Application of Aspisin as molecular marker in regulation and control of brown adipocyte ferroptosis

Abstract

The invention provides application of Asprosin (ASP) as a molecular marker in regulation and control of brown adipocyte ferroptosis, and relates to the technical field of biological medicine. A CRISPR-Cas9 technology is adopted to establish an ASP defect model with brown adipocytes as objects, it is found that ASP defect intervention can affect the level of ferroptosis related indexes, and meanwhile it is found that Notch3 as a downstream target of ASP plays an important role in the ferroptosis process of the brown adipocytes. The ASP and Notch3 are prompted to provide a new thought for developing a treatment strategy for brown adipocyte ferroptosis.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to the application of Asprosin as a molecular marker in regulating ferroptosis of brown fat cells. Background Art

[0002] Ferroptosis is an emerging mode of programmed cell death (PCD), and its core feature is the iron-dependent lipid peroxidation process of cell membrane. Specifically, under the catalysis of divalent iron ions or esteroxygenase, reactive oxygen species (ROS) are generated in large quantities, which then catalyze the lipid peroxidation reaction of polyunsaturated fatty acids rich in cell membranes, generating toxic metabolites including malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE), and finally inducing cell death. In this process, glutathione peroxidase 4 (GPX4) and superoxide dismutase (SOD) play a key role in inhibiting ferroptosis, while prostaglandin endoperoxide synthase 2 (PTGS2) plays a promoting role.

[0003] Ferroptosis is closely related to a variety of disease states, including neurodegenerative diseases (such as Parkinson's disease and Alzheimer's disease), tissue ischemia-reperfusion injury, and certain types of cancer (such as diffuse large B-cell lymphoma and hepatocellular carcinoma). Although ferroptosis has been observed in a variety of cells, it is still unclear whether adipocytes undergo ferroptosis, the specific molecular markers of adipocyte ferroptosis, and whether this molecular marker can serve as an early warning indicator to prevent adipocyte ferroptosis.

[0004] Asprosin (ASP), an adipokine encoded by the fibrinogen-1 (FBN1) gene, is mainly distributed in adipose tissue, especially white adipose tissue (WAT), and plays a key role in regulating energy metabolism and glucose homeostasis. Recent studies have shown that ASP is not only involved in the pathogenesis of obesity, insulin resistance and diabetes, but also affects the function and fate of WAT, specifically by inhibiting the browning of white fat, promoting lipid production and accumulation, and impairing mitochondrial function and heat production. However, the mechanism of action of ASP in brown adipocytes is still unclear. In addition, the specific molecular markers of ferroptosis in brown adipocytes have not yet been determined, and whether ASP can be used as an early sensitive marker for monitoring ferroptosis in brown adipocytes and using it as a target to prevent the occurrence of ferroptosis is still unknown. Therefore, it is urgent to develop a solution that can effectively solve the above problems. Summary of the invention

[0005] The present invention provides an application of Asprosin (ASP) as a molecular marker in regulating ferroptosis of brown adipocytes. The present invention adopts CRISPR-Cas9 technology to establish an ASP deficiency model with brown adipocytes as the object, and finds that ASP deficiency intervention affects the levels of ferroptosis-related indicators. Meanwhile, it is found that Notch3, as a downstream target of ASP, plays an important role in the ferroptosis process of brown adipocytes, suggesting that ASP and Notch3 can provide new ideas for developing treatment strategies for ferroptosis of brown adipocytes.

[0006] In a first aspect, the present invention provides an application of ASP as a molecular marker in regulating ferroptosis of brown adipocytes, specifically, an application of ASP deficiency intervention in inhibiting ferroptosis of brown adipocytes.

[0007] Optionally, the ASP deficiency intervention model is established through CRISPR-Cas9 technology.

[0008] Alternatively, free Fe in brown adipocytes was regulated by ASP deficiency. 2+ The content, ROS level, MitoSOX level and MDA content were decreased, while the SOD content was increased.

