OBJECTIVES: The purinergic receptor P2X2 (P2RX2) encodes an ATP-gated ion channel permeable to Na⁺, K⁺, and especially Ca²⁺. Loss-of-function mutations in P2RX2 are known to cause autosomal dominant nonsyndromic deafness 41 (DFNA41), which manifests as high-frequency hearing loss, accelerated presbycusis, and increased susceptibility to noise-induced damage. Zebrafish, owing to their small size, rapid development, high fecundity, transparent embryos, and high gene conservation with humans, provide an ideal model for studying human diseases and developmental mechanisms. This study aims to generate a p2rx2 knockout zebrafish model using CRISPR/Cas9 gene editing system to investigate the effect of p2rx2 deficiency on the auditory system, providing a basis for understanding P2RX2-related hearing loss and developing gene therapy strategies. METHODS: Two CRISPR targets (sgRNA1 and sgRNA2) spaced 47 bp apart were designed within the zebrafish p2rx2 gene. Synthesized sgRNAs and Cas9 protein were microinjected into single-cell stage Tübingen (TU)-strain zebrafish embryos. PCR and gel electrophoresis verified editing efficiency at 36 hours post-fertilization (hpf). Surviving embryos were raised to adulthood (F0), tail-clipped, genotyped, and screened for positive mosaics. F1 heterozygotes were generated by outcrossing, and F2 homozygous mutants were obtained by intercrossing. Polymerase chain reaction (PCR) combined with sequencing verified mutation type and heritability. At 5 days post-fertilization (dpf), YO-PRO-1 staining was used to examine hair cell morphology and count in lateral line neuromasts and the otolith region. Auditory evoked potential (AEP) thresholds at 600, 800, 1 000, and 2 000 Hz were measured in nine 4-month-old wild type and mutant zebrafish per group. RESULTS: A stable p2rx2 knockout zebrafish line was successfully established. Sequencing revealed a 66 bp insertion at the first target site introducing a premature stop codon (TAA), leading to early termination of protein translation and loss of function. Embryos developed normally with no gross malformations. At 5 dpf, mutants exhibited significantly reduced hair cell density in the otolith region compared with wild type, although lateral line neuromasts were unaffected. AEP testing showed significantly elevated auditory thresholds at all 4 frequencies in homozygous mutants compared with wild type (all P<0.001), indicating reduced hearing sensitivity. CONCLUSIONS We successfully generated a p2rx2 loss-of-function zebrafish model using CRISPR/Cas9 technology. p2rx2 deficiency caused hair cell defects in the otolith region and increased auditory thresholds across frequencies, indicating its key role in maintaining zebrafish auditory hair cell function and hearing perception. The phenotype's restriction to the otolith region suggests tissue-specific roles of p2rx2 in sensory organs. This model provides a valuable tool for elucidating the molecular mechanisms of P2RX2-related hearing loss and for screening otoprotective drugs and developing gene therapies.
Animals ; Zebrafish/genetics* ; Receptors, Purinergic P2X2/deficiency* ; CRISPR-Cas Systems/genetics* ; Gene Knockout Techniques ; Phenotype ; Zebrafish Proteins/genetics* ; Disease Models, Animal

Objective To investigate the protective effect of recombinant Schistosoma japonicum cystatin(rSj-Cys)against acute liver injury induced by lipopolysaccharide(LPS)and D-GalN in mice.Methods Adult male C57BL/6J mice with or without LPS/D-GaIN-induced acute liver injury were given intraperitoneal injections of rSj-Cys or PBS 30 min after modeling(n=18),and serum and liver tissues samples were collected from 8 mice in each group 6 h after modeling.The survival of the remaining 10 mice in each group within 24 h was observed.Serum levels of ALT,AST,TNF-α and IL-6 of the mice were measured,and liver pathologies was observed with HE staining.The hepatic expressions of macrophage marker CD68,Bax,Bcl-2 and endoplasmic reticulum stress(ERS)-related proteins were detected using immunohistochemistry or immunoblotting,and TUNEL staining was used to detect hepatocyte apoptosis.Results The survival rates of PBS-and rSj-Cys-treated mouse models of acute liver injury were 30%and 80%at 12 h and were 10%and 60%at 24 h after modeling,respectively;no death occurred in the two control groups within 24 h.The mouse models showed significantly increased serum levels of AST,ALT,IL-6 and TNF-α and serious liver pathologies with increased hepatic expressions of CD68 and Bax,lowered expression of Bcl-2,increased hepatocyte apoptosis,and up-regulated expressions of ERS-related signaling pathway proteins GRP78,CHOP and NF-κB p-p65.Treatment of the mouse models significantly lowered the levels of AST,ALT,IL-6 and TNF-α,alleviated liver pathologies,reduced hepatic expressions of CD68,Bax,GRP78,CHOP and NF-κB p-p65,and enhanced the expression of Bcl-2.In the normal control mice,rSj-Cys injection did not produce any significant changes in these parameters compared with PBS.Conclusion rSj-Cys alleviates LPS/D-GalN-induced acute liver injury in mice by suppressing ERS,attenuating inflammation and inhibiting hepatocyte apoptosis.

