Abstract: Objective To investigate the effect and underlying mechanism of Shizao decoction ( SZD) on inducing apoptosis in breast cancer transplants in nude mice. Methods SKBR3 cells were cultured and transplanted into nude mice to construct tumor models. SZD was applied by continuous intragastric administration for 4 weeks and its effects on tumor growth were observed. After administration, the mice were killed and the transplanted tumor tissues were separated. Lactate dehydrogenase ( LDH) levels in tumor tissues were detected using an LDH kit. Morphological changes in transplanted tumor tissues were observed by Hoechst33342-propidium iodide staining,reactive oxygen species ( ROS ) were evaluated by 2 ’, 7 ’-dichlorodihydrofluorescein diacetate staining, and mitochondrial membrane potential was evaluated by JC-1 assay. Caspase3, Caspase8, and Caspase9 activities in the transplanted tumor cells were detected by enzyme-linked immunosorbent assay. Fas and Fas ligand ( FasL) protein expression were detected by Western blot. Caspase3, Caspase8, Caspase9, Fas, and FasL gene expression were detected by quantitative polymerase chain reaction. Results Compared with the model group, SZD significantly increased LDH production (P<0. 05, P<0. 01) by transplanted breast cancer cells in nude mice, induced apoptosis,increased the ROS content (P<0. 05) and mitochondrial depolarization ( P<0. 05, P<0. 01), increased the gene expression and enzyme activities of Caspase3, Caspase8, and Caspase9 (P<0. 05, P<0. 01), and increased the gene /protein expression of Fas and FasL (P<0. 05, P<0. 01). Conclusions SZD can induce apoptosis in transplanted breast tumors, possibly via the apoptotic caspase and Fas/ FasL pathways tumor.

Abstract: Objective To investigate the molecular mechanism by which long-chain non-coding RNA(lncRNA) X-inactive specific transcript ( XIST) regulates the biological activity of trophoblast cells. Methods Human HTR-8/ Svneo trophoblast cells were cultured in vitro and separated into control, interference empty (transfected with small interfering RNA-normal control (si-NC)), si-XIST-1 (transfected with si-XIST-1), si-XIST-1+anti-NC (co-transfected with si-XIST-1 and anti-NC), and si-XIST-1+anti-182-5p (co-transfected with si-XIST-1 and anti-182-5p) groups. XIST, miR-182-5p, and hypoxia inducible factor-2α (HIF-2α) mRNA expression levels were measured by real-time fluorescence quantitative polymerase chain reaction (RT-qPCR). Cell proliferation was detected by Cell Counting Kit-8 (CCK-8) and 5-ethynyl-2’-deoxyuridine (EdU) assays. Apoptosis was detected by flow cytometry. Cell migration and invasion abilities were measured by scratch-healing and Transwell experiments. HIF-2α, cleaved-caspase-3, Bcl-2, Bax, matrix metalloproteinase (MMP)-2, and MMP-9 protein levels in cells were measured by Western blot. The targeting relationship of miR-182-5p with XIST and HIF-2α was verified by dualluciferase reporter assay. Results XIST and HIF-2α mRNA levels, apoptosis rate, and HIF-2α, cleaved-caspase-3,and Bax protein levels in the si-XIST-1 group were lower than those in the control and interference empty groups,while miR-182-5p, cell viability, EdU-positive rate, scratch-healing rate, number of invaded cells, and Bcl-2, MMP-2, and MMP-9 protein levels were higher (P<0. 05). HIF-2α mRNA level, apoptosis rate, and HIF-2α, cleavedcaspase-3, and Bax protein levels in the si-XIST-1+anti-182-5p group were higher than those in the si-XIST-1 and siXIST-1+ anti-NC groups, while miR-182-5p, cell viability, EdU-positive rate, scratch-healing rate, number of invaded cells, and Bcl-2, MMP-2, and MMP-9 protein levels were lower (P<0. 05). LncRNA XIST was able to target miR-182-5p in HTR-8/ Svneo cells, and HIF-2α was the target of miR-182-5p. Conclusions Knocking-down lncRNA XIST may inhibit HIF-2α expression by competitively binding to miR-182-5p, thereby enhancing the proliferation, migration, and invasion abilities of HTR-8/ Svneo cells and inhibiting cell apoptosis.

