ObjectiveThis study aims to explore the mechanism by which Yishen Huoxue Tongqiao Formula improves metabolism-neuroplasticity and treats unilateral vestibular labyrinth destruction by regulating the metabolic balance of glutamate （Glu）/γ-aminobutyric acid （GABA）. Methods48 Sprague-Dawley （SD） adult rats were randomly divided into the sham operation group， model group， Yishen Huoxue Tongqiao Formula groups with low， medium， and high doses （9.20， 18.39， 36.78 g·kg^-1）， and betahistine group （1.62 mg·kg^-1）. A unilateral vestibular labyrinth destruction （vestibular dysfunction） model was established by intratympanic injection of chloroform into the right ear， while the control group received intratympanic injection of normal saline. Drugs were administered once daily for seven consecutive days. During the period， behavioral tests were performed to evaluate the behaviors of rats after unilateral vestibular labyrinth destruction. Hematoxylin-eosin （HE） staining and Nissl staining were used to observe the neuronal morphology in the medial vestibular nucleus. Golgi staining was employed to assess the number of dendritic spines of neurons in the medial vestibular nucleus. Ultra-performance liquid chromatography-tandem mass spectrometry （LC-ESI-MS/MS） was utilized to detect Glu/GABA. Immunofluorescence and immunohistochemistry were used to detect the expressions of neuronal nuclei （NeuN）， growth-associated protein 43 （GAP-43）， and glial fibrillary acidic protein （GFAP）. Western blot and real-time fluorescent quantitative polymerase chain reaction （Real-time PCR） were applied to determine the expressions of glutamate-immunoreactive （Glu-IR）， GABA， GFAP， postsynaptic density protein 95 （PSD-95）， and GAP-43. ResultsCompared with the sham operation group， the model group presented with head deviation， balance disorder， increased tail suspension score， nuclear consolidation of medial vestibular nerve neurons， and decreased Nissl bodies （P<0.01）. The number of dendritic spines in neurons and NeuN-positive cells decreased. The content of Glu decreased. The content of GABA increased （Glu/GABA decreased）. The expression of GAP-43 was down-regulated， and GFAP was up-regulated （P<0.05， P<0.01）. The expressions of Glu-IR， PSD-95， and GAP-43 proteins， as well as Glu-IR mRNA decreased， while the expressions of GABA and GFAP proteins and mRNA increased （P<0.05， P<0.01）. Compared with those in the model group， the head deviation， imbalanced behavior， and tail suspension scores in each treatment group decreased， with alleviated neuronal injury and recovered Nissl bodies （P<0.01）. The number of dendritic spines of neurons increased， and the number of NeuN-positive cells rebounded. The content of Glu increased， and the content of GABA decreased （Glu/GABA increased）. GFAP was down-regulated， and GAP-43 was up-regulated （P<0.05， P<0.01）. The expressions of Glu-IR， PMD-95， and GAP-43 proteins， as well as Glu-IR mRNA increased， while the expressions of GABA and GFAP proteins and mRNA decreased. The effect was more significant in the high-dose group （P<0.01）. ConclusionThe Yishen Huoxue Tongqiao Formula can alleviate vestibular dysfunction， and its mechanism may be associated with regulating the metabolic balance of Glu/GABA， mitigating neural damage， improving synaptic plasticity （promoting GAP-43 expression and inhibiting GFAP expression）， and facilitating vestibular compensation.

OBJECTIVES: To investigate the effect of orexin-A-mediated regulation of ionotropic glutamate receptors for promoting motor function recovery in rats with spinal cord injury (SCI). METHODS: Thirty-six newborn SD rats (aged 7-14 days) were randomized into 6 groups (n=6), including a normal control group, a sham-operated group, and 4 SCI groups with daily intrathecal injection of saline, DNQX, orexin-A, or orexin-A+DNQX for 3 consecutive days after PCI. Motor function of the rats were evaluated using blood-brain barrier (BBB) score and inclined plane test 1 day before and at 1, 3, and 7 days after SCI. For patch-clamp experiment, spinal cord slices from newborn rats in the control, sham-operated, SCI, and SCI+orexin groups were prepared, and ventral horn neurons were acutely isolated to determine the reversal potential and dynamic indicators of glutamate receptor-mediated currents under glutamate perfusion. RESULTS: At 3 and 7 days after SCI, the orexin-A-treated rats showed significantly higher BBB scores and grip tilt angles than those with other interventions. Compared with those treated with DNQX alone, the rats receiving the combined treatment with orexin and DNQX had significantly higher BBB scores and grip tilt angles on day 7 after PCI. In the patch-clamp experiment, the ventral horn neurons from SCI rat models exhibited obviously higher reversal potential and greater rise slope of glutamate current with shorter decay time than those from sham-operated and orexin-treated rats. CONCLUSIONS Orexin-A promotes motor function recovery in rats after SCI possibly by improving the function of the ionotropic glutamate receptors.
Animals ; Spinal Cord Injuries/drug therapy* ; Rats ; Rats, Sprague-Dawley ; Receptors, Ionotropic Glutamate/metabolism* ; Recovery of Function/drug effects* ; Orexins/pharmacology* ; Male ; Female ; Animals, Newborn ; Neuropeptides/pharmacology* ; Intracellular Signaling Peptides and Proteins/pharmacology*

