Objective To investigate the associations of immune cells,CD8+and CD4+T cells,and high-sensitivity C-reactive protein(hs-CRP)with cognitive function,and to explore the relation-ships among immunity,chronic inflammation,and Alzheimer's disease-related cognitive impair-ment.Methods A cross-sectional study was conducted on 101 patients with primary complaints of memory decline who visited the Alzheimer's Disease Clinic of Dongzhimen Hospital from June to December 2024.Mini-Mental State Examination(MMSE)and Delayed Story Recall Task(DSR)were performed to assess their cognitive function,and according to the results,they were divided into observation group(cognitively impaired,60 cases)and control group(cognitively normal,41 cases).Peripheral blood levels of CD8+T cells,CD4+T cells,and hs-CRP were compared between the two groups.Results The observation group exhibited significantly lower total scores and scores of different dimensions of MMSE and DSR scores,but notably higher activities of daily liv-ing scores than the control group(P＜0.05,P＜0.01).Serum hs-CRP level was obviously elevated in the observation group than the control group(P＜0.05).Binary logistic regression analysis revealed that CD8+T cells(OR=0.998,95％CI:0.996-1.000,P=0.038)and body mass index(OR=0.843,95％CI:0.719-0.990,P=0.037)were protective factors,while hs-CRP(OR=2.004,95％CI:1.215-3.306,P=0.006)was an independent risk factor for cognitive impairment.Spearman's rank correlation analysis showed a significant positive correlation between hs-CRP and CD4+T cells(P=0.011),but no significant association with CD8+T cells(P＞0.05).Conclusion Chronic inflammation and immune dysregulation synergistically contribute to cogni-tive decline.Hs-CRP may serve as a potential screening biomarker for cognitive impairment in pri-mary care settings.

Objective To investigate the associations of immune cells,CD8+and CD4+T cells,and high-sensitivity C-reactive protein(hs-CRP)with cognitive function,and to explore the relation-ships among immunity,chronic inflammation,and Alzheimer's disease-related cognitive impair-ment.Methods A cross-sectional study was conducted on 101 patients with primary complaints of memory decline who visited the Alzheimer's Disease Clinic of Dongzhimen Hospital from June to December 2024.Mini-Mental State Examination(MMSE)and Delayed Story Recall Task(DSR)were performed to assess their cognitive function,and according to the results,they were divided into observation group(cognitively impaired,60 cases)and control group(cognitively normal,41 cases).Peripheral blood levels of CD8+T cells,CD4+T cells,and hs-CRP were compared between the two groups.Results The observation group exhibited significantly lower total scores and scores of different dimensions of MMSE and DSR scores,but notably higher activities of daily liv-ing scores than the control group(P＜0.05,P＜0.01).Serum hs-CRP level was obviously elevated in the observation group than the control group(P＜0.05).Binary logistic regression analysis revealed that CD8+T cells(OR=0.998,95％CI:0.996-1.000,P=0.038)and body mass index(OR=0.843,95％CI:0.719-0.990,P=0.037)were protective factors,while hs-CRP(OR=2.004,95％CI:1.215-3.306,P=0.006)was an independent risk factor for cognitive impairment.Spearman's rank correlation analysis showed a significant positive correlation between hs-CRP and CD4+T cells(P=0.011),but no significant association with CD8+T cells(P＞0.05).Conclusion Chronic inflammation and immune dysregulation synergistically contribute to cogni-tive decline.Hs-CRP may serve as a potential screening biomarker for cognitive impairment in pri-mary care settings.

BACKGROUND:Enhancing the differentiation of induced pluripotent stem cells into lung stem cells is crucial for repairing lung injuries.NKX2.1 is the earliest marker of lung epithelial differentiation and plays a significant regulatory role in lung development.However,the impact of its expression on the differentiation of induced pluripotent stem cells into lung stem cells remains inadequately understood.OBJECTIVE:To investigate the effect of NKX2.1 on the differentiation of induced pluripotent stem cells into lung stem cells.METHODS:Induced pluripotent stem cells were cultured in vitro.The expression of specific pluripotent stem cell genes was assessed using real-time fluorescence quantitative PCR.NKX2.1 was overexpressed in induced pluripotent stem cells,which were then induced to differentiate into lung stem cells.The expression of FoxA2,SOX9,and P63 was determined via quantitative PCR and immunofluorescence on day 7 of induction of differentiation.The expression of the alveolar marker SPB and SPC was evaluated through immunofluorescence staining on day 7 of induction of differentiation.RESULTS AND CONCLUSION:(1)Induced pluripotent stem cells in vitro were tightly packed and showed typical clonoid growth and significantly expressed stem cell-specific genes OCT-4,SOX2,and NANOG.(2)Compared with the non-transfected control group,the expression of NKX2.1 in human induced pluripotent stem cells was significantly increased in the NKX2.1 overexpression group(P＜0.000 1).(3)Seven days after induction of differentiation,compared with the non-transfected control group,the expression of lung stem cell-related markers FoxA2,SOX9,and P63 was significantly increased in the NKX2.1 overexpression group(P＜0.000 1).(4)Thirteen days after induction of differentiation,compared with the non-transfected control group,the fluorescence intensity of alveolar cell marker molecules SPB and SPC increased significantly in the overexpression NKX2.1 group.The results show that NKX2.1 can promote the differentiation of induced pluripotent stem cells into lung stem cells.

