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Objective To evaluate the comparative clinical outcomes of zero-profile interbody fusion (Zero-P) and traditional anterior cervical discectomy and fusion with titanium plate (ACDF- plate) for three-level cervical spondylosis, providing a reference to optimize surgical strategies for three-level cervical spondylosis. Methods In this retrospective comparative study, 113 patients were included and assigned to either the Zero-P group (n=51) or the ACDF-plate group (n=62) according to their surgical approaches. Clinical outcomes were evaluated and compared between the two groups using operative time, intraoperative blood loss, length of hospital stay, visual analog scale (VAS) scores, cervical range of motion (ROM), neck disability index (NDI), Cervical Disability Index (CDI), Japanese orthopaedic association (JOA) scores, and postoperative complication rates. Results Compared with the ACDF-plate group, the Zero-P group had a shorter operative time and less intraoperative blood loss (P<0.05); the neck pain VAS score was lower on the 1st day and 1st week after surgery (P<0.05); the cervical flexion range of motion was higher at 1 month after surgery (P<0.05); the JOA score was higher at 3 months after surgery, while the NDI and CDI scores were lower (P<0.05); the total incidence of complications (11.76%) was significantly lower than that in the plate fixation group (35.48%), and the incidence of neck foreign body sensation/stiffness was lower (P<0.05). Conclusion Zero-P not only demonstrates safety and efficacy comparable to the ACDF-plate, but also highlights the significant value of the minimally invasive concept in facilitating early recovery. Both procedures have their respective emphasis on biomechanical maintenance and minimally invasive benefits, providing a clear evidence-based reference for individualized and precise surgical decision-making in three-level cervical spondylosis.

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Objectivec To investigate the therapeutic effect of wall-broken Ganoderma lucidum spore powder (GLS) on a mouse model of hepatocellular carcinoma and to explore its underlying mechanism. Methods Fifty 14-day-old C57BL/6J mice were divided into a control group, a model group and low, medium and high-dose GLS groups(0.25, 0.5, 1.0 g/kg ), with 10 mice in each group. A DEN/CCL4 mouse hepatocellular carcinoma model was established, and GLS was administered by gavage starting from the 8th week, once a day for 14 weeks. At the end of the final administration, the number of liver nodules was observed , and the serum alpha-fetoprotein (AFP) was detected using enzyme-linked immunosorbent assay. The content of catalase (CAT) in serum and liver tissue was determined by ammonium molybdate method. The content of reduced glutathione (GSH) in serum and liver tissue was measured by microplate assay. The content of glutathione peroxidase (GSH-PX) in serum and liver tissue was detected by colorimetric method. Immunohistochemistry (IHC-P) was employed to detect the protein expression of proliferating cell nuclear antigen (PCNA). R eal-time fluorescence quantitative polymerase chain reaction was utilized to detect the relative expression of nuclear factor E2-related factor 2 (Nrf2), Kelch-like epichlorohydrin-associated protein 1 (Keap1),NAD(P)H quinone oxidoreductase 1 (Nqo1), heme oxygenase-1 ( Ho-1 ) and glutamate cysteine ligase catalytic subunit (Gclc) mRNA. Results Compared with the control group, the content of serum AFP were significantly higher in the model group (P<0.01), while the contents of CAT, GSH and GSH-PX in liver and serum were significantly lower in the model group (P<0.01). The relative expression of Keap1 and Nqo1 mRNA was significantly higher (P<0.01), the relative expression of Nrf2, Ho-1, Gclc mRNA relative expression was significantly lower (P<0.01), and the positive expression rate of PCNA was significantly higher (P<0.01). Compared with the model group, the total number of liver nodules and content of serum AFP in the administered group were significantly lower (P<0.01), and the contents of CAT, GSH, and GSH-PX in the liver and serum of mice significantly increased (P<0.05 or P<0.01). The expression of Keap1 and Nqo1 mRNA significantly decreased (P<0.05). The expression of Nrf2, Ho-1 and Gclc mRNA significantly increased (P<0.05), and positive expression rate of PCNA significantly decreased (P<0.01). Conclusion GLS has a significant protective effect on DEN/CCL4 -induced mouse hepatocellular carcinoma model, and the mechanism of action is related to the regulation of oxidative stress in mouse liver.

Abstract:
Objective To explore the ameliorative effect of the endoplasmic reticulum stress inhibitor 4-phenylbutyric acid (4-PBA) on bile acid metabolism disorders induced by bacterial lipopolysaccharide (LPS) in late-pregnant mice, and to clarify the regulatory mechanism of endoplasmic reticulum stress (ERS) in intrahepatic cholestasis of pregnancy (ICP). Methods Twenty-four pregnant mice were divided into control group, LPS group, 4-PBA group, and LPS+4-PBA group. On the 16th day of pregnancy, the LPS group received intraperitoneal injection of LPS (200 μg/kg) and the 4-PBA group received injection of 4-PBA (150 mg/kg). In the LPS+4-PBA group, 4-PBA was intraperitoneally injected first, followed by LPS administration 1 h later. Serum total bile acid (TBA), liver injury markers, bile acid profiles, and the expression of key genes and proteins were detected 6 h after LPS treatment. Results LPS exposure increased serum TBA levels in pregnant mice(P<0.05), activated ERS markers and inflammatory factors, and downregulated bile acid transporters and the expression of the rate limiting enzyme CYP7A1(all P<0.05). 4-PBA intervention effectively inhibited ERS, reduced TBA levels(P<0.05), and upregulated Ntcp, Bsep, Mdr3, and Mrp3 gene expression(all P<0.05). Conclusion ERS plays an important role in LPS interference with the homeostasis of bile acid metabolism during pregnancy. Inhibiting ERS can improve bile stasis by regulating bile acid transporters, providing a theoretical basis for targeted therapy of ICP.