OBJECTIVE To establish an ultra performance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS)method for determination of dexmedetomidine(DEX)in plasma of beagle dogs and evaluate the pharmacokinetics and sedation after nasal,buccal and sublingual mucosal admin-istration.METHODS A UPLC-MS/MS method was established and validated for dertermination of DEX in plasma of beagle dogs.DEX was administered to the nasal cavity,buccal and sublingual mucous membranes of beagle dogs,respectively.Blood samples were collected at different time points.The plasma concentration of DEX was measured by the established UPLC-MS/MS method.Pharmacokinetic parameters were fitted by Phoenix software and the sedative effect at different mucous membrane sites was evaluated in conjunction with behavioral and Ramsay scores.RESULTS The linearity of DEX was fine within the range of 0.05-100 μg·L-1(r>0.999),which was validated methodologically to meet the requirements of quantitative detection.The plasma concentration of the drug peaked the fastest with nasal administration.Tmax was 0.25 h,Cmax(4.43±1.19)μg·L-1,and the AUC0-6h was(8.92±2.07)μg·h·L-1,compared with 0.92 and 1 h,(2.87±0.69),(2.70±0.41)μg·L-1,and(7.99±1.77),(7.01±2.09)μg·h·L-1 with buccal and sublingual administration.Nasal administration had the fastest onset at 7 min,with a Ramsay score of 4,and sedation lasted for 36 min,compared with 33 and 35 min,and 38 and 37 min for buccal and sublingual administration.CONCLUSION The proposed method is sensitive,reliable and applicable to quantitative analysis of DEX in plasma of beagle dogs.Administration of DEX to the nasal cavity mucosa has a faster onset and a better sedative effect than to the buccal and sublingual mucosa.

Hot melt extrusion(HME)technology employs thermodynamic and kinetic principles to mix pharmaceutical polymers with crystalline drugs at high temperatures and extrude them,embedding drug molecules within the polymer matrix to form solid dispersions.Due to its solvent-free nature,capability for one-step processing,and support for continuous operation,HME has garnered significant attention in the pharmaceutical industry in recent years.This article introduced the basic principles and development history of HME technology and its marketed drugs.It reviewed the research progress of HME technology in improving drug solubility,masking taste,controlled release,targeted release,oral dispersible films,implant formulations,semi-solid formulations,and 3D printed formulations.Additionally,the article summarized the advantages and limitations of HME technology and provided an outlook on its future development.

