ObjectiveTo investigate whether Huanglian Jiedutang can inhibit neutrophil infiltration in the brains of middle cerebral artery occlusion （MCAO） mice by regulating the expression of neutrophil-related chemokines in exosomes， thereby achieving therapeutic effects. MethodsA total of 130 male specific pathogen-free （SPF） C57BL/6J mice were randomly divided into four groups： Sham-operated group， MCAO model group， Huanglian Jiedutang group （6 g·kg^-1）， and Ginaton group （21.6 mg·kg^-1）， with 10 mice in the Ginaton group and 40 mice in each of the remaining three groups. Mice in the Huanglian Jiedutang group and the Ginaton group were administered the corresponding drugs by oral gavage once daily at a volume of 0.15 mL·（10 g）^-1 for 7 consecutive days， while the sham-operated and model groups received an equal volume of saline via the same route. After 7 days， MCAO surgery was performed. The distal and proximal ends of the right common carotid artery （CCA） were ligated， a small incision was made between the two ligatures， and a silicone rubber-coated monofilament with a rounded tip was inserted into the lumen to occlude the CCA. The filament was left in place for 1 h to establish a focal cerebral ischemia model. At 24 h after modeling， mice were evaluated. Neurological function was assessed using the Longa score. Cerebral infarct volume was measured by 2，3，5-triphenyltetrazolium chloride （TTC） staining. Cerebral blood flow was observed by laser speckle imaging. Hematoxylin and eosin （HE） staining and Nissl staining were used to observe pathological changes in brain tissues. Exosomes were isolated from mouse plasma and brain tissues by ultracentrifugation and molecular size exclusion and identified by electron microscopy， particle size analysis， and protein blotting. Long-chain RNA libraries of exosomes were constructed and sequenced. Real-time quantitative reverse transcription polymerase chain reaction （Real-time PCR） was used to detect the mRNA expression of inflammatory factors and neutrophil-related chemokines in exosomes from plasma and brain tissues of each group. Enzyme-linked immunosorbent assay （ELISA） was used to detect the protein expression of inflammatory factors and neutrophil-related chemokines in exosomes from brain tissues of each group. Immunohistochemistry was used to detect the expression of the neutrophil-specific protein myeloperoxidase （MPO） in the brains of mice in each group. ResultsCompared with the sham-operated group， the model group showed decreased neurological function scores （P<0.01）， obvious cerebral infarction （P<0.01）， reduced cerebral blood flow （P<0.01）， neuronal necrosis in the brain， and decreased numbers of Nissl bodies （P<0.01）. The mRNA expression levels of IL-1β， MPO， CXCL1， CXCL2， CXCL3， CXCL10， CCL2， and CCL3 in exosomes from plasma and brain tissues were significantly increased （P<0.05， P<0.01）. The protein expression levels of IL-1β， MPO， CXCL2， and CXCL10 in exosomes from brain tissues were increased （P<0.05， P<0.01）， and MPO-positive rates and mean optical density values in brain tissues were elevated （P<0.01）. Compared with the model group， the Huanglian Jiedutang group and the Ginaton group showed increased neurological function scores （P<0.05）， reduced cerebral infarct volume （P<0.01）， restored cerebral blood flow （P<0.01）， reduced necrotic cells in the brain， and increased numbers of Nissl bodies （P<0.01）. In the Huanglian Jiedutang group， the mRNA expression levels of IL-1β， MPO， CXCL1， CXCL2， CXCL3， CXCL10， CCL2， and CCL3 in exosomes from plasma and brain tissues were decreased （P<0.05， P<0.01）. The protein expression levels of IL-1β， MPO， CXCL2， and CXCL10 in exosomes from brain tissues were reduced （P<0.05， P<0.01）， and MPO-positive rates and mean optical density values in brain tissues were decreased （P<0.01）. ConclusionHuanglian Jiedutang can effectively regulate the expression of neutrophil-related chemokines in exosomes from plasma and brain tissues of MCAO mice， thereby reducing neutrophil infiltration in the brain and achieving therapeutic effects.

