ICR mice (8 weeks old) were purchased from the Beijing Vital River Laboratory Animal Center (Beijing, China), whose foundation colonies were all introduced from the Charles River Laboratories (Boston, MA, USA) and allowed to acclimatize in the animal facility for 1 week. Throughout the experiments, animals were maintained with free access to water and food under 12 h light/12 h dark cycle, and followed the guidelines for relative humane treatment. Following 1-week acclimation to the colony room, the male and female mice were paired in breeders. The existence of a vaginal plug was designated as gestational day (GD) 1. Pregnant females (n = 40) were treated with either normal saline/low/ middle/high doses of SMM sodium with a purity of 99% (CAS: 38006-08-5; Anhui HuaAo Biotechnology Co., Ltd.), namely control group, low- [10 mg/(kg·day)], middle - [50 mg/(kg·day)], and high-dose [200 mg/(kg·day)] groups. And the treated groups received SMM by gavage daily from GD 1 to 18. Parturition before 8 am was designated as postnatal day (PND) 1. Within 24 h after birth, excess pups were removed so that only 5 male pups were kept with the dam (1 litter). In addition, maternal feces, hippocampus and feces of the male pups were collected during the proper period. This study was approved by the ethical committee of Anhui Medical University and the ethical clearance number for the animal study is LISC 20170323.
Fecal samples from the male pups were collected on PND 22 and 56, also the dams upon cessation of SMM administration (about GD 18). For each mouse, the samples were collected into Petri dishes through gently stretching the mice or rubbing their abdomen, transferred into microcentrifuge tubes, immediately frozen and stored at -80 ℃ until analysis. When prepared, each sample was weighted (approximately 60 mg) and dipped in 600 μL deionized water for 12 h at 4 ℃. Then the mixture was centrifuged at 13, 000 r/m for 15 min at 4 ℃, subsequently, a small amount of 5 mol/L HCl was added to 100 μL of the supernatant, and homogenated. Finally, the internal standard, n-hexanoic acid solution, was spiked into the supernatant and then the chromatographic analysis was carried out using an Agilent 7890 GC system (Agilent, USA). The experimental conditions of the gas chromatographic analysis were described briefly as the following. Helium was supplied as the carrier gas at a flow rate of 20.0 mL/min. The initial oven temperature was 80 ℃, maintained for 3.0 min, raised to 120 ℃ at 5 ℃/min and held for 6.0 min. The temperature of the flame ionization detector and the injection port was 240 and 200 ℃, respectively. The injected sample volume for GC analysis was 1 μL and data processing was carried out with a HP Chem Station Plus software.
In order to observe the long-term effects of SMM exposure on hippocampal mTOR signaling pathway, we collected the pup hippocampus on PND 22 and PND56, respectively. The experimental steps were performed as the preceding literature described but made some changes. After the male pups were sacrificed under anesthesia, the hippocampus were quickly dissected and stored at -80 ℃ until further processed. Following tissue homogenization, total RNA was extracted using TRI-zol Regent (Molecular Research Center, Cincinnati, Ohio, USA) according to the manufacturer's instructions. And the RNA was transcribed into cDNA (A3500, Promega, Fitchburg, Wisconsin, USA). The expression of mRNA was measured by RT-qPCR using the RealStar Power SYBR mixture (GenStar BioSolutionsCo. Ltd., Beijing, China), and the levels were determined through the 2-ΔΔCT method. The primers were synthesized by Sangon Biological Technology (Shanghai, China). And the key genes measured, primer pairs and annealing temperatures are shown in Table 1.
