Application of selenoprotein F expression agent in preparation of drugs for treating oxidative stress diseases

Abstract

The invention relates to the technical field of bioengineering, in particular to application of a selenoprotein F expression agent in preparation of drugs for treating oxidative stress diseases. The invention provides an application of a selenoprotein F expression agent in preparation of drugs for oxidative stress diseases in an optional embodiment. The drugs for oxidative stress diseases comprise the selenoprotein F expression agent. The invention finds that selenoprotein F can influence the expression of an antioxidant gene by regulating an Nrf2 signal channel, the cell injury caused by oxidative stress is remarkably improved, the research direction of the development of antioxidant related drugs is widened, and the selenoprotein F expression agent is prepared into a drug, so that the occurrence of oxidative stress related diseases can be remarkably treated or inhibited.

Description

[0001] This invention claims priority from application number 2023113269496 Technical Field

[0002] The present invention relates to the field of bioengineering technology, and in particular to application of a selenoprotein F expression agent in the preparation of oxidative stress disease drugs. Background Art

[0003] Oxidative stress refers to the imbalance between oxidation and antioxidant defense in the body, the excessive production of reactive oxygen species (ROS) and reactive nitrogen species (RNS), which exceeds the body's antioxidant capacity, leading to the accumulation of free radicals, attacking intracellular phospholipids, proteins, nucleic acids and other biological macromolecules, causing a variety of oxidative damage, and destroying the normal function of cells. As a high-metabolism cell, the maintenance of the basic functions of neurons depends on the energy supplied by the mitochondrial respiratory chain and oxidative phosphorylation. Therefore, neurons are more susceptible to damage by free radicals, accelerating neuronal damage and cell death. Oxidative stress is an important therapeutic target for many nervous system diseases, especially neurodegenerative diseases.

[0004] The transcription factor Nrf2 (Nuclear factor erythroid 2-related factor 2) is an important regulator of cellular antioxidant responses. It protects cells from oxidative stress damage mainly by regulating the expression of antioxidant defense genes in cells. Under normal circumstances, Keap1 (Kelch-like ECH-related protein 1) binds to Nrf2, causing Nrf2 to be ubiquitinated and degraded in the cytoplasm; however, under oxidative stress, the thiol group of Keap1 undergoes oxidative modification, causing Nrf2 to dissociate from Keap1 and translocate to the nucleus, initiating the expression of a series of antioxidant enzymes and detoxification enzyme genes, such as superoxide dismutase (SOD), glutathione peroxidase (GPx), NAD(P)H quinone reductase 1 (NQO1), etc., enhancing the body's antioxidant capacity.

[0005] Selenium (Se) is an essential trace element in the human body and is particularly important for maintaining good health. After various forms of selenium enter the human body, its main physiological function is mainly through selenoproteins (proteins containing selenocysteine). Selenoprotein is a special protein that contains the 21st amino acid - selenocysteine. Selenoprotein F (SELENOF, a 15kDa selenoprotein, also known as Sep15) is an endoplasmic reticulum resident protein. However, the biological function of selenoprotein F is still unclear. Summary of the invention

[0006] In order to solve the above technical problems, the present invention provides an application of a selenoprotein F expression agent in the preparation of drugs for oxidative stress diseases. The present invention finds that selenoprotein F can affect the expression of antioxidant genes by regulating the Nrf2 signaling pathway, significantly improve cell damage caused by oxidative stress, broaden the research direction of the development of antioxidant-related drugs, and prepare the selenoprotein F expression agent into a drug, which can significantly treat or inhibit the occurrence of oxidative stress-related diseases.

[0007] To this end, the present invention provides the following technical solutions:

[0008] First, the present invention provides, in an optional embodiment, a use of a selenoprotein F expression agent in the preparation of a drug for oxidative stress diseases, wherein the drug for oxidative stress diseases comprises a selenoprotein F expression agent.

[0009] Preferably, the selenoprotein F expression agent is selected from one or more of a gene vector (such as a plasmid or a viral vector), a small molecule agonist, other compounds or biological agents that upregulate the expression of selenoprotein F. In the oxidative stress disease drug, the concentration of the selenoprotein F expression agent is 200ng / μL-1000ng / μL. In the oxidative stress disease drug, the concentration of the selenoprotein F expression agent is 600ng / μL.

