Application of aquaporin AQP7 in inhibition of pancreatic beta cell ferroptosis

Abstract

The invention discloses an application of aqua glycerol channel protein AQP7 in inhibition of pancreatic beta cell ferroptosis. According to the application disclosed by the invention, the change of intracellular H2O2 amount and the change of autophagy related proteins (LC3II / I and P62) are studied by overexpressing and knocking out an AQP7 gene in a rat beta cell INS-1 by utilizing a lentivirus, the expression change condition of ferroptosis related proteins (ACSL4 and GPX4) is detected, and it is determined that AQP7 overexpression can inhibit decline of an autophagy function and occurrence of ferroptosis in the INS-1 cell under the condition of high fat and high glucose; after the AQP7 is knocked down, the autophagy of the cells is reduced, the amount of H2O2 in the cells is increased, and ferroptosis is activated. Researches show that change of AQP7 expression may become a feasible strategy for preventing beta cell ferroptosis in type 2 diabetes mellitus.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly relates to an application of aquaglyceroporin AQP7 in inhibiting ferroptosis of pancreatic islet β cells. Background Art

[0002] The modern sedentary lifestyle and high-calorie eating habits have led to a continuous increase in the obesity rate of the population in recent years. Excessive nutrient intake (e.g., glucose, fatty acids) can trigger chronically elevated insulin secretion and insulin resistance, and these factors promote the functional damage of pancreatic islet β cells to varying degrees. Palmitic acid (PA), as one of the most abundant fatty acids in the body, an excessive amount in the body easily leads to lipid metabolism disorders in the body, causes insulin resistance in multiple organs, and induces the damage and even death of pancreatic islet β cells. Pancreatic islet B cells, namely pancreatic islet β cells, are a type of pancreatic islet cells and a type of endocrine cells, which can secrete insulin and play a role in regulating blood glucose together with glucagon secreted by pancreatic islet A cells. The damage of pancreatic islet β cells is considered to be a key link in the disease progression, especially the damage of β cells is often closely related to the defect of insulin secretion. This process involves changes in insulin secretion, depletion of insulin reserves, and dedifferentiation of β cells, and the death of β cells may play a key role in the disease progression.

[0003] Hydrogen peroxide (H2O2), as a main form of intracellular ROS, has been proven to play an important role in the oxidative stress response of pancreatic islet β cells. Due to the low expression of antioxidant enzymes in pancreatic β cells themselves, they are more vulnerable to oxidative stress than other cell types. The accumulation of H2O2 not only destroys the intracellular antioxidant system but also induces apoptosis of pancreatic islet β cells. During the process of type 2 diabetes, H2O2 is generated in pancreatic islet β cells through different biological pathways and affects the survival and function of cells. Although many studies have described the generation of H2O2 in pancreatic islet β cells and its impact on cell function, the fine regulatory mechanism of H2O2 homeostasis has not been fully revealed, and the research on these mechanisms is still one of the important directions in the current diabetes research field.

[0004] Aquaporins (AQP) are a class of transmembrane proteins, and their main function is to regulate the entry and exit of water molecules through cell membranes. Recent studies have further shown that some AQP subtypes are not only water and small molecule transport channels but also capable of transporting hydrogen peroxide. Although H2O2 and water have similar physicochemical properties, the diffusion efficiency of H2O2 is relatively lower than that of water. Therefore, more effective regulation is required to improve the membrane permeability of H2O2. As an important signaling molecule, H2O2 plays an important role in the processes of cellular oxidative stress, immune response, cell death, and repair. The H2O2 transport by AQP3 can affect chemokine-dependent T cell migration, regulate the immune response, and play a key role in the process of immune diseases such as psoriasis. AQP5 promotes the adaptation and migration of cancer cells by transporting H2O2 and may play a positive role in the process of tumor growth and metastasis. In addition, AQP9 can play a regulatory role in the regeneration process of the liver by transporting H2O2, especially after partial hepatectomy, AQP9 promotes the proliferation and regeneration of hepatocytes. These studies indicate that aquaporins are not only water transport channels but also participate in the cellular redox balance, with the functions of regulating intracellular oxidative stress, promoting cell repair, and responding to external stimuli. AQP7 is a water glycerol channel protein highly expressed in pancreatic islet β cells and plays a key role in cell proliferation. Previous studies have found that the application of shRNA to downregulate the expression level of AQP7 can downregulate the insulin secretion of pancreatic islet β cells. In addition, AQP7 promotes the proliferation and adipogenic differentiation of mouse bone marrow mesenchymal stem cells by regulating hydrogen peroxide transport. However, the transport and influence of AQP7 on intracellular hydrogen peroxide in pancreatic islet β cells remain unknown.