Objective To investigate the protective effect of recombinant Schistosoma japonicum cystatin(rSj-Cys)against acute liver injury induced by lipopolysaccharide(LPS)and D-GalN in mice.Methods Adult male C57BL/6J mice with or without LPS/D-GaIN-induced acute liver injury were given intraperitoneal injections of rSj-Cys or PBS 30 min after modeling(n=18),and serum and liver tissues samples were collected from 8 mice in each group 6 h after modeling.The survival of the remaining 10 mice in each group within 24 h was observed.Serum levels of ALT,AST,TNF-α and IL-6 of the mice were measured,and liver pathologies was observed with HE staining.The hepatic expressions of macrophage marker CD68,Bax,Bcl-2 and endoplasmic reticulum stress(ERS)-related proteins were detected using immunohistochemistry or immunoblotting,and TUNEL staining was used to detect hepatocyte apoptosis.Results The survival rates of PBS-and rSj-Cys-treated mouse models of acute liver injury were 30%and 80%at 12 h and were 10%and 60%at 24 h after modeling,respectively;no death occurred in the two control groups within 24 h.The mouse models showed significantly increased serum levels of AST,ALT,IL-6 and TNF-α and serious liver pathologies with increased hepatic expressions of CD68 and Bax,lowered expression of Bcl-2,increased hepatocyte apoptosis,and up-regulated expressions of ERS-related signaling pathway proteins GRP78,CHOP and NF-κB p-p65.Treatment of the mouse models significantly lowered the levels of AST,ALT,IL-6 and TNF-α,alleviated liver pathologies,reduced hepatic expressions of CD68,Bax,GRP78,CHOP and NF-κB p-p65,and enhanced the expression of Bcl-2.In the normal control mice,rSj-Cys injection did not produce any significant changes in these parameters compared with PBS.Conclusion rSj-Cys alleviates LPS/D-GalN-induced acute liver injury in mice by suppressing ERS,attenuating inflammation and inhibiting hepatocyte apoptosis.

Objective:To explore the potent effects of denatonium benzoate(DB)on Mas-related G protein-coupled receptor-B2(MrgprB2)-mediated rat mast cell degranulation.Methods:RBL-2H3 cells were treated with DB overnight,before challenged with MrgprB2 ligands substance P(SP).The release of β-hex from MrgprB2-activated RBL-2H3 was detected by substrate method.Detec-tion of LTC4,IL-6,TNF-α and cPLA2 activity were performed by ELISA.The Ca2+influx and the expression of RBL-2H3 MrgprB2 re-ceptors were measured by fluorescence assay.Results:The results showed 10 μmol/L,50 μmol/L,80 μmol/L,100 μmol/L DB treat-ments promoted β-hex and LTC4 releases from activated RBL-2H3,accompanied by increased Ca2+mobilization and cPLA2 activa-tion.DB treatments did not affect IL-6 and TNF-α LTC4 releases in MrgprB2-activated RBL-2H3,as well as the levels of MrgprB2 ex-pression in mast cells.Conclusion:Taken together,DB enhanced the MrgprB2-mediated RBL-2H3 degranulation in vitro,probably by up-regulating Ca2+mobilization in activated cells.

