Quetiapine Reverses the Behavior and Myelination in Alcohol-Exposed Gestational Diabetes Mellitus Offspring Mice via ERK1/2 Signaling

Abstract

Gestational diabetes mellitus (GDM) is a glucose metabolism abnormality that first emerges during pregnancy and may negatively affect the behavioral and neurodevelopmental outcomes of offspring. Quetiapine (QUE) has been shown to promote differentiation of oligodendrocyte precursor cells (OPCs) and protect oligodendrocytes and myelination. To explore the effects of QUE on improving the expression of conditioned place preference (CPP) and myelination in the infralimbic cortex (IL) of the medial prefrontal cortex in alcohol-exposed GDM offspring mice, we evaluated CPP expression in 5-week-old alcohol-exposed GDM offspring and treated them with QUE and the extracellular-regulated protein kinase (ERK) inhibitor U0126. Immunohistochemical staining compared the numbers of mature oligodendrocytes, OPCs, and myelin expression levels. Immunofluorescence staining was employed to examine OPC differentiation and the activation of the ERK1/2 signaling pathway. In GDM offspring, CPP expression increased considerably following alcohol exposure, whereas early treatment with QUE or U0126 significantly decreased CPP expression. Meanwhile, alcohol exposure resulted in substantial activation of the ERK1/2 signaling pathway within OPCs in the IL region, as well as a substantial reduction in OPC differentiation, mature oligodendrocyte count, and myelin expression. QUE or U0126 inhibited the activation of the ERK1/2 signaling pathway within OPCs in the IL region of alcohol-exposed GDM offspring and markedly restored OPC differentiation, mature oligodendrocyte numbers, and myelin expression. Collectively, QUE enhanced the differentiation of OPCs in the IL region of GDM offspring after alcohol exposure by regulating the overactivation of the ERK1/2 signaling pathway, thus partially reversing myelination loss and ultimately improving CPP expression.

INTRODUCTION

The American Diabetes Association defines gestational diabetes mellitus (GDM) as a specific type of diabetes that manifests as impaired glucose tolerance or diabetes initiated during pregnancy, irrespective of the requirement for insulin or dietary management. It is also independent of the presence or duration of hyperglycemia after delivery.^1,2) Studies, following the International Association of Diabetes and Pregnancy Study Group criteria, reported GDM prevalence rates as high as 24.24% in certain regions.³⁾

In recent years, the potential long-term effects of GDM on the neurodevelopment of offspring have sparked extensive interest and investigation. Evidence suggests that GDM or intrauterine exposure to inflammatory mediators may adversely affect offspring’s behavior and cognitive functions, persisting into adolescence or adulthood.⁴⁾ Mouse model experiments indicate that maternal diabetes can disrupt epigenetic regulation, affecting transcription factors and signaling pathways, and resulting in neurodevelopmental anomalies.⁵⁾ Alcohol dependence, associated with the brain’s reward system, increases the risk of anxiety, depression, and cognitive deficits.^6,7) Research connects changes in the medial prefrontal cortex (mPFC)’s cognitive and executive functions with alcohol dependence development and its significant role in relapse post-abstinence.^8,9) Yet, studies on the heightened vulnerability of GDM offspring to alcohol dependence are limited. Recent findings suggest that these offspring face a higher risk of metabolic complications and are more prone to neuropsychiatric developmental disorders and other conditions after high prenatal glucose exposure.^10,11) Thus, the link between GDM and offspring’s increased susceptibility to alcohol dependence warrants further study. Alcohol exposure is linked to various neurobehavioral and brain tissue disorders, including reward-related behaviors and myelination damage in the mPFC,^12,13) which is crucial for reward processing and decision-making. Disruption in myelination can have lasting impacts on cognitive and emotional functions.^14,15) Additionally, the infralimbic cortex (IL) region is noted for its critical role in impulse control^16–18) and its association with alcohol dependence.¹⁹⁾

Quetiapine (QUE), an atypical antipsychotic, represents the second-generation antipsychotics, which are extensively utilized in the clinical management of mental disorders.^20,21) Recent investigations have highlighted that QUE facilitates oligodendrocyte differentiation and myelination by promoting cell morphological transformation.²²⁾ In models of cuprizone-induced demyelination, QUE treatment has been shown to enhance spatial working memory during recovery and support the development and remyelination of oligodendrocytes.^23,24) Furthermore, in vascular depression animal models, pre-treatment with QUE for 2 weeks before bilateral carotid artery occlusion and reperfusion mitigates myelin damage, oligodendrocyte loss, and fosters oligodendrocyte maturation.²⁵⁾ Additionally, QUE has been reported to reduce CPP expression in rats when co-administered with (+)-amphetamine.²⁶⁾ It can be seen that QUE plays a significant role in promoting oligodendrocyte precursor cell (OPC) differentiation and offering a protective effect on oligodendrocytes and myelination.

