Polycystic ovary syndrome （PCOS）， a common reproductive endocrine disorder in women， is one of the leading causes of ovulatory infertility in women of reproductive age. Due to its heterogeneous etiology， complex symptoms， and challenging treatment， PCOS has become a focal point of research in gynecological and reproductive medicine globally. The pathogenesis of PCOS is complex and may involve regulatory mechanisms such as inflammatory responses， oxidative stress， and cellular autophagy. Crown-like structures （CLSs） refer to pro-inflammatory microenvironments formed by macrophages engulfing adipocytes. The inflammatory disorders induced by CLSs are one of the key factors contributing to the development of PCOS and its complications. Current studies have indicated that the obese status in PCOS accelerates the formation of CLSs， and the density of CLSs can predict the progression of metabolic disorders and influence the outcomes of various metabolic diseases. Traditional Chinese Medicine （TCM） offers the unique advantages of a holistic view， four diagnostic methods， and syndrome differentiation and treatment to ameliorate the symptoms and signs of PCOS through multiple levels， pathways， and targets. Although studies on the mechanisms of metabolic diseases and CLS formation have been reported in China and abroad， there is still a lack of literature on the correlation between CLSs and PCOS， as well as reviews on TCM interventions targeting CLSs for treating this disease. Therefore， this paper summarized the correlation between obese PCOS and CLSs and reviewed recent studies on TCM interventions based on CLS formation （adipose tissue-macrophage inflammatory crosstalk） in the treatment of obese PCOS， aiming to provide new research perspectives for the prevention and treatment of PCOS using TCM.

Polycystic ovary syndrome （PCOS）， a common reproductive endocrine disorder in women， is one of the leading causes of ovulatory infertility in women of reproductive age. Due to its heterogeneous etiology， complex symptoms， and challenging treatment， PCOS has become a focal point of research in gynecological and reproductive medicine globally. The pathogenesis of PCOS is complex and may involve regulatory mechanisms such as inflammatory responses， oxidative stress， and cellular autophagy. Crown-like structures （CLSs） refer to pro-inflammatory microenvironments formed by macrophages engulfing adipocytes. The inflammatory disorders induced by CLSs are one of the key factors contributing to the development of PCOS and its complications. Current studies have indicated that the obese status in PCOS accelerates the formation of CLSs， and the density of CLSs can predict the progression of metabolic disorders and influence the outcomes of various metabolic diseases. Traditional Chinese Medicine （TCM） offers the unique advantages of a holistic view， four diagnostic methods， and syndrome differentiation and treatment to ameliorate the symptoms and signs of PCOS through multiple levels， pathways， and targets. Although studies on the mechanisms of metabolic diseases and CLS formation have been reported in China and abroad， there is still a lack of literature on the correlation between CLSs and PCOS， as well as reviews on TCM interventions targeting CLSs for treating this disease. Therefore， this paper summarized the correlation between obese PCOS and CLSs and reviewed recent studies on TCM interventions based on CLS formation （adipose tissue-macrophage inflammatory crosstalk） in the treatment of obese PCOS， aiming to provide new research perspectives for the prevention and treatment of PCOS using TCM.

Acute kidney injury (AKI) has high morbidity and mortality, but effective clinical drugs and management are lacking. Previous studies have suggested that macrophages play a crucial role in the inflammatory response to AKI and may serve as potential therapeutic targets. Emerging evidence has highlighted the importance of forkhead box protein O1 (FoxO1) in mediating macrophage activation and polarization in various diseases, but the specific mechanisms by which FoxO1 regulates macrophages during AKI remain unclear. The present study aimed to investigate the role of FoxO1 in macrophages in the pathogenesis of AKI. We observed a significant upregulation of FoxO1 in kidney macrophages following ischemia-reperfusion (I/R) injury. Additionally, our findings demonstrated that the administration of FoxO1 inhibitor AS1842856-encapsulated liposome (AS-Lipo), mainly acting on macrophages, effectively mitigated renal injury induced by I/R injury in mice. By generating myeloid-specific FoxO1-knockout mice, we further observed that the deficiency of FoxO1 in myeloid cells protected against I/R injury-induced AKI. Furthermore, our study provided evidence of FoxO1's pivotal role in macrophage chemotaxis, inflammation, and migration. Moreover, the impact of FoxO1 on the regulation of macrophage migration was mediated through RhoA guanine nucleotide exchange factor 1 (ARHGEF1), indicating that ARHGEF1 may serve as a potential intermediary between FoxO1 and the activity of the RhoA pathway. Consequently, our findings propose that FoxO1 plays a crucial role as a mediator and biomarker in the context of AKI. Targeting macrophage FoxO1 pharmacologically could potentially offer a promising therapeutic approach for AKI.

