Pregnant Sprague Dawley (SD) rats, weighing 220 $\pm$ 20 g, were procured from the Animal Experimental Center of Chongqing Medical University (production license number: SCXK (Yu) 2018-0003, usage license number: SYXK (Yu) 2017-0012). All animal procedures were carried out at the Animal Experiment Center of Chongqing Medical University under specific pathogen-free conditions. The experimental protocols involving animals were approved by the Ethics Committee of the Children’s Hospital of Chongqing Medical University (IACUC Issue No: CHCMU-IACUC20221122004).

The female and male rats were paired overnight in a 2:1 ratio (1 male and 2 females), and the females with vaginal plugs observed the following morning were designated as being at day 0.5 of pregnancy. Subsequently, pregnant rats were randomly assigned to one of four groups (n = 5 each): the control group (pregnant rats received no specific intervention, and offspring were administered the same volume of physiological saline on the 1st and 3rd weeks post-birth); the PH group (pregnant rats were exposed to hypoxia for 6 h on the 17th day of pregnancy, and offspring received the same volume of physiological saline on the 1st and 3rd weeks post-birth); the PH and PX-478 group (PH+1 w inhibitor; pregnant rats exposed to hypoxia for 6 h on the 17th day of pregnancy, and offspring were orally administered PX-478 (B6004, APExBIO, Houston, TX, USA; 30 mg/kg, continuously for one week) starting at one week post-birth); and the PH and PX-478 administration at 3rd-week groups (PH+3 w; pregnant rats were exposed to hypoxia for 6 h on the 17th day of pregnancy, and offspring were orally administered PX-478 (30 mg/kg, continuously for one week) starting at the 3rd week post-birth).

According to a previous study [11], a hypoxic box containing padding, food, and water was prepared in advance for PH treatment. Then, the 17-day pregnant rats were placed in the hypoxic box, and the nitrogen and oxygen mixture gas (10% O₂ and 90% N₂) was pumped into the box. Following air removal, an oxygen concentration detector (CY-12C, Jinan Jincheng Security Equipment Co., Ltd, Jinan, Shandong, China) was used to monitor the oxygen concentration in the hypoxic box, which was maintained at 10%. The pregnant rats stayed in a hypoxia box for 6 h.

At the 6th week post-birth, behavioral tests were conducted on offspring from each group. Five offspring were randomly chosen from each group, comprising 2 males and 3 females. Behavioral tests were conducted between 9:00 and 10:00 in the morning. With the exception of the open field test, all other experiments required a one-day adaptation period before commencement. Following the completion of each behavioral experiment, rats were given a 24-hour rest period.

According to previous study [15], a spherical toy was positioned inside the toy box, while untreated offspring of matching age and sex were placed within an unfamiliar mouse box. Subsequently, the offspring were positioned in the middlebox. The durations of playtime for the offspring in both the unfamiliar mouse box and toy box were recorded.

According to previous study [16], a cubic open field box constructed of gray wood (100 cm in length, 100 cm in width, and 45 cm in height) was utilized for the test. The bottom of the open field box was partitioned into 25 square grids delineated by black lines, each measuring 20 $\times$ 20 cm. A camera was positioned directly above the open field box. Throughout the experiment, the animals were situated in the center grid at the bottom of the box, and a 5-minute video recording was initiated. During this recording, the duration of self-grooming and the time spent in the central area by the offspring were observed and recorded.

According to previous study [17], the target box was positioned at the entrance of the experimental platform and was appropriately marked for subsequent examination. Offspring were gently situated at the center of the platform. If an offspring failed to locate the target box within 1 minute, they were gently guided to the target box and allowed to remain there for 15 seconds. Conversely, if an offspring successfully located the target box within the specified time, they were permitted to remain inside for 15 seconds. Offspring underwent training twice daily, and after four days of training, they were subjected to the assessment. The activity of the offspring within a 4-minute period was recorded without any guidance. Finally, the number of trial and error attempts and the latency to escape of the offspring were tallied.

After eight weeks, euthanasia of the offspring was performed using an overdose of pentobarbital sodium injections (30–50 mg/kg; 230301, New Asia Pharma, Shanghai, China), as described in a prior study [18]. Subsequently, hippocampus and blood samples were collected from all animals. For serum separation, blood samples underwent centrifugation at 2500 rpm for 10 minutes, and the resulting supernatant was collected [19]. The VEGF, alkaline phosphatase (ALP), and aspartate transaminase (AST) levels in the serum were determined using a commercial kit (Nanjing Jiangcheng Bioengineering Institute, Nanjing, China) following the manufacturer’s instructions.