Name Sequence Denaturation (℃) Annealing (℃) Extension (℃) Size (bp) GAPDH Forward: 5'-ACCCCAGCAAGGACACTGAGCAAG-3' 94 95 96 97 Reverse: 5'-GGCCCCTCCTGTTATTATGGGGGT-3' PI3K3ca Forward: 5'-TGTGTTCTCTGCTCGTCAGG-3' 94 95 96 97 Reverse: 5'-GAAACACAGCGAAGTCCACG-3' AKT1 Forward: 5'-CCGCCTGATCAAGTTCTCCT-3' 95 96 97 98 Reverse: 5'-TTCAGATGATCCATGCGGGG-3' mTOR Forward: 5'-CAAGATGCTTGGGACGGGT-3' 96 97 98 99 Reverse: 5'-CATTCCGGCTCTTCAGTCCA-3' RpS6Kb1 Forward: 5'-ATTGAGCTTAAGCAGCCGGT-3' 97 98 99 100 Reverse: 5'-GTCCTCAGCTTCCCTGTGTC-3' eIF4EBP1 Forward: 5'-GCACATACCTCCTTGTGCCT-3' 94 95 96 97 Reverse: 5'-TCCCAGGTAACCCAGCCTAA-3' Note. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PI3K3ca, phosphatidylinositol 3-kinase, catalytic, alpha polypeptide; AKT1, thymoma viral proto-oncogene 1; mTOR, mechanistic target of rapamycin; RpS6Kb1, ribosomal protein S6 kinase, polypeptide 1; eIF4EBP1, eukaryotic translation initiation factor 4E binding protein 1.
Table 1. Primers and Annealing Temperature for RT-PCR
On PND 22 and 56, brains in the high-dose group were placed into a pre-cooled mice brain matrix, cut into 3 coronal blacks of 4 mm each and fixed with 4% paraformaldehyde for 24 h. The blocks were dehydrated in ascending concentration of ethanol, embedded in paraffin, and sectioned 5 μm thickness. For immunohistochemistry, the sections were deparaffinized and dehydrated. The sections were immersed in 0.1 mol/L citric acid and steamed in a high-pressure cooker for 10 min for antigen retrieval. Endogenous non-specific biding sites were blocked using 3% hydrogen peroxide at 37% for 60 min. The sections were incubated with anti-mTOR antibody (dilution 1:500, abcam) at 4 ℃ overnight. After sequential incubation with peroxidase conjugated donkey anti-rabbit IgG, 3, 3'-Diaminobenzidine (DAB) was then used as a chromogen to view the reaction using a light microscope. The sections were counterstained by hematoxylin. All incubations were performed in a humidified chamber. The software of Image-Pro Plus (IPP) used to analyze the results of immunohistochemistry.
As described in the previous literature with modification, on PND 22 and 56, the hippocampus of male pups were separated on ice-cold plate, frozen in liquid nitrogen and stored in -80 ℃ freezer until measured. Hippocampus samples were homogenized in 600 μL RIPA lysis buffer and 6 μL PMSF. Samples were centrifuged at 14, 000 ×g for 15 min and then taked 300 μL supernatants centrifuged at 14, 000 ×g for 10 min. Finally, 200 μL supernatants were collected and the protein concentration was measured by BCA protein assay kit (Pierce, Rockford, IL, USA). After the SDS-PAGE gel electrophoresis, theproteins were transferred to PVDF membranes (Millipore). The membranes were then incubated with mouse anti-PI3K (1:1, 000, Abcam, ab22653) and mouse anti-β-actin (1:1, 000, SantaCruz, sc-81178), rabbit anti-AKT (1:5, 000, Abcam, ab179463), rabbit anti-mTOR (1:2, 000, Abcam, ab2732), rabbit anti-S6K1 (1:2, 500, Abcam, ab32359), rabbit anti-eIF4EBP1 (1:10, 000, Abcam, ab32024) and rabbit anti-SPR (1:500, Abcam, ab103353) primary antibodies overnight at 4 ℃. After washes in TBST three times for 10 min each, the membranes were incubated with goat anti-rabbit IgG antibody and goat anti-mouse IgG antibody for 1.5 h with shaking at room temperature. The membranes were then washed three times in TBST for 10 min each. Finally, membranes were processed for detection using the ECL system.
All of the data were expressed as the mean ± SEM. The results of the immunohistochemistry were analyzed with the independent samples t-test. Other data were analyzed using one-way ANOVA. Specific post hoc comparisons between groups were performed using Fisher's protected least significant difference (LSD) tests. Results were considered statistically significant when P < 0.05 (*P < 0.05, **P < 0.01, ***P < 0.001) as compared with controls. Statistics were performed using SPSS Version 23.0 software (SPSS, Chicago, IL, USA).
