ObjectiveTo optimize and validate the optimal sequential alcohol-water extraction process of modified Banxia Xiexintang（MBXT） based on pharmacodynamic evaluation， combined with the G1-entropy weight method and Box-Behnken response surface methodology， and to systematically and comprehensively analyze the material basis of this formula， providing a scientific basis for its quality control and industrial production. MethodsRats were randomly divided into the normal group， model group， metformin group， and MBXT water extraction， water extraction and ethanol precipitation， sequential ethanol-water extraction groups. Except for the normal group， a polycystic ovary syndrome with insulin resistance（PCOS-IR） model was established in all rats via a high-fat diet combined with letrozole induction. Enzyme-linked immunosorbent assay（ELISA） biochemical assay kits and hematoxylin-eosin（HE） staining were used to compare sex hormone levels in serum and ovarian histopathology， thereby screening extraction process routes. Based on this， a comprehensive score was constructed using the G1-entropy weight method based on the transfer rates of index components（berberine hydrochloride and baicalin） and the dry extract rate. Box-Behnken response surface methodology was then utilized to optimize the extraction process parameters. Finally， the chemical constituents of the sample from the optimal process were qualitatively analyzed by ultra-performance liquid chromatography-quadrupole-electrostatic field orbitrap mass spectrometry（UPLC-Q-Orbitrap-MS）. ResultsPharmacodynamic findings revealed that compared with the normal group， serum testosterone（T） and luteinizing hormone（LH） levels were significantly elevated in the model group， while estradiol（E₂） and follicle-stimulating hormone（FSH） levels were significantly decreased（P<0.01）， with polycystic changes observed in ovarian tissues. Compared with the model group， all treatment groups significantly reversed the changes in sex hormone levels， with the sequential ethanol-water extraction group showing the optimal effect in improving the aforementioned indicators and pathological morphology， followed by subsequent process optimization. The optimized process involved adding 12 times the amount of 70% ethanol for extracting twice， each lasting 120 min， and adding 12 times the amount of water for extracting thrice， each lasting 90 min. Validation test results showed that under optimal process conditions， the average transfer rates of berberine hydrochloride and baicalin were 76.05% and 93.38%， respectively. MS analysis identified a total of 377 compounds， including 112 flavonoids， 41 terpenoids， 28 organic acids， 22 coumarins， and 8 alkaloids， while elucidating the cleavage patterns of key components. ConclusionThe optimized sequential ethanol-water extraction process is stable and feasible， effectively preserving the material basis of MBXT for treating PCOS-IR. It further clarifies the main chemical composition of this formula， providing a scientific basis for the development and quality control of its preparations.

ObjectiveTo evaluate the therapeutic effects of modified Banxia Xiexintang（MBXT） on polycystic ovary syndrome with insulin resistance（PCOS-IR） rats and reveal its potential mechanisms based on the integrated analysis of transcriptomics and metabolomics. MethodsFemale SD rats were selected， and a PCOS-IR model was established by intragastric administration of letrozole combined with a high-fat diet for 21 days. The modeled rats were randomly divided into the model group， MBXT low-， medium-， and high-dose groups（6.62， 13.23， 26.46 g·kg^-1）， and metformin group（0.158 g·kg^-1）， with a normal group set up separately. After 14 days of administration， the estrous cycle was observed， ovarian morphology was examined by hematoxylin-eosin（HE） staining， and the levels of testosterone（T）， estradiol（E₂）， follicle-stimulating hormone（FSH）， and luteinizing hormone（LH） in serum were detected by enzyme-linked immunosorbent assay（ELISA）. Serum metabolites and ovarian tissue gene expression were detected using ultra-performance liquid chromatography-quadrupole-electrostatic orbitrap mass spectrometry（UPLC-Q-Orbitrap-MS） and RNA-Seq technology， respectively， followed by multi-omics integrated analysis. ResultsPharmacodynamic findings revealed that all MBXT dose groups could reversed abnormal estrous cycles in PCOS-IR rats， improve polycystic ovarian lesions， and normalize dysregulated serum hormone levels（T， LH， E₂， FS， P<0.05， P<0.01）. Metabolomic analysis revealed that compared with the model group， MBXT reversed 278 differential metabolites such as estrone and S-formylglutathione， mainly involving pathways such as steroid hormone biosynthesis， glutathione metabolism， and lipid peroxidation regulation. Transcriptomic analysis identified 434 differentially expressed genes， and enrichment analysis revealed that MBXT significantly regulated lipid peroxidation defense systems， including glutathione metabolism， peroxisome function， and fatty acid metabolism， thereby intervening in ferroptosis processes. It also engaged in inflammation-related pathways such as the chemokine signaling pathway. Integrated analysis revealed that both metabolomics and transcriptomics co-enriched metabolic pathways associated with ferroptosis and fatty acid metabolism. And key Hub genes［such as Ras-related C3 botulinum toxin substrate 2 gene（Rac2） and Fas ligand gene（Faslg）］ showed significant correlations with differential metabolites. ConclusionMBXT can effectively ameliorate reproductive dysfunction and metabolic disorders in PCOS-IR rats. Its mechanism may be related to remodeling the immune-metabolism network， particularly by regulating MHC-mediated immune responses， inhibiting local ovarian ferroptosis， and enhancing steroid hormone synthesis pathways.

