ObjectiveA mouse model of vaccinia virus Tiantan strain (VTT)-induced encephalopathy was developed using AG6 mice. MethodsVTT was amplified by infecting Vero cells at a multiplicity of infection (MOI) of 0.01, followed by concentration and titration. After 72 h of incubation, virus-containing cells were collected and subjected to concentration. The concentrated viral suspension was serially diluted (10-fold dilutions) and added to 6-well plates containing confluent Vero cell monolayers for plaque assay. The number of plaques formed in each well was counted, and the virus titer was calculated based on the dilution factor. Fourteen 5-6-week-old AG6 mice (half male and half female, housed separately by sex) were randomly divided into a control group (n=3, PBS), a low-dose group (n=6, 1×10⁵ PFU), and a high-dose group (n=5, 5×10⁵ PFU). The mice were anesthetized by isoflurane inhalation and then infected via intranasal instillation. The mental state of the mice in each group was observed daily, and the body weight and mortality were recorded. On day 13 post-infection, 2% Evans Blue (4 mL/kg body weight) was administered via tail vein injection to assess blood-brain barrier (BBB) disruption. Subsequently, brain tissue samples were collected for immunofluorescence analysis to evaluate the activation of astrocytes and microglia. ResultsThe titer of purified VTT was 1×10⁷ PFU/mL. Compared with the control group, mice in the low-dose group showed no significant change in body weight, and no lethality was observed. In contrast, mice in the high-dose group exhibited significant weight loss starting on day 5 post-infection (P<0.05), accompanied by lethality. On day 13 post-infection, no Evans Blue extravasation was detected in the brain tissues of the low-dose group, while the olfactory bulb region of the high-dose group displayed distinct blue staining, indicating disruption of the BBB. Immunofluorescence analysis revealed no significant proliferation of astrocytes and microglia in the olfactory bulb region of the low-dose group on day 13 post-infection. In contrast, marked activation of glial cells was observable in the high-dose group. ConclusionAn animal model of VTT-induced encephalopathy in AG6 mice is successfully established, characterized by BBB disruption and reactive gliosis specifically localized to the olfactory bulb region, manifested as astrocytic and microglial proliferation.

Diabetic retinopathy(DR)has emerged as the leading cause of vision loss among working-age people in many countries under the increasing prevalence of diabetes and the longevity of the population. The pathogenesis of DR is complicated and has not been fully elucidated at present, while the treatment methods of DR have not been greatly improved, mainly retinal laser photocoagulation, anti-vascular endothelial growth factor(VEGF)treatment and vitrectomy surgery. The current treatment methods not only have shortcomings, but also bring serious economic burden to patients. Therefore, new methods are needed to explore the pathogenesis of DR, discover new treatments or improve current treatments, and improve the satisfaction of DR patients. In recent years, the identification and quantification of proteins expressed in blood, retina, vitreous humor, aqueous humor, and tears of all observable DR patients and DR rats and differentially expressed proteins after drug intervention have provided new ideas for further exploring the pathogenesis, diagnosis and treatment of DR with the rise of proteomics, which put forward new insights into early detection and treatment.The proteomics of DR in recent years are reviewed, in order to provide new ideas for the diagnosis and treatment of DR.

Diabetic retinopathy(DR)has emerged as the leading cause of vision loss among working-age people in many countries under the increasing prevalence of diabetes and the longevity of the population. The pathogenesis of DR is complicated and has not been fully elucidated at present, while the treatment methods of DR have not been greatly improved, mainly retinal laser photocoagulation, anti-vascular endothelial growth factor(VEGF)treatment and vitrectomy surgery. The current treatment methods not only have shortcomings, but also bring serious economic burden to patients. Therefore, new methods are needed to explore the pathogenesis of DR, discover new treatments or improve current treatments, and improve the satisfaction of DR patients. In recent years, the identification and quantification of proteins expressed in blood, retina, vitreous humor, aqueous humor, and tears of all observable DR patients and DR rats and differentially expressed proteins after drug intervention have provided new ideas for further exploring the pathogenesis, diagnosis and treatment of DR with the rise of proteomics, which put forward new insights into early detection and treatment.The proteomics of DR in recent years are reviewed, in order to provide new ideas for the diagnosis and treatment of DR.