Animals and Treatment
Measurement of Fecal SCFAs
RT-qPCR Analysis of Key Genes in the mTOR Pathway
Immunohistochemical Expression of mTOR in Pups Hippocampus
We collected the fecal samples of dams and male offspring and detected SCFA levels. In dams, the acetate concentration in the SMM-treated groups was generally elevated, especially in the low-dose group (Figure 1A, P < 0.05). Notably, the propionate concentration in the middle- and high-dose groups was markedly reduced compared with that in the control group (Figure 1B, P < 0.001). Concurrently, in exposed dams, butyrate levels and total SCFA (sum of butyrate, propionate, and acetate) levels were significantly decreased compared with those in controls (Figure 1C, 1D, P < 0.001).
Figure 1. Fecal SCFAs levels in the dams. The fecal SCFAs levels were detected on pregnant day 18. (A) Acetate, (B) Propionate, (C) Butyrate, (D) Total SCFAs levels. Data are presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, compared with controls.
In contrast to dams, the total SCFA levels in male offspring showed an increasing trend on PND 22 (Figure 2D). Moreover, the acetate concentration in the high-dose group (Figure 2A), the propionate concentration in the middle-dose group (Figure 2B), and SCFA levels in the high-dose group were increased compared with those in controls (P < 0.01). However, butyrate, which is known for its anti-inflammatory activity, was markedly reduced in both the middle- and high-dose groups (Figure 2C).
Figure 2. Fecal SCFAs levels in the male offspring on PND 22. The fecal SCFA levels were detected on postnatal day 22. (A) Acetate, (B) Propionate, (C) Butyrate, (D) Total SCFA levels. Data are presented as mean ± SEM. *P < 0.05, **P < 0.01, compared with controls.
On PND 56, the acetate concentration in the middle-dose group (Figure 3A), the propionate concentration in the low-dose group (Figure 3B), and the butyrate concentration and the total SCFA levels in both the low- and middle-dose groups were increased compared with those in controls (Figure 3C, D, P < 0.01). Thus, exposure to SMM early in life not only changed the SCFA level in dams but also had long-term effects on SCFA metabolism in male offspring. According to our study results, the compensatory increase in total SCFA levels in male offspring might have been due to the exposure of male offspring to lower doses of SMM in utero.
Table 2 presents the mRNA expression of key genes implicated in the mTOR pathway on PND 22. The mRNA expression of PI3K3ca and AKT1 in the hippocampus was significantly elevated in the SMM-treated groups (P < 0.05), and the mRNA expression of mTOR and RpS6Kb1 was increased.
mRNA Control Low Middle High F P PI3K3ca 1.00 ± 0.04 1.30 ± 0.12** 1.20 ± 0.17 1.26 ± 0.04* 4.72 0.035 AKT1 1.00 ± 0.05 1.13 ± 0.10 1.24 ± 0.06** 1.22 ± 0.07** 6.85 0.013 mTOR 1.00 ± 0.47 1.31 ± 0.32 1.23 ± 0.33 1.32 ± 0.23 0.56 0.659 RpS6Kb1 1.00 ± 0.39 1.46 ± 0.54 1.27 ± 0.46 1.53 ± 0.10 1.08 0.419 eIF4EBP1 1.00 ± 0.19 1.18 ± 0.59 0.90 ± 0.15 0.88 ± 0.05 0.53 0.673 Note. SMM, sulfamonomethoxine; mRNA, messenger RNA; mTOR, mammalian target of rapamycin. Data were presented as mean ± SEM. *P < 0.05, **P < 0.01, compared with controls.