[0010] Preferably, the oxidative stress disease drug also includes an excipient. The excipient is selected from one or more of a filler, a disintegrant, a lubricant, a suspending agent, a binder or a sweetener. The filler is selected from one or more of starch, lactose, microcrystalline cellulose or sucrose; and / or, the disintegrant is selected from one or more of starch, sodium carboxymethyl starch, pregelatinized starch, low-substituted hydroxypropyl cellulose or cross-linked sodium carboxymethyl cellulose; and / or, the lubricant is selected from one or more of magnesium stearate, silicon dioxide or sodium lauryl sulfate; and / or, the suspending agent is selected from one or more of polyvinyl pyrrolidone, sucrose or hydroxypropyl methylcellulose; and / or, the binder is selected from one or more of hydroxypropyl methylcellulose, starch slurry or polyvinyl pyrrolidone; and / or, the sweetener is selected from one or more of saccharin sodium, glycyrrhetinic acid, sucrose, aspartame or sodium cyclamate. The excipient can also be selected from water for injection or sodium chloride solution for injection. The oxidative stress disease drug is an oral preparation or an injection preparation; and / or, the oral preparation includes capsules, tablets or granules.

[0011] Preferably, the oxidative stress disease comprises a neurodegenerative disease; and the neurodegenerative disease comprises Alzheimer's disease.

[0012] In a second aspect, the present invention provides, in an optional embodiment, the use of a gene editing tool for regulating the expression of selenoprotein F in drugs for oxidative stress diseases, wherein the gene editing tool is selected from CRISPR / Cas9 or RNA interference technology.

[0013] In a third aspect, the present invention provides, in an optional embodiment, a system for screening selenoprotein F expression agents, wherein the system regulates the expression level of selenoprotein F in an in vitro cell model or an in vivo animal model to screen selenoprotein F expression agents that can inhibit oxidative stress disease drugs.

[0014] In a fourth aspect, the present invention provides, in an optional embodiment, the use of a selenoprotein F expression agent as an active ingredient in an antioxidant supplement, wherein the antioxidant supplement includes an antioxidant dietary supplement or a functional food.

[0015] Compared with the prior art, the present invention has one of the following beneficial effects:

[0016] 1. The present invention found that selenoprotein F can affect the expression of antioxidant genes by regulating the Nrf2 signaling pathway, significantly improve cell damage caused by oxidative stress, and broaden the research direction of antioxidant-related drug development. The selenoprotein F expression agent is prepared into a drug, which can significantly treat or inhibit the occurrence of oxidative stress-related diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is a graph showing the results of lipid peroxidation-related level detection in HT22 cells with selenoprotein F silenced in the embodiment of the present invention, wherein: Figure 1 a is the Western blot result of Nrf2, Keap1, HO-1, GPX4, ACSL4, 4-HNE and β-actin proteins in HT22 cells with selenoprotein F silenced. Figure 1 b- Figure 1 g is the protein gray value analysis corresponding to Western blot (n=3, One way ANOVA analysis, *P<0.05), Figure 1 h is the analysis of Nrf2 mRNA expression level in HT22 cells with selenoprotein F silenced. Figure 1 i: ROS content in HT22 cells with silenced selenoprotein F was measured by a multifunctional microplate reader (n=5, One way ANOVA analysis, ***P<0.001).

[0018] Figure 2 This is a graph showing the results of the detection of oxidative stress-related levels in primary neuronal cells of selenoprotein F knockout mice in the embodiment of the present invention, wherein: Figure 2a is the Western blot result of Nrf2, HO-1, GPX4, SELENOF and α-Tubulin proteins in primary neuronal cells of selenoprotein F knockout mice. Figure 2 b- Figure 2 e is the protein gray value analysis corresponding to Western blot (n=3, One way ANOVA analysis, *P<0.05), Figure 2 f is the analysis of the mRNA expression levels of Nrf2, HO-1 and GPX4 in primary neuronal cells of selenoprotein F knockout mice (n=3, One way ANOVA analysis, **P<0.01).