The hippocampi were fixed in 4% paraformaldehyde (Sigma-Aldrich, St. Louis, MO, USA) at room temperature for 48 hours, followed by conventional paraffin embedding and sectioning. Subsequently, the paraffin sections were dewaxed and rehydrated using xylene (Sigma-Aldrich, St. Louis, MO, USA) and ethanol (Sigma-Aldrich, St. Louis, MO, USA). Hematoxylin and eosin (HE; 023J5431, Sigma-Aldrich, St. Louis, MO, USA) staining was conducted, and the sections were sealed with neutral gum. The structure of the hippocampus was observed under an optical microscope (KB0801003, Olympus, Tokyo, Japan) in each group.

The proteins from the hippocampus were extracted using radioimmunoprecipitation lysis buffer (RIPA; P0013B, Beyotime, Shanghai, China). In brief, hippocampus were transferred to pre-cooled microtubes containing an appropriate volume of chilled RIPA buffer. Tissue disruption was achieved using a gentle tissue homogenizer or motorized pestle under continuous cooling. Then, the homogenate was incubated on ice for 30 minutes. Gentle agitation during this period facilitated the release of proteins from subcellular compartments. Post-lysis, samples were centrifuged at 14,000 rpm for 15 minutes at 4 °C to pellet insoluble debris and intact organelles. The resultant supernatant, containing the soluble protein fraction, was carefully aspirated. Following protein concentration determination with the BCA protein colorimetric assay kit (P0009, Beyotime, Shanghai, China), the proteins were separated on Sodium dodecyl sulfate (SDS)-polyacrylamide gels (MP10W12, Sigma-Aldrich, USA) and transferred to polyvinylidene fluoride (PVDF) membranes (03010040001, Sigma-Aldrich, USA). Subsequently, the membranes were blocked with 5% bovine serum albumin (BSA) blocking buffer (V900933, Sigma-Aldrich, USA) and then incubated with primary antibodies (anti-PTEN and HIF-1$\alpha$) (Abcam Limited, Cambridge, UK) overnight at 4 °C. Afterward, the membranes were incubated with secondary antibodies at 37 °C for 1 hour. The protein bands were visualized using the Enhanced Chemiluminescence (ECL) kit (P0018S, Beyotime, Shanghai, China), with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) utilized as the housekeeping control. The original figures of Western Blot can be found in the Supplementary Materials.

SPSS 25.0 (IBM Corp., Chicago, IL, USA) and GraphPad Prism 8.0 software (GraphPad Software, Inc., San Diego, CA, USA) were used to analyze and plot the experimental data. The obtained data were described by mean $\pm$ standard deviation ($\overline{x}$ $\pm$ SD). One-way analysis of variance (ANOVA) was used to analyze the results of the weight, behavior tests T-test, VEGF, ALP, and AST levels, and the protein levels of PTEN and HIF-1$\alpha$. p 0.05 is statistically significant.

The body weights of offspring rats in each group were analyzed initially, and the results are shown in Fig. 1A,B. The results reveal that offspring in the PH, PH+1 w, and PH+3 w inhibitor groups exhibited significantly lower body weights compared to the control group. Furthermore, offspring in the PH+1 w and PH+3 w inhibitor groups showed a significant decrease in body weight compared to the PH group. These findings suggest that both PH and the HIF-1$\alpha$ inhibitor influenced offspring growth, with the inhibitor exacerbating weight loss. However, after seven weeks, there was no significant difference in body weight among offspring in the four groups (Fig. 1C), indicating that PX-478-treated offspring could catch up to normal levels in the later stages of growth. Additionally, PX-478 treatment at one week significantly increased the ALP and Alanine aminotransferase (ALT) levels (Fig. 1D,E) in offspring, whereas treatment at three weeks showed no effect on ALP levels (Fig. 1D,E). These results suggest that PX-478 treatment may induce liver function damage and impact physical development in the early stages of offspring, although they can normalize in the later stages of growth.

Fig. 1.

