Background Prenatal exposure to fine particulate matter (PM_2.5) is closely associated with cortical damage and neuroinflammation in offspring. The cyclic guanosine monophosphate–adenosine monophosphate synthase (cGAS)–stimulator of interferon genes (STING) signaling pathway is a key regulator of inflammation and may be subject to epigenetic regulation. Objective To investigate the role of cGAS-STING pathway activation in PM_2.5-induced cortical damage in offspring mice during pregnancy and the underlying epigenetic regulatory mechanisms. Methods Open field tests were used to assess depressive-like behavior in offspring mice. Morphological analysis was conducted to evaluate cortical damage and microglial activation in offspring brains. Real-time fluorescent quantitative PCR (RT-qPCR) and Western blot (WB) were performed to detect changes in the expression of key molecules in the cGAS-STING pathway in cortical tissue. A PM_2.5-induced microglial cell injury model was established in BV2 cells. Microglial activation was observed, cell viability was measured using the Cell Counting Kit-8 (CCK-8), and the expression levels of inducible nitric oxide synthase (iNOS) and key molecules in the cGAS-STING pathway were detected by RT-qPCR and WB. Bioinformatics analysis was performed to explore the epigenetic regulatory association between the STING signaling pathway and lysine-specific demethylase 5A (KDM5A). Changes in KDM5A mRNA and protein expression, as well as the protein level of histone H3 lysine 4 trimethylation (H3K4me3), were detected in an in vitro PM_2.5 injury model. Using small interfering RNA (siRNA) technology, the KDM5A gene was silenced in BV2 cells exposed to PM_2.5. The protein expression of H3K4me3 was detected to evaluate improvements in microglial activation, changes in inflammatory markers such as iNOS and mannose receptor (CD206), and alterations in the cGAS-STING pathway. Results Compared with the control group, the total distance of offspring mice in the PM_2.5 group was significantly reduced, and both the distance traveled and the time spent in the central area of the open field were significantly decreased (P<0.01, P<0.001), indicating depressive-like behavior in the offspring mice. Compared with the control group, the offspring mice in the PM_2.5 group exhibited disorganized cortical structure and significantly activated microglia (P<0.01), with significantly increased mRNA and protein levels of cGAS and STING (P<0.05, P<0.01, or P<0.001). The in vitro experiments demonstrated that the PM_2.5 treatment induced BV2 cells to polarize toward the M1 phenotype, exhibiting a distinct amoeboid morphology, with upregulated expression of the pro-inflammatory factor iNOS (P<0.05, P<0.01, or P<0.001) and activation of the cGAS-STING pathway (P<0.05, P<0.01). The analysis of RNA-seq data from KDM5A knockout cells revealed significantly downregulated STING expression, suggesting that KDM5A may activate the STING signaling pathway. The in vitro experiments further confirmed that the PM_2.5-treated BV2 cells exhibited significantly elevated mRNA and protein levels of KDM5A (P<0.01), while the H3K4me3 protein levels were markedly reduced (P<0.05). After silencing KDM5A in BV2 cells exposed to PM_2.5, compared with the PM_2.5+siNC group, the PM_2.5+siKDM5A group showed no obvious microglial activation and polarized toward the M2 phenotype, with significantly decreased expression levels of iNOS, cluster of differentiation 16 (CD16), and interleukin-1β (P<0.05, P<0.01), and significantly increased expression levels of anti-inflammatory factors CD206, YM1, and interleukin-10 (P<0.01, P<0.001). Meanwhile, the expression levels of cGAS and STING were also reduced (P<0.05, P<0.01). Conclusion KDM5A activates microglia through the cGAS-STING pathway, thereby contributing to PM_2.5-induced cortical damage in offspring mice during pregnancy.

