[Objective] To explore the feasibility of using whole blood as a source of NK cells for allogeneic CAR NK cell therapy and activated NK cell reinfusion therapy, and initially construct a technical system for the separation and purification of NK cells from whole blood. [Methods] All peripheral blood mononuclear cells (PBMCs) were enriched from 400 mL of whole blood by manual separation and machine separation, respectively. The erythrocyte loss rate, PBMCs number, NK cell purity of the two methods were compared. NK cells were sorted from PBMCs by three separation and enrichment methods as immunomagnetic bead negative selection method, platelet lysate culture expansion and PERCOLL density gradient separation method, and the purity and yield of NK cells, the activity of NK cells and the tumor-killing ability of the three separation and enrichment methods were compared. [Results] The proportion of NK cells in the lymphocyte population was higher in the manual separation method than in the machine separation method[(13.16±5.16)% vs (8.56±3.92)%, P<0.05]; the number PBMCs was lower in the manual separation method than in the machine separation method[(4.09±1.80)×10⁸vs (6.49±2.16)×10⁸, P<0.05], and there was no difference in the red blood cell loss between the two methods (P>0.05). The purity of NK cells isolated and enriched from PBMCs by manual separation method using immunomagnetic was (96.77±2.31)%; the yield was (56.27±10.47)%; the inhibition of tumor proliferation was (38.67±14.05)%; and the tumor killing rate was (19.90±8.05)%. The purity of NK cells isolated and enriched from PBMCs by manual separation method using platelet lysis culture expansion method was the highest at day 7, which was (54.84±15.80)%; the cell expansion multiple could reach 16.92±6.28 at day 7; the in vitro tumor killing rate of NK cells was (15.83±5.5)%; the tumor inhibition rate was (44.33±13.5)%; and there was no difference in the toxicity and activity of NK cells between the two methods (P>0.05). The purity of NK cells isolated and enriched by PERCOLL density gradient separation method was (15.83±5.82)%, and the yield was (14±6.25)%, which was significantly lower than the other two methods. [Conclusion] PBMCs isolated from whole blood by manual separation and NK cells enriched by negative selection with immunomagnetic beads have the potential to provide NK cell materials for CAR-NK cell therapy, and NK cells enriched by platelet lysate-conditioned medium have the potential to provide NK cells for large-scale NK cell activation reinfusion therapy.

Aim To explore the molecular mechanism of luteolin against acute lung injury by network phar-macology and molecular docking technology,and to conduct experimental verification.Methods The re-lated targets of luteolin were predicted by PubChem and Swiss Target Prediction databases.Acute lung in-jury-related targets were collected through the Gene-Cards database.Venny 2.1 was used to draw the Venn diagram,and the common targets of drug and disease were obtained.The protein interaction network(PPI)was established by String online platform,and the core targets were screened by Cytoscape 3.8.2 software.The functional enrichment analysis of Gene Ontology(GO)and pathway enrichment analysis of Kyoto Ency-clopedia of Gene and Genome(KEGG)were per-formed on the common targets using the DAVID data-base,and the results were visualized.Finally,molecu-lar docking was performed by Auto Dock software,and the molecular results were visualized by Pymol.The mouse acute lung injury model was constructed.HE staining was used to detect histopathology,and Western blot was used to detect lung tissue related proteins.Results After screening,85 common targets were ob-tained.Among them,the core targets were AKT1,EG-FR,SRC,MMP9,ESR1,PTGS2,etc.GO enrichment analysis obtained 265 biological processes,including signal transduction,protein phosphorylation,and nega-tive regulation of apoptosis.There were 48 cells,main-ly including plasma membrane,cell solute,cytoplasm,etc.There are 107 molecular functions,mainly inclu-ding ATP binding,protein serine/threonine/tyrosine ki-nase activity,protein kinase activity and so on.A total of 92 signaling pathway were obtained by KEGG path-way enrichment analysis,which mainly acted on PI3 K-AKT signaling pathway,ErbB signaling pathway,VEGF signaling pathway,etc.Molecular docking results showed that luteolin had good docking activity with core targets AKT1,EGFR,SRC,MMP9,ESR1,PTGS2,MMP2,GSK3 B,KDR and PARP1.The binding ener-gy of ERS1,GSK3B and MMP2 was lower than-5.0 kal·mol-1,and the affinity with luteolin was stronger.The pathological results of lung tissue showed that lute-olin could inhibit inflammatory infiltration and had a strong anti-inflammatory effect in LPS-induced acute lung injury model in mice.Western blot experiments showed that luteolin might alleviate the inflammatory response by inhibiting the phosphorylation of AKT.Conclusions Luteolin can play an anti-acute lung in-jury role through multi-target and multi-channel mecha-nisms,which may be closely related to the inhibition of AKT phosphorylation.

