Objective: To identify an optimal back-flush solution for leukocyte-depleted filters that maximizes peripheral blood mononuclear cell (PBMC) recovery with high viability, long-term storage stability, and sterility of the harvested residues, thereby providing a clinically translatable strategy. Methods: Three sterile bag-packaged solutions—Saline, Solvent, and Hanks' balanced salt solution (HBSS)—were used to back-flush randomly assigned leukocyte-depleted filters. Nucleated cell recovery rate and viability of the harvested residues were compared. The optimal solution identified was applied to an expanded sample set. PBMC viability and yield were evaluated after 1h vs 48h storage of the residues. PBMCs isolated from the residues were cryopreserved in liquid nitrogen for 1 month, followed by post-thaw comparisons of viability and T-cell expansion capacity. Results: The Solvent group achieved the highest and most consistent nucleated cell recovery rate. Post-flush recovery rate from filters after 400 mL whole blood processing was (21.3±1.6)% for the Solvent group, significantly higher than Saline group (19.2±6.3)% and HBSS group (11.2±5.0)%, with residues from all groups maintaining viability >90%. No biologically significant difference in residue viability was observed between 48h vs 1h storage groups (93.3±2.3)% vs (95.7±1.8)%). PBMC recovery rates from residues showed no statistical difference between 48h vs 1h storage groups [(48.2%±9.5%)vs (40.41%±8.35%), P>0.05], with (17.7±2.6)×10 cells. After 1-month cryopreservation and 10-day expansion, PBMCs isolated from 48-hour-stored residues retained (91.2±3.2)% viability and achieved a (61.9±15.9)-fold expansion. Conclusion: The bag-packaged Solvent, as a back-flush solution, enables sterile acquisition of leukocyte-depleted filter residues through closed-system tubing connections. These residues maintained PBMC viability and recovery rates after 48h storage at 2℃-8℃, with post-cryopreservation (1-month liquid nitrogen) viability and expansion capacity remaining stable. This protocol complies with blood bank regulatory criteria, addresses the concerns about the infectious window period in cell therapy raw materials, and provides a clinically translatable strategy for PBMC-based applications.

Background::Thoracic aortic aneurysm (TAA) is a fatal cardiovascular disease, the pathogenesis of which has not yet been clarified. This study aimed to identify and validate the diagnostic markers of TAA to provide a strong theoretical basis for developing new methods to prevent and treat this disease.Methods::Gene expression profiles of the GSE9106, GSE26155, and GSE155468 datasets were acquired from the Gene Expression Omnibus (GEO) database. Differentially expressed genes (DEGs) were identified using the "limma" package in R. Least absolute shrinkage and selection operator (LASSO), support vector machine-recursive feature elimination (SVM-RFE), random forest, and binary logistic regression analyses were used to screen the diagnostic marker genes. Single-sample gene set enrichment analysis (ssGSEA) was used to estimate immune cell infiltration in TAA.Results::A total of 16 DEGs were identified. The enrichment and functional correlation analyses showed that DEGs were mainly associated with inflammatory response pathways and collagen-related diseases. Collagen type I alpha 1 chain ( COL1A1) and synaptotagmin like 2 ( SYTL2) were identified as diagnostic marker genes with a high diagnostic value for TAA. The expression of COL1A1 and SYTL2 was considerably higher in TAA vascular wall tissues than in the corresponding normal tissues, and there were significant differences in the infiltration of immune cells between TAA and normal vascular wall tissues. Additionally, COL1A1 and SYTL2 expression were associated with the infiltration of immune cells in the vascular wall tissue. Single-cell analysis showed that COL1A1 in TAA was mainly derived from fibroblasts and SYTL2 mainly from cluster of differentiation (CD)8 + T cells. In addition, single-cell analysis indicated that fibroblasts and CD8 + T cells in TAA were significantly higher than those in normal arterial wall tissue. Conclusions::COL1A1 and SYTL2 may serve as diagnostic marker genes for TAA. The upregulation of SYTL2 and COL1A1 may be involved in the inflammatory infiltration of the vessel wall and poor extracellular matrix remodeling, promoting the progression of TAA.