OBJECTIVES: To investigate the mechanism by which transforming growth factor‑β (TGF‑β) regulates major histocompatibility complex class I (MHC-I) expression in hepatocellular carcinoma (HCC) cells and its role in immune evasion of HCC. METHODS: HCC cells treated with TGF‑β alone or in combination with SB-431542 (a TGF-β type I receptor inhibitor) were examined for changes in MHC-I expression using RT-qPCR and Western blotting. A RNA interference experiment was used to explore the role of miR-23a-3p/IRF1 signaling in TGF‑β‑mediated regulation of MHC-I. HCC cells with different treatments were co-cultured with human peripheral blood mononuclear cells (PBMCs), and the changes in HCC cell proliferation was assessed using CCK-8 and colony formation assays. T-cell cytotoxicity in the co-culture systems was assessed with lactate dehydrogenase (LDH) release and JC-1 mitochondrial membrane potential assays, and T-cell activation was evaluated by flow cytometric analysis of CD69 cells and ELISA for TNF-α secretion. RESULTS: TGF‑β treatment significantly suppressed MHC-I expression in HCC cells and reduced T-cell activation, leading to increased tumor cell proliferation and decreased HCC cell death in the co-culture systems. Mechanistically, TGF-β upregulated miR-23a-3p, which directly targeted IRF1 to inhibit MHC-I transcription. Overexpression of miR-23a-3p phenocopied TGF‑β‑induced suppression of IRF1 and MHC-I. CONCLUSIONS We reveal a novel immune escape mechanism of HCC, in which TGF‑β attenuates T cell-mediated antitumor immunity by suppressing MHC-I expression through the miR-23a-3p/IRF1 signaling axis.
Humans ; MicroRNAs/genetics* ; Carcinoma, Hepatocellular/metabolism* ; Liver Neoplasms/metabolism* ; Interferon Regulatory Factor-1/metabolism* ; Transforming Growth Factor beta/metabolism* ; Signal Transduction ; Histocompatibility Antigens Class I/metabolism* ; Cell Line, Tumor ; Tumor Escape ; Coculture Techniques

This study is designed to address the development, synthesis, and screening of non-animal-derived nanoantibody libraries. Furthermore, it seeks to elucidate the impact of framework region selection and complementarity-determining region (CDR) design on the characteristics of synthesized nanoantibody libraries. These investigations aim to establish a robust theoretical and technical foundation for enhancing the efficacy, diversity, and practical applicability of synthetic nanoantibody libraries. In this study, a new framework (IGHV3S65*01-IGHJ4*01) was identified based on the high-throughput sequencing results of natural nanobodies, and degenerate primers were designed based on the frequency of amino acids at each position in the complementarity-determining region (CDR) region to synthesize the coding fragments of nanobodies by overlap PCR. After 40 times of electro-transformation, a single-frame synthesized nanobody library (SS-Library) containing 6×10⁹ clones was obtained, and the titer of the library was demonstrated to be 10¹³ PFU/mL after rescue by the helper phage M13K07. Random 48 single colonies were picked for PCR, which revealed an insertion rate of 95.8%. Sanger sequencing results showed that 38 clones had complete sequences, none of which showed cysteines or stop codons, and no identical sequences appeared, suggesting that the library had higher diversity. The library was screened and validated with three antigens, including bovine serum albumin (BSA), acetylcholinesterase (AchE), and immunoglobulin G (IgG). Finally, 2 nanobodies against BSA, 10 against AchE, and 15 against IgG were obtained. One positive clone of each antigen was singled out for recombinant expression, and the results showed that all the three nanobodies were expressed in a soluble form. The binding activity of recombinantly expressed nanobodies was evaluated using indirect enzyme-linked immunosorbent assay (ELISA) and bio-layer interferometry (BLI). The results demonstrated that the anti-AChE and anti-IgG nanobodies exhibited specific binding to their respective antigens, with affinity constants (KD) of 294 nmol/L and 250 nmol/L, respectively. The nanobody synthetic library preparation method proposed in this study is simple and easy to use with low preference, and it is expected to be a universal nanobody discovery platform for the preparation and development of lead specific nanobodies.
Single-Domain Antibodies/biosynthesis* ; Peptide Library ; Complementarity Determining Regions/immunology* ; Animals