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Objective This study aims to construct and evaluate various machine learning models for predicting the risk of chronic obstructive pulmonary disease (COPD) in individuals, thereby providing data support for early screening and intervention.Methods A total of 823 subjects were selected for this study, comprising l42 individuals in the high-risk group for COPD and 68l individuals in the low-risk group. Data collected included demographic characteristics, smoking history, symptoms (such as cough and shortness of breath), and scores from the Chronic obstructive pulmonary disease screening questionnaire. Four machine learning algorithms—Logistic Regression, Random Forest, Support Vector Machine, and XGBoost—were utilized to construct risk prediction models. The performance of these models was assessed using 5-fold cross-validation, with evaluation metrics including accuracy, precision, recall, Fl-score, area under the receiver operating characteristic curve (AUC), and average precision (AP). Furthermore, a feature importance analysis was performed.Results The Logistic Regression model exhibited superior performance, achieving an AUC of 0.982 and an AP of 0.939. This was closely followed by the Random Forest model, which recorded an AUC of 0.975 and an AP of 0.890. Feature importance analysis revealed that smoking history, symptoms of shortness of breath, and body weight were significant predictors. All models demonstrated robust performance in identifying low-risk populations, with precision values exceeding 0.93; however, variations were observed in their efficacy in identifying high-risk populations.Conclusion Machine learning models have proven effective in identifying individuals at high risk for COPD. Among these, the logistic regression model exhibits the best overall performance, efficiently identifying high-risk populations and serving as a valuable clinical auxiliary screening tool. Various models, each with distinct performance characteristics, are suited to different clinical screening scenarios, thereby offering targeted decision-making support for the establishment of a hierarchical and intelligent COPD screening pathway.

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Objective To investigate the effect of Semaphorin 3A (Sema3A) on the progression of pathological cardiac hypertrophy. Methods Wild-type C57BL 6J mice, Sema3A global overexpressing (Sema3A/Cre-ERTM) mice, and their littermate controls (Cre-ERTM) were used, with 6 mice per group. A pressure-overload induced cardiac hypertrophy model was established via transverse aortic constriction (TAC), with a sham-operated group (Sham) as the control. Cardiac hypertrophy was assessed by gross morphology, heart weight/body weight ratio (HW/BW), heart weight/tibia length ratio (HW/HL), and heart weight/lung weight ratio (HW/PW). Cardiac function was evaluated by measuring ejection fraction (EF) and fractional shortening (FS) via echocardiography. In heart tissues, the mRNA level of atrial natriuretic peptide (ANP) and Sema3A were detected by RT-qPCR, Sema3A protein level was detected by western blot, morphological structure was detected by hematoxylin and eosin (HE) staining, level of fibrosis was detected by Masson’s trichrome staining, and angiogenesis was detected by immunohistochemical detection of CD31.Forin vitroexperiments, rat H9C2 cardiomyocytes were treated with angiotensin II (Ang II) to induce hypertrophy, with PBS-treated cells as the control. After 48 hours, the mRNA expression of ANP, B-type natriuretic peptide (BNP) and Sema3A were measured by RT-qPCR, and Sema3A protein level was assessed by western blot.Results At the animal level, compared with the sham group, mice subjected to TAC showed significantly increased mRNA levels of ANP in cardiac tissue, as well as markedly elevated mRNA and protein levels of Sema3A (P<0.05). Compared with the Cre-ERTM + TAC group, the Sema3A/Cre-ERTM + TAC group exhibited more pronounced cardiac hypertrophy, greater ventricular wall thickening, higher HW/BW, HW/HL, and HW/PW ratios, more significant reductions in EF and FS, more severe cardiac fibrosis, and a significant decrease in cardiac CD31 expression levels (P<0.05).At the cellular level, compared with the control group, H9C2 cells treated with Ang II displayed significantly increased mRNA levels ofANP and BNP, along with markedly elevated mRNA and protein levels of Sema3A (P<0.05).ConclusionSema3A promotes the progression of pressure overload-induced cardiac hypertrophy by inhibiting angiogenesis.

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Objective To explore the risk factors for liver injury in sepsis, so as to provide a reference for the diagnosis and treatment of sepsis patients with liver injury. Methods A retrospective analysis was conducted on the clinical data of 402 patients with sepsis. According to the occurrence of acute liver injury during ICU stay, the patients were divided into a sepsis with liver injury group and a sepsis without liver injury group. Univariate analysis was used to screen clinical indicators potentially associated with liver injury, and a multivariate Logistic regression model was then established to identify the independent risk factors for acute liver injury in sepsis patients. Results A total of 229 sepsis patients who met the inclusion criteria were enrolled, among whom 110 cases (48.03%) had liver injury, and 52 patients (47.27%) in the liver injury group died. There were statistically significant differences between the two groups in APACHE II score, SOFA score, platelet count, lactate level, fibrinogen content, diabetes mellitus, abdominal infection, vasoactive drug therapy, CRRT therapy, and mechanical ventilation therapy (P < 0.05). Multivariate Logistic regression analysis showed that SOFA score (OR = 1.19, 95%CI: 1.06 ~ 1.33), diabetes mellitus (OR = 3.27, 95%CI: 1.65 ~ 6.48), abdominal infection (OR = 2.74, 95%CI: 1.33 ~ 5.62), and mechanical ventilation therapy (OR = 5.24, 95%CI: 1.58 ~ 17.41) were independent risk factors. Conclusion Diabetes, mellitus, abdominal infection, SOFA score, and mechanical ventilation therapy are independent risk factors for acute liver injury in sepsis patients.

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Objective To focused on patients with unexplained repeated implantation failure (URIF) and compare the clinical efficacy of different administration routes of granulocyte colony- stimulating factor (G-CSF) in patients undergoing frozen-thawed embryo transfer cycles.Methods A retrospective case-control study was conducted. A total of 187 patients with unexplained recurrent implantation failure (URIF) who received medical treatment were enrolled in this study. According to whether G-CSF was used on the day of endometrial transformation and its administration route in frozen-thawed embryo transfer cycles, the patients were divided into three groups: control group (without G-CSF, n = 71), subcutaneous injection group (subcutaneous G- CSF, n = 72), and intrauterine infusion group (intrauterine G-CSF infusion, n = 44). The pregnancy outcomes among the three groups were compared, and Logistic regression analysis was performed. Results There were significant differences in biochemical pregnancy rates among the three groups (P < 0.05), indicating statistical significance in the overall comparison. In terms of numerical trend, the biochemical pregnancy rate was in the order: subcutaneous injection group > intrauterine infusion group > control group. Further pairwise comparison showed that only the difference between the subcutaneous injection group and the control group was statistically significant (P < 0.017), while no significant differences were found between the other groups (P > 0.017). The overall comparison of early miscarriage rates among the three groups also showed a significant difference (P < 0.05), indicating a statistically significant difference in the distribution among the groups. The numerical trend was: intrauterine perfusion group > subcutaneous injection group > control group. Pairwise comparison results showed that only the difference between the intrauterine perfusion group and the control group was statistically significant (P < 0.017), while there was no statistically significant difference between any other two groups (P > 0.017).Multivariate Logistic regression analysis showed that endometrial thickness on the day of transformation, number of transferred embryos, and subcutaneous G-CSF injection were independent factors associated with an increased biochemical pregnancy rate, where as age was an independent factor for a decreased biochemical pregnancy rate. For clinical pregnancy outcome, endometrial thickness on the day of transformation, number of transferred embryos, and subcutaneous G-CSF injection were also independent factors for an increased clinical pregnancy rate, while BMI was an independent factor for a decreased clinical pregnancy rate. Conclusion Subcutaneous injection of G-CSF only improved the biochemical pregnancy rate in URIF patients. Multivariate Logistic regression analysis showed that subcutaneous G-CSF injection was a favorable factor for clinical pregnancy rate in URIF patients; however, no significant improvement in clinical pregnancy rate was observed among the three groups. Both the comparison of pregnancy outcomes and the results of multivariate logistic regression analysis indicates that intrauterine perfusion of G-CSF has no significant therapeutic effect in the treatment of patients with URIF.

