ObjectivePancreatic ductal adenocarcinoma (PDAC) exhibits a limited response to current treatments due to its dense fibrotic stroma and highly immunosuppressive tumor microenvironment. In recent years, advancements in cellular immunotherapy, particularly chimeric antigen receptor macrophage (CAR-M) therapy, have offered new hope for pancreatic cancer treatment. Although CAR-M therapy demonstrates dual potential in directly killing tumor cells and remodeling the immune microenvironment, it still faces challenges such as complex in vitro preparation processes and low in vivo targeting and delivery efficiency. Therefore, developing strategies for efficient and targeted in vivo delivery of CAR genes has become crucial for overcoming current therapeutic limitations. This study aims to develop an orally administrable nano-gene delivery system for the targeted delivery of CAR genes to pancreatic tumor sites. MethodsCore nano-gene particles (PNP/pCAR) were constructed by loading plasmid DNA encoding CAR (pCAR) with cationic polypeptides (PNP). Subsequently, PNP/pCAR was surface-modified with β-glucan to prepare the targeted nanoparticles (βGlus-PNP/pCAR). The loading efficiency of PNP for pCAR was quantitatively assessed by gel retardation assay. The particle size, Zeta potential, morphology, and storage stability of PNP/pCAR were characterized using a Malvern particle size analyzer and transmission electron microscopy. At the cellular level, RAW 264.7 macrophages were selected. The cytotoxicity of PNP/pCAR was evaluated using the CCK-8 assay. The cellular uptake efficiency and lysosomal escape ability of the nanoparticles were assessed via flow cytometry and confocal microscopy. Transfection efficiency was quantitatively evaluated by detecting the expression of the reporter gene GFP using flow cytometry. At the in vivo level, an orthotopic pancreatic cancer mouse model was established. Cy7-labeled βGlus-PNP/pCAR nanoparticles were administered orally, and the fluorescence distribution in mice was dynamically monitored at 1, 2, 4, 8, and 16 h post-administration using a small animal in vivo imaging system. Forty-eight hours after oral gavage, the mice were euthanized, and pancreatic tumor tissues were collected for further analysis of intratumoral fluorescence signals using the imaging system. Additionally, βGlus-PNP/pCAR-GFP nanoparticles loaded with the reporter gene (GFP) were administered orally. Forty-eight hours post-administration, pancreatic tumor tissues were harvested to prepare frozen sections, and GFP expression was observed and analyzed under a fluorescence microscope. ResultsThe PNP carrier exhibited a high loading capacity for pCAR. The successfully prepared PNP/pCAR nanoparticles were regular spheres with a hydrodynamic diameter of approximately (120±10) nm and a Zeta potential of about +(6±1) mV. They maintained good structural stability after incubation in PBS buffer for 7 d. Cell experiments demonstrated that PNP/pCAR exhibited no significant cytotoxicity in RAW 264.7 cells while being efficiently internalized and effectively escaping lysosomal degradation. The transfection positive rate of PNP/pCAR-GFP in RAW 264.7 cells reached (25±3)%, surpassing that of Lipofectamine 2000-loaded pCAR-GFP (Lipo/pCAR-GFP), which was (20±1)%.In vivo experiments revealed that, compared to unmodified PNP/pCAR, βGlus-PNP/pCAR exhibited strongerin situ pancreatic tumor targeting ability after oral administration. Furthermore, oral administration of βGlus-PNP/pCAR-GFP resulted in significant GFP protein expression detectable within pancreatic tumor tissues. ConclusionThis study successfully constructed and validated an orally administrable, pancreatic cancer-targeting polypeptide-based nano-gene delivery system. It provides an important technological foundation in delivery systems and experimental basis for the subsequent development of in situ CAR-M-based therapeutic strategies for pancreatic cancer.

