Animal experiments

The experimental protocol was approved by the Laboratory Animal Center of the Third Xiangya Hospital, Central South University (Ethics number: CSU-2023–0244). A total of Sixteen 9-week-old Sprague-Dawley rats were selected, including 8 male and 8 female rats. The experimental animals were placed in a temperature-controlled room with a 12-hour light/dark cycle, and after 7 days of acclimation to the laboratory environment, the male and female rats were placed in a single cage in a 1:1 ratio, and the day when the vaginal plug appeared was designated as the first day of pregnancy.

The pregnant rats were randomly assigned to either the IUGR group or the control (CON) group using a random number generator. We induced IUGR models using maternal calorie and protein restriction, which has been previously validated [18]. In the IUGR group, pregnant rats were fed a 10% protein diet throughout gestation (protein-restricted diet), while those in the CON group received a 21% protein (normal) diet. Both groups received the 21% protein diet during lactation (S1 Table).

There was no difference in litter size between control and food-restricted pregnancies (15.8 ± 1.0 vs 16.0 ± 0.8 pups/litter). Offspring with a birth weight lower than two standard deviations from the mean of the CON group were classified as IUGR. The incidence rate of IUGR in control and IUGR groups was 1.6% and 85%, respectively. To investigate the effects of postnatal FA intervention, a subset of the offspring received a diet supplemented with FA, establishing four experimental groups for study: CON, CON + FA, IUGR, and IUGR+FA. Offspring in the IUGR+ folic acid (IUGR+FA) group and the CON + FA group were fed with folic acid 5 mg/kg for 3 weeks after weaning at 21 days, and then fed with a normal diet for 3 months (Also see S2A Fig). FA supplementation was initiated at weaning (postnatal day 21) to model an early postnatal intervention during a period of high developmental plasticity, and the timing and dose (5 mg/kg) were selected based on previous studies [14].

The rats were monitored at least twice daily by trained personnel for health status and behavioral changes. Parameters for assessment included, but were not limited to: general activity levels, food and water consumption, coat condition, and any signs of distress (such as vocalization, self-isolation, or aggressive behavior).

If rats exhibited any of the following irreversible and severe signs of distress that could not be alleviated through intervention, such as weight loss exceeding 20% of initial body weight, inability to eat or drink voluntarily, or the development of severe neurological symptoms or organ failure, euthanasia was performed immediately in accordance with the method described below. All other offspring were euthanized at the planned experimental endpoints: postnatal day (P) 1, P7, P21, P60, and P90. At these time points, offspring were administered a deep anesthetic overdose via intraperitoneal injection of pentobarbital sodium at a dose of 150 mg/kg. This ensured the animals rapidly reached a state of deep anesthesia and pain insensibility. Death was confirmed by the absence of corneal reflex, loss of response to toe/tail pinch, and cessation of respiration, followed by cervical dislocation as a secondary physical method of euthanasia. The liver index was calculated as (liver weight / body weight) × 100%. To minimize procedural stress, all interventions were performed in a quiet environment by personnel trained in humane animal handling techniques.

Histological analysis

The liver tissues were embedded in paraffin and 5 μM sections. Paraffin-embedded sections were heated at 60°C for 2 hours and dewaxed by xylene, followed by serial rehydration steps (100% ethanol, 80% ethanol, 50% ethanol; each 5 min). Then, sections were stained with hematoxylin and eosin using a standard staining technique. Finally, images were captured on a fluorescent microscope (Olympus BX5, Tokyo, Japan).

ELISA

Venous blood samples for rats were drawn and stored in the ethylene diamine tetraacetate acid (EDTA) containing tubes, all the blood samples were centrifuged for 15 minutes at 3000 rpm to gain the plasma. The plasma concentration of free fatty acid (A042-2–1, Nanjing Institute of Biological Engineering) and triglyceride (F001-2, Nanjing Institute of Biological Engineering) were measured respectively according to the manufacturer’s instructions.

Quantitative real-time polymerase chain reaction

The total RNA of the liver was extracted with Trizol reagent (Invitrogen, Shanghai, China), and was reverse transcribed using Superscript II reverse transcriptase kits (both from Invitrogen). Diluted cDNAs were used as templates for qPCR with TAKARA gene expression assays (TAKARA Biotechnology Co., LTD. Dalian, China) and PCR mastermix in ABI7300 thermal cycler according to the manufacturer’s instructions (Applied Biosystems, Foster City, CA). The primer sequences used were shown as follows:

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https://doi.org/10.1371/journal.pone.0346676.t001

Western blot analysis

Livers were homogenized in proteinase inhibitor-containing RIPA lysis solution before being centrifuged at 4 °C for 15 min, and the supernatants were collected. Each sample, containing 20 µg of protein, was boiled in the loading buffer before being transferred to 10% SDS-polyacrylamide gels for electrophoresis and then added to a PVDF membrane.

