Objective To investigate the role of the endothelin-1/nitric oxide （ET-1/NO） system in neurological function and cerebral hemodynamics after interventional surgery for cerebral infarction， as well as the association of the levels of ET-1 and NO with neurological recovery and cerebral blood flow after surgery. Methods A total of 108 patients with cerebral infarction who were treated in our hospital from January 2022 to June 2023 were enrolled， and serum samples were collected before surgery and on day 30 after surgery. ELISA was used to measure the levels of ET-1 and NO；National Institutes of Health Stroke Scale（NIHSS） was used to assess neurological function； transcranial Doppler was used to measure cerebral hemodynamic parameters， including mean blood flow velocity， peak systolic velocity， and pulsatility index. A Spearman correlation analysis was used to investigate the correlation of ET-1 and NO with neurological function and cerebral hemodynamics，and the receiver operating characteristic （ROC） curve was used to assess the value of ET-1 and NO in predicting poor neurological function. Results After interventional surgery， there was a significant reduction in the level of ET-1 and a significant increase in the level of NO（both P<0.001）. The patients were divided into good recovery group （NIHSS≤5） and poor recovery group.Compared with the poor recovery group， the good recovery group had a significant reduction in serum ET-1 and a significant increase in serum NO （both P<0.001）. The Spearman correlation analysis showed that the serum level of ET-1 was negatively correlated with neurological recovery and the improvement in cerebral hemodynamics， while the level of NO was positively correlated with these two indicators. The ROC curve analysis showed that ET-1 and NO could effectively predict poor neurological recovery after interventional surgery， with an area under the ROC curve of 0.881 and 0.981，respectively. Conclusion The ET-1/NO system plays an important role in neurological function and cerebral hemodynamics after interventional surgery for cerebral infarction. Reducing ET-1 and increasing NO can facilitate the recovery of neurological function and cerebral blood flow after surgery. The levels of ET-1 and NO can be used as biomarkers for predicting poor postoperative neurological function and provide a scientific basis for optimizing postoperative treatment strategies.
Endothelin-1

OBJECTIVE: To investigate the antioxidant and osteogenic induction capabilities of calcium phosphate nanoflowers (hereinafter referred to as nanoflowers) in vitro at different concentrations. METHODS: Nanoflowers were prepared using gelatin, tripolyphosphate, and calcium chloride. Their morphology, microstructure, elemental composition and distribution, diameter, and molecular constitution were characterized using scanning electron microscopy, transmission electron microscopy, Fourier transform infrared spectroscopy, and energy-dispersive spectroscopy. Femurs and tibias were harvested from twelve 4-week-old Sprague Dawley rats, and bone marrow mesenchymal stem cells (BMSCs) were isolated and cultured using the whole bone marrow adherent method, followed by passaging. The third passage cells were identified as stem cells by flow cytometry and then co-cultured with nanoflowers at concentrations of 0, 0.4, 0.8, 1.2, 1.6, 2.0, 2.4, 2.8, 3.2, and 3.6 mg/mL. Cell counting kit 8 (CCK-8) assay was performed to screen for the optimal concentration that demonstrated the best cell viability, which was subsequently used as the experimental concentration for further studies. After co-culturing BMSCs with the screened concentration of nanoflowers, the biocompatibility of the nanoflowers was verified through live/dead cell staining, scratch assay, and cytoskeleton staining. The antioxidant capacity was assessed by using reactive oxygen species (ROS) fluorescence staining. The in vitro osteoinductive ability was evaluated via alkaline phosphatase (ALP) staining, alizarin red staining, and immunofluorescence staining of osteocalcin (OCN) and Runt-related transcription factor 2 (RUNX2). All the above indicators were compared with the control group of normally cultured BMSCs without the addition of nanoflowers. RESULTS: Scanning electron microscopy revealed that the prepared nanoflowers exhibited a flower-like structure; transmission electron microscopy scans discovered that the nanoflowers possessed a multi-layered structure, and high-magnification images displayed continuous atomic arrangements, with the nanoflower diameter measuring (2.00±0.25) μm; energy-dispersive spectroscopy indicated that the nanoflowers contained elements such as C, N, O, P, and Ca, which were uniformly distributed across the flower region; Fourier transform infrared spectroscopy analyzed the absorption peaks of each component, demonstrating the successful