ObjectiveTo investigate the effects of Qizhujianwei granules （QZJW） on abnormal proliferation and malignant transformation of gastric mucosal cells in rats with gastric intraepithelial neoplasia （GIN） and to explore the related mechanisms. MethodsA total of 80 SPF male Wistar rats were used. A GIN rat model was established using a four-factor comprehensive method consisting of methylnitronitrosoguanidine （MNNG）， ranitidine， irregular feeding patterns， and sodium salicylate. Except for the normal group， after successful modeling， the rats were randomly divided according to body weight into a model group， a Moluodan group （0.55 g·kg^-1）， and a QZJW group （7.34 g·kg^-1）， with 12 rats in each group. All groups were treated for 8 weeks. The general characteristics of the rats and morphological changes of the gastric mucosa were observed. Histopathological changes of the gastric mucosa were examined by hematoxylin-eosin （HE） staining. Enzyme-linked immunosorbent assay （ELISA） was used to detect serum levels of pepsinogenⅠ （PGⅠ）， pepsinogenⅡ （PGⅡ）， and gastrin （G-17）， as well as the expression level of transforming growth factor-β₁ （TGF-β₁） in gastric mucosal tissue， and the PGⅠ/PGⅡ ratio was calculated. Immunohistochemistry （IHC） was used to detect the localization and expression levels of proliferating cell nuclear antigen （Ki-67） and Vimentin in gastric mucosal tissue. Western blot analysis was used to determine the protein expression levels of Wnt family member 3A （Wnt3a）， β-catenin， CyclinD₁， proto-oncogene Cmyc， transforming growth factor-β receptor Ⅰ （TGFβRⅠ）， intracellular signaling transducers Smad2/3， phosphorylated （p）-Smad2/3， twist family transcription factor （Twist1）， and Vimentin in gastric mucosal tissue. ResultsCompared with the normal group， the model group showed characteristic changes including dim eyes， pale ears and claws， dark-red tongue， and reduced luster of the tail. The gastric mucosa appeared pale， with surface congestion and erosion. The gastric mucosal glands were disordered， the nuclear-to-cytoplasmic ratio increased， and local tumor cells were observed. Serum PGⅠ and PGⅡ levels and the PGⅠ/PGⅡ ratio were significantly decreased （P<0.01）， while the level of G-17 was significantly increased （P<0.01）. The protein expression levels of Ki-67， Wnt3a， β-catenin， CyclinD₁， Cmyc， TGF-β₁， TGFβRⅠ， Smad2/3， Twist1， and Vimentin in gastric mucosal tissue were significantly increased （P<0.05， P<0.01）， whereas the ratio of p-Smad2/3 to Smad2/3 was significantly decreased （P<0.05）. Compared with the model group， the general characteristics and gastric mucosal conditions of rats in the Moluodan group and the QZJW group were improved. HE staining showed that QZJW could effectively block the malignant progression of GIN. Serum PGⅠ and PGⅡ levels and the PGⅠ/PGⅡ ratio were significantly increased （P<0.05， P<0.01）， while the level of G-17 was significantly decreased （P<0.01）. The protein expression levels of Ki-67， Wnt3a， β-catenin， CyclinD₁， Cmyc， TGF-β₁， TGFβRⅠ， Smad2/3， Twist1， and Vimentin in gastric mucosal tissue were significantly decreased （P<0.05， P<0.01）. ConclusionQZJW have a therapeutic effect on rats with GIN. The mechanism may involve inhibition of the Wnt/β-catenin signaling pathway to regulate the cell cycle and suppress abnormal cell proliferation. Meanwhile， it may inhibit epithelial-mesenchymal transition by suppressing the TGF-β₁/Smad/Twist1 signaling pathway， thereby blocking the malignant progression of GIN.