ObjectiveTo investigate whether Huanglian Jiedutang can inhibit neutrophil infiltration in the brains of middle cerebral artery occlusion （MCAO） mice by regulating the expression of neutrophil-related chemokines in exosomes， thereby achieving therapeutic effects. MethodsA total of 130 male specific pathogen-free （SPF） C57BL/6J mice were randomly divided into four groups： Sham-operated group， MCAO model group， Huanglian Jiedutang group （6 g·kg^-1）， and Ginaton group （21.6 mg·kg^-1）， with 10 mice in the Ginaton group and 40 mice in each of the remaining three groups. Mice in the Huanglian Jiedutang group and the Ginaton group were administered the corresponding drugs by oral gavage once daily at a volume of 0.15 mL·（10 g）^-1 for 7 consecutive days， while the sham-operated and model groups received an equal volume of saline via the same route. After 7 days， MCAO surgery was performed. The distal and proximal ends of the right common carotid artery （CCA） were ligated， a small incision was made between the two ligatures， and a silicone rubber-coated monofilament with a rounded tip was inserted into the lumen to occlude the CCA. The filament was left in place for 1 h to establish a focal cerebral ischemia model. At 24 h after modeling， mice were evaluated. Neurological function was assessed using the Longa score. Cerebral infarct volume was measured by 2，3，5-triphenyltetrazolium chloride （TTC） staining. Cerebral blood flow was observed by laser speckle imaging. Hematoxylin and eosin （HE） staining and Nissl staining were used to observe pathological changes in brain tissues. Exosomes were isolated from mouse plasma and brain tissues by ultracentrifugation and molecular size exclusion and identified by electron microscopy， particle size analysis， and protein blotting. Long-chain RNA libraries of exosomes were constructed and sequenced. Real-time quantitative reverse transcription polymerase chain reaction （Real-time PCR） was used to detect the mRNA expression of inflammatory factors and neutrophil-related chemokines in exosomes from plasma and brain tissues of each group. Enzyme-linked immunosorbent assay （ELISA） was used to detect the protein expression of inflammatory factors and neutrophil-related chemokines in exosomes from brain tissues of each group. Immunohistochemistry was used to detect the expression of the neutrophil-specific protein myeloperoxidase （MPO） in the brains of mice in each group. ResultsCompared with the sham-operated group， the model group showed decreased neurological function scores （P<0.01）， obvious cerebral infarction （P<0.01）， reduced cerebral blood flow （P<0.01）， neuronal necrosis in the brain， and decreased numbers of Nissl bodies （P<0.01）. The mRNA expression levels of IL-1β， MPO， CXCL1， CXCL2， CXCL3， CXCL10， CCL2， and CCL3 in exosomes from plasma and brain tissues were significantly increased （P<0.05， P<0.01）. The protein expression levels of IL-1β， MPO， CXCL2， and CXCL10 in exosomes from brain tissues were increased （P<0.05， P<0.01）， and MPO-positive rates and mean optical density values in brain tissues were elevated （P<0.01）. Compared with the model group， the Huanglian Jiedutang group and the Ginaton group showed increased neurological function scores （P<0.05）， reduced cerebral infarct volume （P<0.01）， restored cerebral blood flow （P<0.01）， reduced necrotic cells in the brain， and increased numbers of Nissl bodies （P<0.01）. In the Huanglian Jiedutang group， the mRNA expression levels of IL-1β， MPO， CXCL1， CXCL2， CXCL3， CXCL10， CCL2， and CCL3 in exosomes from plasma and brain tissues were decreased （P<0.05， P<0.01）. The protein expression levels of IL-1β， MPO， CXCL2， and CXCL10 in exosomes from brain tissues were reduced （P<0.05， P<0.01）， and MPO-positive rates and mean optical density values in brain tissues were decreased （P<0.01）. ConclusionHuanglian Jiedutang can effectively regulate the expression of neutrophil-related chemokines in exosomes from plasma and brain tissues of MCAO mice， thereby reducing neutrophil infiltration in the brain and achieving therapeutic effects.

