[Role and mechanism of epithelial-mesenchymal transition in a rat model of bronchopulmonary dysplasia induced by hyperoxia exposure]. / 上皮-间å è´¨è½¬åŒ–åœ¨é«˜æ°§è¯±å¯¼å¤§é¼ æ”¯æ°”ç®¡è‚ºå‘è‚²ä¸è‰¯æ¨¡åž‹ä¸­çš„ä½œç”¨åŠæœºåˆ¶ç ”ç©¶.

OBJECTIVES: To investigate the role and mechanism of epithelial-mesenchymal transition (EMT) in a rat model of bronchopulmonary dysplasia (BPD). METHODS: The experiment consisted of two parts. (1) Forty-eight preterm rats were randomly divided into a normoxia group and a hyperoxia group, with 24 rats in each group. The hyperoxia group was exposed to 85% oxygen to establish a BPD model, while the normoxia group was kept in room air at normal pressure. Lung tissue samples were collected on days 1, 4, 7, and 14 of the experiment. (2) Rat type II alveolar epithelial cells (RLE-6TN) were randomly divided into a normoxia group (cultured in air) and a hyperoxia group (cultured in 95% oxygen), and cell samples were collected 12, 24, and 48 hours after hyperoxia exposure. Hematoxylin-eosin staining was used to observe alveolarization in preterm rat lungs, and immunofluorescence was used to detect the co-localization of surfactant protein C (SPC) and α-smooth muscle actin (α-SMA) in preterm rat lung tissue and RLE-6TN cells. Quantitative real-time polymerase chain reaction and protein immunoblotting were used to detect the expression levels of EMT-related mRNA and proteins in preterm rat lung tissue and RLE-6TN cells. RESULTS: (1) Compared with the normoxia group, the hyperoxia group showed blocked alveolarization and simplified alveolar structure after 7 days of hyperoxia exposure. Co-localization of SPC and α-SMA was observed in lung tissue, with decreased SPC expression and increased α-SMA expression in the hyperoxia group at 7 and 14 days of hyperoxia exposure compared to the normoxia group. In the hyperoxia group, the mRNA and protein levels of TGF-ß1, α-SMA, and N-cadherin were increased, while the mRNA and protein levels of SPC and E-cadherin were decreased at 7 and 14 days of hyperoxia exposure compared to the normoxia group (P<0.05). (2) SPC and α-SMA was observed in RLE-6TN cells, with decreased SPC expression and increased α-SMA expression in the hyperoxia group at 24 and 48 hours of hyperoxia exposure compared to the normoxia group. Compared to the normoxia group, the mRNA and protein levels of SPC and E-cadherin in the hyperoxia group were decreased, while the mRNA and protein levels of TGF-ß1, α-SMA, and E-cadherin in the hyperoxia group increased at 48 hours of hyperoxia exposure (P<0.05). CONCLUSIONS: EMT disrupts the tight connections between alveolar epithelial cells in a preterm rat model of BPD, leading to simplified alveolar structure and abnormal development, and is involved in the development of BPD. Citation:Chinese Journal of Contemporary Pediatrics, 2024, 26(7): 765-773.

Involvement of miR-495 overexpression in the pathogenesis of bronchopulmonary dysplasia in preterm infants via the targeting of NEDD4L-ENaC pathway.