Abstract: Objective To investigate the mechanism of remifentanil (Rem) on myocardial fibrosis in rats with dilated cardiomyopathy ( DCM) in relation to the interleukin-6 ( IL-6) / signal transducer and activator of transcription 3 (STAT3) / glutathione peroxidase 4 (GPX4) pathway. Methods A DCM rat model was established by intraperitoneal injection of 2. 5 mg / kg doxorubicin. The rats were then divided into Model, low-dose (Rem-L, 2μg / kg), medium-dose (Rem-M, 4 μg / kg), and high-dose Rem groups (Rem-H, 8 μg / kg), and a high-dose Rem+Colivelin (STAT3 activator, Rem-H+Colivelin, 8 μg / kg Rem+1 mg / kg Colivelin) group (n = 12 rats per group).Another 12 normal Wistar rats were set as a Control group. After continuous administration for 4 weeks, left ventricular end systolic diameter (LVESD), left ventricular ejection fraction (LVEF), left ventricular end diastolic diameter ( LVEDD), and left ventricular fractional shortening ( LVFS) were measured by ultrasound. Type Ⅰ collagen ( Col Ⅰ), transforming growth factor-β1 ( TGF-β1), and type Ⅲ collagen ( Col Ⅲ) expression in myocardial tissue were detected by immunohistochemistry. Pathological changes in myocardial tissue were detected by Masson and hematoxylin / eosin staining. IL-6/ STAT3 / GPX4 pathway-related proteins were detected by Western blot. Results Compared with the Control group, rats in the Model group showed obvious increases in LVEDD, LVESD,ColⅠ((123. 62±10. 78) vs (76. 53±6. 12)), ColⅢ, TGF-β1, IL-6, and phospho-STAT3^Tyr705/ STAT3((0. 95±0.05) vs (0. 46±0. 92)) in myocardial tissue, and decreases in LVEF(( 52. 19± 4. 88)% vs ( 76. 78± 6. 97)%),LVFS, SLC7A11, and GPX4 ((0. 11± 0. 01) vs ( 0. 43± 0. 04)) ( P<0. 05), inflammatory infiltration, collagen accumulation, and myocardial fibrosis. Compared with the Model group, rats in the Rem-M and Rem-H groups showed reduced LVEDD, LVESD, ColⅠ((98. 74±7. 28)、(84. 27±7. 13) vs (123. 62±10. 78)), ColⅢ, TGF-β1,IL-6, and p-STAT3 ^Tyr705/ STAT3((0. 81±0. 06)、(0. 57±0. 04) vs (0. 95±0. 05)) in myocardial tissue, increases in LVEF((69. 85±6. 13)%、(72. 83±6. 55)% vs(52. 19±4. 88)%), LVFS, SLC7A11, and GPX4((0. 24±0. 02)、(0. 36±0. 02) vs (0. 11± 0. 01)) (P<0. 05), and improvements in myocarditis injury and fibrosis. Colivelin was able to reverse the improving effect of Rem on myocardial fibrosis in DCM rats. Conclusions Rem may reduce collagen accumulation and improve myocardial fibrosis and cardiac function in DCM rats by adjusting the IL-6/STAT3 / GPX4 pathway.

