This study explored the effects and underlying mechanism of Raf kinase inhibitory protein(RKIP)on apoptosis in mast cells sensitized by Echinococcus granulosus cyst fluid.Bone marrow-derived mast cells(BMMCs)were isolated and cultured from RKIP knockout(KO)and wild-type(WT)C57BL/6 mice.Cells were divided into control and sensitized groups.The sensitized group was incubated for 24 h in RPMI1640 medium containing 10%serum from mice infected with E.granulosus,then activated for 3 h or 6 h with E.granulosus cyst fluid.The control group was incubated for 24 h in RPMI1640 medium,and then received an equal vol-ume of PBS.Cells and supernatants were collected for analysis.Flow cytometry was used to detect the expression of CD117 and FcεRⅠα on BMMCs.The levels of β-hexosaminidase,IL-4,and TNF-α in the supernatant were quantified with ELISA.Western blot analy-sis was used to assess expression changes in RKIP,apoptosis-related proteins,and pathway proteins in BMMC before and after sensi-tization.Flow cytometry analysis revealed that after 4 weeks of induction,the CD117 and FcεRⅠα double-positivity rates on both WT and KO BMMC exceeded 90%.ELISA indicated that the E.granulosus cyst fluid resulted in significantly greater β-hexosaminidase re-lease(F=16.88,P<0.05),and levels of IL-4(F=16.51,P<0.05)and TNF-α(F=9.78,P<0.05)in the KO sensitized group than the WT sensitized group.With respect to the WT control group,the WT sensitized group showed significantly down-regulated pro-tein expression levels of RKIP(F=8.20,P<0.05)and Bcl-2(F=101.40,P<0.01)after 3 h,but significantly up-regulated levels of p-PI3K(F=8.04,P<0.05),p-Akt(F=32.52,P<0.01),p-P65(F=13.29,P<0.05),and cleaved-caspase-3(F=46.34,P<0.01).With respect to the WT sensitized group,the KO sensitized group showed significantly up-regulated protein expression of p-PI3K(F=8.45,P<0.05),p-Akt(F=8.58,P<0.05),p-P65(F=11.02,P<0.05),and Bcl-2(F=84.50,P<0.001)after 3 h,but significantly down-regulated expression of cleaved-caspase-3(F=15.66,P<0.05).In conclusion,RKIP may inhibit the PI3K/Akt/NF-κB pathway,thereby inducing apoptosis in mast cells sensitized by E.granulosus cyst fluid.This process may help ease aller-gic reactions caused by mast cells in echinococcosis,thus offering a promising new approach for preventing and treating such reactions.

AIM To evaluate the quality of Gegen Formula Granules.METHODS Linear calibration with two reference substances(LCTRS)was adopted in the predicting of retention time with puerarin and daidzein as internal standards.UPLC characteristic chromatograms were established.The contents of 3'-hydroxy puerarin,puerarin(internal standard),3'-methoxy puerarin,puerarin 6"-O-xyloside,puerarin apioside and daidzin were determined by quantitative determination analysis multi-components by a single marker(QAMS),after which their transfer rates were calculated.RESULTS Compared with relative retention time method,LCTRS demonstrated higher positional accuracy for characteristic peaks and wider application range for columns.There were 9 characteristic peaks in the characteristic chromatograms for 14 batches of formula granules and 15 batches of standard decoctions with the similarities of more than 0.95.The contents and transfer rates of various constituents in formula granules and standard decoctions were basically consistent.CONCLUSION The chemical constituents in formula granules and their standard decoctions of Puerariae lobatae Radix display good consistency,reliable preparation process is observable in the former.

