ObjectiveTo construct and validate a clinical prediction model for inflammatory remission outcomes in rheumatoid arthritis （RA） patients with liver and kidney deficiency syndrome treated with Bushen Zhiwang Decoction （补肾治尪汤， BZD） based on metabolomics. MethodsA prospective cohort study was conducted， enrol-ling 60 RA patients with liver and kidney deficiency syndrome. All patients were treated with BZD and conventional-dose oral conventional synthetic disease-modifying antirheumatic drugs （csDMARDs） for 12 months. Clinical data were collected， and the change in disease activity score in 28 joints （DAS28） after treatment compared with baseline （△DAS28） was used as the primary outcome and grouping criterion. Peripheral blood samples were collected before treatment to analyze plasma metabolites. Differential analysis and least absolute shrinkage and selection operator （LASSO） regression were used to preliminarily screen differential metabolites， followed by machine learning algorithms to further identify a core metabolite combination. Based on the expression levels of the core metabolite combination， a novel metabolite index， namely the metabolomics-based inflammatory remission score （Met-IRS）， was calculated using standar-dized metabolite values， and its clinical applicability was evaluated. A clinical prediction model was constructed by integrating clinical characteristics and Met-IRS， and the model performance was assessed. ResultsAmong the 60 patients， those with △DAS28 ≥ 0.27 were assigned to the high inflammatory remission group， while those with △DAS28 ＜ 0.27 were assigned to the low inflammatory remission group， with 30 cases in each group. Compared to the low inflammatory remission group， the high inflammatory remission group showed a higher frequency of methotrexate use and a lower positive rate of rheumatoid factor （RF） （P＜0.05）. Seven core metabolites were identified as the optimal combination， including mangiferic acid， fatty acid-hydroxy fatty acid ester 40∶6， fatty acid-hydroxy fatty acid ester 18∶0， fatty acid-hydroxy fatty acid ester 36∶1， glucosylceramide， lysophosphatidylcholine 22∶5， and pregnanetriol ketone. The calculated Met-IRS comprehensively reflected the characteristics of differential metabolites and demonstrated clinical applicability. Met-IRS was significantly higher in the high inflammatory remission group than in the low inflammatory remission group， and was positively correlated with high inflammatory remission outcomes （P＜0.05）. Based on the variables Met-IRS， methotrexate use， leflunomide use， and RF positivity， a clinical prediction model for inflammatory remission in RA treatment （Cj-RTRM） was constructed. Model performance evaluation demonstrated that the model had good clinical predictive ability， with an area under the receiver operating characteristic curve （AUC） of 0.880， sensitivity 0.967， specificity 0.700 and Youden's index 0.667. ConclusionThe clinical prediction model Cj-RTRM constructed based on the metabolomics-based inflammatory remission score Met-IRS can effectively predict clinical inflammatory remission outcomes in RA patients treated with BZD and accurately identify the advantageous population for this treatment. This model provides guiding evidence for dynamic inflammation monitoring， targeted management， and identification of populations with advantages in traditional Chinese medicine.

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.

OBJECTIVE To explore the improvement effect and potential mechanism of ginsenoside F1 （GF1） on allergic rhinitis （AR） rats based on the mitogen-activated protein kinase （MAPK）/signal transducer and activator of transcription 3 （STAT3） pathway. METHODS Male SD rats were divided into a control group （ n ＝15） and a modeling group （ n ＝55）. The AR rat model was established by basic sensitization with ovalbumin followed by local challenge. The successfully modeled rats were randomly assigned to the AR group， low-dose GF1 group [2.5 mg/（kg·d） ] ， high-dose GF1 group [5 mg/（kg·d） ] ， loratadine group （positive control， 1.5 mg/（kg·d） ] ， and high-dose GF1+MAPK activator group [5 mg/（kg·d） GF1+0.2 nmol C16-PAF ] ， with 10 rats in each group. Another 10 rats from the control group were randomly selected as the blank control group. Rats in each group received corresponding drug solutions or normal saline via intranasal instillation and/or oral gavage once daily for 28 consecutive days. After the final administration， AR symptoms were observed and rhinitis symptom scores were assessed. Histopathological changes of the nasal mucosa were examined. The number of inflammatory cells in nasal lavage fluid， serum levels of inflammatory cytokines [interleukin-4 （IL-4）， IL-17， interferon-γ （IFN-γ） ] and immunoglobulin E （IgE）， as well as the density of goblet cells， the number of mast cells， and the expression of MAPK/STAT3 pathway-related proteins in nasal mucosal tissues were measured. RESULTS Compared with the blank control group， the AR group exhibited marked pathological damage to the nasal mucosal epithelium， characterized by interstitial edema and inflammatory cell infiltration. The rhinitis symptom score， the number of macrophages， lymphocytes， eosinophils， neutrophils and mast cells， the density of goblet cells， the levels of IL-4， IL-17 and IgE， as well as the protein phosphorylation levels of p38 MAPK and STAT3 were significantly increased or elevated， while the level of IFN-γ was significantly decreased in the AR group （ P ＜0.05）. Compared with the AR group， all administration groups showed recovery of nasal mucosal epithelial pathological damage and significant improvements in all quantitative indicators， with the high-dose GF1 group and loratadine group showing more pronounced improvements than the low-dose GF1 group （ P ＜0.05）. C16-PAF significantly reversed the ameliorative effects of GF1 on all quantitative indicators （ P ＜0.05）. CONCLUSIONS GF1 can alleviate rhinitis symptoms in AR rats and inhibit inflammatory responses， which may be related to the inhibition of the MAPK/STAT3 pathway activation.

