Use of piRNA-145836 preparation in the preparation of drugs for preventing and treating high homocysteine-induced type 2 diabetes

Abstract

The application discloses application of a piRNA-145836 preparation in preparation of a medicine for preventing and treating type 2 diabetes induced by high homocysteine, and belongs to the technical field of biological medicine and molecular biology. The nucleotide sequence of the piRNA-145836 is shown as SEQ ID No. 1. The piRNA-145836 can play a key role in type 2 diabetes induced by high homocysteine, and can regulate a mechanism, promote recovery of pancreatic function, restore insulin secretion capacity, reduce abnormal activation of copper death-related proteins, reduce accumulation of copper ions in islet beta cells, and improve sugar metabolism disorder induced by high homocysteine. The application solves the technical problems of lack of cognition of the role of piRNA in copper ion homeostasis and beta cell fate determination, ambiguity of the role target of resveratrol, and unknown expression regulation mechanism of key copper transport proteins in a high homocysteine environment in the prior art.

Description

Technical Field

[0001] This invention belongs to the fields of biomedicine and molecular biology, specifically relating to the application of piRNA-145836 formulation in the preparation of drugs for the prevention and treatment of type 2 diabetes induced by high homocysteine. Background Technology

[0002] As research into the pathogenesis of type 2 diabetes deepens, the academic community has gradually recognized that pancreatic β-cell dysfunction is not only a late-stage manifestation of the disease but also a core component throughout its course. Hyperhomocysteinemia, a common metabolic disorder in diabetic patients, has been proven to not only promote vascular complications but also directly induce peripheral tissue insulin resistance and β-cell damage. Existing studies have shown that homocysteine ​​can interfere with the insulin signaling pathway and trigger β-cell apoptosis by inducing oxidative stress, endoplasmic reticulum stress, and chronic inflammation. However, the recently discovered copper death (cuproptosis), a copper-dependent programmed cell death mechanism, provides a novel perspective for understanding hyperhomocysteinemia-induced β-cell damage. Copper death is triggered by intracellular copper ion overload, leading to abnormal aggregation of esterified proteins in the tricarboxylic acid cycle and instability of iron-sulfur cluster proteins, ultimately resulting in proteotoxic stress and mitochondrial dysfunction. Clinical data show that type 2 diabetes patients often have elevated serum copper levels, suggesting that copper homeostasis imbalance may play a key role in disease progression, but the synergistic pathogenic mechanism between copper and hyperhomocysteinemia remains unclear.

[0003] Resveratrol, a natural polyphenol compound, has been widely reported to improve insulin sensitivity, provide antioxidant effects, and protect β-cells. Recent studies have found that resveratrol can alleviate metabolic disorders caused by high homocysteine ​​levels, but whether it achieves β-cell protection by regulating novel cell death pathways (such as copper death) currently lacks systematic evidence. More importantly, although the role of non-coding RNAs in metabolic regulation is receiving increasing attention, Piwi-interacting RNAs (piRNAs), a class of short non-coding RNAs that mainly maintain genomic stability in the germline, have also been detected in metabolic tissues such as the pancreas and liver in recent years, suggesting their potential involvement in glucose and lipid metabolism regulation. Preliminary evidence suggests that specific piRNAs can target and regulate insulin secretion-related genes, but whether piRNAs respond to high homocysteine ​​stimulation, mediate copper death pathways, and can act as molecular hubs for resveratrol's protective effects remains completely unknown.

[0004] However, existing technologies have several gaps in this field: First, no studies have linked high homocysteine, copper death, and piRNA, and there is a lack of understanding of the role of piRNA in copper ion homeostasis and β-cell fate determination; second, although resveratrol has protective potential, its target is unclear, and a regulatory axis of "small molecules - non-coding RNA - copper death effector proteins" has not been established; third, the expression regulation mechanism of key copper transporters such as SLC31A1 (copper uptake) and SLC3A2 (amino acid transport complex subunit) in a high homocysteine ​​environment is unclear, especially lacking explanation at the upstream non-coding RNA level.

[0005] Based on this, the present invention proposes the application of piRNA-145836 formulation in the preparation of drugs for the prevention and treatment of type 2 diabetes induced by high homocysteine, in order to solve the problems existing in the prior art. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention provides the application of piRNA-145836 formulation in the preparation of drugs for the prevention and treatment of type 2 diabetes induced by high homocysteine ​​levels. This addresses the existing technical issues of a lack of understanding of the role of piRNA in copper ion homeostasis and β-cell fate determination, the ambiguity of the target of resveratrol, and the unclear regulatory mechanism of key copper transporter expression in a high homocysteine ​​environment.

[0007] To achieve the above objectives, the present invention provides the following technical solution:

[0008] The present invention provides a first technical solution: the application of piRNA-145836 preparation in the preparation of drugs for the prevention and treatment of type 2 diabetes induced by high homocysteine, wherein the nucleotide sequence of piRNA-145836 is shown in SEQ ID NO.1.

[0009] In a preferred embodiment of the present invention, the nucleotide sequence of the piRNA-145836 is TGTGTAACAACTCACCTGCCGAATCA.

[0010] In a preferred embodiment of the invention, the drug inhibits copper death in pancreatic β cells.

[0011] In a preferred embodiment of the present invention, the drug inhibits the accumulation of intracellular copper ions caused by the increase of FDX1, SLC31A1, and HSP70.