Abstract: Objective To observe colon tumor status in mice with azoxymethane / dextran sodium sulfate (AOM/ DSS)-induced colitis-associated colorectal cancer (CAC) following autologous fecal microbiota transplantation (AFMT), and to investigate the anti-tumor effects and underlying mechanism of AFMT through its influence on tight junction proteins. Methods Female specific-pathogen-free BALB/ c mice were divided randomly into blank control,CAC model, and AFMT intervention groups ( n= 8 per group). CAC model mice received a single intraperitoneal injection of AOM (10 mg / kg), followed by drinking water containing 3. 5% DSS for 7 days, followed by 0% DSS for 14 days, constituting one cycle; three cycles were performed to establish the CAC model. Mice in the AFMT group received daily AFMT ( 0. 1 mL autologous fecal suspension) via gavage every other day concurrent with the CAC modeling protocol until cycle completion. Control mice received no special treatment. The general condition and body weight of the mice were monitored. Upon completion of the experiment, colon length was measured, tumor numbers were recorded, and colon pathology was examined. mRNA and protein expression levels of the tight junction proteins Occludin ( OCLN) and Claudin-1 ( CLDN1 ) in colorectal tumor tissue were detected by reverse transcriptionpolymerase chain reaction and Western blot, respectively. The fecal microbiota composition in each group was analyzed using 16S rRNA gene sequencing. Results Compared with the blank control group, the CAC model group showed significantly shortened colon length ( P<0. 0001) and a marked increase in tumor number. After AFMT intervention, the shortening of colon length was alleviated (P<0. 01), and the number of tumors was reduced (P<0. 05). Histological examination of mouse colon tissues revealed distorted crypt structures, reduced goblet cells, and increased inflammatory cell infiltration in the CAC model group ( P<0. 01). After AFMT intervention, the crypt structure and goblet cells improved, and inflammatory cells decreased (P<0. 05). Compared with the blank control group, the protein expression levels of Occludin and Claudin-1 in colorectal tumor tissues of the CAC model group were significantly decreased (P<0. 05, P<0. 01). After AFMT intervention, the protein expression levels of Occludin and Claudin-1 in colorectal tumor tissues significantly increased (P<0. 05). As compared to the blank control group,the mRNA expression levels of OCLN and CLDN1 in the CAC model group were significantly downregulated ( P<0. 05); after AFMT intervention, their mRNA expression levels significantly recovered (P<0. 05). Compared with the blank control group, there was no statistically significant difference in the relative abundance of Firmicutes and Bacteroidota in the AFMT intervention group ( P<0. 05). In the CAC model group, the relative abundance of Firmicutes decreased (P<0. 05), while the relative abundance of Bacteroidota increased (P<0. 05). Conclusions AFMT intervention can restore the relative abundance of Firmicutes and reduce the relative abundance of Bacteroroidota in the gut, effectively improve the aberrant expression of tight junction proteins within the tumor microenvironment, repair the intestinal barrier, regulate intestinal barrier function, alleviate intestinal inflammation,and consequently mitigate the pathological progression of CAC.

Abstract: Objective To explore the mechanism by which melanotan-II (MT-II) improves social deficits in a Shank3 gene-deficient autism model. Methods Rats were divided into control and Shank3-deficient model groups (n= 18 per group) treated by microinjection of empty or Shank3-interfering lentivirus, respectively, into the right lateral ventricle of neonatal rats. Shank3-deficient rats were further divided randomly into two groups: Shank3+saline (Sh3-Sal) and Shank3+ MT-II ( Sh3-MT-II) groups ( n= 9 per group). Similarly, control rats were divided into control+saline (V-Sal) and control+MT-II (V-MT-II) groups (n= 9 per group). On day 28, rats in the V-MT-II and Sh3-MT-II groups received intraperitoneal (i. p. ) injections of MT-II (3. 3 mg / kg), while rats in the V-Sal and Sh3-Sal groups received i. p. saline (3. 3 mL / kg). Behavioral changes were assessed using the open-field test, grooming behavior analysis, three-chamber social test, and the Morris water maze test. mRNA and protein expression levels of hypothalamic oxytocin (OXT), OXT receptor (OXTR), and melanocortin receptor 4 ( MC4R) were detected by reverse transcription-polymerase chain reaction and Western blot, respectively. Results In the three-chamber social test, Sh3-Sal group rats showed no significant social preference compared with the time spent with stranger rat 1 (P>0. 05). In contrast, after MT-II intervention, Sh3-MT-II group rats spent significantly longer with stranger rat 2 (P<0. 01). In the Morris water maze test, rats in the Sh3-Sal group exhibited significant learning and memory impairments compared with the V-Sal group (P<0. 05), while MT-II intervention significantly improved the learning and memory performance of the Sh3-MT-II group (P<0. 01). The open field and grooming tests revealed that Sh3-Sal group rats spent significantly longer in the peripheral zone of the open field and exhibited increased grooming behavior compared with the V-Sal group (P<0. 01). However, MT-II did not significantly alter the center time or self-grooming behavior compared with the Sh3-Sal group (P>0. 05). mRNA expression levels of OXT, OXTR, and MC4R were significantly higher in the Sh3-MT-II group than in the Sh3-Sal group (P<0. 05,P<0. 01). Hypothalamic OXT protein expression was significantly increased in the Sh3-MT-II group compared with the Sh3-Sal group (P<0. 05), while hypothalamic SHANK3 protein expression was significantly decreased in both the Sh3-Sal and Sh3-MT-II groups compared with the V-Sal group (P<0. 05,P<0. 01), and protein expression levels of OXTR and MC4R showed no significant changes (P>0. 05). Conclusions The melanocortin receptor agonist MT-II may ameliorate social deficits in Shank3-deficient autistic rats by activating the hypothalamic OXT system. This suggests that targeting the OXT / MC4R pathway could be a potential therapeutic strategy for social deficits in patients with autism spectrum disorder.