To determine the optimal cultivation duration and harvest period for cultivated Notopterygium incisum and promote its industrial development, this study established a characteristic chromatographic profile of cultivated N. incisum and employed chemometrics combined with entropy-weighted grey correlation analysis to assess differences in agronomic traits and quality indicators across different cultivation years and harvest periods. By comparing with reference substances, ten common peaks were identified, including chlorogenic acid, p-coumaric acid, ferulic acid, marmesinin, nodakenin, isochlorogenic acid B, notopterol, phenethyl ferulate, isoimperatorin, and falcarindiol. The similarity between the characteristic chromatographic profiles of N. incisum at different cultivation years and the reference profile was all above 0.932. Principal component analysis(PCA) and orthogonal partial least squares discriminant analysis(OPLS-DA) revealed that the quality of 1-to 3-year-old cultivated N. incisum was highly dispersed and unstable, whereas the quality of 4-year-old cultivated N. incisum remained relatively stable across different harvest periods. This suggests that the accumulation of relevant compounds in the medicinal material had reached a plateau, confirming that the optimal cultivation period for N. incisum is four years. Entropy-weighted grey correlation analysis indicated that the quality of 4-year-old cultivated N. incisum across different harvest periods ranked from highest to lowest as follows: November, December, October, August, July, and September, demonstrating that November is the optimal harvest time. The findings of this study establish the suitable cultivation duration and optimal harvest period for N. incisum, providing a scientific basis for cultivation guidance and quality standardization.
Chromatography, High Pressure Liquid/methods* ; Apiaceae/chemistry* ; Entropy ; Chemometrics/methods* ; Drugs, Chinese Herbal/chemistry* ; Principal Component Analysis ; Quality Control

Drug administration via hollow microneedles (HMN) have the advantages of painlessness, avoidance of first-pass effect, capability of sustained infusion, and no need for professional personnel operation. In addition, HMN can also be applied in the fields of body fluid extraction and biosensors, showing broad application prospects. However, traditional manufacturing technologies cannot meet the demand for low-cost mass production of HMN, limiting its widespread application. This paper reviews the main manufacturing technologies used for HMN in recent years, which include photolithography and etching, laser etching, sputtering and electroplating, micro-molding, three-dimensional (3D) printing and drawing lithography. It further analyzes the characteristics and limitations of existing manufacturing technologies and points out that the combination of various manufacturing technologies can improve production efficiency to a certain extent. In addition, this paper looks forward to the future trends of HMN manufacturing technology and proposes possible directions for its development. In conclusion, it is expected that this review can provide new ideas and references for follow-up research.
Printing, Three-Dimensional ; Needles ; Humans ; Drug Delivery Systems/methods* ; Equipment Design ; Microinjections/methods*