[0012] In a preferred embodiment of the present invention, the drug inhibits the accumulation of intracellular copper ions and reduces copper death in pancreatic β cells.

[0013] The present invention provides a second technical solution: the application of piRNA-145836 and its target gene SLC3A2 inhibitor in the preparation of a drug for the prevention and treatment of type 2 diabetes induced by high homocysteine.

[0014] The present invention provides a third technical solution: a drug for treating type 2 diabetes induced by hyperhomocysteine, wherein the active ingredients of the drug include an inhibitor of piRNA-145836 and an inhibitor of SLC3A2.

[0015] In a preferred embodiment of the present invention, the sequence of piRNA-145836 is as follows:

[0016] TGTGTAACAACTCACCTGCCGAATCA.

[0017] Compared with the prior art, the present invention provides the application of piRNA-145836 formulation in the preparation of drugs for the prevention and treatment of hyperhomocysteine-induced type 2 diabetes, which has the following beneficial effects:

[0018] This invention screened and identified piRNA-145836 by high-throughput RNA sequencing, which showed the most significant upregulation of differential expression in pancreatic β cells treated with homocysteine. The study found that piRNA-145836 plays an important role in copper death in pancreatic β cells, demonstrating that piRNA-145836 can induce copper death in cells by regulating its downstream target gene SLC3A2.

[0019] In a hyperhomocysteinemic environment, elevated SLC3A2 expression affects intracellular copper homeostasis, leading to copper death. Resveratrol intervention can alleviate oxidative stress damage and reduce the degree of copper death. This study confirms that resveratrol can protect pancreatic β-cells by precisely regulating the piRNA-145836 / SLC3A2 signaling axis. This piRNA-145836 / SLC3A2 signaling axis reveals a previously unrecognized regulatory pathway in pancreatic β-cells. Inhibition of piRNA-145836 can reduce pancreatic β-cell copper death and has a protective effect on the pancreas. These findings provide new therapeutic ideas and potential targets for the treatment of hyperhomocysteinemia-induced type 2 diabetes, possessing significant theoretical implications and potential practical value. Attached Figure Description

[0020] Figure 1 The bar chart shows how the resveratrol diet can alleviate metabolic abnormalities in mice caused by a high-methionine diet, according to the present invention.

[0021] Figure 2The figure shows the non-coding piRNA-145836 differentially expressed in homocysteine-treated pancreatic β cells identified by high-throughput RNA sequencing in this invention.

[0022] Figure 3 This invention verifies, through animal and cellular studies, that resveratrol can alleviate homocysteine-induced copper death.

[0023] Figure 4 This invention illustrates how resveratrol regulates the expression of piRNA-145836 to alleviate homocysteine-induced copper death in pancreatic β-cells.

[0024] Figure 5 The diagram shows the screening and validation of the piRNA-145836 target gene SLC3A2 for this invention.

[0025] Figure 6 This diagram illustrates how the target gene SLC3A2 of this invention regulates homocysteine-induced copper death in pancreatic β-cells.

[0026] Among them, Figure 1 In the diagram, Figures A, B, C, and D show the serum levels of homocysteine ​​(Hcy), blood glucose, insulin, and glycated hemoglobin (HbA1c) in mice from the normal diet group, the high methionine diet group, and the high methionine + resveratrol diet group, respectively, as detected by an automated biochemical analyzer. Figure E shows the body weight of mice from the normal diet group, the high methionine diet group, and the high methionine + resveratrol diet group. Figures F, G, and I show the serum levels of total cholesterol (TC), triglycerides (TG), and creatinine (Crea) in mice from the normal diet group, the high methionine diet group, and the high methionine + resveratrol diet group, respectively, as detected by an automated biochemical analyzer.

[0027] exist Figure 2 In the figure, Figure A is a volcano plot showing the differential piRNA expression; Figures B and C are the results of GO and KEGG enrichment analyses; Figure D is a qRT-PCR plot showing the expression level of piRNA-145836 in pancreatic β-cells in the normal control group, homocysteine ​​treatment group, and homocysteine ​​+ resveratrol treatment group; Figure E is a plot showing the expression level of piRNA-145836 in pancreatic tissue of mice in the normal diet group, high methionine diet group, and high methionine + resveratrol diet group.

[0028] exist Figure 3In the diagram, Figures A, B, and C show the expression levels of copper death-related proteins HSP70, FDX1, and SLC31A1 in the pancreatic tissues of mice in the normal diet group, high methionine diet group, and high methionine + resveratrol diet group, respectively, detected by Western blot. Figure D shows the expression levels of copper death-related proteins HSP70, FDX1, and SLC31A1 in the pancreatic tissues of mice in the normal diet group, high methionine diet group, and high methionine + resveratrol diet group, respectively, detected by immunohistochemistry. Figure E shows the effect of different concentrations of homocysteine ​​on pancreatic β-cell viability detected by CCK8 assay. Figure F shows the effect of different concentrations of resveratrol on pancreatic β-cell viability detected by CCK8 assay. Figures G, H, and I show the expression levels of copper death-related proteins HSP70, FDX1, and SLC31A1 in the pancreatic tissues of mice in the normal diet group, high methionine diet group, and high methionine + resveratrol diet group, respectively, detected by Western blot. Figure 1 shows the expression levels of copper death-related proteins HSP70, FDX1, and SLC31A1 in pancreatic β-cells in the normal control group, homocysteine-treated group, and homocysteine ​​+ resveratrol-treated group, as detected by blot. Figure 2 shows the copper ion content in pancreatic β-cells in the normal control group, homocysteine-treated group, and homocysteine ​​+ resveratrol-treated group, as detected by colorimetric assay. Figure 3 shows the insulin secretion level in pancreatic β-cells in the normal control group, homocysteine-treated group, and homocysteine ​​+ resveratrol-treated group, as detected by enzyme-linked immunosorbent assay (ELISA).