Objective: To investigate the daily learning time status of elementary school students and understand the implementation of Health Requirements of Daily Learning Time for Secondary and Elementary School Students (GB/T 17223-2012) in schools, so as to provide a reference for strengthening and improving school health and health education in the new era. Methods: A stratified cluster random sampling method was used to select 7 776 primary school students in Shandong Province, and a survey questionnaire was designed based on the Health Requirements for Dayily Learning Time for Secondary and Elementary School Students(GB/T 17223-2012) standard to investigate their daily learning arrangements, sleep and physical activities, and breaks between classes. Comparison of intergroup differences were used by Chi square test and Kruskal-Wallis H test. Results: About 55.88 % of primary school students for daily learning time met the standard. There was a statistically significant difference in the daily learning time achievement rate among primary school students in three grades for first and second grade, third and fourth grade and fifth and sixth grade ( Z=1 629.47, P <0.01), and the fifth and sixth grade had the highest proportion of achieving the standard (85.92%). Specifically, the proportions of students whose class hours, class numbers and morning reading time meeting the standards were 30.07%, 10.20% and 42.19%, respectively. The sleep deficiency rate of primary school studnets was 58.69%, and the physical activity deficiency rate was 65.78%; and there was a statistically significant difference in the rate of insufficient sleep time and physical activity time among primary school students of different grades ( χ 2=56.39, 95.95, P <0.01), with sixth grade students showing the highest rates for both sleep and physical activity deficiencies (64.35%, 73.37%). Additionally, 49.78% of students had recess time below the standard requirements. Conclusion The health status of daily learning time among primary school students in Shandong Province is poor, with insufficient implementation of school standards, and the implementation of standards needs to be further strengthened.

Programmed death-1 (PD-1) is an inhibitory immune checkpoint that binds to programmed death-ligand 1 (PD-L1) to regulate the immune response and maintain immune system homeostasis of the immune system. Through overexpression of PD-L1, tumor cells bind to PD-1 on the surface of immune cells, inhibiting the activity and function of immune cells, leading to immune escape of cancer cells and tumor progression. Gastrointestinal cancer is a common malignancy with a high mortality rate worldwide, and the effectiveness of current systematic treatment options is limited. In recent years, immune checkpoint inhibitors (ICIs) such as PD-1/PD-L1 inhibitors have attracted much attention in cancer therapy. Immunotherapy has been incorporated into the treatment of some gastrointestinal malignancies. Different from traditional treatment, it uses various means to stimulate and enhance the immune function of the body to achieve the therapeutic purpose of controlling and eliminating tumor cells. However, although PD-1/PD-L1 inhibitors have shown potential in the treatment of gastrointestinal tumors, the efficacy of single inhibitor therapy is limited, which may be due to the ability of tumors to escape immune attack through other pathways after inhibitor treatment, or the presence of other immunosuppressive factors. For example, PD-1 and PD-L1 inhibitors can be combined with other immune checkpoint drugs, molecularly targeted drugs, or chemotherapy drugs to simultaneously act on different immune pathways and improve the comprehensive effect of immunotherapy. However, to achieve an effective combination therapy, we need to delve into the specific mechanisms of action of the PD-1/PD-L1 axis in the development and progression of gastrointestinal tumors, which can help to develop the best treatment strategy and provide individualized treatment options for the appropriate patient population. Therefore, future studies should focus on the regulatory mechanisms of PD-1/PD-L1 axis and evaluate the therapeutic effects of different treatment combinations on gastrointestinal tumors. In this paper, we review the research progress of PD-1/PD-L1 axis in tumorigenicity and its mechanism, and review the single and combined treatment strategies of PD-1 and PD-L1 inhibitors in gastrointestinal tumors.

Objective: To understand the implementation of daily study time standard among secondary school students in Shandong Province, so as to provide scientific basis for the formulation and implementation of relevant policies. Methods: From January to May 2023, a multi stage random sampling method was used to select 8 725 middle school students in Shandong Province. A survey questionnaire was designed based on the Requirements for Daily Study Time of Primary and Secondary School Students(GB/T 17223-2012), to investigate indicators such as students daily learning schedule, sleep and physical activity time, break time and scheduling requirements. Results: The compliance rates for daily study time in junior and senior high school students in Shandong Province were 29.2% and 23.6%, respectively, with a statistically significant difference ( χ 2=33.63, P <0.01). Compliance rates for sleep duration, physical activity and recess time, morning and afternoon class hours, and class duration were 19.3%, 26.2%, 30.5%, 73.2% and 16.2%. Class duration compliance was relatively high, with rates of 96.7% in junior high and 94.4% in senior high school students. There was a statistically significant difference in compliance rates for extended class breaks between different educational stages ( χ 2= 81.78, P <0.01), with rates of 84.6% in junior high and 83.4% in senior high school students. As students progressed through their educational stages, compliance rates for physical activities, class breaks, consecutive classes, and total weekly class hours showed a decreasing trend, with rates of 31.8% and 18.3%, 35.7% and 23.1%, 60.5% and 29.6%, 55.2% and 35.1% in junior and senior high school students, respectively. Conclusions The revised standard of Requirements for Daily Study Time of Primary and Secondary School Students(GB/T 17223-2012) optimizes the daily study and life schedule of middle school students to a certain extent. However, daily study time for middle school students in Shandong Province exceeds standard. Relevant departments need to enhance their ability to implement standards and strengthen the supervision of policy standards implementation.