Objective:To conduct a nationwide physician survey to better understand clinicians’ disease awareness, treatment patterns, and experience of Waldenstr?m macroglobulinemia (WM) in China.Methods:This cross-sectional study was conducted from February 2022 to July 2022 by recruiting clinicians with WM treatment experience from hematology, hematology-oncology, and oncology departments throughout China. Quantitative surveys were designed based on the qualitative interviews.Results:The study included 415 clinicians from 219 hospitals spread across thirty-three cities and twenty-two provinces. As for diagnosis, the laboratory tests prescribed by physicians for suspected WM patients were relatively consistent (92% -99% recommendation for laboratory, 79% -95% recommendation for pathology, 96% recommendation for gene testing, and 63% -83% recommendation for imaging examination). However, from a physician's perspective, there was 22% misdiagnosis occurred in clinical practice. The rate of misdiagnosis was higher in lower-level hospitals than in tertiary grade A hospitals (29% vs 21%, P<0.001). The main reasons for misdiagnosis were that WM was easily confused with other diseases, and physicians lacked the necessary knowledge to make an accurate diagnosis. In terms of gene testing in clinical practice, 96% of participating physicians believed that WM patients would require gene testing for MYD88 and CXCR4 mutations because the results of gene testing would aid in confirming diagnosis and treatment options. In terms of treatment, 55% of physicians thought that the most important goal was to achieve remission, while 54% and 51% of physicians wanted to improve laboratory and/or examination results and extend overall survival time, respectively. Among patients with treatment indications, physicians estimated that approximately 21% of them refused to receive treatment, mainly owing to a lack of affordable care and disease awareness. When selecting the most appropriate treatment regimens, physicians would consider patient affordability (63% ), comorbidity (61% ), and risk level (54% ). Regimens containing Bruton tyrosine kinase inhibitor (BTKi) were most widely recommended for both treatment-na?ve and relapsed/refractory patients (94% for all patients, 95% for treatment-na?ve patients, and 75% for relapsed/refractory patients), and most physicians recommended Ibrutinib (84% ). For those patients who received treatment, physicians reported that approximately 23% of patients did not comply with the treatment regimen due to a lack of affordability and disease awareness. Furthermore, 66% of physicians believe that in the future, increasing disease awareness and improving diagnosis rates is critical. Conclusions:This study is the first national physician survey of WM conducted in China. It systematically describes the issues that exist in WM diagnosis and treatment in China, such as a high rate of misdiagnosis, limited access to gene testing and new drugs, and poor patient adherence to treatment. Chinese doctors believe that improving doctors’ and patients’ understanding of WM is one of the most urgent issues that must be addressed right now.

Objective To understand the disease acceptance status and related factors in human immunodeficiency virus(HIV)-infected/acquired immunodeficiency syndrom(AIDS)patients,so as to guide the clinical development of intervention measures,and to provide empirical evidence for improving clinical outcomes.Methods Convenience sampling method was used to select 555 HIV-infected/AIDS patients who received treatment in the designated AIDS treatment clinic of a hospital.General data,disease acceptance,disease self-management efficacy and clinical out-comes(such as quality of life,CD4+T lymphocyte count and HIV viral load)of the studied subjects were collected.Results The average disease acceptance of HIV-infected/AIDS patients was(26.08±5.34)points.Multiple linear regression analysis showed that religious belief and self-management efficacy were related factors affecting the di-sease acceptance of patients(both P＜0.05),which could explain the 30.4％variation in disease acceptance of HIV-infected/AIDS patients,and the disease acceptance of patients was closely related to their quality of life(P＜0.001).Conclusion HIV-infected/AIDS patients have a moderate level of disease acceptance.Medical staff should fully consider patients'religious beliefs and self-management efficacy,so as to formulate targeted intervention mea-sures to improve patients'acceptance of disease,and further promote patients'quality of life.