Abstract:
Objective To construct a chimeric antigen receptor macrophage (CAR-M) targeting interleukin-13 receptor α (IL-13Rα) and intracellular domain integration, which can double activate the signal adaptor protein OX40 L of macrophages and T cells. Methods The CAR molecule targeting IL- 13Rα (IL-13Rα-OX40L-CAR) with OX40L as the intracellular signal domain was designed and synthesized, and it was cloned into the adenovirus expression vector. After THP-1 cells were induced to differentiate into macrophages (THP-1-M) by phorbol ester, the cells were infected with recombinant adenovirus to construct THP-1 macrophages stably expressing green fluorescent protein (GFP) labeled anti-IL- 13RαCAR. The expression efficiency of CAR molecules was monitored by flow cytometry. The empty vector group was used as the negative control, the CD3ζ group was used as the positive control, and the CD3ζ-OX40L group was used as the experimental group. The phagocytic function of macrophages in each group on IL- 13Rα high expression glioma cells (U251) and low expression glioma cells (T98G) was detected by in vitro co-culture experiments. Results CAR adenovirus was efficiently transfected into THP-1-M to obtain anti-IL- 13RαCAR-M. After co-cultured with glioma cells with different expression levels of IL- 13Rα, the results of flow cytometry showed that there was no significant difference among the three groups for T98G cells with low expression of IL- 13Rα . After co-culture with U251 cells with high expression of IL- 13Rα, the CD3ζ group showed enhanced phagocytosis compared with the empty vector group (P<0.05). Compared with the CD3ζ group, the phagocytosis ability of the CD3ζ-OX40L group was further enhanced (P<0.05). Conclusion Anti-IL13Rα-OX40L-CAR-M is successfully constructed by constructing IL- 13Rα-OX40L-CAR adenovirus and transfecting PMA-induced differentiated THP-1-M. It can engulf glioma cells with high expression of IL- 13Rα by specific targeting, and shows activation of intracellular domain superior to traditional CD3ζ .

Abstract:
Objective To evaluate the efficacy of immediate postoperative oral administration of 2 mg estradiol/estradiol dydrogesterone tablets in reducing secondary surgery for retained products of conception(RPOC), preventing intrauterine adhesions(IUA), and promoting endometrial repair following induced abortion. Methods A retrospective analysis was conducted on 494 patients who underwent induced abortion. The control group (n=214) received conventional postoperative treatment with antibiotics and Yimucao granules, while the observation group (n=280) received immediate administration of estradiol/dydrogesterone tablets (each tablet contains 2 mg of estradiol),combined with antibiotics and Yimucao granules. The two groups were compared for the secondary surgery rate for RPOC, incidence of IUA, endometrial thickness at 2 weeks postoperatively, duration of postoperative vaginal bleeding, time to menstrual resumption, incidence of reduced menstrual volume after resumption, and postoperative complications (pelvic infection, intrauterine fluid accumulation). Results The observation group showed a lower rate of secondary surgery for RPOC compared to the control group (0.4% vs 2.3%), but the difference was not statistically significant (P=0.13, Fisher’s exact test). The observation group also had a significantly lower incidence of IUA (P<0.05), greater postoperative endometrial thickness at 2 weeks (P<0.05), and significantly shorter duration of postoperative vaginal bleeding and time to menstrual resumption. The incidence of reduced menstrual volume postoperatively and complications (pelvic infection, intrauterine fluid accumulation) were also significantly lower in the observation group (all P<0.05). Conclusion Immediate oral administration of estradiol/dydrogesterone tablets after induced abortion significantly prevents intrauterine adhesions, shortens postoperative vaginal bleeding duration, promotes endometrial repair, improves menstrual recovery, and potentially reduces the risk of secondary surgery for retained products of conception.

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Objective To investigate the effect of hypoxia on pemetrexed (PE)-mediated apoptosis in non-small cell lung cancer cells. Methods A549 cells were cultured under 21% O₂ (normoxia) or 1% O₂ (hypoxia), and treated with 8 μmol/L PE for 48 h. The cells were divided into normoxic control group, normoxic vehicle group, normoxic drug-treated group, hypoxic control group, hypoxic vehicle group and hypoxic drug-treated group. Trypan blue staining was used to observe cell death after PE intervention under normoxia and hypoxia. Flow cytometry was adopted to detect cell apoptosis rate. Wound healing assay was used to assess cell migration. Western blot was performed to detect the protein levels of MRP-1, Cleaved Caspase-3, E-Cadherin and N-Cadherin. Results Trypan blue staining results showed that the cell death rate was reduced in PE-treated cells under hypoxia compared with the normoxic PE group (P<0.001). Flow cytometry results indicated that PE induced cell apoptosis under both normoxic and hypoxic conditions.However, the apoptosis rate of the normoxic PE group was significantly higher than that of the hypoxic PE group (P<0.000 1). Wound healing assay showed that the migration distance of A549 cells decreased after PE treatment for 24 h and 48 h under normoxia and hypoxia when compared with the control group (P<0.05), while no significant difference was found between the normoxic PE group and the hypoxic PE group. Western blot results revealed that MRP-1 expression was markedly higher in hypoxic PE-treated cells than in normoxic PE-treated cells (P<0.000 1). Compared with the control group, Cleaved Caspase-3 expression was upregulated in A549 cells following PE intervention under normoxia (P<0.05), whereas no obvious alteration in Cleaved Caspase-3 expression was detected in hypoxic PE-treated cells. Under both normoxic and hypoxic conditions, PE treatment downregulated the expression of E-Cadherin (P<0.000 1 ，P<0.001) and N-Cadherin (P<0.05 ，P<0.000 1). In addition, the expression levels of E-Cadherin and N-Cadherin in the hypoxic PE group were significantly lower than those in the normoxic PE group (P<0.000 1). Conclusion Hypoxia significantly attenuates the pro-apoptotic effect of PE on A549 cells and blocks Cleaved Caspase-3 activation. This mechanism may be related to hypoxia-induced MRP-1 overexpression and reduced drug accumulation. Interfering with MRP-1 or restoring the activity of the apoptosis pathway may serve as a novel strategy to overcome hypoxia-induced drug resistance.