[0005] Autophagy is a key metabolic process that degrades cellular components through the lysosomal pathway and plays a crucial role in maintaining cell growth, development, and homeostasis. It plays an important role in balancing the synthesis, degradation, and recycling of cellular components. In most cases, autophagy helps to inhibit or delay cell death; however, under long-term or severe stress, excessive autophagy may lead to cell death. β cells in the islets are responsible for the synthesis and secretion of insulin and have a highly developed autophagy system that can clear excess insulin granules, thus maintaining normal function and survival. Studies have shown that dysfunction of autophagy in β cells leads to a decrease in cell mass, partly due to an increase in apoptosis. Other studies have found that chronic lipotoxicity damages lysosomal function and disrupts autophagic flux, further promoting cell apoptosis.

[0006] Ferroptosis is a special form of programmed cell death. The key mechanism lies in the excessive generation of iron-dependent reactive oxygen species, which then triggers irreversible lipid peroxidation. This process involves cellular metabolic imbalance and the disruption of redox balance. Ferroptosis plays an important pathophysiological role in the development of diabetes and its complications. Recent studies have shown that pancreatic islet β-cells are sensitive to ferroptosis. Ferroptosis affects the function and survival of pancreatic islet β-cells through multiple mechanisms and may even trigger the onset of diabetes. Summary of the Invention

[0007] The object of the present invention is to address the above problems and provide an application of aquaglyceroporin AQP7 in inhibiting ferroptosis of pancreatic islet β-cells.

[0008] To achieve its object, the present invention adopts the following technical solutions:

[0009] The first aspect of the present invention provides the application of AQP7 as a target in screening drugs for inhibiting ferroptosis of pancreatic islet β-cells.

[0010] The drug promotes the expression of AQP7.

[0011] Preferably, the drug is an overexpression vector of AQP7.

[0012] Preferably, the overexpression vector is a lentivirus.

[0013] In the above application technical solution, AQP7 inhibits the decline of autophagy function and the occurrence of ferroptosis in pancreatic islet β-cells.

[0014] In the above application technical solution, after AQP7 is knocked down, autophagy in the cells decreases, the amount of intracellular H2O2 increases, and ferroptosis is activated.

[0015] The second aspect of the present invention provides the application of AQP7 or an AQP7 expression promoter in the preparation of a drug for inhibiting ferroptosis of pancreatic islet β-cells.

[0016] Furthermore, the application of AQP7 or an AQP7 expression promoter in the preparation of a drug for inhibiting ferroptosis of pancreatic islet β-cells in diabetes.

[0017] Preferably, the AQP7 expression promoter is an overexpression vector of AQP7.

[0018] The beneficial effects of the present invention are as follows: By overexpressing and knocking out the AQP7 gene in rat β-cells INS-1 using lentivirus, the changes in the intracellular H2O2 content and the changes in autophagy-related proteins (LC3Ⅱ / Ⅰ, P62) were studied, and the expression changes of ferroptosis-related proteins (ACSL4, GPX4) were detected. It was determined that overexpression of AQP7 could inhibit the decline of autophagy function and the occurrence of ferroptosis in INS-1 cells under high-fat and high-sugar conditions. After knocking down AQP7, autophagy in the cells decreased, the intracellular H2O2 content increased, and ferroptosis was activated. The research of the present invention shows that changing the expression of AQP7 may become a feasible strategy for preventing the occurrence of β-cell ferroptosis in type 2 diabetes. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The results of the Western blot experiment showing the expression levels of AQP7 in INS-1 pancreatic β-cells with AQP7 knocked down (Figure A - B) or overexpressed (Figure C - D) are presented: In Figures A - B, the grayscale images and bar charts of AQP7 expression in the normal control group (CTRL), the virus control group (KDc), and the AQP7 knockdown group (KD) are shown; in Figures C - D, the grayscale images and bar charts of AQP7 expression in the normal control group (CTRL), the virus control group (OEc), and the AQP7 overexpression group (OE) are shown.