ObjectiveTo investigate the effects of Congrong Zonggan capsules （CRZG） on cognitive impairment in the Alzheimer's disease （AD） model of mice and its related mechanisms. MethodsSPF grade 4-week-old 5×FAD mice were divided into a model group， low-dose CRZG （0.819 g·kg^-1） and high-dose CRZG （1.638 g·kg^-1） groups， and Donepezilepezil hydrochloride group （2 mg·kg^-1）， with eight mice in each group. Eight C57 mice with the same background were set as the normal group. After one week of adaptive feeding， mice were orally administered continuously for six months. On the 5th month of drug administration， Y maze， new object recognition， and Morris water maze tests were conducted separately. After administration， mouse brain tissue was taken， and the levels of tumor necrosis factor-α （TNF-α） and interleukin-6 （IL-6） in brain tissue were detected by enzyme-linked immunosorbent assay （ELISA）. Immunofluorescence （IF） was used to detect the expression of small glial cell markers Iba1， astrocyte markers GFAP， and amyloid protein 1-42 （Aβ_1-42） in the hippocampus of the brain tissue. The hematoxylin-eosin （HE） staining was used to detect pathological changes in the hippocampus of brain tissue. Western blot was used to detect the expression of nuclear factor-κB （NF-κB） p65， NOD-like receptor protein 3 （NLRP3）， cleaved Caspase-1， apoptosis-associated speck-like protein containing a CARD （ASC）， and other proteins in the brain tissue. ResultsCompared with those in the normal group， the mice in the model group had obvious cognitive impairment. The spontaneous alternation rate of the Y maze was decreased， and the discrimination index of novel object recognition was decreased significantly （P<0.01）. The escape latency in the water maze was shortened significantly （P<0.01）. The contents of IL-6 and TNF-α in brain tissue were increased. The fluorescence levels of Iba1 and Aβ_1-42 in the hippocampus were significantly increased （P<0.01）. There was a significant increase in neuronal lesions， neuronal atrophy， loose arrangement of tissue structure， and abnormal erythrocyte aggregation in the hippocampus. The protein expressions of p-NF-κB p65/NF-κB p65， cleaved Caspase-1， ASC， IL-6， and IL-1β were significantly increased （P<0.05， P<0.01）. Compared with the model group， the spontaneous alternation rate and discrimination index of the high-dose CRZG group were increased significantly （P<0.01）， and the escape latency was shortened significantly （P<0.05， P<0.01）. The content of IL-6 decreased in the brain， and that of TNF-α dropped significantly （P<0.01）. The expression of Iba1 protein and Aβ_1-42 in the hippocampus decreased significantly （P<0.05， P<0.01）. The hippocampal neurons were densely arranged， and the pyramidal nuclei were clear and centered. The abnormal aggregation of red blood cells was alleviated. The value of p-NF-κB/NF-κB proteins and the expression of ASC， cleaved Caspase-1， IL-6， and IL-1β were significantly decreased （P<0.05， P<0.01）. ConclusionCRZG can effectively improve cognitive impairment in 5×FAD mice with Alzheimer's disease， and its mechanism may be related to the regulation of the NF-κB/NLRP3 pathway to reduce the abnormal activation of microglia and inhibit neuroinflammation.

Objective:To evaluate the prevalence, potential risk factors, and correlation between coronary and peripheral microvascular dysfunction in heart failure with non-reduced ejection fraction (nHFrEF) patients.Methods:This was a prospective registry study. nHFrEF patients admitted to Zhongshan Hospital affiliated with Fudan University from December 2021 to December 2023 were enrolled. According to coronary flow reserve (CFR) or reactive congestion index (RHI), enrolled patients were divided into coronary microvascular endothelial dysfunction (CMD) group (CFR<2.5) and no CMD group (CFR≥2.5) or peripheral microvascular endothelial dysfunction (MED) group (RHI<1.67) and no MED group (RHI≥1.67). Patients′ general information, laboratory and auxiliary examination data were collected. Univariate and multivariate logistic regression were used to analyze the influencing factors of CMD and MED in nHFrEF patients, and Spearman correlation analysis was used to evaluate the correlation between MED and CMD.Results:A total of 142 nHFrEF patients were enrolled, aged 69.0 (59.0, 74.0) years, with a male proportion of 66.9% (95/142). The grouping results were as follows: (1) According to CFR, there were 73 cases in the CMD group and 69 cases in the no CMD group; (2) According to RHI, there were 57 cases in the MED group and 85 cases in the no MED group. The prevalence of CMD and MED in this study was 51.4% (73/142) and 40.1% (57/142), respectively. Univariate logistic regression analysis showed that increased heart rate, chronic kidney disease, atrial fibrillation, elevated N-terminal pro-B type natriuretic peptide levels, and increased urinary albumin/creatinine ratio were risk factors for CMD, while increased RHI was a protective factor for CMD; Atrial fibrillation is a risk factor for MED, while increased CFR is a protective factor for MED. Incorporating clinically significant variables from univariate analysis into multivariate analysis, the results showed that increased heart rate and elevated RHI remained risk and protective factors for CMD, respectively; increased CFR remains a protective factor for MED. Spearman correlation analysis showed that CFR was negatively correlated with lg urinary albumin/creatinine ratio, lg cardiac troponin T, lg N-terminal pro-B type natriuretic peptide, and heart rate; RHI is positively correlated with CFR.Conclusions:The prevalence of CMD and MED in nHFrEF patients is high, and the two have a certain positive correlation. Increased heart rate and RHI are risk and protective factors for CMD, respectively, while increased CFR is a protective factor for MED. MED may be a potential therapeutic target for nHFrEF patients.

