Objective To explore the effect of hypoxia-inducible factor-1α(HIF-1α)inhibitor LW6 on ferroptosis in septic cardiomyopathy rats.Methods Rat septic cardiomyopathy model was prepared using cecal ligation and puncture(CLP)method.Thirty-six specific pathogen-free(SPF)6-8 weeks male SD rats were randomly divided into the sham-operated group,CLP group,CLP+solvent group,LW6 group,ferrostatin-1(Fer-1)group,and LW6+Fer-1 group.The degree of myocardial damage in each group was evaluated through hematoxylin-eosin stai-ning and detection of lactate dehydrogenase and creatine kinase content in cardiac tissue.Myocardial mitochondrial damage was observed by transmission electron microscopy.Ferroptosis level was determined by detecting iron ion concentration,reduced glutathione,malondialdehyde,and reactive oxygen species.Protein expression levels of HIF-1α,solute carrier family 7 member 11(SLC7A11),and glutathione peroxidase 4(GPX4)in cardiac tissue were detected by Western blotting.Results Compared with the CLP group and the CLP+solvent group,the LW6 group could ameliorate myocardial damage,alleviate mitochondrial damage,inhibit ferroptosis-related indicators(all P＜0.05),reduce HIF-1α protein expression levels(P＜0.05),and enhance SLC7A11 and GPX4 protein expression levels(both P＜0.05).Conclusion LW6 decreases HIF-1α expression and ferroptosis levels through the SLC7A11/GPX4 pathway,and ameliorates sepsis-induced cardiomyopathy.

Objectives:To evaluate the impact of hemoglobin concentration on 5-year cardiovascular mortality in patients with hypertrophic obstructive cardiomyopathy(HOCM).Methods:This study retrospectively analyzed 325 non-surgically treated HOCM patients hospitalized at Fuwai Hospital from October 2009 to December 2014.Baseline information was compared between patients with or without cardiac death.The impact of hemoglobin concentration on 5-year cardiovascular mortality in HOCM patients was analyzed.Results:The median follow-up time was(43.55±19.70)months.During the follow-up period,a total of 29 patients(8.9%)experienced cardiac death.Univariate Cox regression analysis demonstrated that hemoglobin concentration was significantly associated with 5-year cardiac mortality in HOCM patients(P<0.001).After adjusting for potential cardiovascular risk factors in multivariate Cox regression analysis,hemoglobin concentration(P=0.011)remained negatively associated with 5-year cardiac mortality in HOCM patient.HOCM patients with decreased hemoglobin level faced a 3.118-fold increase in 5-year cardiac mortality(HR=4.118,95%CI:1.114-14.822,P=0.030).Kaplan-Meier survival analysis showed that HOCM patients with decreased hemoglobin levels had a significantly higher risk of 5-year cardiac mortality(log-rank test,χ2=24.38,P<0.001).Conclusions:Lower hemoglobin concentration is an independent risk factor for 5-year cardiac mortality in patients with HOCM.Compared to patients with normal hemoglobin levels,HOCM patients with decreased hemoglobin level face a 3-fold increase in 5-year cardiac mortality.

Objectives:To evaluate the impact of hemoglobin concentration on 5-year cardiovascular mortality in patients with hypertrophic obstructive cardiomyopathy(HOCM).Methods:This study retrospectively analyzed 325 non-surgically treated HOCM patients hospitalized at Fuwai Hospital from October 2009 to December 2014.Baseline information was compared between patients with or without cardiac death.The impact of hemoglobin concentration on 5-year cardiovascular mortality in HOCM patients was analyzed.Results:The median follow-up time was(43.55±19.70)months.During the follow-up period,a total of 29 patients(8.9%)experienced cardiac death.Univariate Cox regression analysis demonstrated that hemoglobin concentration was significantly associated with 5-year cardiac mortality in HOCM patients(P<0.001).After adjusting for potential cardiovascular risk factors in multivariate Cox regression analysis,hemoglobin concentration(P=0.011)remained negatively associated with 5-year cardiac mortality in HOCM patient.HOCM patients with decreased hemoglobin level faced a 3.118-fold increase in 5-year cardiac mortality(HR=4.118,95%CI:1.114-14.822,P=0.030).Kaplan-Meier survival analysis showed that HOCM patients with decreased hemoglobin levels had a significantly higher risk of 5-year cardiac mortality(log-rank test,χ2=24.38,P<0.001).Conclusions:Lower hemoglobin concentration is an independent risk factor for 5-year cardiac mortality in patients with HOCM.Compared to patients with normal hemoglobin levels,HOCM patients with decreased hemoglobin level face a 3-fold increase in 5-year cardiac mortality.