ObjectiveTo investigate the effects of Qizhujianwei granules （QZJW） on abnormal proliferation and malignant transformation of gastric mucosal cells in rats with gastric intraepithelial neoplasia （GIN） and to explore the related mechanisms. MethodsA total of 80 SPF male Wistar rats were used. A GIN rat model was established using a four-factor comprehensive method consisting of methylnitronitrosoguanidine （MNNG）， ranitidine， irregular feeding patterns， and sodium salicylate. Except for the normal group， after successful modeling， the rats were randomly divided according to body weight into a model group， a Moluodan group （0.55 g·kg^-1）， and a QZJW group （7.34 g·kg^-1）， with 12 rats in each group. All groups were treated for 8 weeks. The general characteristics of the rats and morphological changes of the gastric mucosa were observed. Histopathological changes of the gastric mucosa were examined by hematoxylin-eosin （HE） staining. Enzyme-linked immunosorbent assay （ELISA） was used to detect serum levels of pepsinogenⅠ （PGⅠ）， pepsinogenⅡ （PGⅡ）， and gastrin （G-17）， as well as the expression level of transforming growth factor-β₁ （TGF-β₁） in gastric mucosal tissue， and the PGⅠ/PGⅡ ratio was calculated. Immunohistochemistry （IHC） was used to detect the localization and expression levels of proliferating cell nuclear antigen （Ki-67） and Vimentin in gastric mucosal tissue. Western blot analysis was used to determine the protein expression levels of Wnt family member 3A （Wnt3a）， β-catenin， CyclinD₁， proto-oncogene Cmyc， transforming growth factor-β receptor Ⅰ （TGFβRⅠ）， intracellular signaling transducers Smad2/3， phosphorylated （p）-Smad2/3， twist family transcription factor （Twist1）， and Vimentin in gastric mucosal tissue. ResultsCompared with the normal group， the model group showed characteristic changes including dim eyes， pale ears and claws， dark-red tongue， and reduced luster of the tail. The gastric mucosa appeared pale， with surface congestion and erosion. The gastric mucosal glands were disordered， the nuclear-to-cytoplasmic ratio increased， and local tumor cells were observed. Serum PGⅠ and PGⅡ levels and the PGⅠ/PGⅡ ratio were significantly decreased （P<0.01）， while the level of G-17 was significantly increased （P<0.01）. The protein expression levels of Ki-67， Wnt3a， β-catenin， CyclinD₁， Cmyc， TGF-β₁， TGFβRⅠ， Smad2/3， Twist1， and Vimentin in gastric mucosal tissue were significantly increased （P<0.05， P<0.01）， whereas the ratio of p-Smad2/3 to Smad2/3 was significantly decreased （P<0.05）. Compared with the model group， the general characteristics and gastric mucosal conditions of rats in the Moluodan group and the QZJW group were improved. HE staining showed that QZJW could effectively block the malignant progression of GIN. Serum PGⅠ and PGⅡ levels and the PGⅠ/PGⅡ ratio were significantly increased （P<0.05， P<0.01）， while the level of G-17 was significantly decreased （P<0.01）. The protein expression levels of Ki-67， Wnt3a， β-catenin， CyclinD₁， Cmyc， TGF-β₁， TGFβRⅠ， Smad2/3， Twist1， and Vimentin in gastric mucosal tissue were significantly decreased （P<0.05， P<0.01）. ConclusionQZJW have a therapeutic effect on rats with GIN. The mechanism may involve inhibition of the Wnt/β-catenin signaling pathway to regulate the cell cycle and suppress abnormal cell proliferation. Meanwhile， it may inhibit epithelial-mesenchymal transition by suppressing the TGF-β₁/Smad/Twist1 signaling pathway， thereby blocking the malignant progression of GIN.