Objective:To construct a machine learning model based on localization CT-based radiomics, dosiomics and clinical features for predicting radiotherapy response in non-small cell lung cancer (NSCLC) and validate its application value.Methods:A retrospective case series study was conducted. A total of 138 NSCLC patients who received radiotherapy at the Affiliated Hospital of Jining Medical University from January 2016 to December 2022 were selected. The efficacy was evaluated according to the Response Evaluation Criteria in Solid Tumors (RECIST) 1.1, and the patients were stratified according to the objective remission (complete remission+partial remission). Random stratified sampling was used to divide the 138 patients into a training group (96 cases) and an internal validation group (42 cases) at a ratio of 7∶3. Additionally, 33 patients who received radiotherapy at Jining Cancer Hospital from January 2019 to December 2022 were included as the external validation group. Based on the pre-radiotherapy data of the radiotherapy planning system, PyRadiomics software package was used to extract 107 radiomics features and 107 dosiomics features for each patient. Pearson correlation analysis and LASSO regression analysis were used for dimensionality reduction screening; the final selected features were weighted and integrated to generate radiomics-dosiomics scores (RDS), which were then input into logistic regression (LR), support vector machine (SVM), extremely randomized forest (Extra Trees), K-nearest neighbor algorithm (KNN), lightweight gradient boosting machine (Light GBM), and multi-layer perceptron (MLP) machine learning algorithms to construct 6 radiomics-dosiomics models (RDM) for predicting the objective remission. RECIST 1.1 standard was used to evaluate objective remission as the gold standard, receiver operating characteristic (ROC) curve of 6 RDM for predicting objective remission was plotted, and the optimal algorithm for RDM was selected. Univariate and multivariate logistic regression were performed on demographic characteristics, hematological indicators and radiotherapy parameters of the training group to screen independent risk factors for NSCLC patients who received radiotherapy but did not achieve objective remission. These factors were input into the optimal machine learning algorithm to construct a clinical model (CM). Combined with features from RDS and CM, the clinical feature-radiomics-dosiomics combined model (CRDM) was established, and the nomogram of the model for predicting objective remission in NSCLC patients with radiotherapy was drawn. ROC curves were used to evaluate the efficacy of CM, RDM and CRDM in predicting the objective remission in NSCLC patients with radiotherapy in the training group, internal validation group and external validation group.Results:Four radiomics features (including grayscale variance, low grayscale long-range operation emphasis, low grayscale area emphasis, and small area low grayscale area emphasis, all of which were texture features) and 6 dosiomics features [including 1 first-order feature (robust mean absolute deviation), 4 texture features (grayscale non-uniformity, large area emphasis, large area high grayscale emphasis, contrast) and 1 shape feature (shortest axis length)] were selected. ROC curve analysis showed that the area under the curve (AUC) of the RDM constructed using SVM algorithm for judging the objective remission in the training group and the internal validation group was 0.907 (95% CI: 0.836-0.977) and 0.822 (95% CI: 0.685-0.959), which were higher than RDM constructed using other algorithms, and the sensitivity (96.2% and 91.7%), specificity (78.6% and 76.7%) and accuracy (83.3% and 81.0%) at the optimal cut-off values were all higher. Considering the stability and generalization ability of the model, SVM algorithm was ultimately used to construct RDM, CM and CRDM uniformly. Based on training group data, univariate and multivariate logistic regression analysis showed that elevated platelet-to-lymphocyte ratio (PLR) ( OR = 1.001, 95% CI: 1.000-1.003, P = 0.035) and increased target volume of radiotherapy plan ( OR = 1.001, 95% CI: 1.000-1.001, P = 0.008) were independent risk factors for failure to achieve objective remission. ROC curve analysis showed that in the training group and the internal validation group, the AUC of CRDM predicting objective remission were 0.914 (95% CI: 0.856-0.972) and 0.864 (95% CI: 0.754-0.974), respectively, which were better than CM [AUC were 0.735 (95% CI: 0.612-0.857) and 0.697 (95% CI: 0.507-0.888)] and RDM, respectively. In the external validation group, the AUC of CRDM, CM and RDM were 0.778 (95% CI: 0.500-1.000), 0.667 (95% CI: 0.434-0.899) and 0.741 (95% CI: 0.463-1.000), respectively. Conclusions:The CRDM constructed by combining radiomics, dosiomics and clinical features can comprehensively and accurately evaluate the radiotherapy response of NSCLC patients, and may have important clinical application value in achieving precision medicine and optimizing treatment strategies.