ObjectivePancreatic ductal adenocarcinoma (PDAC) exhibits a limited response to current treatments due to its dense fibrotic stroma and highly immunosuppressive tumor microenvironment. In recent years, advancements in cellular immunotherapy, particularly chimeric antigen receptor macrophage (CAR-M) therapy, have offered new hope for pancreatic cancer treatment. Although CAR-M therapy demonstrates dual potential in directly killing tumor cells and remodeling the immune microenvironment, it still faces challenges such as complex in vitro preparation processes and low in vivo targeting and delivery efficiency. Therefore, developing strategies for efficient and targeted in vivo delivery of CAR genes has become crucial for overcoming current therapeutic limitations. This study aims to develop an orally administrable nano-gene delivery system for the targeted delivery of CAR genes to pancreatic tumor sites. MethodsCore nano-gene particles (PNP/pCAR) were constructed by loading plasmid DNA encoding CAR (pCAR) with cationic polypeptides (PNP). Subsequently, PNP/pCAR was surface-modified with β-glucan to prepare the targeted nanoparticles (βGlus-PNP/pCAR). The loading efficiency of PNP for pCAR was quantitatively assessed by gel retardation assay. The particle size, Zeta potential, morphology, and storage stability of PNP/pCAR were characterized using a Malvern particle size analyzer and transmission electron microscopy. At the cellular level, RAW 264.7 macrophages were selected. The cytotoxicity of PNP/pCAR was evaluated using the CCK-8 assay. The cellular uptake efficiency and lysosomal escape ability of the nanoparticles were assessed via flow cytometry and confocal microscopy. Transfection efficiency was quantitatively evaluated by detecting the expression of the reporter gene GFP using flow cytometry. At the in vivo level, an orthotopic pancreatic cancer mouse model was established. Cy7-labeled βGlus-PNP/pCAR nanoparticles were administered orally, and the fluorescence distribution in mice was dynamically monitored at 1, 2, 4, 8, and 16 h post-administration using a small animal in vivo imaging system. Forty-eight hours after oral gavage, the mice were euthanized, and pancreatic tumor tissues were collected for further analysis of intratumoral fluorescence signals using the imaging system. Additionally, βGlus-PNP/pCAR-GFP nanoparticles loaded with the reporter gene (GFP) were administered orally. Forty-eight hours post-administration, pancreatic tumor tissues were harvested to prepare frozen sections, and GFP expression was observed and analyzed under a fluorescence microscope. ResultsThe PNP carrier exhibited a high loading capacity for pCAR. The successfully prepared PNP/pCAR nanoparticles were regular spheres with a hydrodynamic diameter of approximately (120±10) nm and a Zeta potential of about +(6±1) mV. They maintained good structural stability after incubation in PBS buffer for 7 d. Cell experiments demonstrated that PNP/pCAR exhibited no significant cytotoxicity in RAW 264.7 cells while being efficiently internalized and effectively escaping lysosomal degradation. The transfection positive rate of PNP/pCAR-GFP in RAW 264.7 cells reached (25±3)%, surpassing that of Lipofectamine 2000-loaded pCAR-GFP (Lipo/pCAR-GFP), which was (20±1)%.In vivo experiments revealed that, compared to unmodified PNP/pCAR, βGlus-PNP/pCAR exhibited strongerin situ pancreatic tumor targeting ability after oral administration. Furthermore, oral administration of βGlus-PNP/pCAR-GFP resulted in significant GFP protein expression detectable within pancreatic tumor tissues. ConclusionThis study successfully constructed and validated an orally administrable, pancreatic cancer-targeting polypeptide-based nano-gene delivery system. It provides an important technological foundation in delivery systems and experimental basis for the subsequent development of in situ CAR-M-based therapeutic strategies for pancreatic cancer.

Background: Surgeons face challenges in selecting cost-effective and biologically active agents for rotator cuff healing, given the numerous commercial products available, such as polydeoxyribonucleotide (PDRN) and atelocollagen (ATC). However, the precise efficacy of PDRN and ATC in rotator cuff healing remains debatable, and there is currently a lack of studies directly comparing the effects of the 2 agents on repaired cuff tendons. Therefore, the purpose of this study was to compare the efficacy of PDRN and ATC on bone-to-tendon interface (BTI) healing using a chronic rotator cuff tear (RCT) model in rabbits. Methods: Forty-eight rabbits were randomly divided into 3 groups. To create chronic RCT models, transected tendons were left untreated for 6 weeks, and then were repaired in a transosseous manner with PDRN and ATC injection into the repair site according to group allocation (group A: saline, group B: PDRN, group C: ATC; n = 16 per group). Genetic and immunofluorescence analyses were performed at 4 weeks after surgery. Furthermore, genetic, histologic, and biomechanical analyses were performed at 12 weeks after surgery. Results: At 4 weeks after surgery, ATC-injected shoulders showed the highest mRNA expression levels of collagen type I alpha 1 and aggrecan compared to the other 2 groups (p < 0.001 and p = 0.002, respectively). Meanwhile, there was more preliminary fibrocartilaginous matrix formation in the ATC-injected group. At 12 weeks after surgery, ATC-injected shoulders demonstrated better collagen fiber continuity and orientation, denser collagen fibers, a more mature bone-to-tendon junction, and greater fibrocartilage layer formation compared to the other 2 groups (all p < 0.001). Furthermore, ATC-injected shoulders also demonstrated a significantly higher load-to-failure value (40.4 ± 4.5 N/kg) than the remaining groups (group A, 26.7 ± 3.0 N/kg; group B, 32.8 ± 4.2 N/kg; p < 0.001). Conclusions ATC demonstrated superior efficacy in promoting BTI healing following surgical repair in a chronic RCT model of rabbits.