[0029] exist Figure 4 In the figure, Figures A and B are the validation diagrams of qRT-PCR detection of the effects of knockdown and overexpression of piRNA-145836 in pancreatic β cells; Figures C, D, and E are the expression levels of copper death-related proteins HSP70, FDX1, and SLC31A1 in pancreatic β cells after knockdown of piRNA-145836 under homocysteine ​​and homocysteine ​​+ resveratrol interventions; Figures F, G, and H are the Western blot results of Western blot analysis. Figure 1 shows the expression levels of copper death-related proteins HSP70, FDX1, and SLC31A1 in pancreatic β cells after overexpression of piRNA-145836, as detected by blot under homocysteine ​​and homocysteine ​​+ resveratrol interventions. Figure 2 shows the copper ion levels in pancreatic β cells after knockdown of piRNA-145836, as detected by colorimetric assay, as detected by colorimetric assay, as detected by overexpression of piRNA-145836, as detected by homocysteine ​​and homocysteine ​​+ resveratrol interventions. Figure K shows the insulin secretion level of pancreatic β cells after knocking down piRNA-145836 under the intervention of homocysteine ​​and homocysteine ​​+ resveratrol, as detected by enzyme-linked immunosorbent assay (ELISA); Figure L shows the insulin secretion level of pancreatic β cells after overexpression of piRNA-145836 under the intervention of homocysteine ​​and homocysteine ​​+ resveratrol, as detected by ELISA.

[0030] exist Figure 5 In the figure, Figure A shows the protein binding to piRNA-145836 detected by RNA pull-down assay; Figure B shows the mass spectrometry peaks of SLC3A2 bound to piRNA-145836; Figure C shows the targeting relationship between piRNA-145836 and SLC3A2 detected by dual-luciferase reporter gene system; Figure D shows the SLC3A2 protein expression level detected by Western blot in normal control group, homocysteine ​​treatment group, and homocysteine ​​+ resveratrol treatment group of pancreatic β cells; Figures E and F are the SLC3A2 expression levels in cells after interference / overexpression of piRNA-145836 detected by Western blot, respectively.

[0031] exist Figure 6 In the figure, Figures A and B are the validation diagrams of the effects of SLC3A2 knockdown and overexpression in pancreatic β cells detected by qRT-PCR, respectively; Figures C, D, and E are the expression levels of copper death-related proteins HSP70, FDX1, and SLC31A1 in pancreatic β cells after SLC3A2 knockdown under homocysteine ​​and homocysteine ​​+ resveratrol intervention, respectively; Figures F, G, and H are the Western blot diagrams of the expression levels of copper death-related proteins HSP70, FDX1, and SLC31A1 in pancreatic β cells after SLC3A2 knockdown, respectively. Figure 1 shows the expression levels of copper death-related proteins HSP70, FDX1, and SLC31A1 in pancreatic β cells after SLC3A2 overexpression, detected by blot analysis under homocysteine ​​and homocysteine ​​+ resveratrol interventions; Figure 2 shows the copper ion levels in pancreatic β cells after SLC3A2 knockdown, detected by colorimetric assay; Figure 3 shows the copper ion levels in pancreatic β cells after SLC3A2 overexpression, detected by colorimetric assay; Figure 4 shows the insulin secretion levels in pancreatic β cells after SLC3A2 knockdown, detected by enzyme-linked immunosorbent assay (ELISA); Figure 5 shows the insulin secretion levels in pancreatic β cells after SLC3A2 overexpression, detected by ELISA. Detailed Implementation

[0032] The following embodiments are used to illustrate the present invention, but are not intended to limit the scope of the invention. Any modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and essence of the invention are within the scope of the invention. Unless otherwise specified, the products and equipment used in the following embodiments are commercially available, and the methods used are consistent with conventional methods unless otherwise specified.

[0033] The technical solution of the present invention will be further described in detail below with reference to the embodiments.

[0034] 1. Experimental subjects;

[0035] (1) Experimental animals: ApoE gene knockout (ApoE- / -) mice; (2) Experimental cells: pancreatic β cells of Min6 mice.

[0036] 2. Instruments, equipment, and experimental reagents;

[0037] 2.1. Main reagents: T25 sterile cell culture flasks (Beaver, China), filters (Corning, USA), fetal bovine serum (Gibco, USA), DMEM high glucose medium (Cellmax, USA), trypsin digestion solution (Solepro, China), Trizol reagent (Invitrogen, USA), reverse transcription kit (Takara, Japan), piRNA-145836 and U6 primers synthesized by Guangzhou Ruibo Biotechnology Co., Ltd., Lipofectamine 2000 transfection aid (Invitrogen, USA), piRNA-145836 recombinant lentivirus (LV-piRNA145836) and empty vector control (LV GFP) provided by Shanghai Jima Pharmaceutical Technology Co., Ltd., dual-luciferase assay kit (Shanghai Jima Pharmaceutical Technology Co., Ltd.), homocysteine ​​(Sigma-Aldrich, Darmstadt, Germany), resveratrol (Sigma-Aldrich, Darmstadt, Germany), etc.