BACKGROUND:Enhancing the differentiation of induced pluripotent stem cells into lung stem cells is crucial for repairing lung injuries.NKX2.1 is the earliest marker of lung epithelial differentiation and plays a significant regulatory role in lung development.However,the impact of its expression on the differentiation of induced pluripotent stem cells into lung stem cells remains inadequately understood.OBJECTIVE:To investigate the effect of NKX2.1 on the differentiation of induced pluripotent stem cells into lung stem cells.METHODS:Induced pluripotent stem cells were cultured in vitro.The expression of specific pluripotent stem cell genes was assessed using real-time fluorescence quantitative PCR.NKX2.1 was overexpressed in induced pluripotent stem cells,which were then induced to differentiate into lung stem cells.The expression of FoxA2,SOX9,and P63 was determined via quantitative PCR and immunofluorescence on day 7 of induction of differentiation.The expression of the alveolar marker SPB and SPC was evaluated through immunofluorescence staining on day 7 of induction of differentiation.RESULTS AND CONCLUSION:(1)Induced pluripotent stem cells in vitro were tightly packed and showed typical clonoid growth and significantly expressed stem cell-specific genes OCT-4,SOX2,and NANOG.(2)Compared with the non-transfected control group,the expression of NKX2.1 in human induced pluripotent stem cells was significantly increased in the NKX2.1 overexpression group(P＜0.000 1).(3)Seven days after induction of differentiation,compared with the non-transfected control group,the expression of lung stem cell-related markers FoxA2,SOX9,and P63 was significantly increased in the NKX2.1 overexpression group(P＜0.000 1).(4)Thirteen days after induction of differentiation,compared with the non-transfected control group,the fluorescence intensity of alveolar cell marker molecules SPB and SPC increased significantly in the overexpression NKX2.1 group.The results show that NKX2.1 can promote the differentiation of induced pluripotent stem cells into lung stem cells.

Objective:To analyze the clinical data and to screen the mortality risk factors of necrotizing fasciitis (NF) secondary to intestinal fistulas (NFsIF).Methods:This study was a retrospective observational study. The data of all NFsIF cases who met the inclusion criteria and were admitted into Shandong Provincial Hospital Affiliated to Shandong First Medical University (hereinafter referred to as our unit) from January 2000 to October 2023, and in PubMed, Web of Science, Scopus, China National Knowledge Infrastructure, and Chinese Medical Journal Network databases from its establishment to October 2023 were retrieved and screened. Based on clinical outcomes, the cases were divided into survival group (47 males and 24 females) and death group (16 males and 7 females), and the mortality rate was calculated. Clinical data of patients in the two groups including age, underlying diseases (most related to NF), symptom duration before presentation, white blood cell count, causes of NF, signs of peritonitis, scope of NF involvement, and intestinal management and wound management measures were compared and analyzed to screen the risk factors of death in 94 patients with NFsIF.Results:A total of 94 valid cases were collected, including 90 patients reported in the literature and 4 patients admitted to our unit, with the mortality rate of patients being 24.5% (23/94). Univariate analysis showed that there were no statistically significant differences in age, underlying diseases, symptom duration before presentation, white blood cell count, causes of NF, signs of peritonitis, scope of NF involvement between patients in the two groups ( P>0.05); there were statistically significant differences in intestinal treatment and wound treatment between the two groups (with χ2 values of 17.97 and 8.33, respectively, P<0.05). Multivariate logistic regression analysis showed that both intestinal treatment measures and wound treatments measures were independent risk factors for death in 94 NFsIF patients, among which first-stage colostomy+late-stage reconstruction and negative presssure therapy had higher protective effects (with odds ratios of 0.05 and 0.27, respectively, 95% confidence intervals of 0.01-0.33 and 0.08-0.88, respectively, P<0.05). Conclusions:The mortality risk of patients with NFsIF is high. Based on comprehensive treatments, active intestinal and wound treatment may be the key to avoid death, with first-stage colostomy+late-stage reconstruction and negative pressure therapy having higher protective effects.