Objective To explore the effect of hypoxia-inducible factor-1α(HIF-1α)inhibitor LW6 on ferroptosis in septic cardiomyopathy rats.Methods Rat septic cardiomyopathy model was prepared using cecal ligation and puncture(CLP)method.Thirty-six specific pathogen-free(SPF)6-8 weeks male SD rats were randomly divided into the sham-operated group,CLP group,CLP+solvent group,LW6 group,ferrostatin-1(Fer-1)group,and LW6+Fer-1 group.The degree of myocardial damage in each group was evaluated through hematoxylin-eosin stai-ning and detection of lactate dehydrogenase and creatine kinase content in cardiac tissue.Myocardial mitochondrial damage was observed by transmission electron microscopy.Ferroptosis level was determined by detecting iron ion concentration,reduced glutathione,malondialdehyde,and reactive oxygen species.Protein expression levels of HIF-1α,solute carrier family 7 member 11(SLC7A11),and glutathione peroxidase 4(GPX4)in cardiac tissue were detected by Western blotting.Results Compared with the CLP group and the CLP+solvent group,the LW6 group could ameliorate myocardial damage,alleviate mitochondrial damage,inhibit ferroptosis-related indicators(all P＜0.05),reduce HIF-1α protein expression levels(P＜0.05),and enhance SLC7A11 and GPX4 protein expression levels(both P＜0.05).Conclusion LW6 decreases HIF-1α expression and ferroptosis levels through the SLC7A11/GPX4 pathway,and ameliorates sepsis-induced cardiomyopathy.