Humans ; Asthma/drug therapy* ; Biological Therapy

BACKGROUND: The presence of fibrosis is a criterion for subtype classification in the newly updated hypersensitivity pneumonitis (HP) guidelines. The present study aimed to summarize differences in clinical characteristics and prognosis of non-fibrotic hypersensitivity pneumonitis (NFHP) and fibrotic hypersensitivity pneumonitis (FHP) and explore factors associated with the presence of fibrosis. METHODS: In this prospective cohort study, patients diagnosed with HP through a multidisciplinary discussion were enrolled. Collected data included demographic and clinical characteristics, laboratory findings, and radiologic and histopathological features. Logistic regression analyses were performed to explore factors related to the presence of fibrosis. RESULTS: A total of 202 patients with HP were enrolled, including 87 (43.1%) NFHP patients and 115 (56.9%) FHP patients. Patients with FHP were older and more frequently presented with dyspnea, crackles, and digital clubbing than patients with NFHP. Serum levels of carcinoembryonic antigen, carbohydrate antigen 125, carbohydrate antigen 153, gastrin-releasing peptide precursor, squamous cell carcinoma antigen, and antigen cytokeratin 21-1, and count of bronchoalveolar lavage (BAL) eosinophils were higher in the FHP group than in the NFHP group. BAL lymphocytosis was present in both groups, but less pronounced in the FHP group. Multivariable regression analyses revealed that older age, <20% of lymphocyte in BAL, and ≥1.75% of eosinophil in BAL were risk factors for the development of FHP. Twelve patients developed adverse outcomes, with a median survival time of 12.5 months, all of whom had FHP. CONCLUSIONS Older age, <20% of lymphocyte in BAL, and ≥1.75% of eosinophil in BAL were risk factors associated with the development of FHP. Prognosis of patients with NFHP was better than that of patients with FHP. These results may provide insights into the mechanisms of fibrosis in HP.
Humans ; Bronchoalveolar Lavage Fluid ; Prospective Studies ; Alveolitis, Extrinsic Allergic/diagnosis* ; Fibrosis ; Carbohydrates

Objective:To investigate the value of 68Ga-1, 4, 7, 10-tetraazacyclododecane-1, 4, 7, 10-tetraacetic acid- D-Phe1-Tyr3-Thr8-octreotide (DOTATATE) combined with 18F-FDG total-body PET/CT imaging in the diagnosis and heterogeneity assessment of primary foci of neuroendocrine neoplasms (NEN). Methods:Clinical, imaging and pathological data of 39 patients with pathological diagnosis (30 NEN and 9 non-NEN, 18 males and 21 females, age (54.0±11.4) years) who underwent 1/10 activity 18F-FDG and 1/2 activity 68Ga-DOTATATE total-body PET/CT combined imaging in Zhongshan Hospital, Fudan University from August 2020 to March 2023 were retrospectively analyzed. The NEN primary foci were classified as neuroendocrine tumor (NET) G1, G2, G3, and neuroendocrine carcinoma (NEC). Diagnostic efficacy of combined dual-low activity dual-tracer imaging for NEN primary foci and its value for evaluating tumor heterogeneity were analyzed. Results:The sensitivities, specificities, and accuracies of 68Ga-DOTATATE alone and in combination with 18F-FDG total-body PET/CT for the diagnosis of NEN primary foci were 81.2%(26/32), 7/9, 80.5%(33/41) and 90.6%(29/32), 7/9, 87.8%(36/41), respectively. Ten NET G1 and seven NET G2 lesions showed 68Ga-DOTATATE uptake and no 18F-FDG uptake; two NET G2 lesions showed no 68Ga-DOTATATE uptake but 18F-FDG uptake; and two NET G1 and six NET G2 lesions showed both 68Ga-DOTATATE and 18F-FDG uptake. The radiation doses of 68Ga-DOTATATE, 18F-FDG and a single examination of CT were (1.59±0.50), 0.49(0.44, 0.58) and 11.46(10.53, 12.85) mSv, respectively. Conclusion:Combining total-body PET/CT imaging with dual tracers can effectively diagnose NEN primary foci and assess inter-tumor heterogeneity.