Mitogen-activated protein kinases (MAPKs), a group of intracellular serine/threonine protein kinases,²⁷⁾ include the extracellular-regulated protein kinases (ERKs), specifically ERK1 and ERK2, as critical members. These kinases are intricately involved in various biological processes such as cell proliferation, differentiation, morphology maintenance, cytoskeleton organization, apoptosis, and malignant transformation.^28–30) The phosphorylated-activated ERK (p-ERK) translocation from the cytoplasm to the nucleus mediates transcriptional activation and regulates cellular responses.^31,32) Khezri et al. found that QUE attenuates morphine-induced CPP acquisition by inhibiting ERK1/2 phosphorylation in the hippocampus and cerebral cortex.³³⁾ QUE improves collagen-induced arthritis in DBA/1J mice by inhibiting the ERK1/2 signaling pathway.³⁴⁾ It has also been reported that QUE stimulates the differentiation of oligodendrocytes by promoting the phosphorylation of ERK1/2.²⁴⁾

This study aimed to investigate whether QUE intervention promotes the differentiation of OPCs in the IL region and restores the number of mature oligodendrocytes and myelin expression by modulating the ERK1/2 signaling pathway in alcohol-exposed GDM offspring mice, thereby improving CPP expression.

MATERIALS AND METHODS

Animals

C57BL/6J background db/m mice were acquired from The Jackson Laboratory (Bar Harbor, ME, U.S.A.), and were maintained and bred at the Experimental Animal Center of the Second Affiliated Hospital of Army Medical University (Chongqing, China). db/m female mice were fed a high-fat diet from 7 d before mating to the end of gestation to establish a gestational diabetes model, which was used to obtain GDM offspring db/m mice. Male offspring were selected for subsequent experiments and housed in a pathogen-free environment with controlled temperature, humidity, and 12-h light–dark cycles, with unrestricted access to food and water. The experimental design, procedures, and handling of animals adhered to the ethical standards set by the ethical review committee (AMUWEC202051). The standard feed was provided by Chongqing Watson Biotechnology Co., Ltd. (Chongqing, China), and the high-fat feed (45 kcal/d, MD12032) was provided by Jiangsu Medison Biomedical Co., Ltd. (Jiangsu, China).

Experimental Grouping and Intervention

Fasting blood glucose was measured in several db/m female mice before mating (embryonic day 0, E0). After mating with male mice of the same genus, female mice were considered pregnant if a vaginal plug was visible, and after excluding pregnant mice due to accidental abortion or death, the fasting blood glucose of 12 db/m pregnant mice was measured at embryonic day 10 (E10) and embryonic day 20 (E20).

Eighty-four 5-week-old GDM offspring mice were randomly divided into 7 groups: (i) GDM + SAL, (ii) GDM + ETOH, (iii) GDM + ETOH + SAL, (iv) GDM + ETOH + QUE, (v) GDM + ETOH + U0126, (vi) GDM + SAL + QUE, and (vii) GDM + SAL + U0126. The groups designated GDM + ETOH, GDM + ETOH + SAL, GDM + ETOH + QUE, and GDM + ETOH + U0126 received intraperitoneal injections of a 20% alcohol solution on even days and saline on odd days during the CPP experiment’s conditioning phase. The GDM + SAL, GDM + SAL + QUE, and GDM + SAL + U0126 groups received saline injections throughout the same period. The 20% alcohol solution was diluted to 20% (v/v) with absolute ethanol in 0.9% saline and administered intraperitoneally at a dose of 12.6 mL/kg, which corresponds to an injectable dose of 2 g/kg.³⁵⁾ Additionally, the GDM + ETOH + QUE and GDM + SAL + QUE groups were pre-treated with QUE (10 mg/kg/d, Solarbio, Beijing, China) 20 min before each alcohol or saline injection.²⁵⁾ The GDM + ETOH + U0126 and GDM + SAL + U0126 groups were pre-treated with the ERK1/2 inhibitor U0126 (30 mg/kg/d, Selleck, Houston, TX, U.S.A.) 20 min before each injection,³⁶⁾ while the GDM + ETOH + SAL group received a comparable volume of saline as a pre-treatment.

Fasting Blood Glucose Testing

Fasting blood glucose levels were measured at 3 time points: E0, E10, and E20. Twelve hours before testing, the mice were fasted with only water available. A 1–2 mm section of the tail was cut, the first drop of blood was discarded, and the second drop was used to measure glucose levels with a blood glucose test strip.

Conditioned Place Preference

The conditioned place preference (CPP) paradigm, a standard model for assessing drug dependence, was utilized to determine the mice’s CPP expression. The CPP protocol followed established guidelines^37,38) with modifications, encompassing preadaptation, conditioning, and testing phases.

Preadaptation phase (Days 1–3): The movable baffle in the center of the CPP device was opened, allowing mice to move freely for 15 min over 3 consecutive days. On Day 3, the time spent in the 2 compartments within a 15-min interval was recorded as the pretest baseline time, indicating the mice’s inherent preferences. The preferred compartment was the side with longer residence time, and the non-preferred compartment was the side with shorter residence time. Meanwhile, mice were excluded from the study if they spent more than 70% of the time in any compartment.

Conditioning phase (Days 4–11): Mice underwent daily training. Mice in the alcohol group were placed in a non-preferred compartment for 15 min after intraperitoneal injection of a 20% alcohol solution on Days 4, 6, 8, and 10. Mice were placed in a preferred compartment for 15 min after receiving an equivalent dose of saline on alternate days (3, 5, 7, and 9). The control group received saline injections only. Four training cycles were completed in a row, with the daily training time fixed.