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Objective. , To evaluate the mechanical properties and biocompatibility of a 3 mol% yttria-stabilized tetragonal zirconia polycrystal (3Y-TZP)-polymethyl methacrylate (PMMA) biomimetic layered composite and to explore the feasibility of using this composite for ceramic dental crowns. Methods The micro-morphology of 3Y-TZP powder was observed using scanning electron microscopy and metallographic microscopy. The ball-milled 3Y-TZP powder was stirred together with bacterial cellulose to obtain a homogeneous ceramic slurry. Uniform ceramic films were prepared by tape casting. After stacking the ceramic films, the composite was finally obtained through pressure-less sintering, silane coupling, and organic matter infiltration. The microstructure of the composite was observed using scanning electron microscopy. The mechanical properties of the composite were measured using a universal testing machine, and the energy dissipation rate and impact toughness of the samples were evaluated through ball drop and falling weight impact tests. The biocompatibility of the composite was investigated using CCK-8 assay, live/dead cell staining, cell adhesion assay, and Transwell cell migration assay. Results The composite exhibited a distinct layered structure. As the thickness of the ceramic layer decreased, the bending and compressive strengths of the material gradually decreased, while its impact resistance was significantly improved. The maximum energy dissipation rate and impact toughness can reach 75.8% and 44.1 kJ/m²respectively. No significant differences were observed in the CCK-8 assay, live/dead cell staining, or cell migration assay results between the blank group and the material group, and cells were able to adhere well to the composite. Conclusion This study successfully fabricates 3Y-TZP-PMMA composite materials, which possess both excellent mechanical properties and biocompatibility, demonstrating promising prospects for clinical application.

Abstract:
Objective To optimize the flow cytometric sorting workflow for myeloid-derived suppressor cells (MDSCs) from murine Lewis lung carcinoma (LLC) subcutaneous tumor tissues, improving single-cell suspension preparation efficiency and sorting purity, thereby providing high-quality cells for subsequent functional studies. Methods Subcutaneous LLC tumor tissues were harvested from C57BL/6 mice, and two enzymatic digestion protocols were first compared to evaluate their effects on single-cell suspension preparation. After staining with a viability dye (Live/Dead) and fluorochrome-conjugated anti-mouse antibodies against CD45, CD11b, and Gr-1, cells were loaded onto a Beckman CytoFLEX cell sorter. Next, conventional and optimized gating strategies were applied. In the optimized strategy, the CD45+ population was used as the initial gate to directly define an MDSC-enriched region, followed by selection of CD11b+Gr-1+ cells. Finally, purity was assessed by flow cytometry, viability was determined by trypan blue staining, and RT-qPCR was performed to measure the expression of MDSC signature genes, including Arg1, Nos2, IL-10, and S100A8/A9. Results The optimized enzymatic digestion protocol significantly increased the yield and viability of single cells. After optimization of the gating strategy, the post-sort positivity rate (purity) was markedly improved. Arg1, Nos2, IL-10and S100A8/A9 were highly expressed in the sorted cells, indicating that the isolated cells retained characteristic MDSC features and functional signatures. Conclusion By optimizing both the tissue digestion protocol and the flow cytometric gating strategy, we establish an efficient workflow for isolating MDSCs from tumor tissues. This method improves the yield and purity of single-cell preparations while preserving antigen integrity, providing a reliable methodological foundation for subsequent studies on MDSC function and metabolism.

Abstract:
Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by persistent synovial inflammation and progressive joint destruction. Recent studies have shown that lactate metabolic reprogramming plays a key role in the pathological process of RA, and the accumulation of lactate in the synovial microenvironment triggers a cascade of immune regulatory effects. These metabolic changes create a self-sustaining cycle of inflammation and tissue damage, leading to progressively worsening disease. Therefore, lactate metabolism has been identified as a therapeutic target. Therapeutic strategies targeting lactate metabolism, such as LDHA inhibitors, targeting PKM2, MCT inhibitors, GPR81 antagonists, and lactylation modification regulators, have shown great potential in restoring immune balance and protecting joint integrity. This review summarizes the mechanisms of lactate metabolism in RA and its therapeutic applications, providing a theoretical basis for the development of novel metabolic intervention therapies.

Abstract:
Objective To investigate the relationship between Type ⅠHelicobacter pylori (H. pylori) infection and the risk of colorectal neoplasms (CRN) in patients attending gastroenterology clinics, and to evaluate its predictive value for CRN in this population. Methods We included 2,148 patients presenting to the Department of Gastroenterology, with 677 assigned to the CRN group and 1,471 to the non-CRN group.H.pylori antibody typing was performed using Western blot, with type Ⅰ defined as positivity for CagA and/or VacA antibodies, and type II as positivity only for UreA/B antibodies. Statistical analyses included univariate screening, multivariate logistic regression, and receiver operating characteristic (ROC) curve evaluation. Results Type ⅠH.pylori seropositivity was significantly more prevalent in the CRN group than in the non-CRN group (54.51% vs 45.62%, P<0.0167). No significant differences were observed for type II H.pylorior seronegative subjects. The CRN group had higher proportions of males, hypertension, diabetes, smoking, and alcohol use, as well as elevated body mass index, fasting plasma glucose, triglycerides, and uric acid, along with lower high-density lipoprotein levels (all P<0.05). Multivariate analysis confirmed type Ⅰ H.pylori (OR=1.303), alcohol consumption (OR=2.315), male sex (OR=1.592), and age (OR=1.048) as independent risk factors for CRN. Although type I H.pylori alone showed limited predictive value (AUC=0.544), a combined model incorporating alcohol use, age, and sex demonstrated significantly improved discriminative ability (AUC=0.681, sensitivity 59.1%, specificity 68.2%). Conclusion Type ⅠH. pylori infection predicts the risk of CRN in gastroenterology patients. Assessing it can help to optimize the identification of high-risk individuals and inform targeted screening strategies.

