Methods for assessing the risk of type 2 diabetes from elevated acrylamide exposure and uses

Abstract

This invention relates to the fields of environmental medicine and molecular biology, and discloses a method and application for assessing the increased risk of type 2 diabetes due to acrylamide exposure. The method involves obtaining in vitro biological samples, detecting the expression level of miR-128-1, and comparing it with a reference value. A level higher than the reference value suggests a tendency for increased type 2 diabetes due to acrylamide exposure. This method can also detect acrylamide exposure biomarkers and oxidative stress or NRF2 pathway-related factors for non-diagnostic risk assessment. The invention also provides the application of miR-128-1 inhibitors in the preparation of related drugs. This invention integrates the assessment strategy of exposure and effect biomarkers and provides a clear molecular intervention target, solving the problem of the lack of integrated assessment methods and intervention targets in existing technologies.

Description

Technical Field

[0001] This invention relates to the fields of environmental medicine and molecular biology, and in particular to a method and application for assessing the increased risk of type 2 diabetes due to acrylamide exposure. Background Technology

[0002] Type 2 diabetes (T2D) is a serious metabolic disease characterized by pancreatic β-cell dysfunction and insulin resistance. Its global prevalence continues to rise, posing a significant public health threat to human health. Besides genetic factors, exposure to environmental pollutants has been proven to be a major risk factor for T2D. Acrylamide (ACR), a widely present environmental pollutant in tobacco smoke, food processing products, and everyday consumer goods, can be continuously exposed to throughout life through ingestion, inhalation, and skin contact. Existing toxicological studies have shown that ACR has a diabetic effect, inducing pathological changes such as hyperglycemia, impaired glucose tolerance, and pancreatic β-cell damage in experimental animals, with oxidative stress considered a key pathogenic mechanism. However, epidemiological evidence regarding the association between ACR exposure and T2D in the general population is insufficient and controversial, with different studies showing inconsistent results, including positive, negative, or no association. The underlying mechanisms of action are also not fully elucidated.

[0003] In recent years, microRNAs (miRNAs), as post-transcriptional regulators of gene expression, have increasingly attracted attention for their role in the pathogenic mechanisms of environmental pollutants. miR-128-1, a highly conserved miRNA, has recently been identified as an emerging therapeutic target for type 2 diabetes (T2D) by regulating glucose homeostasis. Existing research indicates that miR-128-1 can target and inhibit nuclear factor E2-related factor 2 (NRF2)—a core transcription factor regulating the antioxidant defense of pancreatic β-cells, playing a crucial role in resisting oxidative stress damage. However, current technologies lack reports on the association between ACR exposure and miR-128-1 expression levels, and systematic studies on whether miR-128-1 mediates the risk of T2D induced by ACR exposure are also lacking. Furthermore, existing risk assessment methods often rely on single exposure or outcome indicators, lacking an assessment strategy that integrates "environmental exposure-molecular biomarkers-disease risk," making it difficult to accurately identify high-risk individuals for ACR-induced T2D and failing to provide clear molecular targets for intervention in ACR-related T2D.

[0004] To address the aforementioned shortcomings, there is an urgent need to establish an assessment strategy that can integrate exposure and effect biomarkers and reveal the key molecular mechanisms by which ACR exposure leads to T2D, providing new technical solutions for related risk assessment and drug intervention. Summary of the Invention

[0005] The purpose of this invention is to provide a method and application for assessing the increased risk of type 2 diabetes due to acrylamide exposure, which solves the problems in the prior art of lacking risk assessment methods that can integrate exposure and effect biomarkers and lacking clear molecular intervention targets for acrylamide-induced type 2 diabetes.