Testing phase (Days 12): Also known as the post-test. The movable baffle was reopened, and mice were placed at the CPP device’s center to move freely. The time spent in the non-preferred compartment during a 15-min session was recorded, and the time difference between the residence time in the non-preferred compartment in the posttest and pretest was calculated, reflecting the establishment of CPP following alcohol treatment.

Sample Preparation

Post-behavioral testing, mice were fully anesthetized with 1% sodium pentobarbital (70 mg/kg, Sigma-Aldrich, St. Louis, MO, U.S.A.) and perfused with 0.9% saline via the left ventricle, facilitating blood expulsion from the right atrium before complete exsanguination. Subsequently, mice were fixed in 4% paraformaldehyde (Sangon Bioengineering Co., Ltd., Shanghai, China) until rigidity was observed. Brain tissues were then fixed in 4% paraformaldehyde for 24 h, dehydrated in a graded sucrose series (10, 20, and 30%), and prepared for histological examination. Reference was made to the entire range of the mPFC, with Bregma coordinates between +1.98 and +1.78 mm.³⁹⁾ Brain tissues from 6 mice were embedded in paraffin, sectioned at 3.5 μm, and stored at room temperature. Brain tissues from 6 mice were sliced into 20-μm-thick frozen slices and stored at −20°C.

Immunohistochemical Staining

The paraffin sections were heated to 56°C for 2 h, followed by antigen retrieval in a microwave on low heat and natural cooling to room temperature. Sections were then washed with 0.1 M phosphate-buffered saline (PBS) buffer, dewaxed, and hydrated, with the washing procedure repeated thrice, each for 3 min. Incubation with an endogenous peroxidase blocker (PV-6000, ZSGB, Beijing, China) for 10 min at room temperature preceded another series of washes in 0.1 M PBS buffer, repeated 3 times, for 3 min each. Next, sections were incubated with 2′, 3′-cyclic nucleotide 3′-phosphodiesterase (CNPase) antibody (1 : 500, Abcam, Cambridge, U.K., Catalog #ab6319), platelet-derived growth factor receptor α (PDGFRα) antibody (1 : 500, Abcam, Catalog #ab203491), and MBP antibody (1 : 1200, CST, Danvers, MA, U.S.A., Catalog #78896S) in a humidified chamber at 4°C overnight. The following day, sections were rewarmed to room temperature for 30 min, washed 3 times with 0.1 M PBS buffer, and incubated with goat anti-mouse/rabbit immunoglobulin G (IgG) polymer (PV-6000, ZSGB) for 20 min at room temperature. Diaminobenzidine solution (ZLL-9018, ZSGB) was applied for 5–8 min for staining. Sections were then dehydrated in ascending alcohol concentrations (70, 80, 90, and 100%) and cleared in xylene before being sealed with neutral gum. Referring to Allen’s Brain Atlas,⁴⁰⁾ to determine the extent of the IL subregion of the mPFC. Observations were made using an Olympus orthotopic fluorescence microscope (Olympus, Tokyo, Japan), and images were analyzed with ImageJ software (National Institutes of Health, Bethesda, Maryland, U.S.A.). For all measurements, 6 mice per group and 3 sections were analyzed per mice.

Immunofluorescence Staining

Frozen sections of brain tissue were submerged in PBS with Triton X-100 (PBST) 2 times for 5 min each. This step was followed by 3 washes in the PBS buffer, each lasting 5 min. Subsequently, the sections underwent antigen retrieval in an antigen repair solution for 30 min, followed by 2 additional 5-min immersions in PBST and three 5-min washes in PBS buffer. The sections were then incubated in a goat serum blocking solution for 1 h at room temperature on a shaker. Following blocking, the sections were rinsed in PBS, placed in primary antibody solution (anti-oligodendrocyte transcription factor 2 (Olig2) antibody, CST, Catalog #65915S; anti-CC-1 antibody, Merck-Millipore, Burlington, MA, U.S.A., Catalog #OP80; anti-PDGFRα antibody, Merck-Millipore, Catalog #OP80; anti-NG2 antibody, Sigma-Aldrich, Catalog #MAB5384-I; anti-p-Erk1/2 antibody, CST, Catalog #9524) for co-staining, incubated overnight at 4°C, and then rewarmed for 30 min at room temperature on a shaker. After rewarming, the sections were washed 2 times in PBST and 3 times in PBS, each for 5 min. The sections were then incubated with secondary antibodies (Alexa Fluor 488 Goat Anti-Rabbit IgG antibody, Invitrogen, Waltham, MA, U.S.A., Catalog #A-11008; Cy3–conjugated Affinipure Goat Anti-Mouse antibody, Proteintech, Rosemont, IL, U.S.A., Catalog #SA00009-1; Alexa Fluor 555 Donkey Anti-rabbit antibody, Abcam, Catalog #ab150074; Alexa Fluor 488 Goat Anti-Rat IgG antibody, Invitrogen, Catalog #A-11006) for 1 h in conditions protected from light. Following secondary antibody incubation, the sections were washed 2 times in PBST and 3 times in PBS, each for 5 min, under light-protected conditions, mounted on slides, and air-dried to a slightly damp state. DAPI-containing antifade was applied, and the slides were cover-slipped. Referring to the Allen’s Brain Atlas,⁴⁰⁾ to determine the extent of the IL subregion of the mPFC. Representative images were captured using an Olympus orthotopic fluorescence microscope (Olympus), and image analysis was conducted using ImageJ software. For all measurements, 6 mice per group and 3 sections were analyzed per mice.