[0020] Figure 2 The results of the Western blot experiment showing the protein expression levels of LC3Ⅱ / Ⅰ and P62 in cells after knocking down AQP7 are presented: In Figures A - C, the grayscale images and bar charts of LC3Ⅱ / Ⅰ and P62 expression in the normal control group (CTRL), the virus control group (KDc), and the AQP7 knockdown group (KD) are shown.

[0021] Figure 3 Bar charts of the H2O2 and GSH levels in the normal control group (CTRL), the virus control group (KDc), and the AQP7 knockdown group (KD).

[0022] Figure 4 Grayscale images and bar charts of the expression of ACSL4 and GPX4 in the normal control group (CTRL), the virus control group (KDc), and the AQP7 knockdown group (KD).

[0023] Figure 5 For: In Figures A - B, fluorescence images and bar charts of the lipid peroxidation level in the normal control group (CTRL), the virus control group (KDc), and the AQP7 knockdown group (KD) (C11 - BODIPY represents unoxidized lipid (red), ox C11 - BODIPY represents oxidized lipid (green)); in Figures C - D, for the normal control group (CTRL), the virus control group (KDc), and the AQP7 knockdown group (KD), Fe 2+Horizontal fluorescence images and bar charts.

[0024] Figure 6 Gray-scale images and bar charts of LC3Ⅱ / Ⅰ and P62 expression for the control group (CTRL), AQP7 overexpression group (OE), high-fat and high-sugar group (GP), and AQP7 overexpression + high-fat and high-sugar group (GP+OE).

[0025] Figure 7 Bar charts of H2O2 (A) and GSH (B) levels for the control group (CTRL), AQP7 overexpression group (OE), high-fat and high-sugar group (GP), and AQP7 overexpression + high-fat and high-sugar group (GP+OE).

[0026] Figure 8 Gray-scale images and bar charts of ACSL4 and GPX4 expression for the control group (CTRL), AQP7 overexpression group (OE), high-fat and high-sugar group (GP), and AQP7 overexpression + high-fat and high-sugar group (GP+OE).

[0027] Figure 9 Showed that: A - B are fluorescence images and bar charts representing the lipid peroxidation levels of the control group (CTRL), AQP7 overexpression group (OE), high-fat and high-sugar group (GP), and AQP7 overexpression + high-fat and high-sugar group (GP+OE) (C11-BODIPY represents unoxidized lipids (red), oxC11-BODIPY represents oxidized lipids (green)); Figures C - D are fluorescence images and bar charts of the Fe 2+ Horizontal fluorescence images and bar charts. Detailed implementation methods

[0028] The present invention will be further described below in conjunction with embodiments, but the present invention is not limited thereby.

[0029] The experimental methods in the following embodiments are all conventional methods unless otherwise specified.

[0030] Example 1

[0031] 1. Experimental materials and reagents

[0032] 1. Experimental materials

[0033] The rat pancreatic islet β-cell line INS-1 was purchased from the Peking Union Medical College Cell Center.