Background: Bronchopulmonary dysplasia (BPD) is a severe pulmonary complication causing morbidity and mortality in preterm infants. A key histopathological feature of BPD is late lung growth retardation, in which the process of alveolarization is hindered and the mechanism of which is unclear. Emerging evidence indicates that microRNAs (miRNAs) promote the development of BPD via the inhibition of their target genes. MiR-495 has been reported to be involved in various lung diseases. However, the physiological function of miR-495 in BPD has not yet been fully understood. Methods: Differentially expressed miRNAs in peripheral blood of patients with BPD were compared with those of normal controls. A dual-luciferase reporter assay was performed to identify the target genes of miR-495. A BPD neonatal rat model was established by injecting lipopolysaccharide (LPS) in the amniotic sac of pregnant rats. The morphology of the lungs was observed using hematoxylin and eosin (HE) staining. The expression of miR-495, neural precursor cell expressed developmentally down-regulated 4-like (NEDD4L), and epithelial Na+ channel (ENaC) was tested using quantitative reverse transcription-polymerase chain reaction (qRT-PCR), Western blot analysis, and immunofluorescent (IF) staining. Results: The expression of miR-495 was significantly increased in the peripheral blood samples of premature infants with BPD and verified using qRT-PCR. NEDD4L was proven to be the target gene of miR-495. Additionally, miR-495 expression was also increased in the lungs of rat pups with BPD at postnatal day (P) 3 compared with the control group. qRT-PCR and Western blot results showed that NEDD4L expression was decreased while ENaC expression was increased at the transcriptional and translational levels. IF staining results showed that NEDD4L level was decreased while ENaC level was increased in the LPS-induced BPD rat model, which was consistent with abnormal changes in alveolar structure. Conclusions: The aberrant overexpression of miR-495 may contribute to the development of BPD by targeting NEDD4L-ENaC pathway, implying an imbalance in lung fluid clearance.

Loss of Protein Function Causing Severe Phenotypes of Female-Restricted Wieacker Wolff Syndrome due to a Novel Nonsense Mutation in the ZC4H2 Gene.

Pathogenic variants of zinc finger C4H2-type containing (ZC4H2) on the X chromosome cause a group of genetic diseases termed ZC4H2-associated rare disorders (ZARD), including Wieacker-Wolff Syndrome (WRWF) and Female-restricted Wieacker-Wolff Syndrome (WRWFFR). In the current study, a de novo c.352C>T (p.Gln118*) mutation in ZC4H2 (NM_018684.4) was identified in a female neonate born with severe arthrogryposis multiplex congenita (AMC) and Pierre-Robin sequence (cleft palate and micrognathia). Plasmids containing the wild-type (WT), mutant-type (MT) ZC4H2, or GFP report gene (N) were transfected in 293T cell lines, respectively. RT-qPCR and western blot analysis showed that ZC4H2 protein could not be detected in the 293T cells transfected with MT ZC4H2. The RNA seq results revealed that the expression profile of the MT group was similar to that of the N group but differed significantly from the WT group, indicating that the c.352C>T mutation resulted in the loss of function of ZC4H2. Differentially expressed genes (DEGs) enrichment analysis showed that c.352C>T mutation inhibited the expression levels of a series of genes involved in the oxidative phosphorylation pathway. Subsequently, expression levels of ZC4H2 were knocked down in neural stem cells (NSCs) derived from induced pluripotent stem cells (iPSCs) by lentiviral-expressed small hairpin RNAs (shRNAs) against ZC4H2. The results also demonstrated that decreasing the expression of ZC4H2 significantly reduced the growth of NSCs by affecting the expression of genes related to the oxidative phosphorylation signaling pathway. Taken together, our results strongly suggest that ZC4H2 c.352C>T (p.Gln118*) mutation resulted in the loss of protein function and caused WRWFFR.

Severe hyperbilirubinemia in a neonate with hereditary spherocytosis due to a de novo ankyrin mutation: A case report.

BACKGROUND: Hereditary spherocytosis (HS) is a common type of hemolytic anemia caused by a red cell membrane disorder. HS type 1 (HS1) is mostly caused by mutations in ankyrin (ANK1). Newborns with HS1 usually only exhibit anemia and mild jaundice. We herein report a case of HS1 and discuss its clinical characteristics. CASE SUMMARY: A 2-d-old male full-term newborn was admitted to our hospital with severe, intractable neonatal jaundice. Laboratory investigations showed hemolytic anemia and hyperbilirubinemia and excluded immune-mediated hemolysis. The patient underwent two exchange transfusions and one plasmapheresis resulting in significantly reduced serum bilirubin. Hematologic analyses and genomic DNA sequencing studies were performed. The trio clinical exome sequencing revealed a de novo null heterozygous mutation in the patient's ANK1 gene: c.841C > T(p.Arg281Ter). This mutation results in the premature termination of the ANK1 protein. CONCLUSION: Our case demonstrates that genetic analysis can be an essential method for diagnosing HS when a newborn has severe hyperbilirubinemia.