Abstract:
Objective To investigate the distribution characteristics and correlation of paired oral and esophageal microbiota in patients with esophageal squamous cell carcinoma (ESCC). Methods A total of 46 pathologically confirmed ESCC patients were enrolled, and oral swabs and cancerous tissue samples were collected. The microbial composition was analyzed using 16S rRNA gene sequencing, and bioinformatics methods were employed to compare the microbiota diversity, compositional differences, and correlations between the two sites. Results The Alpha diversity of the oral microbiota in ESCC patients was significantly higher than that of their esophageal microbiota, though the overall community structure was similar between the two sites. LEfSe analysis revealed that Spirochaetes, Prevotella, Catonella, Enterococcus, Fusobacterium periodonticum, Veillonella parvula, Capnocytophaga sputigena, and Neisseria subflava were significantly enriched in ESCC tumor tissues. In contrast, Actinobacteria, Proteobacteria, Streptococcus, Neisseria, Haemophilus, Streptococcus mitis, Haemophilus parainfluenzae, Porphyromonas gingivalis, Haemophilus haemolyticus, and Prevotella tannerae were significantly enriched in the oral cavity. Correlation analysis demonstrated significant associations between the two sites at taxonomic levels such as Spirochaetes, Treponema, Granulicatella elegans, Neisseria macacae, Treponema amylovorum, Fusobacterium periodonticum, and Neisseria meningitidis (P<0.05). Conclusion The oral and esophageal microbiota in ESCC patients exhibit both distinct differences and similarities. Oral microbiota may influence the esophageal microenvironment through migration or local immune modulation.

Abstract:
Objective To construct B cell conditional knockout Spi1 gene mice and analysis their genotypes to provide an animal model basis for disease pathogenesis and drug target research. Methods Mb1-cre transgenic mice were crossbred with Spi1flox/flox mice,. The genotypes of the offspring mice were identified using polymerase chain reaction (PCR) and agarose gel electrophoresis. The mice with the genotype of Mb1-cre×Spi1flox/flox that were screened out will be the homozygous mice with B cell-specific conditional knockout of the Spi1 gene. Results PCR identification results showed that when using flox primers for detection, only a 220 bp band was amplified, indicating a genotype of Spi1flox/flox in the mice. When using Mb1-Cre primers for detection, a 383 bp band was amplified, indicating a genotype of Mb1-cre×Spi1flox/flox in the mice; Flow cytometry results showed that compared to Spi1flox/flox mice, Mb1-cre×Spi1flox/flox mice exhibited significantly reduced PU.1 expression levels in bone marrow-derived mononuclear cells, peripheral blood mononuclear cells, and spleen-derived B cells. Moreover, PU.1 exhibited normal expression in other immune cells, such as CD4+ T cells and macrophages. Conclusion By utilizing the Cre/LoxP system and CRISPR/Cas9 technology, this study successfully generates mice with B cell-specific conditional knockout of the Spi1 gene, providing a reliable animal model for in-depth exploration of the specific role of PU.1 in B cell-related diseases.

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Objective To investigate whether the expression of Piezo1 in bulbar conjunctival fibroblasts contributes to the development of conjunctivochalasis (CCH). Methods Primary bulbar conjunctival fibroblasts from CCH patients and normal controls (NC) were obtained by tissue block applanation, purified and then subjected to fibroblast identification. Immunohistochemistry was used to detect the expression of Piezo1 in the NC and CCH groups. Immunofluorescence was used to detect the expression of Piezo1 in bulbar conjunctival fibroblasts in the CCH and NC groups, as well as the expression of MMP-9 after the intervention of the Piezo1 agonist, Yoda1. qRT-PCR was used to detect the expression of mRNA of Piezo1 and MMP-9 in the CCH and NC groups, as well as the expression of mRNA of MMP-9 after the intervention of Yoda1. In addition, confocal microscopy was used to detect Ca2+influx in bulbar conjunctival fibroblasts in the NC group after Yoda1 intervention. Membrane clamp was used to detect the current amplitude of NC bulbar conjunctival fibroblasts after Yoda1 intervention to verify the functional expression of Piezo1. Results Primary bulbar conjunctival fibroblasts were successfully cultured. Immunohistochemistry revealed that Piezo1 protein expression was elevated in the CCH group (P<0.01). qRT-PCR assay revealed that mRNA expression of Piezo1 was elevated in the CCH group (P<0.01), mRNA expression of MMP-9 was elevated in the CCH group (P<0.01), and mRNA expression of MMP-9 was elevated in the CCH+Yoda1 group (P<0.01). Immunofluorescence detection of Piezo1 revealed enhanced fluorescence in the CCH group relative to the NC group (P<0.05), and the fluorescence of MMP-9 was enhanced by the addition of Yoda1 stimulation compared with that of the CCH group (P<0.05). Confocal microscopy of NC group bulbar conjunctival fibroblasts after the addition of Yoda1 showed enhanced fluorescence of Piezo1 (P<0.05). The addition of Yoda1 activated enhanced current amplitude, showed by membrane clamp detection of NC group bulbar conjunctival fibroblasts (P<0.000 1). Conclusion Mechanical damage produced by conjunctival tissues in conjunctivochalasis upregulates Piezo1 expression, and Piezo1 activation may amplify the mechanical damage caused by CCH.