Abstract: Objective Lung adenocarcinoma (LUAD) has a poor prognosis. This study aimed to screen core genes associated with the tumor microenvironment ( TME) and programmed cell death ( PCD) to provide new prognostic markers and therapeutic targets for LUAD, and to validate their cross-species conservation. Methods Based on RNA-seq data for LUAD and normal lung tissue in The Cancer Genome Atlas ( TCGA) database, we assessed the TME and screened for differentially expressed genes (DEGs) using the ESTIMATE algorithm. Functional enrichment analysis (Gene Ontology / Kyoto Encyclopedia of Genes and Genomes), protein-protein interaction (PPI) networks, and univariate Cox regression analysis were performed on the DEGs. Cross-screening combining PPI networks, univariate Cox regression, and the PCD genome was employed to identify core prognostic genes, and their prognostic value and TME association were validated by survival analysis, gene set enrichment analysis (GSEA), and immune infiltration (CIBERSORT) analysis. Results High immune / ESTIMATE scores were significantly associated with prolonged patient survival. The selected shared DEGs were primarily enriched in immune-related pathways. Cross-analysis identified CD19 and CD79A as core prognostic genes associated with PCD. Clinical-feature analysis showed that CD19 and CD79A expression were significantly higher in LUAD tumors than in normal lung tissues, but their expression levels decreased significantly with advancing TNM staging, closely associated with advanced staging, distant metastasis, and poor prognosis. Patients with high CD19 / CD79A expression had significantly longer overall survival than those with low expression. Animal-model validation confirmed the cross-species conserved role of CD19 /CD79A in tumor progression. GSEA indicated that the high-CD19 / CD79Aexpression group was significantly enriched in immune activation pathways (e. g. , allograft rejection, complement response), while the low-expression group was enriched in metabolic ( glycolysis, oxidative phosphorylation ) and oncogenic pathways. CIBERSORT analysis confirmed that their expression levels were significantly positively correlated with TME immune activity. Conclusions CD19 and CD79A are overexpressed in LUAD tumor tissues, but their expression levels decrease with disease progression. High expression of CD19 / CD79A is closely associated with a favorable prognosis and immune-activated TME, while low expression suggests a poor prognosis and immune suppression / oncogenic states. As genes associated with PCD, CD19 / CD79A may serve as potential protective prognostic biomarkers and immunotherapeutic targets in LUAD, thereby providing a basis for understanding the immune mechanisms of LUAD and guiding the development of B cell-targeted therapeutic strategies.