OBJECTIVE To optimize the extraction technology for the raw drugs of Sanse powder gel paste. METHODS SD rats were divided into blank group， model group， traditional technology group， water extraction group and ethanol extraction group， with 5 rats in each group. Anterior cruciate ligament transection was used to construct knee osteoarthritis model， and the pharmacodynamic effects of different extraction methods on arthritic rats were investigated. Analgesic experiments were conducted using cold and hot pain thresholds and pain mediators calcitonin gene-related peptide （CGRP）， cyclooxygenase-2 （COX-2）， substance P （SP）， and prostaglandin E2 （PGE2） contents as indicators. HE staining was performed on the synovial membrane of rats to observe the degree of synovial cell proliferation， inflammatory infiltration and vascular invasion， and anti-inflammatory experiments were conducted using protein and mRNA expressions of interleukin-1β （IL-1β）， tumor necrosis factor-α （TNF-α） and IL-6 as indicators. The analgesic and anti-inflammatory effects were compared among those groups. In the orthogonal test， ethanol dosage， extraction time and extraction times were used as evaluation factors， and the contents of casticin， strychnine and toxiferine were taken as evaluation indicators； comprehensive score was calculated. The validation experiments were carried out after optimizing the extraction technology of the raw drugs of Sanse powder gel paste. RESULTS Compared with the model group， the cold and heat pain thresholds of drug administration groups （except for the traditional technology group） were all increased significantly （P＜0.05）， while the contents of pain （No.Y2021rc02） mediators CGRP， COX-2， SP and PGE2 were all decreased significantly （P＜0.05）. HE staining showed that inflammatory cell infiltration， fibrosis and collagen deposition were 炎。E-mail：liuzixiu3221@126.com decreased in the administration groups； a small amount of capillary proliferation could be found； the protein and mRNA expressions of inflammatory factors such as IL-1β， IL-6 and TNF-α were decreased significantly in synovial tissue of rats in administration groups （P＜0.05）. Compared with the traditional technology group， most indicators of the ethanol extraction group were significantly reduced （P＜0.05）， and only heat pain threshold and mRNA expression of IL-6 in rats were decreased significantly in the water extraction group （P＜0.05）. The optimal extraction technology of the raw drugs of Sanse powder gel paste included suitable dose of Sanse powder， 8-fold 55% ethanol， heating reflux extraction for 90 minutes， extracting twice. The results of 3 times of verification experiments showed that the average contents of casticin， strychnine and toxiferine were 0.007%， 0.092%， and 0.214%， respectively； RSD were all less than 5%. CONCLUSIONS The optimized extraction technology for the raw drugs of Sanse powder gel paste is stable and feasible， which can improve the efficacy of the preparation.

OBJECTIVE To establish a method for concentration determination of caffeine and its three metabolites， theophylline， paraxanthine and theobromine in urine， and apply it in clinical practice. METHODS Using caffeine-13C3-d3 as internal standard （IS）， and the urine samples were protein precipitated with acetonitrile； HPLC-MS/MS method was adopted to determine the concentrations of caffeine and its three metabolites. The determination was performed on Waters ACQUITY UPLC® BEH HILIC column with mobile phase consisting of 60 mmol/L ammonium acetate （A）-acetonitrile （B） （gradient elution） at the flow rate of 0.5 mL/min. The column temperature was set at 38 ℃ ， and the sample size was 2 μL. The electrospray ionization detection was operated in a positive mode by multiple reaction monitoring. The detection ions for quantitative analysis were m/z 195.1→110.0 for caffeine， m/z 181.1→124.0 for theophylline， m/z 181.1→124.0 for paraxanthine， m/z 181.1→138.0 for theobromine， and m/z 198.1→ 140.1 for IS. The above method was used to determine the concentrations of caffeine and its three metabolites in the urine of 19 infants with apnea of prematurity （AOP）. RESULTS The linear ranges of mass concentration of caffeine， theophylline， paraxanthin and theobromine were 0.200-200， 0.050-50.0，0.050 0-50.0， and 0.100-100 μg/mL， respectively. The lower limits of quantification were 0.200， 0.050， 0.050 and 0.100 μg/mL （r＞0.990）， respectively. RSDs of intra-day and intra- day precision were not above 10.37%， and matrix factors were 85.68%-109.90%； extraction recoveries were 93.53%-109.40% （RSD≤15%）， and RSDs of stability tests were all lower than 15%. The concentrations of caffeine and its three metabolites in the urine of 19 cases were （27.346±7.951）， （0.351±0.223）， （0.428±0.395） and （0.472±0.374） μg/mL， respectively. CONCLUSIONS The established HPLC-MS/MS method is simple， sensitive and can be used for the determination of caffeine and its three metabolites in urine samples of AOP.