This study explored the effects and underlying mechanism of Raf kinase inhibitory protein(RKIP)on apoptosis in mast cells sensitized by Echinococcus granulosus cyst fluid.Bone marrow-derived mast cells(BMMCs)were isolated and cultured from RKIP knockout(KO)and wild-type(WT)C57BL/6 mice.Cells were divided into control and sensitized groups.The sensitized group was incubated for 24 h in RPMI1640 medium containing 10%serum from mice infected with E.granulosus,then activated for 3 h or 6 h with E.granulosus cyst fluid.The control group was incubated for 24 h in RPMI1640 medium,and then received an equal vol-ume of PBS.Cells and supernatants were collected for analysis.Flow cytometry was used to detect the expression of CD117 and FcεRⅠα on BMMCs.The levels of β-hexosaminidase,IL-4,and TNF-α in the supernatant were quantified with ELISA.Western blot analy-sis was used to assess expression changes in RKIP,apoptosis-related proteins,and pathway proteins in BMMC before and after sensi-tization.Flow cytometry analysis revealed that after 4 weeks of induction,the CD117 and FcεRⅠα double-positivity rates on both WT and KO BMMC exceeded 90%.ELISA indicated that the E.granulosus cyst fluid resulted in significantly greater β-hexosaminidase re-lease(F=16.88,P<0.05),and levels of IL-4(F=16.51,P<0.05)and TNF-α(F=9.78,P<0.05)in the KO sensitized group than the WT sensitized group.With respect to the WT control group,the WT sensitized group showed significantly down-regulated pro-tein expression levels of RKIP(F=8.20,P<0.05)and Bcl-2(F=101.40,P<0.01)after 3 h,but significantly up-regulated levels of p-PI3K(F=8.04,P<0.05),p-Akt(F=32.52,P<0.01),p-P65(F=13.29,P<0.05),and cleaved-caspase-3(F=46.34,P<0.01).With respect to the WT sensitized group,the KO sensitized group showed significantly up-regulated protein expression of p-PI3K(F=8.45,P<0.05),p-Akt(F=8.58,P<0.05),p-P65(F=11.02,P<0.05),and Bcl-2(F=84.50,P<0.001)after 3 h,but significantly down-regulated expression of cleaved-caspase-3(F=15.66,P<0.05).In conclusion,RKIP may inhibit the PI3K/Akt/NF-κB pathway,thereby inducing apoptosis in mast cells sensitized by E.granulosus cyst fluid.This process may help ease aller-gic reactions caused by mast cells in echinococcosis,thus offering a promising new approach for preventing and treating such reactions.

AIM To evaluate the quality of Gegen Formula Granules.METHODS Linear calibration with two reference substances(LCTRS)was adopted in the predicting of retention time with puerarin and daidzein as internal standards.UPLC characteristic chromatograms were established.The contents of 3'-hydroxy puerarin,puerarin(internal standard),3'-methoxy puerarin,puerarin 6"-O-xyloside,puerarin apioside and daidzin were determined by quantitative determination analysis multi-components by a single marker(QAMS),after which their transfer rates were calculated.RESULTS Compared with relative retention time method,LCTRS demonstrated higher positional accuracy for characteristic peaks and wider application range for columns.There were 9 characteristic peaks in the characteristic chromatograms for 14 batches of formula granules and 15 batches of standard decoctions with the similarities of more than 0.95.The contents and transfer rates of various constituents in formula granules and standard decoctions were basically consistent.CONCLUSION The chemical constituents in formula granules and their standard decoctions of Puerariae lobatae Radix display good consistency,reliable preparation process is observable in the former.

Idiosyncratic drug-induced liver injury (IDILI) has long posed a challenging and pivotal concern in pharmaceutical research. The complex composition of traditional Chinese medicine (TCM) has introduced a bottleneck in current research, hindering the elucidation of the component basis associated with IDILI in TCM. Using Epimedii Folium (EF) and Psoraleae Fructus (PF) as illustrative examples, this study endeavors to establish an in vitro evaluation model, providing a high-throughput and preliminary assessment method for screening components related to TCM-induced IDILI. A TNF-α-mediated HepG2 susceptible model was first established in this study, with the focus on the index components present in EF and PF. The release of lactate dehydrogenase (LDH) in the cell supernatant served as the detection index. A concentration-toxicity response curve was constructed, and the hepatotoxic components of EF and PF were identified utilizing the synergistic toxicity index. The LDH results unveiled the hepatotoxic effects of bavachin, backuchiol, isobavachin, neobavaisoflavone, psoralidin, isobavachalcone, icarisid I, and icarisid II on both normal and susceptible cells, categorizing these 8 components as both direct hepatotoxicity components and idiosyncratic hepatotoxicity components. Bavachin and neobavaisoflavone exhibited no hepatotoxicity on normal cells but demonstrated significant effects on susceptible cells, designating them as potential idiosyncratic susceptible hepatotoxicity components. The study further delineated that 10 EF components and 3 PF components were direct immune-promoting hepatotoxicity components. Additionally, 14 idiosyncratic immune-promoting hepatotoxicity components were identified, encompassing 10 EF components and 4 PF components, with neobavaisoflavone, bavachinin, and isobavachin being potential idiosyncratic susceptible immune-promoting hepatotoxicity components. Synergistic toxicity index results indicated that 13 idiosyncratic immune-promoting hepatotoxicity components (except anhydroicaritin) combined with bavachin demonstrated synergistic hepatotoxicity on susceptible cells. Notably, 3 idiosyncratic susceptible immune-promoting hepatotoxicity components combined with bavachin exhibited synergistic hepatotoxicity, with neobavaisoflavone displaying the highest synergistic toxicity index and bavachinin the lowest. In summary, this methodology successfully screens hepatotoxic and immune-promoting hepatotoxic components in EF and PF, distinguishing the types of components inducing hepatotoxicity, evaluating the hepatotoxicity degree of each component, and elucidating the synergistic relationships among them. Importantly, these findings align with the characteristics of IDILI. The method provides an effective model tool for the fundamental research of TCM-related IDILI components.