Abstract:
Objective To investigate the association between insulin resistance (IR) and cardiovascular metabolic indicators, including blood pressure, blood lipids, and serum uric acid, in a middle-aged population. Methods Based on the baseline survey of the Hong Sifang cohort of China National Salt Industry Group, a total of 575 participants were included. According to the homeostasis model assessment of insulin resistance, the participants were divided into an IR group (n = 116) and a non-IR group (n = 459). Differences between the two groups were compared using the t-test and chi-square test. Multivariate logistic regression analysis was performed to evaluate the associations between IR and abnormalities in metabolic indicators. Results A total of 575 participants were included, including 390 men and 185 women, with a mean age of (48.58 ± 5.14) years and a mean body mass index (BMI) of (24.16 ± 2.97) kg/m². Blood pressure, serum uric acid, and blood lipid levels were significantly higher in the IR group than in the non-IR group (P<0.001). After adjusting for potential confounders including age, sex, and BMI, IR was independently associated with hypertension, dyslipidemia, and hyperuricemia, with odds ratios (OR) and 95% confidence intervals (CI) of 2.57 (1.61-4.09), 3.81 (2.36-6. 17), and 2.68 (1.53-4.71), respectively (all P<0.001). Conclusion Insulin resistance is significantly associated with multiple cardiovascular metabolic risk factors in the middle-aged population. Monitoring IR may contribute to the early prevention and control of cardiovascular diseases.

Abstract:
Objective To investigate the effects of Hand-Arm Bimanual Intensive Training (HABIT) based on the FITT-CORRECT principles on upper limb function and functional independence in children with spastic hemiplegic cerebral palsy. Methods A total of 87 children with spastic hemiplegia were enrolled and randomly divided into an observation group (n=44), which received HABIT based on the FITT-CORRECT principles, and a control group (n=43), which received conventional HABIT. Both groups received intervention for 6 weeks. The Quality of Upper Extremity Skills Test (QUEST), Peabody Developmental Motor Scales–Fine Motor (PDMS-FM), and the Functional Independence Measure for Children (WeeFIM) were administered before and after treatment. The root mean square (RMS) values of the biceps and triceps brachii during maximum isometric contraction of elbow flexion and extension on the affected side were also measured. Results Before treatment, there were no significant differences between the two groups in QUEST, PDMS-FM, WeeFIM scores, or RMS values of the biceps and triceps during maximum isometric contraction of the affected elbow (P>0.05). After 6 weeks of treatment, no significant change was observed in the RMS values of the triceps during elbow flexion in either group (P>0.05). All other outcome measures improved significantly in both groups (P<0.05). The observation group showed significantly greater improvements in dissociated movement and grasp scores of QUEST, grasp and visual-motor integration scores of PDMS-FM, WeeFIM scores, as well as RMS values of the biceps during elbow flexion and both the biceps and triceps during elbow extension on the affected side (P<0.05). Conclusion Compared with conventional HABIT, HABIT based on the FITT-CORRECT principles is more effective in improving upper limb function and functional independence in children with spastic hemiplegia.

Abstract:
Macropinocytosis (MP) is an actin-dependent process by which cells internalize large volumes of extracellular fluid. This engulfed material is subsequently digested and degraded within lysosomes, providing essential nutrients that enable cell survival and growth under hypoxic and nutrient-scarce microenvironments. In the pathogenesis of hepatocellular carcinoma (HCC), sustained liver inflammation plays a critical role. Chronic liver inflammation not only creates favorable conditions for MP activation but also triggers relevant signaling pathways, thereby establishing a persistent positive feedback loop. This review aims to systematically elucidate the mechanistic basis of MP, summarize recent advances in its regulation via relevant signaling pathways during HCC progression, and discuss the potential value of targeting MP as a therapeutic strategy for HCC, thereby providing insights for future clinical research.

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Objective To investigate the differential expression of LEFTY1 in peritoneal dialysis (PD)-associated peritoneal fibrosis (PF) and its clinical value. Methods Levels of LEFTY1, transforming growth factor-β (TGF-β), and fibronectin (FN) in peritoneal dialysis effluent were measured in 72 PD patients, and clinical data were collected. Differences in these indicators were compared among groups with different dialysis durations, and the correlation between LEFTY1 and clinical parameters was analyzed to evaluate its predictive value for ultrafiltration insufficiency. Results LEFTY1 levels decreased progressively with longer dialysis duration: short-term group [(2 262.49±270.47) ng/mL] > mid-term group [(1 853.52±226.06) ng/mL] > long-term group [(1 767.24±264.57) ng/mL], with significant differences among groups (P<0.001). TGF-β and FN levels increased with prolonged dialysis duration (P<0.05). Correlation analysis revealed that LEFTY1 was significantly negatively correlated with FN (r=-0.327, P=0.004), dialysis duration (r=-0.496,P<0.001), peritoneal Kt/V (r=-0.333, P=0.004), peritoneal creatinine clearance (r=-0.239, P=0.043), and serum calcium (r=-0.410, P<0.001), while positively correlated with total creatinine clearance (r=0.283, P=0.016). The area under the ROC curve for LEFTY1 combined with peritoneal Kt/V and peritoneal Ccr in predicting ultrafiltration insufficiency was 0.739. Multivariate regression analysis identified dialysis duration as an independent influencing factor for LEFTY1 expression (P=0.001). Conclusion The expression of LEFTY1 in the peritoneal dialysis effluent of PD patients decreases with prolonged dialysis duration and is negatively correlated with pro-fibrotic indicators, suggesting that it may play a potential anti-fibrotic role in the progression of PF. The combined detection of LEFTY1 and solute removal indicators holds value for the early identification of ultrafiltration dysfunction.

Abstract:
Objective To develop an early - warning approach for influenza in Anhui Province using the Moving Epidemic Method (MEM), offering a scientific basis for influenza prevention and control in the province. Methods Based on the surveillance data reported weekly by influenza sentinel hospitals and network laboratories in the “China Influenza Surveillance Information System”, the weekly influenza virus detection positive rate (positive rate, PR) across the province was analyzed. The MEM was used to estimate the epidemic threshold and classification intensity threshold of influenza in Anhui Province before and after the COVID-19 pandemic, respectively. The early warning effect of the MEM was evaluated using cross-validation. Results Epidemic and graded intensity thresholds of influenza were derived using PR and MEM. Prior to the COVID - 19 pandemic, the starting, medium - intensity, high - intensity, extremely - high - intensity, and ending thresholds of influenza epidemics were 17.08%, 38.47%, 53.20%, 59.71%, and 12.34% respectively. At this point, the model exhibited sensitivity of 0.98, specificity of 0.86, Matthews Correlation Coefficient of 0.84, and Youden Index of 0.84. After the COVID - 19 pandemic, these thresholds were 25.61%, 43.81%, 63.29%, 74.46%, and 17.82% respectively. At this point, the model demonstrates sensitivity of 0.86, specificity of 0.92, Matthews Correlation Coefficient of 0.78, and Youden Index of 0.77. Conclusion MEM shows good applicability in estimating the epidemic and graded intensity thresholds of influenza during the winter and spring in Anhui Province. Both the influenza epidemic threshold and graded intensity thresholds after the COVID- 19 pandemic are higher than those before the pandemic.