Objective:To investigate the changes and significance of granulocyte-like myeloid-derived suppressor cells (G-MDSC) in the acute phage of Kawasaki disease (KD).Methods:Forty-two children with acute KD were enrolled in the present study and 32 age-matched healthy children were selected as control group. The proportion of HLA-DR -CD11b + CD33 + CD14 -CD15 + G-MDSC, the concentration of reactive oxygen species (ROS) and the expression of arginase-1 (Arg-1), programmed death-ligand 1 (PD-L1), cytotoxic T lymphocyte associated protein 4 (CTLA4), glycoprotein 130 (gp130) and phosphorylated signal transducer and activator of transcription 3 (pSTAT3) at protein level were detected by flow cytometry. Quantitative real-time PCR was used to measure the expression of inducible nitric oxide synthase (iNOS), interferon regulatory factor 8 (IRF-8), IL-6 receptor α subunit (IL-6Rα), granulocyte colony-stimulating factor receptor (G-CSFR), CCAAT/enhancer binding protein β (C/EBPβ), suppressor of cytokine signaling 1 (SOCS1) and SOCS3 at mRNA level in G-MDSC. Chromatin immunoprecipitation was performed to detect the acetylation of histone H3 at the promoters of SOCS1 and SOCS3 genes. Plasma concentrations of IL-6 and granulocyte colony-stimulating factor (G-CSF) and protein levels of IL-10, transforming growth factor-β (TGF-β) and nitric oxide (NO) in the culture supernatant of G-MDSC stimulated with LPS were measured by ELISA. Results:(1) Compared with the control group, the proportion of HLA-DR -CD11b + CD33 + CD14 -CD15 + G-MDSC as well as the concentration of ROS and the expression of inhibitory molecules (Arg-1, PD-L1 and CTLA4) in G-MDSC increased significantly in patients with acute KD ( P<0.05). Moreover, the concentrations of IL-10 and TGF-β in culture supernatant of G-MDSC were also higher than those of the control group after stimulation with lipopolysaccharide for 48 h ( P<0.05). All of the seven afore-mentioned indexes in KD patients with coronary artery lesion (CAL group ) were lower than those in patients without coronary artery lesion (NCAL group) ( P<0.05), and restored to some extent after IVIG therapy ( P<0.05). There were no statistical differences in iNOS expression or NO concentration in culture supernatant of G-MDSC among different groups ( P<0.05). (2) Plasma concentrations of IL-6 and G-CSF, and the expression of IL-6Rα, gp130, G-CSFR, pSTAT3 and C/EBPβ increased remarkably during acute phase of KD ( P<0.05). The expression of IRF-8 at transcription level in patients with acute KD was found to be lower than that of healthy controls ( P<0.05), and restored significantly after IVIG therapy ( P<0.05). Moreover, the plasma concentrations of IL-6 and G-CSF and the expression of IL-6Rα, gp130, G-CSFR and IRF-8 in the CAL group were higher than those in the NCAL group ( P<0.05), while the expression of pSTAT3 and C/EBPβ was lower in the CAL group ( P<0.05), which were restored by IVIG therapy ( P<0.05). (3) In patients with acute KD, the expression of SOCS1 and SOCS3 at mRNA level and histone acetylation at the promoters of SOCS1 and SOCS3 genes were reduced significantly in comparison with those in healthy controls ( P<0.05) , but were increased remarkably after IVIG treatment( P<0.05). The four indexes were higher in the CAL group than in the NCAL group ( P<0.05). Pearson correlation analysis showed the expression of SOCS1 and SOCS3 was negatively correlated with the protein level of pSTAT3 in G-MDSC of patients with acute KD ( r=-0.46 and -0.32, P<0.05). Conclusions:Changes in the number and function of G-MDSC caused by aberrant histone acetylation at SOCS1 and SOCS3 genes might contribute to the immune dysfunction and vascular damage in patients with KD.