preparation of the nanoflowers. Through CCK-8 screening, the concentrations of 0.8, 1.2, and 1.6 mg/mL were selected for subsequent experiments. The live/dead cell staining showed that nanoflowers at different concentrations exhibited good cell compatibility, with the 1.2 mg/mL concentration being the best (P<0.05). The scratch assay results indicated that the cell migration ability in the 1.2 mg/mL group was superior to the other groups (P<0.05). The cytoskeleton staining revealed that the cell morphology was well-extended in all concentration groups, with no significant difference compared to the control group. The ROS fluorescence staining demonstrated that the ROS fluorescence in all concentration groups decreased compared to the control group after lipopolysaccharide induction (P<0.05), with the 1.2 mg/mL group showing the weakest fluorescence. The ALP staining showed blue-purple nodular deposits around the cells in all groups, with the 1.2 mg/mL group being significantly more prominent. The alizarin red staining displayed orange-red mineralized nodules around the cells in all groups, with the 1.2 mg/mL group having more and denser nodules. The immunofluorescence staining revealed that the expressions of RUNX2 and OCN proteins in all concentration groups increased compared to the control group, with the 1.2 mg/mL group showing the strongest protein expression (P<0.05). CONCLUSION The study successfully prepares nanoflowers, among which the 1.2 mg/mL nanoflowers exhibits excellent cell compatibility, antioxidant properties, and osteogenic induction capability, demonstrating their potential as an artificial bone substitute material.
Animals ; Osteogenesis/drug effects* ; Mesenchymal Stem Cells/drug effects* ; Calcium Phosphates/pharmacology* ; Rats, Sprague-Dawley ; Rats ; Antioxidants/chemistry* ; Cells, Cultured ; Cell Differentiation/drug effects* ; Nanostructures/chemistry* ; Tissue Engineering/methods* ; Bone Marrow Cells/cytology* ; Coculture Techniques ; Tissue Scaffolds/chemistry* ; Male ; Biocompatible Materials/chemistry* ; Cell Survival ; Core Binding Factor Alpha 1 Subunit/metabolism* ; Cell Proliferation

OBJECTIVE: To investigate the effect of mild hypothermia on neurological function in rats with spinal cord injury (SCI) based on the adenosine monophosphate activated protein kinase (AMPK)/Nod-like receptor protein 3 (NLRP3) pathway. METHODS: Fifty 7-8 weeks old SPF male Sprague Dawley rats were used to establish rat model of SCI by Allen's method. Among them, 48 successfully modeled rats were randomly divided into SCI group, mild hypothermia group (SCI+mild hypothermia treatment), and Compound C group (SCI+mild hypothermia+intraperitoneal injection of 20 mg/kg AMPK/NLRP3 pathway inhibitor Compound C), with 16 rats in each group. Another 16 normal rats with laminectomy were selected as sham-operation group. Basso-Beattie-Bresnahan (BBB) score was used to evaluate the motor ability of rats at 1, 3, 7, 14 days after treatment. After 14 days, the rats were sacrificed, and the spinal cord histopathological morphology was observed by HE staining, the neuronal apoptosis in spinal cord tissue was detected by TUNEL assay, and the serum levels of interleukin 2 (IL-2), IL-6, transforming growth factor β ₁ (TGF-β ₁), malondialdehyde (MDA), and superoxide dismutase (SOD) were detected by ELISA. The expressions of AMPK/NLRP3 pathway proteins in spinal cord tissue, including B-cell lymphoma 2 (Bcl-2), Bcl-2-associated X protein (Bax), and cleaved-Caspase-9 were detected by Western blot. RESULTS: At 1 day after treatment, the rats in SCI group, mild hypothermia group, and Compound C group did not recover their motor ability. With the prolongation of time, the motor function of rats in each group gradually recovered. Among them, the BBB score of SCI group was significantly lower than that of sham-operation group and mild hypothermia group ( P<0.05), and the BBB score of Compound C group was significantly lower than that of mild hypothermia group ( P<0.05). Compared with the sham-operation group, the SCI group displayed obvious pathological changes in the spinal cord tissue, with disordered tissue architecture, inflammatory infiltration, and blurred interstitial boundaries. The neuronal apoptosis rate, Bax/Bcl-2 ratio, cleaved Caspase-9 expression, NLRP3 protein expression, serum IL-2, IL-6, and MDA levels were elevated, whereas serum TGF-β ₁, SOD levels, and spinal cord phosphorylation AMPK/AMPK protein expression significantly decreased ( P<0.05). Compared with the SCI group, the above phenomena significantly improved in the mild hypothermia group ( P<0.05), while the Compound C group showed the opposite trend of change compared to the mild hypothermia group ( P<0.05). CONCLUSION Mild hypothermia can attenuate neurological dysfunction after SCI in rats, potentially by activating the AMPK/NLRP3 pathway.