Objective To analyze the practical value of spiral CT three-dimensional reconstruction techniquein evaluating triplane fractures of the distal tibia.Methods A retrospective analysis was conducted on183 patients with triplane fractures of the distal tibia admitted to Sichuan Orthopedic Hospital from January 2021 to March 2023.All patients underwent both X-ray and spiral CT examinations.Taking surgical reduction results as the gold standard for diagnosis,the diagnostic accuracies of X-ray examination and spiral CT three-dimensional reconstruction technique for triplane fractures of the distal tibia were analyzed.Results Fracture classification according to the number of fracture fragments showed that among the 183 patients with triplane fractures of the distal tibial,there were 44 cases of four-part fractures,62 cases of three-part fractures,and 77 cases of two-part fractures.The classification by the location of epiphyseal injury in the distal tibia showed 175 cases of lateral type and 8 cases of medial type.According to whether the fracture line involved the articular surface,they were categorized into 94 cases of type I,60 cases of type II,and 29 cases of type III.For the classification of the number of fracture fragments,X-ray misdiagnosed 9 cases of four-part fractures as three-part or two-part fractures,and 21 cases of three-part fractures as two-part fractures,resulting in a diagnostic accuracy of 83.60%.For theclassification of fracture line and articular surface position,X-ray led to misdiagnosis or inaccurate diagnosis in 39 cases,with a diagnostic accuracy of 78.69%.When spiral CT three-dimensional reconstruction technique was used to classify the number of fracture fragments,only 1 case of four-part fracture was misdiagnosed as three-part fracture,and 2 cases of three-part fractures were misdiagnosed as four-part fractures or two-part fractures,yielding a diagnostic accuracy of 98.36%.For the diagnosis of the positional relationship of the fracture line to the articular surface,spiral CT three-dimensional reconstruction technique had 8 misdiagnoses,with a corresponding diagnostic accuracy of 95.63%.Conclusion Spiral CT three-dimensional reconstruction technique can stereoscopically display the spatial information of the triplane fractures of the distal tibia,such as the location,shape,type,and articular surface,exhibiting high accuracy for classification diagnosis and significant application value in the reduction and treatment of triplane fractures of the distal tibia.