Objective:To explore the effect of Notch1 signaling on regulatory T cells and its roles in vascular damage in patients with Kawasaki disease (KD).Methods:A total of 42 children with KD were enrolled in the present study from March 2019 to June 2020, as 32 age-matched healthy children were recruited as control. The proportions of CD4 +CD25 hiFoxp 3+ regulatory T cells (Treg) and expressions of transcription factor forkhead box protein 3 (Foxp3), cytotoxic T lymphocyte associated antigen-4 (CTLA4), glucocorticoid-induced tumor necrosis factor receptor (GITR), and Notch1 protein were evaluated by flow cytometry. Chromatin immunoprecipitation was conducted to detect acetylation level of histone H4 (H4Ac) associated with the promoter of Foxp3 gene and its binding abilities of Notch1 intracellular domain 1 (NICD1), recombination signal binding protein for immunoglobulin kappa J region (RBP-J) and p300 in CD4 + T cells. Transcription levels of Foxp3, presenilin 1 (PSEN1), mastermind like transcriptional coactivator 1 (MAML1), and RBP-J in CD4 + T cells were determined by real-time polymerase chain reaction (PCR). Concentrations of interleukin (IL)-10 and transforming growth factor-β (TGF-β) in plasma and culture supernatant stimulated with Jagged1 were measured by enzyme linked immunosorbent assay. Independent-sample t-test, Pearson correlation analysis was used as the statistical method in this study. Results:① The frequencies of Treg in acute KD patients decreased significantly [(4.3±1.5)% vs (7.9±2.9)%; t=6.41, P<0.001], as protein levels of Foxp3, CTLA4 and GITR and concentrations of IL-10 and TGF-β in plasma reduced remarkably in acute KD patients ( t=6.87, P<0.001; t=4.26, P<0.001; t=7.88, P<0.001; t=8.42, P<0.001; t=13.01, P<0.001). All parameters afore-mentioned in patients combined with coronary artery lesions (CAL) were lower than those of patients without coronary artery lesions (NCAL) ( t=5.83, P<0.001; t=3.83, P<0.001; t=3.28, P=0.002; t=5.05, P<0.001; t=5.96, P<0.001; t=5.17, P<0.001), and increased after therapy ( t=7.13, P<0.001; t=6.10, P<0.001; t=4.31, P<0.001; t=6.55, P<0.001; t=7.40, P<0.001; t=7.84, P<0.001). ② H4Ac associated with promoter of Foxp3 gene and the binding abilities of NICD1 and p300 in acute KD patients were lower than those of the controls ( t=10.25, P<0.001; t=6.93, P<0.001; t=6.75, P<0.001), and increased remarkably after therapy ( t=7.72, P<0.001; t=4.16, P<0.001; t=5.76, P<0.001). Meanwhile, the three items in CAL group were found to be less than those of NCAL group ( t=6.08, P<0.001; t=2.66, P=0.011; t=6.02, P<0.001). Pearson correlation analysis showed a positive correlation between H4Ac associated with Foxp3 promoter and its mRNA level in acute KD patients ( r=0.47, P<0.001). No statistical significant difference about the binding ability of RBP-J with Foxp3 promoter were found among the groups ( t=0.57, P>0.05; t=0.61, P>0.05; t=1.20, P>0.05). ③ Protein level of Notch1 and the expressions of PSEN1, MAML1 and RBP-J mRNA in CD4 + T cells from acute KD patients were down-regulated remarkably ( t=5.28, P<0.001; t=6.31, P<0.001; t=11.78, P<0.001; t=8.06, P<0.001), and restored after therapy ( t=4.77, P<0.001; t=6.43, P<0.001; t=11.95, P<0.001; t=7.79, P<0.001). In parallel, the four indexes aforementioned of CAL group were lower than those of NCAL group ( t=3.16, P=0.003; t=4.13, P<0.001; t=5.42, P<0.001; t=4.05, P<0.001). Upon rhJagged1 stimulation for 48 hours, H4Ac level of Foxp3 promoter and its binding abilities with NICD1 and p300 in CD4 + T cells in KD patients and control group was significantly higher than those of untreated group [(KD: t=15.36, P<0.001; t=7.25, P<0.001; t=14.29, P<0.001), (Ctrl: t=7.87, P<0.001; t=5.71, P<0.001; t=8.74, P<0.001)], as the binding ability of RBP-J with Foxp3 promoter increased slightly without statistically significant difference (KD: t=1.11, P>0.05; Ctrl: t=1.37, P>0.05). Simultaneously, H4Ac level of Foxp3 promoter and its binding abilities with NICD1 and p300 in KD group were still lower than those of the control group after stimulation ( t=3.86, P<0.001; t=3.42, P=0.001; t=2.85, P=0.006). ④ After incubation of PBMC from heathy children with KD serum, the proportion of Treg cells, protein level of Foxp3 and expressions of Notch1 and RBP-J in CD4 + T cells in the group treated with IVIG increased significantly compared with the untreated group ( t=7.10, P<0.001; t=10.16, P<0.001; t=8.06, P<0.001; t=9.77, P<0.001), as well as H4Ac level of Foxp3 promoter and its binding abilities with NICD1 in the group treat with IVIG were also higher than the latter ( t=7.24, P<0.001; t=8.24, P<0.001). Conclusion:Insufficiency and impaired function of Treg caused by aberrant Notch1 signaling may be the important factor contributing to immune dysfunction and vascular damage in KD.