[0006] To achieve the above objectives, the present invention provides a method for assessing the increased risk of type 2 diabetes due to acrylamide exposure, comprising the following steps:

[0007] Obtain ex vivo biological samples of the object to be tested;

[0008] The expression level of miR-128-1 in the biological samples was detected;

[0009] The miR-128-1 expression level was compared with a preset reference value. If the miR-128-1 expression level was higher than the reference value, it indicated that the subject had a tendency to have an increased risk of type 2 diabetes due to acrylamide exposure.

[0010] Specifically, obtaining ex vivo biological samples of the object to be tested includes: The biological sample is selected from at least one of blood, plasma, serum, urine exosomes, or pancreatic tissue.

[0011] Specifically, detecting the expression level of miR-128-1 in the biological sample includes: The method for detecting miR-128-1 expression level is selected from at least one of quantitative reverse transcription polymerase chain reaction, digital PCR, nucleic acid hybridization, gene chip or high-throughput sequencing.

[0012] The method of detecting the expression level of miR-128-1 in the biological sample further includes: The method involves detecting acrylamide exposure biomarkers, wherein the acrylamide exposure biomarkers are selected from at least one of AAMA, GAMA, and ∑UAAM in urine.

[0013] The method of detecting the expression level of miR-128-1 in the biological sample further includes: The oxidative stress index or NRF2 signaling pathway-related factors were detected. The oxidative stress index included ROS or MDA, and the NRF2 signaling pathway-related factors included NRF2, HO-1, or NQO1.

[0014] The method is used for environmental exposure risk assessment, drug screening effect evaluation, population cohort studies, or diabetes risk prediction model construction.

[0015] Application of miR-128-1 inhibitors in the preparation of drugs for the prevention or treatment of type 2 diabetes caused by acrylamide exposure.

[0016] Specifically, miR-128-1 mediates the association between acrylamide exposure and type 2 diabetes by targeting and inhibiting the NRF2 antioxidant pathway, thereby causing oxidative damage to pancreatic β cells.

[0017] This invention discloses a method and application for assessing the increased risk of type 2 diabetes due to acrylamide exposure. First, an in vitro biological sample is obtained from the subject, and the expression level of miR-128-1 is detected. The detection result is compared with a preset reference value. If the miR-128-1 expression level is higher than the reference value, it indicates that the subject has a tendency to have an increased risk of type 2 diabetes due to acrylamide exposure. This method can also further detect acrylamide exposure biomarkers (AAMA, GAMA, ∑UAAM) and oxidative stress indicators (ROS, MDA) or NRF2 signaling pathway-related factors (NRF2, HO-1, NQO1) for non-diagnostic purposes such as environmental exposure risk assessment, drug screening, population cohort studies, or the construction of diabetes risk prediction models. Furthermore, this invention also provides the application of miR-128-1 inhibitors in the preparation of drugs for the prevention or treatment of type 2 diabetes caused by acrylamide exposure. This invention addresses the lack of effective risk assessment methods that integrate exposure and effect biomarkers by integrating an assessment strategy of "environmental exposure-molecular biomarkers-disease risk". At the same time, based on the mechanism by which miR-128-1 targets and inhibits the NRF2 antioxidant pathway to cause β-cell oxidative damage, it provides a clear molecular intervention target, thus providing a complete technical solution for risk assessment and drug intervention of type 2 diabetes caused by acrylamide exposure. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below.

[0019] Figure 1 This is a graph showing the statistical analysis results of the association between acrylamide exposure biomarkers and type 2 diabetes in Example 1 of the present invention.

[0020] Figure 2 This is a graph showing the statistical analysis results of the association between acrylamide exposure biomarkers and miR-128-1 in Example 1 of the present invention.

[0021] Figure 3 This is a graph showing the statistical analysis results of the association between miR-128-1 and type 2 diabetes in Embodiment 1 of the present invention.

[0022] Figure 4 This is a graph illustrating the mediating role of miR-128-1 in the association between acrylamide exposure biomarkers and type 2 diabetes in Example 1 of this invention.

