High-performance liquid chromatography(HPLC) was used to determine the content of three major components in Citri Reticulatae Semen(CRS), including limonin, nomilin, and obacunone. The chromaticity of the CRS sample during salt processing and stir-frying was measured using a color difference meter. Next, the relationship between the color and content of the salt-processed CRS sample was investigated through correlation analysis. By integrating the oil bath technique for processing simulation with HPLC, the changes in the relative content of nomilin and its transformation products were analyzed, with its structural transformation pattern during processing identified. Additionally, RAW264.7 cells were induced with lipopolysaccharides(LPSs) to establish an inflammatory model, and the anti-inflammatory activity of nomilin and its transformation product, namely obacunone was evaluated. The results indicated that as processing progressed, E~*ab and L~* values showed a downward trend; a~* values exhibited a slow increase over a certain period, followed by no significant changes, and b~* values remained stable with no significant changes over a certain period and then started to decrease. The limonin content remained barely unchanged; the nomilin content decreased, and the obacunone increased significantly. The changing trends in content and color parameters during salt-processing and stir-frying were basically consistent. The content of nomilin and obacunone was significantly correlated with the colorimetric values(L~*, a~*, b~*, and E~*ab), while limonin content showed no significant correlation with these values. By analyzing HPLC patterns of nomylin at different heating temperatures and time, it was found that under conditions of 200-250 ℃ for heating of 5-60 min, the content of nomilin significantly decreased, while the obacunone content increased pronouncedly. The in vitro anti-inflammatory activity results indicated that compared to the model group, the group with a high concentration of nomilin and the groups with varying concentrations of obacunone showed significantly reduced release of nitric oxide(NO)(P<0.01). When both were at the same concentration, obacunone showed better performance in inhibiting NO release. In this study, the obvious correlation between the color and content of major components during the processing of CRS samples was identified, and the dynamic patterns of quality change in CRS samples during processing were revealed. Additionally, the study revealed and confirmed the transformation of nomilin into obacunone during processing, with the in vitro anti-inflammatory activity of obacunone significantly greater than that of nomilin. These findings provided a scientific basis for CRS processing optimization, tablet quality control, and its clinical application.
Mice ; Animals ; Drugs, Chinese Herbal/pharmacology* ; RAW 264.7 Cells ; Limonins/chemistry* ; Chromatography, High Pressure Liquid ; Citrus/chemistry* ; Color ; Benzoxepins/chemistry* ; Anti-Inflammatory Agents/chemistry*

To investigate the mechanism of action of Syngnathus extract in treating knee osteoarthritis of rats, forty-eight male SD rats were randomly divided into the blank group, model group, positive drug group, as well as low-dose, medium-dose, and high-dose groups of Syngnathus extract. The rat model of knee osteoarthritis was constructed by intra-articular injection of sodium iodoacetate. After successful modeling, celecoxib(18 mg·kg~(-1)·d~(-1)) and Syngnathus extract(0.4, 0.8, and 1.6 g·kg~(-1)·d~(-1)) were given in different groups by gavage intervention for two weeks. Hematoxylin-eosin(HE) staining was used to observe the histopathological changes of cartilage in knee joints, and enzyme-linked immunosorbent assay(ELISA) was used to detect the expression level of inflammatory factors in serum. Real-time fluorescence quantitative PCR, Western blot, and immunohistochemistry were used to detect the levels of phosphatidylinositol 3-kinase(PI3K)/protein kinase B(Akt)/mammalian target protein of rapamycin(mTOR) pathway-related mRNA and protein expression. The results showed that, comparied with the blank group, the cartilage surface of the knee joints of rats in the model group was uneven, with disorganized levels and defective cartilage tissue. The serum levels of interleukin-1β(IL-1β), interleukin-6(IL-6), and tumor necrosis factor-α(TNF-α) and the mRNA levels of PI3K, Akt, and mTOR in cartilage tissue, as well as the protein expression levels of phosphorylated PI3K(p-PI3K)/PI3K, phosphorylated Akt(p-Akt)/Akt, phosphorylated mTOR(p-mTOR)/mTOR, and P62 were significantly increased. Beclin1 protein expression was decreased. Comparied with the model group, the number of chondrocytes in the knee joint of rats in each group of Syngnathus extract increased, and the arrangement of chondrocytes was relatively neat. The cartilage layer was restored, and the serum levels of IL-1β, IL-6, and TNF-α, as well as the mRNA expression levels of PI3K, Akt, and mTOR in cartilage tissue were significantly reduced. The protein expression levels of p-PI3K/PI3K, p-Akt/Akt, p-mTOR/mTOR, and P62 were significantly reduced in the rats in the middle-dose and high-dose groups of Syngnathus extract, and the Beclin1 protein expression was significantly increased. The protein expression levels of p-PI3K/PI3K, p-Akt/Akt, and P62 in rats in the low-dose group of Syngnathus extract were significantly reduced. In summary, Syngnathus extract may be used to treat knee osteoarthritis by inhibiting the expression of PI3K/Akt/mTOR signaling pathway, so as to alleviate the inflammatory response in the organism, enhance the autophagy activity of chondrocytes, and reduce the apoptosis of chondrocytes.
Animals ; TOR Serine-Threonine Kinases/genetics* ; Male ; Rats, Sprague-Dawley ; Signal Transduction/drug effects* ; Proto-Oncogene Proteins c-akt/genetics* ; Rats ; Osteoarthritis, Knee/metabolism* ; Drugs, Chinese Herbal/administration & dosage* ; Phosphatidylinositol 3-Kinases/genetics* ; Humans