OBJECTIVE: To explore gender difference in the clinical manifestations of two children with Keishi-Bukuryo-Gan syndrome (KBGS). METHODS: Clinical manifestations of the two children were reviewed. Genetic testing was carried out through next generation sequencing (NGS). Treatment was summarized, and the prognosis was followed up. RESULTS: Both children showed particular appearance including megatooth, abnormal hair distribution, hands' abnormality and language development delay. NGS revealed that both children have carried pathogenic variants of the ANKRD11 gene (c.1903_1907del and c.4911delT), which resulted in shifting of amino acid sequences starting from the Lysine and Proline at positions 635 and 1638, respectively. The female patient exhibited central precocious puberty. Her height has increased by 13 cm, and sex characteristics has retracted after treatment with leuprorelin for 23 months and recombinant human growth hormone for 1 month. CONCLUSION Comparison of the two cases with different genders and summary of previously reported cases found that male KBGS patients have more obvious dysmorphisms such as triangular face, synophrys, ocular hypertelorism and vertebral body abnormality, with higher morbidity of epilepsy, mental retardation, autism, congenital heart disease, immune thrombocytopenia and other complications. KBGS is an autosomal dominant disease featuring more evident peculiar appearance and global development delay. Male patients often have multi-system involvement, and multidisciplinary cooperation is required for early recognition of particular features in order to improve the prognosis.
Abnormalities, Multiple ; Bone Diseases, Developmental ; Child ; Facies ; Female ; Humans ; Intellectual Disability ; Male ; Phenotype ; Repressor Proteins/genetics* ; Sex Characteristics ; Tooth Abnormalities

OBJECTIVE:To establish a method for the concentration determination of voriconazole in human plasma.METHODS:Plasma samples were precipitated with acetonitrile.Using ketoconazole as internal standard,HPLC method was adopted.The determination was performed on Dionex U-3000 Dimonsil C18 column with mobile phase consisted of triethylamine-glacial acetic acid-water mixed solution (1 ∶ 1∶98,V/V/V,pH was about 4.0)-acetonitrile (40∶60,V/V) at the flow rate of 1.0 mL/min.The detection wavelength was set at 255 nm.The column temperature was 40 ℃,and sample size was 20 μL.RESULTS:The linear range of voriconazole was 0.2-20.0 μg/mL.The limits of quantification was 0.2 μg/mL,and the minimum detection limit was 0.03 μg/mL.RSDs of inter-day and intra-day were lower than 10％.The method recoveries were 92.06％-106.26％ (RSD＜5％,n=5),and extraction recoveries were 75.62 ％-90.59 ％ (RSD ＜ 5 ％,n=5).The plasma concentration of voriconazole in 10 children ranged 0.22-4.90 μg/mL (n =10).CONCLUSIONS:The method is simple,rapid,specific and can be used for drug monitoring of voriconazole.

Objective To investigate the effects of polycyclic aromatic hydrocarbons ( PAHs) on regulatory T ( Treg) cells in newborns.Methods Blood samples were taken from the umbilical cord of sev-en newborn babies.CD4+CD25+T cells were isolated and treated with or without 300 nmol/L of phenan-threne for 72 hours in the presence of 100 IU/ml of IL-2.The expression of forkhead box P3 (Foxp3), sig-nal transducer and activator of transcription 3 (STAT3) and STAT5 were analyzed by 8-color flow cytometry. RT-PCR was performed to detect the expression of DNA ( cytosine-5 )-methyltransferase 1 ( DNMT1 ) , DNMT3a, DNMT3b and IL-4 at mRNA level.Pyrosequencing in combination with bisulphite sequencing was used to evaluate the methylation within the promoter and the Treg-specific demethylated region ( TSDR) of Foxp3 locus.Treg cells were cultured for 7 hours with autologous Tresp at Tresp/Treg ratios of 1 ∶1, 2 ∶1, 4 ∶1 and 8 ∶1 and stimulated with anti-CD3/CD28 beads and IL-2 for the evaluation of the immunosuppres-sive activities of Treg cells.Results (1) PAHs inhibited the expression of Foxp3 and the function of Treg cells collected from newborns.(2) PAHs significantly decreased the expression of STAT5 and Foxp3, but increased the expression of STAT3 (P<0.05).(3)PAHs enhanced the methylation of the promoter and the TSDR within Foxp3 gene.(4) The transcription levels of DNMT1, DNMT3a and DNMT3b in PAHs treated group were significantly higher than those of the control group (P<0.05).(5) More IL-4 was secreted by PAHs treated CD4+CD25+T cells, indicating that IL-4 was negatively correlated with STAT5, but positively correlated with STAT3.Conclusion PAHs decreased the number and inhibited the function of Treg cells in newborns.The possible mechanism might be related to the abnormal expression of STAT3 and STAT5 in-duced by IL-4 as well as the methylation within Foxp3 gene.

