OBJECTIVE: To comprehensively evaluate the effect of icariin in alleviating reproductive function damage (RFD) in male mice via in vitro and in vivo experiments. METHODS: We isolated Leydig cells from 60 KM male mice in vitro, and examined the toxic effect of icariin on the Leydig cells using Cell Counting Kit-8 (CCK-8). We equally randomized the mice into six groups: normal control, RFD model control (made by intraperitoneal injection of busulfan at 10 mg/kg combined with cyclophosphamide (CP) at 120 mg/kg), positive control, and low-, medium- and high-dose icariin. After modeling, we treated the mice in the positive control group with Wuziyanzong Pills and those in the low-, medium- and high-dose icariin groups by intragastrical administration of icariin at 20, 40 and 80 mg/kg-1, respectively, for 30 successive days. Then we obtained the weight and visceral coefficients of the reproductive organs, calculated the sperm count, observed the pathological changes in the testis tissue by HE staining, measured the serum testosterone (T) level by ELISA, determined the indexes of testicular oxidative stress and nitric oxide (NO) signaling pathway by colorimetric assay, and detected the expression levels of the pro-apoptotic genes Fas and Bax by qRT-PCR. RESULTS: CCK-8 assay confirmed that icariin had no toxic effect on the isolated Leydig cells of the mice, and could effectively reduce busulfan- and CP-induced cytotoxicity and promote the secretion of serum T. Icariin at 80 mg/kg significantly increased the visceral coefficient of the testis and promoted spermatogenesis (P<0.05), but had little effect on the visceral coefficient of the epididymis in the RFD model mice. Testicular histomorphometric observation revealed significantly improved testis structure, intact boundary membrane of seminiferous tubules and increased numbers of various types of spermatogenic cells of the model mice after treated with icariin. Compared with the mice in the model control group, those treated with high-dose icariin showed a significantly reduced content of malondialdehyde (MDA) (by 35.3%, P<0.01), elevated total antioxidant capacity (TAOC) and superoxide dismutase (T-SOD) activity (P<0.05), and decreased NO content and nitric oxide synthase (NOS) activity in the testis tissue (P<0.01). In addition, icariin exhibited an evident inhibitory effect on the expressions of the pro-apoptotic genes Bax and Fas. CONCLUSION Icariin can ameliorate oxidative stress-induced damage to the testicular function and protect spermatogenesis of male mice by elevating TAOC, decreasing NOS activity, inhibiting the NO level in the testis, and suppressing busulfan- and CP-induced apoptosis of testicular cells.
Animals ; Male ; Cyclophosphamide/adverse effects* ; Mice ; Busulfan/adverse effects* ; Flavonoids/pharmacology* ; Leydig Cells/drug effects* ; Oxidative Stress/drug effects* ; Testis/drug effects* ; Apoptosis/drug effects* ; Testosterone/blood*