This study predicts the potential mechanism of Hippocampus in the treatment of knee osteoarthritis(KOA) through network pharmacology, with preliminary verification using molecular docking and animal experiments. The database was used to screen the active chemical components of Hippocampus and the targets of KOA, and Gene Ontology(GO) functional analysis, Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway enrichment analysis, and molecular docking were performed on the relevant core targets to preliminarily explore the potential targets and mechanisms of Hippocampus in the treatment of KOA. A rat KOA model was constructed by intra-articular injection of sodium iodoacetate, and the rats were intervened with different doses of Hippocampus decoction and celecoxib. The expression of relevant targets was detected through hematoxylin-eosin(HE) staining, enzyme-linked immunosorbent assay(ELISA), RT-qPCR, and Western blot to further validate the network pharmacology results. A total of 23 drug-like components of the Hippocampus were screened, and 128 common targets with KOA were identified, involving interleukin-17(IL-17) signaling pathway, transcription factor(FoxO) signaling pathway, tumor necrosis factor(TNF) signaling pathway. Molecular docking results showed that the screened core chemical components exhibited good affinity with key targets. HE staining demonstrated that Hippocampus improved the morphology of the cartilage layer. ELISA confirmed that Hippocampus significantly reduced the levels of IL-6 and TNF-α in the serum of KOA rats. Western blot and RT-qPCR analysis showed that Hippocampus significantly reduced the expression of IL-6, TNF-α, matrix metalloproteinase(MMP) 13, IL-17A, nuclear factor κB activator 1(ACT1), tumor necrosis factor receptor-associated factor 6(TRAF6) and nuclear factor κB(NF-κB) in cartilage tissue. The results suggest that Hippocampus can alleviate the degree of joint damage in the KOA rat model induced by sodium iodoacetate. The mechanism of action is related to the inhibition of the IL-17 signaling pathway, reduction of inflammation, and inhibition of extracellular matrix(ECM) degradation.
Animals ; Molecular Docking Simulation ; Rats ; Drugs, Chinese Herbal/administration & dosage* ; Network Pharmacology ; Male ; Osteoarthritis, Knee/metabolism* ; Rats, Sprague-Dawley ; Signal Transduction/drug effects* ; Humans ; Interleukin-17/metabolism* ; Tumor Necrosis Factor-alpha/metabolism* ; Disease Models, Animal ; Hippocampus/chemistry*