[0023] Figure 5 The figure shows the results of the effects of acrylamide exposure on miR-128-1, NRF2 signaling pathway, oxidative stress, and glucose-stimulated insulin secretion in INS-1 cells in Example 2 of this invention.

[0024] Figure 6 The figure shows the effect of miR-128-1 overexpression on the NRF2 signaling pathway, oxidative stress, and glucose-stimulated insulin secretion capacity of acrylamide-induced INS-1 cells in Example 2 of this invention.

[0025] Figure 7 This figure shows the effect of miR-128-1 inhibition on the NRF2 signaling pathway, oxidative stress, and glucose-stimulated insulin secretion in acrylamide-induced INS-1 cells in Example 2 of this invention.

[0026] Figure 8 This is a flowchart of the steps in the method of assessing the increased risk of type 2 diabetes due to acrylamide exposure according to the present invention. Detailed Implementation

[0027] The embodiments of the present invention are described in detail below. Examples of the embodiments are shown in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and intended to explain the present invention, but should not be construed as limiting the present invention.

[0028] Please refer to Figures 1 to 8 This invention provides a method for assessing the increased risk of type 2 diabetes due to acrylamide exposure, comprising the following steps: S101: Obtain an ex vivo biological sample of the object to be tested; S102: Detect the expression level of miR-128-1 in the biological sample; S103: Compare the detected miR-128-1 expression level with the preset reference value. If the miR-128-1 expression level is higher than the reference value, it indicates that the subject has a tendency to have an increased risk of type 2 diabetes due to acrylamide exposure.

[0029] Specifically, the biological samples are selected from at least one of blood, plasma, serum, urine exosomes, or pancreatic tissue. Studies have shown that miR-128-1 can be stably detected in all of these biological samples. Blood, plasma, or serum samples are easy to collect and suitable for large-scale population screening; urine exosomes are rich in miRNA and have high detection sensitivity; pancreatic tissue samples are suitable for mechanistic studies or animal experiments. The method for detecting miR-128-1 expression levels is selected from at least one of quantitative reverse transcription polymerase chain reaction (RTRP), digital PCR, nucleic acid hybridization, gene chip, or high-throughput sequencing. These methods are all conventional techniques for detecting miRNA expression in this field, among which RTRP has advantages such as high sensitivity, strong specificity, and ease of operation, making it the preferred detection method.

[0030] Furthermore, the detection of miR-128-1 expression levels in the biological samples also includes the detection of acrylamide exposure biomarkers, which are selected from at least one of AAMA, GAMA, and ∑UAAM in urine. Epidemiological studies have found a significant positive correlation between ACR exposure biomarkers and miR-128-1 expression levels. Combined detection of both can more accurately assess the risk of T2D caused by ACR exposure. Specifically, AAMA is N-acetyl-S-[2-carbamoylethyl]-L-cysteine, GAMA is N-acetyl-S-[2-carbamoyl-2-hydroxyethyl]-L-cysteine, and ∑UAAM is the sum of AAMA and GAMA. These three substances are all metabolites of ACR in the human body and can reflect the internal exposure level of ACR in the body. Their detection can be performed using conventional methods in the field, such as liquid chromatography-mass spectrometry.

[0031] Furthermore, in addition to detecting the expression level of miR-128-1 in the biological samples, the study also included detecting oxidative stress indicators or NRF2 signaling pathway-related factors. The oxidative stress indicators included ROS or MDA, and the NRF2 signaling pathway-related factors included NRF2, HO-1, or NQO1. Toxicological experiments confirmed that ACR exposure leads to increased intracellular reactive oxygen species and malondialdehyde levels, while simultaneously activating the NRF2 signaling pathway and upregulating the expression of HO-1 and NQO1. The combined detection of these indicators can further verify the extent of oxidative damage caused by ACR exposure and the response status of the NRF2 pathway, providing a more comprehensive molecular basis for risk assessment.