ObjectiveThe nucleolar protein PES1 (Pescadillo homolog 1) plays critical roles in ribosome biogenesis and cell cycle regulation, yet its involvement in cellular senescence remains poorly understood. This study aimed to comprehensively investigate the functional consequences of PES1 suppression in cellular senescence and elucidate the molecular mechanisms underlying its regulatory role. MethodsInitially, we assessed PES1 expression patterns in two distinct senescence models: replicative senescent mouse embryonic fibroblasts (MEFs) and doxorubicin-induced senescent human hepatocellular carcinoma HepG2 cells. Subsequently, PES1 expression was specifically downregulated using siRNA-mediated knockdown in these cell lines as well as additional relevant cell types. Cellular proliferation and senescence were assessed by EdU incorporation and SA-β-gal staining assays, respectively. The expression of senescence-associated proteins (p53, p21, and Rb) and SASP factors (IL-6, IL-1β, and IL-8) were analyzed by Western blot or qPCR. Furthermore, Northern blot and immunofluorescence were employed to evaluate pre-rRNA processing and nucleolar morphology. ResultsPES1 expression was significantly downregulated in senescent MEFs and HepG2 cells. PES1 knockdown resulted in decreased EdU-positive cells and increased SA‑β‑gal-positive cells, indicating proliferation inhibition and senescence induction. Mechanistically, PES1 suppression activated the p53-p21 pathway without affecting Rb expression, while upregulating IL-6, IL-1β, and IL-8 production. Notably, PES1 depletion impaired pre-rRNA maturation and induced nucleolar stress, as evidenced by aberrant nucleolar morphology. ConclusionOur findings demonstrate that PES1 deficiency triggers nucleolar stress and promotes p53-dependent (but Rb-independent) cellular senescence, highlighting its crucial role in maintaining nucleolar homeostasis and regulating senescence-associated pathways.

Investigating the correlation between micronucleus formation and male infertility has the potential to improve clinical diagnosis and deepen our understanding of pathological progression. Our study enrolled 2252 male patients whose semen was analyzed from March 2023 to July 2023. Their clinical data, including semen parameters and age, were also collected. Genetic analysis was used to determine whether the sex chromosome involved in male infertility was abnormal (including the increase, deletion, and translocation of the X and Y chromosomes), and subsequent semen analysis was conducted for clinical grouping purposes. The participants were categorized into five groups: normozoospermia, asthenozoospermia, oligozoospermia, oligoasthenozoospermia, and azoospermia. Patients were randomly selected for further study; 41 patients with normozoospermia were included in the control group and 117 patients with non-normozoospermia were included in the study group according to the proportions of all enrolled patients. Cytokinesis-block micronucleus (CBMN) screening was conducted through peripheral blood. Statistical analysis was used to determine the differences in micronuclei (MNi) among the groups and the relationships between MNi and clinical data. There was a significant increase in MNi in infertile men, including those with azoospermia, compared with normozoospermic patients, but there was no significant difference between the genetic and nongenetic groups in azoospermic men. The presence of MNi was associated with sperm concentration, progressive sperm motility, immotile spermatozoa, malformed spermatozoa, total sperm count, and total sperm motility. This study underscores the potential utility of MNi as a diagnostic tool and highlights the need for further research to elucidate the underlying mechanisms of male infertility.
Humans ; Male ; Infertility, Male/genetics* ; Adult ; Micronucleus Tests ; Semen Analysis ; Oligospermia/genetics* ; Azoospermia/genetics* ; Chromosome Aberrations ; Sperm Count ; Micronuclei, Chromosome-Defective ; Middle Aged