[0038] 2.2. Major experimental instruments: constant temperature cell culture incubator (Thermo Fisher Scientific, USA), ultra-clean workbench (AnTai Technology Co., Ltd., Suzhou, China), 5415D micro-volume benchtop centrifuge (Eppendorf, Germany), BS1105 precision electronic balance (Sartorius, Germany), ice maker (AF1OSCOTSMAN, USA), real-time fluorescence quantitative PCR instrument (analytik-jena, Germany), conventional PCR instrument (Bio-Rad, USA), micropipette (Eppendorf, Germany), three-channel pure water system (HealForce, China), fully automated microplate reader (Bio-Tek, USA), high-speed centrifuge (Thermo Fisher, USA), vortex mixer (Dalong Xingchuang Experimental Instrument Co., Ltd., China), constant temperature metal bath (Coyote, China), etc.

[0039] 3. Experimental methods;

[0040] 3.1. Establish an animal model of hyperhomocysteinemia;

[0041] Six-week-old apolipoprotein E knockout (ApoE- / -) mice were purchased from Beijing Vesunlide Biotechnology Co., Ltd. and housed at the Experimental Animal Center of Ningxia Medical University. Mice were housed in a specific animal room at a temperature of 22±1℃ and humidity of 40%-70%, using a 12-hour light / 12-hour dark cycle, with free access to food and water. After one week of acclimatization, the mice were randomly divided into three groups: a normal diet group (NC, n=6), a high-methionine diet group (HMD, containing 1.8% methionine, n=6), and a high-methionine + resveratrol group (HMD+Res, resveratrol dose of 200 mg / kg, n=6). Mice were sacrificed at week 10 for serological analysis.

[0042] The serological analysis results are as shown in the attached instructions. Figure 1 As shown in the attached document. Figure 1 As shown in Figures A, B, D, F, and G, compared with the normal control group, mice in the high-methionine diet group had significantly higher serum homocysteine ​​concentration, glycated hemoglobin level, blood glucose concentration, total cholesterol, triglycerides, and creatinine levels. In contrast, mice in the high-methionine diet + resveratrol diet group showed lower levels of all these indicators compared to mice in the high-methionine diet group. (See attached figure.) Figure 1 As shown in Figures C and E, compared with the normal control group, the high-methionine diet group mice had lower insulin levels and body weight. The high-methionine diet + resveratrol diet group mice had higher levels of relevant indicators compared with the high-methionine diet group mice.

[0043] The above experimental results indicate that high serum homocysteine ​​levels can induce endothelial cell damage, oxidative stress, and chronic inflammation, all of which are key factors in insulin resistance, and that a resveratrol diet can alleviate these effects.

[0044] 3.2. Cell culture;

[0045] 3.2.1. Resuscitation and culture of pancreatic β cells;

[0046] Cells were removed from the -80°C cryopreservation freezer and quickly thawed in a 37°C water bath. After sterilization, they were transferred to the cell culture medium for resuscitation. Cells containing cryopreservation solution were aspirated into 15mL centrifuge tubes, 3mL of complete culture medium containing 10% serum was added, and the tubes were centrifuged (1000rpm / min, 5min). The supernatant was discarded, and the cells were resuspended in 3mL of culture medium containing 10% serum. The cells were mixed by pipetting and transferred to culture flasks and cultured in a 37°C CO2 incubator with 5% CO2.

[0047] 3.2.2. Grouping of pancreatic β cells;

[0048] Healthy pancreatic β cells were collected, and when the cell fusion rate reached 70%-80%, the cells were treated with 140 μM homocysteine ​​and 10 μM resveratrol for 48 hours. The control group received no drug intervention. The cells were divided into a control group, a homocysteine ​​group, and a homocysteine ​​+ resveratrol group.

[0049] 3.3. Cell transfection;

[0050] When the pancreatic β-cell density reaches 85%, digested pancreatic β-cells are seeded in T25 culture flasks at a 1:3 ratio. When the cell density reaches 60%, transfection is performed. 30 μM nucleic acid and transfection aid are mixed 1:1, and the mixture is pipetted 10-15 times, then incubated at room temperature for 15 min. The mixture is then added to the cell culture flask, gently mixed, and placed in an incubator for further culture. 24 hours after transfection, the culture medium is replaced with normal culture medium.

[0051] 3.4. Cell infection;

[0052] A certain number of doses (approximately 5 x 10) were administered. 5 Pancreatic β-cells were cultured in T25 flasks. When the cell confluence reached approximately 60%, the culture medium was replaced with fresh medium before infection. 150 μL of virus was added to each flask (MOI = 30), and the complete culture medium was brought to a final volume of 5 mL. Polybrene was added to each flask to a final concentration of 5 μg / mL. The flasks were gently shaken to mix, and the cells were returned to the incubator. After 36 hours, the culture medium was replaced with fresh medium.