Objective:To investigate the differential expression of Nuclear Factor Kappa B(NF-κB)and Tumor Necrosis Factor-Alpha(TNF-α)in endometrial polyp tissues,polyp-adjacent endometrium,and normal endometrium,and to analyze their correlation with local inflammatory responses,thereby elucidating the pathogenesis of endometrial polyps.Methods:Thirty-six patients with hysteroscopically confirmed endometrial polyps at Qingdao University Affiliated Hospital(September 2023-December 2024)were enrolled.Polyp tissues and adjacent endometrial tissues(0.5 cm-1 cm from polyps)were collected during surgery.Twenty-six patients undergoing hysterectomy for uterine fibroids,with pathologically confirmed normal endometrium,served as controls.The expression of NF-κB and TNF-α proteins was assessed using immunohistochemistry(IHC).Real-time quantitative PCR(RT-qPCR)was employed to detect the mRNA expression levels of NF-κB and TNF-α,and Western blot analysis was used to measure their protein expression.Differences among the three groups were compared.Results:Immunohistochemical analysis demonstrated:NF-κB p65 was predominantly localized to the cytoplasm and/or nucleus;TNF-α-positive expression was principally observed in the cytoplasmic compartment and/or cell membrane,manifesting as brown-yellow to brownish granules.The positive expression rate of NF-κB protein was significantly elevated in endometrial polyp tissues compared to both adjacent endometrial tissues and normal endometrial tissues(P＜0.05),whereas no statistically significant difference was observed between adjacent and normal endometrial tissues(P＞0.05).TNF-α protein expression exhibited a gradient pattern:Endometrial polyp tissues＞Adjacent endometrial tissues＞Normal endometrial tissues(with all inter-group comparisons demonstrating statistically significant differences,P＜0.05).Real-time quantitative PCR analysis revealed a significant gradient trend in the mRNA expression of NF-κB and TNF-α in endometrial polyp tissues:NF-κB mRNA:Polyp group(7.15±3.15)＞parapolyp group(4.38±2.01)＞normal group(1.03±0.30),with significant intergroup differences(all P＜0.05).TNF-α mRNA:Polyp tissue(17.66±9.25)＞parapolyp tissue(9.35±5.75)＞normal endometrium(1.16±0.58),with statistically significant differences among all groups(all P＜0.05).Western blot quantification further confirmed this trend:NF-κB protein:Expression in the polyp group(0.87±0.11)was 2.23-fold higher than in the normal group(0.39±0.02)(P=0.0007),while the parapolyp group(0.59±0.07)showed a 51％increase compared to the normal group(P=0.0435).TNF-α protein:Expression in the polyp group(1.13±0.21)was elevated by 94.8％versus the parapolyp group(0.58±0.03,P=0.0036)and by 494.7％versus the normal group(0.19±0.03,P=0.0002).One-way ANOVA indicated significant intergroup differences(F=43.56,P＜0.05).Conclusion:The TNF-α-mediated NF-κB signaling pathway is abnormally activated in endometrial polyps,exhibiting its highest expression at the polyp site.This suggests that the localized chronic inflammatory microenvironment may promote polyp formation through persistent stimulation of the NF-κB pathway.Targeted regulation of this pathway offers a novel strategy for preventing polyp recurrence.

AIM To optimize the extraction process for Jiangzhi Ligan Decoction,and to investigate its effects on improving lipid deposition in HepG2 cells.METHODS With extraction time,extraction frequency and solvent consumption as influencing factors,comprehensive score for tanshensu,lotine,catechin contents and extract yield as an evaluation index,the extraction process was optimized by Box-Behnken response surface method on the basis of single factor test.Palmitic acid-induced HepG2 cells were adopted in the establishment of non-alcoholic fatty liver disease model,after which the effect of drug-containing serum on TC and TG levels was investigated.RESULTS The optimal conditions were determined to be 90 min for extraction time,three times for extraction frequency,and 11 times for solvent consumption.Compared with the control group,the model group demonstrated increased TG,TC levels(P＜0.01);compared with the model group,the Jiangzhi Ligan Decoction high-dose group displayed decreased TG,TC levels(P＜0.05,P＜0.01),especially for those after optimization(P＜0.05,P＜0.01).CONCLUSION Jiangzhi Ligan Decoction can alleviate lipid deposition in HepG2 cells induced by palmitic acid,whose efficacy after extraction process optimization is better than that reported in literature.