[0034] 2. Main experimental reagents

[0035] Antibody Name Manufacturer Catalog Number Mouse anti-α-Tubulin monoclonal antibody ABclonal AC012 Rabbit anti-β-Actin monoclonal antibody ABclonal AC006 Rabbit anti-LC3-I / II monoclonal antibody Abmart MA8198S Rabbit anti-p62 / SQSTM1 monoclonal antibody Abmart T55546S Rabbit anti-GPX4 monoclonal antibody Abmart T56959S Rabbit anti-FACL4 monoclonal antibody Abmart T510198S Mouse anti-AQP7 monoclonal antibody Santa Cruz sc-376407 Horseradish peroxidase-labeled goat anti-rabbit IgG (H+L) ZSGB-BIO ZB-5301 Horseradish peroxidase-labeled goat anti-mouse IgG (H+L) ZSGB-BIO ZB-2305

[0036] 2. Experimental methods

[0037] 1 Cell culture

[0038] INS-1 cells were cultured routinely in 1640 (Gibco) medium supplemented with 10% fetal bovine serum (Hyclone), penicillin (10 7 / L), and streptomycin (10 mg / L) in a humidified incubator containing 5% carbon dioxide at a constant temperature of 37 °C.

[0039] 2 Lentiviral infection

[0040] 2.1 Preliminary experiment

[0041] (1) Seeding cells: When the cell confluence reached about 80%, the cells were digested and counted, seeded in a 96-well plate with 104 cells per well, a total of 12 wells, and placed in a cell incubator for 24 h.

[0042] (2) Infection process: The 12 wells were divided into three groups with MOIs of 1, 10, and 100, and three wells were set for each MOI group. Three wells in each group were added with complete medium, complete medium + virus infection reagent solution A, and complete medium + virus infection reagent solution P, respectively, and the remaining three wells were used as control groups without adding virus. First, the virus was taken out from the ultra-low temperature freezer at -80 °C and thawed on ice. The medium in each well was discarded, and the medium was changed according to the virus titer to calculate the required virus amount. After mixing evenly, it was placed in a cell incubator for culture. The cell status was observed after 8 h. If there was no change in the cell status, the medium was changed after 16 h; if there was a slight change in the cell status, the medium should be changed immediately.

[0043] (3) Confirmation of infection effect: After 72 h of infection, the cell morphology and growth status were observed under a white light microscope, and photos were taken and observed using a fluorescence microscope. By comparing the white light and green light, the proportion of green light (representing the infection marker) was evaluated to judge the infection efficiency. If the infection efficiency reached about 80% and the cell growth status was good, the infection conditions of this group could be used for the subsequent formal experiment.

[0044] 2.2 Formal experiment

[0045] (1) Seeding cells: When the cell confluence reached about 85%, the cells were digested and counted. The digestion of the cells should be terminated immediately by adding the termination digestion solution. Then, about 2×105 cells were seeded in each well of a six-well plate for culture. The medium was changed the next day and the cell status was observed. When the cell confluence reached about 60%, infection was carried out.

[0046] (2) Virus infection procedure: To establish INS-1 cells stably overexpressing AQP7, INS-1 cells were infected with the Ubi-AQP7MCS-3FLAG-SV40-EGFP-IRES-puromycin lentivirus encoding the full-length gene of rat AQP7 (Gene ID number in NCBI: 29171), and Ubi-MCS-3FLAG-SV40-EGFP-IRES-puromycin was used as the empty vector control; the AQP7 gene was knocked out using the hU6-AQP7-MCS-CBH-gcGFP-IRES-puromycin lentivirus, and HU6-MCS-CBH-gcGFP-IERS-puromycin was used as the negative control. First, take out the virus from the ultra-low temperature freezer at -80°C, place it on ice to melt, and aliquot the virus according to the MOI value of 10 determined by preliminary experiments. After aliquoting, take out enough virus for one infection for subsequent experiments, seal the rest with parafilm, label clearly and store it in the -80°C refrigerator. For formal infection, take a 4 ml EP tube, place it on ice, and add successively: 1 ml of medium, 40 μl of virus infection enhancer P solution, and calculate the required inoculated virus volume according to the virus titer and MOI. After mixing evenly, add it to the corresponding wells of the six-well plate in the way of changing the medium, make marks, and put it back into the cell culture incubator for continuous culture. After 16 hours, change to normal medium, and take pictures with a fluorescence microscope after 48 hours to confirm the infection efficiency. After ensuring that the lentivirus has been expressed in the cells, start screening with puromycin at a concentration of 2 μg / ml for 2 weeks. After screening out INS-1 cells stably overexpressing or knocking down AQP7, immunoblotting and immunofluorescence assays were performed to confirm the efficiency of gene knockdown or overexpression. At the same time, freeze the cells for subsequent experiments. The lentiviruses for overexpression and AQP7 gene knockout were provided by Tsingke Biotechnology Co., Ltd. (China).