OBJECTIVE: To investigate the clinical features and risk factors associated with cutaneous chronic graft-versus-host disease (cGVHD) after allogeneic hematopoietic stem cell transplantation (allo-HSCT) in children. METHODS: A retrospective analysis was conducted on the clinical data of children who underwent allo-HSCT in the Wuhan Children's Hospital from August 1, 2016, to December 31, 2023, and were regularly followed up for 1 year or more. The differences in clinical features between children with and without cutaneous cGVHD were compared, and the risk factors affecting the occurrence of cutaneous cGVHD were analyzed. RESULTS: During the study period, 296 children received allo-HSCT. Until December 31, 2024, follow-up showed that 20 children (6.8%) developed cutaneous cGVHD, which manifested as cutaneous lichenification, hyperpigmentation, keratosis pilaris, sclerotic changes, and hair or nail involvement. According to their skin lesion area and degree of grading, 5 cases were mild, 10 cases were moderate, and 5 cases were severe. Multivariate logistic regression analysis revealed that female donors and previous acute GVHD were risk factors for the development of cutaneous cGVHD after allo-HSCT. All 20 children were treated with glucocorticoid ± calcineurin inhibitors (tacrolimus/cyclosporine) as first-line therapeutic agents. Only 1 child improved after first-line treatment. The remaining 19 children treated with a second-line regimen of combination interventions based on individualized status, including 10 children who could not tolerate hormonotherapy or first-line treatment, and showed no significant improvement after 3 months, as well as 9 children with multi-organ cGVHD. After comprehensive second-line treatment, 17 children showed improvement in cutaneous symptoms. There were 3 deaths, including 1 due to primary disease recurrence and 2 due to pulmonary infections. CONCLUSION The skin is the first manifestation and most common organ involved in cGVHD in children. Cutaneous cGVHD severely affects the daily activities of transplanted children and requires prolonged immunosuppressive therapy, but has a favorable prognosis. First-line treatments for adults are not applicable to children who usually require a combination treatment with multiple drugs.
Humans ; Graft vs Host Disease/etiology* ; Hematopoietic Stem Cell Transplantation/adverse effects* ; Retrospective Studies ; Risk Factors ; Female ; Child ; Skin Diseases/etiology* ; Chronic Disease ; Transplantation, Homologous ; Male ; Child, Preschool ; Adolescent

Mutations in the cyclin-dependent kinase-like 5 gene (CDKL5) cause a severe neurodevelopmental disorder, yet the impact of truncating mutations remains unclear. Here, we introduce the Cdkl5^492stop mouse model, mimicking C-terminal truncating mutations in patients. 492stop/Y mice exhibit altered dendritic spine morphology and spontaneous seizure-like behaviors, alongside other behavioral deficits. After creating cell lines with various Cdkl5 truncating mutations, we found that these mutations are regulated by the nonsense-mediated RNA decay pathway. Most truncating mutations result in CDKL5 protein loss, leading to multiple disease phenotypes, and offering new insights into the pathogenesis of CDKL5 disorder.
Animals ; Disease Models, Animal ; Mice ; Protein Serine-Threonine Kinases/deficiency* ; Mutation/genetics* ; Epileptic Syndromes/genetics* ; Humans ; Dendritic Spines/pathology* ; Spasms, Infantile/genetics* ; Male ; Seizures/genetics* ; Mice, Inbred C57BL

