[Objective] To study the molecular biological mechanism for a case of ABO blood group B subtype, and perform three-dimensional modeling of the mutant enzyme. [Methods] The ABO phenotype was identified by the tube method and microcolumn gel method; the ABO gene of the proband was detected by sequence-specific primer polymerase chain reaction (PCR-SSP), and the exon 6 and 7 of the ABO gene were sequenced and analyzed. Homologous modeling of Bw.03 glycosyltransferase (GT) was carried out by Modeller and analyzed by PyMOL2.5.0 software. [Results] The weakening B antigen was detected in the proband sample by forward typing, and anti-B antibody was detected by reverse typing. PCR-SSP detection showed B, O gene, and the sequencing results showed c.721 C>T mutation in exon 7 of the B gene, resulting in p. Arg 241 Trp. Compared with the wild type, the structure of Bw.03GT was partially changed, and the intermolecular force analysis showed that the original three hydrogen bonds at 241 position disappeared. [Conclusion] Blood group molecular biology examination is helpful for the accurate identification of ambiguous blood group. Homologous modeling more intuitively shows the key site for the weakening of Bw.03 GT activity. The intermolecular force analysis can explain the root cause of enzyme activity weakening.

This study investigated the effect of Wuling San on transforming growth factor-β1(TGF-β1)-induced fibrosis, inflammation, and oxidative stress in human renal tubular epithelial cells(HK-2) and its mechanism of antioxidant stress injury. HK-2 cells were cultured in vitro and divided into a control group, a TGF-β1 model group, and three treatment groups receiving Wuling San-containing serum at low(2.5%), medium(5.0%), and high(10.0%) doses. TGF-β1 was used to establish the model in all groups except the control group. CCK-8 was used to analyze the effect of different concentrations of Wuling San on the activity of HK-2 cells with or without TGF-β1 stimulation. The expression of key fibrosis molecules, including actin alpha 2(Acta2), collagen type Ⅰ alpha 1 chain(Col1α1), collagen type Ⅲ alpha 1 chain(Col3α1), TIMP metallopeptidase inhibitor 1(Timp1), and fibronectin 1(Fn1), was detected using qPCR. The expression levels of inflammatory cytokines, including tumor necrosis factor-α(TNF-α), interleukin-1β(IL-1β), interleukin-6(IL-6), interleukin-8(IL-8), and interleukin-4(IL-4), were measured using ELISA kits. Glutathione peroxidase(GSH-Px), malondialdehyde(MDA), catalase(CAT), and superoxide dismutase(SOD) biochemical kits were used to analyze the effect of Wuling San on TGF-β1-induced oxidative stress injury in HK-2 cells, and the expression of nuclear factor E2-related factor 2(Nrf2), heme oxygenase 1(HO-1), and NAD(P)H quinone oxidoreductase 1(NQO1) was analyzed by qPCR and immunofluorescence. The CCK-8 results indicated that the optimal administration concentrations of Wuling San were 2.5%, 5.0%, and 10.0%. Compared with the control group, the TGF-β1 model group showed significantly increased levels of key fibrosis molecules(Acta2, Col1α1, Col3α1, Timp1, and Fn1) and inflammatory cytokines(TNF-α, IL-1β, IL-6, IL-8, and IL-4). In contrast, the Wuling San administration groups were able to dose-dependently inhibit the expression levels of key fibrosis molecules and inflammatory cytokines compared with the TGF-β1 model group. Wuling San significantly increased the activities of GSH-Px, CAT, and SOD enzymes in TGF-β1-stimulated HK-2 cells and significantly inhibited the level of MDA. Furthermore, compared with the control group, the TGF-β1 model group exhibited a significant reduction in the expression of Nrf2, HO-1, and NQO1 genes and proteins. After Wuling San intervention, the expression of Nrf2, HO-1, and NQO1 genes and proteins was significantly increased. Correlation analysis showed that antioxidant stress enzymes(GSH-Px, CAT, and SOD) and Nrf2 signaling were significantly negatively correlated with key fibrosis molecules and inflammatory cytokines in the TGF-β1-stimulated HK-2 cell model. In conclusion, Wuling San can inhibit TGF-β1-induced fibrosis in HK-2 cells by activating the Nrf2 signaling pathway, improving oxidative stress injury, and reducing inflammation.
Humans ; Oxidative Stress/drug effects* ; Transforming Growth Factor beta1/metabolism* ; Fibrosis/genetics* ; Cell Line ; Drugs, Chinese Herbal/pharmacology* ; Epithelial Cells/immunology* ; Inflammation/metabolism*