Abstract: Objective To investigate the effect of free Heme on sepsis-induced immunosuppression model mice based on regulation of macrophage PANoptosis by NLR family pyrin domain containing 12 (NLRP12). Methods A sepsis mouse model was constructed and the mice were divided into Model, Heme, Heme+empty plasmid (Heme+si-NC), and Heme+NLRP12 low-expression plasmid groups (Heme+si-NLRP12) (n= 12 mice per group). Another 12 mice with exposed cecum without ligation or puncture were considered as the sham surgery group (Sham). Serum levels of inflammatory factors were detected by enzyme-linked immunosorbent assay (ELISA). The CD4⁺/ CD8⁺ratio in the blood was detected by flow cytometry, pathological changes in lung tissue were detected by hematoxylin / eosin staining, apoptosis was detected by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) staining, and expression levels of PANoptosis-related proteins in lung tissue were detected by Western blot. To clarify the mechanism by which heme regulates NLRP12, sepsis cell models were induced using lipopolysaccharide (LPS)- infected mouse bone marrow-derived macrophages (BMDM). Model mice were divided into LPS, LPS+Heme, LPS+Heme+si-NC group, and LPS+Heme+si-NLRP12 groups, with mice administered normally cultivated BMDMs as the Control group. Levels of inflammatory factors in the BMDM supernatant were detected by ELISA, cell viability was detected by Cell Counting Kit-8 assay, apoptosis rate was detected by TUNEL staining, and expression levels of PANoptosis-related proteins were detected by Western blot. Results Compared with the Model group, rats in the heme group showed increased serum levels of inflammatory factors, aggravated lung tissue injury and cell apoptosis, and progression of cell PANoptosis, while the CD4⁺/ CD8⁺ratio was reduced (P<0. 05). Compared with the Heme+si-NC group, pathological lung tissue damage and the process of PANoptosis were alleviated in the Heme+si-NLRP12 group, serum levels of inflammatory factors, and the apoptosis rate of lung tissue cells were decreased, while theCD4⁺/ CD8⁺ratio was increased ( P<0. 05). Cell experiments showed that the cell survival rate was significantly decreased in the LPS group, while the process of cell PANoptosis and levels of inflammatory factors in the supernatant were increased (P<0. 05). Compared with the LPS group, the apoptosis rate, process of PANoptosis, and levels of inflammatory factors were further increased in the LPS+Heme group (P<0. 05). Compared to the LPS+Heme+si-NC group, the LPS + Heme + si-NLRP12 group exhibited significantly increased cell viability, reduced apoptosis and supernatant inflammatory factor levels, along with decreased expression of NLRP12, Bax, Caspase-8, p-RIPK3, and GSDMD-N(P<0. 05). Conclusions Heme may induce macrophage PANoptosis by upregulating NLRP12, thereby enhancing immune suppression in sepsis.

Abstract:Animal experiment courses are a crucial practical component in the life sciences field. Traditional teaching, however, often overemphasizes theoretical instruction, leading to insufficient skill development, poor ethical and safety awareness, detachment from real-world applications, and a lack of innovation training. To address these challenges, this study introduced an outcome-based education (OBE) concept, constructing a student-ability-centered animal experimentation curriculum system. Three dimensional Objective were established based on this OBE:mastering foundational knowledge, proficiency in experimental skills, and enhancing comprehensive qualities. A closed-loop framework of “content conception-program design-process implementation-experience summarization” was adopted. Using authentic research projects as the vehicle, theoretical knowledge, practical skills, and research capability cultivation were integrated through project database construction, process-oriented evaluation, and teamcollaboration models. Practice demonstrated that the OBE model significantly enhanced students’ knowledge mastery,skill proficiency, and teaching satisfaction, effectively fostering their team collaboration, problem-solving, and innovative thinking abilities. This study shows that project-driven teaching under the OBE concept can resolve the structural dilemmas of traditional experimental teaching, thus providing an effective pathway for enhancing the quality of animal experimentation courses and cultivating high-caliber scientific research talent.

Abstract:Post-translational modifications ( PTMs) are crucial for regulating protein functions. As a novel PTM, lactylation has emerged as a recent research hotspot, participating in pathological processes such as tumor progression and inflammation; it dynamically regulates gene expression and protein activity, which is significant for metabolic-immune coordination. In degenerative osteoarthropathy ( such as osteoarthritis and rheumatoid arthritis),cartilage metabolic imbalance is closely associated with dynamic changes in lactylation, involving pathological links such as cartilage matrix degradation, synovial inflammation, and abnormal bone remodeling. Current clinical interventions cannot block disease progression. This review systematically summarizes the molecular mechanisms of lactylation (including lactate metabolism as well as histone and non-histone lactylation), regulatory factors (enzymes,environment, and molecular tools), and its role in degenerative osteoarthropathy, covering expression changes in cartilage tissues, correlation with arthritis progression, and biomarker studies in clinical models. Lactylation affects joint degeneration by regulating glycolysis-oxidative phosphorylation balance, inflammatory factor expression, and extracellular matrix metabolism. Targeted regulation strategies ( such as CRISPR editing and small-molecule inhibitors) hold promise as novel therapeutic approaches. Future research needs to overcome technical limitations in detection, decipher the dynamic lactylation network through interdisciplinary collaboration, and promote its clinical translation in early diagnosis, precise intervention, and personalized treatment.