OBJECTIVE To investigate the mechanism by which Ginkgo flavone aglycone （GA） reduces the cardiotoxicity of doxorubicin （DOX） based on transcriptomics and proteomics. METHODS Thirty-six mice were randomly assigned to control group （CON group， tail vein injection of equal volume of physiological saline every other day+daily intragastric administration of an equal volume of physiological saline）， DOX group （tail vein injection of 3 mg/kg DOX every other day）， and GDOX group （daily intragastric administration of 100 mg/kg GA+tail vein injection of 3 mg/kg DOX every other day）， with 12 mice in each group. The administration of drugs/physiological saline was continued for 15 days. Mouse heart tissues were collected for RNA-Seq transcriptomic sequencing and 4D-Label-free quantitative proteomic analysis to screen differentially expressed genes and proteins， which were then subjected to Kyoto encyclopedia of genes and genomes （KEGG） enrichment analysis. The expression levels of Apelin peptide （Apelin）， phosphatidylinositol 3-kinase （PI3K）， and protein kinase B （Akt） mRNA and protein in mouse heart tissues， as well as the phosphorylation levels of PI3K and Akt proteins， were verified. H9c2 cardiomyocytes were divided into control group （CON group）， DOX group （2 μmol/L）， and GDOX group （2 μg/mL GA+2 μmol/L DOX） to determine cell viability and the levels of key glycolytic substances in the cells. RESULTS Six common pathways were identified from transcriptomics and proteomics， including the Apelin signaling pathway， the PI3K-Akt signaling pathway， and insulin resistance. Among them， the Apelin and PI3K-Akt signaling pathways were the most enriched in terms of gene numbers. Target validation experiments showed that compared to the CON group， the relative expression of Apelin， PI3K and Akt mRNA and protein levels， as well as the phosphorylation levels of PI3K and Akt proteins， were significantly decreased in the DOX group （P＜0.05 or P＜0.01）. The relative expression of Apelin， PI3K and Akt mRNA and the phosphorylation levels of PI3K and Akt proteins were significantly increased in the GDOX group as compared with the DOX group （P＜0.05 or P＜0.01）. Cellular experiments indicated that compared to the CON group， cell viability in the DOX group was significantly decreased （P＜0.05）， the relative uptake of glucose and the relative production of pyruvate and lactate were significantly increased （P＜0.05）， and the relative production of ATP was significantly reduced （P＜0.05）. Compared to the DOX group， cell viability in the GDOX group was significantly increased （P＜ 0.05）， and the relative production of pyruvate and lactate was significantly reduced （P＜0.05）. CONCLUSIONS GA may alleviate DOX-induced cardiotoxicity by upregulating the mRNA and protein expression of Apelin， PI3K， and Akt in heart tissues， and regulating glycolytic processes.

Objective To comprehensively analyze the clinical outcomes of total cavopulmonary connection (TCPC) in the treatment of functional single ventricle combined with heterotaxy syndrome (HS). Methods A retrospective analysis was conducted on the patients with functional single ventricle and HS who underwent TCPC (a HS group) in Guangdong Provincial People's Hospital between 2004 and 2021. The analysis focused on postoperative complications, long-term survival rates, and identifying factors associated with patient survival. Early and late postoperative outcomes were compared with matched non-HS patients (a non-HS group). Results Before propensity score matching, 55 patients were collected in the HS group, including 42 males and 13 females, with a median age of 6.0 (4.2, 11.8) years and a median weight of 17.0 (14.2, 28.8) kg. Among the patients, there were 53 patients of right atrial isomerism and 2 patients of left atrial isomerism. Eight patients underwent TCPC in one stage. TCPC procedures included extracardiac conduit (n=39), intracardiac-extracardiac conduit (n=14), and direct cavopulmonary connection (n=2). Postoperative complications included infections in 27 patients, liver function damage in 19 patients, and acute kidney injury in 11 patients. There were 5 early deaths. The median follow-up time was 94.7 (64.3, 129.8) months. The 1-year, 5-year, and 10-year survival rates were 87.2%, 85.3%, and 74.3%, respectively. After propensity score matching, there were 45 patients in the HS group and 81 patients in the non-HS group. Compared to the non-HS group, those with HS had longer surgical and mechanical ventilation time, higher infection rates (P<0.05), and a 12.9% lower 10-year survival rate. Multivariate Cox regression analysis identified asplenia was a risk factor for mortality (HR=8.98, 95%CI 1.86-43.34, P=0.006). Conclusion Compared to non-HS patients, patients with HS have lower survival rates after TCPC, and asplenia is an independent risk factor for the survival of these patients.