ObjectiveTo investigate the mechanism of modified Banxia Xiexintang（MBXT） in improving ovarian dysfunction in polycystic ovary syndrome with insulin resistance（PCOS-IR） rats by inhibiting ferroptosis through the adenosine monophosphate（AMP）-activated protein kinase（AMPK）/fatty acid synthase（FASN）/glutathione peroxidase 4（GPX4） signaling pathway. MethodsSeventy-six female SD rats were randomly divided into a normal group（n=13） and a modeling group（n=63）. The modeling group established a PCOS-IR model by intragastric administration of letrozole combined with a high-fat diet for 21 days. After successful modeling， these rats were randomly divided into the model group， MBXT low-， medium-， and high-dose groups（6.62， 13.23， 26.46 g·kg^-1）， metformin group（0.158 g·kg^-1）， and high-dose of MBXT combined with ferroptosis inducer Erastin group（15 mg·kg^-1）， with 10 rats in each group. After 14 days of intervention， ovarian pathological morphology was observed by hematoxylin-eosin（HE） staining， the mitochondrial ultrastructure of granulosa cells was observed by transmission electron microscopy（TEM）， ovarian reactive oxygen species（ROS） levels were detected by dihydroethidium（DHE） probe， biochemical methods were used to detect Fe²⁺， malondialdehyde（MDA）， glutathione（GSH） and other indicators in ovarian tissues， serum sex hormone and insulin levels were measured by enzyme-linked immunosorbent assay（ELISA）， and the protein expressions of AMPK， FASN， acyl-CoA synthetase long-chain family member 4（ACSL4）， GPX4， and solute carrier family 7 member 11（SLC7A11） in ovarian tissues were detected by Western blot. ResultsCompared with the normal group， the model group showed polycystic changes in the ovaries， with atrophy of mitochondria in granulosa cells and increased membrane density. Serum levels of testosterone（T）， luteinizing hormone（LH）， and insulin were significantly increased（P<0.01）. The levels of ROS， MDA， 4-hydroxynonenal（4-HNE）， and Fe²⁺ in ovarian tissues were significantly elevated（P<0.01）， while adenosine triphosphate（ATP）， GSH， and reduced nicotinamide adenine dinucleotide phosphate （NADPH） levels were significantly decreased（P<0.01）. The phosphorylation levels of AMPK and acetyl-CoA carboxylase （ACC）， as well as the protein expressions of SLC7A11， GPX4， and ferroptosis suppressor protein 1（FSP1） were significantly downregulated（P<0.01）， whereas the expressions of FASN， ACSL4， and nuclear receptor coactivator 4（NCOA4） were significantly upregulated（P<0.01）. Compared with the model group， MBXT intervention at various doses improved the above pathological changes and biochemical indicators in a dose-dependent manner， with the high-dose group showing the most significant effect（P<0.01）. Compared with the MBXT high-dose group， the high-dose of MBXT combined with ferroptosis inducer Erastin group restored ovarian ferroptosis characteristics in rats， with increased ROS and lipid peroxidation products， and altered expressions of key proteins（P<0.05， P<0.01）. ConclusionMBXT can effectively improve ovarian function and metabolic disorders in PCOS-IR rats. Its mechanism may be related to activating the AMPK/ACC signaling pathway， downregulating FASN and ACSL4 to reduce lipid peroxidation substrates， and restoring glucose-6-phosphate dehydrogenase/phosphoglycerate dehydrogenase（G6PD/PHGDH） metabolic flux to enhance the GPX4/FSP1 antioxidant defense system， thereby inhibiting ferroptosis in ovarian granulosa cells.

