The study investigated the specific mechanism by which oxocrebanine, the anti-hepatic cancer active ingredient in Stephania hainanensis, inhibits the proliferation of hepatic cancer cells. Firstly, methyl thiazolyl tetrazolium(MTT) assay, 5-bromodeoxyuridine(BrdU) labeling, and colony formation assay were employed to investigate whether oxocrebanine inhibited the proliferation of HepG2 and Hep3B2.1-7 cells. Propidium iodide(PI) staining was used to observe the oxocrebanine-induced apoptosis of HepG2 and Hep3B2.1-7 cells. Western blot was employed to verify whether apoptotic effector proteins, such as cleaved cysteinyl aspartate-specific protease 3(c-caspase-3), poly(ADP-ribose) polymerase 1(PARP1), B-cell lymphoma-2(Bcl-2), Bcl-2-associated X protein(Bax), Bcl-2 homologous killer(Bak), and myeloid cell leukemia-1(Mcl-1) were involved in apoptosis. Secondly, HepG2 cells were simultaneously treated with oxocrebanine and the autophagy inhibitor 3-methyladenine(3-MA), and the changes in the autophagy marker LC3 and autophagy-related proteins [eukaryotic translation initiation factor 4E-binding protein 1(4EBP1), phosphorylated 4EBP1(p-4EBP1), 70-kDa ribosomal protein S6 kinase(P70S6K), and phosphorylated P70S6K(p-P70S6K)] were determined. The results of MTT assay, BrdU labeling, and colony formation assay showed that oxocrebanine inhibited the proliferation of HepG2 and Hep3B2.1-7 cells in a dose-dependent manner. The results of flow cytometry suggested that the apoptosis rate of HepG2 and Hep3B2.1-7 cells increased after treatment with oxocrebanine. Western blot results showed that the protein levels of c-caspase-3, Bax, and Bak were up-regulated and those of PARP1, Bcl-2, and Mcl-1 were down-regulated in the HepG2 cells treated with oxocrebanine. The results indicated that oxocrebanine induced apoptosis, thereby inhibiting the proliferation of hepatic cancer cells. The inhibition of HepG2 cell proliferation by oxocrebanine may be related to the induction of protective autophagy in hepatocellular carcinoma cells. Oxocrebanine still promoted the conversion of LC3-Ⅰ to LC3-Ⅱ, reduced the phosphorylation levels of 4EBP1 and P70S6K, which can be reversed by the autophagy inhibitor 3-MA. It is prompted that oxocrebanine can inhibit the proliferation of hepatic cancer cells by inducing autophagy. In conclusion, oxocrebanine inhibits the proliferation of hepatic cancer cells by inducing apoptosis and autophagy.
Humans ; Apoptosis/drug effects* ; Autophagy/drug effects* ; Cell Proliferation/drug effects* ; Hep G2 Cells ; Liver Neoplasms/genetics* ; Carcinoma, Hepatocellular/genetics* ; Caspase 3/genetics*

OBJECTIVE: To investigate the molecular alterations of splenocytes associated with anti-factor Ⅷ (FⅧ) immune response and the underlying mechanisms based on hemophilia A (HA) murine model via RNA sequencing (RNA-seq) technology. METHODS: Severe HA mice were immunized with recombinant human factor Ⅷ (rhF8) weekly for 4 weeks to establish an FⅧ inhibitor model. High quality raw data were obtained by using bulk RNA-seq and CASAVA base identification technology, and the differentially expressed genes (DEGs) were identified. The DEGs were statistically classified by gene ontology (GO) annotation to obtain information on the major signaling pathways and biological processes involved in anti-FⅧ immune response in HA mouse splenocytes. The cell clusters, genes, and signaling pathway datasets were comprehensively analyzed by GO, Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis and single cell RNA-seq (ScRNA-seq) analysis, respectively. Flow cytometry analysis was used to verify the changes in T follicular helper cells (Tfh) and regulatory T cells (Treg). RESULTS: A total of 3731 DEGs was identified, including 2275 genes with up-regulated expression and 1456 genes with down-regulated expression. The DEGs were enriched in helper T cell differentiation, cytokine receptor, T cell receptor signaling pathway, ferroptosis, etc. Uniform Manifold Approximation and Project (UMAP) downscaling and visualization analysis yielded a total number of 11 T/NK cell subsets, visualizing the overall expression distribution of C-X-C chemokine-specific receptor gene cxcr5 among these T/NK cell subsets. Higher expression of cxcr5 was found in activated Tfh from FⅧ inhibitor mice, in comparison to the control group. The visualization using Upset plot R language showed a close interaction between Tfh and Treg. Moreover, the increased frequencies of Tfh and the decreased frequencies of Treg in inhibitor mouse splenocytes were further verified by flow cytometry analysis. CONCLUSION Multiple immune cell subsets, signaling pathways, and characteristic genes may be involved in the process of anti-FⅧ immune response in HA mouse splenocytes. The molecules involved in the regulation of Tfh/Treg may play key roles, which provide potential biological targets and therapeutic strategies for HA patients with inhibitors in the future.
Animals ; Hemophilia A/genetics* ; Mice ; Sequence Analysis, RNA ; Disease Models, Animal ; Spleen/cytology* ; T-Lymphocytes, Regulatory/immunology* ; Humans ; Signal Transduction ; Factor VIII/immunology* ; T-Lymphocytes, Helper-Inducer/immunology*

