This study aims to reveal the effect and mechanism of Gentianella turkestanorum total extract(GTI) in ameliorating non-alcoholic steatohepatitis(NASH). UPLC-Q-TOF-MS was employed to identify the chemical components in GTI. SwissTarget-Prediction, GeneCards, OMIM, and TTD were utilized to screen the targets of GTI components and NASH. The common targets shared by GTI components and NASH were filtered through the STRING database and Cytoscape 3.9.0 to identify core targets, followed by GO and KEGG enrichment analysis. AutoDock was used for molecular docking of key components with core targets. A mouse model of NASH was established with a methionine-choline-deficient high-fat diet. A 4-week drug intervention was conducted, during which mouse weight was monitored, and the liver-to-brain ratio was measured at the end. Hematoxylin-eosin staining, Sirius red staining, and oil red O staining were employed to observe the pathological changes in the liver tissue. The levels of various biomarkers, including aspartate aminotransferase(AST), alanine aminotransferase(ALT), hydroxyproline(HYP), total cholesterol(TC), triglycerides(TG), low-density lipoprotein cholesterol(LDL-C), high-density lipoprotein cholesterol(HDL-C), malondialdehyde(MDA), superoxide dismutase(SOD), and glutathione(GSH), in the serum and liver tissue were determined. RT-qPCR was conducted to measure the mRNA levels of interleukin 1β(IL-1β), interleukin 6(IL-6), tumor necrosis factor α(TNF-α), collagen type I α1 chain(COL1A1), and α-smooth muscle actin(α-SMA). Western blotting was conducted to determine the protein levels of IL-1β, IL-6, TNF-α, and potential drug targets identified through network pharmacology. UPLC-Q-TOF/MS identified 581 chemical components of GTI, and 534 targets of GTI and 1 157 targets of NASH were screened out. The topological analysis of the common targets shared by GTI and NASH identified core targets such as IL-1β, IL-6, protein kinase B(AKT), TNF, and peroxisome proliferator activated receptor gamma(PPARG). GO and KEGG analyses indicated that the ameliorating effect of GTI on NASH was related to inflammatory responses and the phosphoinositide 3-kinase(PI3K)/AKT pathway. The staining results demonstrated that GTI ameliorated hepatocyte vacuolation, swelling, ballooning, and lipid accumulation in NASH mice. Compared with the model group, high doses of GTI reduced the AST, ALT, HYP, TC, and TG levels(P<0.01) while increasing the HDL-C, SOD, and GSH levels(P<0.01). RT-qPCR results showed that GTI down-regulated the mRNA levels of IL-1β, IL-6, TNF-α, COL1A1, and α-SMA(P<0.01). Western blot results indicated that GTI down-regulated the protein levels of IL-1β, IL-6, TNF-α, phosphorylated PI3K(p-PI3K), phosphorylated AKT(p-AKT), phosphorylated inhibitor of nuclear factor kappa B alpha(p-IκBα), and nuclear factor kappa B(NF-κB)(P<0.01). In summary, GTI ameliorates inflammation, dyslipidemia, and oxidative stress associated with NASH by regulating the PI3K/AKT/NF-κB signaling pathway.
Animals ; Non-alcoholic Fatty Liver Disease/genetics* ; Mice ; Network Pharmacology ; Male ; Drugs, Chinese Herbal/administration & dosage* ; Chromatography, High Pressure Liquid ; Liver/metabolism* ; Mice, Inbred C57BL ; Humans ; Mass Spectrometry ; Tumor Necrosis Factor-alpha/metabolism* ; Disease Models, Animal ; Molecular Docking Simulation