[0053] 3.5. RNA pull-down and mass spectrometry analysis;

[0054] RNA precipitation was performed using streptavidin magnetic beads. Biotin-labeled RNA probes were incubated with pre-washed magnetic beads at room temperature for 30 minutes to immobilize them on the bead surface. Simultaneously, total protein was extracted from pancreatic β-cells using lysis buffer supplemented with protease and nuclease inhibitors, and the supernatant was collected by centrifugation. The RNA-bound magnetic beads were mixed with protein lysis buffer and incubated overnight at 4°C with gentle shaking. The magnetic beads were then thoroughly washed with washing buffer to remove non-specifically bound proteins. Specifically bound proteins were eluted with elution buffer at 37°C. The eluent was diluted with loading buffer and then subjected to SDS-PAGE electrophoresis, silver staining, and mass spectrometry analysis.

[0055] 3.6. CCK-8 assay for pancreatic β-cell proliferation;

[0056] Pancreatic β cells were seeded in 96-well plates and pre-cultured overnight. After cell adhesion, homocysteine ​​and resveratrol of different concentration gradients were added and cultured for another 48 hours. 10 μL of CCK-8 solution was added to each well, and the culture plate was incubated in an incubator for 2 hours. The absorbance at 450 nm was measured using a microplate reader.

[0057] 3.7. Real-time quantitative PCR;

[0058] According to the RNA extraction instructions, pancreatic β cells from each group were extracted, and the concentration of extracted RNA was measured. The RNA was then reverse transcribed into cDNA and amplified. The qRT-PCR conditions were: 95℃ pre-denaturation for 30 s; 95℃ denaturation for 5 s; Tm annealing for 34 s; and amplification cycle number of 45. After the reaction, according to 2... -△△CT Legal analysis results.

[0059] 3.8. Immunohistochemical staining;

[0060] Mouse pancreatic tissue specimens were dehydrated, embedded, and then prepared into tissue sections. The sections were dewaxed with xylene, hydrated with a gradient of ethanol, and thoroughly washed with phosphate buffer. HSP70 antibody, FDX1 antibody, and SLC31A1 antibody were added, and the sections were incubated overnight at 4°C, followed by standing at room temperature for 1 hour. Unbound antibodies were removed by washing with PBS, and the sections were reacted with diaminobenzidine (DAB) chromogenic solution for 1 minute. Staining was terminated with distilled water. The cell nuclei were counterstained with hematoxylin, differentiated with ethanol, dehydrated with a gradient of ethanol, and washed with PBS until clear. The sections were mounted with neutral resin, and the expression and localization of the target protein were observed under a light microscope.

[0061] 3.9. Enzyme-linked immunosorbent assay (ELISA);

[0062] Collect cell culture medium using sterile tubes, centrifuge at 2500 rpm / min for 25 minutes, collect the supernatant, and add standards. Add 50 μL of different concentrations of standards (40, 20, 10, 5, 3.5, 0 mIU / L) to each well. Then add the test samples by adding the samples to the bottom of the wells of the ELISA plate and gently shaking to mix. Add 100 μL of enzyme-labeled reagent to each well (except for blank wells); seal the plate with sealing film and incubate at 37°C for 60 min; dilute 20× concentrated washing buffer with enzyme-free water to make 1× washing buffer for later use; remove the sealing film, discard the liquid in the wells, shake dry, fill each well with washing buffer, let stand for 15 s and discard, repeat the above steps 5 times, and pat dry; under light-protected conditions, add 50 μL of chromogenic reagent A to each well, then add 50 μL of chromogenic reagent B, gently shake to mix, and develop color at 37°C for 15 min; add 50 μL of stop solution to each well (blue immediately turns yellow) to stop the reaction, and use an enzyme-linked immunosorbent assay (ELISA) reader to detect at a wavelength of 450 nm.

[0063] 3.10. Western Blotting: Total protein was extracted from pancreatic tissue and pancreatic β-cells using RIPA lysis buffer, and Western blot analysis was performed according to standard procedures. The antibodies used were: HSP70 (70 kDa, 1:1000, Abcam), FDX1 (19 kDa, 1:1000, Abcam), SLC31A1 (26 kDa, 1:1500, Abways), and SLC3A2 (75 kDa, 1:1000, Abcam). Signal intensity was analyzed using ImageLab software, and relative protein expression levels were normalized using β-actin as an internal control.

[0064] 3.11. Dual-luciferin reporter gene assay;

[0065] After digestion, cells were seeded into 24-well plates. When the cell density reached 70%, transfection was performed. 2 μL of transfection aid, 0.5 μg of plasmid, and 2.5 μL of Loligo were added to each well. After 24 hours of transfection, the culture medium was discarded, and the cells were washed once with PBS. 250 μL of cell lysis buffer was added to each well, and lysis was performed for 45 min to ensure complete lysis. The lysate was collected and centrifuged (4000 rpm / 5 min). The supernatant was collected, and 50 μL of cell lysis buffer was added to 100 μL of firefly luciferase reaction solution. The firefly luciferase activity was then measured. 100 μL of Renilla luciferase reaction solution was added, and the Renilla luciferase activity was measured. Finally, the data were analyzed.

[0066] Example 1: A resveratrol diet can alleviate metabolic abnormalities caused by a high-methionine diet-induced hyperhomocysteinemia.