Objective:To explore the clinical efficacy of the sixth generation CyberKnife (M6) in treating patients with brain metastases, and analyze clinical characteristics and dosimetric factors.Methods:Clinical data of patients with brain metastases who received CyberKnife treatment at Sichuan Cancer Hospital from April 2023 to March 2024 were retrospectively analyzed. All patients were treated with CyberKnife with 6 MV X-ray. According to the maximum diameter of brain metastases, the radiation prescription dose of brain metastases was adjusted. The tumor remission, recurrence, 6-month and 1-year overall survival (OS), local control (LC) of intracranial target lesions, progression-free survival (PFS), distant metastasis-free survival (DMFS) of intracranial brain metastases and adverse reactions were evaluated. According to the median biological dose, the survival difference between the groups was compared. Survival analysis was conducted by Kaplan-Meier method. Survival differences among different groups were analyzed by log-rank test.Results:A total of 63 eligible patients with brain metastases were enrolled, with a median age of 59 years (rang: 36-80 years). Among them, 47 patients were diagnosed with primary tumors originating from the lungs, 16 patients with primary tumors originating from other organs; 44 patients with single brain metastases, and 19 patients with 2-3 lesions, respectively. The median biological dose was 67.2 Gy (rang: 47.4-86.4 Gy), and the median single dose was 8 Gy/F (rang: 4-24 Gy/F). The follow-up was conducted until July 15, 2024. The median follow-up time for the entire group was 9 months (rang: 2-15 months). Among the 87 target lesions treated with CyberKnife, 11 patients corresponding to 14 target lesions experienced local recurrence. And the 6-month and 1-year LC rates were 92.5% and 70.9%, respectively. Ten patients corresponding to 16 target lesions died. And the 6-month and 1-year OS rates were 92.7% and 74.8%, respectively. Thirty-five patients corresponding to 50 target lesions experienced disease progression. And the 6-month and 1-year PFS rates were 64.3% and 25.5%, respectively. Thirty-three patients corresponding to 48 target lesions showed distant metastasis outside the target lesions, with a 6-month DMFS of 67.0% and a 1-year DMFS of 33.9%. Group comparison showed that 43 target lesions in the group receiving ≤67.2 Gy irradiation and 44 in the group receiving >67.2 Gy irradiation. The 6-month LC, OS, PFS, and DMFS rates between two groups were 89.8% vs. 97.7% ( P=0.127), 89.8% vs. 95.4% ( P=0.305), 65.4% vs. 68.5% ( P=0.514), 65.4% vs. 68.5% ( P=0.516), respectively. The 1-year LC, OS, PFS, and DMFS rates between two groups were 54.1% vs. 89.5% ( P=0.003), 67.3% vs. 82.9% ( P=0.219), 19.2% vs. 32.7% ( P=0.370) and 23.3% vs. 33.0% ( P=0.533). During the follow-up, only 2 patients (3.2%) were found to have grade 1-2 radiation-induced brain injury (asymptomatic brain injury) by MRI examination, and there were no other radiotherapy related adverse reactions. Conclusions:CyberKnife therapy is clinically effective for brain metastases, with mild adverse reactions. Increasing the tumor irradiation dose can improve local tumor control and is expected to further improve the OS of patients.