Association of Thyroid Function During Pregnancy With the Risk of Pre-eclampsia and Gestational Diabetes Mellitus.

OBJECTIVE: To estimate the association of maternal thyroid dysfunction with the risk of gestational hypertension and diabetes. Whether the association was affected by gestational age at diagnosis and thyroid autoimmunity was further explored. METHODS: A cohort study of 41 647 participants was conducted. Thyroid function (ie, thyroid-stimulating hormone [TSH] and free thyroxine [FT4]) was measured by electrochemiluminescence immunoassay. Thyroid antibody positivity (eg, thyroperoxidase, thyroglobulin, and TSH receptor antibody) was indicated if the values of these antibodies exceeded the upper targets of the reference range. The relationship between maternal thyroid dysfunction and the risk of pre-eclampsia (PE) and gestational diabetes mellitus (GDM) was assessed by multivariate logistic regression. RESULTS: Isolated hypothyroxinemia (defined as 5th ≤ TSH ≤ 95th percentile, FT4 < 5th percentile) was associated with the risk of PE (odds ratio [OR], 1.32; 95% CI, 1.10-1.58). Overt hypothyroidism (TSH > 95th percentile; FT4 < 5th percentile) was related to the risk of severe PE (OR, 2.59; 95% CI, 1.05-6.37). Being positive for TSH receptor antibody was associated with a decreased risk of GDM (OR, 0.49; 95% CI, 0.35-0.70). A marginally significant association between overt hypothyroidism detected at the first trimester and the risk of GDM was found (OR, 1.60; 95% CI, 1.00-2.83). The association of thyroid dysfunction with the risk of PE and GDM was stronger among pregnant women who were negative for autoantibodies. CONCLUSION: Some types of thyroid dysfunction during pregnancy were associated with the risk of PE and GDM. The associations varied by gestational age at diagnosis and by thyroid autoantibody status.

[Expression of microRNA-495-5p in preterm infants with bronchopulmonary dysplasia: a bioinformatics analysis].

OBJECTIVE: To study the expression of microRNA-495-5p (miRNA-495-5p) in the serum of preterm infants with bronchopulmonary dysplasia (BPD) based on a bioinformatics analysis, and to provide a theoretical basis for further research on the association between miRNA-495-5p and BPD. METHODS: A total of 40 preterm infants who were admitted to the neonatal intensive care unit from January 2015 to December 2016 were enrolled. Among these infants, 20 with early clinical manifestations of BPD were enrolled as the BPD group, and 20 without such manifestations were enrolled as the control group. Peripheral blood samples were collected. The miRNA microarray technique was used to screen out differentially expressed miRNAs in serum between the two groups. RT-PCR was used for validation of results. TargetScan, miRDB, and miRWalk databases were used to predict the target genes of miRNA-495-5p. The DAVID database was used to perform gene ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of the target genes. RESULTS: Compared with the control group, the BPD group had a significant increase in the expression of miRNA-495-5p in serum (P<0.05). A total of 117 target genes of miRNA-495-5p were predicted by the above three databases and they were involved in several molecular functions (including transcriptional regulatory activity, transcriptional activation activity, and transcription cofactor activity), biological processes (such as metabolic regulation, DNA-dependent transcriptional regulation, and vascular pattern), and cell components (including nucleoplasm, membrane components, and insoluble components) (P<0.05). As for signaling pathways, these genes were significantly enriched in the mTOR signaling pathway (P<0.05). CONCLUSIONS: MiRNA-495-5p may be involved in the development and progression of BPD by regulating angiogenesis, stem cell differentiation, apoptosis, and autophagy, which provides clues for further research on the role and functional mechanism of miRNA-495-5p in BPD.

Novel PITX2c loss-of-function mutations associated with complex congenital heart disease.