This study was aimed at analyzing the changes in the cell proteome of HTR-8/Svneo human villous trophoblasts during infection with Listeria monocytogenes,to explore the mechanisms involved in L.monocytogenes infection of the placenta at the molecular level in host cells.Tandem Mass Tag(TMT)proteomics was used to compare the quantitative proteome be-tween the HTR-8/Svneo infection group and control group.The Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)databases were used to analyze enrichment in the molecular functions,biological processes,and metabolic pathways of differentially expressed proteins.A total of 76 285 peptides and 6 979 quantifiable proteins were identified.A total of 356 proteins showed significant differential expression:153 up-regulated and 203 down-regulated.The protein with the high-est fold change was microtubule motor protein(B4DYE2),and the protein with the lowest fold change was translation initia-tion factor 1(Q6IAV3).GO functional enrichment and KEGG metabolic pathway enrichment analyses indicated that the up-regulated proteins were involved primarily in biological processes and metabolic pathways such as the movement of microtubules and microfilament as well as autophagy,whereas the down-regulated proteins were involved mainly in primary metabolic pathways,such as carbon metabolism,nitrogen metabolism,amino acid biosynthesis,and nucleic acid metabolism,as well as ubiquitin mediated protein hydrolysis.In the process of infection of HTR-8/Svneo by L.monocytogenes,the basal metabolism of host cells may be significantly diminished,and the autophagy level may be high.The significant up-regulation of cell migration-inducing and hyaluronan-binding protein CEMIP(Q8WUJ3)suggested an important role of this protein in regulating the migration and invasion of trophoblast cells,thus causing adverse pregnancy outcomes.Our findings may provide new ideas for the study of the molecular mechanism of L.monocytogenes infection of the placenta.

Objective To investigate the clinical characteristics and prognosis of pneumococcal meningitis(PM),and drug sensitivity of Streptococcus pneumoniae(SP)isolates in Chinese children.Methods A retrospective analysis was conducted on clinical information,laboratory data,and microbiological data of 160 hospitalized children under 15 years old with PM from January 2019 to December 2020 in 33 tertiary hospitals across the country.Results Among the 160 children with PM,there were 103 males and 57 females.The age ranged from 15 days to 15 years,with 109 cases(68.1% )aged 3 months to under 3 years.SP strains were isolated from 95 cases(59.4% )in cerebrospinal fluid cultures and from 57 cases(35.6% )in blood cultures.The positive rates of SP detection by cerebrospinal fluid metagenomic next-generation sequencing and cerebrospinal fluid SP antigen testing were 40% (35/87)and 27% (21/78),respectively.Fifty-five cases(34.4% )had one or more risk factors for purulent meningitis,113 cases(70.6% )had one or more extra-cranial infectious foci,and 18 cases(11.3% )had underlying diseases.The most common clinical symptoms were fever(147 cases,91.9% ),followed by lethargy(98 cases,61.3% )and vomiting(61 cases,38.1% ).Sixty-nine cases(43.1% )experienced intracranial complications during hospitalization,with subdural effusion and/or empyema being the most common complication[43 cases(26.9% )],followed by hydrocephalus in 24 cases(15.0% ),brain abscess in 23 cases(14.4% ),and cerebral hemorrhage in 8 cases(5.0% ).Subdural effusion and/or empyema and hydrocephalus mainly occurred in children under 1 year old,with rates of 91% (39/43)and 83% (20/24),respectively.SP strains exhibited complete sensitivity to vancomycin(100% ,75/75),linezolid(100% ,56/56),and meropenem(100% ,6/6).High sensitivity rates were also observed for levofloxacin(81% ,22/27),moxifloxacin(82% ,14/17),rifampicin(96% ,25/26),and chloramphenicol(91% ,21/23).However,low sensitivity rates were found for penicillin(16% ,11/68)and clindamycin(6% ,1/17),and SP strains were completely resistant to erythromycin(100% ,31/31).The rates of discharge with cure and improvement were 22.5% (36/160)and 66.2% (106/160),respectively,while 18 cases(11.3% )had adverse outcomes.Conclusions Pediatric PM is more common in children aged 3 months to under 3 years.Intracranial complications are more frequently observed in children under 1 year old.Fever is the most common clinical manifestation of PM,and subdural effusion/emphysema and hydrocephalus are the most frequent complications.Non-culture detection methods for cerebrospinal fluid can improve pathogen detection rates.Adverse outcomes can be noted in more than 10% of PM cases.SP strains are high sensitivity to vancomycin,linezolid,meropenem,levofloxacin,moxifloxacin,rifampicin,and chloramphenicol.[Chinese Journal of Contemporary Pediatrics,2024,26(2):131-138]