[0032] The method of this invention can be used for non-diagnostic purposes such as environmental exposure risk assessment, drug screening efficacy evaluation, population cohort studies, or diabetes risk prediction model construction. For example, in environmental exposure risk assessment, the potential impact of ACR exposure on population health can be assessed by detecting the expression level of miR-128-1 in population samples; in drug screening, compounds with the potential to intervene in ACR-induced T2D can be screened by detecting the regulatory effect of candidate drugs on miR-128-1 expression; in population cohort studies, miR-128-1 can be used as an exposure-effect biomarker to explore the association mechanism between ACR exposure and T2D; and in diabetes risk prediction model construction, miR-128-1 can be used as a predictor to construct a risk assessment model.

[0033] Furthermore, this invention also provides the use of miR-128-1 inhibitors in the preparation of medicaments for the prevention or treatment of type 2 diabetes caused by acrylamide exposure. The miR-128-1 inhibitor is selected from at least one of antisense oligonucleotides, small interfering RNA, short hairpin RNA, CRISPR / Cas9-mediated gene editing elements, or small molecule compounds. The inventors confirmed through dual-luciferase assays that NRF2 is a direct target gene of miR-128-1, and miR-128-1 mediates the association between acrylamide exposure and type 2 diabetes by targeting and inhibiting the NRF2 antioxidant pathway, leading to oxidative damage to pancreatic β-cells. Therefore, by inhibiting the expression of miR-128-1, its inhibitory effect on NRF2 can be relieved, enhancing the antioxidant function of the NRF2 signaling pathway, thereby alleviating β-cell oxidative damage and functional impairment caused by ACR exposure. Preferably, the medicament further comprises an NRF2 activator to synergistically enhance the antioxidant stress effect and better prevent or treat type 2 diabetes caused by ACR exposure.

[0034] Compared with existing technologies, this invention reveals for the first time the key mediating role of miR-128-1 in the association between ACR exposure and T2D, and provides an assessment method with miR-128-1 as the core biomarker, which can integrate exposure and effect biomarkers, making up for the lack of effective integrated assessment methods in existing technologies. Based on the mechanism by which miR-128-1 targets and inhibits the NRF2 antioxidant pathway to cause β-cell oxidative damage, this invention provides a clear molecular intervention target, offering a new technical solution for drug intervention of T2D caused by ACR exposure. The method of this invention can be applied to various non-diagnostic scenarios such as environmental exposure risk assessment, drug screening, population cohort studies, and risk prediction model construction, and has broad application prospects.

[0035] Example 1: Epidemiological Study

[0036] This study selected adult residents of a city community as research subjects. Participants underwent physical examinations and face-to-face questionnaires to obtain covariate data including age, body mass index, gender, alcohol consumption, smoking status, dietary patterns, physical activity levels, education level, income level, and family history of diabetes. Morning urine and fasting peripheral blood samples were collected on the day of the physical examination, aliquoted, and cryopreserved at -20℃ and -80℃ respectively for later use.

[0037] Forty-two individuals with complete ACR exposure, T2D outcome, and covariate data were randomly selected from participants without severe cardiovascular disease, lung disease, or cancer. Plasma miR-128-1 levels were measured using quantitative reverse transcription polymerase chain reaction (RT-PCR). Urinary ACR exposure biomarkers, including AAMA, GAMA, and ∑UAAM (the sum of AAMA and GAMA), were measured using liquid chromatography-mass spectrometry (LC-MS). T2D was diagnosed according to the American Diabetes Association criteria: fasting blood glucose ≥7.0 mmol / L, or glycated hemoglobin ≥6.5%, or currently receiving hypoglycemic medication.