For blocking 5% dry non-fat milk dissolved in Tris-buffered saline with 0.05% Tween 20 as detergent (TBST) was used. Membranes were then incubated with antibodies (β-actin,1:5000; Acox1,1:1000; Acox3,1:1000; CPT1, 1:1000; PPARα,1:500) at 4 °C overnight, followed by peroxidase-conjugated secondary antibody, or incubated with HRP-conjugated beta-actin monoclonal antibody. Blots were detected by an imaging system (ChemiScope6100, Shanghai, China).

Statistical analysis

The test data were initially processed by SPSS22.0 statistical software (Point Richmond, CA). A Kolmogorov's-Smirnov test was used to test the normality of the data, and all normally distributed continuous variables were described as mean and standard deviation. For comparisons between 2 groups, statistical significance was calculated using Student’s t-test. Differences between the 4 groups were evaluated using one-way ANOVA. For longitudinal data, a repeated-measures ANOVA or mixed-effects model was employed to account for within-subject correlations over time. * P < 0.05 was considered statistically significant.

IUGR induces persistent lipid metabolism dysfunction in offspring

A total of 146 rats were used in this experiment. 141 of them were euthanized and 5 died during the perinatal period. As previously reported, the body weights of the IUGR group were significantly lower compared to the normal group from birth to 21 days (S1 Fig B). After 3 weeks of age, IUGR offspring exhibited catch-up growth, and the differences in body weight were normalized at 60 days after birth (S1 Fig B).

Given that the liver is the principal target organ in response to adverse intrauterine conditions, we then examine the effect of IUGR on the liver. Our results demonstrated that liver weight and liver index levels are decreased in infant IUGR rats compared to control rats, while no significant differences are observed during adolescence (S1 Fig C-D). Consistent with previous studies [19], levels of serum triglycerides (TG) and free fatty acids (FFA) significantly increased in IUGR rats compared to control rats during the early stages. Furthermore, we found that these lipid alterations persisted into adulthood in IUGR rats, at the 90-day age the TG and FFA were significantly higher when compared to controls (TG: 2.9-fold; FFA: 4.1-fold) (S1 Fig E-F). Compared to the normal structure in control rats, H&E staining exhibited enlarged hepatocytes, intercellular spaces, and extensive hepatocellular vacuoles in the liver of IUGR rats at both 1 day and 90 days after birth, indicative of the abnormalities in hepatic lipid metabolism (S1 Fig G).

At 90 days after birth, IUGR downregulated the expression of lipid oxidation-related genes, including PPARα, as well as Carnitine palmitoyl transferase 1 (Cpt1), acyl-CoA oxidase 1 (Acox1) and Acox3, which are known to be components in the regulatory network of fatty acid β-oxidation (PPARα: 0.12-fold; Ctp1:0.10-fold; Acox1:0.18-fold; Acox3: 0.11-fold) (S1 Fig H). These results suggest the IUGR-induced deficiency of the PPARα network and dysfunction of lipid metabolism persists into adulthood.

The impact of early postnatal folic acid supplementation on IUGR-induced lipid metabolic dysfunction in adulthood

To determine if folic acid supplementation in the early postpartum period could counteract IUGR-induced metabolic dysfunction, offspring were fed either a control or folic acid-supplemented diet for 3 weeks after weaning (S2 Fig A). Folic acid intervention did not affect the general appearance or weight of both IUGR and control rats (S2 Fig B). However, folic acid addition acutely reduced serum TG and FFA levels in adult IUGR rats at 60 days and 90 days of age (S2 Fig C-D). The IUGR-induced hepatic changes were reversed by folic acid supplementation. Compared to livers from the IUGR group, livers from the IUGR+FA group showed clear tissue structure and less vacuolization, as revealed by H&E staining (S2 Fig E). These results suggest that folic acid supplementation can mitigate IUGR-induced lipid metabolic dysfunction and hepatic abnormalities in adulthood.

Folic acid upregulates lipid-related genes in IUGR Rats

Then we determined the effect of folic acid on lipid-related genes in the liver. As shown in S3 Fig A-D, early postnatal folic acid supplementation significantly upregulated the expression of lipid-related genes in the liver from adult IUGR rats, including PPARα, CPT1, Acox1 and Acox3, while not affecting control rats (S3 Fig A-D). Correspondingly, the expression levels of these proteins were also significantly upregulated after folic acid treatment (S3 Fig E-I). Furthermore, to explore a potential epigenetic mechanism, we assessed global DNA methylation levels in the liver. IUGR was associated with significantly elevated DNA methylation at 90 days. Postnatal FA supplementation effectively reduced this IUGR-induced hypermethylation in the IUGR+FA group (S3 Fig J, K). Overall, folic acid improves hepatic lipid metabolism by upregulating PPARα levels and β-oxidation related genes.