[0067] Through the experimental procedures of enzyme-linked immunosorbent assay (ELISA) and the determination by a fully automated biochemical analyzer, the following results were obtained: Figure 1 The results shown; in this embodiment, homocysteine, blood glucose, glycated hemoglobin, low-density lipoprotein, triglyceride, total cholesterol, and creatinine levels in mouse serum were measured using a fully automated biochemical analyzer. Figure 1 As shown in Figures A, B, D, F, G, H, and I, it can be concluded that the biochemical indicators of mice on a high-methionine diet were all higher than those in the normal group, while the levels of resveratrol combined with a high-methionine diet were lower. In this example, the serum insulin level of mice was measured using an enzyme-linked immunosorbent assay (ELISA). Figure 1 As shown in Figure C, insulin levels in mice on a high-methionine diet were lower than those on a normal diet, while insulin levels in mice on a high-methionine diet plus resveratrol diet increased. The body weights of mice in each group were obtained by weighing them using a balance. Figure 1As shown in Figure E, the mice in the high-methionine diet group had a lower body weight than the normal diet group, and the resveratrol diet reversed this weight loss. In conclusion, resveratrol has a certain ameliorative effect on metabolic disorders induced by a high-methionine diet in mice.

[0068] Example 2: Screening and identification of differentially expressed non-coding piRNA-145836 in hyperhomocysteinemia.

[0069] After total RNA sample detection, library construction, library testing, and sequencing, piRNA information was obtained. Bioinformatics analysis was performed on the piRNAs, including branching and multi-dimensional analysis. The results of piRNA clustering analysis, GO enrichment analysis, and KEGG enrichment analysis are as follows: Figure 2 As shown. Finally, differentially expressed piRNAs were identified in pancreatic β-cells. RNA sequencing analysis revealed that 143 piRNAs were differentially expressed in homocysteine-treated pancreatic β-cells. Compared to the control group, piRNA-145836 showed the most significant upregulation. Figure 2 As shown in Figure A. Then, KEGG and GO analyses were performed on these piRNAs, and the results are as follows. Figure 2 As shown in Figures B and C, it was found that it mainly participates in the copper death signaling pathway. Subsequently, real-time quantitative PCR was used to extract RNA and reverse transcribe it into cDNA. After amplification, the results were obtained and analyzed, yielding... Figure 2 The results of D and E verified the mRNA expression levels of piRNA-145836 in pancreatic tissues of mice in the normal control group, high-methionine diet group, and high-methionine diet + resveratrol diet group, as well as in pancreatic β cells of the normal control group, high-homocysteine ​​treatment group, and high-homocysteine ​​+ resveratrol treatment group. The results showed that the expression of piRNA-145836 was upregulated after high-homocysteine ​​intervention, while its expression was decreased after resveratrol intervention.

[0070] The sequence of piRNA-145836 obtained by sequencing is shown in SEQ ID NO.1, specifically: TGTGTAACAACTCACCTGCCGAATCA.

[0071] Example 3: Animal and cell experiments verify that resveratrol alleviates homocysteine-induced copper death.

[0072] To further clarify the role of resveratrol in alleviating homocysteine-induced copper death in pancreatic β-cells, this embodiment conducted validation experiments at both the animal tissue and cellular levels. At the animal tissue level, Western blotting was used to validate copper death-related proteins. Protein extraction, electrophoresis, membrane transfer, incubation with primary and secondary antibodies, and imaging were performed on pancreatic tissues from the high homocysteine ​​treatment group, resveratrol intervention group, and control group. The results were as follows: Figure 3 The results shown are obtained through Figure 3 As shown in Figures A, B, and C, compared to the normal control group, the expression levels of copper death-related proteins HSP70, FDX1, and SLC31A1 were increased in the pancreatic tissue of mice in the high-methionine diet group, while the expression levels of these proteins were decreased in the pancreatic tissue of mice in the high-methionine diet + resveratrol diet group. This indicates that resveratrol can alleviate homocysteine-induced copper death. This is corroborated by immunohistochemical staining experiments, and the results are consistent with those of protein immunoblotting experiments. Figure 3 As shown in Figure D. In cell experiments, to ensure the scientific validity of the experimental drug concentration, this embodiment uses a CCK8 assay to screen drug concentrations. Figure 3 As shown in E and F, the optimal intervention concentrations for homocysteine-induced copper death in pancreatic β-cells and for resveratrol to exert a protective effect can be determined. Simultaneously, Western blotting was used to further validate the expression of copper death-related proteins at the cellular level, such as... Figure 3 As shown in Figures G, H, and I, the levels of copper death-related proteins HSP70, FDX1, and SLC31A1 in pancreatic β-cells of the normal control group, the high homocysteine ​​treatment group, and the high homocysteine ​​+ resveratrol treatment group showed trends consistent with those observed in animal studies. Furthermore, the levels of copper ions in cells were detected using colorimetric methods, such as... Figure 3 As shown in Figure J, this embodiment shows that the copper ion level in cells of the homocysteine ​​group was significantly higher than that in the control group, while the copper ion level in cells of the homocysteine ​​+ resveratrol group was lower than that in the homocysteine ​​group; insulin secretion was detected by enzyme-linked immunosorbent assay (ELISA). Figure 3 As shown in the K-plot, insulin secretion was significantly reduced in the homocysteine ​​group, while insulin secretion recovered in the homocysteine ​​+ resveratrol group. These results indicate that elevated homocysteine ​​levels lead to increased expression of copper death-related proteins in tissues and cells, copper accumulation, and impaired pancreatic function. Resveratrol intervention can reverse these phenomena, thus exerting a protective effect on pancreatic tissue and islet β-cells in a hyperhomocysteinemic environment. Therefore, animal and cellular experiments validate that resveratrol alleviates homocysteine-induced copper death.