[0038] A generalized linear model was used to assess the association between ACR exposure biomarkers and miR-128-1 levels and type 2 diabetes mellitus (T2D), while the association between miR-128-1 and T2D was also analyzed. The linearity of the associations was tested using a restricted cubic spline model. Mediation analysis was used to assess the mediating role of miR-128-1 in the association between ACR exposure biomarkers and T2D, and the mediation ratio was calculated. All models were adjusted for confounding factors such as age, sex, body mass index, smoking status, alcohol consumption, education level, income, physical activity, family history of diabetes, diet, and city.

[0039] The results are as follows Figures 1 to 4 As shown, ACR exposure biomarkers were linearly and positively associated with T2D. For every 1 unit increase in AAMA, GAMA, and ∑UAAM (after natural logarithmic transformation), the risk of T2D increased by 58%, 42%, and 63%, respectively. Figure 1 ACR exposure biomarkers showed a linear positive association with miR-128-1; for every unit increase in AAMA, GAMA, and ∑UAAM, the β values ​​of miR-128-1 were 0.77, 0.63, and 0.82, respectively. Figure 2 miR-128-1 showed a linear positive correlation with T2D, with an OR of 1.34. Figure 3 Mediation analysis showed that miR-128-1 significantly mediated the association between ACR exposure biomarkers and T2D, with mediation rates of 24.54%, 22.82%, and 24.05%, respectively. Figure 4 ).

[0040] Example 2: Cytotoxicology Experiment

[0041] In this embodiment, INS-1 rat pancreatic β cells were used for in vitro experiments. The cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum at 37°C and 5% CO2.

[0042] Cell viability was assessed using a cell counting kit-8. INS-1 cells were counted at 1 × 10⁶ cells per well. 4 INS-1 cells were seeded in 96-well plates and cultured overnight. Then, 0, 0.5, 1, 2, and 4 mM ACR were added to each well for 24 hours. 10 μL of CCK-8 reagent was added to each well, and the cells were incubated at 37°C for 1.5 hours. The absorbance was measured at 450 nm. The results showed that ACR reduced INS-1 cell viability in a dose-dependent manner within the concentration range of 0.5–4 mM.

[0043] Intracellular reactive oxygen species (ROS) levels were measured using a reactive oxygen species (ROS) assay kit. Cells were co-incubated with a 10 μM DCFH-DA probe at 37°C for 20 minutes. After washing, fluorescence intensity was measured at an excitation wavelength of 488 nm and an emission wavelength of 525 nm. Malondialdehyde (MDA) levels were measured using a malondialdehyde (MDA) assay kit. The results showed that ACR exposure dose-dependently increased intracellular ROS and MDA levels.

[0044] β-cell function was assessed using a glucose-stimulated insulin secretion assay. Cells were first washed with KRBH buffer and incubated for 1.5 hours. Then, they were stimulated for 1.5 hours in KRBH buffer containing either 3.3 mM or 16.7 mM glucose. The supernatant was collected, and insulin levels were detected using a rat insulin ELISA kit. The results showed that ACR exposure dose-dependently reduced GSIS capacity.

[0045] The expression of miR-128-1 and NRF2 signaling pathway-related factors was detected by qRT-PCR and Western blot. The results showed that ACR exposure dose-dependently upregulated miR-128-1 expression and simultaneously activated the NRF2 pathway, leading to increased mRNA and protein levels of NRF2, HO-1, and NQO1. Figure 5 ).

[0046] Dual-luciferase assays were used to verify that NRF2 is a target gene of miR-128-1. Wild-type and mutant sequences of the NRF2 gene 3'-UTR were inserted into reporter plasmids and co-transfected with miR-128-1 mimics or negative controls into 293T and INS-1 cells. Luciferase activity was measured after 24 hours of culture. Results showed that in cells transfected with wild-type NRF2, the miR-128-1 mimic significantly reduced luciferase activity, while the mutant showed no significant change, confirming that NRF2 is a direct target gene of miR-128-1.