Statistical Analysis

Statistical analysis was performed using SPSS 26.0 software, with all data presented as mean ± standard error of the mean (S.E.M.). Data were initially subjected to tests for normality and homogeneity of variance. If these criteria were met, comparisons between 2 groups were made using the Student’s t-test, and comparisons among multiple groups utilized one-way ANOVA followed by Tukey’s post hoc test. A p-value <0.05 was considered statistically significant.

RESULTS

Establishment of a GDM Model

Db/m mice were fed a high-fat diet from 7 d before mating until the birth of their offspring to simulate abnormal glucose tolerance during pregnancy. To verify the successful establishment of the GDM model, fasting blood glucose levels were measured in db/m mice at E0, E10, and E20, respectively. Referring to previous studies, mice with fasting blood glucose ≥11.1 mmol/L can be diagnosed as diabetes mellitus.^41,42) As shown in Fig. 1, fasting blood glucose in db/m mice was 4.8 ± 0.7 mmol/L at E0 and increased significantly to 11.5 ± 0.3 mmol/L by E10, meeting the diagnostic criteria for diabetes mellitus. By E20, fasting blood glucose further increased to 16.2 ± 0.2 mmol/L. These results confirmed the successful establishment of a GDM model using db/m mice on a C57BL/6J background with a high-fat diet, providing a basis for subsequent analysis of GDM offspring.

Fig. 1. Fasting Blood Glucose in db/m Mice during Pregnancy

Fasting blood glucose in db/m mice at E0, E10, and E20 (E0: 4.8 ± 0.2 mmol/L; E10: 11.5 ± 0.3 mmol/L; E20: 16.2 ± 0.2 mmol/L). Data are expressed as mean ± S.E.M. (n = 12 samples per group).

Effect of QUE on CPP Expression following Alcohol Exposure

Figure 2A showed that the initial residence time in the non-preferred compartment was essentially similar across the 7 groups of mice (one-way ANOVA, F(6, 77) = 1.043, p = 0.405). Following alternating alcohol and saline injections during the conditioning phase, the GDM + ETOH, GDM + ETOH + SAL, GDM + ETOH + QUE, and GDM + ETOH + U0126 groups showed a substantial increase in residence time within the non-preferred compartment at the posttest (p < 0.001, p < 0.001, p < 0.001, and p < 0.001). In contrast, no significant change in residence time was observed in the GDM + SAL, GDM + SAL + QUE, and GDM + SAL + U0126 groups (p = 0.056, p = 0.057, and p = 0.201).

Fig. 2. Effect of QUE on CPP Expression Following Alcohol Exposure in GDM Offspring Mice

(A) Time spent in the non-preferred compartment in pretest and posttest (before and after the conditioning phase), ^##p < 0.01, time spent in the non-preferred compartment in posttest compared with pretest of all 7 groups. (B) Time difference, **p < 0.01, compared with the GDM + SAL group, ⁺⁺p < 0.01, compared with the GDM + ETOH group. Data are expressed as mean ± S.E.M. (n = 12 samples per group).

As depicted in Fig. 2B, the time difference following alcohol exposure was significantly different across the 7 groups of mice (one-way ANOVA, F(6, 77) = 56.328, p < 0.001). Compared to the GDM + SAL group, the time difference of the GDM + ETOH group significantly increased following alcohol exposure (p < 0.001), whereas the GDM + SAL + QUE and GDM + SAL + U0126 groups showed no significant change in time difference (p = 0.999 and p = 1.000). Compared with the GDM + ETOH group, the GDM + ETOH + QUE group exhibited a substantial reduction in time difference following QUE intervention (p < 0.001). Similarly, the GDM + ETOH + U0126 group showed a substantial decrease in time difference following ERK inhibitor U0126 intervention (p < 0.001), while the GDM + ETOH + SAL group showed no significant change (p = 0.874).

Effect of QUE on the Number of Mature Oligodendrocytes and Myelin Expression in the IL Subregion following Alcohol Exposure

The functionality of the brain is intimately linked to the integrity of myelin and mature oligodendrocytes with myelin-forming capabilities, which are abundant in the central nervous system (CNS).^43,44) Using myelin basic protein (MBP) and CNPase as markers, we evaluated changes in myelination and mature oligodendrocytes within the IL region following alcohol exposure in GDM offspring mice.^45,46)

Figures 3A and 3B indicated that the average optical density (AOD) of myelin in the IL region was significantly different in the 7 groups of mice following alcohol exposure (one-way ANOVA, F(6, 119) = 3.724, p = 0.002). Compared to the GDM + SAL group, there was no significant change in the AOD of myelin in the IL region of the GDM + ETOH, GDM + SAL + QUE and GDM + SAL + U0126 groups (p = 0.671, p = 0.988, and p = 1.000). In contrast, compared with the GDM + ETOH group, the AOD of myelin was significantly restored in the GDM + ETOH + QUE group following QUE intervention (p = 0.003). No significant change was observed in the GDM + ETOH + U0126 group and the GDM + ETOH + SAL group (p = 0.065 and p = 1.000).

