Effects Of Dietary Hydrogen Sulfide Donor Diallyl Trisulfide On Reproductive Performance Of Obese Maternal

Placenta is a temporary organ for the exchange of nutrients and metabolites between mother and fetus during pregnancy.The vascular development of placenta is very important to the growth and development of fetus.Studies have shown that maternal obesity can lead to impaired placental function,limited placental angiogenesis and fetal development disorders.Previous studies in our laboratory have found that excessive fat of sows during pregnancy can cause ectopic deposition of placental fat,which aggravate oxidative stress and inflammation of placental tissue,and significantly reduce the density of placental blood vessels,which may be one of the reasons for the occurrence of weak piglets.Hydrogen Sulfide（H₂S）is a ubiquitous second messenger in cells,and has many biological effects such as promoting lipid metabolism,anti-inflammatory,anti-oxidative stress,promoting/anti-apoptotic activity,promoting vasodilation and angiogenesis.However,in the case of maternal obesity,whether H₂S can be used as an important signal molecule to regulate body’s lipid metabolism and oxidative stress response,alleviate placental inflammation and promote placental angiogenesis remains to be further studied.Therefore,two experiments were conducted to investigate the effects of H₂S donor Diallyl trisulfide（DATS）on reproductive performance of obese sows during pregnancy,and to explore the mechanism of H₂S promoting placental vascular development and improving reproductive performance of obese female mice induced by high-fat diet.Experiment 1:Effects of dietary diallyl trisulfide on reproductive performance of pregnant obese sowsA total of 155 intercalated cross-breeding of Danish line sows（landrace and Yorkshire）with similar first fetuses and backfat thickness greater than 16 mm at 60 days of gestation were randomly divided into 4 groups with 36-42 sows in each group.Each sow in each group was fed 0 g/d（control group）,2 g/d（treatment 1）,4 g/d（treatment 2）and 6 g/d（treatment 3）H₂S donor diallyl trisulfide,respectively.Backfat,feed intake at60,90 and 109 days of pregnancy and reproductive performance of sows were measured and recorded to study the effects of backfat change,feed intake and reproductive performance.The main results are as follows:1.Feed intake and backfat:Compared with the control group,different DATS treatment had no significant effect on feed intake and backfat thickness of sows during pregnancy（P>0.05）.2.Reproductive performance:Compared with the control group and treatment 1,the total number of born（18.49 VS 15.80 VS 16.93）,number of born alive（17.76 VS 15.00VS 16.10）and number of born healthy（16.46 VS 13.97 VS 15.30）in treatment 2 were significantly increased（P<0.05）.The total number of born of treatment 3（18.73 VS15.80 VS 16.93）was significantly higher than that of control group and treatment 1（P<0.05）.Compared with the control group,the number of born alive（17.27 VS 15.00）and number of born healthy（15.76 VS 13.97）in treatment 3 were significantly increased（P<0.05）.In addition,compared with the control group,the number of weaned in treatment 1,2 and 3（11.85 VS 12.00 VS 11.84 VS 11.03）was significantly increased（P<0.05）.The weight of weaned at 26 d（81.33 kg VS 71.60 kg）in treatment 2 was significantly higher than that in control group（P<0.05）.Experiment 2 The mechanism of diallyl trisulfide promoting placental vascular development and improving reproductive performance of obese female miceSixty female C57BL/6J mice aged 6-8 weeks were fed a high-fat diet（60%kcal fat,High-fat diet,HFD）for 13 weeks,and then obese mice and Normal female mice of the same age（10%kcal fat,Normal-fat diet,NFD）mated with adult male mice.After pregnancy,according to a 2×2 experimental design,the mice were divided into 4 groups,with 9-11 mice in each group,and randomly received the following treatments:group 1（NFD+PBS,NFD with PBS administered by gavage）;group 2（NFD+DATS,NFD with50 mg/kg/d DATS administered by gavage）;group 3（HFD+PBS,NFD with PBS administered by gavage）;group 4（HFD+DATS,HFD with 50 mg/kg/d DATS administered by gavage）.The female mice were sacrificed on day 18.5 of gestation.Maternal body weight,inguinal white adipose tissue,reproductive performance,fetal and placental weight were collected.Body fat percentage and placental efficiency were calculated.Acquisition mice serum,inguinal white adipose and placenta,detection of mice serum and placental tissue H₂S level,lipid metabolism,oxidative stress,maternal serum insulin and glucose levels,placental inflammation,placenta angiogenesis related genes and related indicators,and to study the effects on lipid metabolism,oxidative stress,inflammatory response,placental structure and placental angiogenesis.The main results are as follows:1.Body weight and body composition:Compared with the normal diet group,the body weight of obese mice induced by high fat significantly increased（33.39 g VS 21.96g）（P<0.05）,and gained more than 25%of the initial body weight.Compared with normal diet,the average daily feed intake,total feed intake and weight gain of pregnant mice fed with high-fat diet decreased during pregnancy（P<0.05）,but the body weight,body fat percentage and inguinal white adipose tissue content increased at 0.5 and 18.5days of pregnancy（P<0.05）.DATS treatment reduced inguinal white adipose content（1.03 g VS 1.35 g）（P<0.05）and body fat percentage（2.87%VS 3.47%）（P<0.05）.There was significant interaction between diet and DATS on fat content in inguinal white adipose（P<0.05）,on a high fat diet,compared with group 3,inguinal white adipose content in group 4 was significantly decreased（P<0.05）.2.Reproductive performance:Compared with normal diet,high fat diet increased the litter size（9.04 VS 8.19）（P<0.05）,while DATS increased the litter size（9.02 VS 8.21）and the alive litter size of female mice（8.45 VS 7.39）（P<0.05）.Compared with normal diet,fetal weight