OBJECTIVE To extract the essential oil of “Wenyujin Rhizoma Concisum-Angelica Sinensis Radix”, and to qualitatively analyze the chemical composition of the extracted essential oil by LC-MS. Network pharmacology was used to explore the mechanism of the essential oil acting on knee osteoarthritis(KOA). METHODS The essential oil of “Wenyujin Rhizoma Concisum-Angelicae Sinensis Radix” was extracted by steam distillation, the components of the essential oil were analyzed by LC-MS. PubChem, Swiss Target Prediction, GeneCards, DAVID and other databases were used to predict disease targets and pathways of active component in prevention and treatment of KOA. The key genes were analyzed by Gene ontology(GO) analysis and Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway enrichment analysis. Cytoscape was used to construct a visual network map of “active component-disease targets”. AutoDock and Pymol were used to verify the molecular docking between the target with the highest degree target and the active components. RESULTS A total of 59 compounds were identified in the “Wenyujin Rhizoma Concisum-Angelicae Sinensis Radix” essential oil, and the main components were curzerenone, isocurcumenol, ligustilide, etc. Through network pharmacology, 28 potential targets of essential oil acting on KOA were screened, including CTSK, PTGS1, PTGS2 and ESR1, etc. KEGG enrichment analysis predicted that “Wenyujin Rhizoma Concisum-Angelicae Sinensis Radix” essential oil mainly targeted TNF signaling pathway, osteoclast differentiation, IL-17 signaling pathway, relaxin signaling pathway, MAPK signaling pathway to treat KOA. The results of molecular docking showed that the screened active components and their corresponding target proteins had positive binding activity. CONCLUSION LC-MS combined with network pharmacology is used to preliminaries explore the main active components and the potential targets and mechanism of action of “Wenyujin Rhizoma Concisum-Angelicae Sinensis Radix” essential oil in the intervention of KOA, providing ideas for the further study of “Wenyujin Rhizoma Concisum-Angelicae Sinensis Radix” essential oil.

Objective:To design a knowledge-based cervical cancer planning model and apply it to cases of endometrial cancer and rectal cancer in order to explore the generalization of the model.Methods:A total of 179 cases of pelvic regions with different prescribed doses of dual-arc volumetric modulated arc therapy clinical plans were collected, of which 99 cases of cervical cancer clinical plans with a prescribed dose of 50.4 Gy were used as the training set to establish the RapidPlan model, and the remaining clinical plans were divided into 4 validation groups with 20 cases in each group. The clinical plans for cervical cancer and endometrial cancer with a prescription dose of 50.4 Gy were named groups A and B, while the clinical plan for endometrial cancer and rectal cancer with a prescription dose of 45 Gy were named groups C and D. The model was used to redesign the clinical plans in the 4 groups and the automatic plans were obtained. The planning target volume (PTV) and organ at risk (OAR) dosimetry parameters were compared between automatic plans and clinical plans.Results:The conformity index (CI) of the automatic plans in the A, B, C, and D groups were equivalent to that of the clinical plans ( P>0.05). The homogeneity index (HI) and D2% of the automatic plans in groups A, B, and C were all lower than those in clinical plans(HI, Z=-3.248, -3.360, -2.329, P<0.05; D2%, Z=-2.987, -3.397, -2.442, P<0.05). The HI and D2% of the automatic plans in group D were similar those in the clinical plans ( P>0.05). While ensuring the PTV coverage, the average value of OAR dosimetry parameters in all automatic plans groups were lower than that of the clinical plans. Conclusions:The RapidPlan model established by the cervical cancer clinical plans can complete the automatic plan design for endometrial cancer and rectal cancer under different prescription doses, which preliminarily proves the possibility of the generalization of the RapidPlan model.