OBJECTIVE: To reveal the effects and potential mechanisms by which synaptic vesicle glycoprotein 2A (SV2A) influences the distribution of amyloid precursor protein (APP) in the trans-Golgi network (TGN), endolysosomal system, and cell membranes and to reveal the effects of SV2A on APP amyloid degradation. METHODS: Colocalization analysis of APP with specific tagged proteins in the TGN, ensolysosomal system, and cell membrane was performed to explore the effects of SV2A on the intracellular transport of APP. APP, β-site amyloid precursor protein cleaving enzyme 1 (BACE1) expressions, and APP cleavage products levels were investigated to observe the effects of SV2A on APP amyloidogenic processing. RESULTS: APP localization was reduced in the TGN, early endosomes, late endosomes, and lysosomes, whereas it was increased in the recycling endosomes and cell membrane of SV2A-overexpressed neurons. Moreover, Arl5b (ADP-ribosylation factor 5b), a protein responsible for transporting APP from the TGN to early endosomes, was upregulated by SV2A. SV2A overexpression also decreased APP transport from the cell membrane to early endosomes by downregulating APP endocytosis. In addition, products of APP amyloid degradation, including sAPPβ, Aβ _1-42, and Aβ _1-40, were decreased in SV2A-overexpressed cells. CONCLUSION These results demonstrated that SV2A promotes APP transport from the TGN to early endosomes by upregulating Arl5b and promoting APP transport from early endosomes to recycling endosomes-cell membrane pathway, which slows APP amyloid degradation.
Amyloid beta-Protein Precursor/genetics* ; Membrane Glycoproteins/genetics* ; Animals ; Protein Transport ; Nerve Tissue Proteins/genetics* ; Humans ; Mice ; Endosomes/metabolism* ; trans-Golgi Network/metabolism*

A liquid chromatography-tandem mass spectrometry method was established and validated for determining the concentrations of costunolide(CO), piperine(PI), agarotetrol(AG), glycyrrhizic acid(GL), vanillic acid(VA), and glycyrrhetinic acid(GA) in rat plasma. This method was then applied to the toxicokinetic study of these six compounds in rats with chronic cerebral ischemia(CCI) following multiple oral doses of Zhachong Shisanwei Pills. Finally, the effects of continuous multiple-dose administration of Zhachong Shisanwei Pills on the liver of CCI rats were investigated. The results showed that after oral administration of different doses of Zhachong Shisanwei Pills, the in vivo exposure of AG, VA, and GA was relatively high, with AUC_(0-∞) values ranging from 604.0-2 494.2, 1 305.4-4 634.5, and 2 177.5-4 045.7 h·ng·mL~(-1), respectively, while the exposure of CO, PI, and GL was relatively low, with AUC_(0-∞) values ranging from 37.8-238.2, 2.4-17.0, and 146.9-408.5 h·ng·mL~(-1), respectively. The C_(max) and AUC_(0-∞) of the six compounds were positively correlated with the administered dose. The T_(max) of PI and AG ranged from 0.3 to 2.0 h, their T_(1/2) ranged from 0.8 to 2.9 h, and their mean residence time(MRT) ranged from 1.0 to 3.7 h. The T_(max) of GL and VA was shorter(0.4-1.9 h), while their T_(1/2)(2.6-5.9 h) and MRT(2.5-8.5 h) were longer. Both CO and GA exhibited a bimodal phenomenon, with T_(max) ranging from 1.6 to 6.6 h, T_(1/2) ranging from 2.8 to 7.7 h, and MRT ranging from 4.1 to 12.9 h. Liver histopathology after 28 days of continuous multiple-dose administration of Zhachong Shisanwei Pills showed that the liver tissue remained normal at a low dose(crude drug 0.8 g·kg~(-1), approximately 5 times the clinical equivalent dose). However, as the dose increased(crude drug 1.1-3.0 g·kg~(-1), 6.9-18.8 times the clinical equivalent dose), varying degrees of liver damage were observed. Blood biochemical tests revealed no significant changes in the serum levels of alanine aminotransferase(ALT), aspartate aminotransferase(AST), alkaline phosphatase(ALP), and total bile acid(TBA) in CCI rats from administration groups 1 to 3(crude drug 0.8, 1.1, 1.5 g·kg~(-1)). However, ALT, AST, ALP, and TBA levels in groups 4 and 5(crude drug 2.1, 3.0 g·kg~(-1)) showed significant increases. This study preliminarily elucidated the toxicokinetic characteristics of the six compounds in Zhachong Shisanwei Pills and their effects on liver tissue in CCI rats, providing data as a reference for clinical use.
Animals ; Tandem Mass Spectrometry/methods* ; Rats ; Drugs, Chinese Herbal/toxicity* ; Male ; Rats, Sprague-Dawley ; Brain Ischemia/blood* ; Chromatography, Liquid/methods* ; Polyunsaturated Alkamides/blood* ; Piperidines/toxicity* ; Benzodioxoles/toxicity* ; Alkaloids/blood* ; Liquid Chromatography-Mass Spectrometry