ObjectiveTo investigate whether Huanglian Jiedutang （HLJD） and its major active constituents （geniposide， baicalin， and berberine） can inhibit the inflammatory response of BV2 cells under lipopolysaccharide （LPS） stimulation via the high-mobility group protein B1 （HMGB1）/Toll-like receptor 4 （TLR4）/nuclear factor-κB （NF-κB） signaling pathway， and to explore differences in therapeutic efficacy among the three monomers， their combined formula， and HLJD under equal content ratios. MethodsBV2 microglial cells were used as the primary experimental model. Cell viability was assessed using the cell counting kit-8 （CCK-8） method to examine the effects of different concentrations of dimethyl sulfoxide （DMSO， 0.8%， 0.4%， 0.2%， 0.1%， and 0.05%） on cell viability. IncuCyte was employed to monitor the growth of cells under different concentrations of HLJD （200， 100， 50， 25， 12.5， 6.25 mg·L^-1）. Nitric oxide （NO） assay was used to screen the optimal HLJD concentration. High-performance liquid chromatography （HPLC） determined the content of geniposide， baicalin， and berberine in HLJD， and experimental groups were subsequently established according to the relative proportions of these constituents. CCK-8 assay evaluated cell viability under different treatments. Enzyme-linked immunosorbent assay （ELISA） measured levels of inflammatory factors （TNF-α， IL-1β， IL-6， IL-10） in the supernatant. Flow cytometry assessed the effects of treatments on M1-type polarization of BV2 cells. Western blot determined the expression levels of HMGB1， TLR4， and NF-κB-related proteins. ResultsCompared with the blank group， DMSO at concentrations ≤0.2% did not affect cell viability within 48 h. BV2 cell growth plateaued at 24 h after treatment with 200 mg·L^-1 HLJD. Under stimulation with 2 mg·L^-1 LPS， this concentration of HLJD effectively reduced NO release， and 6 h pre-treatment had a stronger inhibitory effect on NO than direct administration. HPLC results showed that 1 mg of HLJD freeze-dried powder contained approximately 24 μg of geniposide， 15 μg of baicalin， and 30 μg of berberine. Based on these ratios， experimental groups were blank， LPS （2 mg·L^-1）， HLJD （200 mg·L^-1）， monomer combination， geniposide （4.8 mg·L^-1）， baicalin （3 mg·L^-1）， and berberine （6 mg·L^-1）. The monomer combination group consisted of all three active constituents dissolved together. LPS and HLJD or its active constituents did not affect cell viability compared with the blank group. LPS significantly increased TNF-α， IL-1β， IL-6， and IL-10 in the supernatant （P<0.01）. HLJD and its active constituents significantly reduced pro-inflammatory factors TNF-α， IL-1β， and IL-6 （P<0.05， P<0.01） while upregulating anti-inflammatory IL-10 （P<0.01）， with the monomer combination showing the strongest effect （P<0.05， P<0.01）. Compared with the blank group， LPS significantly increased the proportion of CD80⁺CD86⁺ （M1-type） BV2 cells （P<0.01）. HLJD and its constituents partially inhibited M1 polarization （P<0.05， P<0.01）， with the monomer combination exhibiting the most pronounced effect （P<0.05， P<0.01）. Compared with the blank group， LPS upregulated HMGB1， TLR4， and NF-κB-related proteins （P<0.01）， whereas HLJD and its active constituents significantly reduced their expression （P<0.05， P<0.01）， with the monomer combination having the strongest regulatory effect （P<0.05， P<0.01）. ConclusionHLJD and its major active constituents （geniposide， baicalin， berberine） can inhibit LPS-induced inflammatory responses in BV2 cells. The combination of the three active constituents demonstrates the most potent anti-inflammatory effect， significantly attenuating M1-type polarization of BV2 cells via the HMGB1/TLR4/NF-κB signaling pathway.