Objective To investigate the effects of peripheral blood neutrophil infiltration on the polarization regulation of cerebral resident microglia under a permanent ischemic stroke model.Methods Fifty-eight C57BL/6 mice were divided into two groups.One group was sham group,and the other group of mice was subjected to permanent middle cerebral artery occlusion surgery.Mice were euthanized 48 hours,7 days,14 days,and 30 days after surgery for tissue collection.Western blotting was used to detect expression levels of M1 microglia markers CD 16,M2 microglia marker arginase 1(Arg1),inflammatory cytokine interleukin-1 β(IL-1β),and neutrophil marker myeloperoxidase(MPO)in brain tissue.Immunofluorescence histochemical staining was used to assess neutrophil infiltration and M2 microglial distribution around the infarct area in brain sections.In vitro,purified neutrophils were co-cultured with BV2 microglial cells.After lipopolysaccharide stimulation,the phagocytosis of neutrophils by BV2 cells was observed,and the expression levels of CD16 and Arg1 proteins in BV2 cells were detected.Results Western blotting showed that the levels of CD16(P＜0.05),IL-1β(P＜0.001),and MPO(P＜0.05)in brain tissue increased significantly 48 hours and 7 days after surgery,then decreased,with MPO expression returning to normal levels 30 days after surgery.Immunofluorescence showed a significant increase of MPO-positive cells around the infarct area of the mouse cerebral cortex 48 hours after surgery(P＜0.001),followed by a decrease(P＜0.05).The number of ionized calcium binding adapter molecule 1(Iba1)and MPO double-positive cells gradually increased after surgery,and reached their peak at 14 days(P＜0.05).Iba1 and Arg1 double-positive cells also increased significantly 7 days(P＜0.05)and 14 days(P＜0.01)after surgery.In vitro,co-culture experiments showed that after BV2 phagocytosing neutrophils,CD 16(P＜0.05)significantly decreased and Arg1 significantly upregulated(P＜0.05).Conclusion In a permanent ischemic stroke model,microglia transition from M1 to M2 type after phagocytosing neutrophils,and the injured brain area changes from pro-inflammatory state to anti-inflammatory state.

This study identified 8 members including NjBIN2 of the GSK3 family in Nardostachys jatamansi by bioinformatics analysis. Moreover, the phylogenetic tree revealed that the GKS3 family members of N. jatamansi had a close relationship with those of Arabidopsis. RT-qPCR results showed that NjBIN2 presented a tissue-specific expression pattern with the highest expression in roots, suggesting that NjBIN2 played a role in root growth and development. In addition, the application of epibrassinolide or the brassinosteroid(BR) synthesis inhibitor(brassinazole) altered the expression pattern of NjBIN2 and influenced the photomorphogenesis(cotyledon opening) and root development of N. jatamansi, which provided direct evidence about the functions of NjBIN2. In conclusion, this study highlights the roles of BIN2 in regulating the growth and development of N. jatamansi by analyzing the expression pattern and biological function of NjBIN2. It not only enriches the understanding about the regulatory mechanism of the growth and development of N. jatamansi but also provides a theoretical basis and potential gene targets for molecular breeding of N. jatamansi with improved quality in the future.
Brassinosteroids/metabolism* ; Steroids, Heterocyclic/metabolism* ; Gene Expression Regulation, Plant/drug effects* ; Plant Proteins/metabolism* ; Phylogeny ; Nardostachys/metabolism* ; Plant Growth Regulators/pharmacology* ; Plant Roots/drug effects*

Descurainiae Semen Lepidii Semen, also known as Tinglizi, originates from Brassicaceae plants Descurainia sophia or Lepidium apetalum. The former is commonly referred to as "Southern Tinglizi(Descurainiae Semen)", while the latter is known as "Northern Tinglizi(Lepidii Semen)". To scientifically and accurately identify the origin of Tinglizi medicinal materials and traditional Chinese medicine products, this study developed a specific DNA mini-barcode based on chloroplast genome sequences. By combining the DNA mini-barcode with DNA metabarcoding technology, a method for the qualitative and quantitative identification of Tinglizi medicinal materials and Chinese patent medicines was established. In this study, chloroplast genomes of Southern Tinglizi and Northern Tinglizi and seven commonly encountered counterfeit products were downloaded from the GenBank database. Suitable polymorphic regions were identified to differentiate these species, enabling the development of the DNA mini-barcode. Using DNA metabarcoding technology, medicinal material mixtures of Southern and Northern Tinglizi, as well as the most common counterfeit product, Capsella bursa-pastoris seeds, were analyzed to validate the qualitative and quantitative capabilities of the mini-barcode and determine its minimum detection limit. Additionally, the mini-barcode was applied to Chinese patent medicines containing Tinglizi to authenticate their botanical origin. The results showed that the developed mini-barcode(psbB) exhibited high accuracy and specificity, effectively distinguishing between the two authentic origins of Tinglizi and commonly encountered counterfeit products. The analysis of mixtures demonstrated that the mini-barcode had excellent qualitative and quantitative capabilities, accurately identifying the composition of Chinese medicinal materials in mixed samples with varying proportions. Furthermore, the analysis of Chinese patent medicines revealed the presence of the adulterant species(Capsella bursa-pastoris) in addition to the authentic species(Southern and Northern Tinglizi), indicating the occurrence of adulteration in commercially available Tinglizi-containing products. This study developed a method for the qualitative and quantitative identification of multi-origin Chinese medicinal materials and related products, providing a model for research on other multi-origin Chinese medicinal materials.
DNA Barcoding, Taxonomic/methods* ; Drugs, Chinese Herbal/chemistry* ; Drug Contamination ; Genome, Chloroplast ; Medicine, Chinese Traditional