[0047] 3 Western blot assay

[0048] After washing the well plates 2 times with PBS, add lysis buffer and shake on ice for 30 minutes to lyse. Detect and adjust the protein concentration according to the BCA kit instructions, and perform steps such as loading-sodium dodecyl sulfate polyacrylamide gel electrophoresis-transferring membrane-blocking-primary antibody-secondary antibody, etc. Use the Bio-Rad gel imaging system to expose and save the data.

[0049] 4 Lipid peroxidation detection

[0050] The cells were cultured in 6-well plates, washed 2 times with PBS, 1 ml of BODIPY 581 / 591 C11 staining working solution was added to each well, incubated in the cell culture incubator at 37°C for 30 minutes. After the incubation at 37°C ended, aspirate the supernatant and wash 2 times with PBS. Add 2 ml of PBS and observe under a fluorescence microscope.

[0051] 5 Detection of iron content

[0052] The cells were cultured in a fluorescence culture dish and washed 3 times with HBSS. The working solution of FerroOrange at a concentration of 1 μmol / l was added, and the cells were cultured in an incubator at 37 °C with 5% CO2 for 30 minutes, and then observed under a fluorescence microscope.

[0053] 6 Data analysis and statistics

[0054] The data were expressed as mean ± s.d. All experiments were performed at least 3 times. Statistical analysis was performed using GraphPad Prism 8.0 software. The statistical differences between groups were determined by two-tailed Student's t-test. Significance was designated as follows: *, P < 0.05.

[0055] III Results

[0056] 1 Establishment and verification of AQP7 knockdown or overexpression INS-1 pancreatic islet β cell lines

[0057] To verify the knockdown or overexpression effect of AQP7, the expression level of AQP7 in cells of each group was detected by Western blot experiment, demonstrating the successful establishment of INS-1 pancreatic islet β cell lines with AQP7 knockdown ( Figure 1 A-B) or overexpression ( Figure 1 C-D).

[0058] 2 Autophagy in cells was reduced after AQP7 knockdown, the amount of intracellular H2O2 increased, and ferroptosis was activated

[0059] To verify the effect of AQP7 knockdown on autophagy, the protein expression levels of LC3Ⅱ / Ⅰ and P62 in cells of each group were detected by Western blot experiment. LC3Ⅱ / Ⅰ and P62 were upregulated after AQP7 knockdown ( Figure 2 A-C). This indicates that although the number of autophagosomes in cells increased after AQP7 knockdown, the autophagic flux was inhibited, suggesting that autophagy was inhibited.

[0060] To study the effect of AQP7 on the total amount of intracellular H2O2, we applied Elisa to detect the levels of intracellular H2O2 and GSH. The total intracellular H2O2 content increased in pancreatic islet β cells with AQP7 knockdown, while the GSH level decreased, suggesting that the decrease in AQP7 led to the accumulation of intracellular H2O2 and downregulated the intracellular glutathione (GSH) content ( Figure 3 A-B).

[0061] To study the effect of AQP7 knockdown on ferroptosis, the protein expression levels of ACSL4 and GPX4 were detected by Western blot experiment. The expression level of ACSL4 was significantly upregulated and the expression level of GPX4 was significantly downregulated in pancreatic islet β cells with AQP7 knockdown, (Figure 4 A - C). Cellular lipid peroxidation staining showed that under high - fat and high - sugar conditions, the oxidized lipids in cells were significantly increased compared with non - oxidized lipids. Figure 5 A - B). In addition, the intracellular ferrous ion content is also an important indicator of ferroptosis in cells. Detection of intracellular ferrous ion content in cells found that in islet β - cells with AQP7 knockdown, there was a significant accumulation of intracellular ferrous ions. Figure 5 C - D).