ObjectiveBased on literature data mining， this study explores the modeling elements of diarrhea-predominant irritable bowel syndrome （IBS-D） animal models in China and abroad， providing references and suggestions for improving modeling methods and evaluation indicators. MethodsRelevant literature on IBS-D animal experiments from 2014 to 2024 was retrieved through computer searches in databases such as China National Knowledge Infrastructure （CNKI）， Wanfang Data， VIP， Chinese Medical Journals Full-text Database， and PubMed. Information on experimental animal species， gender， body weight， modeling methods， modeling periods， intervention controls， modeling standards， and detection indicators was organized. Microsoft Excel 2021 software was used to establish a database and perform statistical analysis to examine the characteristics of IBS-D animal models. ResultsA total of 398 articles that met the inclusion criteria were reviewed. The IBS-D animal models were predominantly established using SD rats， Wistar rats， and C57BL/6 mice. Male animals were more commonly used， with rats typically aged 6-8 weeks and mice aged 4-6 weeks. In terms of interventions， piverium bromide was the main Western medicine， Tongxieyaofang was the primary Chinese medicine， and electroacupuncture was the primary acupuncture method. Among the modeling methods， the multi-factor combined composite modeling approach was the most common. Modeling periods were mainly concentrated between 1-14 days and 15-30 days. The success criteria for modeling were mainly evaluated based on the animal's general condition， fecal appearance， visceral sensitivity， gastrointestinal motility， behavior， and pathology. Detection indicators included apparent indexes， pathological markers， biochemical indicators， oxidative stress， brain-gut peptides， neurotransmitters， inflammatory factors， immune function， intestinal permeability， autophagy， apoptosis， proteins related to relevant signaling pathways， intestinal microbiota and its metabolites， etc. ConclusionThere are various methods for establishing IBS-D animal models， but no unified and universally accepted method has been established. The operation of the same modeling methods and the evaluation standards of the models vary across studies. Based on the results of data mining， the authors suggest that the multi-factor combined composite modeling approach most closely reflects the pathophysiological processes of IBS-D， better simulating the complex clinical symptoms of IBS-D patients， such as abdominal pain and diarrhea， and has a high degree of clinical relevance. This method is relatively recommended. While animal models in general align with Western medicine standards， models incorporating traditional Chinese medicine （TCM） syndromes are relatively few. Therefore， one of the future directions for research is to establish IBS-D animal models that meet the combined clinical disease and syndrome requirements of both Western and Chinese medicine.

[Objective] To investigate the factors influencing the detection of HLA-C expression by flow cytometry. [Methods] A total of 12 hematopoietic stem cell suspension samples from peripheral hematopoietic stem cell volunteer donors were randomly collected after CD34⁺ cell counting detection. The influence of detecting different number of nucleated cell (500 000, 50 000 and 5 000), sequential order of red blood cell lysis and antibody incubation, and the HLA-C antibody with varied remaining time from the expiration date on the detection results of HLA-C expression by flow cytometry were investigated, respectively. The significance of differences between different groups was analyzed through Student t test. [Results] There was no significant difference in the proportion of HLA-C positive cells and mean fluorescence intensity (MFI) among the three groups with different nucleated cell numbers detected (500 000, 50 000 and 5 000) (P>0.05). The sequential order of red blood cell lysis and antibody incubation had no influence on the proportion of HLA-C positive cells (P>0.05), but HLA-C MFI value was significantly lower when antibody incubation was performed after red blood cell lysis than that when antibody incubation was performed before red blood cell lysis (P<0.05). The proportion of HLA-C positive cells and MFI value detected by HLA-C antibody remaining 24 months from the expiration date were significantly higher than those detected by HLA-C antibody remaining only 5 months from the expiration date (P<0.05). [Conclusion] The present study has investigated the factors of influencing HLA-C expression level by flow cytometry, the results have important reference and application value for standardizing the experimental operation of HLA-C expression and improving the accuracy and comparability of detection results.

Neuroendocrine carcinoma (NEC) represents a category of malignant tumors originating from neuroendocrine cells. Given that NEC cells exhibit characteristics of both neural and endocrine cells, they can hijack neuronal signaling pathways and dynamically regulate the expression of neuronal lineage markers during tumor metastasis, thereby constructing a microenvironment conducive to tumor growth and metastasis. Conversely, alterations in the tumor microenvironment can enhance the interactions between neurons and tumor cells, ultimately synergistically promoting the metastasis of NEC. This review highlights recent advancements in the field of cancer neuroscience, uncovering neuronal lineage markers in NEC that facilitate tumor dissemination through mediating crosstalk, bidirectional communication, and synergistic interactions between tumor cells and the nervous system. Consequently, the latest findings in tumor neuroscience have enriched our understanding of the biological mechanisms underlying tumor metastasis, opening new research avenues for a deeper comprehension of the complex biological processes involved in tumor metastasis, particularly brain metastasis. This review provides a comprehensive review of the crosstalk between tumor cells and neural signaling in the metastasis of NEC.