[0073] Example 4: Resveratrol alleviates high homocysteine-induced copper death in pancreatic β cells by regulating piRNA-145836 expression.

[0074] In the above experiments, this embodiment showed that both piRNA-145836 and resveratrol affect homocysteine-induced copper death. To explore the potential mechanism of piRNA-145836 in resveratrol's alleviation of homocysteine-induced copper death in pancreatic β cells, Shanghai Gemma Technology Co., Ltd. was commissioned to construct a small interfering RNA (siRNA) fragment of piRNA-145836 and an overexpression lentiviral vector. The siRNA fragment was transfected into pancreatic β cells at a cell density of 60%-70%. The transfection efficiency was verified by real-time quantitative polymerase chain reaction (qRT-PCR), and the results were as follows: Figure 6 The results shown indicate that, through Figure 6 Figures A and B show that the mRNA expression level of piRNA-145836 in the interference group was significantly lower than that in the control group, while the mRNA expression level of piRNA-145836 in the overexpression group was significantly higher than that in the control group. Secondly, this embodiment obtained the following results through Western blotting experiments: Figure 6 Figures C, D, and E show the results. As indicated, in pancreatic β-cells treated with homocysteine, compared to the interference control group, the expression levels of copper death-related proteins HSP70, FDX1, and SLC31A1 were significantly reduced in the piRNA-145836 interference group. The addition of resveratrol further reduced the levels of these copper death-related proteins. Conversely, the piRNA-145836 overexpression group showed the opposite trend, as shown in the attached figure. Figure 6 As shown in F, G, and H. Subsequently, in this embodiment, the level of intracellular copper ions was detected by colorimetry, and the results were obtained. Figure 6 The results shown are obtained through Figure 6 As shown in Figures F and G, the intracellular copper ion levels in the piRNA-145836 interference group were significantly reduced, and the copper ion level further decreased after resveratrol intervention. The piRNA-145836 overexpression group showed the opposite trend, with increased intracellular copper ion levels, but decreased after resveratrol supplementation. Simultaneously, to verify pancreatic β-cell function, this embodiment used an enzyme-linked immunosorbent assay (ELISA) to detect insulin secretion, obtaining... Figure 6 The results shown are obtained by Figure 6 As shown in Figures H and I, it can be seen that the insulin secretion capacity of pancreatic β cells in the piRNA-145836 interference group was significantly improved, and the secretion level was further improved after resveratrol supplementation; the insulin secretion level of pancreatic β cells in the piRNA-145836 overexpression group decreased, but the insulin level showed a recovery trend after resveratrol intervention.

[0075] Therefore, this embodiment concludes that piRNA-145836 participates in and regulates homocysteine-induced copper death in pancreatic β cells, thereby regulating homocysteine-induced type 2 diabetes.

[0076] Example 5: Screening and verification of the downstream target gene SLC3A2 of piRNA-145836.

[0077] To investigate the molecular mechanism by which resveratrol alleviates homocysteine-induced copper death in pancreatic β-cells, this example first conducted a verification experiment on the regulatory role of piRNA. Through relevant experimental procedures, the following results were obtained: Figure 4 The results shown; via Figure 4 As shown in the sub-figures, it can be seen that piRNA-145836 participates in and regulates the resveratrol-mediated protective mechanism, thereby protecting pancreatic β-cell function.

[0078] It is worth noting that non-coding RNAs influence gene expression, RNA modification, DNA damage repair, and cell proliferation, migration, and metabolism through interactions with proteins, mRNA, and DNA. Therefore, in-depth analysis of downstream target genes of piRNAs is necessary. To screen the interacting target genes of piRNA-145836, this embodiment performed an RNA pull-down / MS experiment. By using piRNA-145836 to target its interacting genes, the following results were obtained: Figure 5 The results shown; via Figure 5 As shown in Figure A, compared with the negative control group, the piRNA-145836 group showed a distinct specific band (silver staining result). To identify the protein corresponding to the specific band, this embodiment performed mass spectrometry analysis on the above two groups of experimental products. Through the experimental operation of mass spectrometry detection and result analysis, the following results were obtained: Figure 5 The results shown; via Figure 5 As shown in Figure B, SLC3A2 scored highly, suggesting a high probability of binding between this protein and piRNA-145836. To further confirm the targeting effect of piRNA-145836 on SLC3A2, a dual-luciferase reporter gene assay was performed. Wild-type (WT) and mutant (MUT) SLC3A2 reporter plasmids were constructed, and luciferase activity was detected after co-transfecting pancreatic β cells with the piRNA-145836 mimic and the two SLC3A2 plasmids. The results are shown below. Figure 5 The results shown; via Figure 5As shown in Figure C, the results indicate that luciferase activity significantly increased when piRNA-145836 was co-expressed with the WT reporter plasmid; however, luciferase activity did not change significantly when co-expressed with the MUT reporter plasmid, suggesting that piRNA-145836 directly acts on SLC3A2. Furthermore, to investigate the effects of high homocysteine ​​and resveratrol on SLC3A2 protein expression, this example performed a Western blotting experiment. Protein extraction, electrophoresis, transfer, incubation with primary and secondary antibodies, and development were performed on pancreatic β cells from the high homocysteine ​​treatment group, resveratrol intervention group, and control group, yielding the following results: Figure 5 The results shown; via Figure 5 As shown in Figure D, SLC3A2 protein expression in pancreatic β cells of the high homocysteine ​​treatment group was significantly increased, while this increase was inhibited after intervention with resveratrol. Furthermore, to clarify the regulatory role of piRNA-145836 on SLC3A2 protein expression, pancreatic β cells were transfected with the piRNA-145836 interference sequence, overexpression vector, and negative control, respectively. Western blotting was then used to detect SLC3A2 protein expression, yielding the following results: Figure 5 The results shown; via Figure 5 As shown in Figures E and F, it can be concluded that interfering with piRNA-145836 can inhibit SLC3A2 protein expression, while overexpressing piRNA-145836 can promote SLC3A2 protein expression.