[0019] Figure 3 This is a graph showing the results of the detection of lipid peroxidation and related levels in the cortex of selenoprotein F knockout mice in the embodiment of the present invention, wherein: Figure 3 a is the Western blot result of Nrf2, Keap1, GPX4, 4-HNE, ACSL4, β-actin and α-Tubulin proteins in mouse cortical tissue. Figure 3 b- Figure 3 f is the protein gray value analysis corresponding to Western blot (n=3, One way ANOVA analysis, *P<0.05), Figure 3 g is the analysis of the mRNA expression levels of Nrf2, GPX4, GCLC, GLU, GCLM, GSR and HO-1 in the cortex tissue of selenoprotein F knockout mice (n=3, One way ANOVA analysis, *P<0.05), Figure 3 h- Figure 3 i is the content of total glutathione (T-GSH), the content of reduced glutathione (GSH) and the content ratio of oxidized glutathione (GSSG) in mouse cortical tissue (n=3, One way ANOVA analysis, *P<0.05), Figure 3 j is the relative content of malondialdehyde (MDA) in mouse cortical tissue (n=3, One way ANOVA analysis, *P<0.05).

[0020] Figure 4 This is a result diagram of the effect of selenoprotein F on Nrf2 and its downstream genes in the embodiment of the present invention, wherein: Figure 4 a is the interaction result diagram of Nrf2 and selenoprotein F immunoprecipitation. Figure 4 b is the immunofluorescence staining of selenoprotein F and Nrf2 in HT22 cells, image scale: 5 μm, Figure 4 c is the Western blot result of nuclear and cytoplasmic SELENOF, β-actin, and Lamin B proteins in HT22 cells. Figure 4d is the Western blot result of Nrf2 and Lamin B proteins in HT22 cell nuclei knocked down by selenoprotein F. Figure 4 e is the analysis of mRNA expression levels of GCLC, GCLM, GLU, GSR and GPX4 in primary neuronal cells of selenoprotein F knockout mice (n=3, One way ANOVA analysis, *P<0.05), Figure 4 f is the Western blot result of Nrf2 and β-actin after resveratrol (Nrf2 agonist) treated the primary neuronal cells of selenoprotein F knockout mice. Figure 4 g is the gray value analysis of Nrf2 in primary neurons of selenoprotein F knockout mice after treatment with resveratrol (n=3, One way ANOVA analysis, *P<0.05). DETAILED DESCRIPTION

[0021] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0022] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0023] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0024] Example 1

[0025] Animal experiments

[0026] Four-month-old C57B6N mice with selenoprotein F knockout were selected as model animals to create a selenoprotein F gene whole-body knockout mouse model; C57B6N served as the control group, the knockout mice were purchased from Biocytogen Jiangsu Gene Biotechnology Co., Ltd., and C57B6N were purchased from China Vital Gene.

[0027] C57B6N mice and corresponding selenoprotein F knockout mice were randomly divided into 2 groups, corresponding to the model group (abbreviated as NC) and selenoprotein F knockout group (KO). All mice were housed in a controlled environment with a 12-h light-dark cycle, a temperature of 23±2℃, and had free access to standard laboratory food and water.

[0028] Table 1 Mouse model information

[0029] name Breeding rat describe Age NC C57B6N Control 4 months old KO C57B6N <![CDATA[SelenoF- / -]]> 4 months old

[0030] The mice in each group were subjected to the following experiments:

[0031] (1) Protein extraction and western blotting

[0032] Referring to the instructions of each reagent, lysis buffer (including ice-cold RIPA lysis buffer and protease and phosphatase inhibitors) was used to prepare lysis buffer in proportion for protein lysis. Protein concentration was determined using a BCA protein assay kit (Cat#23235, Thermo Scientific, USA). According to the molecular weight of the target protein, a suitable SDS-PAGE gel was selected for electrophoresis. The sample loading amount per well was 40-60ug. Then, the protein bands were electrotransferred to polyvinylidene fluoride (PVDF, Cat#ISEQ00010, Millipore, USA). After transfer, the PVDF membrane was removed, soaked with 1×TBST, and then moved to a box containing a fast blocking solution (Beyotime), shaken on a decolorizing shaker at room temperature for 0.5-1h or blocked overnight at 4°C. The PVDF membrane was then removed from the blocking solution, placed in a primary antibody diluent, and incubated overnight at 4°C. The next day, the primary antibody dilution was recovered, and the PVDF membrane incubated with the primary antibody was placed in 1×TBST and washed 3 times at room temperature on a decolorizing shaker, each time for 10-15 minutes. The secondary antibody dilution (Cat#ab288151, Abcam, USA) was prepared in the same way and contacted with the PVDF membrane. After incubation at room temperature for 1-2 hours, the secondary antibody dilution was discarded. The PVDF membrane incubated with the secondary antibody was then placed in 1×TBST and washed 3 times at room temperature on a decolorizing shaker, each time for 10-15 minutes. After the above was completed, the PVDF membrane was evenly immersed in the developer and developed in the Tanon-5200 chemiluminescent development system.

[0033] ImageJ or quantityone analysis software was used to adjust the images obtained after development to the most appropriate contrast, and the grayscale values ​​of protein bands and fluorescent images were analyzed. Graphpad Prism8 software was used to analyze the data, and one-way ANOVA was performed, with significant differences at p < 0.05.

[0034] (2) Determination of GSH levels

[0035] GSH levels were determined using the reduced glutathione (GSH) colorimetric test kit (Elabscience, E-BC-K030-M) and the total glutathione (T-GSH) / oxidized glutathione (GSSG) colorimetric test kit (Elabscience, E-BC-K097-M).

[0036] (3) Determination of MDA levels

[0037] NDA levels were determined using the malondialdehyde (MDA) colorimetric test kit (Elabscience, E-BC-K028-M).

[0038] Example 2

[0039] Cell experiments

[0040] (1) Cell culture and treatment

[0041] HT22 and HEK293 cells were used for cell experiments. The cells were cultured in a medium containing 50% Dulbecco's modified Eagle's medium (DMEM) (Gibico, USA) supplemented with 50% Opti-MEM, 5% fetal bovine serum, 0.5% penicillin and streptomycin, and with or without 200 μg / mL G418 (Invitrogen). The cells were incubated at 37°C and 5% CO 2 When the cell confluence is about 60-80%, selenoprotein F silencing virus or overexpression plasmid transfection is performed as follows:

[0042] a. Virus transfection: dilute the virus to the required concentration with serum-free or low-serum medium to ensure that the final MOI value is 220. Before cell transfection, wash the cells with PBS buffer for 3 times to ensure that there is no original culture medium residue, then add the virus diluent to infect the cells for 30 minutes, then add an equal volume of the corresponding complete culture medium, place in a constant temperature incubator at 37°C, 5% CO2 for 24 hours, and finally collect the cells for use.

[0043] b. Plasmid transfection: Prepare the endotoxin-free plasmid into working solution according to the instructions of the transfection reagent (Invigentech INVIDNA RNA Transfection ReagentTM, IV1216100), pipette and mix, let stand at room temperature for 15 minutes, then drop into the cells in the well, mix, and place at 37°C, 5% CO 2 The cells were cultured in a constant temperature incubator for 24 hours and finally the cells were collected for later use.

[0044] (2) RT-qPCR detection of gene mRNA expression levels

[0045] Real-time fluorescence quantitative PCR (RT-qPCR) is a method for quantitatively analyzing DNA amplification in real time by adding fluorescently labeled primers or probes to the PCR reaction and monitoring the accumulation of these fluorescent labels in each cycle, including the following steps.

[0046] 1) Extract RNA: Extract RNA from cells or tissues according to the experimental requirements of the kit.

[0047] 2) Reverse transcription: reverse transcription was performed according to the instructions of the reverse transcription kit (yeasen, 11141ES10) to synthesize cDNA.

[0048] 3) Perform RT-qPCR detection. The reaction system and procedure are shown in reference (yeasen, 11184ES08).