Animals ; SARS-CoV-2 ; Antibodies, Neutralizing ; Macaca mulatta ; COVID-19/prevention & control* ; Antibodies, Viral ; Vaccines

OBJECTIVE: To explore the association between rs2587552 polymorphism (has a strong lin-kage disequilibrium with rs1800497 which had been found in many studies to be related to obesity, r²=0.85) of DRD2 gene and the effect of a childhood obesity intervention in Chinese population, and provide a scientific basis for future personalized childhood obesity intervention based on genetic background. METHODS: From a multi-center cluster randomized controlled trial studying the effect of a childhood obesity intervention, we enrolled 382 children from 8 primary schools (192 and 190 children from intervention and control groups, respectively) in Beijing as study subjects. Saliva was collected and DNA was extracted to detect the rs2587552 polymorphism of DRD2 gene, and the interactions between the gene and study arms on childhood obesity indicators [including body weight, body mass index (BMI), BMI Z-score, waist circumference, hip circumference, waist-to-hip ratio, waist-to-height ratio, and body fat percentage] were analyzed. RESULTS: No association was found between rs2587552 polymorphism and the changes in hip circumference or body fat percentage in the intervention group (P>0.05). However, in the control group, children carrying the A allele at DRD2 rs2587552 locus showed a greater increase in hip circumference and body fat percentage compared with those not carrying A allele (P < 0.001). There were interactions between rs2587552 polymorphism of DRD2 gene and study arms on the changes in hip circumference and body fat percentage (P=0.007 and 0.015, respectively). Compared with the control group, children in the intervention group carrying the A allele at DRD2 rs2587552 locus showed decrease in hip circumference by (-1.30 cm, 95%CI: -2.25 to -0.35, P=0.007) and decrease in body fat percentage by (-1.34%, 95%CI: -2.42 to -0.27, P=0.015) compared with those not carrying A allele. The results were consistent between the dominant model and the additive model (hip circumfe-rence: -0.66 cm, 95%CI: -1.28 to -0.03, P=0.041; body fat percentage: -0.69%, 95%CI: -1.40 to 0.02, P=0.056). No interaction was found between rs2587552 polymorphism and study arms on the changes in other childhood obesity-related indicators (P>0.05). CONCLUSION Children carrying the A allele at rs2587552 polymorphism of DRD2 gene are more sensitive to intervention and showed more improvement in hip circumference and body fat percentage after the intervention, suggesting that future personalized childhood obesity lifestyle intervention can be carried out based on the rs2587552 polymorphism of DRD2 gene.
Humans ; Child ; Pediatric Obesity/therapy* ; Prospective Studies ; Polymorphism, Genetic ; Body Mass Index ; Waist Circumference ; Receptors, Dopamine D2/genetics*