[0009] Fe 2+ The ROS generated by catalysis attack the polyunsaturated fatty acids on the cell membrane, amplify lipid peroxidation, form electrophilic, oxidative truncated phospholipid variants, react with amino acid residues, induce protein and lipid oxidation, generate toxic metabolites such as MDA, destroy cell membrane structure and function, and induce cell ferroptosis. In this process, SOD catalyzes superoxide anion free radicals (O 2- ) into hydrogen peroxide (H2O2) and oxygen (O2) to alleviate oxidative stress.

[0010] Alternatively, upon ASP deficiency intervention, the expression level of GPX4 was increased while the expression level of PTGS2 was decreased in brown adipocytes.

[0011] During ferroptosis, GPX4 can reduce lipid peroxides to harmless alcohol compounds, preventing the accumulation of lipid peroxides from causing irreversible damage to the cell membrane, while the expression and activity of PTGS2 are positively correlated with the stimulation of ferroptosis activators (such as RSL3 and erastin).

[0012] In a second aspect, the present invention provides an application of a Notch3 molecule in preparing a method for regulating ASP to promote ferroptosis of brown adipocytes. Specifically, Notch3 is a downstream target of ASP, and ASP cannot function when Notch3 is silenced.

[0013] Notch3 is a key mediator of ASP's role in regulating ferroptosis in brown adipocytes.

[0014] In a third aspect, the present invention provides a use of a Notch3 activator in the preparation of an inhibitor for enhancing ferroptosis of brown adipocytes.

[0015] In a fourth aspect, the present invention provides a method for using a Notch3 molecule as a downstream target of ASP in preparing a method for antagonizing ASP-promoted ferroptosis of brown adipocytes.

[0016] Notch3 silencing blocked the promoting effect of ASP on lipid peroxidation.

[0017] In a fifth aspect, the present invention provides a use of a ferroptosis inhibitor in developing a drug for preventing ferroptosis of brown adipocytes.

[0018] Optionally, the drug includes an ASP molecular marker and a Notch3 activator.

[0019] The beneficial effects of the present invention include:

[0020] (1) The ASP molecular marker provided by the present invention can be used for the detection and prevention of ferroptosis of brown adipocytes, and can be applied to reagents, kits, and the preparation of drugs for preventing ferroptosis of brown adipocytes;

[0021] (2) The present invention found that silencing Notch3, a downstream target of ASP, can avoid the effect of ASP on brown adipocyte ferroptosis. Utilizing this target can further develop methods to resist brown adipocyte ferroptosis, providing theoretical and experimental basis for the development of biological treatments for brown adipocyte ferroptosis and new small molecule antagonists. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly introduce the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for describing the embodiments.

[0023] Figure 1 The Fe of different groups after ASP intervention in the brown adipocytes model established in Example 1 2+ content;

[0024] Figure 2 The ROS levels, MitoSOX levels, MDA contents and SOD contents of different groups after ASP intervention in the ASP-deficient model established in brown adipocytes in Example 1;

[0025] Figure 3 The GPX4 and PTGS2 levels of different groups after ASP intervention in the ASP-deficient model established in brown adipocytes in Example 1;

[0026] Figure 4 The Fe of different groups after ASP intervention and / or Notch3 gene silencing in brown fat cells in Example 2 2+ content;

[0027] Figure 5 The ROS levels, MDA contents and SOD contents of different groups after ASP intervention and / or Notch3 gene silencing in brown adipocytes in Example 2;

[0028] Figure 6 The GPX4 and PTGS2 levels of different groups after ASP intervention and / or Notch3 gene silencing in brown adipocytes in Example 2;

[0029] Figure 7 The mechanism by which ASP and its downstream target Notch3 regulate ferroptosis of brown adipocytes. DETAILED DESCRIPTION

[0030] In order to make the purpose, technical solutions and advantages of the present invention clearer, the present invention will be further described through the following embodiments in conjunction with the accompanying drawings. However, the following embodiments are illustrative and are only used to specifically describe the present invention rather than to limit the present invention.

[0031] The present invention provides an application of ASP as a molecular marker in regulating ferroptosis of brown adipocytes, specifically, an application of ASP deficiency intervention in inhibiting ferroptosis of brown adipocytes.