Aim To investigate the mechanism of py-roptosis mediated by the NLRP3/caspase-1/GSDMD signaling pathway and the intervention effect of Radix Angelica Sinensis and Radix Astragalus ultrafiltration extract(RAS-RA)in radiation-induced pulmonary fi-brosis.Methods Fifty Wistar rats were randomly di-vided into five groups,with ten rats in each group.Ex-cept for the blank control group,all other groups of rats were anesthetized and received a single dose of 40 Gy X-ray local chest radiation to establish a radiation-in-duced pulmonary fibrosis rat model.After radiation,the rats in the RAS-RA intervention groups were orally administered doses of 0.12,0.24 and 0.48 g·kg-1 once a day for 30 days.The average weight and lung index of the rats were observed after 30 days of contin-uous administration.Hydroxyproline(HYP)content in lung tissue was determined by hydrolysis method.The levels of IL-18 and IL-1 β in serum were detected by ELISA.Lung tissue pathological changes were ob-served by HE and Masson staining.Ultrastructural changes in lung tissue were observed by transmission e-lectron microscopy.The expression levels of NLRP3/caspase-1/GSDMD pyroptosis pathway-related proteins and fibrosis-related proteins in lung tissue were detec-ted by Western blot.Results Compared with the blank group,the HYP content in lung tissue and the levels of IL-18 and IL-1 β in serum significantly in-creased in the model group(P＜0.01).HE and Mas-son staining showed inflammatory cell infiltration and collagen fiber deposition.Transmission electron mi-croscopy revealed increased damaged mitochondria,disordered arrangement,irregular morphology,shallow matrix,outer membrane rupture,mostly fractured and shortened cristae,mild expansion,increased electron density of individual mitochondrial matrix,mild sparse structure of lamellar bodies,partial disorder,unclear organelles,and characteristic changes of pyroptosis.Western blot analysis showed increased expression of caspase-1,GSDMD,NLRP3,CoL-Ⅰ,α-SMA,and CoL-Ⅲ proteins(P＜0.01).Compared with the model group,the RAS-RA intervention group showed signifi-cant improvement in body mass index and lung index of rats,decreased levels of IL-18 and IL-1 β inflammatory factors(P＜0.01),improved mitochondrial structure,reduced degree of fibrosis,and decreased expression of caspase-1,GSDMD,NLRP3,COL-Ⅰ,COL-Ⅲ,and α-SMA proteins in lung tissue(P＜0.01).Conclusion RAS-RA has an inhibitory effect on radiation-in-duced pulmonary fibrosis,and its mechanism may be related to the inhibition of pyroptosis through the regu-lation of the NLRP3/caspase-1/GSDMD signaling pathway.

To understand the distribution of ticks in the Wuwei Region,enrich tick species data,and provide a basis for the prevention of tick-borne diseases,tick were collected using flagging and tick-picking methods during the highest activity period from April to September in 2021 and 2022 in the mountainous areas of Wuwei City.The ticks were identified based on morpho-logical and molecular biological characteristics,and characteristic sequences were obtained.A systematic evolutionary tree was constructed using the neighbor-joining method in MEGA 11.0 software.In total,7 342 ticks collected in Wuwei,which be-longed to 5 species from 4 genera with in the Ixodidae family,which included Dermacentor nuttalli,Hyalomma asiaticum,Ixodes canisuga,Haemaphysalis longicornis and Haema-physalis danieli.Ticks of the same species clustered together into the same branch of an evolutionary tree.In the Wuwei Re-gion,five common tick species are found across various habi-tats,with each habitat featuring different distributions of tick species and populations.The Dermacentor nuttalli is the dom-inant tick species in this area.