Objective To explore the application value of multi-model adaptive statistical iterative reconstruction-Veo (ASiR-V) in ultra-low dose chest CT examination of children in the plateau area. Methods The children who underwent chest CT examination in Xizang Autonomous Region People's Hospital were enrolled in this study and assigned into two groups according to the scanning conditions.Group A underwent scanning at a tube voltage of 100 kV and ASiR-V 50% reconstruction,and group B underwent scanning at a tube voltage of 80 kV and ASiR-V 0 (Group B1) and ASiR-V 50% (Group B2) reconstruction.The image quality of each group was evaluated objectively and subjectively.The radiation dose and image quality were compared between groups. Results Groups A and B showed the volume CT dose indexes of (2.33±0.62) mGy and (0.86±0.01) mGy and the dose length products of (65.01±25.12) mGy·cm and (23.55±3.38) mGy·cm,respectively,which presented differences between groups (both P<0.001).The image noise in the bilateral upper and middle lung areas in group B2 was lower than that in group B1 but higher than that in group A (all P<0.001).There was no significant difference in image quality score of the lung window among groups (all P>0.05).Groups A,B1,and B2 had no significant differences in ascending aorta (P=0.538) or liver CT value (P=0.175) in the mediastinal window.The signal-to-noise ratios and contrast-to-noise ratios of ascending aorta and liver in group B2 were higher than those in group B1 (all P<0.001) and lower than those in group A (all P<0.05).The image quality score of the mediastinal window followed a descending order of group A>group B2>group B1 (all P<0.001)。Conclusion ASiR-V combined with low tube voltage can effectively reduce the radiation dose and guarantee the image quality of chest CT of children in the plateau area.
Humans ; Radiation Dosage ; Tomography, X-Ray Computed/methods* ; Child ; Male ; Female ; Child, Preschool ; Radiography, Thoracic/methods* ; Infant ; Models, Statistical ; Image Processing, Computer-Assisted/methods*

Objective To investigate the clinical characteristics of patients with clinical and subclinical Cushing's syndrome caused by primary bilateral macronodular adrenal hyperplasia(PBMAH).Methods A retrospective analysis was performed on the clinical data of 198 patients with Cushing's syndrome caused by PBMAH diagnosed in the First Medical Center of Chinese PLA General Hospital from January 2004 to October 2024.According to clinical manifestations,the patients were classified into clinical type Cushing's syndrome(n=61)and subclinical type Cushing's syndrome(n=137),and the clinical characteristics of the two types were compared.Results The mean age at diagnosis of patients with PBMAH-induced Cushing's syndrome was(53.5±10.4)years,including 118 males and 80 females,with a male-to-female ratio of 1.475:1.Compared with the subclinical type,the clinical type had a higher proportion of females,higher levels of serum cortisol,24-hour urine free cortisol(24 h UFC),and inhibited serum cortisol after low-dose dexamethasone suppression.Additionally,the clinical type had lower plasma ACTH,larger adrenal nodules and a higher risk of surgery(P<0.05)compared with those in subclinical type.The incidences of hypertension,dyslipidemia,obesity,diabetes mellitus,hypokalemia,vitamin D deficiency,osteoporosis,coronary heart disease,and cerebrovascular disease in patients with Cushing's syndrome caused by PBMAH were 87.9%,50.5%,37.1%,36.9%,27.8%,25.9%,18.7%,18.7%and 12.1%,respectively.Among them,compared with subclinical type patients,clinical type patients had higher incidence of hypokalaemia,vitamin D deficiency and osteoporosis(P<0.05),while there were no statistically significant differences in the incidences of other comorbidities between the two types(P>0.05).The results of postoperative follow-up for PBMAH patients showed that the short-term biochemical remission rate of unilateral total adrenalectomy was 41.5%(22/53)and the long-term biochemical remission rate was 32.0%(8/25).The short-term biochemical remission rate of unilateral partial(or nodular)adrenalectomy was 52.9%(9/17),and the long-term biochemical remission rate was 14.3%(1/7).All patients who underwent unilateral total adrenalectomy plus contralateral partial resection developed adrenal insufficiency(3/3),and 1 patient(1/3)relapsed 3.4 years after surgery.Conclusion Clinical and subclinical types of Cushing's syndrome caused by PBMAH have their distinct clinical characteristics.Surgery is an effective treatment for PBMAH,but a certain proportion of patients fail to achieve biochemical remission after non-bilateral total adrenalectomy.

