AIM:To investigate the function and mechanism of the smooth muscle relaxant choline theophylli-nate(CT)in activating primordial follicles in mice.METHODS:Experiments were conducted using in vitro culture of 3-day postpartum(dpp)neonatal SPF-grade female mice,intraperitoneal injection in 3-dpp neonatal mice,and oral adminis-tration in 21-dpp adolescent female mice.The mice were divided into control and CT groups.The ovaries were isolated from 3-dpp neonatal mice for the in vitro culture.Hematoxylin staining was used to count the number of primordial and growing follicles,with 10 mice in each group.qPCR was performed to analyze the expression levels of genes related to fol-licle growth,proliferation,and apoptosis,including growth differentiation factor 9(Gdf9),zona pellucida glycoprotein 3(Zp3),proliferating cell nuclear antigen(PCNA),Ki-67,B-cell lymphoma 2(BCL2),BCL2-associated X protein(BAX),and caspase-3,with 9 mice in each group.Immunofluorescence staining was used to detect the expression of pro-liferation-and apoptosis-related proteins,including PCNA,Ki-67,5-bromo-2'-deoxyuridine(BrdU),cleaved caspase-3,and forkhead box O3a(FOXO3a),with 15 mice in each group.Western blot was performed to measure the expression of DEAD-box helicase 4(DDX4),phosphorylated mammalian target of rapamycin(p-mTOR),and phosphorylated protein kinase B(p-Akt),with 9 mice in each group.Intraperitoneal injection of CT was administered to 3-dpp mice,and follicle counting was performed with 10 mice in each group.Western blotting was used to detect p-mTOR and p-Akt expression,with 9 mice in each group.Immunofluorescence was employed to assess FOXO3a nuclear export,with 15 mice in each group.For the oral administration of CT in drinking water to 21-dpp mice,immunofluorescence and hematoxylin staining was used to count follicles,with 9 mice in each group.RESULTS:Compared with control group,CT treatment signifi-cantly increased the number of growing follicles in mice.The mRNA and/or protein levels of Gdf9,Zp3,Ki-67,PCNA and DDX4 were markedly elevated.Further studies revealed that CT treatment significantly increased p-Akt levels in the ovaries but had no significant effect on p-mTOR levels.The PI3K/Akt inhibitor LY294002 also reversed the choline the-ophyllinate-induced increase in growing follicles.CONCLUSION:Choline theophyllinate promotes the activation of pri-mordial follicles in mice via the PI3K/Akt signaling pathway in oocytes.

Alzheimer's disease(AD)is the most common neurodegenerative disease in the elderly,and its main pathological manifestations include senile plaques formed by β-amyloid deposition and neuronal fibrillar nodules formed by hyperphosphorylation of tau proteins.Lysosome is an important organelle in eukaryotic cells,containing a variety of hydrolytic enzymes that can break down proteins and other biomolecules.It is closely related to intracellular transport and autophagy,and is important for maintaining cellular homeostasis.This review summarizes the interaction between lysosomal dysfunction and the development and progression of AD and the potential therapeutic mechanisms in treating AD by regulating and restoring the functions of lysosomes.Lysosomal dysfunction can lead to neurodegenerative diseases such as AD.Modulation of lysosomal function is a promising treatment strategy for AD.It is expected that more drugs and therapeutic regimens based on this mechanism can be used in the clinical treatment for AD patients in the future.

