[0026] Figure 8 The results of Western blot analysis are as described in the embodiments of the present invention, which include the autophagy-lysosomal pathway inhibitor chloroquine and the proteasome inhibitor MG132. Detailed Implementation

[0027] To more clearly illustrate the technical solutions of the embodiments of the present invention, the present invention will be described in detail below with reference to the accompanying drawings.

[0028] The present invention will now be described in conjunction with specific embodiments:

[0029] Example 1

[0030] Constructing AIC animal models and AIC cell models, such as Figure 1 As shown, in the AIC animal model, the iron ion solubility in myocardial tissue increased in the DOX-treated group. Figure 1 A), MDA levels increased ( Figure 1 B), GPX4 and FTHI protein expression levels decreased ( Figure 1 C). In the AIC cell model, with increasing DOX dosage, NRCM cardiomyocyte viability decreased and intracellular iron ion fluorescence increased (C). Figure 1 D), MDA content increased ( Figure 1 E), the expression levels of GPX4 and FTHI proteins also decreased (E), Figure 1 F).

[0031] Example 2

[0032] SLC7A11 was expressed at low protein levels and showed no change in mRNA levels in both animal and cell models of AIC, confirming that SLC7A11 is associated with AIC and that SLC7A11 undergoes post-translational modifications.

[0033] like Figure 2 As shown, in the AIC animal model, the expression level of SLC7A11 was significantly lower than that in the normal treatment group, while the mRNA expression level did not change significantly. Figure 2 A, B). In the AIC cell model, the expression level of SLC7A11 was significantly lower than that of the normal treatment group, while the mRNA expression level did not change significantly. Figure 2 C, D).

[0034] Example 3

[0035] The ubiquitin level of SLC7A11 was increased in the AIC cell model, confirming that AC can lead to an increase in the ubiquitination level of SLC7A11.

[0036] like Figure 3 As shown, IP was used to detect the ubiquitination level of SLC7A11;

[0037] Example 4

[0038] Mass spectrometry and immunoprecipitation analysis revealed the interaction between P62 and SLC7A11, confirming the existence of this interaction. Figure 4 A) In NRCM cardiomyocytes, IP experiments revealed that p62 may interact with SLC7A11. Figure 4 B).

[0039] like Figure 4 As shown, Figure 4 A. Mass spectrometry analysis revealed that P62 may bind to SLC7A11; B. In NRCM cells, P62 binds to SLC7A11.

[0040] Example 5

[0041] In both the AIC animal and AIC cell models, p62 was highly expressed, while SLC7A11 was lowly expressed, indicating that p62 may regulate SLC7A11 expression levels. In the AIC animal model, p62 expression levels were significantly higher than in the normal treatment group, while SLC7A11 expression levels were significantly lower than in the normal treatment group. Figure 5 In the AIC cell model, the expression level of P62 was significantly higher than that of the normal treatment group, while the expression level of SLC7A11 was significantly lower than that of the normal treatment group, and the two expression levels were negatively correlated. Figure 5 .B).

[0042] Figure 5 middle, Figure 5 A. Western blot was used to detect the expression of P62 and SLC7A11 in the AIC animal model, with Tublin as the loading reference; B. Western blot was used to detect the expression of P62 and SLC7A11 in the AIC cell model, with Tublin as the loading reference.

[0043] Example 6

[0044] Downregulation of p62 expression inhibited ferroptosis in an AIC cell model, indicating that p62 is involved in regulating ferroptosis in AIC. Using p62-interfering lentivirus to inhibit p62 expression in NRCM cells, followed by co-incubation with 1 μmol / L DOX for 24 h, Western blot analysis showed that, compared with the control group and the DOX-only treatment group, downregulation of p62 expression significantly increased the expression of DOX-induced ferroptosis-related proteins GPX4 and FTH1. Figure 6 A). MDA biochemical assay results showed that, compared with the control group and the DOX-treated group alone, downregulation of P62 expression significantly reduced the DOX-induced increase in MDA expression levels. Figure 6 B).

[0045] Figure 6 middle, Figure 6 A. Aesthetically blotting was used to detect the expression of GPX4 and FTH1 in the AIC cell model, with GAPDH as the loading reference; B. Biochemical analysis was used to detect the expression of MDA in the AIC cell model *p<0.05.

