The Effect And Mechanism Of Enzyme Arg1 In Coordination Of Fetal Lung Development And Parturition

Preterm birth is one of the most common obstetric complications and the leading cause of neonatal death and teratogenesis.However,the incidence of preterm birth has not been effectively controlled in recent decades.The fundamental reason is that the initiation mechanism of delivery has not been fully elucidated.Therefore,clear mechanisms about onset of labor to drop the incidence of preterm birth without delay.The previous work of our group found that steroid hormone receptor co-activator(SRC)-deficient mice had delayed fetal lung growth and delayed delivery,suggesting that the abnormal start of labor may be related to the maturation of fetal lungs and the secretion of fetal factors.So we performed RNA-seq analysis and verification on SRC-deficient and wild-type(WT)mouse fetal lungs at 18.5 days post coitum(18.5 dpc),and found the transcription levels of arginase(Arg1)and argininosuccinate synthase(Ass1)reduced in SRC1/2 double gene homozygous knockout(dKO)mice.As Arg1 and Ass1 are metabolic enzymes of the urea cycle,they are related to the synthesis and metabolism of arginine.Therefore,we further tested the levels of small molecular metabolites such as amino acids in the fetal lungs and found that the arginine content was significantly increased in the dKO group,while the level of the metabolite ornithine of arginine showed an opposite trend,which consistent with the changes in the expression levels of Arg1 and Ass1.Since Arg1 is a key gene in the lung development pathway and affects cell proliferation and apoptosis,we propose a scientific hypothesis: Arg1 coordinates fetal development and initiation of labor by affecting fetal lung development and the synthesis of related small molecule metabolites.This study explores the effects of Arg1 on the growth and development of fetal lungs and the initiation of labor.We detected the expression of Arg1 and Ass1 in the fetal lungs in the middle and late stages of normal pregnancy,and then used placental injection to interfere with the mouse fetal lungs to observe the start time of parturition and the growth and development of the fetal lung.Through the above experiments,it is clear that Arg1 plays a role in fetal lung development and labor initiation,which provides a new target for exploring the mechanism of preterm birth and preventing preterm birth.Main Results18.5 dpc fetal lungs were collected from SRC-deficient mice and WT mice.RNAseq analysis found that the transcription levels of Arg1 and Ass1 were lower in dKO mice.RT-PCR,Western blotting and immunohistochemical experiments showed that the expressions of Arg1 and Ass1 in fetal lungs of dKO mouse were down-regulated compared with WT group.The mRNA and protein level of Arg1 and Ass1 were significantly up-regulated in the middle and late stages of pregnancy in WT mice,and reached a peak at 19.5 dpc.Immunohistochemistry results demonstrated that both Ass1 and Arg1 expressed in fetal lungs from 15.5,17.5 and 19.5 days post-coitum WT mice,and the lowest expression occurred at 15.5 dpc,the highest expression occurred at 19.5 dpc.Multiplex immunofluorescence assay showed that Arg1 was expressed in type Ⅱ alveolar epithelial cells,while Ass1 was mainly expressed in type Ⅰ alveolar epithelial cells.LCMS-MS combined with MRM technology was used to perform broad-spectrum targeted metabolomics detection of WT and 18.5 dpc dKO fetal lung tissues,a total of200 small molecule metabolites were detected.KEGG pathway enrichment analysis results revealed that arginine biosynthesis pathway and arginine and proline metabolic pathways are significantly enriched.Arginine and ornithine are directly related metabolites of Arg1,the concentration of arginine was significantly up-regulated in dKO group,while that of ornithine was reversed.There was no significant difference in the content of citrulline as a substrate in the metabolic reaction catalyzed by Ass1,and the reaction product arginine-succinic acid was not detected due to its low concentration.Either AAV that interferes with Arg1 expression(AAV-Arg1)or unloaded AAV(AAV-2/9)was injected into the placenta of WT mice at 13 dpc,both AAVs successfully infected fetal lungs.AAV-Arg1 injection can significantly decrease the protein expression of Arg1 in fetal lungs,but the expression of Arg1 in fetal livers,placentas and myometrium were not significantly changed.Compared with mice injected with AAV-2/9,the activation of NF-κB and the expression of contractile related protein were both downregulated in 18.5 dpc myometrium of mice injected with AAV-Arg1.Finally,the delayed delivery occurred in AAV-Arg1-injected pregnant mice.After placentas were injected with different viruses,we found the expression of the Ki67 and precursors of Caspase3 in the lung tissues of the parturient fetuses did not change significantly between the AAV-Arg1 mice and AAV-2/9 mice,but the expression of two Caspase3 splicing bodies was increased significantly in the AAV-Arg1 group.TUNEL assay and immunofluorescence test also showed that cell apoptosis was increased in the lung tissues of fetuses after injecting AAV-Arg1 compared with that of AAV-2/9 group during labor,however,there was no significant difference in cell proliferation.Conclusions:The expression of Arg1 and Ass1 specifically decreased in dKO fetal lungs,but they were significantly increased in the fetal lungs of WT mice in the middle and late stages of pregnancy,and reached a peak during labor.Ass1 and Arg1 were expressed in type Ⅰ and type Ⅱ alveolar epithelial cells,respectively.These two key enzymes are closely related to the synthesis and metabolism of arginine and citrulline.The changes in the levels of arginine and citrulline in the fetal lungs are consistent with the changes in the protein expression of Arg1.Interfering with the expression of Arg1 in fetal lungs aggravated the apoptosis of fetal lung cells,suggesting that Arg1 may promote cell apoptosis by altering amino acid metabolism in fetal lungs and affect fetal lung development.Furthermore,knockdown of Arg1 in fetal lungs significantly inhibited the activation of NF-κB and down-regulated the expression of contraction-related proteins in myometrium,causing a dramatically delayed delivery in mice.The results of the study suggest Arg1 may have dual roles in coordinating the fetal lung development and signaling the initiation of labor by affecting amino acid metabolism,which means fetusderived factors are expected to be potential targets for the treatment of fetal organ immaturity and the prevention of preterm labor.