OBJECTIVES: To explore the therapeutic mechanism of Guizhi Fuling (GZFL) Pellets against cervical cancer. METHODS: Publicly available databases were used to identify the targets of GZFL Pellets and cervical cancer to construct the protein-protein interaction (PPI) network, followed by GO biological process and KEGG pathway enrichment analysis of the hub genes. The "Traditional Chinese Medicine-Active Ingredients-Targets-Pathways" network for GZFL Pellets in cervical cancer treatment was generated using Cytoscape v10.0.0, and molecular docking of the drug and potential targets was performed to predict the specific targets of active components in Guizhi Fuling Pellets. The inhibitory effects of hederagenin, an active ingredient in GZFL Pellets, was tested in cultured cervical cancer cells and in nude mice bearing cervical cancer xenografts. RESULTS: GZFL Pellets contain 338 active components targeting 247 action sites. A total of 10127 cervical cancer-related targets were obtained, and among them 195 were identified as potential therapeutic targets of GZFL Pellets for cervical cancer treatment, including the key targets of GABRA1, PTK2, JAK2, HTR3A, GSR, and IL-17. Molecular docking study showed low binding energies of the active components such as hederagenin, campesterol, and stigmasterol for protein-molecule interaction. GO enrichment analysis suggested that GZFL Pellets inhibited cervical cancer primarily by regulating responses to steroid hormones, oxidative stress, and lipopolysaccharides. Among the active components of GZFL Pellets, hederagenin was found to inhibit cervical cancer cells in vitro and significantly reduced STAT3 phosphorylation level in the cancer cells. In nude mice bearing cervical cancer xenografts, hederagenin effectively inhibited tumor growth rate without causing obvious adverse effects. CONCLUSIONS GZFL Pellets inhibit cervical cancer cell growth through its multiple active components that target different pathways. Among these components, hederagenin inhibits tumor cell growth possibly by directly binding to JAK2 protein to inhibit STAT3 phosphorylation.
Female ; Animals ; Uterine Cervical Neoplasms/pathology* ; Mice, Nude ; Humans ; Mice ; Oleanolic Acid/therapeutic use* ; Drugs, Chinese Herbal/therapeutic use* ; Molecular Docking Simulation ; Xenograft Model Antitumor Assays ; Cell Line, Tumor ; STAT3 Transcription Factor/metabolism* ; Protein Interaction Maps ; Janus Kinase 2/metabolism*

OBJECTIVES: This study aimed to observe the effects of initial periodontal therapy on the level of neutrophil extracellular traps (NETs) in gingival crevicular fluid (GCF) of patients with severe periodontitis and to analyze the factors related to the formation of NETs. METHODS: Thirty-one patients with stage Ⅲ-Ⅳ periodontitis were recruited. Clinical periodontal parameters, including plaque index (PLI), gingival index (GI), probing depth (PD), and clinical atta-chment loss (CAL), were recorded before and 6-8 weeks after initial periodontal therapy. Levels of NETs in GCF were detected by immunofluorescence staining. Quantities of total bacteria, Porphyromonas gingivalis (P. gingivalis), Aggregatibacter actinomycetemcomitans (A. actionomycetemcomitans) and Prevotella intermedia (P. intermedia)in unattached subgingival plaque were determined by real-time quantitative PCR, and levels of tumor necrosis factor-α (TNF-α) and interleukin-8 (IL-8) in GCF were explored by enzyme-linked immunosorbent assay. In addition, the correlations between the level of NETs and the above indicators were analyzed. RESULTS: After initial periodontal therapy, the level of NETs in GCF, PLI, GI, PD, and CAL; quantities of total bacteria, P. gingivalis, A. actinomycetemcomitans, and P. itermedia; and levels of IL-8 and TNF-α significantly decreased (P<0.05). We observed strong positive correlations between the level of NETs and PLI, GI, PD, CAL, the amount of total bacteria, P. gingivalis, TNF-α, and IL-8 (P<0.05). CONCLUSIONS Initial periodontal therapy might decrease the level of NETs in GCF from patients with severe periodontitis, which might be positively correlated with the quantities of P. gingivalis andthe levels of TNF-α and IL-8 in GCF.
Humans ; Gingival Crevicular Fluid ; Extracellular Traps/metabolism* ; Porphyromonas gingivalis/isolation & purification* ; Aggregatibacter actinomycetemcomitans/isolation & purification* ; Periodontitis/metabolism* ; Tumor Necrosis Factor-alpha/analysis* ; Prevotella intermedia/isolation & purification* ; Interleukin-8/analysis* ; Male ; Female ; Middle Aged ; Periodontal Index ; Adult