[0079] Example 6: SLC3A2 participates in and regulates homocysteine-induced copper death in pancreatic β cells.

[0080] In this embodiment, a small interfering RNA fragment targeting SLC3A2 and an overexpression lentiviral vector were constructed and transfected into pancreatic β cells. First, the transfection efficiency was verified using real-time quantitative polymerase chain reaction (qRT-PCR), yielding the following results: Figure 6 The results shown indicate that, through Figure 6 Figures A and B show that the mRNA expression level of SLC3A2 in the interference group was significantly lower than that in the control group, while the mRNA expression level of SLC3A2 in the overexpression group was significantly higher than that in the control group. Secondly, this embodiment obtained the following results through Western blotting experiments: Figure 6 Figures C, D, and E show the results. As indicated, in pancreatic β-cells treated with homocysteine, compared to the interference control group, the expression levels of copper death-related proteins HSP70, FDX1, and SLC31A1 were significantly reduced in the SLC3A2 interference group. The addition of resveratrol further reduced the levels of these copper death-related proteins. Conversely, the SLC3A2 overexpression group showed the opposite trend, as shown in the attached figure. Figure 6As shown in F, G, and H. Subsequently, in this embodiment, the level of intracellular copper ions was detected by colorimetry, and the results were obtained. Figure 6 The results shown are obtained through Figure 6 As shown in Figures F and G, the intracellular copper ion levels in the SLC3A2 interference group were significantly reduced, and further decreased after resveratrol intervention. The SLC3A2 overexpression group showed the opposite trend, with increased intracellular copper ion levels, but decreased after resveratrol supplementation. Simultaneously, to verify pancreatic β-cell function, this embodiment used an enzyme-linked immunosorbent assay (ELISA) to detect insulin secretion, obtaining... Figure 6 The results shown are obtained by Figure 6 As shown in Figures H and I, it can be seen that the insulin secretion capacity of pancreatic β cells in the SLC3A2 interference group was significantly improved, and the secretion level was further improved after resveratrol supplementation; the insulin secretion level of pancreatic β cells in the SLC3A2 overexpression group decreased, but the insulin level showed a recovery trend after resveratrol intervention.

[0081] Therefore, based on the above results, it can be seen that the downstream target gene SLC3A2 of piRNA-145836 participates in and regulates homocysteine-induced copper death in pancreatic β cells, thereby regulating homocysteine-induced type 2 diabetes.

[0082] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. The application of piRNA-145836 formulation in the preparation of drugs for the prevention and treatment of hyperhomocysteine-induced type 2 diabetes, characterized in that, The nucleotide sequence of the piRNA-145836 is shown in SEQ ID NO.

1.

2. The application of the piRNA-145836 formulation as described in claim 1 in the preparation of drugs for the prevention and treatment of hyperhomocysteine-induced type 2 diabetes, characterized in that, The nucleotide sequence of the piRNA-145836 is TGTGTAACAACTCACCTGCCGAATCA.

3. The application of the piRNA-145836 formulation as described in claim 1 in the preparation of drugs for the prevention and treatment of hyperhomocysteine-induced type 2 diabetes, characterized in that, The drug inhibits copper death in pancreatic β cells.

4. The application of the piRNA-145836 formulation as described in claim 3 in the preparation of drugs for the prevention and treatment of hyperhomocysteine-induced type 2 diabetes, characterized in that, The drug inhibits the accumulation of intracellular copper ions caused by the increase of FDX1, SLC31A1, and HSP70.

5. The application of the piRNA-145836 formulation as described in claim 4 in the preparation of a drug for preventing and treating type 2 diabetes induced by high homocysteine ​​levels, characterized in that... The drug inhibits the accumulation of copper ions in cells and reduces copper death in pancreatic β cells.

6. Application of piRNA-145836 and its target gene SLC3A2 inhibitor in the preparation of drugs for the prevention and treatment of type 2 diabetes induced by high homocysteine.

7. A drug for treating type 2 diabetes induced by hyperhomocysteinemia, characterized in that, The active ingredients of the drug include a piRNA-145836 inhibitor and an SLC3A2 inhibitor.

8. The medicament for treating type 2 diabetes induced by hyperhomocysteinemia as described in claim 7, characterized in that, The sequence of piRNA-145836 is as follows: TGTGTAACAACTCACCTGCCGAATCA.

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