Improvement Of Pregnant Outcomes In Preeclampsia-like Mice By L-Carnitine: Alterations In Circulation And Decidual Monocytes/Macrophages And The Underlying Mechanisms

Objective: Preeclampsia(PE)is not only the leading cause of maternal death and perinatal mortality,but also significantly increases the incidence of cardiovascular disease(CVD)in patients and their offspring.In recent years,studies have shown that circulation and decidual monocytes(DMs)/ macrophages are involved in immunosuppression and helical arterial remodeling during pregnancy,which are the key to successful pregnancy.L-Carnitine(LC)is an anti-inflammatory and antioxidant.It has been used in many CVD and can promote the development of embryos in vitro,increase the success rate of in vitro fertilization(IVF)and up-regulate the expression of PPAR-?.Therefore,this study was to investigate the effect of LC on pregnant outcomes and DMs in PE-like mice by L-NAME,to explore the application prospect and potential mechanism of LC in PE and the effect of p Stat6/Stat6-PPAR-? pathway on the intervening effect of LC.Methods:(1).7 / 8-week-old male and female C57 BL / 6J mice were randomly divided into 8 groups: control group(group A),PE group [subcutaneous injection of L-NAME on gestation day 7(GD7),group B],PE + LC high dose groups(800mg / kg / d,group C to group E)and PE + LC low dose groups(400 mg / kg / d,group F to group H).Group C or group F,group D or group G and group E or group H were given LC on 1 week before pregnancy,GD0 and GD9,respectively.The blood pressure and 24-hour urine protein were monitored.The blood vessel function was evaluated by high-resolution ultrasonography and the growth and development of placenta and kidney were observed on GD18.The peripheral blood Ly6 Chi monocytes,trimethylamine oxide(TMAO),the phenotype of DMs and microbial community composition of mouse caecums in group A to group E were measured.The degree of atherosclerosis was determined in group B and group C.(2).L-NAME,LC and GW9662(antagonist of PPAR-?)were used to investigate the expression of i NOS,IL-1?,TNF-?,CD206,ARG1,IL-10 and PPAR-? m RNA inRAW 264.7 cell line.Results:(1).L-NAME can induce PE-like performance,increase circulation Ly6 Chi monocyte subset and decrease M2 phenotype in decidual macrophages in C57 BL / 6J mice.Ly6 Chi was positively correlated with blood pressure,24-hour proteinuria(r=0.6363,r=0.6752,all P<0.001)and negatively correlated with fetal weight,crown-rump length and placenta weight(r=-0.6694,r=-0.6623,r=-0.6623,all P<0.001).The M2 phenotype was negatively correlated with blood pressure,24-hour proteinuria(r=-0.5214,r=-0.6562,all P<0.05)and positively correlated with fetal weight,crown-rump length and placenta weight(r=0.4882,r=0.5701,r=0.5701,all P<0.05).(2).LC can dose-dependently reduce the blood pressure,24-hour urine protein,improve vascular endothelial function,intrauterine growth restriction,and decrease plusatility index(PI)and resistance index(RI)of umbilical artery and uterine-placental artery.The difference was statistically significant(all P <0.05).(3).Compared with group A,the circulation Ly6 Chi monocyte subset was significantly higher and the macrophage M2 in decidual was significantly lower than that in group B(61.7 ± 8.66% vs.72.2 ± 6.02%,65.4 ± 9.66 vs.53.7 ± 4.08,all P <0.01).Given LC on 1 week before pregnancy can decrease Ly6 Chi and increase M2 phenotype in decidual macrophages(72.2 ± 6.02% vs.61.25 ± 3.21%,53.7 ± 4.08 vs.65.8 ± 6.14,all P<0.01).The relative expression of M1-phenotype markers(TNF-?,IL-1?,i NOS)in decidual tissue in group B was significantly higher than that in control group(1.00 vs.1.41 ± 0.15;1.00 vs.5.35 ± 1.14;all P <0.001).The expression of TNF-?,IL-1? and i NOS in group C and group D were significantly lower than that in group B(1.42±0.15 vs.0.34±0.14;5.35±1.14 vs.3.64±1.13;81.3±16.3 vs.8.84±1.41;all P<0.001;1.42±0.15 vs.0.41±0.11;5.35±1.14 vs.4.39±0.89;81.3±16.3 vs.12.32±1.04;all P<0.05).The expression of i NOS gene in GD9 was significantly lower than that in group B(81.3±16.3 vs.20.2±3.37;P<0.001).The relative expression of CD206 in the decidual tissue in group B was lower than that in group A(1.00 vs.0.60 ± 0.17;P <0.001).There was no difference in IL-10 and ARG1 between group A and group B(1.00 vs.1.46±0.31;1.00 vs.1.04±0.15;all P>0.05).Compared with group B,the expression of CD206 and IL-10 were increasedin group C(0.60±0.17 vs.1.00±0.20;1.46±0.31 vs.2.57±0.66;all P<0.001),and IL-10 was increased in group D(1.46±0.31 vs.1.77±0.48;P<0.05).(4).The M2 phenotype was negatively correlated with blood pressure and 24-hour proteinuria(r=-0.5196,r=-0.5132,all P<0.001)and positively correlated with fetal weight and crown-rump length(r=0.5408,r=0.5779,all P <0.001).(5).The total dose of LC was correlated with macrophage M2,the expression of TNF-?,IL-1?,i NOS,CD206?IL-10 in decidual tissue.(6).The m RNA expression levels of TNF-? was decreased and CD206,IL-10 and ARG1 were increased by LC significantly,accompanied with the increased PPAR? m RNA expression level.GW9662(antagonist of PPAR-?)can inhibit the effection of LC.Western Blot shown the expression of PPAR-? and p Stat6 / Stat6 in L-NAME + LC group were significantly higher than that in control group(all P <0.05).After giving GW9662,the expression of those were not significant with control group(all P> 0.05).(7).There was no significant difference in plasma TMAO among group A to E and no difference in the development of atherosclerosis.between group B and group C(all P > 0.05).The results of 16 S r DNA sequencing of intestinal microflora in group A to group E showed that Bacteriodes and Firmicute were dominant floras in phylum level,and there were no differences among groups.No correlations were found between dominant floras with TMAO or the total dose of LC.Unclassified_Clostridia,Unclassified_Alphaproteobacteria,Clostridium_Xl Va,Anaerovorax,Anaerorhabdus,Olsenella and Sporobacter were significantly different among groups in genus level.Olsenella was negatively correlated with the total dose of LC(r=-0.7989,P<0.01).Conclusions: This study shown that the PPAR-? may be the key of the polarization of DMs induced by LC,which improved the pregnant outcomes in PE-like mice by L-NAME.Oral LC(800 mg / kg / d)in perinatal period had no significant impact on the development of atherosclerosis,suggesting that LC may be a new method to prevent and treat PE.