AIM:To investigate the function and mechanism of the smooth muscle relaxant choline theophylli-nate(CT)in activating primordial follicles in mice.METHODS:Experiments were conducted using in vitro culture of 3-day postpartum(dpp)neonatal SPF-grade female mice,intraperitoneal injection in 3-dpp neonatal mice,and oral adminis-tration in 21-dpp adolescent female mice.The mice were divided into control and CT groups.The ovaries were isolated from 3-dpp neonatal mice for the in vitro culture.Hematoxylin staining was used to count the number of primordial and growing follicles,with 10 mice in each group.qPCR was performed to analyze the expression levels of genes related to fol-licle growth,proliferation,and apoptosis,including growth differentiation factor 9(Gdf9),zona pellucida glycoprotein 3(Zp3),proliferating cell nuclear antigen(PCNA),Ki-67,B-cell lymphoma 2(BCL2),BCL2-associated X protein(BAX),and caspase-3,with 9 mice in each group.Immunofluorescence staining was used to detect the expression of pro-liferation-and apoptosis-related proteins,including PCNA,Ki-67,5-bromo-2'-deoxyuridine(BrdU),cleaved caspase-3,and forkhead box O3a(FOXO3a),with 15 mice in each group.Western blot was performed to measure the expression of DEAD-box helicase 4(DDX4),phosphorylated mammalian target of rapamycin(p-mTOR),and phosphorylated protein kinase B(p-Akt),with 9 mice in each group.Intraperitoneal injection of CT was administered to 3-dpp mice,and follicle counting was performed with 10 mice in each group.Western blotting was used to detect p-mTOR and p-Akt expression,with 9 mice in each group.Immunofluorescence was employed to assess FOXO3a nuclear export,with 15 mice in each group.For the oral administration of CT in drinking water to 21-dpp mice,immunofluorescence and hematoxylin staining was used to count follicles,with 9 mice in each group.RESULTS:Compared with control group,CT treatment signifi-cantly increased the number of growing follicles in mice.The mRNA and/or protein levels of Gdf9,Zp3,Ki-67,PCNA and DDX4 were markedly elevated.Further studies revealed that CT treatment significantly increased p-Akt levels in the ovaries but had no significant effect on p-mTOR levels.The PI3K/Akt inhibitor LY294002 also reversed the choline the-ophyllinate-induced increase in growing follicles.CONCLUSION:Choline theophyllinate promotes the activation of pri-mordial follicles in mice via the PI3K/Akt signaling pathway in oocytes.

【Objective】 To establish and verify a new nucleic acid extraction method for OBI detection with large volume and high sensitivity, and apply it in the quantitative determination of OBI samples with low viral load. 【Methods】 The method for nucleic acid extraction with large volume was established based on the method of Roche nucleic acid detection kit. HBV standards were configured into 10 000 IU/mL, 1 000 IU/mL, 100 IU/mL, 10 IU/mL and 1 IU/mL respectively, and nucleic acid was extracted from the 10 mL standards by magnetic beads. CT values of each concentration were detected by fluorescence quantitative PCR and each concentration gradient was detected in parallel duplicates. The logarithm of virus concentration was taken as the X-axis and the average CT values of two tests were taken as the Y-axis to construct the fluorescence quantitative standard curve and regression equation. Three repeated experiments were conducted to verify the stability of the method. This method was used to extract nucleic acid from OBI samples with low viral load, and fluorescence quantification was performed. 【Results】 The amplification efficiency of fluorescence quantitative standard curves ranged from 90% to 105%, and the regression equation was greater than 0.99. The variation coefficients of variation of CT values were 0.63%, 0.78%, 1.52%, 1.36% and 0.78%, respectively. This method can extract nucleic acid from OBI samples with viral load of 1 IU/mL for quantification. 【Conclusion】 The detection limit of HBV nucleic acid quantitative detection system can reach 1 IU/mL, and it has strong stability and high sensitivity, which can be used for the quantitative detection of OBI with low viral load.

