Objective To establish an ion chromatography method to determine the content of galactosamine in heparin sodium sample. Methods The content of galactosamine was determined by the ratio of response value of galactosamine and glucosamine.The determination was performed on an Dionex ICS, and the separation was carried out on a Amino acids capture column (30 mm ×3 mm), Series protect column (30 mm × 3 mm)and analytical column CarboPac PA20 (150 mm ×3 mm).The mobile phase was 14 mM potassium hydroxide solution at a flow rate of 0.4 mL/min; the column tempertature was at 30℃; the injection volume was 10μL.Results Glucosamine hydrochloride had good linearity within the range of 1.013 -16.211μg/mL(Y=2.303 4X+0.824 2,r=0.998 3), the average accuracy was 92.7%, and RSD was 3.2%(n=9), the limit of detection was 0.101 3μg/mL, and the limit of quantitation was 0.337 7μg/mL.D-Galactosamine hydrochloride had good linearity within the range of 0.010 2 -0.162 5 g/mL, (Y=31.157X-0.114 4,r=0.999 3).The accuracy was 102.1%, RSD was 2.4%(n=9).The limit of detection was 0.001 0μg/mL, and the limit of quantitation was 0.003 4μg/mL.The determination of galactosamine in 3 batches of heparin sodium raw material was not detected, (0.02 ±2.1)%, (0.03 ±1.5)%, respectively, which were all lower than the limit value (1%) of United States Pharmacopeia regulation.Conclusion The method for the determination of galactosamine in total hexose amine is successfully developed , which could be used as reference for improvement of the quality standard of heparin sodium.

OBJECTIVE To explore the effect of aqueous extract of Zheng Chaihu Yin(ZCH)on paracetamol(acetaminophen,APAP)-induced hepatotoxicity. METHODS Male ICR mice were divided into three scenarios randomly:the single treatment dose of ZCH,multiple treatment or pretreatment dose of ZCH. Each scenario had a up control group and an APAP model group,while single treatment dose of ZCH group had a ZCH group at the same time. The dose of APAP and ZCH was 500 mg·kg-1 and 36 g · kg- 1,respectively. 24 h after the last administration,plasma and liver samples were prepared. Ultra- performance liquid chromatography/quadrupole- time- of- flight mass spectrometry (UPLC-Q-TOF-MS)based metabolomics profiling was used to examine changes in plasma after expo?sure to ZCH,APAP or co-exposure to ZCH and APAP. Glutamic pyruvic transaminase (GPT) and glutamic oxaloacetic transaminas (GOT) values were determined by a biochemical auto analyzer in plasma. Histopathologic changes in the liver were observed and the area was calculated after HE staining. The data were analyzed with SPSS16.0 statistical software and the results were compared with the test between the two groups to find biomarkers. Also,SIMCA software was used for partial least squares-discriminant analysis (PLS-DA) pattern recognition. RESULTS Compared to control group, APAP dosing alone caused an increase in plasma transaminases and alterations in multiple metabolic pathways. Compared to APAP group,decrease in plasma transaminases was noted when ZCH was administered after or prior to APAP. Histopathologic results showed that in the single treatment group, multiple treatment group and pretreatment group,ZCH could alleviate the liver damage induced by APAP from (32.3 ± 12.0)% to (14.2 ± 9.9)%,(8.6 ± 7.9)% to (5.2 ± 1.7)% and (32.5 ± 10.0)% to (5.2 ± 6.4)%(P<0.05). Similarly,the PLS-DA of the LC-MS data showed that the groups dosed with APAP alone were the most distinct from controls,while animals dosed with ZCH prior to or after APAP treatment were located near control group. Metabolic spectrum results showed that ZCH could restore the changes in endogenous substances including lipid metabolism,amino acid metabolism,sugar metabolism and energy metabolism induced by APAP to normal. CONCLUSION ZCH water-extraction plays major roles in the regulation of metabolism on APAP-induced liver injury. These studies demonstrate that UPLC-Q-TOF-MS-based metabolomic analysis can be sensitively and accurately predict the initiation and progres?sion of liver injury and greatly contribute to a better understanding of the hepatoprotective effects of ZCH in a clinical environment.

Background::Heterogeneity of tumor cells and the tumor microenvironment (TME) is significantly associated with clinical outcomes and treatment responses in patients with urothelial carcinoma (UC). Comprehensive profiling of the cellular diversity and interactions between malignant cells and TME may clarify the mechanisms underlying UC progression and guide the development of novel therapies. This study aimed to extend our understanding of intra-tumoral heterogeneity and the immunosuppressive TME in UC and provide basic support for the development of novel UC therapies.Methods::Seven patients with UC were included who underwent curative surgery at our hospital between July 2020 and October 2020. We performed single-cell RNA sequencing (scRNA-seq) analysis in seven tumors with six matched adjacent normal tissues and integrated the results with two public scRNA-seq datasets. The functional properties and intercellular interactions between single cells were characterized, and the results were validated using multiplex immunofluorescence staining, flow cytometry, and bulk transcriptomic datasets. All statistical analyses were performed using the R package with two-sided tests. Wilcoxon-rank test, log-rank test, one-way analysis of variance test, and Pearson correlation analysis were used properly.Results::Unsupervised t-distributed stochastic neighbor embedding clustering analysis identified ten main cellular subclusters in urothelial tissues. Of them, seven urothelial subtypes were noted, and malignant urothelial cells were characterized with enhanced cellular proliferation and reduced immunogenicity. CD8 + T cell subclusters exhibited enhanced cellular cytotoxicity activities along with increased exhaustion signature in UC tissues, and the recruitment of CD4 + T regulatory cells was also increased in tumor tissues. Regarding myeloid cells, coordinated reprogramming of infiltrated neutrophils, M2-type polarized macrophages, and LAMP3 + dendritic cells contribute to immunosuppressive TME in UC tissues. Tumor tissues demonstrated enhanced angiogenesis mediated by KDR + endothelial cells and RGS5 +/ACTA2 + pericytes. Through deconvolution analysis, we identified multiple cellular subtypes may influence the programmed death-ligand 1 (PD-L1) immunotherapy response in patients with UC. Conclusion::Our scRNA-seq analysis clarified intra-tumoral heterogeneity and delineated the pro-tumoral and immunosuppressive microenvironment in UC tissues, which may provide novel therapeutic targets.