Antibiotic resistance is spreading at a faster rate than new antibiotic agents applied for clinical remedies. It is an urgent need to discover potential compounds to combat multidrug-resistant (MDR) bacteria. Marine fungi offer a promising avenue for mining antibiotic-like molecules with chemical diversity. To discover structurally novel and antibiotic metabolites, we screened the in-house marine fungus genome library and found a fungus Stephanonectria keithii LZD-10-1 containing a non-ribosomal peptide synthetase (NRPS) cluster with 18 modules to synthesize a new subfamily of peptaibols with effective eradication against MDR pathogens. Targeting isolation of the cultured fungus afforded six new peptaibols, which exhibit the ability to kill MDR bacteria by targeting bacterial membrane phospholipids, especially phosphatidylglycerol (PG), leading to the dysfunction of bacterial membranes. Furthermore, their efficacies against methicillin-resistant Staphylococcus aureus (MRSA) in both Galleria mellonella and mouse wound infection models were observed. This study underscores the significance of employing genome-guided approaches to identify untapped marine fungi as potential sources for novel antibiotic candidates with unique scaffolds.

Cardiac fibrosis is characterized by an elevated amount of extracellular matrix (ECM) within the heart. However, the persistence of cardiac fibrosis ultimately diminishes contractility and precipitates cardiac dysfunction. Circular RNAs (circRNAs) are emerging as important regulators of cardiac fibrosis. Here, we elucidate the functional role of a specific circular RNA CELF1 in cardiac fibrosis and delineate a novel feedback loop mechanism. Functionally, circ-CELF1 was involved in enhancing fibrosis-related markers' expression and promoting the proliferation of cardiac fibroblasts (CFs), thereby exacerbating cardiac fibrosis. Mechanistically, circ-CELF1 reduced the ubiquitination-degradation rate of BRPF3, leading to an elevation of BRPF3 protein levels. Additionally, BRPF3 acted as a modular scaffold for the recruitment of histone acetyltransferase KAT7 to facilitate the induction of H3K14 acetylation within the promoters of the Celf1 gene. Thus, the transcription of Celf1 was dramatically activated, thereby inhibiting the subsequent response of their downstream target gene Smad7 expression to promote cardiac fibrosis. Moreover, Celf1 further promoted Celf1 pre-mRNA transcription and back-splicing, thereby establishing a feedback loop for circ-CELF1 production. Consequently, a novel feedback loop involving CELF1/circ-CELF1/BRPF3/KAT7 was established, suggesting that circ-CELF1 may serve as a potential novel therapeutic target for cardiac fibrosis.

Objective To investigate if Shikonin(SKI)can induce ferroptosis via the ROS/JNK pathway to inhibit the malignant behavior of pancreatic cancer.Methods Human pancreatic cancer PANC-1 or BxPC-3 cells were selected.Drug efficacy experiments were established with a blank control group(Con group)and low,medium,and high dose SKI groups(2,4,8 μmol/L).JNK-related mechanism experiments were categorized into a blank control group(Con group),SKI group,and SKI+JNK inhibitor group(SKI+SP600125 group).ROS-related mechanism experiments were divided into a blank control group(Con group),SKI group,and SKI+ROS scavenger group(SKI+NAC group).Cell viability was assessed using the CCK-8 method to calculate IC50;Transwell experiments evaluated cell migration and invasion capabilities;the C11 BODIPY 581/591 probe was utilized for flow cytometry to detect lipid peroxidation levels,while the FerroOrange fluorescent probe measured ferrous ion levels;ROS levels were determined using a ROS detection kit;the Western blot method identified ferroptosis-related key proteins(SLC7A11,GPX4),apoptosis-related proteins(Caspase3,PARP),and JNK pathway proteins(JNK,p-JNK);an in vivo xenograft tumor model was employed to assess tumor proliferation.Results SKI treatment significantly and dose-dependently inhibited PANC-1 cell viability(IC50:6.04 μmol/L,P<0.0001)and BxPC-3 cell viability(IC50:12.27 μmol/L,P<0.0001),and significantly reduced migrating and invasive cell numbers(P<0.0001),with migration cell numbers dropping to about 30%of the control group at 8 μmol/L SKI treatment(P<0.0001).Mechanistically,SKI induced increased intracellular lipid peroxidation,Fe2+accumulation,and significant ROS production(P<0.0001),significantly downregulated SLC7A11 and GPX4 protein expression(GPX4 protein expression reduced to 40%of that in the control group,P<0.0001),and activated JNK phosphorylation(p-JNK/JNK ratio increased to 2.8-fold,P<0.0001).Pretreatment with the JNK-specific inhibitor SP600125 or ROS scavenger NAC effectively reversed SKI's inhibition of cell viability and downregulation of SLC7A11/GPX4 protein(all P<0.01).SKI also inhibited pancreatic cancer tumor cell proliferation in vivo(P<0.0001).Conclusion SKI induces ferroptosis by activating the ROS/JNK pathway,thereby inhibiting pancreatic cancer proliferation,migration,and invasion.