Background::Although overnight fasting is recommended prior to endoscopic retrograde cholangiopancreatography (ERCP), the benefits and safety of high-carbohydrate fluid diet (CFD) intake 2 h before ERCP remain unclear. This study aimed to analyze whether high-CFD intake 2 h before ERCP can be safe and accelerate patients’ recovery.Methods::This prospective, multicenter, randomized controlled trial involved 15 tertiary ERCP centers. A total of 1330 patients were randomized into CFD group ( n = 665) and fasting group ( n = 665). The CFD group received 400 mL of maltodextrin orally 2 h before ERCP, while the control group abstained from food/water overnight (>6 h) before ERCP. All ERCP procedures were performed using deep sedation with intravenous propofol. The investigators were blinded but not the patients. The primary outcomes included postoperative fatigue and abdominal pain score, and the secondary outcomes included complications and changes in metabolic indicators. The outcomes were analyzed according to a modified intention-to-treat principle. Results::The post-ERCP fatigue scores were significantly lower at 4 h (4.1 ± 2.6 vs. 4.8 ± 2.8, t = 4.23, P <0.001) and 20 h (2.4 ± 2.1 vs. 3.4 ± 2.4, t= 7.94, P <0.001) in the CFD group, with least-squares mean differences of 0.48 (95% confidence interval [CI]: 0.26–0.71, P <0.001) and 0.76 (95% CI: 0.57–0.95, P <0.001), respectively. The 4-h pain scores (2.1 ± 1.7 vs. 2.2 ± 1.7, t = 2.60, P = 0.009, with a least-squares mean difference of 0.21 [95% CI: 0.05–0.37]) and positive urine ketone levels (7.7% [39/509] vs. 15.4% [82/533], χ2 = 15.13, P <0.001) were lower in the CFD group. The CFD group had significantly less cholangitis (2.1% [13/634] vs. 4.0% [26/658], χ2 = 3.99, P = 0.046) but not pancreatitis (5.5% [35/634] vs. 6.5% [43/658], χ2 = 0.59, P = 0.444). Subgroup analysis revealed that CFD reduced the incidence of complications in patients with native papilla (odds ratio [OR]: 0.61, 95% CI: 0.39–0.95, P = 0.028) in the multivariable models. Conclusion::Ingesting 400 mL of CFD 2 h before ERCP is safe, with a reduction in post-ERCP fatigue, abdominal pain, and cholangitis during recovery.Trail Registration::ClinicalTrials.gov, No. NCT03075280.

Epigenomic imbalance drives abnormal transcriptional processes,promoting the onset and progression of cancer.Although defective gene regulation generally affects carcinogenesis and tumor suppression networks,tumor immunogenicity and immune cells involved in antitumor responses may also be affected by epigenomic changes,which may have significant implications for the development and application of epigenetic therapy,cancer immunotherapy,and their combinations.Herein,we focus on the impact of epigenetic regulation on tumor immune cell function and the role of key abnormal epigenetic processes,DNA methylation,histone post-translational modification,and chromatin structure in tumor immunogenicity,and introduce these epigenetic research methods.We emphasize the value of small-molecule inhibitors of epigenetic modulators in enhancing antitumor immune responses and discuss the challenges of developing treatment plans that combine epigenetic therapy and immuno-therapy through the complex interaction between cancer epigenetics and cancer immunology.

Purpose: The causal relationship between the incidence and prognosis of chronic kidney disease (CKD) and serum testosterone levels in patients is not yet fully understood. This study aims to use the National Health and Nutrition Examination Survey (NHANES), a large-scale nationally representative sample, to investigate the relationship between CKD and testosterone. Materials and Methods: This study included six NHANES cycles for linear regression analysis, verified by multiple imputation methods. Stratified analysis and subgroup analysis were used to demonstrate the stability of CKD’s effect on testosterone. Furthermore, we used Kaplan-Meier plots and log-rank tests to evaluate differences in survival rates between CKD male patients with low and normal levels of testosterone. Results: From a total of 71,163 subjects, the cohort selected 28,663 eligible participants. Results showed that CKD patients had testosterone levels 28.423 ng/mL (24.762, 32.083) lower than non-CKD patients. The results of multiple imputations (β=27.700, 95% confidence interval: 23.427, 31.974) were consistent with those of linear regression analysis, and the numerical match was good. Stratified regression analysis, and subgroup analysis results showed that CKD had a significant impact on testosterone at different dimensions. Kaplan-Meier plots showed significantly reduced survival rates in low testosterone CKD male patients (p<0.0001). Conclusions The results of this big data analysis suggest that there may be a two-way risk between low levels of testosterone and CKD. The testosterone levels of CKD patients were significantly lower than those of the non-CKD population, and CKD patients with low testosterone levels had poorer prognoses. These results suggest that correcting testosterone levels in a timely manner can have preventive and therapeutic effects on the progression of CKD.