Medicinal plants， with diverse species， high heterozygosity， and special breeding objectives， can be hardly bred with conventional hybridization techniques. Plant genetic transformation is highly selective and can specifically change the traits of plants， serving as an important technical means for the breeding of medicinal plants. The commonly used plant genetic transformation technologies include Agrobacterium-mediated transformation and particle bombardment. Agrobacterium-mediated transformation is the most widely used method， while it is not applicable to all medicinal plants due to the high specificity. Although not specific， particle bombardment is limited in application due to the low conversion efficiency and external force damage to cells and tissue. With the rise and development of nanotechnology， the emerging nanomaterial-mediated transformation has solved the problems of the above two technologies. However， limited by its late development， the mechanism of nanomaterial-mediated introduction of genetic materials into plant cells remains unclear， and thus this technology is rarely used in medicinal plants. This article summarizes the development status of several commonly used or emerging plant genetic transformation technologies such as Agrobacterium-mediated transformation， particle bombardment， and nanomaterial-mediated transformation， as well as their application in different medicinal plants. Furthermore， this article looks forward to the development trend of genetic transformation technologies for plants and their application prospects in medicinal plants and Chinese materia medica resources， aiming to provide new technical ideas for the genetic improvement and germplasm innovation of medicinal plants and inject new impetus into the sustainable development of Chinese materia medica resources.

BACKGROUND:Gradient artificial bone repair scaffolds can mimic unique anatomical features in musculoskeletal tissues,showing great potential for repairing injured musculoskeletal tissues. OBJECTIVE:To review the latest research advances in gradient artificial bone repair scaffolds for tissue engineering in the musculoskeletal system and describe their advantages and fabrication strategies. METHODS:The first author of the article searched the Web of Science and PubMed databases for articles published from 2000 to 2023 with search terms"gradient,bone regeneration,scaffold".Finally,76 papers were analyzed and summarized after the screening. RESULTS AND CONCLUSION:(1)As an important means of efficient and high-quality repair of skeletal system tissues,gradient artificial bone repair scaffolds are currently designed bionically for the natural gradient characteristics of bone tissue,bone-cartilage,and tendon-bone tissue.These scaffolds can mimic the extracellular matrix of native tissues to a certain extent in terms of structure and composition,thus promoting cell adhesion,migration,proliferation,differentiation,and regenerative recovery of damaged tissues to their native state.(2)Advanced manufacturing technology provides more possibilities for gradient artificial bone repair scaffold preparation:Gradient electrospun fiber scaffolds constructed by spatially differentiated fiber arrangement and loading of biologically active substances have been developed;gradient 3D printed scaffolds fabricated by layered stacking,graded porosity,and bio-3D printing technology;gradient hydrogel scaffolds fabricated by in-situ layered injections,simple layer-by-layer stacking,and freeze-drying method;and in addition,there are also scaffolds made by other modalities or multi-method coupling.These scaffolds have demonstrated good biocompatibility in vitro experiments,were able to accelerate tissue regeneration in small animal tests,and were observed to have significantly improved histological structure.(3)The currently developed gradient artificial bone repair scaffolds have problems such as mismatch of gradient scales,unclear material-tissue interactions,and side effects caused by degradation products,which need to be further optimized by combining the strengths of related disciplines and clinical needs in the future.

