Our previous research demonstrated that the Wenxia Changfu Formula (WCF), as a neoadjuvant therapy, inhibits M2 macrophage infiltration in the tumor microenvironment and prevents lung cancer metastasis. Given tumor-associated macrophages (TAMs) in epithelial-mesenchymal transition (EMT), this study investigated whether WCF impedes lung cancer metastasis by attenuating TAM-induced EMT in non-small cell lung cancer (NSCLC) cells. Utilizing a co-culture model treated with or without WCF, we observed that WCF downregulated cluster of differentiation 163 (CD163) expression in macrophages, reduced CCL18 levels in the conditioned medium, and inhibited the growth, invasion, and EMT of NSCLC cells induced by macrophage co-culture. Manipulation of CCL18 levels and Src overexpression in NSCLC cells revealed that WCF's effects are mediated through CCL18 and Src signaling. In vivo, WCF inhibited recombinant CCL18 (rCCL18)-induced tumor metastasis in nude mice by blocking Src signaling. These findings indicate that WCF inhibits NSCLC metastasis by impeding TAM-induced EMT via antagonistic modulation of CCL18, providing evidence for its potential development and clinical application in NSCLC patients.
Epithelial-Mesenchymal Transition/drug effects* ; Carcinoma, Non-Small-Cell Lung/metabolism* ; Humans ; Animals ; Lung Neoplasms/metabolism* ; Chemokines, CC/antagonists & inhibitors* ; Mice ; Mice, Nude ; Drugs, Chinese Herbal/administration & dosage* ; Cell Line, Tumor ; Neoplasm Metastasis ; Tumor-Associated Macrophages/drug effects* ; Mice, Inbred BALB C ; Signal Transduction/drug effects*

Objective:To validate and optimize the interpretation methods and cut-off values of prolonged activated partial thromboplastin time (APTT) mixing test, in order to elevate the clinical applicational value in differentiating single factor deficiency, positive lupus anticoagulant (LA) and FⅧ inhibitors.Methods:This cross-sectional retrospective study involved 130 cases with single APTT prolongation for unknown reasons who underwent mixing tests in the First Hospital of Jilin University from January 1st, 2022 to December 31st, 2023, including 84 males and 46 females. Rosner index (RI), Percentage correction ("Chang") method, Normal reference interval (NRI) method, differential analysis, post-incubation prolongation time and percentage (including mixed immediately and ) were respectively calculated and analyzed for both immediate mixing and 2-hour post-mixing incubation. According to clinical diagnosis, patients were divided into single factor deficiency (51 cases), positive lupus anticoagulant (LA) (56 cases), and positive factor Ⅷ (FⅧ) inhibitor (23 cases). Receiver operation characteristic (ROC) curve analysis was used to determine the cut-off values for APTT mixing test methods in patients with different reasons for prolonged APTT, which were evaluted and adjusted by AUC, sensitivity, and specificity, and compared with the methods currently used for interpretations.Results:For the single factor deficiency cases, the AUCs of immediate RI index <7.5% and incubated RI index <13.9% were 0.87 and 0.90, respectively, with sensitivities at 87.8% and 83.8%, and specificities at 72.0% and 80.0%, whose combination had sensitivity and specificity at 87.3%, and 94.0%, respectively; the judging accuracy increased from 84.6% (110/130) in currently used rule to 93.1% (121/130). For the positive lupus anticoagulant (LA) cases, the combination of immediate RI index >10.3% and prolonged post-incubation clotting time percentage <9.1% showed 88.5% sensitivity and 99.8% specificity; the judging accuracy improved from 80.0% (104/130) in currently used rule to 86.2% (112/130). For positive FⅧ inhibitor cases, the combination of incubated RI index >17.0% and prolonged post-incubation clotting time percentage >9.1% had 99.8% sensitivity and 100.0% specificity; the judging accuracy increased from 89.2% (116/130) in currently used rule to 94.6% (123/130).Conclusion:This study validated and optimized the interpretation methods and cut-off values for the prolonged APTT mixing test in differentiating single factor deficiencies, prositive FⅧ inhibitors and LA positive cases, significantly improving the judging sensitivity and specificity.