OBJECTIVESepsis is the major cause of death in pediatric intensive care unit (PICU). The clinical manifestations of early sepsis is very similar to systemic inflammatory response syndrome (SIRS) caused by non-infectious reason. This study aimed to investigate the expression of miRNA and inflammatory cytokines in plasma in pediatric sepsis patients and its clinical significance.

METHODForty children with sepsis seen in Shenzhen children's hospital PICU from April 2012 to March 2013 were enrolled in this study, the median age was 0.75 (0.52, 1.90) years; 27 were males and 13 females, of whom 16 had severe sepsis. We selected 20 postsurgical patients with SIRS and 15 healthy children as a control group. The expression levels of plasma miR-21, miR-125b, miR-132, miR-146a, miR-155 and miR-223 were detected by real-time quantitative PCR (qRT-PCR). The predictive value of miRNA, PCT and CRP for sepsis were evaluated by Receiver operating characteristic curve (ROC). TNF-α and IL-10 levels in plasma detected by Cytometric Beads Array (CBA). Quantitative data of normal distribution was compared with ANOVA among the three groups and LSD-t test between two groups. To non-normal distribution of data, multiple comparisons among three groups were conducted by Kruskal-Wallis H test and differences between two groups were assessed by Mann-Whitney U test for post hoc analysis.

RESULTThere were no significant differences between the age and gender of each group. Expression of miR-21, miR-125b, miR-132 and miR-155 in plasma had no significant difference in each group (all P > 0.05). MiR-146a and miR-223 levels in sepsis were upregulated compared with SIRS group and control group [(5.7 ± 3.5)×10(-5) vs. (2.4 ± 1.6)×10(-5) and (2.6 ± 1.2)×10(-5), (12.5 ± 7.7)×10(-4) vs. (8.3 ± 3.4)×10(-4) and (5.3 ± 2.2)×10(-4), all P < 0.01], expression levels of miR-223 in SIRS increased as compared to control group (P < 0.01). MiR-146a levels in severe sepsis were higher than those of the general sepsis [ (7.1 ± 3.3)×10(-5) vs. (4.6 ± 2.6)×10(-5), P < 0.01]. CRP and PCT levels are all higher in sepsis and SIRS groups than control group (all P < 0.01). The area under ROC curve (AUC) of miR-146a, miR-223, PCT and CRP to predict sepsis were 0.815 (95%CI: 0.708-0.922), 0.678(95%CI: 0.537-0.818), 0.706 (95%CI: 0.571-0.842) and 0.588 (95%CI: 0.427-0.748). Expression levels of IL-10 and IL-10/TNF-α in sepsis were upregulated compared with and SIRS group and the control group (all P < 0.01). There was a positive correlation between miR-146a, miR-223 and IL-10 and IL-10/TNF-α (r = 0.545, 0.305, 0.562, 0.373, all P < 0.01).

CONCLUSIONThe expression levels of miR-146a and miR-223 in plasma in pediatric patients with sepsis was significantly upregulated, and had a positive correlation with IL-10 and IL-10/TNF-α, which may be used as early diagnostic markers and can reflect the severity of condition to a certain degree.

Biomarkers ; blood ; Calcitonin ; blood ; Case-Control Studies ; Female ; Humans ; Infant ; Infant, Newborn ; Intensive Care Units, Pediatric ; Interleukin-10 ; blood ; Male ; MicroRNAs ; blood ; Prognosis ; ROC Curve ; Sepsis ; blood ; diagnosis ; Severity of Illness Index ; Systemic Inflammatory Response Syndrome ; blood ; diagnosis ; Tumor Necrosis Factor-alpha ; blood