Objective To investigate the Gram-positive coccus resistance in nationwide's tertiary hospitals and understand the trend of antimicrobial resistance.Methods All the clinical isolates were collected from 19 hospitals and the minimal inhibitory concentrations(MICs)were tested using agar/broth dilution method recommended.Results A total of 1 974 pathogenic Gram-positive coccus from 19 tertiary hospitals in 19 cities nationwide over the period from July 2021 to June 2022 were studied.Based on the MIC results,the prevalence of methicillin resistant Stapylococcus aureus(MRSA)and methicillin resistant Stapylococcus epidermidis(MRSE)were 36.4％and 79.9％respectively.No vancomycin insensitivity Staphylococcus was detected.Staphylococcus aureus were 100％susceptibility to linezolid and teicoplanin.Antibiotic resistance rate of Enterococcus faecalis and Enterococcus faecium to ampicillin were 3.1％and 92.9％.The detectation rate of vancomycin resistant Enterococcus(VRE)was 1.6％.Nonsusceptibility rate of Enterococcus faecalis to linezolid was 32.2％,two consecutive monitoring rises and nonsusceptibility rate of Enterococcus faecium(12.5％)was also significantly increased.The prevalence of penicillin non-susceptible Streptococcus pneumoniae(PNSSP)was 0.8％based on non-meningitis and parenteral administration criterion,decrease of nearly 30 percentage points from the previous surveillance.While for cases of oral penicillin,the rate was 71.8％,showing similar to last time.The results indicated that the number of strains with higher MIC value of penicillin(MIC ≥4 mg·L-1)decreased significantly.There were no significant differences of resistance rates of Stapylococcus aureus,Stapylococcus epidermidis,Enterococcus faecalis,Enterococcus faecium and Streptococcus pneumoniae among various groups such as different department,age,or specimen source.Conclusion VRE detection ratio stablized at a relatively low level.The number of Streptococcus pneumoniae with higher MIC value of penicillin decreased significantly compared with the previous monitoring.The increase of linezolidin-insensitive Enterococcus was noteworthy.

Objective To investigate the Gram-negative bacteria resistance in nationwide's tertiary hospitals and understand the trend of antimicrobial resistance.Method All the clinical isolates were collected from 19 hospitals and the minimal inhibitory concentrations(MICs)were tested using agar/broth dilution method recommended.Results A total of 4 066 pathogenic isolates from 19 tertiary hospitals in 19 cities nationwide over the period from July 2021 to June 2022 were studied.Based on the MIC results,Escherichia coli and Klebsiella pneumoniae showed extended spectrum β-lactamase(ESBLs)phenotype rates of 55.0％and 21.0％,respectively,ESBLs phenotype rate of Klebsiella pneumoniae keep going down.The ratios of carbapenems resistance Klebsiella pneumoniae increased by 5 percentage points compared with the previous monitoring.Carbapenems,moxalactam,sitafloxacin,β-lactam combination agents,fosfomycin trometamol,and amikacin displayed desirable antibacterial activity against Enterbacterales,susceptibal rates were above 75％.In addition,tigacycline,omacycline,colistin and fluoxefin maintained good antibacterial activity against their respective effective bacteria/species,and the bacterial sensitivity rates by more than 80％.Resistance rates of Pseudomonas aeruginosa and Acinetobacter baumannnii to imipenem were 26.3％and 72.1％and multidrug-resistant(MDR)detection rates were 41.1％and 77.3％,extensively drug-resistant(XDR)were 12.0％and 71.8％,respectively.Comparison of drug resistance rates from different wards,ages and specimen sources indicated that the proportion of resistance in Klebsiella pneumoniae and Acinetobacter baumannii isolated from intensive care unit(ICU)were significantly higher than non-ICU.Carbapenem resistance rates of Klebsiella pneumoniae isolated from ICU were more than 35％.Resistance rates of Haemophilus influenzae isolated in children to β-lactam,macrolide,clindamycin and ESBLs detection rate in Klebsiella pneumoniae isolated from children were more than those from adults and the old people,so bacterial resistance in children is an important problem in China.Conclusion ESBLs detection rate of Escherichia coli increased slightly after years of continuous decline.The proportion of carbapenem resistant Pseudomonas aeruginosa was stable,but the resistance rate of Klebsiella pneumoniae and Acinetobacter baumannii to carbapenems was still increased,which should be paid more attention.