Fig. 3. Effect of QUE on Myelin Expression in the IL Subregion Following Alcohol Exposure in GDM Offspring Mice

(A) Immunohistochemical staining of myelin in the IL region of the GDM + SAL, GDM + ETOH, GDM + ETOH + SAL, GDM + ETOH + QUE, GDM + ETOH + U0126, GDM + SAL + QUE, and GDM + SAL + U0126 groups. Scale bar = 20 μm. (B) Histogram of AOD value of MBP in the IL region of all 7 groups. ⁺⁺p < 0.01, compared with the GDM + ETOH group. Data are expressed as mean ± S.E.M. (n = 18 images, from 6 mice per group).

Figures 4A and 4B indicated that the number of CNPase⁺ cells in the IL region was significantly different in the 7 groups of mice following alcohol exposure (one-way ANOVA, F(6, 119) = 16.493, p < 0.001). Compared to the GDM + SAL group, the number of CNPase⁺ cells in the IL region significantly decreased in the GDM + ETOH group (p < 0.001), whereas there was no significant difference in the number of CNPase⁺ cells in the GDM + SAL + QUE and GDM + SAL + U0126 groups (p = 0.999 and p = 0.912). Compared with the GDM + ETOH group, the number of CNPase⁺ cells significantly increased in the GDM + ETOH + QUE and GDM + ETOH + U0126 groups (p < 0.001 and p < 0.001), with no significant change in the GDM + ETOH + SAL group (p = 0.969).

Fig. 4. Effect of QUE on the Number of Mature Oligodendrocytes in the IL Subregion Following Alcohol Exposure in GDM Offspring Mice

(A) Immunohistochemical staining of CNPase⁺ cells in the IL region of the GDM + SAL, GDM + ETOH, GDM + ETOH + SAL, GDM + ETOH + QUE, GDM + ETOH + U0126, GDM + SAL + QUE, and GDM + SAL + U0126 groups. Scale bar = 20 μm. (B) Histogram of the number of CNPase⁺ cells per unit area in the IL region of all 7 groups. **p < 0.01, compared with the GDM + SAL group. ⁺⁺p < 0.01, compared with the GDM + ETOH group. Data are expressed as mean ± S.E.M. (n = 18 images, from 6 mice per group).

Effect of QUE on the Number of OPCs in the IL Subregion following Alcohol Exposure

The CNS is known to harbor a significant number of OPCs, which constitute a major portion of the cell population.^47,48) These OPCs are capable of proliferating, migrating, and differentiating into mature oligodendrocytes in vivo, thereby contributing to myelin formation.⁴⁹⁾ PDGFRα) serves as a specific marker for OPCs and reflects changes in their quantities.⁵⁰⁾ Consequently, we investigated the impact of QUE on the number of OPCs using immunohistochemical staining.

Results illustrated in Fig. 5 revealed that the number of PDGFRα⁺ cells in the IL region was significantly different across the 7 groups of mice following alcohol exposure (one-way ANOVA, F(6, 119) = 5.142, p < 0.001). Compared to the GDM + SAL group, the number of PDGFRα⁺ cells in the IL region after alcohol exposure was significantly reduced in the GDM + ETOH group (p = 0.030), whereas there was no significant difference in the number of PDGFRα⁺ cells of the GDM + SAL + QUE and GDM + SAL + U0126 groups (p = 0.950 and p = 0.996). Additionally, compared with the GDM + ETOH group, there was no significant difference in the number of PDGFRα⁺ cells in the IL region of the GDM + ETOH + QUE group treated with QUE and the GDM + ETOH + U0126 group treated with U0126 (p = 0.974 and p = 0.932). The GDM + ETOH + SAL group also showed no significant difference (p = 0.999).

Fig. 5. Effect of QUE on the Number of OPCs in the IL Subregion Following Alcohol Exposure in GDM Offspring Mice

(A) Immunohistochemical staining of PDGFRα⁺ cells in the IL region of the GDM + SAL, GDM + ETOH, GDM + ETOH + SAL, GDM + ETOH + QUE, GDM + ETOH + U0126, GDM + SAL + QUE, and GDM + SAL + U0126 groups. Scale bar = 20 μm. (B) Histogram of the number of PDGFRα⁺ cells per unit area in the IL region of all 7 groups. *p < 0.05, compared with the GDM + SAL group. Data are expressed as mean ± S.E.M. (n = 18 images, from 6 mice per group).

Effect of QUE on the Differentiation of OPCs in the IL Subregion following Alcohol Exposure

QUE may promote the differentiation of OPCs by regulating the cell cycle and cellular morphological transformation.⁵¹⁾ Olig2 is expressed throughout the entire process of oligodendrocyte differentiation and maturation and has been proven to be a suitable marker for oligodendrocyte lineage cells.^52,53) Cyclic nucleotide phosphodiesterase 1 (CC1) is highly expressed in mature oligodendrocytes, and CC1 antibodies are often used to characterize mature oligodendrocytes.⁵⁴⁾ PDGFRα is one of the markers specifically expressed by OPCs.⁵⁵⁾ Therefore, we used the immunofluorescence double-labeling technique to assess OPC differentiation. The ratio of CC1⁺/Olig2⁺ cells to Olig2⁺ cells reflects the proportion of mature oligodendrocytes, and the ratio of PDGFRα⁺/Olig2⁺ cells reflects the proportion of immature oligodendrocytes.