The objective of this study was to evaluate the toxicity and safety of novel Mycobacterium tuberculosis fusion strain protein derivatives,referred to as B/R strain active proteins.In cellular experiments,RAW264.7 cells were treated with each vaccine preparation,and apoptosis rates were measured.In subsequent animal experiments,C57BL/6 mice were immunized via subcutaneous injection,and their survival and body weight changes were monitored and recorded at 2,4,8,12,and 16 weeks.The lungs and spleens were harvested to calculate organ coefficients,and pathological examinations were conducted.At the eighth week of immunization,the mice were infected with high concentrations of BCG,and pathological changes in the lungs and spleens were observed 4 weeks post-infection.The apoptosis rate at 6 hours was significantly higher in the experimental group than the PBS group(P<0.05).At 12 and 24 hours,the apoptosis rate in the experimental group remained higher than that in the PBS group,although this difference was not statistically significant.After immunization,mice in all four groups exhibited normal growth patterns,as indicated by stable body weight changes.At 4 and 12 weeks post-immunization,the lung coefficients in the protein group were significantly higher than those in the PBS group at the same time points.Additionally,the lung coefficients in the BCG group were significantly elevated across all time periods(P<0.05).The spleen coefficients in the protein and BCG groups were significantly higher than those in the PBS group at 2,4,8,12,and 16 weeks,whereas the ICD B/R group showed higher spleen coefficients than the PBS group only at week 8(P<0.05).Pathological examination revealed normal lung and spleen tissues in the PBS group.However,during the 2-8 weeks immunization period,lung and spleen tissues in all experimental groups exhibited varying degrees of damage,which gradually diminished by 12-16 weeks.Notably,no tuberculosis nodules were observed in any experimental group.After infection with high concentrations of BCG,no overt pathological changes were observed on the surfaces of the lungs and spleens in any group.Microscopic examination revealed less severe pathological changes in the lungs and spleens of mice in the experimental groups than the PBS group.Furthermore,no statistically significant differences were observed between the protein group and the BCG group.Our findings suggested that the B/R strain active proteins'toxicity and safety profiles were comparable to those of BCG,and showed immunoprotective effects.This study provides an experimental foundation for the development of a novel tuberculosis vaccine.

Objective Cigarette smoke(CS)exposure combined with Klebsiella pneumoniae(KP)infection in mice was used to establish a model of chronic obstructive pulmonary disease(COPD)to investigate the mechanism of airway remodeling.Methods Male BALB/c mice were randomly divided into a Control group,CS group,KP group,and CS+KP group.The mice were exposed to CS,KP,and CS+KP from weeks 1 to 8,and were sacrificed in weeks 4,8,16,and 24.MV,Penh,MLI,MAN,and changes in lung pathological structure were detected.The expression levels of IL-1β and TNF-α in lung tissue were detected by ELISA.Collagen deposition was observed by Masson staining and immunohistochemistry.α-SMA and TGF-β1 expression in lung tissue was detected by immunofluorescence.Human bronchial epithelioid cells(16HBE)were also stimulated by CS and lipopolysaccharide(LPS)in vitro,and the expression levels of airway epithelial junction proteins,autophagy-related protein,and mTOR signaling proteins were detected.Results Compared with the Control group,the CS+KP group mice had significantly decreased MV from weeks 4 to 24(P＜0.05 or P＜0.01)and significantly increased Penh from weeks 8 to 24(P＜0.05 or P＜0.01);while the CS group had markedly decreased MV and markedly increased Penh from weeks 8 to 16(P＜0.05 or P＜0.01).Compared with the Control group,massive inflammatory cell infiltration,alveolar wall thickening,alveolar rupture and fusion,and airway wall thickening were observed by HE staining in CS+KP group from weeks 4 to 24.The CS+KP group mice had significantly decreased MAN and significantly increased MLI,IL-1β and TNF-α in their lung tissue from weeks 4 to 24(P＜0.05 or P＜0.01).The aforementioned inflammation and tissue damage were observed in the CS group and the KP group from week 8 to 16.Compared with the Control group,COL Ⅰ,COL Ⅲ,α-SMA,and TGF-β1 were significantly increased in lung tissue of mice in the CS+KP group from weeks 8 to 16(P＜0.01);COL Ⅰ was significantly increased in the CS group and KP group from weeks 8 to 16(P＜0.01).In addition,increased E-cad and decreased N-cad(P＜0.05);significantly decreased LC3B and Beclin-1(P＜0.05);and significantly increased p-mTORC1,p-P70-S6K,and p-4E-BP1 expression were observed in 16HBE cells exposed to CS and LPS(P＜0.05 or P＜0.01).Conclusion Pulmonary functional decline,pathological changes in lung tissue,and airway remodeling appeared to occur early and persist in COPD mice induced by CS and KP.The mechanisms may be related to the activation of mTORC1 signaling pathway and subsequent inhibition of autophagy.

Objective A new occipital bone removal technology was applied to improve the success rate of brain removal.Methods The skull was sawed based on the traditional brain removal technology,and part of the occipital bone was removed downward centered in external occipital protuberance to the foramen magnum,then exposed the telencephalon,cerebellum and posterior medulla oblongata.After that,removed the tentorium cerebelli and cut down the medulla oblongata and the related cranial nerves at the skull base,then removed the brain tissues.Results The removed brain tissues had structurally intact telencephalon,cerebellum and brain stem,clear vessels in the cerebral sulci,and relatively intact optic chiasm,olfactory tracts and vertebro-basilar arteries.Conclusion Brain removal through a fenestration on the external occipital protuberance can effectively preserve the integrity of brain specimens,and improve the success rate of brain removal,which is of great significance for central nervous system teaching and improvement of human brain tissue repositories.