Objective:To validate and optimize the interpretation methods and cut-off values of prolonged activated partial thromboplastin time (APTT) mixing test, in order to elevate the clinical applicational value in differentiating single factor deficiency, positive lupus anticoagulant (LA) and FⅧ inhibitors.Methods:This cross-sectional retrospective study involved 130 cases with single APTT prolongation for unknown reasons who underwent mixing tests in the First Hospital of Jilin University from January 1st, 2022 to December 31st, 2023, including 84 males and 46 females. Rosner index (RI), Percentage correction ("Chang") method, Normal reference interval (NRI) method, differential analysis, post-incubation prolongation time and percentage (including mixed immediately and ) were respectively calculated and analyzed for both immediate mixing and 2-hour post-mixing incubation. According to clinical diagnosis, patients were divided into single factor deficiency (51 cases), positive lupus anticoagulant (LA) (56 cases), and positive factor Ⅷ (FⅧ) inhibitor (23 cases). Receiver operation characteristic (ROC) curve analysis was used to determine the cut-off values for APTT mixing test methods in patients with different reasons for prolonged APTT, which were evaluted and adjusted by AUC, sensitivity, and specificity, and compared with the methods currently used for interpretations.Results:For the single factor deficiency cases, the AUCs of immediate RI index <7.5% and incubated RI index <13.9% were 0.87 and 0.90, respectively, with sensitivities at 87.8% and 83.8%, and specificities at 72.0% and 80.0%, whose combination had sensitivity and specificity at 87.3%, and 94.0%, respectively; the judging accuracy increased from 84.6% (110/130) in currently used rule to 93.1% (121/130). For the positive lupus anticoagulant (LA) cases, the combination of immediate RI index >10.3% and prolonged post-incubation clotting time percentage <9.1% showed 88.5% sensitivity and 99.8% specificity; the judging accuracy improved from 80.0% (104/130) in currently used rule to 86.2% (112/130). For positive FⅧ inhibitor cases, the combination of incubated RI index >17.0% and prolonged post-incubation clotting time percentage >9.1% had 99.8% sensitivity and 100.0% specificity; the judging accuracy increased from 89.2% (116/130) in currently used rule to 94.6% (123/130).Conclusion:This study validated and optimized the interpretation methods and cut-off values for the prolonged APTT mixing test in differentiating single factor deficiencies, prositive FⅧ inhibitors and LA positive cases, significantly improving the judging sensitivity and specificity.

Objective:To explore the role of tumor necrosis factor receptor-associated factor 4 (TRAF4) in promoting the abnormal activation of epidermal growth factor receptor (EGFR) and its effect on lung cancer cell proliferation, migration and invasion.Methods:Tumor tissues from patients who underwent lung adenocarcinoma resection at The First Affiliated Hospital of Second Military Medical University, from January 2015 to May 2017 were collected, and the expressions of TRAF4 and Ki-67 in lung cancer tissues were detected by immunohistochemistry, the mRNA levels of Cyclin D and Vimentin were detected by real-time fluorescence quantitative PCR (qRT-PCR). The effect of TRAF4 on the tumor growth ability of lung cancer A549 cells was investigated by the xenograft model, the effect of TRAF4 or EGFR on the tumor proliferation ability was detected by using cell counting kit 8 (CCK8) and BrdU assay, and the migration and invasion abilities of tumor cells were detected by Transwell assay. Different structural domain deletion expression vectors of TRAF4 and EGFR were constructed to transfect cells, and the interaction mode of TRAF4 and EGFR was investigated by immunoprecipitation assay.Results:The expression of TRAF4 in non-small cell lung cancer (NSCLC) tissues was positively correlated with the expressions of Ki-67, cyclin D, and vimentin ( r2: 0.438, 0.695, and 0.736, respectively, all P＜0.01). Immunohistochemical assay of tumor tissues from NSCLC patients showed that tissues with high expression of TRAF4 were also high in Ki-67. Patients with high TRAF4 expression (TRAF4 positivity >30%) had a shorter progression-free survival (PFS) time than that of patients with low TRAF4 expression (TRAF4 positivity ≤30%) (median PFS of 12 and 19 months, respectively; P=0.034). Traf4 -/- cells had a weakened proliferative capacity than traf4+/+ cells and formed tumors with smaller size ( P＜0.05). The expression level of Ki-67 in the tumor tissues formed by traf4 -/- cells [(45.6±8.7)%] was lower than that in the tumor tissues formed by traf4+/+ cells [(62.3±10.3)%, P=0.015], the mRNA levels of cyclin D (1.01±0.15) and vimentin (1.01±0.12) in the traf4 -/- cells were lower than those of the traf4+/+ cells (3.41±0.32 and 3.12±0.18, respectively, both P＜0.05).The western blot results showed that, with the elevated intracellular expression level of TRAF4, phosphorylation level of EGFR was significantly increased in both wild-type EGFR and activation mutant EGFR-expression cells. The capacities of proliferation, migration and invasion of A549 cells was weakened after EGFR knockdown (all P＜0.01). Immunoprecipitation experiments showed that TRAF4 binds to the peptide segment of the near-membrane region of EGFR through the TRAF structural domain, and the mutual binding between EGFR molecules was enhanced under TRAF4 overexpression conditions. Increasing TRAF4 expression promoted EGFR molecular phosphorylation and activation of downstream signaling. Conclusions:TRAF4 expression is elevated in NSCLC tissues and tumor cells, which promotes tumor proliferation, migration and invasion. TRAF4 directly binds to EGFR molecules, enhances its own phosphorylation and activates the downstream signaling pathway by promoting the interaction between EGFR molecules.