Figure 6 shows that the ratio of CC1⁺/Olig2⁺ cells to Olig2⁺ cells in the IL region was significantly different among the 7 groups of mice following alcohol exposure (one-way ANOVA, F(6, 119) = 9.585, p < 0.001). Compared to the GDM + SAL group, the ratio of CC1⁺/Olig2⁺ cells to Olig2⁺ cells in the IL region post-alcohol exposure was significantly reduced in the GDM + ETOH group (p = 0.001), whereas the GDM + SAL + QUE and GDM + SAL + U0126 groups showed no significant change in the ratio of CC1⁺/Olig2⁺ cells to Olig2⁺ cells (p = 0.966 and p = 0.992). Compared with the GDM + ETOH group, the ratio of CC1⁺/Olig2⁺ cells to Olig2⁺ cells in the IL region was significantly restored in the GDM + ETOH + QUE group treated with QUE and the GDM + ETOH + U0126 group treated with U0126 (p = 0.030 and p = 0.025). No significant restoration was noted in the GDM + ETOH + SAL group (p = 0.994).

Fig. 6. Effect of QUE on the Proportion of Mature Oligodendrocytes in the IL Subregion Following Alcohol Exposure in GDM Offspring Mice

(A) Immunofluorescence staining of CC1⁺/Olig2⁺ cells in the IL region of the GDM + SAL, GDM + ETOH, GDM + ETOH + SAL, GDM + ETOH + QUE, GDM + ETOH + U0126, GDM + SAL + QUE, and GDM + SAL + U0126 groups. Scale bar = 20 μm. (B) Histogram of the ratio of CC1⁺/Olig2⁺ cells to Olig2⁺ cells per unit area in the IL region of all 7 groups. **p < 0.01, compared with the GDM + SAL group, ⁺p < 0.05, compared with the GDM + ETOH group. Data are expressed as mean ± S.E.M. (n = 18 images, from 6 mice per group).

Additionally, Fig. 7 shows that the ratio of PDGFRα⁺/Olig2⁺ cells to Olig2⁺ cells in the IL region was significantly different among the 7 groups of mice following alcohol exposure (one-way ANOVA, F(6, 119) = 9.003, p < 0.001). Compared to the GDM + SAL group, the ratio of PDGFRα⁺/Olig2⁺ cells to Olig2⁺ cells in the IL region post-alcohol exposure significantly increased in the GDM + ETOH group (p < 0.001), whereas the GDM + SAL + QUE and GDM + SAL + U0126 groups showed no significant change in the ratio of PDGFRα⁺/Olig2⁺ cells to Olig2⁺ cells (p = 0.997 and p = 1.000). Compared with the GDM + ETOH group, the ratio was significantly reduced in the IL region of the GDM + ETOH + QUE group following QUE intervention (p < 0.001). A similar reduction was observed in the GDM + ETOH + U0126 group following U0126 intervention (p < 0.001). No significant change was observed in the GDM + ETOH + SAL group (p = 0.657).

Fig. 7. Effect of QUE on the Proportion of Immature Oligodendrocytes in the IL Subregion Following Alcohol Exposure in GDM Offspring Mice

(A) Immunofluorescence staining of PDGFRα⁺/Olig2⁺ cells in the IL region of the GDM + SAL, GDM + ETOH, GDM + ETOH + SAL, GDM + ETOH + QUE, GDM + ETOH + U0126, GDM + SAL + QUE, and GDM + SAL +U0126 groups. Scale bar = 20 μm. (B) Histogram of the ratio of PDGFRα⁺/Olig2⁺ cells to Olig2⁺ cells per unit area in the IL region of all 7 groups. **p < 0.01, compared with the GDM + SAL group, ⁺⁺p < 0.01, compared with the GDM + ETOH group. Data are expressed as mean ± S.E.M. (n = 18 images, from 6 mice per group).

Effect of QUE on ERK1/2 Signaling in the IL Subregion following Alcohol Exposure

ERK, as a key molecule in signaling, mainly includes ERK1 and ERK2. p-ERK, the active form of ERK, is directly involved in the regulation of a variety of biological processes.^56,57) Therefore, the level of p-ERK can reflect the activation state of ERK signaling pathway. In the CNS, chondroitin sulfate proteoglycan (NG2) is mainly expressed by OPCs.^50,58) To explore the impact of alcohol exposure on the ERK1/2 signaling pathway in OPCs within the IL region of the mPFC in GDM offspring mice and to evaluate the effect of QUE intervention on this pathway, we quantified the number of p-ERK⁺/NG2⁺ cells per unit area using double-labeled immunofluorescence staining.

