A sensitive and accurate GC-MS method for analyzing microbial metabolites short chain fatty acids and their hydroxylated derivatives in newborn fecal samples.

Short chain fatty acids (SCFAs), crucial intestinal bacterial metabolites, have been widely accepted as potential diagnostic markers in neonatal medicine. Nevertheless, it is still a great challenge to accurately quantify SCFAs in newborn fecal samples due to the huge variation of water content, limited commercial isotope-labeled internal standards and poor sensitivity. In this study, Na2CO3 solution (50 µg/mL) was applied to convert the free SCFAs to SCFA sodium salts, which could prevent the loss of violate SCFAs during lyophilization process. Furthermore, N-methylbenzylamine-d0/d3 was applied as the chemical derivatization regent to enhance the sensitivity and accuracy. Based on this method, the SCFA contents in meconium and neonatal fecal samples were analyzed to illustrate the change of SCFAs during the gut microbiome development. Chemical derivatization based on N-methylbenzylamine-d0/d3 could not only significantly promote the sensitivity (323-1280 folds compared to free SCFAs) by promoting the ionization efficiency, but also provide one-to-one isotope internal standards. Moreover, 7 SCFAs, including acetic acid (2), n-butyric acid (4), isobutyric acid (5), 2-hydroxybutyric acid (11), 2-hydroxy-3-methylbutyric acid (13), 3-hydroxybutyric acid (14), 2-hydroxy-2-methylbutyric acid (17) were found to be significantly increased in neonatal fecal samples compared to the meconium fecal samples. All these results proved that this method could be applied for SCFA analysis in newborn fecal samples with perfect accuracy and sensitivity.

Oridonin enhances γ­globin expression in erythroid precursors from patients with ß­thalassemia via activation of p38 MAPK signaling.

Upregulation of fetal hemoglobin expression can alleviate the severity of ß­thalassaemia. This study aimed to investigate the effects of Oridonin (ORI, a diterpenoid compound) on γ­globin expression in human erythroid precursor cells and the potential underlying mechanisms. Erythroid precursor cells were enriched from 12 patients with ß­thalassaemia by two­phase culture. The cells were then treated with different doses of ORI and the survival of erythroid precursor cells was determined. In addition, the expression levels of γ­globin and potential mechanisms were analyzed by reverse transcription­quantitative PCR, western blotting and chromatin immunoprecipitation. Treatment with 0.5 µM ORI preferably enhanced γ­globin expression and exhibited little cytotoxicity. Similar to sodium butyrate (NaB, a histone deacetylase inhibitor), ORI significantly increased p38 mitogen­activated protein kinase (MAPK) activation, γ­globin expression, histone H3 and H4 acetylation at the Gγ­ and Aγ­globin promoters, and cAMP­response element binding protein 1 (CREB1) phosphorylation. These effects were significantly mitigated by treatment with SB23580, a p38 MAPK inhibitor, in erythroid precursor cells. Therefore, ORI may effectively enhance γ­globin expression by activating p38 MAPK and CREB1, leading to histone modification in γ­globin gene promoters during the maturation of erythroid precursor cells. These findings suggested that ORI may be a novel and potential therapeutic agent for the treatment of ß­thalassaemia.

WITHDRAWN:Radix astragali induces in vitro and in vivo synthesis of fetal hemoglobin in non­transfusion­dependent ß­thalassemia: A preliminary study.

Ahead of Print article withdrawn by publisher.

Plastrum testudinis induces γ-globin gene expression through epigenetic histone modifications within the γ-globin gene promoter via activation of the p38 MAPK signaling pathway.

The pharmacologically-induced expression of the γ-globin gene, to increase fetal hemoglobin (HbF) production, is a therapeutic strategy used for the treatment of ß-thalassemia and sickle cell anemia (SCA). The aim of this study was to investigate the effects of Plastrum testudinis (PT) on differentiation, proliferation, γ-globin gene expression and HbF synthesis in human erythroid cells. For this purpose, we used the K562 human leukemia cell line and human erythroid progenitor cells from normal donors and patients with ß-thalassemia cultured using the two-phase liquid culture system. The effects of PT on erythroid differentiation, proliferation, γ-globin gene expression and HbF synthesis, as well as the involvement of epigenetic histone modifications within the γ-globin gene promoter via activation of the p38 mitogen-activated protein kinase (MAPK) signaling pathway, were assessed by benzidine staining, trypan-blue dye exclusion, quantitative real-time RT-PCR (qRT-PCR), western blot analysis and chromatin immunoprecipitation (ChIP). PT promoted the erythroid differentiation of K562 cells, and increased γ-globin mRNA accumulation and HbF synthesis without inhibiting cell proliferation in K562 cells and human erythroid progenitors. PT exerted no effect on α- and ß-globin gene expression. In human erythroid cells, PT activated the p38 MAPK signaling pathway, and enhanced the acetylation of histone H3 and H4, the phosphorylation of histone H3 within the Gγ- and Aγ-globin gene promoter regions, γ-globin mRNA accumulation and HbF synthesis. These effects were suppressed by pre-treatment with the p38 MAPK inhibitor, SB203580. Epigenetic histone modifications within γ-globin gene promoter regions, via activation of the p38 MAPK signaling pathway, are important for the induction of γ-globin gene expression in human erythroid cells by PT. PT may be a novel potential therapeutic agent for ß-hemoglobinopathies, including ß-thalassemia and SCA.

[Prospective clinical study of radix astragali and its compound prescription for treatment of ß-thalassemia in children].

OBJECTIVE: To evaluate the efficacy and safety of radix astragali and its compound prescription for treatment of ß-thalassemia in children. METHODS: This study was a randomized, controlled, double-blind clinical trial. Fifty-seven children with ß-thalassemia were randomly assigned to radix astragali, compound prescription (radix astragali+ codonopsis pilosula + tortoise plastron) and placebo control groups after stratifying the patients according to disease type (intermedia and major). The parameters of hematology and safety were assessed after 12 weeks of treatment. RESULTS: After 12 weeks of treatment, the mean Hb elevation levels in children with ß-thalassemia intermedia from the compound prescription and the radix astragali groups were 1.21±1.12 and 1.05±0.80 g/dL respectively compared with -(0.28±0.51) g/dL in the placebo control group (P<0.01). Mean Hb levels in the compound prescription and radix astragali groups were significantly higher than in the placebo control group (P<0.05). Therapy with both radix astragali and its compound prescription increased fetal hemoglobin, red blood cell, mean corpuscular hemoglobin and reticulocyte levels in children with ß-thalassemia intermedia. The total effective rates were 64% and 62% in children with ß-thalassemia intermedia from the compound prescription and radix astragali groups respectively, which was significantly higher than in the placebo control group (9%; P<0.01). Therapy with radix astragali or its compound prescription in children with ß-thalassemia major had similar but less favourable effects than the same therapy in children with ß-thalassemia intermedia. White blood cell, neutrophil, platelet and hepatic and renal functions were not adversely affected by the medicines. CONCLUSIONS: Therapy with radix astragali or its compound prescription is effective and safe in children with ß-thalassemia.