Animals ; Spinal Cord Injuries/physiopathology* ; Rats, Sprague-Dawley ; Male ; Rats ; NLR Family, Pyrin Domain-Containing 3 Protein/metabolism* ; AMP-Activated Protein Kinases/metabolism* ; Hypothermia, Induced ; Signal Transduction ; Spinal Cord/pathology* ; Apoptosis ; Interleukin-6/metabolism* ; Disease Models, Animal ; bcl-2-Associated X Protein/metabolism* ; Superoxide Dismutase/metabolism* ; Proto-Oncogene Proteins c-bcl-2/metabolism* ; Caspase 9/metabolism*

OBJECTIVE: To summarize recent research progress in hydrogel-based growth factors for treatment of intervertebral disc degeneration (IDD). METHODS: The relevant literature on hydrogel-based growth factors for IDD treatment at home and abroad was extensively reviewed, and their advantages and therapeutic effects in repairing IDD were analyzed and summarized. RESULTS: Hydrogels exhibit high hydration, biocompatibility, and biodegradability, enabling targeted delivery and sustained release of growth factors such as growth differentiation factors and transforming growth factors. This facilitates enhanced efficacy in promoting cell proliferation, extracellular matrix synthesis, and reducing inflammatory responses. Consequently, hydrogels demonstrate broad application prospects in the repair of IDD. CONCLUSION Research on hydrogel-based growth factors for treating IDD demonstrates advantages such as avoiding disc damage caused by repeated injections and controlling growth factor release concentrations. However, drawbacks include the limited variety of loaded growth factors and the need to verify the long-term stability and biocompatibility of hydrogels. Therefore, further research is required on aspects such as the types of loaded growth factors and the long-term stability and biocompatibility of hydrogels to establish an experimental foundation for their clinical application.
Intervertebral Disc Degeneration/therapy* ; Hydrogels/chemistry* ; Humans ; Intercellular Signaling Peptides and Proteins/administration & dosage* ; Biocompatible Materials/chemistry* ; Animals ; Tissue Engineering/methods* ; Cell Proliferation/drug effects* ; Drug Delivery Systems

Objective This study investigated the regulatory effect of high mobility group protein B1 (HMGB1) in the peripheral blood of patients with primary immune thrombocytopenia (ITP) on myeloid dendritic cells (mDC) and Th17/regulatory T cells (Treg) balance. Methods The study enrolled 30 newly diagnosed ITP patients and 30 healthy controls.Flow cytometry was used to measure the proportion of mDC, Th17, and Treg cells in the peripheral blood of ITP patients and healthy controls. ELISA was conducted to quantify the serum levels of HMGB1, interleukin 6 (IL-6), IL-23, IL-17, and transforming growth factor β(TGF-β). The mRNA levels of retinoic acid-related orphan receptor γt(RORγt) and forehead box P3(FOXP3) were detected by real-time PCR. The correlation between the abovementioned cells, cytokines, and platelet count was assessed using Pearson linear correlation analysis. Results The proportion of Th17 cells and the expression levels of HMGB1, IL-6, IL-23, IL-17 and the level of RORγt mRNA in the peripheral blood of ITP patients were higher than those in healthy controls. However, the Treg cell proportion and TGF-β level were lower in ITP patients than those in healthy controls. In patients with ITP, the proportion of mDC and the level of FOXP3 mRNA did not show significant changes. The proportion of mDC cells was significantly correlated with the expression of IL-6 and IL-23. Moreover, the expression of HMGB1 showed a significant correlation with the expression of mDC, IL-6, IL-23, RORγt mRNA, and IL-17. Notably, both the proportion of mDC cells and the expression of HMGB1 were negatively correlated with platelet count. Conclusion The high expression of HMGB1 in peripheral blood of ITP patients may induce Th17/Treg imbalance by promoting the maturation of mDC and affecting the secretion of cytokines, thereby potentially playing a role in the immunological mechanism of ITP.