Objective To explore whether voluntary wheel running affects liver oxidative stress by regulating the Nrf2/HO-1 pathway,thereby alleviating HFFC diet-related lipid deposition in the liver.Methods Eight-week-old C57BL/6J mice were randomly divided into a normal diet group(NC group,n=10)and high-fat,fructose,and cholesterol diet group(HFFC group,n=20)after 1 week of adaptive feeding.Ten weeks of feeding later,mice in the HFFC group were divided into a quiet group(HFFC group,n=10)and HFFC combined with exercise group(HFFC+EX group,n=10).HFFC+EX group mice were caged with voluntary running wheels for free movement,and the number of running wheels was recorded every day for 8 weeks.After the last treatment,the mice were sacrificed by fasting for 12 hours at an interval of 24 hours,and the blood and liver were collected for analysis.Results(1)Body weight,liver weight,and liver index of mice fed the HFFC diet were significantly higher than those of the NC group,which significantly decreased after exercise(P＜0.05).(2)Compared with the NC group,HDL-C and LDL-C in the HFFC group were significantly increased,and the LDL-C level was significantly decreased after 8 weeks of exercise(P＜0.05).(3)The liver fat droplet area and liver TG content in the HFFC group were significantly higher than those in the NC group,whereas those in HFFC+EX group were significantly decreased(P＜0.05).(4)Compared with the NC group,the content of oxidase MDA in the HFFC group were significantly increased,and nuclear translocation and gene expression of Nrf2 were significantly decreased.After exercise,the activities of SOD and T-AOC were significantly increased,and the nuclear translocation and gene expression of Nrf2 and expression levels of HO-1 and SOD-1 were significantly increased(P＜0.05).(5)The number of apoptotic hepatocytes and CHOP expression in the HFFC diet group were significantly higher than those in the NC group,whereas the number of apoptotic hepatocytes,and CHOP and Bax/Bcl-2 expression in the exercise group were significantly lower than those in the NC group(P＜0.05).Conclusions Voluntary wheel can alleviate HFFC diet induced liver lipid deposition by regulating the Nrf2/HO-1 pathway,thereby alleviating oxidative stress and reducing apoptosis in liver cells.

Objective:To investigate the effect of CD8+CD28-T cells on acute graft-versus-host disease(aGVHD)after haploidentical hematopoietic stem cell transplantation(haplo-HSCT).Methods:The relationship between absolute count of CD8+CD28-T cells and aGVHD in 60 patients with malignant hematological diseases was retrospectively analyzed after haplo-HSCT,and the differences in the incidence rate of chronic graft-versus host disease(cGVHD),infection and prognosis between different CD8+CD28-T absolute cells count groups were compared.Results:aGVHD occurred in 40 of 60 patients after haplo-HSCT,with an incidence rate of 66.67％.The median occurrence time of aGVHD was 32.5(20-100)days.At 30 days after the transplantation,the absolute count of CD8+CD28-T cells of aGVHD group was significantly lower than that of non-aGVHD group(P=0.03).Thus the absolute count of CD8+CD28-T cells at 30 days after transplantation can be used to predict the occurrence of aGVHD to some extent.At 30 days after transplantation,the incidence rate of aGVHD in the low cell count group(CD8+CD28-T cells absolute count＜0.06/μl)was significantly higher than that in the high cell count group(CD8+CD28-T cells absolute count ≥0.06/μl,P=0.011).Multivariate Cox regression analysis further confirmed that the absolute count of CD8+CD28-T cells at 30 days after transplantation was an independent risk factor for aGVHD,and the risk of aGVHD in the low cell count group was 2.222 times higher than that in the high cell count group(P=0.015).The incidence of cGVHD,fungal infection,EBV infection and CMV infection were not significantly different between the two groups with different CD8+CD28-T cells absolute count.The overall survival,non-recurrent mortality and relapse rates were not significantly different between different CD8+CD28-T cells absolute count groups.Conclusion:Patients with delayed CD8+CD28-T cells reconstitution after haplo-HSCT are more likely to develop aGVHD,and the absolute count of CD8+CD28-T cells can be used to predict the incidence of aGVHD to some extent.The absolute count of CD8+CD28-T cells after haplo-HSCT was not associated with cGVHD,fungal infection,EBV infection,and CMV infection,and was also not significantly associated with the prognosis after transplantation.

