Abstract
Objective:To explore the specific mechanism of lactic acid(LA)on regulating decidual macrophage differentia-tion in pre-eclampsia(PE)through non-receptor tyrosine kinase sarcoma virus protein/lactate dehydrogenase A(SRC/LDHA)signaling pathway.Methods:Decidual tissues were collected from 16 women with preeclampsia(PE)and 16 women with normal pregnancies during the same period.Immunofluorescence was used to assess macrophage subtypes and LA secretion in decidual tissues.Biochemi-cal assays and Western blot were performed to measure levels of LA,LDHA and MCT-4 in tissues.Biochemical assays were conducted to measure LA level in supernatants of normal trophoblast cells under different cell densities and oxygen conditions.Flow cytometry was used to assess polarization of macrophages in normal decidual tissues under different conditions of LA,oxygen and culture media.qRT-PCR,Western blot,ROS,and NAD+/NADH measurements were performed to evaluate mRNA and protein levels related to gly-colysis and mitochondrial oxidative phosphorylation,as well as proteins associated with SRC/LDHA signaling pathway,ROS level,and NAD+/NADH.Thirty pregnant rats were divided into normal group,model group,low-,medium-,high-dose AZD3965 groups,with 6 rats in each group.PE model was established,and the systolic blood pressure and urinary protein levels of pregnant rats were measured at various time points.On gestational day 20,the placental and fetal development in each group were compared.Immunofluo-rescence was used to assess trophoblast cell invasion and uterine spiral artery remodeling in decidual tissues.Immunohistochemistry and flow cytometry were performed to evaluate fetal vascular development and macrophage polarization.Results:Compared with nor-mal pregnant women,patients with PE exhibited an increased proportion of M1 macrophages,CK7+LDHA+,CK7+MCT-4+cells,LA,LDHA and MCT-4(P<0.05),while proportion of M2 macrophages was decreased(P<0.05).Compared with normoxic conditions,LA level in supernatant of trophoblast cells was significantly increased under hypoxic conditions(P<0.05).Under normoxic condition,compared with control groups,conditioned culture group showed a decreased M1/M2 macrophage(P<0.05).LA group exhibited in-creased expressions of NDUFA1,NDUFA2,UQCRC1,UQCRC2,COX4I1,COX4I2,ATP5F1A,ATP5F1B mRNA,and SDHB,COX4I1,LDHB,p-SRC(Y416),p-LDHA(Y10),VEGF and ARG-1 protein(P<0.05).NAD+/NADH was decreased(P<0.05).PX478 group showed increased expression of p-SRC(P<0.05).LA+PX478 group exhibited increased expressions of p-SRC(Y416)and p-LDHA(Y10)(P<0.05),while VEGF expression was decreased(P<0.05).Under hypoxic conditions,compared with control groups,the conditioned culture group,LA group and LA+PX478 group exhibited an increased M1/M2 macrophage(P<0.05).LA group showed upregulated expressions of LDHA mRNA,HIF-1α,LDHA,p-SRC(Y416),p-LDHA(Y10)and iNOS(P<0.05),while mRNA expression of LDHB and protein expressions of NDUFA1 and UQCRC2 were downregulated(P<0.05).ROS level and NAD+/NADH were decreased(P<0.05).PX478 group demonstrated reduced HIF-1α expression(P<0.05)and increased p-SRC(Y416)expression(P<0.05).LA+PX478 group showed decreased HIF-1α expression(P<0.05)and increased p-LDHA(Y10)ex-pression(P<0.05).Compared with LA group,LA+PX478 group exhibited elevated ROS level(P<0.05)and a reduced NAD+/NADH(P<0.05).Compared with normal group,pregnant rats in model group exhibited increased blood pressure,urine protein levels and an elevated M1/M2 in decidual tissue(P<0.05).The fetal mice exhibited abnormal development,with inadequate trophoblast cell infiltra-tion,impaired uterine spiral artery remodeling,a decreased number of fetal blood vessels in placental labyrinthine layer,and nar-rowed lumens.Compared with model group,the low,medium,and high-dose AZD3965 groups showed decreased blood pressure,urine protein levels and M1/M2(P<0.05).Additionally,there was an improvement in fetal mouse development,trophoblast cell infil-tration and uterine spiral artery remodeling.The number of fetal blood vessels in placental labyrinthine layer increased,and the lu-mens expanded.Conclusion:LA can regulate macrophage polarization in decidua of PE through SRC/LDHA pathway,and blocking LA uptake can alleviate the pathological manifestations in pregnant rats with PE.关键词
乳酸/非受体酪氨酸激酶肉瘤病毒蛋白/乳酸脱氢酶A/子痫前期/巨噬细胞Key words
Lactic acid/Non-receptor tyrosine kinase sarcoma virus protein/Lactate dehydrogenase A/Pre-eclampsia/Macro-phage分类
医药卫生