Objective:To summarized the clinical characteristics and surgical management of anterior cervical enterogenic in pediatric patients. Methods:Clinical data were retrospectively analyzed for 9 children with pathologically confirmed anterior cervical enterogenic cysts（including bronchogenic and esophagogenic subtypes） treated at the Children's Hospital of Shandong University（Jinan Children's Hospital） between January 1, 2020, and November 30, 2023. Results:Nine patients（6 males and 3 females） were involved in this study, aged 14 days to 10 years old. There were 4 cases on the left side, 4 on the right side, and 1 in the middle of the neck. All patients presented with neck masses. The patients were followed up from 3 months to 35 months after surgery and recovered well, with no recurrence or complications observed. Conclusion:①Anterior intestinal cysts in children are rare and easy to be misdiagnosed. ②Concurrent branchial cleft fistulas or associated anomalies may coexist, necessitating comprehensive evaluation. ③Preoperative diagnosis is not easy and mainly depends on pathological diagnosis. ④The treatment of anterior cervical enterogenic cysts in children is surgical resection of the lesion.
Humans ; Male ; Female ; Child ; Retrospective Studies ; Child, Preschool ; Infant ; Neck ; Cysts/surgery*

The specific mechanisms of interferon regulatory factor 8(IRF8)in porcine intestinal in-nate immunity and resistance to enteric virus infection remain to be elucidated.To investigate the immunoregulatory role of IRF8,establishing an IRF8 gene knockout porcine intestinal epithelial cell(IPEC-J2)monoclonal cell line is of significant importance.This study initially aimed to obtain recombinant adeno-associated virus rAAV-sgIRF8-eGFP capable of knocking out the IRF8 gene through co-transfection of HEK-293T cells with three plasmids.Subsequently,IPEC-J2 cells were infected with the virus,and those expressing eGFP were selected by flow cytometry and cultured to form monoclonal cell lines.These cell lines were then identified by Sanger sequencing and West-ern blot techniques.Lastly,qPCR analysis was used to measure the expression levels of interferon factors IFN-α,IFN-β,IFN-γ and IFN-λ,providing preliminary insights into the impact of IRF8 gene knockout on IPEC-J2 cell immunity.The results demonstrated successful generation of rAAV-sgIRF8-eGFP,which successfully infected IPEC-J2 cells leading to eGFP fluorescence.Flow cytometry followed by cell culture led to the establishment of two monoclonal cell lines,IRF8-KO1 and IRF8-KO3.Sanger sequencing revealed a five-base deletion in IRF8-KO1 and a seven-base dele-tion in IRF8-KO3.Western blot confirmed the absence of IRF8 protein expression in IRF8-KO1,making it an ideal candidate monoclonal cell line.qPCR analysis of interferon factors indicated sig-nificant decrease in IFN-γ(P＜0.05)and IFN-λ(P＜0.01)transcription level in IRF8-knockout cells,while the transcription levels of IFN-α and IFN-β remained relatively unchanged.This study successfully established an IRF8 gene knockout IPEC-J2 monoclonal cell line,providing a founda-tion for further research on IRF8-related porcine intestinal immune regulation and mechanisms of intestinal virus infection.