Objective:To explore the feasibility of one-tenth dose 18F-FDG total-body PET/CT (TB PET/CT) in patients with malignant tumors. Methods:A retrospective analysis was carried out on 34 preliminarily diagnosed cancer patients (30 males, 4 females, age (64.0±1.6) years) who underwent one-tenth dose (0.37 MBq/kg) 18F-FDG TB PET/CT examination between April 2020 and September 2022 in Zhongshan Hospital, Fudan University. The raw data were reconstructed into 15 min and initial 2 min PET images (G15 and G2, respectively). A matched cohort of 34 preliminarily diagnosed malignant tumor patients (27 males, 7 females, age (63.3±2.1) years) undergoing full dose (3.70 MBq/kg) 18F-FDG conventional digital PET/CT (C PET/CT) examination with a PET scan rate of 2-3 min/bed position, were analyzed in line with the same pathological types. Signal-to-noise ratios (SNR) of G15, G2 and C PET/CT groups were compared, and based on the pathological results, the detection rates of those 3 groups for lesions were also compared. The χ2 test, independent sample t-test, Mann-Whitney U test, and Wilcoxon rank sum test were used for data analysis. Results:The significant differences in gender, age, body mass index (BMI), blood sugar level and postinjection waiting time between TB PET/CT group and C PET/CT group were not found ( χ2=0.98, t values: 0.08, -1.05, z values: 0.68, 0.41, all P>0.05). The SNR, from G15 to C PET and G2 groups, decreased gradually, which were 16.0(11.3, 20.0), 10.5(8.2, 13.5) and 8.4±0.3 respectively ( z values: 5.09, 3.31, -4.24, all P<0.05). All primary lesions and hepatic metastases were detected by G15 and G2 imaging (100%, 37/37) as well as by C PET/CT (100%, 36/36). The detection rates for lymph node metastasis lesions were 10/15 in the G2/G15 groups, which were higher than the detection rate in the C PET/CT group (64.4%(29/45); χ2=62.03, P=0.002). Conclusion:One-tenth dose 18F-FDG TB PET/CT with a 2-minute acquisition is feasibility in the clinical practice.

Objective: To investigate routine blood test results and secular changes among Tibetan children and adolescents aged from 3 to 19 in the plateau, and to provide the basis for reference range of routine blood test for this population. Methods: A total of 1 568 Tibetan children and adolescents aged from 3 to 19 living in Shigatse, Tibet were selected by cluster random sampling method. Routine blood test results and its secular trends were compared by age and gender. Results: Significantly differences were found in red blood cell(RBC), hemoglobin(HGB), hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin(MCH), white blood cell(WBC), neutrophil(NEU), neutrophil percentage (NEU%), lymphocyte(LYM), lymphocyte percentage(LYM%)，monocyte percentage(MON%)，eosinophil percentage(EOS%),basophil percentage(BAS%) and platelet(PLT) among the four age groups of 3-5, 6-12, 13-15, and 16-19 years ( F/H =60.22, 179.41, 249.45, 115.03, 74.90, 14.33 , 33.46, 78.90, 49.20, 97.29, 24.45,24.28,42.65,20.10, P <0.05). Among red blood cell indexes, RBC, HGB, HCT,MCH increased with age in boys( F =148.77, 493.04, 623.14, 249.92, P <0.05), but there was no similar trend in girls( F =1.37, 0.15, 2.94, 0.11, P >0.05). HCT showed significant sex differences among the four age groups of 3-5 years, 6-12 years, 13-15 years, and 16-19 years [(41.33±2.31)% vs (41.98±2.40)%; (43.28±2.60)% vs ( 43.75 ±2.36)%; (46.20±3.11)% vs (44.83±2.67)%; (51.10±4.15)% vs (43.61±4.70)%, t =-2.10, -2.88, 3.50, 10.82, P <0.05]. WBC, NEU, NEU%, LYM, LYM%, monocyte(MON), and MON% increased significantly with age in both boys and girls ( P <0.05). From the age of 12 to 13, RBC, HGB and HCT in Tibetan male and female adolescents showed an opposite trend and widened gradually. Conclusion Red blood cell index shows significantly different trends among Tibetan adolescents and children of different ages and genders. Regional nationality, age, gender, and other factors should be considered when developing the reference value range of blood routine index.