Objective: To analyze and compare therapy responses, outcomes, and incidence of severe hematologic adverse events of flumatinib and imatinib in patients newly diagnosed with chronic phase chronic myeloid leukemia (CML) . Methods: Data of patients with chronic phase CML diagnosed between January 2006 and November 2022 from 76 centers, aged ≥18 years, and received initial flumatinib or imatinib therapy within 6 months after diagnosis in China were retrospectively interrogated. Propensity score matching (PSM) analysis was performed to reduce the bias of the initial TKI selection, and the therapy responses and outcomes of patients receiving initial flumatinib or imatinib therapy were compared. Results: A total of 4 833 adult patients with CML receiving initial imatinib (n=4 380) or flumatinib (n=453) therapy were included in the study. In the imatinib cohort, the median follow-up time was 54 [interquartile range (IQR), 31-85] months, and the 7-year cumulative incidences of CCyR, MMR, MR(4), and MR(4.5) were 95.2%, 88.4%, 78.3%, and 63.0%, respectively. The 7-year FFS, PFS, and OS rates were 71.8%, 93.0%, and 96.9%, respectively. With the median follow-up of 18 (IQR, 13-25) months in the flumatinib cohort, the 2-year cumulative incidences of CCyR, MMR, MR(4), and MR(4.5) were 95.4%, 86.5%, 58.4%, and 46.6%, respectively. The 2-year FFS, PFS, and OS rates were 80.1%, 95.0%, and 99.5%, respectively. The PSM analysis indicated that patients receiving initial flumatinib therapy had significantly higher cumulative incidences of CCyR, MMR, MR(4), and MR(4.5) and higher probabilities of FFS than those receiving the initial imatinib therapy (all P<0.001), whereas the PFS (P=0.230) and OS (P=0.268) were comparable between the two cohorts. The incidence of severe hematologic adverse events (grade≥Ⅲ) was comparable in the two cohorts. Conclusion: Patients receiving initial flumatinib therapy had higher cumulative incidences of therapy responses and higher probability of FFS than those receiving initial imatinib therapy, whereas the incidence of severe hematologic adverse events was comparable between the two cohorts.
Adult ; Humans ; Adolescent ; Imatinib Mesylate/adverse effects* ; Incidence ; Antineoplastic Agents/adverse effects* ; Retrospective Studies ; Pyrimidines/adverse effects* ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive/drug therapy* ; Treatment Outcome ; Benzamides/adverse effects* ; Leukemia, Myeloid, Chronic-Phase/drug therapy* ; Aminopyridines/therapeutic use* ; Protein Kinase Inhibitors/therapeutic use*

OBJECTIVE: To observe the effects of Xingnao Kaiqiao (regaining consciousness and opening orifices) acupuncture therapy on the expression of hypoxia-inducible factor 1α (HIF-1α) and Nod-like receptor protein 3 (NLRP3) in cerebral ischemia-reperfusion rats, and to explore the mechanism of acupuncture against cerebral ischemia-reperfusion injury. METHODS: Seventy-two male SD rats were randomly divided into a sham-operation group, a model group, an acupuncture group and a non-point acupuncture group, with 18 rats in each one. Using modified Longa thread embolization method, the rat model of acute focal cerebral ischemia was prepared; and after 2 h ischemia, the reperfusion was performed to prepared the model of cerebral ischemia-reperfusion. Immediately after reperfusion, Xingnao Kaiqiao acupuncture method was applied to bilateral "Neiguan" (PC 6) and "Shuigou" (GV 26) in the acupuncture group, while in the non-point acupuncture group, acupuncture was delivered at non-points and all of the needles were retained for 30 min in these two groups. The samples were collected 24 h after reperfusion in the rats of each group. Zea-Longa neurological deficit score was used to evaluate the degree of cerebral neurological impairment, TTC staining was adopted to observe the volume percentage of cerebral infarction, HE staining was provided to observe the morphological changes of brain, and Western blot was applied for detecting the expression of HIF-1α and NLRP3 proteins in the cerebral cortex on the right side. RESULTS: Compared with the sham-operation group, neurological deficit score and volume percentage of cerebral infarction were increased in the model group (P<0.01), and HIF-1α and NLRP3 protein expression was elevated (P<0.01). Compared with the model group, neurological deficit score and volume percentage of cerebral infarction were decreased (P<0.01), and HIF-1α and NLRP3 protein expression was lower (P<0.01) in the acupuncture group. There was no significant difference in above indexes in the non-point acupuncture group compared with the model group (P>0.05). Compared with the sham-operation group, the brain tissue of the rats in the model group and the non-point acupuncture group was loose and edema, and the nuclei were shriveled. The brain tissue morphology in the acupuncture group was similar to that of the sham-operation group. CONCLUSION Acupuncture can alleviate cerebral ischemia-reperfusion injury, and its mechanism may be related to the regulation of HIF-1α/NLRP3 signaling pathway to attenuate inflammatory response.
Male ; Animals ; Rats ; Rats, Sprague-Dawley ; NLR Family, Pyrin Domain-Containing 3 Protein/genetics* ; Acupuncture Therapy ; Reperfusion Injury/therapy* ; Brain Ischemia/therapy* ; Cerebral Infarction/therapy* ; NLR Proteins