Humans ; Nutrition Surveys ; Diabetes Mellitus, Type 2/chemically induced* ; Environmental Exposure/analysis* ; Environmental Monitoring ; Phthalic Acids/toxicity* ; Environmental Pollutants/toxicity*

Objective: To investigate the clinical phenotype and genetic characteristics of disorders of sex development (DSD) caused by Y chromosome copy number variant (CNV). Methods: A retrospective analysis was performed on 3 patients diagnosed with DSD caused by Y chromosome CNV admitted to the First Affiliated Hospital of Zhengzhou University from January, 2018 to September, 2022. Clinical data were collected. Clinical study and genetic test were performed by karyotyping, whole exome sequencing (WES), low coverage whole genome copy number variant sequencing (CNV-seq), fluorescence in situ hybridization (FISH) and gonadal biopsy. Results: The 3 children, aged 12, 9, 9 years, the social gender were all female, presented with short stature, gonadal dysplasia and normal female external genital. No other phenotypic abnormality was found except for case 1 with scoliosis. The karyotype of all cases were identified as 46, XY. No pathogenic vraiants were found by WES. CNV-seq determined that case 1 was 47, XYY,+Y(2.12) and case 2 was 46, XY,+Y(1.6). FISH concluded that the long arm of Y chromosome was broken and recombined near Yq11.2, and then produced a pseudodicentric chromosome idic(Y). The karyotype was reinterpreted as mos 47, X, idic(Y)(q11.23)×2(10)/46, X, idic(Y)(q11.23)(50) in case 1. The karyotype was redefined as 45, XO(6)/46, X, idic(Y)(q11.22)(23)/46, X, del(Y)(q11.22)(1) in case 2. 46, XY, -Y(mos) was found by CNV-seq in case 3, and the karyotype of 45, XO/46, XY was speculated. Conclusions: The clinical manifestations of children with DSD caused by Y chromosome CNV are short stature and gonadal dysgenesis. If there is an increase of Y chromosome CNV detected by CNV-seq, FISH is recommended to classify the structural variation of Y chromosome.
Humans ; Female ; DNA Copy Number Variations ; In Situ Hybridization, Fluorescence ; Retrospective Studies ; Chromosomes, Human, Y ; Turner Syndrome

Aim Human TMPRSS2 is a transmembrane serine protease.In this paper, the structure and func¬tion of the protein were systematically analyzed by bioinformatics, the codon was optimized and the pro- karvotie expression vector was constructed to explore the molecular mechanism of SARS-CoV-2 infecting host cells.Methods The recombinant expression vector pET-22b-TMPRSS2 was generated by molecular clo¬ning technology.The homology, functional sites, sub¬cellular localization, three-dimensional structure and evolutionary characteristics of TMPRSS2 protein were systematically analyzed by using analytical tools such as Protparam, NetPhos3.1, Blast, Clustal X2 and MEGA7.0.Results The prokarvotic expression plas- mid was constructed correctly; TMPRSS2 belongs to medium molecular weight protein, which is composed of 492 amino acid residues.The theoretical isoelectric point is 8.12, the molecular extinction coefficient is 118 145 L • mol~1 • cm"1 , and the half-life is 30 h; TMPRSS2 has 15 potential glycosylation sites and 49 possible phosphorylation sites.It is a transmembrane hydrophilie protein without signal sequenee.In addi¬tion, the protein has 13 potential B-cell epitopes and 7 T-eell epitopes.Seeondarv structure analysis showed that random coil accounted for the highest proportion of TMPRSS2 protein ( 0.453 3) , followed by extended strand (0.252 0).Sequence comparison and evolu¬tionary analysis showed that the highest sequence con¬sistency and closest genetic relationship with human TMPRSS2 was Pan troglodytes, followed by gorilla.Conclusions Human-derived TMPRSS2 protein is ev- olutionarilv conserved and functionally important.Hie results of this study can help to reveal the structure and mechanism of action of TMPRSS2 protein, provide ide¬as for the diagnosis and treatment of COYID-19, and accelerate the research and development process of new drugs targeting TMPRSS2 protein.