[0032] In some embodiments, the ASP deficiency intervention is modeled by CRISPR-Cas9 technology.

[0033] In some embodiments, free Fe in brown adipocytes is increased upon ASP deficiency intervention. 2+ The content, ROS level, MitoSOX level and MDA content were decreased, while the SOD content was increased.

[0034] In some embodiments, upon ASP deficiency intervention, the expression level of GPX4 is increased while the expression level of PTGS2 is decreased in brown adipocytes.

[0035] The present invention also provides an application of a Notch3 molecule in preparing a method for regulating ASP to promote ferroptosis of brown fat cells.

[0036] The present invention also provides an application of a Notch3 activator in the preparation of an inhibitor for enhancing ferroptosis of brown adipocytes.

[0037] The present invention also provides an application of a Notch3 molecule as a downstream target of Asprosin in preparing a method for antagonizing Asprosin's promotion of ferroptosis in brown fat cells.

[0038] The present invention also provides an application of a ferroptosis inhibitor in developing a drug for preventing ferroptosis of brown fat cells.

[0039] In some embodiments, the drug includes an ASP molecular marker and a Notch3 activator.

[0040] In the following examples, unless otherwise specified, raw materials or processing techniques are conventional commercially available raw materials or conventional processing techniques in the art.

[0041] Example 1

[0042] In Example 1 of the present invention, ASP deficiency was intervened in brown fat cells by CRISPR-Cas9 technology, and then ASP was supplemented, and relevant indicators were measured to verify the effect of ASP on ferroptosis of brown fat cells. C3H10T1 / 2 cells were selected for the experiment, and the following steps were included:

[0043] Culture, grouping, treatment and detection of ferroptosis-related indicators of S1 and C3H10T1 / 2 cells

[0044] C3H10T1 / 2 cells were cultured in DMEM medium containing 10% fetal bovine serum (FBS), 10 μg / mL insulin, 100 nM T3, 5 μM rosiglitazone, 10 μM dexamethasone, 0.25 mM indomethacin, and 0.5 mM IBMX for 48 h and then transferred to DMEM medium containing 10% FBS, 10 μg / mL insulin, 5 μM rosiglitazone, and 100 nM T3, and the medium was changed every 2 days for 3 times;

[0045] CRISPR-Cas9 technology was used to introduce Cas9 protein and sgRNA sequence expression frame of target protein ASP into C3H10T1 / 2 cells via lentivirus to establish the ASP deficiency model of C3H10T1 / 2 cells;

[0046] Divided into control group (WT), ASP - / - Groups and ASP - / - +ASP group, WT group were normal C3H10T1 / 2 cells, ASP - / - The ASP-deficient C3H10T1 / 2 cells were - / - +ASP group consisted of ASP-deficient C3H10T1 / 2 cells treated with 1 μM ASP for 48 h;

[0047] The cell culture supernatant and cells of each group were collected for index detection;

[0048] S2. Detection of Fe 2+ content

[0049] Fe was detected using a commercially available detection kit 2+ Assay content, follow the manufacturer's reagent instructions;

[0050] After the cells of each group were lysed and centrifuged, the supernatant was collected. The samples, standard solutions and reagents were added to a 96-well plate. After standing at 37°C for 10 minutes, the absorbance at 593 nm was measured. The corresponding content was calculated using the standard curve. The results are shown in the figure. Figure 1 As shown;

[0051] S3. Detection of ROS and MitoSOX levels

[0052] ROS and MitoSOX levels were detected using commercially available test kits according to the manufacturer’s reagent instructions;

[0053] After the cells were treated in the previous stage, the culture medium was discarded, the cells were rinsed twice with PBS, the dye diluted with serum-free culture medium (1:1000) was added, and the cells were placed in an incubator at 37°C in the dark for 30 minutes. After being washed with PBS, the cells were placed in an ELISA reader for detection and the relative fluorescence intensity of each group was calculated to indicate the level of ROS and MitoSOX. The results are shown in Figure 2 A and Figure 2 As shown in B;

[0054] S4. Detection of MDA and SOD content

[0055] The MDA and SOD contents were detected using commercially available test kits according to the manufacturer's reagent instructions.