[Objective]To investigate the effect of Baoyuan Jiedu Decoction(BJD)on serum lipids and white adipose tissue browning in cancer cachexia mice.[Methods]The specific pathogen free C57BL/6 mice were divided into normal group,model group,BJD group and megestrol acetate(MA)group.After 21 days of intervention,the changes of body weight,food intake,water consumption and tumor volume of the mice were observed,multidimensional mass spectrometry-based shotgun lipidomics(MDMS-SL)was used to determine the content of serum lipid of mice,white adipose tissue morphology and lipid droplet area were detected by hematoxylin-eosin(HE)staining,the expressions of white adipose tissue browning related genes were detected by Real-time quantitative polymerase chain reaction(Real-time PCR);and the protein expression of uncoupling protein 1(UCP1)was detected by Western blot and immunohistochemistry(IHC)staining.[Results]Compared with model group,the mice in BJD group were generally in good condition,and their food intake,water consumption and weight were increased significantly(P＜0.05),and the volumes of tumors were significantly suppressed(P＜0.05).Compared with normal group,there were 61 kinds of abnormal lipids in the serum of model group,while 30 kinds of lipids were influenced by BJD treatment(P＜0.05).Compared with model group,BJD alleviated the mass loss and lipid droplets(P＜0.05),inhibited the mRNA expression of UCP1,Cidea,Prdm16(P＜0.05)and the protein expression of UCP1(P＜0.05)in epididymal white adipose tissue(eWAT)and inguinal white adipose tissue(iWAT)of cancer cachexia mice.[Conclusion]BJD can inhibit weight loss,relieve the disorder of serum lipid,and inhibit the white adipose tissue browning of iWAT and eWAT of cancer cachexia mice.

Objective:To evaluate the effect of lung recruitment maneuvers combined with individualized positive end-expiratory pressure(PEEP) on the degree of postoperative atelectasis in elderly patients undergoing laparoscopic surgery.Methods:One hundred and forty-three elderly patients, aged ≥65 yr, with body mass index of 18.5-30.0 kg/m 2, scheduled for elective laparoscopic surgery, were assigned to either individualized PEEP combined with recruitment maneuvers (group Ⅱ) or fixed PEEP (group Ⅰ) using a random number table method. PEEP was maintained at 6 cmH 2O starting from the beginning of procedure until the end of the procedure in group I. Individualized PEEP titration was performed after induction of anesthesia in group Ⅱ. The primary outcome measure was the 12-zone lung ultrasound score at 15 min after tracheal extubation. Other outcome measures were the occurrence of postoperative pulmonary complications within 7 days after surgery, Quality of Recovery-15 scale score on 3rd day after surgery, rate of unplanned admission to intensive care units, length of hospital stay, incidence of intraoperative hypoxemia, usage rate of intraoperative vasoactive drugs, and incidence of postoperative hypotension. Results:Compared with group Ⅰ, the lung ultrasound score, driving pressure and postoperative pulmonary complications were significantly decreased, the dynamic lung compliance was increased ( P<0.05 or 0.01), and no significant changes were found in the other parameters in group Ⅱ ( P>0.05). Conclusions:Individualized PEEP combined with recruitment maneuvers can reduce the degree of postoperative atelectasis in elderly patients undergoing laparoscopic surgery.