Objective To analyze the practical value of spiral CT three-dimensional reconstruction techniquein evaluating triplane fractures of the distal tibia.Methods A retrospective analysis was conducted on183 patients with triplane fractures of the distal tibia admitted to Sichuan Orthopedic Hospital from January 2021 to March 2023.All patients underwent both X-ray and spiral CT examinations.Taking surgical reduction results as the gold standard for diagnosis,the diagnostic accuracies of X-ray examination and spiral CT three-dimensional reconstruction technique for triplane fractures of the distal tibia were analyzed.Results Fracture classification according to the number of fracture fragments showed that among the 183 patients with triplane fractures of the distal tibial,there were 44 cases of four-part fractures,62 cases of three-part fractures,and 77 cases of two-part fractures.The classification by the location of epiphyseal injury in the distal tibia showed 175 cases of lateral type and 8 cases of medial type.According to whether the fracture line involved the articular surface,they were categorized into 94 cases of type I,60 cases of type II,and 29 cases of type III.For the classification of the number of fracture fragments,X-ray misdiagnosed 9 cases of four-part fractures as three-part or two-part fractures,and 21 cases of three-part fractures as two-part fractures,resulting in a diagnostic accuracy of 83.60%.For theclassification of fracture line and articular surface position,X-ray led to misdiagnosis or inaccurate diagnosis in 39 cases,with a diagnostic accuracy of 78.69%.When spiral CT three-dimensional reconstruction technique was used to classify the number of fracture fragments,only 1 case of four-part fracture was misdiagnosed as three-part fracture,and 2 cases of three-part fractures were misdiagnosed as four-part fractures or two-part fractures,yielding a diagnostic accuracy of 98.36%.For the diagnosis of the positional relationship of the fracture line to the articular surface,spiral CT three-dimensional reconstruction technique had 8 misdiagnoses,with a corresponding diagnostic accuracy of 95.63%.Conclusion Spiral CT three-dimensional reconstruction technique can stereoscopically display the spatial information of the triplane fractures of the distal tibia,such as the location,shape,type,and articular surface,exhibiting high accuracy for classification diagnosis and significant application value in the reduction and treatment of triplane fractures of the distal tibia.

ObjectiveTo study the clinical characteristics and influencing factors of rheumatoid arthritis （RA） in the patients with cold dampness obstruction syndrome. MethodsThe RA patients treated in the Department of Traditional Chinese Medicine and Rheumatology of the China-Japan Friendship Hospital from August 2022 to June 2024 were selected. The demographic information， clinical data， laboratory test results， and traditional Chinese medicine （TCM） symptom information were collected for syndrome differentiation， on the basis of which the characteristics and influencing factors of cold dampness obstruction syndrome were analyzed. ResultsA total of 258 RA patients were selected in this study， including 88 （34.1%） patients with cold dampness obstruction syndrome， 53 （20.5%） patients with dampness and heat obstruction syndrome， 31 （12.0%） patients with wind dampness obstruction syndrome， 29 （11.2%） patients with liver-kidney deficiency syndrome， 19 （7.4%） patients with Qi-blood deficiency syndrome， 14 （5.4%） patients with phlegm-stasis obstruction syndrome， 15 （5.8%） patients with stasis obstructing collateral syndrome and 9 （3.5%） patients with Qi-Yin deficiency syndrome. The patients were assigned into two groups of cold dampness obstruction syndrome and other syndromes. The group of cold dampness obstruction syndrome had lower joint fever， 28-tender joint count （TJC28）， and 28-joint disease activity score （DAS28）-C-reactive protein （CRP） and higher central sensitization， cold feeling of joints， fear of wind and cold， cold limbs， and abdominal distention than the group of other syndromes （P<0.05）. The binary logistic regression analysis showed that central sensitization （OR 5.749， 95%CI 2.116-15.616， P<0.001） and DAS28-CRP （OR 0.600， 95% CI 0.418-0.862， P=0.006） were the independent factors influencing cold dampness obstruction syndrome in RA. ConclusionCold dampness obstruction syndrome is a common syndrome in RA patients. It is associated with central sensitization， cold feeling of joints， abdominal distension and may be a clinical syndrome associated with central sensitization.