[0062] Overexpression of AQP7 can inhibit the decline of autophagy function and the occurrence of ferroptosis in INS - 1 cells under high - fat and high - sugar conditions.

[0063] INS - 1 cells were cultured under high - fat and high - sugar conditions (33 mM glucose and 25 μM sodium palmitate). To verify the effect of AQP7 overexpression on autophagy in cells, the protein expression levels of LC3Ⅱ / Ⅰ and P62 in each group of cells were detected by Western blot. There was no significant change in LC3Ⅱ / Ⅰ and P62 after AQP7 overexpression, but they were up - regulated under high - fat and high - sugar conditions. However, AQP overexpression could relieve the impact of high - fat and high - sugar on cells. Figure 6 A - C). This indicates that after AQP7 overexpression, it can relieve the down - regulation of autophagy function caused by high - fat and high - sugar stimulation.

[0064] To study the effect of AQP7 on the total amount of intracellular H2O2, we detected the levels of intracellular H2O2 and GSH by ELISA. The total intracellular H2O2 content in islet β - cells with AQP7 overexpression did not change significantly, but it was up - regulated under high - fat and high - sugar conditions. AQP overexpression could relieve the accumulation of intracellular H2O2 after high - fat and high - sugar treatment; the total intracellular GSH content in islet β - cells with AQP7 overexpression did not change significantly, but it was down - regulated under high - fat and high - sugar conditions. AQP overexpression could relieve the down - regulation of intracellular GSH after high - fat and high - sugar treatment. This suggests that overexpression of AQP7 can effectively relieve the accumulation of intracellular H2O2 caused by high - fat and high - sugar stimulation and rescue the down - regulation of intracellular glutathione (GSH) content. Figure 7 A - B).

[0065] To study the effect of AQP7 overexpression on ferroptosis in cells, the protein expression levels of ACSL4 and GPX4 were detected by Western blot. Under high - fat and high - sugar conditions, the expression level of ACSL4 was significantly up - regulated, and the expression level of GPX4 was significantly down - regulated. These changes were significantly reversed after AQP7 overexpression. Figure 8 A - C). Cellular lipid peroxidation staining showed that under high - fat and high - sugar conditions, the oxidized lipids in cells were significantly increased compared with non - oxidized lipids, while after AQP7 overexpression, the ratio of oxidized lipids to non - oxidized lipids in cells was significantly decreased. Figure 9(A-B). In addition, the intracellular ferrous ion content was detected. Under high-fat and high-sugar conditions, there was a significant accumulation of intracellular ferrous ions, which was improved after overexpression of AQP7( Figure 9 (C-D).

[0066] IV. Analysis

[0067] Based on the above experimental results, we found that the decrease in the expression of AQP7 in INS-1 cells could increase the total amount of intracellular H2O2, inhibit autophagy, promote lipid peroxidation and intracellular Fe 2+ accumulation, and promote ferroptosis. Overexpression of AQP7 in INS-1 cells could reverse the decrease in autophagy and the occurrence of ferroptosis induced by high fat.

Claims

1. Use of AQP7 as a target in screening drugs for inhibiting ferroptosis of pancreatic islet β cells.

2. The application according to claim 1, characterized in that: The drug promotes the expression of AQP7.

3. The application according to claim 2, wherein: The drug is an overexpression vector of AQP7.

4. The application according to claim 3, wherein: The overexpression vector is a lentivirus.

5. The application according to claim 2 or 3, characterized in that: AQP7 inhibits the decline of autophagy function and the occurrence of ferroptosis in pancreatic islet β cells.

6. The application according to claim 1, wherein: After AQP7 knockdown, autophagy in cells decreases, the amount of intracellular H2O2 increases, and ferroptosis is activated.

7. Use of AQP7 or an AQP7 expression promoter in the preparation of a drug for inhibiting ferroptosis of pancreatic islet β cells.

8. The application according to claim 7, wherein: Use of AQP7 or an AQP7 expression promoter in the preparation of a drug for inhibiting ferroptosis of pancreatic islet β cells in diabetes.

9. The application according to claim 7, wherein: The AQP7 expression promoter is an overexpression vector of AQP7.

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