Purpose To explore the clinicopathological features and potential clinical value of marginal zone lym-phoma(MZL)derived from T-bet positive memory B cell.Methods Clinical data of 67 cases of MZL were collected.Hematoxylin-eosin,immunohistochemistry,and multiple immunofluorescence stains,B cell receptor high throughput sequencing technology were used to study the histology,immunophenotype,and immunoglobulin heavy chain variable gene(IGHV)repertoire.Results T-bet was expressed in some MZL patients(34/67,50.75％),which was correla-ted with clinicopathological characteristics such as gender,clinical stage,and Ki67 proliferation index(P＜0.05),and also the progression-free survival was poor(P=0.012 2).T-bet positivity was a risk factor affecting the progres-sion of MZL.Microscopically,T-bet positive MZL frequently presented T-bet positive tumor B cells surrounded follicu-lar germinal center,"MALT ball"-type lymphoepithelial lesions,and IgG positive neoplastic plasma cells(P＜0.05).T-bet had no biased influence on the VH gene usage(P＞0.05).The common VH families were IGHV4 and IGHV3,and the segments were IGHV4-34 and IGHV3-30.The positivity of T-bet was associated with somatic hypermutation(SHM)state(P=0.014 9).The SHM was mainly in the range of 2％-4.9％in T-bet positive MZL,while in T-bet negative MZL the SHM was mostly greater than 5％.The VH gene usage was not correlated with the clinicopathological features of patients(P＞0.05).IGHV4 was correlated with progression-free survival in T-bet positive MZL(P=0.038 2).Conclusion The expression of T-bet in MZL is closely related to the clinicopathological features such as histology,plasma cell immunophenotype and IGHV gene repertoire,and the prognosis of patients is poor,which may be a potential molecular marker affecting the progression of MZL.

This paper analyzes the treatment process of a patient who developed severe acute respiratory distress syndrome (ARDS) following a chemical leak of dimethyl sulfate caused by factory operational error in February 2023, and explores the efficacy of extracorporeal membrane oxygenation (ECMO) in patient management. The patient developed severe respiratory symptoms five hours after chemical exposure and received sequential treatment with nasal high-flow oxygen therapy and invasive mechanical ventilation upon admission. When the oxygenation index dropped below 100 mmHg, veno-venous (VV) ECMO was initiated alongside 12-hour daily prone position ventilation to improve oxygenation and lung compliance. The patient recovered and was discharged following treatment. For patients with dimethyl sulfate poisoning complicated by severe ARDS, early implementation of VV-ECMO significantly improves treatment outcomes and prognosis.

The specific mechanisms of interferon regulatory factor 8(IRF8)in porcine intestinal in-nate immunity and resistance to enteric virus infection remain to be elucidated.To investigate the immunoregulatory role of IRF8,establishing an IRF8 gene knockout porcine intestinal epithelial cell(IPEC-J2)monoclonal cell line is of significant importance.This study initially aimed to obtain recombinant adeno-associated virus rAAV-sgIRF8-eGFP capable of knocking out the IRF8 gene through co-transfection of HEK-293T cells with three plasmids.Subsequently,IPEC-J2 cells were infected with the virus,and those expressing eGFP were selected by flow cytometry and cultured to form monoclonal cell lines.These cell lines were then identified by Sanger sequencing and West-ern blot techniques.Lastly,qPCR analysis was used to measure the expression levels of interferon factors IFN-α,IFN-β,IFN-γ and IFN-λ,providing preliminary insights into the impact of IRF8 gene knockout on IPEC-J2 cell immunity.The results demonstrated successful generation of rAAV-sgIRF8-eGFP,which successfully infected IPEC-J2 cells leading to eGFP fluorescence.Flow cytometry followed by cell culture led to the establishment of two monoclonal cell lines,IRF8-KO1 and IRF8-KO3.Sanger sequencing revealed a five-base deletion in IRF8-KO1 and a seven-base dele-tion in IRF8-KO3.Western blot confirmed the absence of IRF8 protein expression in IRF8-KO1,making it an ideal candidate monoclonal cell line.qPCR analysis of interferon factors indicated sig-nificant decrease in IFN-γ(P＜0.05)and IFN-λ(P＜0.01)transcription level in IRF8-knockout cells,while the transcription levels of IFN-α and IFN-β remained relatively unchanged.This study successfully established an IRF8 gene knockout IPEC-J2 monoclonal cell line,providing a founda-tion for further research on IRF8-related porcine intestinal immune regulation and mechanisms of intestinal virus infection.