Objective : To explore the utility of a nomogram integrating contrast-enhanced CT radiomics with clinical features in the preoperative prediction of WHO/ISUP grade for clear cell renal cell carcinoma(ccRCC). Methods: A total of 214 patients with pathologically proven ccRCC who underwent enhanced CT scan before surgery were retrospectively included. According to the WHO/ISUP grade system, the cases were classified into low-grade(grades Ⅰ-Ⅱ) and high-grade(grades Ⅲ-Ⅳ), and then randomly divided into training and test set with a ratio of 4 ∶1. Regions of interest were segmented from both unenhanced and three-phase enhanced images, and radiomic features were extracted. Feature selection and dimensionality reduction were performed using Spearman rank correlation coefficients and LASSO regression, followed by the construction of the radiomic model with the KNN algorithm. Clinical and semantic imaging features were selected through univariate and multivariate analyses, and a clinical model was developed using the KNN algorithm. The clinical and radiomics signatures were used to construct a combined model and a nomogram was developed. The ROC curve and delong test were used to evaluate the diagnostic performance of the model, while calibration and decision curve analyses assessed its accuracy and clinical applicability. Results: 8 clinical features and 11 radiomic features were selected. The combined model, integrating these clinical and radiomics signatures, exhibited robust predictive performance with AUC values of 0.887 in the training set and 0.800 in the test set. The calibration curve demonstrated good consistency between the nomogram model and actual outcomes, while decision curve analysis indicated a favorable net benefit for the nomogram. Conclusion The nomogram constructed by combining radiomics and clinical signatures can provide evidence for preoperative prediction of ccRCC grade and guide clinical decision-making.

Objective To analyze the burden and changing trend of testicular cancer in China from 1990 to 2019.Methods Based on the 2019 Global Burden of Disease Database(GBD 2019),analyze the incidence,mortality,disability-adjusted life years(DALYs),years of life lost(YLLs),years lived with disability(YLDs)and their variation trend of testicular cancer in Chinese population from 1990 to 2019.Evaluating changes in age standardized rate(ASR)by calculating annual estimated percentage change(EAPC).According to the age grouping,analyze the age distribution characteristics of testicular cancer disease burden by age group.Results In 2019,the incident cases,deaths,age-standardized incidence rate,and age-standardized mortality rate of testicular cancer in China were 17.17×103,1.21×103,2.39/105,and 0.16/105,respectively.Compared to 1990,incident cases,deaths,and age-standardized incidence rate increased obviously in China,which was consistent with the global change trend,while the increase was higher than the global level.However,both Chinese and global age-standardized mortality rate showed a downward trend.From 1990 to 2019,DALYs,YLLs and YLDs of testicular cancer increased by 29.66%,9.83%and 720.91%respectively in China.The two age groups,0-15 years group and 30-35 years group,were with highest incidence of testicular cancer,while the highest disease burden of testicular cancer was 30-35 years.Conclusion From 1990 to 2019,the disease burden of testicular cancer in China showed an upward trend.Adolescents and young adults should be the priority population for screening and prevention due to their higher incidence and disease burden.

Objective To generate minichromosome maintenance protein 2(MCM2)gene knockout cervi-cal cancer HeLa cell lines using inducible CRISPR/Cas9 technology,and to explore the effect of MCM2 on DNA replication and replication stress.Methods The inducible CRISPR/Cas9 system,TLCV2,was used to construct MCM2 knockout HeLa cell lines.And the cell lines were divided into control group(Control),knockout group 1(KO1),and knockout group 2(KO2).Western blot,Edu incorporation experiment,real-time quantitative PCR(qPCR),immunofluorescence and MTT assay were used to analyze the effects of MCM2 knockout on DNA replica-tion and replication stress induced by hydroxyurea.Results The CRISPR/Cas9 system successfully knocked out the MCM2 gene after induction,and MCM2 knockout affected the stability of MCM2-7 complex.Compared with the control cells,MCM2 knockout cells had a dramatic decrease in the capacity of DNA replication,and the mRNA levels of Cyclin A1,Cyclin E1 and CDK4.Under DNA replication stress,MCM2 knockout cells decreased cell viability,DNA damage repair capacity,and increased genomic instability compared with control cells.Conclusion Knockout of MCM2 gene reduces the DNA replication capacity of HeLa cells under normal conditions and cell viability under replication stress.This study successfully generates MCM2 gene inducible knockout HeLa cell lines,laying the foundation for further research on the role and biological function of MCM2 gene in the occurrence and progression of cervical cancer.