[0049] (3) Co-immunoprecipitation

[0050] Co-immunoprecipitation is a classic method for studying protein interactions based on the specific interaction between antibodies and antigens. It is an effective method to determine the physiological interaction between two proteins in intact cells. 2 HT22 cells in the culture flask, add 1ml lysis buffer, add protease inhibitors (100:1), collect the lysis buffer, centrifuge (4℃, 13000rpm, 10mins) and collect the supernatant and put it on ice for later use. Magnetic beads (Protein G Magnetic Beads, MCE) were premixed with anti-Sep15 antibody at 4℃ for 2 hours in advance. The lysis buffer was added to the magnetic beads and incubated in a flip mixer (4℃ overnight), magnetic separation, supernatant was discarded, 100μL 1×SDS-PAGE Loading Buffer was added to the magnetic beads and mixed evenly, and heated at 95℃ for 5mins. The magnetic beads were separated, the supernatant was collected, and SDS-PAGE detection was performed.

[0051] (4) Cell Immunofluorescence

[0052] HT22 cells were cultured at 6 × 10 5 Cells were seeded into 60 mm 2In the confocal culture dish, after the cells are completely attached to the wall, the cells are synchronized with serum-free medium, and then the cells are incubated with fresh HT 22 complete medium for 1 hour, and then transfected with a plasmid containing the selenoprotein F expression gene (containing a flag tag) for 24 hours. The cells are rinsed 3 times with pre-cooled PBS and fixed with 4% PFA solution at room temperature for 15 minutes. After fixation, the cells are then rinsed 3 times with pre-cooled PBS, and the membrane is broken with PBST (0.1% Triton-X in PBS) and incubated at room temperature for 10 minutes. The cells are then blocked at room temperature for 1 hour. According to the instructions, the rabbit species Nrf2 and sheep species flag primary antibodies are prepared and mixed, and the cells are incubated with the mixed primary antibodies at 4°C overnight. Wash the cells 3 times with PBS, 5min / time, dilute the corresponding source Alexa 488 and Alexa 594 fluorescent secondary antibodies in advance with immunofluorescence blocking solution, and place the cells in the fluorescent secondary antibodies and incubate them in the dark for 2 hours at room temperature. The cells were rinsed with PBS for 3 times, and then incubated with DAPI for 10 min in the dark for nuclear staining. Finally, the results were observed and recorded.

[0053] (5) Determination of ROS levels

[0054] The ROS level was determined using the reactive oxygen species (ROS) fluorescence test kit (Elabscience, E-BC-K138-F).

[0055] in conclusion:

[0056] 1) Selenoprotein F deficiency can cause lipid peroxidation

[0057] See also Figure 1-3 By detecting lipid peroxidation metabolism-related proteins at the cellular and tissue levels, it was found that the protein levels of ACSL4 (acyl-CoA synthetase long-chain family member 4) and 4-HNE (4-hydroxy-2-nonenal, a lipid peroxidation product) were significantly increased when selenoprotein F was missing. At the same time, the intracellular ROS level and the levels of MDA and GSH in the tissue were detected by the kit. It was found that the ROS level in the cells with silenced selenoprotein F was significantly increased, while the ROS level in the cells overexpressing selenoprotein F was significantly decreased; similarly, the MDA level in the tissues with selenoprotein F deficiency was significantly increased, and the GSH level was significantly decreased. This shows that there is a strong lipid peroxidation phenomenon in the tissue. The above shows that the knockout of selenoprotein F caused lipid peroxidation in cells and tissues, ultimately leading to the death of cells and neurons.

[0058] 2) Selenoprotein F regulates peroxidation in mouse brain via Nrf2-related signaling pathway

[0059] See also Figure 4, the expression of Nrf2 and its downstream related target proteins at the cellular and tissue levels was detected at the protein and mRNA levels. The results showed that the loss of selenoprotein F led to a significant decrease in the Nrf2 protein level, while the mRNA level expression was significantly increased. This phenomenon indicates that the loss of selenoprotein F may cause the degradation of Nrf2, and at this time the body needs to promote the expression of the NRF2 gene to cope with oxidative stress. At the same time, it was found that the protein levels of Nrf2 downstream target proteins such as GPX4 and HO-1 were significantly downregulated, and the mRNA expression levels of other downstream target genes such as GCLC, GCLM, GLU, GSR, and GPX4 were significantly decreased. This phenomenon is consistent with the effect of the decrease in Nrf2 protein levels.