Objective To compare functional differences of CD8+T cells in lung tissues between young and aged C57BL/6J mice during the contraction phase and memory immune response phase after infection with influenza A(H1N1)virus.Methods Lung tissues from young(3-month-old)and aged(24-month-old)C57BL/6J female mice without influenza virus infection were collected to prepare single-cell suspensions,which were stimulated with phorbol 12-myristate 13-acetate(PMA)/ionomycin or cluster of differentiation(CD)3/CD28 antibodies(T-cell antigen receptor/co-stimulatory signals)respectively(non-specific antigens stimulation).Flow cytometry intracellular cytokine staining(ICS)was performed on lung CD8+T cells to detect their secretion capacity of tumor necrosis factor-α(TNF-α)and interferon-γ(IFN-γ).Young and aged C57BL/6J mice were infected intranasally with 490 PFU PR8 influenza virus,and reinfected with homologous influenza virus 28 days later.Lung tissues were isolated on day 28(the contraction phase)and day 32(the memory immune response phase)after primary infection.Influenza virus-specific MHC-Ⅰ tetramer staining was used to detect the proportion of virus-specific CD8+T cells in lung tissue CD8+T cells,and ICS was used to analyze TNF-α,IFN-γ,and granzyme B expression in CD8+CD44high T cell subset.Results After non-specific antigen stimulation,TNF-α and IFN-γ secretion capacity in lung tissue CD8+T cells of aged group mice was significantly higher than that of young group(P＜0.05).After virus-specific antigen stimulation,there were no statistically significant differences in the proportion of virus-specific CD8+T cells and the expression levels of TNF-α,IFN-γ,and granzyme B between the two groups of mice during the contraction phase(P＞0.05),while during the memory immune response phase,the proportion of virus-specific CD8+T cells and the expression levels of TNF-α,IFN-γ,and granzyme B in the aged group mice were significantly lower than those in the young group(P＜0.05).Conclusion CD8+T cells in aged mice maintain normal immune-related factor expression function under non-specific antigen stimulation,but show impaired immune-related factor expression function during antigen-specific memory immune response phase,suggesting that aging leads to defects in the formation or maintenance of CD8+T cell immune memory.

Aim To evaluate the bioequivalence of two oral preparations of rivaroxaban tablets(test preparation T and refe-rence preparation R)in fasting/postprandibular state in healthy Chinese subjects.Methods A randomized,open,single-dose,four-cycle,completely repeated crossover experiment was used in this study.A total of 70 healthy male and female subjects were enrolled,including 38 subjects in the fasting group and 32 sub-jects in the postprandial group.Rivaroxaban tablets(2.5 mg/tablet)were taken orally once per cycle and their reference preparations were tested.The plasma rivaroxaban concentration was determined by LC-MS/MS method.The pharmacokinetic parameters of rivaroxaban tablets were calculated by WinNonlin software,and the parameters were analyzed and processed.Re-sults The PK parameters of rivaroxaban tablets and reference preparations in fasting group were as follows:Cmax was(72.48±17.08)and(66.36±15.64)μg·L-1,respectively.AUC0-t were(383.49±101.06)and(370.43±102.16)h·ng·mL-1,and AUC0-inr were(389.58±102.28)and(375.84±103.01)h·μg·L-,respectively.Main PK parameters of subjects taking rivaroxaban tablets orally after meals:Cmax were(66.48±15.64 and 60.87±13.44)μg·L-1,AUC0-t were(404.44±72.58)and(381.80±79.93)h·μg·L-1,re-spectively.AUC0_inf was(410.88±73.55)and(393.64±69.71)h·μg·L-1,respectively.Under fasting and postmeal conditions,subjects took rivaroxaban test and reference prepara-tion orally,one tablet(2.5 mg/tablet)each time.The geometric mean of the main pharmacokinetic parameters of rivaroxaban in plasma(Cmax,AUC0-t,AUC0-inf)and their corresponding values had a 90％confidence interval ranging from 80.00％to 125.00％.No serious adverse events or unexpected adverse e-vents occurred in both groups.Conclusion Rivaroxaban tablets are bioequivalent and safe in vivo under fasting and postprandial conditions.