A rapid and sensitive method for analyzing trace β-blockers in complex biological samples,which involved magnetic solid-phase extraction(MSPE)coupled with Fourier transform ion cyclotron reso-nance mass spectrometry(FTICR-MS),was developed.Novel nanosilver-functionalized magnetic nano-particles with an interlayer of poly(3,4-dihydroxyphenylalanine)(polyDOPA@Ag-MNPs)were synthesized and used as MSPE adsorbents to extract trace β-blockers from biological samples.After extraction,the analytes loaded on the polyDOPA@Ag-MNPs were desorbed using an organic solvent and analyzed by FTICR-MS.The method was rapid and sensitive,with a total detection procedure of less than 10 min as well as limits of detection and quantification in the ranges of 3.5-6.8 pg/mL and 11.7-22.8 pg/mL,respectively.The accuracy of the method was also desirable,with recoveries ranging from 80.9％to 91.0％following the detection of analytes in human blood samples.All the experimental results demonstrated that the developed MSPE-FTICR-MS method was suitable for the rapid and sensitive analysis of trace β-blockers in complex biological samples.

Objective To evaluate the effectiveness of a whole-process health education model among inpatients with ascites type of advanced schistosomiasis. Methods A “admission-hospitalization-discharge” whole-process health education model was created, 101 inpatients with ascites type of advanced schistosomiasis were given the whole-process health education. The scores of schistosomiasis control knowledge, attitudes towards schistosomiasis control and healthy behaviors, and awareness of schistosomiasis control knowledge, correct rate of attitudes towards schistosomiasis control and correct rate of healthy behaviors were compared among inpatients with ascites type of advanced schistosomiasis before and after implementation of the whole-process health education. Results The scores of schistosomiasis control knowledge, schistosomiasis control attitudes and healthy behaviors were all significantly higher among inpatients with ascites type of advanced schistosomiasis after implementation of the whole-process health education than before implementation (Z = −7.688, −3.576 and −4.328, all P values < 0.01). In addition, the awareness of schistosomiasis control knowledge increased from 54.3% to 82.7% (χ² = 188.886, P < 0.01), and the correct rate of attitudes towards schistosomiasis control increased from 88.4% to 98.0% (χ² = 22.001, P < 0.01), while the correct rate of healthy behaviors increased from 48.2% to 59.7% (χ² = 11.767, P < 0.01). Conclusions The whole-process health education model may remarkably improve the awareness of schistosomiasis control knowledge and promote the formation of positive attitudes towards schistosomiasis control and correct behaviors among inpatients with ascites type of advanced schistosomiasis, which is of great significance to facilitate patients’ cure.