Objective:To develop and validate a combined model integrating radiomics, dosiomics, and clinical parameters based on CT simulation and dosimetric images in order to predict the occurrence of radiation pneumonitis (RP) in patients with non-small cell lung cancer (NSCLC).Methods:A retrospective study was conducted on the clinic data of 143 NSCLC patients who received radiotherapy at the Affiliated Hospital of Jining Medical University from January 2016 to December 2022. Patients were randomly stratified into a training group ( n = 100) and an internal validation group ( n = 43) at a 7∶3 ratio. Moreover, clinic data were collected from 34 NSCLC patients who received radiotherapy at the Jining Cancer Hospital between January 2019 and December 2022 as an external validation group. All three groups (the training group, internal validation, and external validation groups) were further categorized into two groups based on the RP severity (i.e., RP ≥ grade 2 and RP < grade 2). Their radiotherapy dose, CT simulation, and 3D dose distribution images were collected. Then, the total lung minus planning target volume (TL-PTV) was defined as the region of interest (ROI) for radiomics and dosiomic feature extraction, followed by feature dimensionality reduction. Consequently, key features associated with RP were determined. Four predictive models were developed using machine learning approaches (especially multilayer perceptron, MLP): a clinical model (CM), a radiomics model (RM), a dosiomics model (DM), and a radiomics and dosiomics nomogram (RDN), with a nomogram subsequently constructed. Ultimately, the performance and clinical feasibility of these models were assessed using receiver operating characteristic (ROC), area under the curve (AUC), and decision curve analysis (DCA). Results:A total of 1 834 radiomic features and 1 834 dosiomic features were extracted. Using the occurrence of RP ≥ grade 2 as the marker variable, 14 radiomic features, 15 dosiomic features, and three clinical features were selected from the training group to construct the prediction models (CM, RM, DM, and RDN). The performance and generalizability of these models were subsequently validated in both the internal validation and external validation groups. Specifically, the RDN exhibited AUCs of 0.915 (95% CI: 0.852-0.978), 0.879 (95% CI: 0.777-0.982), and 0.838 (95% CI: 0.701-0.975) in the three groups, respectively. A nomogram was established for RDN by integrating the radiomics score (R-score), dosiomics score (D-score), mean lung dose (MLD), V20, and V30. This nomogram allowed for individualized risk estimation of RP and facilitated personalized radiotherapy planning. Conclusions:The RDN model that is developed based on CT simulation and 3D dose distribution images and integrates radiomics, dosiomics, and clinical features can effectively predict the RP risk of NSCLC patients. The integration of multidimensional data contributes to the formation of the optimal predictive model, offering guidance for clinicians.

OBJECTIVE To investigate the impact of vesicular stomatitis virus envelope glycopro-tein-G(VSV-G)modification on the mucosal immune efficacy of antigen-loaded engineered exosome vaccines.METHODS In vitro experiments:Dendritic cells(DCs)were divided into three groups:cell-control(treated with culture medium),receptor binding domain(RBD)(transfected with plasmid RBD),and RBD+VSV-G(co-transfected with plasmids RBD and VSV-G).Expression levels of RBD and VSV-G were assessed using Western blotting,flow cytometry,and immunofluorescence.Exosomes were extracted via ultracentrifugation,whose morphology,size distribution,and marker proteins were analyzed using transmission electron microscopy,nanoparticle tracking analysis,and Western blotting that confirmed the expressions of RBD and VSV-G in the exosomes.In vivo experiments:① Female BALB/c mice were divided into the control group Mock exosomes(Mock-Exo)(derived from the supernatant of cell-control),RBD decorated exosomes(RBD-Exo)(derived from the RBD cell supernatant),and RBD and VSV-G decorated exosomes(RBD+VSV-G-Exo)(derived from RBD+VSV-G cell supernatant).Follow-ing intranasal immunization with the respective vaccines,the nasal retention effects were evaluated using in vivo imaging.Flow cytometry was used to assess the ability to recruit immune cells to the nasal tissue.Serum RBD-specific immunoglobulin G(IgG)and mucosal immunoglobulin A(IgA)(bronchoal-veolar lavage fluid/nasal wash)were quantified at 7 and 21 d post-immunization by enzyme-linked immuno-sorbent assay.Body weight changes were monitored and key serum biochemical parameters along with histopathological damage to major organs were analyzed following immunization.② Female BALB/c mice were divided into the Mock-Exo group(intranasally inoculated with Mock-Exo),RBD+VSV-G-Exo group(intranasally inoculated with RBD+VSV-G-Exo),and RBD+VSV-G-Exo(im)group(intramus-cularly injected with RBD+VSV-G-Exo).RESULTS In vitro experiments:RBD and VSV-G were successfully expressed in cells,with positive rates of RBD+and VSV-G+cells at 64.4%and 31.2%,respectively.The extracted exosomes exhibited regular morphology and qualified purity,with a particle size of approximately 138 nm and successfully loaded RBD and VSV-G proteins.In vivo experiments:Compared to Mock-Exo and RBD-Exo,RBD+VSV-G-Exo prolonged nasal retention time to 96 h and markedly increased the numbers of CD49B+natural killer cells,CD11c+dendritic cells,and F4/80+macrophages in nasal tissues.RBD+VSV-G-Exo induced robust RBD-specific immune responses,with serum IgG titers,BALF IgA titers,and nasal wash IgA titers reaching 1∶5 215,1∶2 560,1∶1 114,respec-tively.In contrast,no RBD-specific IgA antibody titers were detected in the BALF and nasal wash of mice treated with RBD+VSV-G-Exo(im).Mice showed stable body weight gain during 30 d post-immu-nization.Major serum biochemical indices were within normal reference ranges,and no obvious patho-logical changes were observed in major organs or olfactory bulbs 7 d after immunization.CONCLU-SION VSV-G modification extends the retention time of engineered exosome vaccines in nasal tissues,enhance their ability to recruit immune cells,and induce a high-level antigen-specific respiratory mucosal immune response.