OBJECTIVES: To explore the relationship between white matter injury (WMI) and cerebral perfusion in preterm infants using arterial spin labeling (ASL). METHODS: A total of 293 preterm infants (gestational age <34 weeks) hospitalized at the Third Affiliated Hospital of Zhengzhou University between June 2022 and June 2024 were included. After achieving clinical stability, the infants underwent brain magnetic resonance imaging (MRI) and ASL. Based on MRI findings, infants were classified into WMI (n=66) and non-WMI (n=227) groups. Cerebral perfusion parameters were compared between groups, and the association between WMI and perfusion alterations was evaluated. RESULTS: The WMI group showed a higher incidence of mild intraventricular hemorrhage (IVH) than the non-WMI group (P<0.05). Significantly lower cerebral perfusion was observed in the WMI group across bilateral frontal, temporal, parietal, and occipital lobes, as well as the basal ganglia and thalamus (P<0.05). After adjusting for gestational age, corrected gestational age at ASL scan, and mild IVH, WMI remained significantly associated with reduced regional perfusion (P<0.05). CONCLUSIONS WMI in preterm infants correlates with localized cerebral hypoperfusion. ASL-detected perfusion abnormalities may provide novel insights into WMI pathogenesis.
Humans ; White Matter/blood supply* ; Infant, Newborn ; Spin Labels ; Infant, Premature ; Female ; Male ; Cerebrovascular Circulation ; Magnetic Resonance Imaging

Disruption of the intestinal mucosal barrier caused by gut dysbiosis and metabolic imbalance is the underlying pathology of inflammatory bowel disease (IBD). Traditional Chinese medicine Wuji Wan (WJW) is commonly used to treat digestive system disorders and showed therapeutic potential for IBD. In this interdisciplinary study, we aim to investigate the pharmacological effects of WJW against experimental colitis by combining functional metabolomics and gut-microbiota sequencing techniques. Treatment with WJW altered the profile of the intestinal microbiota and notably increased the abundance of Lactobacillus, thereby facilitating the conversion of tryptophan into indole-3-acetic acid (IAA) and indoleacrylic acid (IA). These indole derivatives activated the aryl hydrocarbon receptor (AhR) pathway, which reduced colonic inflammation and restored the expression of intestinal barrier proteins. Interestingly, the beneficial effects of WJW on gut barrier function improvement and tryptophan metabolism were disappeared in the absence of gut microbiota. Finally, pre-treatment with the AhR antagonist CH-223191 confirmed the essential role of IAA-mediated AhR activation in the therapeutic effects of WJW. Overall, WJW enhanced intestinal barrier function and reduced colonic inflammation in a murine colitis model by modulating Lactobacillus-IAA-AhR signaling pathway. This study provides novel insights into colitis pathogenesis and presents an effective therapeutic and preventive approach against IBD.

Objective:To investigate the clinicopathological features, classification, and genetic characteristics of common lymphatic malformation (CLM) in superficial soft tissue.Methods:A retrospective study of 110 patients with the diagnosis of CLM at the Henan Province People′s Hospital, China from August 2019 to August 2022 was performed. The clinicopathological features, relevant immunohistochemical (IHC) staining results, and fluorescence quantitative PCR of PIK3CA mutation were analyzed, and patients were followed up.Results:Among the 110 CLM patients, there were 53 males and 57 females; 65 cases (65/110, 59.1%) were first detected when the patients were≤2 years old. The most common location was the head and neck in 41 cases (41/110, 37.3%). Clinically, 102 cases (102/110, 92.7%) were solitary, 83 cases (83/110, 75.5%) were skin-colored, 69 cases (69/110, 62.7%) had indistinct borders, and 10 cases (10/110, 9.1%) had diffuse and severe macroscopic manifestations. There were 52 macrocystic type (52/110, 47.3%), 23 microcystic type (23/110, 20.9%), and 35 combined type (35/110, 31.8%). The macrocystic CLM presented as soft, translucent masses with large cystic cavities on the cut surface, and histologically they were composed of large, irregularly dilated channels that were thicker with irregular smooth muscle and lymphocytic infiltration. Microcystic CLM showed wartlike projections or translucent blisters on the skin, with small honeycomb structures on the cut surface, and histologically consisted of round or angular dilated small lymphatic vessels with little or no smooth muscle. The combined CLM had both macrocystic and microcystic morphologies. IHC staining showed that the lymphatic endothelial cells were positive for LYVE-1, D2-40, PROX1, CD31, and VEGFR3 but negative for CD34; in the macrocystic and combined CLM vessel walls were positive for SMA. Eight of 13 CLM had PIK3CA mutation. All patients were followed up, and 24 (24/110, 21.8%) had relapses, which more frequently occurred in combined type, followed by microcystic type.Conclusions:CLM is a congenital vascular malformation composed of dilated, abnormal lymphatic channels, with PIK3CA mutation. There are significant differences in clinicopathological characteristics among the different types. Since microcystic and combined CLM are prone to recurrence, accurate pathological subtyping is necessary to guide treatment and to predict prognosis.