Congenital heart disease (CHD) is the most common form of birth defect in humans and is the leading non-infectious cause of infant mortality. Emerging evidence strongly suggests that genetic risk factors play an important role in the pathogenesis of CHD. However, CHD is of pronounced genetic heterogeneity, and the genetic defects responsible for CHD in an overwhelming majority of patients remain unclear. In this study, the entire coding region and splice junction sites of the PITX2c gene, which encodes a paired-like homeodomain transcription factor crucial for proper cardiovascular morphogenesis, was sequenced in 170 unrelated neonates with CHD. The available relatives of the mutation carriers and 200 unrelated ethnically matched healthy individuals were genotyped. The disease-causing potential of the PITX2c sequence variations was predicted by MutationTaster and PolyPhen-2. The functional effect of the mutations was characterized using a luciferase reporter assay system. As a result, 2 novel heterozygous PITX2c mutations, p.R91Q and p.T129S, were identified in 2 unrelated newborns with transposition of the great arteries and ventricular septal defect, respectively. A genetic scan of the pedigrees revealed that each mutation co-segregated with CHD transmitted in an autosomal dominant pattern with complete penetrance. The mutations, which altered the amino acids completely conserved evolutionarily, were absent in 400 normal chromosomes and were predicted to be causative. Functional analysis revealed that the PITX2c mutations were both associated with significantly diminished transcriptional activity compared with their wild-type counterpart. This study demonstrates the association between PITX2c loss-of-function mutations and the transposition of the great arteries and ventricular septal defect in humans, providing further insight into the molecular mechanisms responsible for CHD.

[Rapid screening for MTHFR gene 677C>T polymorphism in Down syndrome using high resolution melting curve and pyrosequencing].

OBJECTIVE: To establish a rapid method for detecting MTHFR gene 677C>T polymorphisms with high-resolution melting curve method (HRM) and pyrosequencing. METHODS: Peripheral blood samples were collected from 155 Down syndrome patients and 182 normal controls from Children's Hospital of Shanghai. The accuracy of three methods including regular HRM, internal control HRM and artificial heterozygosity HRM was compared. Meanwhile, allele frequencies in 10, 30 and 50 mixed samples were measured with pyrosequencing, and the results were compared with that of HRM. RESULTS: Heterozygosity of 677C>T polymorphism could be distinguished by various HRM methods. However, homozygotes CC and TT were only identifiable by internal control HRM and artificial heterozygosity HRM. The accuracy of pyrosequencing for allele frequency has improved with increased sample number. When the number of mixed samples has exceeded 30, the difference between pyrosequencing results and actual values became less than 4%. TT genotype was more frequent in Down syndrome patients than controls (25.2% vs. 14.3%). No significant difference was found in T allele frequency between the two groups (44.9% vs. 40.1%). CONCLUSION: Respectively, internal control HRM and pyrosequencing may be ideal methods for determination of genotypic and allelic frequencies.

[Effects of vasoactive intestinal peptide on Toll-like receptor (TLR) 2 mRNA and TLR4 mRNA expression on acute lung injury induced by lipopolysaccharide in rat].