OBJECTIVE: To identify the blood type of specimens from pregnant patients with difficult-to-type ABO status, and to guide clinical safe blood transfusion. METHODS: The specimens from 36 pregnant patients with suspicious ABO blood group were collected. These specimens were submitted by clinical institutions from various regions to our center's genetic testing platform from January 2021 to December 2022. The blood group phenotypes and genotypes of these specimens were identified by serological method and genetic sequencing. RESULTS: A total of 20 ABO subtypes were detected in the 36 samples, including 10 cases of BA/O, 3 cases of cisAB/O, 2 cases of A/Bw, 1 case of A2/B, 1 case of Aw/B, 1 case of BA/B, 1 case of BA/A, and 1 case of Bw/O. Additionally, 4 cases were identified as para-Bombay blood type, and no specific variations associated with abnormal phenotypes were found in the remaining 12 cases. CONCLUSION ABO subtypes interfere with ABO blood group identification in pregnant patients, and pregnancy status also affects blood group phenotype. Accurate determination of blood group genotype by genetic sequencing technology can guide clinical blood transfusion for pregnant patients, and ensure maternal and infant safety.
Humans ; Female ; Pregnancy ; ABO Blood-Group System/genetics* ; Blood Grouping and Crossmatching ; Blood Transfusion ; Genotype ; Phenotype

OBJECTIVE: To elucidate the modulation mechanism of Suanzaoren Decoction (SZRD) on basolateral amygdala (BLA) neuronal activity to alleviate chronic restraint stress (CRS)-related behavioral deficits. METHODS: The male C57BL/6J mice were assigned to 4 groups using the complete randomization method, including control (CON, n=19), CRS (n=19), SZRD (n=21), and fluoxetine (Flu, n=22) groups. Mice were restrained for 6 h per day, over a 21-d period to establish CRS models. The CON group remained in their cages without food or water during the 6-h matching period. SZRD and Flu groups received intragastric administration of SZRD (4.68 g/kg) and Flu (20 mg/kg) daily, respectively, 30 min before restraint for 21 consecutive days. The therapeutic effects of SZRD were evaluated using behavioral tests including the tail suspension test, elevated plus maze test, and forced swimming test. The cellular Fletcher B. Judson murine osteosarcoma proto-oncogene (c-Fos) expression in the BLA was measured using immunofluorescence, while action potential (AP) firing and synaptic transmission in BLA pyramidal neurons were evaluated using whole-cell patch-clamp recordings. RESULTS: SZRD administration significantly increased time spent in the open arms and open-arm entries while reducing immobility time (P<0.05 or P<0.01). It downregulated CRS-induced c-Fos expression and AP firing of pyramidal neurons in the BLA (P<0.01). Additionally, SZRD selectively attenuated excitatory (P<0.01), but not inhibitory, synaptic transmission onto BLA pyramidal neurons. CONCLUSION SZRD alleviated CRS-induced anxiety- and depression-like behaviors in mice by modulating the excitability and synaptic transmission of BLA pyramidal neurons.
Animals ; Drugs, Chinese Herbal/therapeutic use* ; Depression/complications* ; Pyramidal Cells/pathology* ; Male ; Mice, Inbred C57BL ; Basolateral Nuclear Complex/pathology* ; Restraint, Physical ; Anxiety/complications* ; Behavior, Animal/drug effects* ; Stress, Psychological/physiopathology* ; Mice ; Proto-Oncogene Proteins c-fos/metabolism* ; Action Potentials/drug effects* ; Synaptic Transmission/drug effects*

The p21-activated kinase 4 (PAK4), a key regulator of malignancy, is negatively correlated with immune infiltration and has become an emergent drug target of cancer therapy. Given the lack of high efficacy PAK4 inhibitors, we herein reported the identification of a novel inhibitor 13 bearing a tetrahydrobenzofuro[2,3-c]pyridine tricyclic core and possessing high potency against MIA PaCa-2 and Pan02 cell lines with IC₅₀ values of 0.38 and 0.50 μmol/L, respectively. This compound directly binds to PAK4 in a non-ATP competitive manner. In the mouse Pan02 model, compound 13 exhibited significant tumor growth inhibition at a dose of 100 mg/kg, accompanied by reduced levels of PAK4 and its phosphorylation together with immune infiltration in mice tumor tissue. Overall, compound 13 is a novel allosteric PAK4 inhibitor with a unique tricyclic structural feature and high potency both in vitro and in vivo, thus making it worthy of further exploration.