Effects of PX-478 on the growth of offspring rats. (A) Weight of offspring rats at four weeks. (B) Trend of changes in body weight of rats over four weeks. (C) Weight of offspring rats at seven weeks. (D) ALP levels in the serum of offspring rats. (E) Alanine aminotransferase (ALT) levels in the serum of offspring rats. The significance of differences between groups was analyzed using the one-way ANOVA test. ns, p $>$ 0.05, *p 0.05, ***p 0.001, ****p 0.0001. PH, prenatal hypoxia; ALP, alkaline phosphatase; ANOVA, analysis of variance.

The HE staining results (Fig. 2) revealed that in the control group, brain tissue cells exhibited orderly and continuous arrangement with intact cell morphology. In contrast, brain tissue cells of mice in the PH group displayed disordered arrangement, nuclear pyknosis and disappearance, blurred nuclear structure, vacuoles, and necrotic cells in the cytoplasm. However, these pathological changes were alleviated after treatment with PX-478, suggesting that PH could impair neural cell development in offspring brain, and early inhibitor administration post-birth may mitigate PH-induced damage to hippocampal neurons in offspring.

Fig. 2.

Effects of PX-478 on brain development of PH-treated offspring rats. HE staining of the hippocampal CA1 and CA3 neurons of the offspring rats. The black arrow represented the vacuoles. Magnification 100$\times$: scale bar = 100 μM; 400$\times$: scale bar = 25 μM. HE, hematoxylin and eosin; CA, Cornu Ammonis.

Through behavioral tests on offspring, it was observed that PH treatment notably increased self-grooming time (Fig. 3A), toy area stay time (Fig. 3B), and familiar-area stay time (Fig. 3D), while decreasing central area stay time (Fig. 3C). However, in the PX-478 treated group, self-grooming time (Fig. 3A), toy area stay time (Fig. 3B), and familiar area stay time (Fig. 3D) were significantly reduced, while central area stay time (Fig. 3C) was increased in PH-treated offspring. Similarly, results from the Barnes maze test revealed that trial and error times (Fig. 3E,H) and escape latency (Fig. 3F,G) of offspring in the PH group were markedly elevated, whereas PX-478 1 w or 3 w treatment significantly reduced trial and error times (Fig. 3E,H) and escape latency (Fig. 3F,G) of PH-treated offspring. These findings indicate that PH treatment compromises offspring’s spatial memory ability, which can be mitigated by postnatal administration of PX-478 along with improving learning ability. No significant difference was observed between PX-478 1 w and 3 w treatments.

Fig. 3.

Effects of PX-478 on the behaviors of PH-treated offspring rats. The self-grooming time (A) and central area stay time (C) of offspring rats were measured using the open field test. The toy area stay time (B) and the familiar area stay time (D) of offspring rats were assessed using the three-chamber social interaction test. The trial and error times (E,H) and escape latency (F,G) of offspring rats were evaluated using the Barnes maze test. The one-way ANOVA test was used to analyze the significance of differences between groups. ns, p $>$ 0.05, *p 0.05, **p 0.01, ***p 0.001, ****p 0.0001.

It was observed that PH treatment markedly elevated HIF-1$\alpha$ levels (Fig. 4A,B) and reduced PTEN levels (Fig. 4A,C) in the hippocampus of offspring, while PX-478 treatment led to a significant decrease in HIF-1$\alpha$ and increase in PTEN levels. The effects of PX-478 treatment at one week were particularly noteworthy. Additionally, PH treatment notably increased VEGF concentration in the serum of offspring, which was significantly reduced with PX-478 treatment (Fig. 4D).

Fig. 4.

Effects of PX-478 on the HIF-1$\alpha$ and PTEN protein levels and VEGF concentration of the PH-treated offspring rats. The protein levels of HIF-1$\alpha$ (A,B) and PTEN (A,C) in the hippocampus of offspring rats were detected by western blotting. (D) The VEGF concentration in the serum of offspring rats was detected by Enzyme-Linked Immunosorbent Assay (ELISA). The one-way ANOVA test was used to analyze the significance of differences between groups. ns, p $>$ 0.05, *p 0.05, **p 0.01, ****p 0.0001. HIF-1$\alpha$, Hypoxia-inducible factor-1 alpha; PTEN, phosphatase and tensin homolog; VEGF, vascular endothelial growth factor; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.