Humans ; Th17 Cells/cytology* ; HMGB1 Protein/genetics* ; T-Lymphocytes, Regulatory/cytology* ; Female ; Male ; Dendritic Cells/metabolism* ; Adult ; Middle Aged ; Purpura, Thrombocytopenic, Idiopathic/genetics* ; Nuclear Receptor Subfamily 1, Group F, Member 3/genetics* ; Young Adult ; Interleukin-23/blood* ; Interleukin-17/blood* ; Interleukin-6/blood* ; Forkhead Transcription Factors/genetics* ; Myeloid Cells/cytology* ; Aged

Objective The aim of this study was to investigate the effect of p38 on stem cell maintenance of pancreatic cancer. Methods Human pancreatic cancer cells PANC-1 were treated with different concentrations of 5-fluorouracil(5-FU)(0.5×IC₅₀, IC₅₀, and 2×IC₅₀) for 24 hours, and VX-702 (p38 phosphorylation inhibitor) was added, and the cells were inoculated in 6-well culture dishes with ultra-low adhesion to observe the changes of sphere tumors. The expression levels of cyclin-dependent kinase 2(CDK2), cyclin B1 and D1, Octamer-binding transcription factor 4(OCT4), SRY-box transcription factor 2(SOX2), Nanog and p38 were measured by Western blot. The mRNA expression levels of p38, OCT4, Nanog and SOX2 were tested by RT-PCR. Cell cycle, apoptosis, and the proportion of CD44⁺CD133⁺PANC-1 cells were evaluated by flow cytometry. Results The results showed that 5-FU inhibited the formation of tumor spheres in PANC-1 cells, increased CD44⁺CD133⁺cell fragments, down-regulated the expression of OCT4, Nanog and SOX2, and inhibited the stemness maintenance of PANC-1 tumor stem cells. Phosphorylation of PANC-1 cells was inhibited by a highly selective p38 MAPK inhibitor, VX-702(p38 mitogen-activated protein kinase inhibitor), which had the same effect as 5-FU treatment. When VX-702 combined with 5-FU was used to treat PANC-1 cells, the therapeutic effect was enhanced. Conclusion p38 inhibitors decreased PANC-1 cell activity and increased cell apoptosis. p38 inhibitors inhibit the stemness maintenance of pancreatic cancer stem cells.
Humans ; Phosphorylation/drug effects* ; Cell Line, Tumor ; p38 Mitogen-Activated Protein Kinases/antagonists & inhibitors* ; Neoplastic Stem Cells/metabolism* ; Drug Resistance, Neoplasm/drug effects* ; Fluorouracil/pharmacology* ; Pancreatic Neoplasms/pathology* ; Apoptosis/drug effects* ; SOXB1 Transcription Factors/genetics* ; Octamer Transcription Factor-3/genetics*

Objective To explore the effects of S100 calcium-binding protein A9 (S100A9) gene knockout on the phenotype of systemic lupus erythematosus (SLE) in mice and to clarify the role of S100A9 in the pathogenesis of SLE. Methods Ten female C57BL/6 wild-type and S100A9 knockout (S100A9-KO ) mice were selected, with five wild-type and five S100A9-KO B6 mice receiving imiquimod (IMQ) cream to establish SLE mouse model. The other five wild-type and five S100A9-KO B6 mice were treated as control groups by wiping the skin of the right ear with a cotton swab. After 8 weeks, the mice were sacrificed. The serum was collected from each mouse to detect the levels of anti-double-stranded DNA (dsDNA) antibodies, immunoglobulin G (IgG), B cell activating factor (BAFF), and interleukin 6 (IL-6) using ELISA. The levels of serum creatinine were determined using a sarcosine oxidase method. Urine was collected to measure urinary protein concentration. Kidneys were collected and stained with hematoxylin and eosin (H&E) for evaluating histological changes. Results After IMQ treatment, the length and weight of spleen, levels of serum creatinine, anti-dsDNA antibodies, IgG, BAFF, IL-6, and urinary protein in the IMQ B6 group and IMQ S100A9-KO B6 group were significantly higher than those of the control groups. Lupus-like changes including increased glomerular volume and tubular epithelial swelling were observed in kidneys from the IMQ and IMQ S100A9-KO groups. However, compared with the IMQ B6 group, the IMQ S100A9-KO B6 group exhibited milder levels of serum and urine indicators as well as the lupus-like symptoms. Conclusion IMQ could induce lupus-like symptoms in both wild-type B6 mice and S100A9-KO B6 mice, but the lesions in S100A9 knockout mice are milder. Theses results suggested that S100A9 is involved in and promotes the pathogenesis of SLE.