Abstract:
Objective To investigate the association between single and combined exposure to arsenic (As), mercury (Hg), and cadmium (Cd) in urine and hearing loss in adults. Methods A total of 400 adult patients were recruited for the study, comprising 200 participants in the normal hearing group and 200 in the hearing loss group. General demographic information of the study subjects was collected by a self-administered questionnaire, morning urine samples were tested for As, Hg, and Cd concentrations, and hearing status was assessed by Pure Tone Audiometry. A binary logistic regression model was used to explore the association between the concentrations ofthe three metals in urine and hearing loss, and a Bayesian kernel-machine regression (BKMR) model was used to further explore the association between the concentrations of the three metal mixtures and hearing loss. Results After correction for urinary creatinine, As, Hg, and Cd concentrations were higher in the hearing loss group than those in the normal hearing group (P<0.05). Binary logistic regression indicated that concentrations of As, Hg, and Cd were positively associated with hearing loss, and the risk of hearing loss was significantly higher in the second quartile of Cd concentration compared to the first quartile (OR=2.000, 95% CI: 1.009-3.964). BKMR modeling analysis showed that the combined exposure levels of As, Hg, and Cd significantly increased the risk of hearing loss, with Cd being the most important contributor (posterior inclusion probability=0.412), and that there were potential interactions between As and Cd, and Cd and Hg. Conclusion Combined exposure to As, Hg, and Cd in urine is significantly associated with adult hearing loss, with Cd being the primary contributor in the exposure combination. The interactions between As and Cd, as well as Cd and Hg, may have adverse effects on hearing loss.

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Abstract:
Objective To investigate the mechanism by which activation of α7nicotinic acetylcholine receptors (α7nAChR) optimizes brown adipose tissue (BAT) thermogenesis in obese mice. Methods Obesity was induced in C57BL/6J mice via a high-fat diet. Fifty 8-week-old mice were randomly assigned to five groups: low-fat diet (Control), high-fat diet (HFD), high-fat diet plus a β3-adrenergic receptor agonist (HFD+β3), high-fat diet plus a β3-adrenergic receptor agonist and an α7nAChR selective agonist (HFD+β3+GTS-21), and high-fat diet plus a β3- adrenergic receptor agonist and an α7nAChR selective antagonist (HFD+β3+α-BGT). Haematoxylin and eosin (HE) staining was used to evaluate BAT morphology. Transmission electron microscopy was performed to assess mitochondrial number and lipid droplet morphology in adipocytes. ELISA was used to measure levels of tumour necrosis factor-α (TNF-α), interleukin-1β(IL-1β), interleukin-10(IL-10), transforming growth factor-β (TGF-β), cyclic adenosine monophosphate (cAMP), and norepinephrine (NE). RT-qPCR was conducted to determine mRNA expression levels of vascular endothelial growth factor A (VEGF-A), nitric oxide synthase 2 (NOS2), arginase 1 (Arg1), uncoupling protein 1 (UCP1), PR domain-containing protein 16 (PRDM16), and peroxisome proliferator- activated receptor γ coactivator-1α (PGC-1α) in BAT. Immunohistochemistry was used to detect macrophage markers (CD31, CD86, and CD206). Protein expression levels of UCP1, α7nAChR, nuclear factor-κB p65(NF-κB p65), phosphorylated Janus kinase 2 (p-JAK2), and phosphorylated signal transducer and activator of transcription 3 (p-STAT3) were analyzed by Western blot. Results Compared with the Control group, the HFD group showed enlarged lipid droplets and reduced mitochondrial numbers in adipocytes, accompanied by increased TNF-α and IL-1β and decreased IL-10, TGF-β, Arg1, and VEGF-A (all P<0.01). Compared with the HFD+β3 group, the HFD+β3+GTS-21 group showed a smaller lipid droplet area and a higher mitochondrial number, along with reduced levels of pro-inflammatory factors, NOS2, and NF-κB p65, and increased levels of anti-inflammatory factors, Arg1, and phosphorylation of JAK2 and STAT3 (all P<0.01). Conclusion Activation of α7nAChR in combination with a β3-adrenergic receptor agonist effectively enhances thermogenesis, alleviates local adipose tissue hypoxia, and shifts the local inflammatory phenotype toward an M2-like state, thereby reducing inflammation in brown adipose tissue and improving its thermogenic capacity.

Abstract:
Objective To investigate the molecular mechanisms by which kaempferol induces apoptosis in hepatic stellate cells (HSC). Methods HSC were divided into five groups: blank control, transforming growth factor-β1(TGF-β1) (10 ng/mL) group, and groups treated with different concentrations of kaempferol (8, 16, 32 µmol/L). Cell counting kit-8(CCK-8) assay was used to evaluate chrysin's effect on HSC proliferation. Transwell assay was used to assess chrysin's impact on HSC migration. Flow cytometry was used to detect apoptosis, reactive oxygen species (ROS) expression, and mitochondrial permeability transition pore (mPTP) opening across groups. 5,5,,6,6,-Tetrachloro-1,1,,3,3,-tetraethylimidacarbocyanine iodide(JC-1) assay was used to test mitochondrial membrane potential. Western blot was used to detect apoptosis-related mitochondrial proteins and extracellular matrix protein expression. Transmission electron microscopy was used to examine mitochondrial structural alterations. Molecular docking and kinetic simulations was used to analyze the targeted interaction between kaempferol and NAD-dependent deacetylase sirtuin-3 (SIRT3). Results Kaempferol significantly inhibited the proliferation and migration of HSC while promoting their apoptosis. It markedly increased ROS expression and opened the mPTP in these cells, concurrently reducing their mitochondrial membrane potential. It increased the expression of Bcl-2 associated X protein (Bax), Cleaved-Caspase3, and Cleaved-Caspase9 while decreasing the expression of B lymphocytoma-2 (Bcl2) and SIRT3. Kaempferol promoted mitochondrial fission in HSC. Molecular docking and kinetic simulations revealed that kaempferol exhibits favorable binding affinity and stability with SIRT3. Conclusion Kaempferol induces HSC apoptosis by targeting SIRT3 to mediate ROS regulation of the mitochondrial apoptosis pathway, thereby reducing extracellular matrix deposition. This demonstrates kaempferol's potential anti-fibrotic effects in liver.