Objective:To explore the molecular mechanism of Jiangtang Xiaozhi tablets （JTXZT） in the treatment of non-alcoholic fatty liver disease （NAFLD） by means of network pharmacology and molecular docking. Method:With the help of traditional Chinese medicine （TCM） Systems Pharmacology Database and Analysis Platform （TCMSP）， TCMs Integrated Database （TCMID）， Encyclopedia of TCM （ETCM） and Bioinformatics Analysis Tool for Molecular Mechanism of TCM （BATMAN-TCM）， the chemical compositions of medicinal materials in JTXZT were obtained， the compound targets were predicted in SwissTargetPrediction database and STITCH database. The targets of NAFLD were searched by The Human Gene Database （GeneCards）， Online Mendelian Inheritance in Man （OMIM）， Therapeutic Target Database （TTD） and DisGeNET， and intersection analysis was performed with the targets of the active ingredients to obtain the targets of JTXZT for treatment of NAFLD. Based on STRING 11.0 database， the protein-protein interaction （PPI） network of therapeutic targets was constructed， and the enrichment analysis of therapeutic targets was carried out by DAVID 6.8. Finally， the interaction characteristics of key components and core therapeutic targets of JTXZT for treatment of NAFLD were verified based on molecular docking. Result:The key components of JTXZT for treatment of NAFLD were quercetin， luteolin， kaempferol， berberine， isorhamnetin， betulinic acid， oleanolic acid， ursolic acid. formononetin and hexitol， and the core targets of JTXZT for treatment of NAFLD were mitogen-activated protein kinase 1 （MAPK1）， Jun proto-oncogene， activator protein-1 （AP-1） transcription factor subunit （JUN）， MAPK3， protein kinase B1 （AKT1 or Akt1）， tumor protein p53 （TP53）， E1A binding protein p300 （EP300）， Fos proto-oncogene， AP-1 transcription factor subunit （FOS）， tumor necrosis factor （TNF），amyloid beta precursor protein （APP） and cytochrome P450 family 2 subfamily E member 1 （CYP2E1）. Biological function and pathway enrichment analysis showed that JTXZT mainly through xenobiotic metabolic process， oxidation-reduction process， cholesterol metabolic process and other biological processes， regulating phosphatidylinositol 3-kinase/protein kinase B （PI3K/Akt） signaling pathway， MAPK signaling pathway， NAFLD and insulin signaling pathway to play a role in the treatment of NAFLD. The results of molecular docking showed that the active components of JTXZT had a good affinity with the core targets of JTXZT for the treatment of NAFLD. Conclusion:JTXZT treats NAFLD through multiple active components， multiple key targets and multiple action pathways.

ObjectiveTo observe whether PT109 ［5-（1，2-dithiolan-3-yl）-N-（4-（isoquinolin-5-ylamino）cyclohexyl）pentanamide］ could improve the cognitive dysfunction in sporadic AD mice induced by lateral ventricular injection of streptozotocin and study the underlying mechanisms. MethodsThirty-two seven-week-old male C57BL/6 mice were randomly divided into 4 groups： control group （7 mice）， model group （7 mice）， PT109 low dosage group （9 mice） and PT109 high dosage group （9 mice）. To establish the sporadic Alzheimer’s disease， these mice were injected with intracerebroventricular streptozotocin on the first and third day （3 mg/kg， 5 μL per injection site）. Then PT109 （30， 100 mg·kg^-1·d^-1） was injected intraperitoneally. Two weeks later， Morris water maze and step through test were used to evaluate the effect of PT109 on the learning and memory ability of AD mice. The AD related indexes such as microglia， neurons， dendritic spines and phosphorylated Tau protein were detected by immunofluorescence， immunohistochemistry， western blotting and Golgi staining. ResultsThe behavioral experiments results showed that PT109 could improve the learning and memory impairment. The immunofluorescence and immunohistochemistry staining results showed that compared with model group， PT109 reduced the number of Iba1 positive cell in hippocampus region （low dosage： P <0.001， high dosage： P <0.001） and high dosage PT109 increased the total number of MAP2 and Tuj1 positive cell in hippocampus and cortex region （P <0.05， P <0.01）. The Golgi staining results showed that compared with model group， PT109 increased the density of dendritic spines （low dosage： P <0.001， high dosage： P <0.001）. The western blotting results showed that compared with model group， PT109 decreased the protein levels of NLRP3 （high dosage： P <0.05 ） and phosphorylated Tau protein （low dosage： P <0.05， high dosage： P <0.01 ）， and high dosage PT109 increased the protein levels of PSD95 （P <0.05） and phosphorylated GSK3β （P <0.05）. ConclusionPT109 could improve the learning and memory impairment of icv-STZ mice， which might be related to the regulation of GSK3β/Tau pathway.