Primary ciliary dyskinesia (PCD) is a genetically heterogeneous disorder characterized by impaired motility of cilia and flagella. Mutations in cilia- and flagella-associated protein 300 ( CFAP300 ) are associated with human PCD and male infertility; however, the underlying pathogenic mechanisms remain poorly understood. In a consanguineous Chinese family, we identified a homozygous CFAP300 loss-of-function variant (c.304delC) in a proband presenting with classical PCD symptoms and severe sperm abnormalities, including dynein arm deficiency and acrosomal malformation, as confirmed by transmission electron microscopy (TEM). Histological analysis revealed multiple morphological abnormalities of the sperm flagella in CFAP300 -mutant individual, whereas immunofluorescence demonstrated markedly reduced CFAP300 expression in the spermatozoa of the proband. Furthermore, tandem mass tag (TMT)-based quantitative proteomics showed that the CFAP300 mutation reduced key spermatogenesis proteins (e.g., sperm flagellar 2 [SPEF2], solute carrier family 25 member 31 [SLC25A31], and A-kinase anchoring protein 3 [AKAP3]) and mitochondrial ATP synthesis factors (e.g., SLC25A31, cation channel sperm-associated 3 [CATSPER3]). It also triggered abnormal increases in autophagy-related proteins and signaling mediator phosphorylation. These molecular alterations are likely to contribute to progressive deterioration of sperm ultrastructure and function. Notably, successful pregnancy was achieved via intracytoplasmic sperm injection (ICSI) using the proband's sperm. Overall, this study expands the known CFAP300 mutational spectrum and offers novel mechanistic insights into its role in spermatogenesis.
Humans ; Male ; Infertility, Male/pathology* ; Acrosome/pathology* ; Sperm Tail/pathology* ; Pedigree ; Spermatozoa ; Adult ; Loss of Function Mutation ; Ciliary Motility Disorders/genetics* ; Spermatogenesis/genetics* ; Female

Objective:To investigate the differential expression of Nuclear Factor Kappa B(NF-κB)and Tumor Necrosis Factor-Alpha(TNF-α)in endometrial polyp tissues,polyp-adjacent endometrium,and normal endometrium,and to analyze their correlation with local inflammatory responses,thereby elucidating the pathogenesis of endometrial polyps.Methods:Thirty-six patients with hysteroscopically confirmed endometrial polyps at Qingdao University Affiliated Hospital(September 2023-December 2024)were enrolled.Polyp tissues and adjacent endometrial tissues(0.5 cm-1 cm from polyps)were collected during surgery.Twenty-six patients undergoing hysterectomy for uterine fibroids,with pathologically confirmed normal endometrium,served as controls.The expression of NF-κB and TNF-α proteins was assessed using immunohistochemistry(IHC).Real-time quantitative PCR(RT-qPCR)was employed to detect the mRNA expression levels of NF-κB and TNF-α,and Western blot analysis was used to measure their protein expression.Differences among the three groups were compared.Results:Immunohistochemical analysis demonstrated:NF-κB p65 was predominantly localized to the cytoplasm and/or nucleus;TNF-α-positive expression was principally observed in the cytoplasmic compartment and/or cell membrane,manifesting as brown-yellow to brownish granules.The positive expression rate of NF-κB protein was significantly elevated in endometrial polyp tissues compared to both adjacent endometrial tissues and normal endometrial tissues(P＜0.05),whereas no statistically significant difference was observed between adjacent and normal endometrial tissues(P＞0.05).TNF-α protein expression exhibited a gradient pattern:Endometrial polyp tissues＞Adjacent endometrial tissues＞Normal endometrial tissues(with all inter-group comparisons demonstrating statistically significant differences,P＜0.05).Real-time quantitative PCR analysis revealed a significant gradient trend in the mRNA expression of NF-κB and TNF-α in endometrial polyp tissues:NF-κB mRNA:Polyp group(7.15±3.15)＞parapolyp group(4.38±2.01)＞normal group(1.03±0.30),with significant intergroup differences(all P＜0.05).TNF-α mRNA:Polyp tissue(17.66±9.25)＞parapolyp tissue(9.35±5.75)＞normal endometrium(1.16±0.58),with statistically significant differences among all groups(all P＜0.05).Western blot quantification further confirmed this trend:NF-κB protein:Expression in the polyp group(0.87±0.11)was 2.23-fold higher than in the normal group(0.39±0.02)(P=0.0007),while the parapolyp group(0.59±0.07)showed a 51％increase compared to the normal group(P=0.0435).TNF-α protein:Expression in the polyp group(1.13±0.21)was elevated by 94.8％versus the parapolyp group(0.58±0.03,P=0.0036)and by 494.7％versus the normal group(0.19±0.03,P=0.0002).One-way ANOVA indicated significant intergroup differences(F=43.56,P＜0.05).Conclusion:The TNF-α-mediated NF-κB signaling pathway is abnormally activated in endometrial polyps,exhibiting its highest expression at the polyp site.This suggests that the localized chronic inflammatory microenvironment may promote polyp formation through persistent stimulation of the NF-κB pathway.Targeted regulation of this pathway offers a novel strategy for preventing polyp recurrence.