Objective Vasculogenic mimicry(VM)is a novel vasculogenic process integral to glioma stem cells(GSCs)in glioblastoma(GBM).However,the relationship between VM and ataxia-telangiectasia mutated(ATM)serine/threonine kinase activation,which confers chemoradiotherapy resistance,remains unclear. Methods We investigated VM formation and phosphorylated ATM(pATM)levels by CD31/GFAP-periodic acid-Schiff dual staining and immunohistochemical staining in 145 GBM specimens.Glioma stem-like cells(GSLCs)derived from the formatted spheres of U87 and U251 cell lines and their pATM level and VM formation ability were examined using western blot and three-dimensional culture.For the examination of the function of pATM in VM formation by GSLCs,ATM knockdown by shRNAs and deactivated via ATM phosphorylation inhibitor KU55933 were studied. Results VM and high pATM expression occurred in 38.5%and 41.8%of tumors,respectively,and were significantly associated with reduced progression-free and overall survival.Patients with VM-positive GBMs exhibited higher pATM levels(rs=0.425,P=0.01).The multivariate analysis established VM as an independent negative prognostic factor(P=0.002).Furthermore,GSLCs expressed high levels of pATM and formed vascular-like networks in vitro.ATM inactivation or knockdown hindered VM-like network formation concomitant with the downregulation of pVEGFR-2,VE-cadherin,and laminin B2. Conclusion VM may predict a poor GBM prognosis and is associated with pATM expression.We propose that pATM promotes VM through extracellular matrix modulation and VE-Cadherin/pVEGFR-2 activation,thereby highlighting ATM activation as a potential target for enhancing anti-angiogenesis therapies for GBM.

This paper summarized the experience in treating flat warts by Professor XU Xian based on the theory of Bai Qi （白气） micro-discharge. Flat warts is considered as a minor skin disease. Wind-damp attacking the exterior and contention between the muscles are the prerequisites for its occurrence， and failure to diffuse due to wei （卫） constraint， and qi discharge to striae and interstices are the core pathogenesis. For treatment， the core principle is to treat minor diseases with minor adjustment， and therefore， the method of dispelling wind and overcoming dampness is used to produce slight sweating and relieve the symptoms， and by taking advantage of the situation， the constraint is unblocked with diffusion and dispersion. Simultaneously， it is paid attention to not over-damaging blood vessels during treatment to avoid causing other symptoms. Self-made Modified Maxing Yigan Decoction （麻杏薏甘汤） is recommended as the basic formula for flat warts， together with self-made Xi You Formula （洗疣方） in external administration to restore defense qi， eliminate pathogenic qi and remove the warts.