Objective:Analyze the medical reimbursement rate and influencing factors under the DIP payment method to refine the DIP payment policy,promote the optimization of internal operations in medical institutions,and ensure reasonable compensation.Methods:Based on the 2022 DIP fund settlement data from 196 medical institutions in City A,the study used multiple linear regression to analyze the factors affecting medical reimbursement rate and conducted a heterogeneity analysis for medical institutions of different levels.Results:The medical reimbursement rate for medical institutions in City A in 2022 was 103.32%.Medical institutions with lower CMI standardized inpatient costs,lower rates of deviation cases,tertiary care institutions,lower proportion of level-four surgeries,and lower ratios of resident to employee medical insurance cases have higher medical reimbursement rate(P<0.05).Heterogeneity analysis reveals that therates of deviation cases,the proportion of primary care diseases,the ratio of resident to employee medical insurance cases,and the low-standard admission rate have different impacts on medical institutions of different levels.Conclusion:Medical insurance departments should improve policies for primary care diseases,dynamically adjust disease catalogs and payment standards,optimize funding levels and institutional coefficients,and increase penalties for violations to ensure effective use of funds.Medical institutions need to strengthen their understanding of policies,focus on refined internal management,promote standardized and rational diagnosis and treatment through performance assessment transformation,and leverage their own advantages in medical services to reasonably increase the medical reimbursement rate.

AIM:To investigate the impact of total flavonoids of Pterocarya hupehensis Skan(PHSTF)on the migration,invasion,and ferroptosis of non-small-cell lung cancer A549 cells.METHODS:The A549 cells were divided into control group,low-,medium-and high-dose(100,150 and 200 μg/mL)PHSTF groups,ferroptosis inhibitor liprox-statin-1(Lip-1)group,and high-dose PHSTF combined with Lip-1 group,each cultured in corresponding media.Cell via-bility was assessed using the CCK-8 assay,while cell migration and invasion abilities were determined through scratch and Transwell assays.Cell lipid peroxidation levels were measured using the glutathione(GSH)assay kit.RT-qPCR was em-ployed to assess the mRNA expression of solute carrier family 7 member 11(SLC7A11)and glutathione peroxidase 4(GPX4),while Western blot was utilized to examine the protein expression of SLC7A11,GPX4,Kelch-like epichlorohy-drin-associated protein-1(Keap-1),nuclear factor E2-related factor 2(Nrf2)and heme oxygenase-1(HO-1).RE-SULTS:Compared with control group,PHSTF significantly diminished the viability of A549 cells in a time-and dose-de-pendent manner(P＜0.01),and the cell migration and invasion were also reduced(P＜0.01),along with a significant de-crease in GSH level(P＜0.01).Treatment with PHSTF inhibited the mRNA and protein expression levels of ferroptosis-re-lated proteins,including SLC7A11 and GPX4(P＜0.01),suppressed the protein expression of Nrf2 and HO-1(P＜0.01),and enhanced the expression of Keap-1(P＜0.01).The Lip-1 partially restored the decrease in cell viability in-duced by PHSTF(P＜0.01),significantly up-regulated the protein expression levels of SLC7A11,GPX4,Nrf2 and HO-1,and suppressed the protein expression of Keap-1(P＜0.01).CONCLUSION:Total flavonoids of Pterocarya hupehen-sis Skan can inhibit the migration and invasion of non-small-cell lung cancer A549 cells,and induce the cell ferroptosis by regulating the Keap-1/Nrf2/HO-1 pathway.