Animals ; Lupus Erythematosus, Systemic/chemically induced* ; Female ; Calgranulin B/genetics* ; Mice, Knockout ; Mice, Inbred C57BL ; Phenotype ; Mice ; Interleukin-6/blood* ; Disease Models, Animal ; Antibodies, Antinuclear/blood* ; B-Cell Activating Factor/blood* ; Immunoglobulin G/blood* ; Kidney/pathology*

Objective To explore the effect of miR-582-5p on Mycobacterium tuberculosis (Mtb)-infected macrophages by regulating dual specificity phosphatase 1 (DUSP1). Methods THP-1 macrophages were divided into six groups: control group, Mtb group, inhibitor-NC group, miR-582-5p inhibitor group, miR-582-5p inhibitor+si-NC group, and miR-582-5p inhibitor+si-DUSP1 group. QRT-PCR was applied to detect the gene expression of miR-582-5p and DUSP1 in cells. ELISA kit was used to detect the levels of interferon γ (IFN-γ), interleukin 6 (IL-6), tumor necrosis factor α (TNF-α), and interleukin 1β (IL-1β). CCK-8 method was applied to detect cell proliferation. Flow cytometry was applied to detect cell apoptosis rate. Western blot analysis was used to measure the protein expression levels of B-cell lymphoma 2 (Bcl2), Bcl2-associated X (BAX), and cleaved-caspase 3 (c-caspase-3) in cells. In addition, the target relationship between miR-582-5p and DUSP1 was verified. Results Compared with the control group, the expression of miR-582-5p, levels of IFN-γ, IL-6, TNF-α, IL-1β, bacterial load and OD450 values (24 h, 48 h), and the protein expression of Bcl2 in macrophages were higher in the Mtb group, while the mRNA expression of DUSP1, apoptosis rate, and the protein expression levels of c-caspase-3, BAX and DUSP1 were lower. Compared with the Mtb group and the inhibitor-NC group, the above-mentioned indicators in the miR-582-5p inhibitor group were partially reversed. Down-regulation of DUSP1 expression partially reversed the inhibitory effect of down-regulation of miR-582-5p expression on Mtb-infected macrophages. Conclusion Inhibiting the expression of miR-582-5p can up-regulate DUSP1, thereby inhibiting the proliferation and inflammatory response of Mtb-infected macrophages and promoting cell apoptosis.