in high fat diet group was decreased（1.01 g VS 1.09 g）（P<0.05）.Compared with PBS group,DATS treatment reduced placental weight（0.08 g vs 0.09 g）（P<0.05）and improved placental efficiency（P<0.05）.3.The content of hydrogen sulfide in serum and placental tissues:Compared with normal diet,high-fat diet reduced H₂S levels in serum and placental tissues（P<0.05）;DATS treatment increased H₂S levels in serum and placental tissues of mice compared with PBS group（P<0.05）.4.Placental structure:The results of HE staining for placenta showed that,compared with normal diet,the placenta and labyrinth zone area in high fat diet group decreased（P<0.05）,but had no significant effect on junction area（P<0.05）.Compared with the PBS group,DATS treatment increased placental area and labyrinth zone area（P<0.05）,and there was an interaction between dietary treatment and perfusion DATS（P<0.05）,compared with group 3,the placental area of mice in group 4 was significantly increased under high fat diet（P<0.05）.Correlation analysis showed that H₂S level was significantly positively correlated with placental area and the labyrinth zone area（P<0.05）,and significantly negatively correlated with placental weight（P<0.05）.5.Placental vascular development:Immunohistochemical results showed that high-fat diet significantly inhibited the expression of CD31 protein in labyrinth zone compared with normal diet（P<0.05）.QPCR detected that the m RNA expressions of angiogenesis related factors PLGF,VEGFA,VEGFR2 and HIF1αwere significantly up-regulated（P<0.05）.Compared with PBS group,DATS treatment significantly increased the expression of CD31 protein in the labyrinth zone of the placenta（P<0.05）,and up-regulated the expression of CD31,PLGF,VEGFA,VEGFR2 and HIF1αm RNA（P<0.05）.Moreover,the expression of CD31 protein and m RNA expression of various angiogenesis related factors had interaction between dietary treatment and DATS（P<0.05）,compared with group 3,the expression of CD31 protein and m RNA expression of CD31,PLGF,VEGFA,VEGFR2 and HIF1αin placenta of mice in group 4 were significantly increased in high-fat diet（P<0.05）.In addition,correlation analysis showed that H₂S level was significantly positively correlated with CD31 protein expression in labyrinth zone（P<0.05）.6.Lipid metabolism level:Compared with normal diet,serum triglyceride and low density lipoprotein cholesterol levels were increased in high-fat diet group（P<0.05）.Compared with PBS group,DATS treatment decreased low density lipoprotein cholesterol level（P<0.05）,and high-density lipoprotein cholesterol level were increased（P<0.05）.The m RNA expressions of FABP4 and FATP4 related genes were detected.Compared with normal diet,the m RNA expressions of FABP4 and FATP4 were increased in high-fat diet group（P<0.05）,while compared with PBS group,DATS treatment increased the m RNA expressions of FABP4 and FATP4（P<0.05）.In addition,total cholesterol and low density lipoprotein cholesterol levels,as well as m RNA expression of FABP4 and FATP4,had an interaction between dietary treatment and DATS（P<0.05）.Further multiple comparisons of total and low-density lipoprotein cholesterol levels found that total and low-density lipoprotein cholesterol levels were significantly higher in mice in groups 2 and 3 than in group 1（P<0.05）;under the treatment of high-fat diet,compared with group 3,the levels of total cholesterol and low density lipoprotein cholesterol were significantly decreased in group 4（P<0.05）;FABP4 and FATP4 m RNA expression was significantly increased（P<0.05）.7.the levels of placental inflammatory factors:High-fat diet up-regulate the m RNA relative expressions of IL-10,IL-6,and TNFαm RNA in placenta of mice（P<0.05）.Compared with PBS group,DATS treatment increased the relative expression of IL-10m RNA in mice placenta（P<0.05）,but inhibited IL-6 and TNFαm RNA expression（P<0.05）.Moreover,IL-10 and IL-6 m RNA expression had a significant interaction between diet and treatment（P<0.05）;under the treatment of high-fat diet,the expression of IL-10in group 4 was significantly higher than that in group 3（P<0.05）,the expression of IL-6in placental tissues of mice in group 4 was significantly lower than that in group 3.8.The serum and placental oxidative stress levels of female mice:Compared with normal diet,high-fat diet resulted in decreasing serum total antioxidant capacity and placenta total superoxide dismutase activity levels of mice（P<0.05）,while increasing serum total superoxide dismutase,the lipid peroxidation product malondialdehyde levels and placenta malondialdehyde levels of mice（P<0.05）.Compared with PBS group,DATS treatment increased the activity of total superoxide dismutase in serum of maternal mice and the activity of total superoxide dismutase and glutathione peroxidase in placental tissues（P<0.05）,decreased the serum malondialdehyde level（P<0.05）.In addition,there was an interaction between dietary treatment and DATS administration on glutathione peroxidase and malondialdehyde levels in placental tissues（P<0.05）;under the treatment of normal diet,compared with group 1,the level of glutathione peroxidase in group 2 was significantly increased（P<0.05）;compared with group 3,malondialdehyde level in group 4 decreased（P>0.05）.9.Insulin sensitivity:Compared with normal diet,treatment with high fat diet increased blood glucose level and IPGTT area（P<0.05）.Compared with PBS group,DATS treatment reduced the area under IPGTT curve（P<0.05）.In addition,after 60minutes of glucose injection,blood glucose concentration in group 4 was significantly lower than that in group 3（P<0.05）.In summary,our study indicates that H₂S is an important signal molecule regulating angiogenesis.The addition of H₂S donor diallyl trisulfide during pregnancy reduced the body’s inflammatory response,improved maternal and placental lipid metabolism,oxidative stress level and placental vascular development,alleviated maternal insulin resistance,and improved maternal reproductive performance.