[0046] Example 7

[0047] Downregulating p62 expression increased SLC7A11 protein expression in the AIC cell model, but did not affect SLC7A11 mRNA expression, confirming that p62 is involved in the degradation of ubiquitinated SLC7A11. Using p62-interfering lentivirus to inhibit p62 expression in NRCM cells, followed by co-incubation with 1 μmol / L DOX for 24 h, the results showed that, compared with the control group and the empty vector treatment group, interfering with p62 expression significantly increased SLC7A11 protein levels. Figure 7 AC); however, it does not affect the mRNA level of SLC7A11 (AC); Figure 7 D).

[0048] Figure 7 A, B: Western Blot analysis to detect the expression of P62 and SLC7A11 proteins in the AIC cell model after P62 interference, with GAPDH as the loading reference; C, qRT-PCR analysis to detect the expression of P62 mRNA in the AIC cell model after P62 interference; D, qRT-PCR analysis to detect the expression of SLC7A11 mRNA in the AIC cell model after P62 interference.

[0049] Example 8

[0050] In an AIC cell model, the addition of chloroquine (CQ), an inhibitor of the autophagy-lysosomal pathway, increased SLC7A11 levels, while the addition of the proteasome inhibitor MG132 decreased SLC7A11 levels. This indicates that the p62-autophagy pathway is involved in the degradation of ubiquitinated SLC7A11. Western blotting in the AIC cell model confirmed that the addition of chloroquine increased SLC7A11 levels. Figure 8 A, B), while the addition of the proteasome inhibitor MG132 reduced SLC7A11 levels ( Figure 8 C, D).

[0051] Figure 8 A, B: Western Blot analysis of SLC7A11 expression in AIC cell model after the addition of chloroquine, an inhibitor of the autophagy-lysosome pathway, with GAPDH as the loading reference; C, D: Western Blot analysis of SLC7A11 expression in AIC cell model after the addition of MG132, a proteasome inhibitor, with GAPDH as the loading reference.

[0052] Example 9

[0053] According to the above embodiments, p62 is used as a drug target for screening drugs that prevent, alleviate, or / and treat doxorubicin cardiotoxicity;

[0054] In this embodiment, p62 autophagy regulates SLC7A11 expression, affecting AIC ferroptosis and promoting the cardiotoxicity of anthracycline antibiotics.

[0055] In this embodiment, p62 is used in the preparation of drugs for the prevention, relief, and / or treatment of doxorubicin cardiotoxicity.

[0056] In this embodiment, a drug for preventing, alleviating, and / or treating doxorubicin cardiotoxicity is disclosed, wherein the drug contains a p62 agonist, a p62 gene, or a p62 protein.

[0057] p62 agonists are small molecules, antibodies, or cooperating nucleic acids that target and enhance or increase p62 expression.

[0058] The drug also includes a pharmaceutically acceptable carrier.

[0059] The preferred embodiments of the present invention disclosed above are merely illustrative of the invention. These preferred embodiments do not exhaustively describe all details, nor do they limit the invention to the specific implementations described. Many modifications and variations can be made based on the content of this specification. These embodiments have been selected and specifically described in this specification to better explain the principles and practical applications of the invention, thereby enabling those skilled in the art to better understand and utilize the invention. The invention is limited only by the claims and their full scope and equivalents.

Claims

1. Application of p62 as a drug target for screening drugs to prevent, alleviate, or / and treat doxorubicin cardiotoxicity.

2. The application as described in claim 1, characterized in that: The p62 autophagy regulation of SLC7A11 expression affects AIC ferroptosis and promotes the cardiotoxicity of anthracycline antibiotics.

3. The use of p62 in the preparation of drugs for the prevention, relief, and / or treatment of doxorubicin cardiotoxicity.

4. A drug for preventing, alleviating, and / or treating doxorubicin cardiotoxicity, characterized in that: The drug contains a p62 agonist, the p62 gene, or the p62 protein.

5. The drug as described in claim 4, characterized in that: The p62 agonist is a small molecule, antibody, or cooperating nucleic acid that targets and enhances or increases p62 expression.

6. The drug as described in claim 4, characterized in that: The drug also includes a pharmaceutically acceptable carrier.

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