OBJECTIVE: To explore the genetic etiology of a Chinese pedigree affected with Primary ciliary dyskinesia (PCD). METHODS: A child who presented at the ENT Department of Zhengzhou University Children's Hospital in March 2024 due to secretory otitis media, chronic sinusitis, adenoid hypertrophy, dextrocardia, and bronchiectasis was selected as study subject. Relevant clinical data were collected. Peripheral blood samples from the child and her family members were collected. Following DNA extraction, whole exome sequencing was carried out. Candidate variants were validated by Sanger sequencing, and the correlation between the variants and phenotype was analyzed. This study was approved by the Medical Ethics Committee of the Hospital (Ethics No.: 2024-K-135). RESULTS: The child and her elder siblings exhibited similar clinical manifestations including recurrent cough, secretory otitis media, chronic sinusitis, tracheobronchitis, and pneumonia. The child also presented with bronchiectasis and visceral situs inversus. Genetic testing results indicated that the child and her elder siblings had all harbored compound heterozygous variants of the DNAH11 gene, namely c.3000 1G>A and c.5775C>G (p.Tyr1925*), which were respectively inherited from their phenotypically normal parents. Both variants can affect mRNA splicing and protein translation integrity. Based on the guidelines from the American College of Medical Genetics and Genomics, both variants were classified as likely pathogenic. It was predicted that they may jointly lead to a functional defect in axonemal dynein, resulting in the phenotype of PCD, conforming to an autosomal recessive inheritance. CONCLUSION The compound heterozygous variants c.3000 1G>A and c.5775C>G (p.Tyr1925*) of the DNAH11 gene probably underlay the pathogenesis of PCD in this pedigree. The same variant in different individuals may lead to different clinical phenotypes, which has reflected significant heterogeneity in genetic background and clinical phenotype. Above findings have enriched the mutational spectrum of PCD gene and have important implications for the accurate diagnosis, treatment, prognosis, and genetic counseling.
Humans ; Pedigree ; Female ; Axonemal Dyneins/genetics* ; Male ; Child ; Asian People/genetics* ; Kartagener Syndrome/genetics* ; Mutation ; Phenotype ; China ; Adult ; East Asian People

Objective:To evaluate the clinical performance of high-risk human papillomavirus (HR-HPV) genotyping in cervical cancer screening.Methods:Between June and July 2017, a prospective cervical cancer screening cohort was established in Xiaye Town, Jiyuan City, Henan Province, China by recruiting 3 254 women aged 21 to 64 years. At baseline screening, cervical exfoliated cell specimens were collected for HR-HPV genotyping and liquid-based cytology testing. Follow-ups were conducted over a 3-year period, with cytology testing in the first and second years and both HR-HPV genotyping and cytology testing in the third year. Women meeting the referral criteria were referred for colposcopy, with cervical biopsy and histopathological diagnosis performed as necessary. The endpoint was defined as cervical intraepithelial neoplasia grade 2 (CIN2) or higher confirmed by histopathological diagnosis. The sensitivity and specificity for detecting CIN2 or higher lesions of HR-HPV genotyping were calculated, as well as the cumulative risk of developing CIN2 or higher lesions over the 4-year study period in women with different baseline HR-HPV genotyping results.Results:A total of 2 741 women were included in the statistical analysis. Baseline HR-HPV genotyping detected 453 HR-HPV positive cases (16.53%), including 98 HPV 16/18 positive cases (3.58%) and 355 other HR-HPV positive cases (12.95%). During the 4-year period, 83 cases of CIN2 or higher were diagnosed. The sensitivity and specificity of baseline HR-HPV positivity for CIN2 or higher were 89.16% (95% CI: 80.66%-94.19%) and 85.74% (95% CI: 84.36%-87.02%), respectively. The corresponding rates for HPV 16/18 positivity were 43.37% (95% CI: 33.24%-54.09%) and 97.67% (95% CI: 97.02%-98.18%). The 4-year cumulative absolute risk of CIN2 or higher was highest in the HPV 16/18 positive group (36.73%, 95% CI: 27.85%-46.62%), followed by other HR-HPV positive groups (10.70%, 95% CI: 7.87%-14.38%), and the HR-HPV negative group was the lowest (0.39%, 95% CI: 0.19%-0.76%). Conclusions:HR-HPV genotyping testing exhibits high sensitivity and specificity for detecting CIN2 or higher lesions in cervical cancer screening. It also provides a scientific basis for stratifying the individual risk of developing CIN2 or higher lesions to guide subsequent management. Therefore, the HR-HPV genotyping testing can be considered as an effective method for cervical cancer screening.

