| 2.1 Objective The present study aimed to investigate the exaggerated effects of maternal exposure to ambient levels of PM2.5 in Fuzhou during pregnancy and lactation period on filial cardiovascular malformation induced by homocysteine.2.2 Methods Sixty-four 10-week-old pregnant SD rats on the first gestation day(GO) were randomly divided into four groups(n = 16 per group): filtered air plus normal saline(FA-NS); PM2.5 plus NS(PM2.5-NS); filtered air plus homocysteine(FA-HCY); PM2.5 plus HCY(PM2.5-HCY). Groups PM2.5-NS and PM2.5-HCY were exposed to environmentally relevant PM2.5 in Fuzhou General Hospital throughout the entire pregnancy and lactation period in a "real-world" airborne PM exposure system, while groups FA-NS and FA-HCY exposed to filtered air. Groups FA-HCY and PM2.5- HCY were additionally administered with 33.3 μL homocysteine(HCY) at concentration of 75 μmol/L and groups FA-NS and PM2.5-NS with the same volume of normal saline(NS) daily from gestation days 8 to 10. The neonatal hearts from half of the pregnant rats within 12 hours after spontaneous delivery, were extracted. For the rest of the pregnant rats(n = 8 per group) after spontaneous delivery, litters were maintained with their dams until weaning(postnatal day 21) and the hearts of the young rats were then harvested. The neonatal hearts were histopathologically examined(myocardial pathological section, transmission electron microscope and Tunel examination); The expression of GATA4, NKx2-5 protein and m RNA in the heart tissue of 20 neonatal and young rats respectively were assayed by Western-blotting and Real time PCR. The concentrations of TNF-a, IL-1β in the plasma of 10 young rats’ hearts were measured by Elisa.2.3 Results2.3.1 Under light microscopy, The apoptosis-like changes of myocardial cells, nucleus, and chromatin structure were morphologically appeared in groups FA-HCY, and were more obviously seen in group PM2.5- HCY, which was in accordance with an increased myocardial apoptosis rates in the two groups. Under electron microscopy, groups FA-HCY and PM2.5- HCY exhibited abnormal changes in cell membrane, mitochondria, glycogen to certain degree. There is no obvious structural changes in the neonatal heart tissue of groups FA-NS and PM2.5-NS.2.3.2 The myocardial cell apoptosis detected by Tunel showed both PM2.5 exposure and HCY treatment were effective in elevating myocardial cell apoptosis. There was no interaction in complex intervention in elevating myocardial cell apoptosis.2.3.3 Both PM2.5 exposure and HCY treatment were effective in lowering the levels of Nkx2-5 protein and GATA4, Nkx2-5 m RNA in the heart tissue of neonatal and young rats. HCY treatment was effective in lowering the levels of GATA4 protein in the heart tissue of neonatal and young rats but PM2.5 exposure was not. There was an interaction in complex intervention in lowering the levels of GATA4, Nkx2-5 protein and m RNA in the heart tissue of neonatal rats ?P ? 0.05 and P ? 0.01), however, that was not in young rats. Further detailed comparison shows: There was no statistically significant difference between groups FA-NS and PM2.5-NS ?P ? 0.05?, while there were statistically significant difference between groups FA-NS and FA-HCY, groups PM2.5-NS and PM2.5- HCY, groups FA-HCY and PM2.5- HCY ?P ? 0.05 or P ? 0.01).2.3.4 PM2.5 exposure was effective in increasing the concentrations of TNF-? and IL-1β in plasma of young rats ?P ? 0.01 and P ? 0.05), HCY treatment was effective in increasing the concentrations of TNF-? in plasma of 2 young rats ?P ? 0.01?, but that was not in IL-1β ?P ? 0.05?. There was no interaction in complex intervention in increasing the concentrations of TNF-? and IL-1β in plasma of young rats ?P ? 0.05?.2.4 Conclusions2.4.1 The atmosphere quality of Fuzhou has no obvious adverse effects on fetal heart development, however, it affects adversely on filial cardiovascular malformations induced by HCY.2.4.2 There was no obvious structural changes in the neonatal cardiac tissue of groups FA-NS and PM2.5-NS were seen. While the apoptosis of myocardial cell was seen in groups FA-HCY and PM2.5- HCY, and group PM2.5- HCY was more obvious. Myocardial apoptosis detected by Tunel between FA-HCY and PM2.5-HCY groups was statistically significant(P < 0.05). Suggesting that PM2.5 exposure may cause changes to the mitochondria in structure or function of the fetal heart, then increasing the oxidative stress and myocardial ischemia, thus causing structural abnormalities to the fetal heart tissue.2.4.3 Both PM2.5 exposure and HCY treatment were effective in lowering the levels of Nkx2-5 protein and GATA4, Nkx2-5 m RNA in the heart tissue of neonataand young rats. There were statistically significant difference between groups FA-NS and FA-HCY, groups PM2.5-NS and PM2.5- HCY, groups FA-HCY and PM2.5- HCY ?P < 0.05). Suggesting that PM2.5 affects adversely on filial cardiovascular malformations induced by HCY. The mechanism maybe that PM2.5 exposure may reduce the m RNA and protein levels of GATA4 and Nkx2-5 transcription factor.2.4.4 PM2.5 exposure was effective in increasing the concentrations of TNF-? and IL-1β in plasma of young rats ?P < 0.01 and P P < 0.05), there was no interaction in complex intervention in increasing the concentrations of TNF-? and IL-1β in plasma of young rats ?P ? 0.05?. PM2.5 induces oxidative stress and inflammatory response may be an another mechanism of its effect on heart development in rats. |
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