Figure 8 shows that the number of p-ERK⁺/NG2⁺ cells in the IL region was significantly different among the 7 groups of mice following alcohol exposure (one-way ANOVA, F(6, 119) = 7.136, p < 0.001). Compared to the GDM + SAL group, the number of p-ERK⁺/NG2⁺ cells in the IL region post-alcohol exposure slightly increased in the GDM + ETOH group, but there was no statistically significant difference (p = 0.119), and there was no significant difference in the number of p-ERK⁺/NG2⁺ cells in the GDM + SAL + QUE and GDM + SAL + U0126 groups (p = 0.426 and p = 0.228). Compared with the GDM + ETOH group, the number of p-ERK⁺/NG2⁺ cells in the IL region was significantly reduced in the GDM + ETOH + QUE group treated with QUE and the GDM + ETOH + U0126 group treated with U0126 (p = 0.029 and p = 0.048). No significant change was observed in the GDM + ETOH + SAL group (p = 0.998).

Fig. 8. Effect of QUE on ERK1/2 Signaling in the IL Subregion Following Alcohol Exposure in GDM Offspring Mice

(A) Immunofluorescence staining of p-ERK⁺/NG2⁺ cells in the IL region of the GDM + SAL, GDM + ETOH, GDM + ETOH + SAL, GDM + ETOH + QUE, GDM + ETOH + U0126, GDM + SAL + QUE, and GDM + SAL + U0126 groups. Scale bar = 20 μm. (B) Histogram of the number of p-ERK⁺/NG2⁺ cells per unit area in the IL region of all 7 groups. ⁺p < 0.05, compared with the GDM + ETOH group. Data are expressed as mean ± S.E.M. (n = 18 images, from 6 mice per group).

DISCUSSION

Our findings showed that QUE intervention inhibited the over-activation of the ERK1/2 signaling pathway in the IL region following alcohol exposure in GDM offspring mice, promoted the differentiation of OPCs, restored the number of mature oligodendrocytes and myelin expression, and ultimately improved CPP expression.

Our findings on CPP expression indicated that alcohol exposure significantly induced CPP in GDM offspring mice, while QUE or ERK inhibitor U0126 treatment effectively mitigated CPP expression following alcohol exposure. Echoing previous research, Khezri et al. found that QUE mitigates morphine-induced CPP acquisition,³³⁾ and when co-administered with (+)-amphetamine, it reduces CPP expression in rats.²⁶⁾ These results suggest QUE’s positive influence on the reward circuitry associated with CPP behavior, potentially counteracting the dysregulated behaviors linked to alcohol exposure in GDM offspring.

Further analysis revealed that alcohol exposure notably impacted the number of mature oligodendrocytes and myelination in the IL region of GDM offspring, while QUE or U0126 intervention significantly restored the number of mature oligodendrocytes and myelination. Aligning with prior studies that reported acute alcohol exposure during adolescence disrupts myelination in the mPFC of rats.^12,59) Kim et al. showed that chronic alcohol exposure affects the proliferation, differentiation, and survival of OPCs in the mPFC of Wistar rats, which in turn reduces MBP expression.⁶⁰⁾ QUE treatment promoted the differentiation and maturation of OPCs and somewhat restored the number of mature oligodendrocytes and myelination.²³⁾ Overall, our results underscored the protective effect of QUE intervention on mature oligodendrocytes and myelination.

Our study also discovered that alcohol exposure, while increasing the ratio of OPCs to the total oligodendrocyte population and decreasing the ratio of mature oligodendrocytes, paradoxically led to a reduction in the quantity of OPCs per unit area within the IL region of GDM offspring. Both QUE and U0126 interventions promoted OPC differentiation while causing a slight increase in the quantity of OPCs per unit area within the IL region, which consequently exerted a regulatory and functional improvement effect. The apparent discrepancy between the quantity of OPCs and the trend in OPC differentiation may be attributed to several factors: First, the proliferative capacity of OPCs in the IL region and apoptosis must be considered, as differentiation of OPCs is suppressed post-alcohol exposure in GDM offspring. For instance, studies have indicated that cytotoxicity due to elevated pro-inflammatory cytokines following alcohol exposure can lead to apoptosis.^61,62) Newville et al. also showed that alcohol exposure not only affects myelin composition and structure but may also impair OPCs by diminishing their proliferation.⁶³⁾ Second, the duration of alcohol exposure experienced by the GDM offspring in our study might have been too brief to trigger a compensatory proliferation of OPCs for repair. Previous research demonstrated that alcohol exposure in the first 2 weeks of life in mouse pups results in a substantial reduction, approximately 75%, in the number of OPCs, with proliferating OPCs returning to baseline levels by the 50th day post-birth.⁶³⁾ Third, brain regions do not uniformly respond to external stimuli, exhibiting notable heterogeneity.