This study established the HPLC fingerprints, characteristic chromatograms, and a multi-component content determination method for Bidens bipinnata and B. biternata. The chemical pattern recognition analysis was then employed to clarify the characteristic indexes of quality differences between the two original plants of Bidentis Herba, providing a reference for establishing the quality standards of Bidentis Herba. HPLC was launched on an Agilent Poroshell 120 EC-C_(18) chromatographic column(4.6 mm×250 mm, 4 μm) by gradient elution with a mobile phase of 0.1% aqueous phosphoric acid-acetonitrile at a flow rate of 0.7 mL·min~(-1), detection wavelength of 270 nm, column temperature of 25 ℃, and an injection volume of 5 μL. The similarity between the fingerprints of 18 batches of Bidentis Herba samples and the common pattern(R) ranged from 0.572 to 0.933. A total of 23 chromatographic peaks were calibrated. Through comparison with the reference substances, six components(neochlorogenic acid, chlorogenic acid, isochlorogenic acid A, isochlorogenic acid B, rutin, and hyperoside) were identified and subjected to quantitative analysis. The characteristic fingerprints of B. bipinnata and B. biternata were calibrated with 20 and 17 characteristic peaks, respectively. Among them, peaks 8, 9, 22, and 23 were the characteristic peaks of B. bipinnata, and peak 7 was the characteristic peak of B. biternata, which can be used to distinguish the two original plants of Bidentis Herba. The relative standard deviation of the content of the above-mentioned six components ranged from 36% to 123%. The cluster analysis, principal component analysis, and orthogonal partial least squares-discriminant analysis(OPLS-DA) classified the 18 batches of Bidentis Herba samples into two categories. Additionally, through the analysis of variable importance in projection(VIP) under OPLS-DA, three characteristic indexes, rutin, isochlorogenic acid A, and isochlorogenic acid B, were identified. The analytical method established in this study can comprehensively evaluate the consistency of Bidentis Herba samples derived from different original plants, specifically identify the differential components between them, and effectively distinguish the two original plants of Bidentis Herba, providing a basis for the differentiation between different original plants and the quality control of Bidentis Herba.
Chromatography, High Pressure Liquid/methods* ; Drugs, Chinese Herbal/chemistry* ; Quality Control ; Bidens/chemistry*

OBJECTIVE: Ulcerative colitis is closely associated with intestinal stem cell (ISC) loss and impaired intestinal mucus barrier. Sinisan (SNS), a compound Chinese herbal medicine, has a long history in the treatment of intestinal dysfunction, yet whether SNS can relieve acute experimental colitis by modulating ISC proliferation and secretory cell differentiation has not been studied. Our study tested the effect of SNS against acute colitis and focused on the mechanisms involving intestinal barrier recovery. METHODS: Network pharmacology analysis and blood entry component analysis of SNS were used to explore the underlying mechanism by which SNS affects the acute dextran sulfate sodium (DSS)-induced murine colitis model. RNA-sequencing was used to demonstrate the mechanism. Further, reverse transcription-quantitative polymerase chain reaction, immunofluorescence staining, and alcian blue and periodic acid-Schiff staining were performed in vivo and in the colonic organoids to investigate the cell lineage differentiation-related mechanism of SNS. Furthermore, potential active ingredients from SNS were predicted by network pharmacology analysis. RESULTS: SNS dramatically suppressed DSS-induced acute colonic inflammation in mice. RNA-sequencing analysis revealed downregulation of inflammation and apoptosis-related genes, and upregulation of lipid metabolism and proliferation-related genes, such as Irf7, Pparα, Clspn and Hspa5. Additionally, ISC renewal and intestinal secretory cell lineage commitment were significantly promoted by SNS both in vivo and in vitro in colonic organoids, leading to enhanced mucin expression. Furthermore, potential active ingredients from SNS that mediated inflammation, lipid metabolism, proliferation, apoptosis, stem cells and secretory cells were predicted using a network pharmacology approach. CONCLUSION Our study shed light on the underlying mechanism of SNS in attenuating acute colitis from the perspective of ISC renewal and secretory lineage cell differentiation, suggesting a of novel therapeutic strategy against colitis. Please cite this article as: Cai YJ, Lan JH, Li S, Feng YN, Li FH, Guo MY, et al. Sinisan, a compound Chinese herbal medicine, alleviates acute colitis by facilitating colonic secretory cell lineage commitment and mucin production. J Integr Med. 2025; 23(4): 429-444.
Animals ; Drugs, Chinese Herbal/therapeutic use* ; Mice ; Colon/pathology* ; Mucins/metabolism* ; Mice, Inbred C57BL ; Cell Differentiation/drug effects* ; Male ; Colitis/metabolism* ; Cell Lineage/drug effects* ; Dextran Sulfate ; Stem Cells/drug effects* ; Disease Models, Animal