Poloxamer， as a non-ionic surfactant， exhibits a unique triblock [polyethylene oxide-poly （propylene oxide）-polyethylene oxide] structure， which endows it with broad application potential in various fields， including solid dispersion technology， nanotechnology， gel technology， biologics， gene engineering and 3D printing. As a carrier， it enhances the solubility and bioavailability of poorly soluble drugs. In the field of nanotechnology， it serves as a stabilizer etc.， enriching preparation methods. In gel technology， its self-assembly behavior and thermosensitive properties facilitate controlled drug release. In biologics， it improves targeting efficiency and reduces side effects. In gene engineering， it enhances delivery efficiency and expression levels. In 3D printing， it provides novel strategies for precise drug release control and the production of high-quality biological products. As a versatile material， poloxamer holds promising prospects in the pharmaceutical field.

In modern society, people are increasingly exposed to chronic stress, leading to various mental disorders. However, the activities of brain regions, especially neural firing patterns related to specific behaviors, remain unclear. In this study, we introduce a novel approach, NeuroSync, which integrates open-field behavioral testing with electrophysiological recordings from emotion-related brain regions, specifically the central amygdala and the paraventricular nucleus of the hypothalamus, to explore the mechanisms of negative emotions induced by chronic stress in mice. By applying machine vision techniques, we quantified behaviors in the open field, and signal processing algorithms elucidated the neural underpinnings of the observed behaviors. Synchronizing behavioral and electrophysiological data revealed significant correlations between neural firing patterns and stress-related behaviors, providing insights into real-time brain activity underlying behavioral responses. This research combines deep learning and machine learning to synchronize high-resolution video and electrophysiological data, offering new insights into neural-behavioral dynamics under chronic stress conditions.
Animals ; Mice ; Male ; Emotions/physiology* ; Stress, Psychological/physiopathology* ; Action Potentials/physiology* ; Mice, Inbred C57BL ; Behavior, Animal/physiology* ; Machine Learning ; Amygdala/physiopathology* ; Neurons/physiology* ; Paraventricular Hypothalamic Nucleus/physiopathology* ; Brain/physiology*

Compared with conventional transdermal drug delivery systems, dissolving microneedles significantly enhance drug bioavailability by penetrating the stratum corneum barrier and achieving intradermal drug delivery. In order to improve the transdermal bioavailability of dexmedetomidine hydrochloride, in this study, a novel microneedle delivery system was developed for dexmedetomidine hydrochloride based on 3D printing combined with micro-molding. By systematically optimizing the microneedle geometrical parameters, array arrangement, and preparation process parameters, we determined the optimal ratio of drug-carrying matrix as 15% PVP (polyvinyl pyrrolidone) K90. The microneedles exhibited significant drug loading gradients, with mean content of (209.99±27.56) μg/patch, (405.31±30.31) μg/patch, and (621.61±34.43) μg/patch. They showed a regular pyramidal structure under SEM and handheld electron microscopy, and their mechanical strength allowed effective penetration into the stratum corneum. The surface contact angles were all < 90°, indicating excellent hydrophilicity. The microneedles dissolved completely within 10 min after skin insertion, achieving a cumulative release rate of 90% (Higuchi model, r=0.996) during 2 hours of in vitro transdermal permeation. The cytotoxicity test and hemolysis test verified good biocompatibility. Pharmacodynamic evaluation showed that the microneedle group demonstrated pain-relieving effect within 15 min, with the pain threshold at the time point of 60 min being 3 times that in the transdermal cream group. The microneedle system developed in this study not only offers an efficient drug delivery option for patients but also establishes an innovative platform for rapid percutaneous delivery of hydrophilic drugs, demonstrating significant potential in perioperative pain management.
Dexmedetomidine/pharmacokinetics* ; Printing, Three-Dimensional ; Needles ; Drug Delivery Systems/methods* ; Administration, Cutaneous ; Animals ; Microinjections/instrumentation* ; Skin Absorption ; Skin/metabolism*