As a key component of the MAPK family, ERK1/2 is phosphorylated and activated in response to external stimuli such as growth factors, hormones, and neurotransmitters, playing a pivotal role in cell proliferation and differentiation.^64,65) The effects of QUE on the ERK1/2 signaling pathway have been variedly reported. Khezri et al. observed that QUE inhibited ERK1/2 phosphorylation in the hippocampus and cerebral cortex, diminishing morphine-induced CPP acquisition.³³⁾ Lee et al. found that QUE could reduce liver cancer cell survival and invasion by inducing apoptosis and reducing ERK/Akt-mediated anti-apoptotic and metastatic protein expression.⁶⁶⁾ Xiao et al. reported that QUE promotes oligodendrocyte differentiation through ERK1/2 phosphorylation.²⁴⁾ Our findings indicated that exposure to alcohol in GDM offspring led to the activation of the ERK1/2 signaling pathway in OPCs within the IL region of mice, resulting in a reduced ratio of mature oligodendrocytes to total oligodendrocytes and an elevated ratio of OPCs to total oligodendrocytes. These findings suggested that alcohol exposure activates the ERK1/2 signaling pathway, preventing OPC differentiation in the IL region, whereas QUE treatment promotes OPC differentiation by inhibiting the ERK1/2 pathway. Previous research found that a 2-week oral treatment with QUE significantly improved anxiety-like behavior, learning, and spatial memory deficits in rats exposed to the enhanced single prolonged stress, while also increasing pERK1/2 expression in the prefrontal cortex, medial amygdala, and cingulate gyrus.⁶⁷⁾ Evidently, our results are not consistent with those reported in previous studies. This discrepancy may stem from several factors: First, our research focused on the IL region in GDM offspring exposed to alcohol, contrasting with previous studies^24,67) that examined neural progenitor cells from the fetal rat cortex of Sprague-Dawley rats and different brain regions. Such variations in study regions and comparison methodologies may account for the differing outcomes. Second, our study utilized a mouse model of GDM offspring with concurrent alcohol exposure, where both intrauterine high glucose and alcohol exposure during pregnancy are known to potentially induce ERK activation,^68,69) suggesting that the ERK1/2 pathway may have been overly activated in our model’s IL region, with QUE treatment reverting the pathway to its normal activation state and thus promoting OPC differentiation. Lastly, the differences in findings could be attributed to variations in the experimental model, administration route, dosage and duration of QUE treatment, and comparison methods.

We must consider its association with GDM leading to epigenetic changes in the genes of offspring mice when discussing the results of the current study. Previous studies have shown that altered DNA methylation patterns in the genome of GDM offspring are strongly associated with neonatal hypoglycemia.^70,71) Ding et al. demonstrated that intrauterine hyperglycemic exposure affected methylation changes in adult spermatozoa of the first generation, altering hippocampus development of the second generation and leading to cognitive dysfunction.⁷²⁾ Therefore, our findings may also be relevant to GDM-induced epigenetic changes in offspring mice. This association not only reveals the potential long-term effects of GDM on offspring but also provides new directions and perspectives for future research.

In addition, our study faces certain limitations. Primarily, we utilized only db/m mice on a C57BL/6J background combined with high-fat feed to establish a GDM model. To ensure the reliability and accuracy of the results, validation in another animal model (e.g., drug-induced establishment of GDM) or further clinical settings is required. Second, in this study, we referred to the QUE dose (10 mg/kg/d) used in previous studies,²⁵⁾ which is equivalent to a human dose of 1.1 mg/kg/d (based on 70 kg/person) according to the equivalent dose factor discounting, without investigating the influence of dose-dependent curves and exploring the optimal use of QUE. Although this concentration achieved significant effects under experimental conditions, we recognize that it may differ from the actual concentration used in human treatment. Therefore, when interpreting these experimental results, we need to be cautious about their potential significance for human diseases. Third, we only investigated the ERK1/2 signaling pathway within OPCs in the IL region of GDM offspring mice, which does not imply that QUE specifically regulates the ERK1/2 signaling pathway to play an ameliorative role. Moreover, we have investigated indicators such as the number of oligodendrocyte lineage cells and myelination in the IL subregion of GDM offspring, and have not yet explored alterations in other brain regions or neurodevelopmental indicators, such as neurogenesis, deletion or death, and synapse formation.

CONCLUSION

In conclusion, our study revealed that QUE treatment promotes OPC differentiation and maturation by inhibiting the ERK1/2 signaling pathway within the IL subregion, restoring the number of mature oligodendrocytes and myelin expression, and significantly improving CPP expression in GDM offspring mice following alcohol exposure. Future research should continue to validate the effectiveness and mechanisms of QUE and assess its clinical applicability in additional animal models and human subjects. Additionally, a comprehensive understanding of QUE’s precise mechanisms, effect pathways, optimal dosage, and treatment duration requires further investigation. The inclusion of these research dimensions will provide us with more detailed and comprehensive insights to advance neurodevelopmental protection and therapeutic strategies for GDM offspring.

Acknowledgments

We appreciate the graphical abstract drawn by Figdraw (www.figdraw.com).

Funding

This study was supported by the Chongqing Natural Science Foundation of China (No. CSTC2021jcyj-msxmX0249) and the National Natural Science Foundation of China (No. 81770806).

Author Contributions

All authors participated in the editing and revision of the manuscript. All authors have read and approved the final manuscript. All authors have fully participated in the work and agree to be responsible for all aspects of the work. Dong Huang: conceptualization, investigation, data curation, formal analysis, visualization, and writing—original draft. Maolin Li: data curation and writing—review and editing. Zhifei Qiao: investigation and data curation. Hongli Zhou: conceptualization and methodology. Zuo Zhang: methodology and project administration. Jiyin Zhou: conceptualization, supervision, funding acquisition, resources and writing—review and editing.

Conflict of Interest

The authors declare no conflict of interest.

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