OBJECTIVE: Vasoactive intestinal peptide (VIP) is a neuro-peptide that can modulate immunity. Previous studies indicated that VIP can attenuate the deleterious consequences of severe sepsis and septic shock by regulating production of inflammatory cytokines in immune activated cells. The signaling induced by bacterial components occurs primarily through Toll like receptors (TLRs). TLRs have been recognized to play a key role in pathogen recognition and innate immunity. It was convincingly demonstrated that lung is one of early suffered disaster organ and may trigger multiple organ dysfunction syndrome in sepsis. The present study was conducted to investigate the effects of VIP on TLR2/4 mRNA expressions on acute lung injury of endotoxic shock induced by lipopolysaccharide (LPS) in rat. METHOD: Forty Sprague-Dawley rats were randomly divided into 3 groups, i.e., LPS shock group (n = 16), LPS + VIP group (n = 16), and control group (n = 8). LPS shock model was established by LPS (E. coli O(55)B(5) 10 mg/kg) with tail intravenous injection. The rats in LPS + VIP group were given a bolus of 5 nmol VIP intravenous injection follow by LPS. The rats in control group were given normal saline. The rats were sacrificed at 6 h, 24 h after being injected. The lung tissues were collected. The TLR2 mRNA and TLR4 mRNA expressions were detected by RT-PCR from the lung tissues. Pathological changes of the lungs were observed by light microscope and electron microscope 24 h after LPS injection. RESULT: (1) Lung histopathology: the alveolar space was full with leukocyte, necrotic cells, segmental hemorrhage and protein effusion. Partial alveolar space was enlarged, lung interstitial edema were observed in LPS shock group. However, pathological changes of LPS + VIP group were milder than those in LPS shock group. (2) The expressions of TLR2 mRNA and TLR4 mRNA were significantly higher in LPS shock group compared with those of the control group (F = 16.638, P = 0.000; t = 5.876, P = 0.000), TLR2 mRNA and TLR4 mRNA expression on 24 h was down-regulated in LPS + VIP shock subgroup than those in LPS shock subgroup (F = 16.676, P = 0.000; t = 3.946, P < 0.001). CONCLUSION: Expressions of TLR2 mRNA and TLR4 mRNA were up-regulated on LPS induced lung injury in rats. VIP mitigated lung injury induced by LPS, which may be related to TLR2 mRNA and TLR4 mRNA down-regulation of expression. The effect of VIP may suggest a protective mechanism in sepsis. VIP may play a potential protective role in severe infection.

Regulation of activity of nuclear factor-kappaB and activator protein-1 by nitric oxide, surfactant and glucocorticoids in alveolar macrophages from piglets with acute lung injury.

AIM: To investigate whether acute lung injury (ALI) in ventilated piglets with bacterial infection affects NF-kappaB and AP-1 expression in alveolar macrophages (AM) and whether nitric oxide (NO), surfactant (Surf), glucocorticoids (GC) affect NF-kappaB and AP-1 activation in AM in vivo and in vitro. METHODS: The animals were intraperitoneally injected Escherichia coli, which caused ALI. Nuclear extracts of AM were analyzed by electrophoretic mobility shift assay (EMSA) for the nuclear factor-kappa B (NF-kappaB) and activation protein-1 (AP-1) expression. Detection of IkappaB-alpha protein was from cytoplasmic extract by Western blotting. Immunocytochemistry staining was used for intracellular location of p65 subunits of NF-kappaB. RESULTS: In ex vivo experiments, strikingly higher expression of NF-kappaB and AP-1 by EMSA was found 6 h after bacterial injection in contrast to the Normal group. In the NO, SNO, and GC groups, markedly attenuated NF-kappaB and AP-1 activation was observed. The NF-kappaB and AP-1 activation in Surf group showed lower levels of the expression. Immunoblotting of AM cytoplasmic extract showed low expression of IkappaB-alphaprotein in the Control and Surf groups. The stronger expression was observed in the NO, GC, and SNO groups. AM of the Control and Surf groups showed intense nuclear staining, with decreased nuclear staining in the NO, GC and SNO groups. In in vitro experiment, it caused a significant increase in NF-kappaB and AP-1 activity in AM 1 h after exposure to lipopolysaccharides (LPS). In AM treated by LPS+SNP and LPS+GC, all showed decrease of DNA binding activity of NF-kappaB and AP-1 compared to those exposed to LPS+Surf. Immunoblotting of AM cytoplasmic extract showed that LPS stimulation of AM resulted in the low expression of IkappaB-alpha protein, which was not observed in the presence of SNP and methylprednisolone. However, the surfactant did not show such effect. LPS+Surf-exposed AM had intense nuclear staining, whereas decreased nuclear staining in the LPS+NO and LPS+GC-treated cultures was found, confirming a decrease in NF-kappaB activity. CONCLUSION: Activation of NF-kappaB was found in AM of ventilated piglets with bacterial ALI. NO and GS could prevent NF-kappaB and AP-1 activation in vivo and in vitro. Surfactant has limited effects on NF-kappaB and AP-1 activity.