ObjectiveTo observe the effects of Sargassum and Glycyrrhizae Radix et Rhizoma incompatible pair with the Haizao Yuhutang （HYT） on oxidative stress in the liver of goiter rats under the condition of 2 times the dose limit of the Pharmacopoeia of the People's Republic of China 2020. MethodA total of 128 male Wistar rats were randomly divided into a blank group， a model group， a euthyrox group （20 μg·kg^-1）， a HYT group （12.06 g·kg^-1）， a HYT without Sargassum （HYT-H） group （9.90 g·kg^-1）， a HYT without Glycyrrhizae Radix et Rhizoma （HYT-G） group （10.26 g·kg^-1）， a HYT without Sargassum and Glycyrrhizae Radix et Rhizoma （HYT-HG） group （8.10 g·kg^-1）， and a Sargassum and Glycyrrhizae Radix et Rhizoma （HG） group （3.96 g·kg^-1）. The blank group was given deionized water by gavage， and the others were given propylthiouracil （PTU） to replicate the goiter pathological model. Euthyrox was taken as a positive control drug， and the rest of the Chinese medicine groups were given the corresponding decoction by gavage， the material was collected 12 hours after the last dose. The serum levels of aspartate aminotransferase （AST）， alanine aminotransferase （ALT）， alkaline phosphatase （ALP）， and the contents of superoxide dismutase （SOD）， glutathione peroxidase （GSH-Px）， malondialdehyde （MDA）， reactive oxygen species （ROS） in liver tissue were detected in each group. The pathological changes in the liver were observed via hematoxylin-eosin （HE） staining. Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR） was utilized to detect the mRNA expressions of Kelch-like Ech-associated protein 1 （Keap1）， nuclear factor E₂-related factor 2 （Nrf2）， heme oxygenase-1 （HO-1）， p53 and Caspase-3 in liver tissues. Western blot was adopted to detect the protein expressions of Nrf2 and HO-1 in liver tissues in oxidative stress-related signaling pathways. ResultCompared with control group， the model group showed significantly increased serum ALT level and contents of MDA and ROS in liver tissues （P<0.05， P<0.01）， significantly reduced activities of SOD and GSH-Px in the liver （P<0.01）， significantly increased mRNA expression of Keap1 （P<0.01）， and significantly decreased mRNA and protein expressions of Nrf2 and HO-1 （P<0.05， P<0.01）. Compared with the model group， the HYT group manifested significantly reduced serum levels of AST， ALT， and ALP （P<0.05， P<0.01）， significantly reduced contents of MDA and ROS in liver tissue （P<0.01）， significantly increased the activities of SOD and GSH-Px （P<0.01）， significantly decreased mRNA expressions of Keap1， p53， and Caspase-3 （P<0.01）， and significantly increased mRNA and protein expressions of Nrf2 and HO-1 （P<0.05， P<0.01）. ConclusionUnder the condition of 2 times the dose limit of the Pharmacopoeia of the People's Republic of China 2020， Sargassum and Glycyrrhizae Radix et Rhizoma incompatible pair with the HYT on oxidative stress in the liver of goiter rats had different effects. The HYT that contains Sargassum and Glycyrrhizae Radix et Rhizoma has a protective effect on the liver of goiter rats， and the effect is better than that of the HG group， the euthyrox group， and the incomplete groups. Its mechanism may be related to activating the Nrf2/HO-1 signaling pathway to alleviate liver oxidative stress and inhibiting the p53/Caspase-3 signaling pathway to reduce hepatocyte apoptosis.