Objective To investigate whether norepinephrine (NE) regulates the oxidative stress in human endom-etrial epithelial cells (hEECs) by activating nuclear factor E2-related factor 2(Nrf2)/ heme oxygenase -1(HO-1) signal pathway.Methods Cultured hEECs were used.The expression of α and β adrenergic receptors was detec-ted by reverse transcription-polymerase chain reaction (RT-PCR).Cell counting kit-8(CCK-8) assay was applied to test the effect of NE on cell viability, then the cells were divided into Control group and NE treatment group, and the appropriate concentrations were chosen.The expression of tight junction proteins Occludin and zona occludens-1(ZO-1), apoptosis-related proteins apoptosis-related protein B-cell lymphoma-2 protein(Bcl-2) and Bcl-2 associ-ated X protein(Bax) , antioxidant proteins Nrf2 and HO-1 were examined by Western blot.The apoptosis was de-tected by flow cytometry.The malonaldehyde (MDA) and superoxide dismutase(SOD) in the cell culture medium were detected by enzyme-linked immunosorbent assays kit (ELISA).Results The mRNA expression of α1 a,α1 b,α2 a,α2 b,α2 c,β1, β3 was detected in the hEECs.After the NE treatment, no significant change in cell viability was observed in low concentration (5 μmol/L and 10 μmol/L) groups, while 15 μmol/L and 20μmol/L NE treatments for 6 h or 24 h promoted the cell viability significantly.The expression of ZO-1 and Occlu-din increased significantly in 15 μmol/L group after 24 h treatment, the expression of ZO-1 decreased in 6 h treat-ment group, significant down regulation was observed after 15 μmol/L NE application, the expression of Occludin increased in 6 h group.The cell apoptosis increased compared with the control group after NE stimulation, espe-cially a significantly increase in 6 h 15 μmol/L group was detected,while the fall in increased apoptosis was ob-served after 24 h treatment.The ration of Bcl-2/Bax>1.The expression of Nrf2 and HO-1 was elevated by NE.There was no obvious change in MDA level while significant elevation in SOD was detected in cell culture medium.Conclusion Nrf2/HO-1 signal is activated after application of NE to the hEECs, which may responsible for the upregulation of SOD, antioxidant and anti-apoptotic effect in the hEECs.

Objective:To explore the molecular basis for an individual with Bel subtype of the ABO blood type due to a novel c. 620T>C variant gene, and assess its impact on the structure of GTB transferase.Methods:An individual who had visited the First Affiliated Hospital of Zhengzhou University on February 11, 2023 was selected as the study subject. ABO phenotyping was initially conducted with serological methods, which was followed by direct sequencing of 7 exons of the ABO gene. Subsequently, single-strand sequencing was carried out by using allele-specific primers, and the variant in the B transferase was homology-modeled using the Modeller software. The impact of the variant on the transferase′s spatial structure was analyzed with the PyMOL software. Results:The serological phenotype of the patient was identified as the Bel subtype. Direct sequencing revealed that she has harbored a novel c. 620T>C variant, resulting in a p. Leu207Pro substitution in the polypeptide chain. Combined with single-strand sequencing, her genotype was ultimately determined as ABO* BELnew/ ABO* O.01.02. Three-dimensional protein structure modeling showed that, compared with the wild type, the distance of one hydrogen bond between Proline and Glycine at position 272 has increased, along with disappearance of another hydrogen bond. Conclusion:The novel c. 620T>C (p.Leu207Pro) variant of B allele may affect the structural stability of the glycosyltransferase. The weakened enzyme activity in turn may lead to reduced B antigen expression, manifesting as the Bel subtype by serological analysis.

Objective:To study the molecular basis for a proband with A subtype B of the ABO blood group and explore the influence of amino acid variant on the activity of glycosyltransferase (GT).Methods:A proband who had presented at the First Affiliated Hospital of Zhengzhou University on July 2, 2020 was selected as the study subject. Serological identification of the ABO blood groups of the proband and her family members were performed by gel card and test tube methods. The ABO gene of the proband was identified by PCR-sequence specific primers (PCR-SSP) and DNA sequencing. A 3D molecular homologous model was constructed to predict the impact of the variant on the stability of α-(1→3)-D-N-acetylgalactosamine transferase (GTA). Results:The red blood cells of the proband, her mother and two younger brothers showed weak agglutination with anti-A and strong agglutination with anti-B. The sera showed 1~2+ agglutination with Ac and no agglutination with Bc. Based on the serological characteristics, the proband was identified as AwB subtype. Pedigree analysis suggested that the variant was inherited from her mother. The blood group of the proband was identified as A223B type by PCR-SSP. ABO gene sequencing analysis showed that the proband has harbored heterozygous variants of c. 297A>G, c. 467C>T, c. 526C>G, c. 657C>T, c. 703G>A, c. 796C>A, c. 803G>C, c. 930G>A and c. 1055insA. Based on the results of clone sequencing, it was speculated that the genotype was ABO* A223/ ABO* B.01. There were c. 467C>T and c. 1055insA variants compared with ABO* A1.01, and c. 1055insA variant compared with ABO* A1.02. Homologous modeling showed that the C-terminal of A223 GT was significantly prolonged, and the local amino acids and hydrogen bond network have changed. Conclusion:Above results revealed the molecular genetics mechanism of A223B subtype. The c. 1055insA variant carried by the proband may affect the enzymatic activity of GTA and ultimately lead to weakening of A antigen.