Objective:To explore the role of tumor necrosis factor receptor-associated factor 4 (TRAF4) in promoting the abnormal activation of epidermal growth factor receptor (EGFR) and its effect on lung cancer cell proliferation, migration and invasion.Methods:Tumor tissues from patients who underwent lung adenocarcinoma resection at The First Affiliated Hospital of Second Military Medical University, from January 2015 to May 2017 were collected, and the expressions of TRAF4 and Ki-67 in lung cancer tissues were detected by immunohistochemistry, the mRNA levels of Cyclin D and Vimentin were detected by real-time fluorescence quantitative PCR (qRT-PCR). The effect of TRAF4 on the tumor growth ability of lung cancer A549 cells was investigated by the xenograft model, the effect of TRAF4 or EGFR on the tumor proliferation ability was detected by using cell counting kit 8 (CCK8) and BrdU assay, and the migration and invasion abilities of tumor cells were detected by Transwell assay. Different structural domain deletion expression vectors of TRAF4 and EGFR were constructed to transfect cells, and the interaction mode of TRAF4 and EGFR was investigated by immunoprecipitation assay.Results:The expression of TRAF4 in non-small cell lung cancer (NSCLC) tissues was positively correlated with the expressions of Ki-67, cyclin D, and vimentin ( r2: 0.438, 0.695, and 0.736, respectively, all P＜0.01). Immunohistochemical assay of tumor tissues from NSCLC patients showed that tissues with high expression of TRAF4 were also high in Ki-67. Patients with high TRAF4 expression (TRAF4 positivity >30%) had a shorter progression-free survival (PFS) time than that of patients with low TRAF4 expression (TRAF4 positivity ≤30%) (median PFS of 12 and 19 months, respectively; P=0.034). Traf4 -/- cells had a weakened proliferative capacity than traf4+/+ cells and formed tumors with smaller size ( P＜0.05). The expression level of Ki-67 in the tumor tissues formed by traf4 -/- cells [(45.6±8.7)%] was lower than that in the tumor tissues formed by traf4+/+ cells [(62.3±10.3)%, P=0.015], the mRNA levels of cyclin D (1.01±0.15) and vimentin (1.01±0.12) in the traf4 -/- cells were lower than those of the traf4+/+ cells (3.41±0.32 and 3.12±0.18, respectively, both P＜0.05).The western blot results showed that, with the elevated intracellular expression level of TRAF4, phosphorylation level of EGFR was significantly increased in both wild-type EGFR and activation mutant EGFR-expression cells. The capacities of proliferation, migration and invasion of A549 cells was weakened after EGFR knockdown (all P＜0.01). Immunoprecipitation experiments showed that TRAF4 binds to the peptide segment of the near-membrane region of EGFR through the TRAF structural domain, and the mutual binding between EGFR molecules was enhanced under TRAF4 overexpression conditions. Increasing TRAF4 expression promoted EGFR molecular phosphorylation and activation of downstream signaling. Conclusions:TRAF4 expression is elevated in NSCLC tissues and tumor cells, which promotes tumor proliferation, migration and invasion. TRAF4 directly binds to EGFR molecules, enhances its own phosphorylation and activates the downstream signaling pathway by promoting the interaction between EGFR molecules.

Objective:To analyze the mutations of globin genes among patients suspected for thalassemia from the Shanghai area.Methods:A total of 4 644 patients diagnosed at Ruijin Hospital, Shanghai Jiao Tong University School of Medicine between June 2016 and December 2019 were selected as the study subjects. The patients were tested for common mutations associated with thalassemia gene by Gap-PCR and reverse dot blotting (RDB). Patients were suspected to harbor rare mutations based on the inconsistency between hematological phenotypes and results of common mutation detection, and were further analyzed by Gap-PCR and Sanger sequencing.Results:Among the 4 644 patients, 2 194 (47.24%) were found to carry common thalassemia mutations, among which 701 (15.09%) were α-thalassemia, 1 448 (31.18%) were β-thalassemia, and 45 (0.97%) were both α- and β-thalassemia. Forty six samples were found to harbor rare mutations, which included 17 α-globin gene and 29 β-globin gene mutations. CD77(CCC>ACC) ( HBA2: c. 232C>A) of the α-globin gene, NG_000007.3: g. 70567_71015del449, codon 102(-A) ( HBB: c. 308_308delA) and IVS-Ⅱ-636 (A>G) ( HBB: c. 316-215A>G) of the β-globin gene were previously unreported new types of globin gene mutations. Conclusion:Among the 4 644 patients, the detection rate for common thalassemia mutations was 47.24%, whilst 46 samples were detected with rare gene mutations. The type of gene mutation types were diverse in the Shanghai area. The study has provided more accurate results for genetic diagnosis and counseling.