Objective To observe the pharmacokinetic effect of amoxicillin and clavulanate potassium tablets on amoxicillin in Chinese healthy subjects under fasting and high fat and high calorie diet.Methods 71 healthy subjects were given a single dose of amoxicillin potassium clavulanate tablets(0.375 g)on fasting or high fat diet,and venous blood samples were collected at different time points.The concentrations of amoxicillin in human plasma were determined by HPLC-MS/MS method,and the pharmacokinetic parameters were calculated by non-atrioventricular model using PhoenixWinNonlin 8.0 software.Results The main pharmacokinetic parameters of amoxicillin potassium clavulanate tablets after fasting and high fat diet were(5 105.00±1 444.00),(4 593.00±1 327.00)ng·mL-1,and postprandial-fasting ratio 89.40％,90％confidence interval(79.55％-100.19％);t1/2 were(1.52±0.16),(1.39±0.22)h;AUC0-t were(12 969.00±1 841.00),(11 577.00±1 663.00)ng·mL-1·h,and postdietary/fasting ratio 89.20％,90％confidence interval(83.92％-94.28％);AUC0-∞ were(13 024.00±1 846.00),(11 532.00±1 545.00)ng·mL-1·h,and postprandial-fasting ratio 88.60％,90％confidence interval(83.48％-93.50％).The median Tmax(range)were 1.63(0.75,3.00)and 2.50(0.75,6.00)h,respectively,and the Tmax of postprandial medication was delayed(P＜0.01).Conclusion Compared with fasting condition,amoxicillin Tmax was significantly delayed after high fat diet,while Cmax,AUC0-t and AUC0-∞ were not significantly changed,indicating that food could delay the absorption of amoxicillin,but did not affect the degree of absorption.

The 2-(2-phenylethyl)chromones were separated from agarwood of Aquilaria agallocha Roxb. and their anti-KRAS mutant non-small cell lung cancer (NSCLC) activities were evaluated. 2-(2-Phenyethyl)chromones in agarwood were separated and purified by silica gel, RP-18 reverse phase silica gel, MCI gel CH20P, Diol, and semi preparative HPLC chromatography techniques, while the structures of the compounds were identified by extensive spectroscopic analysis, such as 1D and 2D NMR and ESI-MS. The cell counting kit 8 (CCK-8) assay was used to screen the anti-tumor activity of the isolated monomeric compounds on three KRAS-mutant NSCLC cells. The cell proliferation, cloning formation, adhesion ability, and cell cycle arrest activity of compound 8 were analyzed. Molecular docking and Western blot experiments were used to study the mechanism of compound 8. The in vivo anti-tumor activity of the compound 8 was evaluated by zebrafish cell derived xenograft (CDX) model. Nineteen known 2-(2-phenylethyl)chromones were isolated from the ethyl acetate extract of agarwood. On A549 (KRAS G12S) cells, compounds 8, 10, and 19 showed good inhibitory activity, on H23 (KRAS G12C) cells, compounds 7, 8, and 19 showed good inhibitory activity, and on H358 (KRAS G12C) cells, compounds 8, 10, and 16 showed good inhibitory activity. Compound 8 had the best inhibitory activity in all three cell lines. It effectively inhibited cell proliferation, clone formation, adhesion ability, and arrested the H23 cell cycle at G2/M phase. Compound 8 could also inhibit the expression of c-Met and its downstream signaling pathways, effectively inhibiting tumor growth in zebrafish CDX model. In conclusion, among the nineteen known 2-(2-phenylethyl)chromones, compound 8 had the best activity and significantly inhibited the proliferation of KRAS-mutant NSCLC cells by arresting the cells in the G2/M phase.