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]

Objective:To observe the impacts of using heated and humidified gas sources for delivery room resuscitation on the short-term outcomes of extremely preterm infants.Methods:A retrospective study was conducted on 231 extremely preterm infants admitted to the neonatal intensive care unit of Shenzhen Maternity & Child Healthcare Hospital, the First School of Clinical Medicine, Southern Medical University, from January 2020 to December 2022. The infants were divided into two groups based on whether heated and humidified gas sources were used during delivery room resuscitation: the heated and humidified group (103 cases) and the non-heated and humidified group (128 cases). Independent sample t-tests, Chi-square tests, and Mann-Whitney U tests were used to compare general conditions and short-term outcomes between the groups. Multivariate logistic regression analysis was used to assess the impact of using heated and humidified gas sources during delivery room resuscitation on the short-term outcomes of extremely preterm infants. Results:Compared to the non-heated and humidified group, the heated and humidified group had a lower incidence of intubation resuscitation [28.2% (29/103) vs. 41.4% (53/128), χ 2=4.38], moderate to severe bronchopulmonary dysplasia (BPD)/death [22.3% (23/103) vs. 39.1% (50/128), χ 2=7.39] and low rectal temperature upon admission (<36.7 ℃) [57.3% (59/103) vs. 79.7% (102/128), χ 2=13.57], while the rectal temperature upon admission was higher [36.3 ℃ (36.0-36.7 ℃) vs. 35.9 ℃ (35.5-36.3 ℃), U=－5.05], with all differences being statistically significant (all P<0.05). After adjusting for gender, gestational age, mode of delivery, amniotic fluid condition, weight on admission, maternal premature rupture of membranes, assisted reproductive technology, and full course of prenatal steroid use, multivariate logistic regression analysis showed that the use of heated and humidified gas sources during delivery room resuscitation could increase the hospital admission rectal temperature ( β=0.46, 95% CI: 0.28-0.64), and decrease the risks of severe BPD/death ( aOR=0.39, 95% CI: 0.20-0.75), and low rectal temperature upon admission ( aOR=0.29, 95% CI: 0.16-0.55)(all P<0.05). Compared to the non-heated and humidified group, the heated and humidified group showed no statistically significant differences in the use of pulmonary surfactant [37.9% (39/103) vs. 43.8% (56/128), χ 2=1.45], incidence of stage Ⅲ or higher necrotizing enterocolitis [2.0% (2/103) vs. 5.5% (7/128), χ 2=2.06], grade Ⅲ or higher intracranial hemorrhage [2.9% (3/103) vs. 3.9% (5/128), χ 2=0.22], and retinopathy of prematurity requiring surgical treatment [3.9% (4/103) vs. 10.2% (13/128), χ 2=3.60] (all P>0.05). Conclusion:The use of heated and humidified gas sources during resuscitation of extremely preterm infants can reduce the risk of moderate to severe BPD/death, help maintain warmth during resuscitation, and do not adversely affect other short-term outcomes.