Table 2. Hippocampal mRNA Levels in the mTOR Pathway of Male Offspring on Postnatal Day 22
As shown in Table 3, on PND 56, marked increases in the mRNA expression of PI3K3ca in the low-dose group as well as of AKT1 and mTOR in the high-dose group were detected compared with those in controls (P < 0.01), and the mRNA expression of RpS6Kb1 was increased. The low- and high-dose groups also expressed higher mRNA levels of eIF4EBP1 compared with those in the control group (P < 0.05).
mRNA Control Low Middle High F P PI3K3ca 1.00 ± 0.03 1.25 ± 0.05*** 1.07 ± 0.03 1.07 ± 0.06 17.50 0.0007 AKT1 1.00 ± 0.02 0.99 ± 0.13 1.02 ± 0.13 1.26 ± 0.04* 4.93 0.0379 mTOR 1.00 ± 0.09 1.18 ± 0.01 1.15 ± 0.15 1.47 ± 0.10** 8.93 0.0086 RpS6Kb1 1.00 ± 0.06 1.15 ± 0.04 1.04 ± 0.29 1.25 ± 0.24 0.83 0.5187 eIF4EBP1 1.00 ± 0.10 1.22 ± 0.06* 1.08 ± 0.11 1.31 ± 0.09** 6.40 0.0161 Note. SMM, sulfamonomethoxine; mRNA, messenger RNA; mTOR, mammalian target of rapamycin. Data were presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, compared with controls.
Table 3. Hippocampal mRNA Levels in the mTOR Pathway of Male Offspring on Postnatal Day 56
SPR catalyzes the reduction of sepiapterin to dihydrobiopterin (BH2), which is the precursor for tetrahydrobiopterin (BH4). Our previous study indicated that the serum concentration of BH4 on PND 22 was significantly reduced in male offspring treated with a high dose of SMM. In the current study, we further investigated SPR protein expression in the hippocampus of male offspring. On PNDs 22 (Figure 4A) and 56 (Figure 4B), SPR protein expression was significantly decreased in all SMM-treated groups compared with that in the control group (Figure 4, P < 0.001).
The CA1 region of the hippocampus is closely related to memory and cognitive function of the brain. Therefore, in this study, we assessed mTOR expression in the hippocampal CA1 area. The immunochemistry results (Figure 5) revealed that compared with the control group, mTOR expression in the hippocampal CA1 area increased in the high-dose group on PND 22 (P < 0.01) and PND 56 (P < 0.01).
On PND 22, SMM significantly increased the expression of AKT (Figure 6B, P < 0.01), mTOR (Figure 6C, P < 0.001), and S6K1 (Figure 6D, P < 0.01) in the hippocampus in the SMM-treated groups compared with the control group. However, no difference was observed in the expression of the PI3K (Figure 6A) and 4EBP1 (Figure 6E) between the SMM-treated groups and the control group. These results provided evidence that the mTOR signaling pathway was upregulated on PND 22.
Figure 6. Expression of related proteins of mTOR signaling pathway in male offspring on PND 22. PI3K: Phosphatidylinositol 3-kinase, AKT: protein kinase B, mTOR: mammalian target of rapamycin, S6K1: ribosomal protein S6 kinase 1, 4EBP1: 4E-binding protein 1, PND: postnatal day. (A) PI3K expression, (B) AKT expression, (C) mTOR expression, (D) S6K1 expression, (E) 4EBP1 expression. Data are presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, compared with controls.
On PND 56, the expression levels of PI3K (Figure 7A, P < 0.001), mTOR (Figure 7C, P < 0.001), S6K1 (Figure 7D, P < 0.001), and 4EBP1 (Figure 7E, P < 0.001) were upregulated in SMM-treated groups compared with control group; these results were consistent with those for mRNA expression. These findings indicate that maternal SMM exposure during pregnancy led to the upregulation of the mTOR signaling pathway in the hippocampus of offspring until puberty.
Figure 7. Expression of related proteins of mTOR signaling pathway in male offspring on PND 56. PI3K: Phosphatidylinositol 3-kinase, AKT: protein kinase B, mTOR: mammalian target of rapamycin, S6K1: ribosomal protein S6 kinase 1, 4EBP1: 4E-binding protein 1, PND: postnatal day. (A) expression of PI3K, (B) expression of AKT, (C) expression of mTOR, (D) expression of S6K1, (E) expression of 4EBP1. Data are presented as mean ± SEM. ***P < 0.001, compared with controls.