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Humans ; Carcinoma, Neuroendocrine/metabolism* ; Signal Transduction ; Animals ; Neoplasm Metastasis ; Neurons/pathology* ; Tumor Microenvironment ; Cell Communication

Objective To investigate the expression of ubiquitin binding enzyme E2T (UBE2T) in breast cancer (BRCA) and its role and mechanism in the prognosis of BRCA patients. Methods The Tumor Genome Atlas (TCGA) database was used to analyze UBE2T expression in BRCA tissues, and the effects of UBE2T expression on disease-free survival (DFS) and overall survival (OS) were analyzed by Kaplan-Meier (KM) survival curve. In vitro, real-time quantitative PCR and Western blot were used to confirm the knock-down and overexpression efficiency, to analyze its effect on tumor cell biological behavior. The effect of UBE2T on cell epithelial–mesenchymal transition (EMT) was studied by Western blot. A xenograft tumor model was established to verify the effect of UBE2T knockdown on the growth of BRCA cells in vivo. Results The UBE2T expression levels in BRCA and adjacent tissues were statistically different (P<0.001), and the expression was increased in tissues with distant metastasis or late stage (all P<0.05). The DFS and OS were decreased in the UBE2T high-level group (both P<0.05). UBE2T was highly expressed in MCF-7 and MDA-MB-231 cells and lowly expressed in MDA-MB-361 cells (all P<0.01). After UBE2T was silenced by shRNA, the proliferation ability of tumor cells significantly decreased, whereas it increased after UBE2T up-expression (all P<0.05). The apoptotic rates of MCF-7 and MDA-MB-231 cells in the silent groups were significantly higher than those in the shNC groups, while the apoptotic rates of MAD-MB-361 cells in the overexpression group decreased (all P<0.001). The mobility in the knockdown groups were lower than in the shNC groups, while the mobility in the overexpression group significantly increased (both P<0.01). The migration and invasion cells in the shUBE2T groups were lower than those in the shNC groups, and the migration and invasion cells in the UBE2T group were higher than those in the vector group (all P<0.01). Downregulation of UBE2T decreased the expression levels of N-cadherin, Snail, and Vimentin (all P<0.05) and increased that of E-cadherin; however, the result of UBE2T upregulation was opposite (all P<0.01). TIMER results showed that UBE2T was positively correlated with E-cadherin (P<0.001), N-cadherin (P=0.013), and Snail (P<0.001) and negatively correlated with Vimentin (P<0.001). In vivo experiments showed that downregulation of UBE2T slowed down the growth of transplanted tumors. Conclusion UBE2T is highly expressed in BRCA tissues and may affect the prognosis. UBE2T can promote the proliferation of BRCA cells, inhibit apoptosis, and increase the migration and invasion abilities by changing the expression levels of EMT-related proteins.

