Supplementation with dietary omega-3 PUFA mitigates fetal brain inflammation and mitochondrial damage caused by high doses of sodium nitrite in maternal rats

Abstract

Objective: Food safety and nutrition during pregnancy are important concerns related to fetal brain development. In the present study, we aimed to explore the effects of omega-3 polyunsaturated fatty acids (PUFA ω-3) on exogenous sodium nitrite intervention-induced fetal brain injury in pregnant rats.

Methods: During pregnancy, rats were exposed to water containing sodium nitrite (0.05%, 0.15%, and 0.25%) to establish a fetal rat brain injury model. Inflammatory factors and oxidative stress levels were detected using enzyme-linked immunosorbent assay (ELISA) or flow cytometry. Subsequently, animals were divided into three groups: control, model, and 4% PUFA ω-3. Pregnancy outcomes were measured and recorded. Hematoxylin-eosin (H&E) staining and immunohistochemistry (IHC) were utilized to observe brain injury. ELISA, quantitative real-time PCR (qRT-PCR), western blot, flow cytometry, and transmission electron microscopy (TEM) were adopted to measure the levels of inflammatory factors, the NRF1/HMOX1 signaling pathway, and mitochondrial and oxidative stress damage.

Results: With the increase of sodium nitrite concentration, the inflammatory factors and oxidative stress levels increased. Therefore, the high dose group was set as the model group for the following experiments. After PUFA ω-3 treatment, the fetal survival ratio, average body weight, and brain weight were elevated. The cells in the PUFA ω-3 group were more closely arranged and more round than the model. PUFA ω-3 treatment relieved inflammatory factors, oxidative stress levels, and mitochondria damage while increasing the indicators related to brain injury and NRF1/HMOX1 levels.

Conclusions: Sodium nitrite exposure during pregnancy could cause brain damage in fetal rats. PUFA ω-3 might help alleviate brain inflammation, oxidative stress, and mitochondrial damage, possibly through the NRF1/HMOX1 signaling pathway. In conclusion, appropriately reducing sodium nitrite exposure and increasing PUFA omega-3 intake during pregnancy may benefit fetal brain development. These findings could further our understanding of nutrition and health during pregnancy.

Fig 1. Exposure to sodium nitrite during pregnancy induces inflammation and oxidative stress.

(A) The concentration of plasma peroxynitrite in pregnant rats as detected by ELISA, n = 12 rats/group. (B) Plasma NO levels in pregnant rats from different groups were detected by ELISA, n = 12 rats/group. (C) Effect of sodium nitrite exposure on blood inflammation in placenta tissues as detected by ELISA, n = 3 rats/group. (D) Flow cytometry was used to detect ROS in the fetal cerebral cortex, n = 3 rats/group. (E) Plasma MDA and SOD levels of fetal rats in different groups as detected by ELISA, n = 3 rats/group. *P < 0.05 vs. control group; ^#P < 0.05 vs. low dose group; ^&P < 0.05 vs. medium-dose group. MDA, malondialdehyde; NO, nitric oxide; ROS, reactive oxygen species; SOD, superoxide dismutase.

Fig 2. PUFA ω-3 ameliorates adverse pregnancy outcomes following sodium nitrite exposure during pregnancy.

(A) The number of fetuses per litter, (B) The fetal survived ratio, (C) female rats ratio, (D) average body weight, and (E) average brain weight were measured and recorded. *P < 0.05, n = 3 rats/group.

Fig 3. PUFA ω-3 alleviates fetal brain damage induced by sodium nitrite exposure during pregnancy.

(A) Representative images of brain tissues from each group were analyzed by H&E staining, n = 3 rats/group. (B) Representative images of OCLN and TJP1 expression in brain tissues of each group, as measured by immunohistochemistry, n = 3 rats/group. Blue indicates nuclei. Light red indicates cytoplasm. Red arrows indicate typical nuclear damage. (C) Integrated optical density (IOD) values of representative images of OCLN and TJP1 expression in brain tissues of each group were analyzed by Image-Pro-Plus, n = 3 rats/group. *P < 0.05. H&E, hematoxylin-eosin.

Fig 4. PUFA ω-3 alleviates sodium nitrite exposure-induced cerebral cortical tissue damage in fetal rats via the NRF1/HMOX1 pathway.

(A) TNF and IL-1B concentrations were detected by ELISA, n = 3 rats/group. (B-C) The mRNA and protein expression levels of Tnf, Nos2, and Il1b were detected by qRT-PCR and western blot, n = 3 rats/group. Original blots are presented in S2A Fig. (D) Expression levels of SOX2, ENO2, and TUBB3 in the brain cortical tissue of each group were detected by IF, n = 3 rats/group. (E) IOD data statistics of SOX2, ENO2, and TUBB3 expression in the brain cortical tissue of each group were analyzed by Image-Pro-Plus, n = 3 rats/group. (F-H) Nrf1 and Hmox1 mRNA and protein expression levels were analyzed by qRT-PCR and western blot, n = 3 rats/group. Original blots are presented in S2B Fig. *P < 0.05. IF, immunofluorescence; IL-1B, interleukin-1 beta; IOD, integrated optical density; qRT-PCR, quantitative real-time PCR; TNF, tumor necrosis factor.

Fig 5. PUFA ω-3 reduces mitochondrial damage induced by sodium nitrite exposure in fetal cortical tissue.

(A-B) ROS and JC-1 levels in the cerebral cortex of fetal rats as determined by flow cytometry, n = 3 rats/group. (C) Plasma MDA and SOD levels of fetal rats in different groups were detected by ELISA, n = 3 rats/group. (D-E) Mitochondrial complex II, mitochondrial complex IV, and ATP activity were measured by ELISA, n = 3 rats/group. (F) mt-Co1 expression detected by qRT-PCR, n = 3 rats/group. (G) Mitochondrial damage observed using TEM, n = 3 rats/group. Red arrows indicate mitochondria. Scale bars: 2 μm and 5 μm. *P < 0.05. MDA, malondialdehyde; qRT-PCR, quantitative real-time PCR; ROS, reactive oxygen species; SOD, superoxide dismutase; TEM, transmission electron microscopy.

Fig 6. PUFA ω-3 may alleviate brain damage in fetal rats caused by sodium nitrite exposure during pregnancy via NRF1/HMOX1 signaling.