[0056] The cell suspension was mixed with the reagents in the kit respectively. The MDA detection suspension was placed in a 95°C water bath for 40 minutes and then centrifuged at 3500 rpm for 10 minutes to obtain the supernatant. The absorbance was measured at 532 nm. The results were as follows: Figure 2 C; After the SOD detection suspension was incubated at 37°C for 20 min, the absorbance was measured at 450 nm. Figure 2 As shown in D;

[0057] S5. Detection of GPX4 and PTGS2 levels by Western blotting

[0058] After lysing C3H10T1 / 2 cells with RIPA lysis buffer, centrifugation was performed at 12000 rpm for 15 min at 4°C, and the supernatant was taken to determine the total protein content using a BCA kit;

[0059] 30 μg of protein was loaded and electrophoresed on 10% SDS-PAGE, transferred to a PVDF membrane and blocked with 7% skim milk powder for 2 h. After incubation with the corresponding GPX4 and PTGS2 primary antibody solutions (1:1000) at 4°C overnight, the membrane was incubated in an HRP-conjugated secondary antibody solution (1:2000) for 2 h. The signal was detected using a gel imaging system and an ECL detection kit. The results are shown in Figure 3 As shown in A, Image J software was used to analyze the protein gray value of the bands. Figure 3 As shown in B.

[0060] Results Analysis

[0061] The data obtained in Example 1 are expressed as mean ± standard error (SEM); GraphPad Prism 8.0 was used for data analysis, Shapiro-Wilk normality test was used to test the normality of the data, one-way ANOVA was used to compare the data between different groups, and then Bonferroni multiple comparison test was performed; - / - Compared with the same group, “*” represents P < 0.05, and “**” represents P < 0.01;

[0062] See also Figure 1 Compared with the WT group, ASP - / - Fe in C3H10T1 / 2 cells 2+ Levels dropped significantly; while ASP - / - In the +ASP group, Fe 2+ The level of ASP increased significantly, indicating that the level of ASP can serve as a Fe 2+ Important indicator of content;

[0063] See also Figure 2 A. Figure 2 B. Figure 2 C and Figure 2 D, Compared with the WT group, ASP - / - The ROS level, MitoSOX level and MDA content in C3H10T1 / 2 cells of group A decreased significantly, while the SOD content increased significantly. - / - In the +ASP group, the changing trends of the above indicators were similar to those of ASP - / - The groups were completely opposite, indicating that ASP has the effect of promoting lipid peroxidation;

[0064] See also Figure 3 B, Compared with the WT group, ASP - / - The expression level of PTGS2 in C3H10T1 / 2 cells of group A was significantly decreased, while the expression level of GPX4 was significantly increased;- / - In the +ASP group, the changing trends of the above indicators were similar to those of ASP - / - The results of the two groups were completely opposite, which further confirmed the important regulatory role of ASP in the ferroptosis process;

[0065] In summary, ASP can significantly regulate the ferroptosis process of brown adipocytes. The changes in ferroptosis-related indicators are closely related to ASP levels. ASP can be used as a molecular marker for early monitoring of ferroptosis in brown adipocytes, and its level can be used as a basis for identifying ferroptosis. In addition, ASP can also be used as a potential drug target, providing new ideas for the development of drugs, reagents and kits to prevent ferroptosis in brown adipocytes.

[0066] Example 2

[0067] Example 2 of the present invention performs ASP intervention and / or Notch3 gene silencing in brown adipocytes to study the mechanism of ASP regulating ferroptosis of brown adipocytes. The difference from Example 1 is that:

[0068] In step S1, Notch3 gene needs to be silenced, including: after designing and synthesizing siRNA that specifically binds to Notch3 mRNA, the siRNA is introduced into C3H10T1 / 2 cells through a transfection reagent, the intracellular siRNA is processed into a mature siRNA molecule, and binds to RISC (RNA-induced silencing complex) to form a RISC-siRNA complex, which recognizes and cuts Notch3 mRNA to achieve silencing of Notch3 gene;

[0069] In step S1, the cells were divided into a vehicle group (Vehicle), a negative control group (NC-siRNA), a Notch3-siRNA silencing group (Notch3-siRNA), an ASP intervention group (ASP), an ASP intervention + negative control group (ASP+NC-siRNA), and an ASP intervention + Notch3-siRNA silencing group (ASP+Notch3-siRNA).