Abstract： ObjectiveThe study aims to explore the effects and regulatory roles of glucose transporter 1 （GLUT1） on the proliferation， migration， adhesion， and angiogenesis of human umbilical vein endothelial cells （HUVECs） under ischemia-hypoxic conditions. MethodsIn vitro experiments were conducted to subject HUVECs to an ischemia-hypoxic-mimicking environment （1% O₂， 5% CO₂， 94% N₂）. The biological characteristics of HUVECs under normoxic and ischemia-hypoxic conditions were compared by assessing cell viability， proliferation capacity， and examining the expression changes of GLUT1， HIF-1α， and VEGFA proteins under ischemia-hypoxia using Western blot technology. Further， GLUT1 was overexpressed using plasmid transfection and the proliferation， migration， adhesion， and angiogenic capabilities of HUVECs were evaluated through scratch assays， cell adhesion assays， and tube formation assays. Mitochondrial morphological changes were observed by transmission electron microscopy，and oxygen consumption rate （OCR） was detected by Seahorse metabolic analyzer to evaluate mitochondrial function. ResultsCompared with normoxic conditions， the ischemia-hypoxic environment significantly inhibited the proliferation， cell viability， migration， and adhesion capabilities of HUVECs and impaired their angiogenic potential. The expression levels of GLUT1， HIF-1α and VEGFA proteins were also markedly reduced. However， when GLUT1 expression was upregulated， the migration， adhesion， and angiogenic capabilities of HUVECs were significantly improved， and the protein expression levels of HIF-1α， VEGFA and VEGFR were increased. Transmission electron microscopy revealed that ischemic-hypoxia leads to mitochondrial swelling and matrix damage， while GLUT1 overexpression significantly alleviates mitochondrial morphology abnormalities. OCR results suggest that GLUT1 overexpression may enhance oxidative phosphorylation of endothelial cells in ischemic-hypoxic environments to improve energy metabolism. These results suggest that GLUT1 may influence the function and angiogenic potential of HUVECs by regulating glucose metabolism and energy supply. ConclusionsThis study reveals the significant regulatory role of GLUT1 in the function of HUVECs under ischemia-hypoxic conditions， potentially through modulating cellular energy metabolism and signal transduction pathways， thereby affecting cell proliferation， migration， adhesion， and angiogenesis. These findings provide a new perspective on the role of GLUT1 in cardiovascular diseases and may offer potential targets for the development of new therapeutic strategies.

BACKGROUND: Peripheral helper T (T PH ) cells are uniquely positioned within pathologically inflamed non-lymphoid tissues to stimulate B-cell responses and antibody production. However, the phenotype, function, and clinical relevance of T PH cells in hepatocellular carcinoma (HCC) are currently unknown. METHODS: Blood, tumor, and peritumoral liver tissue samples from 39 HCC patients (Sep 2016-Aug 2017) and 101 HCC patients (Sep 2011-Dec 2012) at the Third Affiliated Hospital of Sun Yat-sen University were used. Flow cytometry was used to quantify the expression, phenotype, and function of T PH cells. Log-rank tests were performed to evaluate disease-free survival and overall survival in samples from 39 patients and 101 patients with HCC. T PH cells, CD19 + B cells, and T follicular helper (T FH ) cells were cultured separately in vitro or isolated from C57/B6L mice in vivo for functional assays. RESULTS: T PH cells highly infiltrated tumor tissues, which was correlated with tumor size, early recurrence, and shorter survival time. The tumor-infiltrated T PH cells showed a unique ICOS hi CXCL13 + IL-21 - MAF + BCL-6 - phenotype and triggered naïve B-cell differentiation into regulatory B cells. Triggering programmed cell death protein 1 (PD-1) induced the production of C-X-C motif chemokine ligand 13 (CXCL13) by T PH cells, which then suppressed tumor-specific immunity and promoted disease progression. CONCLUSION Our study reveals a novel regulatory mechanism of T PH cell-regulatory B-cell-mediated immunosuppression and provides an important perspective for determining the balance between the differentiation of protumorigenic T PH cells and that of antitumorigenic T FH cells in the HCC microenvironment.
Carcinoma, Hepatocellular/metabolism* ; Liver Neoplasms/metabolism* ; Humans ; T-Lymphocytes, Helper-Inducer/metabolism* ; Animals ; Mice ; Male ; Female ; Mice, Inbred C57BL ; Middle Aged ; B-Lymphocytes, Regulatory/metabolism* ; Flow Cytometry ; Interleukin-21 ; Aged ; Chemokine CXCL13/metabolism*