Abstract:The blood-brain barrier (BBB) is a dynamic interface for selective molecular trafficking between blood and brain parenchyma, playing a pivotal role in maintaining central nervous system homeostasis. Recent investigations have uncovered intricate regulatory networks governing BBB function, involving dynamic intercellular tight junction remodeling, signaling cascade activation, and multicomponent interactions within the neurovascular unit. This review systematically integrates current mechanistic understanding of BBB regulation and provides a critical evaluation of emerging interventional strategies, including nanotechnology-based drug delivery systems, gene editing,and traditional Chinese medicine formula interventions, with emphasis on their translational potential in the future. By integrating bench-to-bedside perspectives, this work aims to provide novel theoretical frameworks for developing precision therapies targeting BBB dysfunction in neurological disorders.

Abstract:Inflammatory pain and depression are clinically common disorders with a high comorbidity rate.They mutually exacerbate their pathological processes and severely reduce the quality of life of patients. The pathogenesis of the comorbidity of inflammatory pain and depression is highly complex, posing significant challenges for clinical diagnosis and treatment. Recent studies have revealed that sustained activation of neuroinflammation is a central pathological link in the comorbidity of inflammatory pain and depression. As a non-pharmacological intervention, exercise can effectively improve both pain and depressive symptoms in patients with comorbidity.However, its specific mechanisms of action remain incompletely elucidated. This article summarizes recent research advances on the effects of exercise in inhibiting central inflammation, modulating the balance of microglial M1 / M2 polarization, regulating neuronal excitatory-inhibitory homeostasis, and promoting hippocampal neurogenesis. It systematically reviews the molecular and neural mechanisms by which exercise modulates neuroinflammation to treat the comorbidity of inflammatory pain and depression, with the aim of providing a theoretical basis and practical guidance for clinical exercise therapy.

Abstract:The blood-brain barrier ( BBB) plays a critical role in maintaining brain homeostasis. BBB dysfunction is closely associated with various neurodegenerative diseases and acute brain injuries, which impair brain defense mechanisms and can exacerbate the progression of pathological conditions. Nitric oxide (NO), an important biological signaling molecule, exerts a wide range of effects on the central nervous system, influencing both physiological and pathological states. This review summarizes the production and metabolism of NO and their effects on BBB permeability, followed by an introduction to the role of NO in pathological conditions such as cerebral ischemiareperfusion injury, neurodegenerative diseases, and brain tumors. NO regulates BBB integrity through the regulation of tight junction proteins and vascular endothelial growth factor receptor 2 (VEGFR2). The review also delves into the potential of NO as a therapeutic target in drug delivery and gene therapy, and explores its potential to enhance drug penetration across the BBB. These explorations provide novel perspectives for future therapeutic strategies.

Abstract:We conducted a systematic review of the construction method and pathological mechanisms of animal and cellular models for diminished ovarian reserve (DOR), to provide a theoretical basis for understanding its pathophysiological mechanisms and references for future drug development and therapeutic strategies. We carried out extensive retrieval and analysis of the recent literature to identify the detailed diverse modeling approaches (including pharmaceutical induction, environmental factors, natural factors, and composite factors) and explored the roles of their mechanisms in DOR pathogenesis. The pathogenesis of DOR involves multiple factors, including oxidative stress, apoptosis and autophagy dysregulation, impaired angiogenesis, and imbalances in follicular development and the immune microenvironment. Studies also revealed intrinsic correlations among key signaling pathways in DOR models. Although existing DOR modeling method can effectively replicate Western medicine pathological features,models incorporating traditional Chinese medicine (TCM) syndrome characteristics remain scarce. Given the unique advantages of TCM in regulating the complex pathological network of DOR, future research is needed to prioritize the construction of TCM syndrome-oriented DOR models. This will support investigations into the scientific basis of TCM’s multi-target regulatory mechanisms and provide a robust theoretical foundation for clinical applications.