With the rapid development of generative artificial intelligence(GAI)technologies,their widespread application in academic research and writing is continuously expanding the boundaries of sci-entific inquiry.However,this trend has also raised a series of ethical and regulatory challenges,inclu-ding issues related to authorship,content authenticity,citation accuracy,and accountability.In light of the growing involvement of AI in generating academic content,establishing an open,controllable,and trustworthy ethical governance framework has become a key task for safeguarding research integrity and maintaining trust within the academic community.This expert consensus outlines ethical requirements across key stages of AI-assisted academic writing-including topic selection,data management,citation practices,and authorship attribution.It aims to clarify the boundaries and ethical obligations surrounding AI use in academic writing,ensuring that technological tools enhance efficiency without compromising in-tegrity.The goal is to provide guidance and institutional support for building a responsible and sustainable research ecosystem.

Objective To summarize the changing prevalence of carbapenem resistance in Enterobacterales based on the data of CHINET Antimicrobial Resistance Surveillance Program from 2015 to 2021 for improving antimicrobial treatment in clinical practice.Methods Antimicrobial susceptibility testing was performed using a commercial automated susceptibility testing system according to the unified CHINET protocol.The results were interpreted according to the breakpoints of the Clinical & Laboratory Standards Institute(CLSI)M100 31st ed in 2021.Results Over the seven-year period(2015-2021),the overall prevalence of carbapenem-resistant Enterobacterales(CRE)was 9.43％(62 342/661 235).The prevalence of CRE strains in Klebsiella pneumoniae,Citrobacter freundii,and Enterobacter cloacae was 22.38％,9.73％,and 8.47％,respectively.The prevalence of CRE strains in Escherichia coli was 1.99％.A few CRE strains were also identified in Salmonella and Shigella.The CRE strains were mainly isolated from respiratory specimens(44.23±2.80)％,followed by blood(20.88±3.40)％and urine(18.40±3.45)％.Intensive care units(ICUs)were the major source of the CRE strains(27.43±5.20)％.CRE strains were resistant to all the β-lactam antibiotics tested and most non-β-lactam antimicrobial agents.The CRE strains were relatively susceptible to tigecycline and polymyxins with low resistance rates.Conclusions The prevalence of CRE strains was increasing from 2015 to 2021.CRE strains were highly resistant to most of the antibacterial drugs used in clinical practice.Clinicians should prescribe antimicrobial agents rationally.Hospitals should strengthen antibiotic stewardship in key clinical settings such as ICUs,and take effective infection control measures to curb CRE outbreak and epidemic in hospitals.