[0060] Then, through immunoprecipitation experiments, it was found that selenoprotein F and Nrf2 may interact. For further confirmation, immunofluorescence was also used to detect that there was co-localization between selenoprotein F and Nrf2. It was also found that selenoprotein F and DAPI overlapped, indicating that selenoprotein F may also enter the cell nucleus. Further, nuclear proteins and cytoplasmic proteins in the cells were detected, and selenoprotein F was found in both. Since Nrf2 is a transcription factor, it generally needs to be in the nucleus to exert its effect. In order to confirm the effect of selenoprotein F deficiency on the entry of Nrf2 protein into the nucleus, after silencing selenoprotein F, it was found that the protein level of Nrf2 in the nucleus decreased significantly.

[0061] Finally, in order to test whether selenoprotein F affects the normal function of Nrf2, the Nrf2 agonist, resveratrol, was added for testing. The results showed that after the addition of the agonist, the expression level of NRF2 increased compared with the control group, while no obvious upward trend was seen in the neurons of selenoprotein F knockout mice.

[0062] In general, selenoprotein F may interact with NRF2, affecting the level of Nrf2 protein and its normal nuclear activation, thereby affecting the body's antioxidant capacity.

[0063] Although the principles of the present invention are described in detail above in conjunction with the preferred embodiments of the present invention, those skilled in the art should understand that the above embodiments are merely explanations of the exemplary implementations of the present invention, and are not intended to limit the scope of the present invention. The details in the embodiments do not constitute limitations on the scope of the present invention, and any obvious changes such as equivalent transformations, simple replacements, etc. based on the technical solution of the present invention, without departing from the spirit and scope of the present invention, fall within the protection scope of the present invention.

Claims

1. Use of a selenoprotein F expression agent in the preparation of a drug for oxidative stress diseases, characterized in that: The oxidative stress disease drug includes a selenoprotein F expression agent.

2. Use of the selenoprotein F expression agent according to claim 1 in the preparation of a drug for oxidative stress diseases, characterized in that: The selenoprotein F expression agent is selected from gene vectors, small molecule agonists, other compounds or biological agents that upregulate the expression of selenoprotein F.

3. Use of the selenoprotein F expression agent according to claim 1 in the preparation of a drug for oxidative stress diseases, characterized in that: In the oxidative stress disease drug, the concentration of the selenoprotein F expression agent is 200ng / μL-1000ng / μL.

4. Use of the selenoprotein F expression agent according to claim 1 in the preparation of a drug for oxidative stress diseases, characterized in that: In the oxidative stress disease drug, the concentration of the selenoprotein F expression agent is 600 ng / μL.

5. Use of the selenoprotein F expression agent according to claim 1 in the preparation of a drug for oxidative stress diseases, characterized in that: Also includes auxiliary materials.

6. Use of the selenoprotein F expression agent according to any one of claims 1 to 5 in the preparation of a drug for oxidative stress diseases, characterized in that: The oxidative stress disease drug is an oral preparation or an injection preparation; and / or, The oral preparations include capsules, tablets or granules.

7. Use of the selenoprotein F expression agent according to claim 1 in the preparation of a drug for oxidative stress diseases, characterized in that: The oxidative stress diseases include neurodegenerative diseases; The neurodegenerative diseases include Alzheimer's disease.

8. Application of a gene editing tool for regulating the expression of selenoprotein F in a drug for oxidative stress diseases, characterized in that: The gene editing tool is selected from CRISPR / Cas9 or RNA interference technology.

9. A system for screening selenoprotein F expression agents, characterized in that: The system is used to screen selenoprotein F expression agents that can inhibit oxidative stress disease drugs by regulating the expression level of selenoprotein F in an in vitro cell model or an in vivo animal model.

10. Use of a selenoprotein F expression agent as an active ingredient in an antioxidant supplement, characterized in that: The antioxidant supplement includes an antioxidant dietary supplement or a functional food.

Images