Objective: To analyze the clinical and molecular diagnostic status of Fanconi anemia (FA) in China. Methods: The General situation, clinical manifestations and chromosome breakage test and genetic test results of 107 pediatric FA cases registered in the Chinese Blood and Marrow Transplantation Registry Group (CBMTRG) and the Chinese Children Blood and Marrow Transplantation Registry Group (CCBMTRG) from August 2009 to January 2022 were analyzed retrospectively. Children with FANCA gene variants were divided into mild and severe groups based on the type of variant, and Wilcoxon-test was used to compare the phenotypic differences between groups. Results: Of the 176 registered FA patients, 69 (39.2%) cases were excluded due to lack of definitive genetic diagnosis results, and the remaining 107 children from 15 hospitals were included in the study, including 70 males and 37 females. The age at transplantation treatment were 6 (4, 9) years. The enrolled children were involved in 10 pathogenic genes, including 89 cases of FANCA gene, 7 cases of FANCG gene, 3 cases of FANCB gene, 2 cases of FANCE gene and 1 case each of FANCC, FANCD1, FANCD2, FANCF, FANCJ, and FANCN gene. Compound heterozygous or homozygous of loss-of-function variants account for 69.2% (72/104). Loss-of-function variants account for 79.2% (141/178) in FANCA gene variants, and 20.8% (37/178) were large exon deletions. Fifty-five children (51.4%) had chromosome breakage test records, with a positive rate of 81.8% (45/55). There were 172 congenital malformations in 80 children.Café-au-Lait spots (16.3%, 28/172), thumb deformities (16.3%,28/172), polydactyly (13.9%, 24/172), and short stature (12.2%, 21/172) were the most common congenital malformations in Chinese children with FA. No significant difference was found in the number of congenital malformations between children with severe (50 cases) and mild FANCA variants (26 cases) (Z=-1.33, P=0.185). Conclusions: FANCA gene is the main pathogenic gene in children with FA, where the detection of its exon deletion should be strengthened clinically. There were no phenotypic differences among children with different types of FANCA variants. Chromosome break test is helpful to determine the pathogenicity of variants, but its accuracy needs to be improved.
Male ; Female ; Humans ; Child ; Fanconi Anemia/genetics* ; Chromosome Breakage ; Retrospective Studies ; Exons ; China/epidemiology*

OBJECTIVE: To explore the anti-inflammatory effects of ethyl lithospermate in lipopolysaccharide (LPS)-stimulated RAW 264.7 murine-derived macrophages and zebrafish, and its underlying mechanisms. METHODS: 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazoliumbromide (MTT) assays were performed to investigate the toxicity of ethyl lithospermate at different concentrations (12.5-100 µ mol/L) in RAW 264.7 cells. The cells were stimulated with LPS (100 ng/mL) for 12 h to establish an inflammation model in vitro, the production of pro-inflammatory cytokines interleukin (IL)-6 and tumor necrosis factor α (TNF-α) were assessed by enzyme linked immunosorbent assay (ELISA). Western blot was used to ascertain the protein expressions of signal transducer and activator of transcription 3 (STAT3), nuclear factor kappa B (NF-κB) p65, phospho-STAT3 (p-STAT3, Tyr705), inhibitor of NF-κB (IκB) α, and phospho-I κB α (p-IκB α, Ser32), and confocal imaging was used to identify the nuclear translocation of NF-κB p65 and p-STAT3 (Tyr705). Additionally, the yolk sacs of zebrafish (3 days post fertilization) were injected with 2 nL LPS (0.5 mg/mL) to induce an inflammation model in vivo. Survival analysis, hematoxylin-eosin (HE) staining, observation of neutrophil migration, and quantitative real-time polymerase chain reaction (qRT-PCR) were used to further study the anti-inflammatory effects of ethyl lithospermate and its probable mechanisms in vivo. RESULTS: The non-toxic concentrations of ethyl lithospermate have been found to range from 12.5 to 100 µ mol/L. Ethyl lithospermate inhibited the release of IL-6 and TNF-α(P<0.05 or P<0.01), decreased IκBα degradation and phosphorylation (P<0.05) as well as the nuclear translocation of NF-κB p65 and p-STAT3 (Tyr705) in LPS-induced RAW 264.7 cells (P<0.01). Ethyl lithospermate also decreased inflammatory cells infiltration and neutrophil migration while increasing the survival rate of LPS-stimulated zebrafish (P<0.05 or P<0.01). In addition, ethyl lithospermate also inhibited the mRNA expression levels of of IL-6, TNF-α, IκBα, STAT3, and NF-κB in LPS-stimulated zebrafish (P<0.01). CONCLUSION Ethyl lithospermate exerts anti-Inflammatory effected by inhibiting the NF-κB and STAT3 signal pathways in RAW 264.7 macrophages and zebrafish.
Animals ; Mice ; NF-kappa B/metabolism* ; Lipopolysaccharides ; RAW 264.7 Cells ; Zebrafish ; NF-KappaB Inhibitor alpha/metabolism* ; Interleukin-6/metabolism* ; Tumor Necrosis Factor-alpha/metabolism* ; STAT3 Transcription Factor/metabolism* ; Inflammation/metabolism* ; Anti-Inflammatory Agents/therapeutic use*