[0047] To investigate the role of miR-128-1 in ACR-induced β-cell damage, INS-1 cells were transfected with miR-128-1 mimic (50 nM), an inhibitor (100 nM), and corresponding negative controls, respectively. Twenty-four hours after transfection, cells were treated with 2 mM ACR for 24 hours. After verifying transfection efficiency by detecting miR-128-1 expression, GSIS capacity, ROS and MDA levels, and the mRNA and protein levels of NRF2, HO-1, and NQO1 were measured. Results showed that compared with the negative control group, the miR-128-1 mimic transfection group had decreased GSIS capacity, increased ROS and MDA levels, and decreased NRF2, HO-1, and NQO1 expression; while the miR-128-1 inhibitor transfection group had increased GSIS capacity, decreased ROS and MDA levels, and increased NRF2, HO-1, and NQO1 expression. Figure 6 , Figure 7 ).

[0048] The above examples fully demonstrate that miR-128-1 plays a key mediating role in the increased risk of type 2 diabetes due to acrylamide exposure by targeting and inhibiting the NRF2 antioxidant pathway, thereby causing oxidative damage to pancreatic β-cells. The method and application provided by this invention can effectively assess the risk of T2D caused by ACR exposure and provide a clear molecular target for related drug interventions.

[0049] The above-disclosed embodiments are merely one or more preferred embodiments of this application and should not be construed as limiting the scope of this application. Those skilled in the art can understand that all or part of the processes for implementing the above embodiments and equivalent changes made in accordance with the claims of this application still fall within the scope of this application.

Claims

1. A method for assessing the increased risk of type 2 diabetes due to acrylamide exposure, characterized in that, Includes the following steps: Obtain in vitro biological samples of the object to be tested; The expression level of miR-128-1 in the biological samples was detected; The miR-128-1 expression level was compared with a preset reference value. If the miR-128-1 expression level was higher than the reference value, it indicated that the subject had a tendency to have an increased risk of type 2 diabetes due to acrylamide exposure.

2. The method for assessing the increased risk of type 2 diabetes due to acrylamide exposure as described in claim 1, characterized in that, Obtaining ex vivo biological samples of the object to be tested specifically includes: The biological sample is selected from at least one of blood, plasma, serum, urine exosomes, or pancreatic tissue.

3. The method for assessing the increased risk of type 2 diabetes due to acrylamide exposure as described in claim 1, characterized in that, Detecting the expression level of miR-128-1 in the biological sample specifically includes: The method for detecting miR-128-1 expression level is selected from at least one of quantitative reverse transcription polymerase chain reaction, digital PCR, nucleic acid hybridization, gene chip or high-throughput sequencing.

4. The method for assessing the increased risk of type 2 diabetes due to acrylamide exposure as described in claim 2, characterized in that, Detecting the expression level of miR-128-1 in the biological sample further includes: The method involves detecting acrylamide exposure biomarkers, wherein the acrylamide exposure biomarkers are selected from at least one of AAMA, GAMA, and ∑UAAM in urine.

5. The method for assessing the increased risk of type 2 diabetes due to acrylamide exposure as described in claim 1, characterized in that, Detecting the expression level of miR-128-1 in the biological sample further includes: The oxidative stress index or NRF2 signaling pathway-related factors were detected. The oxidative stress index included ROS or MDA, and the NRF2 signaling pathway-related factors included NRF2, HO-1, or NQO1.

6. The method for assessing the increased risk of type 2 diabetes due to acrylamide exposure as described in claim 1, characterized in that, The method is used for environmental exposure risk assessment, drug screening effect evaluation, population cohort studies, or diabetes risk prediction model construction.

7. The use of miR-128-1 inhibitors in the preparation of drugs for the prevention or treatment of type 2 diabetes caused by acrylamide exposure.

8. The application as described in claim 7, characterized in that, The miR-128-1 mediates the association between acrylamide exposure and type 2 diabetes by targeting and inhibiting the NRF2 antioxidant pathway, which leads to oxidative damage to pancreatic β cells.

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