Abstract:
Objective To explore the effects of Vascular Endothelial Growth Factor A (VEGFA) rs2010963 and rs3025039 polymorphisms on chemotherapy toxicities and clinical prognosis in children with brain tumors. Methods A total of 104 pediatric patients with brain tumors receiving standardized chemotherapy were enrolled. Matrix-assisted laser desorption/ionization time of flight mass spectrometry was used for VEGFA rs2010963 and rs3025039 genotyping. The χ² test was applied to analyze the association between genotypes and chemotherapy_related toxicities. Cox proportional hazards regression was used to evaluate the correlations of clinicopathological characteristics and genotypes with the progression_free survival (PFS). In addition, bioinformatic analyses were conducted to investigate the regulatory factors potentially affected by the two SNP loci. Results The VEGFA expression in brain tumors (5.17±1.81) was significantly higher than that in normal tissues (4.33±1.56, P<0.001). Patients with high VEGFA expression had significantly worse overall survival than patients with low VEGFA expression (P<0.001). Among the 104 children with brain tumors included, the rs2010963 CC, CG, and GG genotypes accounted for 14.42%, 55.77%, and 29.81%, respectively. The frequencies of C and G alleles were 42.31% and 57.69%, respectively. The rs3025039 CC, CT, and TT genotypes accounted for 70.19%, 25.96%, and 3.85%, respectively. The frequencies of C and T alleles were 83.17% and 16.83%, respectively. The children with the rs3025039 CC genotype had significantly higher incidences of thrombocytopenia (46.58%) and gastrointestinal toxicity (56.16%), and significantly lower incidences of coagulation disorders (4.11%) than CT genotype carriers (22.22%, 33.33%, and 50.00%, respectively, P<0.05). They had significantly lower incidences of dyslipidemia (2.74%) than TT genotype carriers (14.82%, P<0.05). The tumor type and the rs2010963 genotype were significantly associated with PFS (P<0.05) in univariable and multivariable Cox regression analysis. Bioinformatic analysis indicated that the rs2010963 and rs3025039 polymorphisms regulated VEGFA expression by affecting the binding of transcription factors and miRNAs to their target gene sequences, respectively. Conclusion The VEGFA rs3025039 CC genotype significantly affects the risk of thrombocytopenia, gastrointestinal toxicity, coagulation disorders, and hyperlipidemia, while the rs2010963 CG genotype significantly affects the risk of brain tumor progression.

Abstract:
Objective To investigate the change of interleukin (IL)-18 and Th1, Th2 of CD3+CD8- T helper lymphocytes in pediatric liver transplantation with cytomegalovirus (CMV) infection.Methods A total of 40 pediatric patients who underwent pediatric liver transplantation were collected. Patients were divided into the CMV group (n = 20) and the non-infected group (n = 20) according to whether CMV infection occurred. The plasma IL-18 levels were determined by enzyme-linked immunosorbent assay (ELISA). The expression percentage of IFN-γ and IL-10 of CD3+CD8- T lymphocytes (Th1 and Th2) in peripheral blood were determined with flow cytometry. Results The expression level of plasma IL-18 significantly increased in the CMV group compared with that in non-infected group (P < 0.01). The expression percentage of Th1 in peripheral blood significantly decreased in the CMV group compared with that in non-infected group (P < 0.01). No significant difference was found in the expression percentage of Th2 between CMV group and non-infected group. IL-18 levels were negatively correlated with the Th1 (P < 0.05). Conclusion The elevated IL-18 levels along with polarized Th 1 subsets’ imbalance involves in the pathogenesis ofCMV infection in pediatric liver transplantation.

Abstract:
Objective To explore the molecular mechanism of the therapeutic effect of total timosaponin on central precocious puberty (CPP) rats based on the RF-amide-related Peptide 3 (RFRP-3)/G protein-coupled receptor 147 (GPR147) signaling pathway. Methods Twenty-six day-old female SD rats were randomly assigned into a control group, a model group, an RFRP-3 up-regulated group (1.5 mg/kg), a total timosaponin group (200 mg/kg), a total timosaponin+empty load group, and a total timosaponin+RFRP-3 knockdown group, with 12 rats in each group. A CPP rat model was prepared by subcutaneous injection of N-methyl-DL-aspartic acid (NMA) (40 mg/kg). After modeling and administration, the opening time of the rat’s vulva and the first estrus interval daily were observed. Administration was stopped when the first estrus interval appeared in the model group. The day of vaginal opening and the time of the first estrus interval of rats were recorded in each group of rats. The wet masses of both ovaries and uterus were measured, and the ovarian and uterine indices were calculated; HE staining was used to observe the morphological changes of rat ovarian and uterine tissues, the number of corpus luteum in the ovaries and the thickness of the uterine wall were determined; ELISA method was applied to detect levels of serum luteinizing hormone (LH), follicle stimulating hormone (FSH), and estradiol (E2); RT-qPCR was used to detect the mRNA expression of hypothalamic gonadotropin-releasing hormone (GnRH), KISS-1, GPR54, RFRP-3, and GPR147. Results Compared with the control group, the day of vaginal opening and the time of the first estrus interval of rats in the model group were advanced (P<0.05), the ovarian index and uterine index, number of ovarian corpus luteum and uterine wall thickness, serum LH, FSH, E2 levels, and the GnRH, KISS-1, and GPR54 mRNA expression levels in the hypothalamus increased (P<0.05), while the RFRP-3 and GPR147 mRNA expression levels decreased (P<0.05). Compared with the model group, the day of vaginal opening and the time of the first estrus interval of rats in the RFRP-3 up-regulated group and the total timosaponin group were delayed (P<0.05), the ovarian index and uterine index, number of ovarian corpus luteum and uterine wall thickness, serum LH, FSH, E2 levels, and the GnRH, KISS-1, and GPR54 mRNA expression levels in the hypothalamus decreased (P<0.05), while the RFRP-3 and GPR147 mRNA expression levels increased (P<0.05). Knocking down RFRP-3 could prominently weaken the delaying effect of total timosaponin on the development of precocious puberty in CPP rats (P<0.05). Conclusion Total timosaponin may inhibit the release of GnRH and premature activation of the hypothalamic pituitary gonadal axis by activating the RFRP-3/GPR147 signaling pathway, thereby alleviating the degree of precocious puberty development in CPP model rats.