Humans ; Macrophages/metabolism* ; Dual Specificity Phosphatase 1/metabolism* ; MicroRNAs/metabolism* ; Mycobacterium tuberculosis/physiology* ; Tuberculosis/microbiology* ; Apoptosis/genetics* ; THP-1 Cells ; Cell Proliferation/genetics* ; Interferon-gamma/genetics* ; Tumor Necrosis Factor-alpha/genetics* ; Interleukin-1beta/genetics*

Objective To explore the effect of the knockdown of transmembrane 9 superfamily protein member 2 (TM9SF2) on the replication of vesicular stomatitis virus (VSV), and investigate its role in the mechanism of antiviral innate immunity. Methods Small interfering RNA (siRNA) was used to knock down the TM9SF2 gene in human non-small cell lung cancer A549 cells. The CCK-8 method was used to assess cell proliferation. A VSV-green fluorescent protein (VSV-GFP) infected cell model was established. The plaque assay was used to measure the viral titer in the supernatant. RT-qPCR and Western blotting were employed to quantify the mRNA and protein levels of VSV genome replication in A549 cells following VSV infection, as well as the expression of interferon β (IFN-β) mRNA and interferon regulatory factor 3 (IRF3) protein phosphorylation following polyinosinic-polycytidylic acid (poly(I:C)) stimulation. Results Compared to the negative control, the knockdown of TM9SF2 exhibited a significant effect, with no observed impact on A549 cell proliferation. The VSV-GFP infected A549 cell model was successfully established. After viral stimulation, fluorescence intensity was reduced following TM9SF2 knockdown, and the mRNA and protein levels of VSV were significantly downregulated. The viral titer of VSV was decreased. After poly(I:C) stimulation, TM9SF2 knockdown significantly upregulated the mRNA level of IFN-β and the phosphorylation level of IRF3 protein. Conclusion The knockdown of TM9SF2 inhibits the replication of vesicular stomatitis virus, and positively regulates the type I interferon signaling pathway, thus enhancing the host's antiviral innate immune response.
Humans ; Virus Replication/genetics* ; Signal Transduction ; Membrane Proteins/metabolism* ; A549 Cells ; Vesiculovirus/physiology* ; Interferon-beta/metabolism* ; Interferon Regulatory Factor-3/genetics* ; Interferon Type I/metabolism* ; Vesicular Stomatitis/immunology* ; Gene Knockdown Techniques ; Vesicular stomatitis Indiana virus/physiology* ; RNA, Small Interfering/genetics*

Objective To explore the characteristics of the inhibitory effect of Evodiamine on the proliferation of activated T cells. Methods Mononuclear cells from peripheral blood (PBMCs) were obtained from healthy donors through density gradient centrifugation, and T cells were subsequently purified by using immunomagnetic bead separation. T cell activation was induced by employing anti-human CD3 and anti-human CD28 antibodies. T cells were treated with different concentrations of EVO (0.37, 1.11, 3.33, and 10)μmol/L. Flow cytometry was applied to evaluate the proliferation index, apoptosis rate, viability, CD25 expression levels, and cell cycle distribution of T cells. The expression levels of cytokines IL-2, IL-17A, IL-4, and IL-10 were quantified by using ELISA. Results 1.11, 3.33 and 10 μmol/L EVO effectively inhibited the proliferation of activated T cells, with an IC₅₀ of (1.5±0.3)μmol/L. EVO did not induce apoptosis in activated T cells and affect the survival rate of resting T cells. EVO did not affect the expression of CD25 and the secretion of IL-2 in activated T cells. EVO arrested the T cell cycle at the G2/M phase, resulting in an increase in G2/M phase cells, and exhibited a concentration-dependent effect. EVO did not affect the secretion of IL-4, IL-10 by activated T cells, but significantly inhibited the secretion of IL-17A. Conclusion EVO did not significantly affect the activation process of T cells but inhibited T cell proliferation by arresting the cell cycle at the G2/M phase and significantly suppressed the secretion of the pro-inflammatory cytokine IL-17A, which suggests that EVO has the potential to serve as a lead compound for the development of low-toxicity and high-efficiency immunosuppressants and elucidates the mechanisms underlying the anti-inflammatory and immunomodulatory effects of the traditional Chinese medicine Evodia rutaecarpa.
Humans ; Cell Proliferation/drug effects* ; Quinazolines/pharmacology* ; T-Lymphocytes/metabolism* ; Lymphocyte Activation/drug effects* ; Apoptosis/drug effects* ; Interleukin-4/metabolism* ; Interleukin-10/metabolism* ; Interleukin-2 Receptor alpha Subunit/metabolism* ; Interleukin-17/metabolism* ; Interleukin-2/metabolism* ; Cell Cycle/drug effects* ; Cells, Cultured