Objective:To discern the association between autism-like behaviors and resilience within the ado-lescent demographic.Methods:A total of 7 063 middle school students were selected to assess ASD-like behaviors and resilience in adolescents using the Autism Spectrum Screening Questionnaire(ASSQ)as well as the Resilience Scale for Chinese Adolescent(RSCA).Subgroups bounded by P5 and P95 of the total ASSQ score,a comparative analysis of the resilience scores between these groups was executed.A correlation evaluation and linear regression a-nalysis was carried out between ASSQ and RSCA scores from all participants.Results:The RSCA scores within the high ASSQ scoring group,were inferior to those in the low scoring group.ASSQ scores were negatively correlated with RSCA scores for the full sample(Ps＜0.01);Social interaction scores on the ASSQ were negatively correlated with the five-factor RSCA scores(β=-0.23,-0.27,-0.11,-0.23,-0.37,Ps＜0.05).Conclusion:There was a negative association between autism spectrum disorder-like behaviors and resilience in adolescents,with more severe social interaction symptoms being associated with poorer resilience.

Objective:To explore the feasibility, practicality, and safety of the day surgery management model for the treatment of patent ductus arteriosus with Da Vinci robotic surgery in children.Methods:A non-randomized controlled study was conducted.The clinical data of children with patent ductus arteriosus who underwent Da Vinci robotic surgery at Children′s Hospital, Zhejiang University School of Medicine from August 2020 to December 2023 were collected and retrospectively analyzed.The patients were divided into day surgery group and non-day surgery group according to whether they had discharged within 24 hours. t-test and Chi-square tests were used to compare the differences in clinical features, perioperative complications and prognosis between the two groups of patients.In addition, the satisfaction of the treatment effect was surveyed. Results:A total of 276 children were included in this study, with an age ranging from 6 to 154 months with a median of 34 months.The weight ranged from 6.2 to 63.2 kilograms, with a median of 13.5 kilograms.The operation time ranged from 23 to 90 minutes, with a median of 45 minutes.One hundred and thirty-five patients underwent day surgery and 141 patients underwent non-day surgery.The average age and average weight of the patients in the day surgery and non-day surgery groups was (43.7±30.8) months vs.(42.4±30.2) months and (15.0±6.4) kg vs.(14.8±6.9) kg, respectively.There was no statistically significant difference in age and weight between the two groups (all P>0.05).There was no difference in perioperative data and incidence of perioperative complications between the two groups (all P>0.05).However, the length of hospital stay of the day surgery group was significantly shorter than that of the non-day surgery group[(1.0±0) days vs.(2.5±0.9) days, t=-20.993, P<0.001], and the hospitalization cost of the day surgery group was also significantly lower than that of the non-day surgery group[(50 800±1 100) yuan vs.(53 500±3 900) yuan, t=-10.040, P<0.001].Both families of the two groups were satisfied with the treatment results, and there was no difference in satisfaction between the two groups ( χ2=0.501, P=0.479). Conclusions:The day surgery management model of Da Vinci robotic surgery for the treatment of pediatric patent ductus arteriosus is safe and feasible.To a certain extent, it can reduce the length of hospital stay, treatment costs, and treatment burden.