Objective To explore characteristics of clinical parameters and cytokines in patients with drug-induced liver injury(DILI)caused by different drugs and their correlation with clinical indicators. Method The study was conducted on patients who were up to Review of Uncertainties in Confidence Assessment for Medical Tests(RUCAM)scoring criteria and clinically diagnosed with DILI.Based on Chinese herbal medicine,cardiovascular drugs,non-steroidal anti-inflammatory drugs(NSAIDs),anti-infective drugs,and other drugs,patients were divided into five groups.Cytokines were measured by Luminex technology.Baseline characteristics of clinical biochemical indicators and cytokines in DILI patients and their correlation were analyzed. Results 73 patients were enrolled.Age among five groups was statistically different(P=0.032).Alanine aminotransferase(ALT)(P=0.033)and aspartate aminotransferase(AST)(P=0.007)in NSAIDs group were higher than those in chinese herbal medicine group.Interleukin-6(IL-6)and tumor necrosis factor alpha(TNF-α)in patients with Chinese herbal medicine(IL-6:P<0.001;TNF-α:P<0.001)and cardiovascular medicine(IL-6:P=0.020;TNF-α:P=0.001)were lower than those in NSAIDs group.There was a positive correlation between ALT(r=0.697,P=0.025),AST(r=0.721,P=0.019),and IL-6 in NSAIDs group. Conclusion Older age may be more prone to DILI.Patients with NSAIDs have more severe liver damage in early stages of DILI,TNF-α and IL-6 may partake the inflammatory process of DILI.

The blood-brain barrier (BBB) plays a crucial role in maintaining the homeostasis of the brain's internal environment, which poses challenges to the treatment of central nervous system diseases. Drug carriers can aid in the delivery of therapeutic agents across the BBB to exert their pharmacological effects. The article reviewed the pathways for drug delivery across the BBB, the intracerebral fate and the classification of drug carriers and focuses on the functions and characteristics of liposomes, exosomes, apoptotic bodies, cell-penetrating peptides, and cell-targeting peptides. The review will provide an outlook on the future and challenge of research in the field of drug delivery across the BBB.

OBJECTIVE: To compare the efficiency and effect of establishing rat peri-implantitis model by traditional cotton thread ligation and local injection of Porphyromonas gingivalis lipopolysaccharide (LPS) around the implant, as well as the combination of the two methods. METHODS: Left side maxillary first molars of 39 male SD rats were extracted, and titanium implants were implanted after four weeks of healing. After 4 weeks of implant osseointegration, 39 rats were randomly divided into 4 groups. Cotton thread ligation (n=12), local injection of LPS around the implant (n=12), and the two methods combined (n=12) were used to induce peri-implantitis, the rest 3 rats were untreated as control group. All procedures were conducted under 5% isoflurane inhalation anesthesia. The rats were sacrificed 2 weeks and 4 weeks after induction through carbon dioxide asphyxiation method. The maxilla of the rats in the test groups were collected and marginal bone loss was observed by micro-CT. The gingival tissues around the implants were collected for further real time quantitative PCR (RT-qPCR) analysis, specifically the expression of tumor necrosis factor-alpha (TNF-α) as well as interleukin-1β (IL-1β). The probing depth (PD), bleeding on probing (BOP) and gingival index (GI) of each rat in the experimental group were recorded before induction of inflammation and before death. RESULTS: After 4 weeks of implantation, the osseointegration of implants were confirmed. All the three test groups showed red and swollen gums, obvious marginal bone loss around implants. After 2 weeks and 4 weeks of inflammation induction, PD, GI and BOP of the three test groups increased compared with those before induction, but only BOP was statistically significant among the three test groups (P < 0.05). At the end of 2 weeks of inflammation induction, marginal bone loss was observed at each site in the cotton thread ligation group and the combined group. At each site, the bone resorption in the combined group was greater than that in the cotton thread ligation group, but the difference was not statistically significant (P > 0.05), bone resorption was observed at some sites of some implants in LPS local injection group. At the end of 4 weeks of inflammation induction, marginal bone loss was observed at all sites in each group. The marginal bone loss in the cotton thread ligation group and the combined group was greater than that in the LPS local injection group, and the difference was statistically significant (P < 0.05). At the end of 2 weeks and 4 weeks of induction, the expression of TNF-α and IL-1β in the test groups were higher than those in the control group (P < 0.05). CONCLUSION Compared with local injection of LPS around the implant, cotton thread ligature and the two methods combined can induce peri-implantitis in rats better and faster.
Animals ; Male ; Rats ; Alveolar Bone Loss/etiology* ; Dental Implants/adverse effects* ; Inflammation ; Lipopolysaccharides ; Peri-Implantitis/pathology* ; Rats, Sprague-Dawley ; Tumor Necrosis Factor-alpha