ObjectiveTo conduct a systematic comparative study on wild and cultivated Codonopsis pilosula（CP） from three aspects， including characters， microscopy， and contents of primary and secondary metabolites. MethodWild and cultivated CP samples were collected， their characters were measured using vernier caliper， tape measure and balance， the paraffin sections were stained with safranin-fixed green dyeing， and their microstructure were observed under the optical microscope. The content of alcohol-soluble extracts in wild and cultivated CP was determined according to the method for determination of extract under CP in the 2020 edition of Chinese Pharmacopoeia， the starch content was determined by anthrone colorimetry， the content of total polysaccharides was determined by kit method， Fiber analyzer was used to determine the content of fiber components， and ultra performance liquid chromatography（UPLC） was used to determine the content of monosaccharides， disaccharides and some secondary metabolites. Multivariate statistical analysis methods such as principal component analysis（PCA） and orthogonal partial least squares-discriminant analysis（OPLS-DA） were employed to screen key differential components between wild and cultivated CP on the basis of variable importance in the projection（VIP） value>1 and P<0.05. ResultIn terms of morphological characteristics， the "lion's head-like" shape， longitudinal wrinkles， and circumferential wrinkles below the root cap of wild CP were more pronounced in wild CP compared to the cultivated ones. Regarding transverse sectional features， wild CP had more fissures on the outer side of the cortex and a larger duramen. Under microscopic examination， wild CP had more stone cells， a larger proportion of xylem， and the presence of cork cells arranged in rings in the xylem， while cultivated CP has a larger proportion of phloem， smaller vessel diameters， and a more loosely arranged vascular system. In terms of primary metabolites， the contents of 45% ethanol-soluble extract and total polysaccharides in cultivated CP were significantly higher than those in the wild ones（P<0.05）， the contents of lignin， hemicellulose， cellulose， fructose and glucose in wild CP were significantly higher than those in the cultivated ones（P<0.05）， while sucrose content in the cultivated CP was significantly higher than that in the wild ones（P<0.05）. Concerning secondary metabolites， the contents of tryptophan and tangshenoside Ⅰ in cultivated CP were significantly higher than those in the wild ones（P<0.05）， whereas the contents of lobetyolinin， lobetyol and atractylenolide Ⅲ in wild CP were significantly higher than those in the cultivated ones（P<0.05）. ConclusionThere are significant differences between wild and cultivated CP in terms of morphological characteristics， microscopic features and chemical composition. Glucose， fructose， sucrose， tangshenoside Ⅰ， tryptophan and cellulose components are the key differential components between wild and cultivated CP. Wild CP contains more polyacetylenes and fructose， whereas cultivated CP has higher levels of tangshenoside Ⅰ and sucrose， with noticeably lower cellulose content. These distinctions may be related to their growth conditions， growth years and cultivation techniques. Based on the results of this study， it is recommended to increase polyacetylenes and the content ratio of fructose to sucrose as an indicators to characterize different production methods of CP， in order to guide the high-quality production of CP.

Objective To explore the effect of Shexiang Baoxin pill on cardiac tissue angiogenesis in spontaneously hypertensive rats(SHR).Method Twenty 12 week old male SHR were randomly divided into experimental group and model group,with 12 week old male SD rats as the normal control group.The experimental group rats were orally administered with Shexiang Baoxin pill(45 mg·kg-1)daily,and their blood pressure was monitored using a non-invasive tail artery blood pressure gauge every four weeks.Eight weeks later,cardiac tissue was taken for platelet endothelial cell adhesion molecule-1(PECAM-1/CD31)immunofluorescence staining to observe CD31 expression level.Use protein blotting to detect the expression levels of myocardial endothelial growth factor(VEGF),myocardial endothelial growth factor receptor 2(VEGF-R2),basic fibroblast growth factor(bFGF)and phosphorylated protein kinase B(p-Akt)/protein kinase B(Akt)proteins.Result There was significant increase in blood pressure between the experimental group,model group and normal group at the same time point(all P＜0.01),but there was no statistically significant difference in blood pressure changes between the experimental group and model group at the same time point(all P＞0.05).The CD31 expression rates of the normal group,model group and experimental group were(3.79±0.84)％,(2.54±0.42)％and(3.56±0.49)％;VEGF levels were 0.95±0.10,0.73±0.08 and 0.94±0.15;VEGF-R2 levels were 0.85±0.10,0.61±0.14 and 0.80±0.10;bFGF levels were 0.84±0.04,0.51±0.21 and 0.74±0.14;p-Akt/Akt levels were 0.85±0.15,0.57±0.13 and 0.80±0.20,respectively.The differences between the normal group and the model group,as well as the experimental group and the model group,were statistically significant(all P＜0.05).Conclusion Shexiang Baoxin pill can promote the neovascularization of microvessels in the heart tissue of spontaneously hypertensive rats,and its mechanism may be related to the activation of PI3K/Akt phosphorylation,upregulation of bFGF,VEGF and their receptor VEGF-R2 in myocardial tissue.