Objective: To analyze the prevalence and the risk factors of fungal sepsis in 25 neonatal intensive care units (NICU) among preterm infants in China, and to provide a basis for preventive strategies of fungal sepsis. Methods: This was a second-analysis of the data from the "reduction of infection in neonatal intensive care units using the evidence-based practice for improving quality" study. The current status of fungal sepsis of the 24 731 preterm infants with the gestational age of <34⁺⁰ weeks, who were admitted to 25 participating NICU within 7 days of birth between May 2015 and April 2018 were retrospectively analyzed. These preterm infants were divided into the fungal sepsis group and the without fungal sepsis group according to whether they developed fungal sepsis to analyze the incidences and the microbiology of fungal sepsis. Chi-square test was used to compare the incidences of fungal sepsis in preterm infants with different gestational ages and birth weights and in different NICU. Multivariate Logistic regression analysis was used to study the outcomes of preterm infants with fungal sepsis, which were further compared with those of preterm infants without fungal sepsis. The 144 preterm infants in the fungal sepsis group were matched with 288 preterm infants in the non-fungal sepsis group by propensity score-matched method. Univariate and multivariate Logistic regression analysis were used to analyze the risk factors of fungal sepsis. Results: In all, 166 (0.7%) of the 24 731 preterm infants developed fungal sepsis, with the gestational age of (29.7±2.0) weeks and the birth weight of (1 300±293) g. The incidence of fungal sepsis increased with decreasing gestational age and birth weight (both P<0.001). The preterm infants with gestational age of <32 weeks accounted for 87.3% (145/166). The incidence of fungal sepsis was 1.0% (117/11 438) in very preterm infants and 2.0% (28/1 401) in extremely preterm infants, and was 1.3% (103/8 060) in very low birth weight infants and 1.7% (21/1 211) in extremely low birth weight infants, respectively. There was no fungal sepsis in 3 NICU, and the incidences in the other 22 NICU ranged from 0.7% (10/1 397) to 2.9% (21/724), with significant statistical difference (P<0.001). The pathogens were mainly Candida (150/166, 90.4%), including 59 cases of Candida albicans and 91 cases of non-Candida albicans, of which Candida parapsilosis was the most common (41 cases). Fungal sepsis was independently associated with increased risk of moderate to severe bronchopulmonary dysplasia (BPD) (adjusted OR 1.52, 95%CI 1.04-2.22, P=0.030) and severe retinopathy of prematurity (ROP) (adjusted OR 2.55, 95%CI 1.12-5.80, P=0.025). Previous broad spectrum antibiotics exposure (adjusted OR=2.50, 95%CI 1.50-4.17, P<0.001), prolonged use of central line (adjusted OR=1.05, 95%CI 1.03-1.08, P<0.001) and previous total parenteral nutrition (TPN) duration (adjusted OR=1.04, 95%CI 1.02-1.06, P<0.001) were all independently associated with increasing risk of fungal sepsis. Conclusions: Candida albicans and Candida parapsilosis are the main pathogens of fungal sepsis among preterm infants in Chinese NICU. Preterm infants with fungal sepsis are at increased risk of moderate to severe BPD and severe ROP. Previous broad spectrum antibiotics exposure, prolonged use of central line and prolonged duration of TPN will increase the risk of fungal sepsis. Ongoing initiatives are needed to reduce fungal sepsis based on these risk factors.
Infant ; Infant, Newborn ; Humans ; Birth Weight ; Intensive Care Units, Neonatal ; Retrospective Studies ; Tertiary Care Centers ; Infant, Extremely Low Birth Weight ; Gestational Age ; Infant, Extremely Premature ; Sepsis/epidemiology* ; Retinopathy of Prematurity/epidemiology* ; Bronchopulmonary Dysplasia/epidemiology*