Objective:To establish a set of artificial intelligence (AI) verification rules for blood routine analysis.Methods:Blood routine analysis data of 18 474 hospitalized patients from the First Hospital of Jilin University during August 1st to 31st, 2019, were collected as training group for establishment of the AI verification rules,and the corresponding patient age, microscopic examination results, and clinical diagnosis information were collected. 92 laboratory parameters, including blood analysis report parameters, research parameters and alarm information, were used as candidate conditions for AI audit rules; manual verification combining microscopy was considered as standard, marked whether it was passed or blocked. Using decision tree algorithm, AI audit rules are initially established through high-intensity, multi-round and five-fold cross-validation and AI verification rules were optimized by setting important mandatory cases. The performance of AI verification rules was evaluated by comparing the false negative rate, precision rate, recall rate, F1 score, and pass rate with that of the current autoverification rules using Chi-square test. Another cohort of blood routine analysis data of 12 475 hospitalized patients in the First Hospital of Jilin University during November 1sr to 31st, 2023, were collected as validation group for validation of AI verification rules, which underwent simulated verification via the preliminary AI rules, thus performance of AI rules were analyzed by the above indicators. Results:AI verification rules consist of 15 rules and 17 parameters and do distinguish numeric and morphological abnormalities. Compared with auto-verification rules, the true positive rate, the false positive rate, the true negative rate, the false negative rate, the pass rate, the accuracy, the precision rate, the recall rate and F1 score of AI rules in training group were 22.7%, 1.6%, 74.5%, 1.3%, 75.7%, 97.2%, 93.5%, 94.7%, 94.1, respectively.All of them were better than auto-verification rules, and the difference was statistically significant ( P<0.001), and with no important case missed. In validation group, the true positive rate, the false positive rate, the true negative rate, the false negative rate, the pass rate, the accuracy, the precision rate, the recall rate and F1 score were 19.2%, 8.2%, 70.1%, 2.5%, 72.6%, 89.2%, 70.0%, 88.3%, 78.1, respectively, Compared with the auto-verification rules, The false negative rate was lower, the false positive rate and the recall rate were slightly higher, and the difference was statistically significant ( P<0.001). Conclusion:A set of the AI verification rules are established and verified by using decision tree algorithm of machine learning, which can identify, intercept and prompt abnormal results stably, and is moresimple, highly efficient and more accurate in the report of blood analysis test results compared with auto-vefication.

Objective:Clot waveform analysis (CWA) of activated partial thromboplastin time (APTT) was used to rapidly identify patients with hemophilia A (HA) and lupus anticoagulant (LA) positive, and to explore the diagnostic efficacy of APTT-CWA in HA and LA-positive patients.Methods:From July 25, 2022, to December 28, 2023, 145 patients with APTT prolongation were admitted to the First Hospital of Jilin University. This group comprised 106 males and 39 females, with an average age of (29.3±15.8) years, among whom there were 94 clinically confirmed HA cases and 51 LA-positive cases. The retrospective analysis employed the Mann-Whitney U test to compare APTT results and CWA parameters between HA patients and LA-positive individuals.CWA parameters including the first derivative of maximum reaction velocity (|min1|), the second derivative of maximum acceleration (|min2|), and maximum deceleration (|max2|) were investigated. Patients were classified based on the duration of APTT prolongation into mild (33 s53 s) groups. A comparison of APTT seconds and CWA parameters |min1|, |min2|, and |max2| was conducted for HA and LA-positive patients between each group. Furthermore, receiver operating characteristic (ROC) curves and area under the curve (AUC) were utilized to assess the diagnostic efficacy of CWA parameters and determine cut-off values. Results:The APTT results for HA and LA-positive patients were 70.2 s and 41.5 s, respectively, exhibiting a statistically significant difference ( P<0.001). CWA parameters |min1| were 1.459 %/s vs 3.868 %/s, |min2| were 0.124 %/s2 vs 0.502 %/s2, and |max2| were 0.054 %/s2 vs 0.388 %/s2in HA and LA patients, respectively ( P<0.001). In all APTT prolongation groups, the CWA parameters |min1|, |min2|and |max2| for HA were significantly lower than those for LA-positive patients ( P<0.05). CWA graph analysis revealed that the peak heights of |min1|, |min2|, and |max2| in HA were lower compared to LA patients. In the severe extension group, HA displayed significantly decreased peak values and a flatter, incomplete curve. ROC curve analysis indicated AUC values were greater than 0.8 ( P<0.01) for |min1|, |min2|, and |max2| in all APTT prolongation groups. Cut-off values for |min1|, |min2|, and |max2| were established for each prolongation group: mild (3.205 %/s, 0.423 %/s2, and 0.300 %/s2), moderate (2.884 %/s, 0.340 %/s2, 0.231 %/s2), and severe (2.209 %/s, 0.190 %/s2, 0.095 %/s2). Conclusion:When APTT was extended within the same range, there were significant differences in CWA parameters between HA patients and LA patients, ROC curve analysis demonstrated the effectiveness of CWA parameters in distinguishing HA from LA-positive patients,APTT-CWA can serve as a low-cost, efficient, and specific method for the rapid identification of hemophilia and lupus anticoagulant-positive patients.