[0070] Finally, the cell culture supernatant and cells of each group were collected and Fe 2+ content( Figure 4 )、ROS content( Figure 5 A) MDA content ( Figure 5 B) and SOD content ( Figure 5 C), GPX4 level and PTGS2 level ( Figure 6 ) and other indicators.

[0071] Results Analysis

[0072] The data obtained in Example 2 are expressed as mean ± standard error (SEM); GraphPad Prism 8.0 was used for data analysis, Shapiro-Wilk normality test was used to test the normality of the data, one-way ANOVA was used to compare the data between different groups, and then Bonferroni multiple comparison test was performed; compared with the Vehicle group, "*" represents P < 0.05, "**" represents P < 0.01, and ns represents no statistical significance;

[0073] See also Figure 4 Compared with the Vehicle group, the Fe 2+ The levels of Fe in brown fat cells increased significantly. 2+ The level was positively correlated with ASP and negatively correlated with Notch3; however, the Fe 2+ The levels of Notch3-siRNA group were not significantly different from those of Notch3-siRNA group, indicating that the two treatments did not produce an additive effect; this finding confirms that Notch3 plays a role in the expression of Fe in brown adipocytes. 2+ It plays an important role in homeostasis and is a key mediator of ASP's effects in brown adipocytes;

[0074] See also Figure 5 A. Figure 5 B and Figure 5 C, Compared with the Vehicle group, the ROS and MDA levels in the ASP group and the Notch3-siRNA group were significantly increased, while the SOD content was significantly decreased; compared with the Notch3-siRNA group, the levels of each index in the ASP+Notch3-siRNA group did not change significantly; this finding indicates that Notch3 silencing blocked the promoting effect of ASP on lipid peroxidation, indicating that Notch3 is also involved in regulating the lipid peroxidation process;

[0075] See also Figure 6 A and Figure 6 B, The ASP group and Notch3-siRNA group showed the same trend in affecting the levels of GPX4 and PTGS2; however, compared with the Notch3-siRNA group, the expression levels of GPX4 and PTGS2 in the ASP+Notch3-siRNA group did not change significantly;

[0076] In summary, Notch3, as a downstream target of ASP, plays an important role in the ferroptosis of brown adipocytes. At the same time, Notch3 silencing can block the effect of ASP. Therefore, Notch3 can also be used as a potential target for drugs, reagents, kits and gene editing, providing new ideas for the development of therapeutic strategies for ferroptosis of brown adipocytes.

[0077] Although the embodiments of the present invention are described in detail above, it is obvious to those skilled in the art that various modifications and variations can be made to these embodiments. However, it should be understood that such modifications and variations are within the scope and spirit of the present invention as described in the claims. Moreover, the present invention described herein may have other embodiments and may be implemented or realized in a variety of ways.

Claims

1. Application of Asprosin as a molecular marker in regulating ferroptosis of brown adipocytes.

2. Application of Asprosin deficiency intervention in inhibiting ferroptosis of brown adipocytes.

3. Application of Notch3 molecules in the preparation and regulation of Asprosin to promote ferroptosis of brown adipocytes.

4. Application of Notch3 activators in the preparation of inhibitors that enhance ferroptosis of brown adipocytes.

5. Application of Notch3 molecule as a downstream target of Asprosin in the preparation of antagonizing Asprosin-promoting ferroptosis of brown adipocytes.

6. Application of ferroptosis inhibitors in the development of drugs to prevent ferroptosis of brown adipocytes.

7. The use according to claim 6, characterized in that: The drug includes an Asprosin molecular marker and a Notch3 activator.

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