OBJECTIVE To investigate the effects and mechanisms of Lycium ruthenicum Murr. in improving sleep. METHODS Network pharmacology was employed to identify the active components of L. ruthenicum and their associated disease targets， followed by enrichment analysis. A caffeine‑induced zebrafish model of sleep deprivation was established ， and the zebrafish were treated with L. ruthenicum Murr. extract （LRME） at concentrations of 0.1， 0.2 and 0.4 mg/mL， respectively； 24 h later， behavioral changes of zebrafish and pathological alterations in brain neurons were subsequently observed. The levels of inflammatory factors [interleukin-6 （IL-6）， IL-1β， IL-10， tumor necrosis factor-α （TNF-α）]， oxidative stress markers [superoxide dismutase （SOD）， malondialdehyde （MDA）， glutathione peroxidase （GSH-Px）， catalase （CAT）]， and neurotransmitters [5- hydroxytryptamine （5-HT）， γ-aminobutyric acid （GABA）， glutamic acid （Glu）， dopamine （DA）， and norepinephrine （NE）] were measured. The protein expression levels of protein kinase B1 （AKT1）， phosphorylated AKT1 （p-AKT1）， epidermal growth factor receptor （EGFR）， B-cell lymphoma 2 （Bcl-2）， sarcoma proto-oncogene，non-receptor tyrosine kinase （SRC）， and heat shock protein 90α family class A member 1 （HSP90AA1） in the zebrafish were also determined. RESULTS A total of 12 active components and 176 intersecting disease targets were identified through network pharmacology analysis. Among these， apigenin， naringenin and others were recognized as core active compounds， while AKT1， EGFR and others served as key targets； EGFR tyrosine kinase inhibitor resistance signaling pathway was identified as the critical pathway. The sleep improvement rates in zebrafish of LRME low-， medium-， and high-dose groups were 54.60%， 69.03% and 77.97%， 开发。E-mail：hjp_yft@163.com respectively， while the inhibition ratios of locomotor distance were 0.57， 0.83 and 0.95， respectively. Compared with the model group， the number of resting counts， resting time and resting distance were significantly increased/extended in LRME medium- and high-dose groups （P＜0.05）. Neuronal damage in the brain was alleviated. Additionally， the levels of IL-6， IL-1β， TNF-α， MDA， Glu， DA and NE， as well as the protein expression levels of AKT1， p-AKT1， EGFR， SRC and HSP90AA1， were markedly reduced （P＜0.05）， while the levels of IL-10， SOD， GSH-Px， CAT， 5-HT and GABA， as well as Bcl-2 protein expression， were significantly elevated （P＜0.05）. CONCLUSIONS L. ruthenicum Murr. demonstrates sleep-improving effects， and its specific mechanism may be related to the regulation of inflammatory responses， oxidative stress， neurotransmitter balance， and the EGFR tyrosine kinase inhibitor resistance signaling pathway.

Background: Influenza A virus (IAV) is a negative-sense RNA virus of the Orthomyxoviridae family and is the etiological agent of a highly contagious acute respiratory disease that can lead to acute lung injury. Objective: To elucidate the molecular mechanisms of IAV infection, an integrative research approach combining gene expression profiling, multinetwork analysis, and in vivo experimental validations was employed. Methods: First, a series of network-based analyses were performed, including protein-protein interaction network construction, weighted gene co-expression network analysis, and subsequent gene set enrichment analysis, to identify the major underlying mechanisms of IAV infection. Following gene expression analysis, core targets, both direct and indirect regulators, were screened. An IAV (H1N1) strain A/PR/8/34-induced acute lung injury mouse model was constructed for in vivo validations. Batch one included two groups to evaluate findings from the multi-network analysis: Mock (n = 10; 5 males and 5 females) and IAV (n = 10; 5 males and 5 females). Batch two included three groups to assess the role of toll-like receptor 3 (TLR3) in IAV infection: Mock (n = 6; 3 males and 3 females), IAV (n = 6; 3 males and 3 females), and TLR3 inhibitor (n = 6; 3 males and 3 females). Body weight was measured on days 0, 3, and 5 after infection. On day 5, lung tissues were collected to assess viral load and histopathological changes. Key targets were examined using enzyme-linked immunosorbent assay, Western blotting, and immunofluorescence staining, both in sera and lung tissues. Results: IAV infection was significantly associated with dysregulation of the immune-inflammation system, such as the LTR, nucle-otide-binding oligomerization domain-(NOD) like receptor, retinoic acid-inducible gene I-like receptor, and nuclear factor kappa-B signaling pathways. Gene set enrichment analysis further indicated that the TLR and necroptosis signaling pathways played crucial roles in the progression of IAV infection (TLR signaling pathway normalized enrichment score = 2.3941, P = 1.00 × 10 ⁻¹⁰; necroptosis normalized enrichment score = 1.9421, P = 6.21 × 10 ⁻⁷). Among the core targets, TLR3 and mixed lineage kinase domain-like protein (MLKL) may regulate gene expression at the transcriptional level (all P < 0.05). In vivo validation using an IAV (PR8) infected acute lung injury mouse model demonstrated increased viral load and lung index, alveolar structural damage, and inflammatory cell infiltration. Immunofluorescence staining exhibited large gaps in Lamin B1 staining and breaches in Emerin signals following IAV-PR8 infection. Expression levels of TLR3, p-receptor-interacting serine/threonine-protein kinase 3 (RIPK3)/RIPK3, and p-mixed lineage kinase domain-like protein (MLKL)/MLKL proteins in lung tissues, as well as proinflammatory factors and mediators in sera, were significantly elevated after IAV infection. Moreover, enhanced neutrophil infiltration (myeloperoxidase) and citrullinated histone H3 (a neutrophil extracellular trap-specific marker), both established indicators of neutrophil extracellular trap formation, were observed. Notably, treatment with a TLR3 inhibitor significantly ameliorated IAV-induced acute lung injury by regulating necroptosis-related targets. Conclusion: Our study provides network-based in vivo evidence that TLR3-receptor-interacting serine/threonine-protein kinase 3-MLKL-mediated necroptosis may underlie IAV-induced acute lung injury and could serve as a potential therapeutic target in severe influenza cases.

Objective: To compare the efficacy of heat and acid elution methods for IgG anti-M and anti-Ku. Methods: Ten samples with IgG anti-M and two samples with IgG anti-Ku were selected and standardized to a titer of 64. These antibodies underwent overnight absorption at 4℃ with O-type MM and kk-type erythrocytes, and then heat and acid elution methods were used on the absorbed sensitized erythrocytes respectively by detecting the titer of anti-M and anti-Ku in the eluate to compare the differences in the elution efficiency of IgG anti-M and anti-Ku between the two elution methods. Results: In heat elution tests, all 10 anti-M samples showed positive results with titers ranging from 8 to 64, while 2 anti-Ku samples yielded negative results. In acid elution tests, all 10 anti-M samples demonstrated negative results, whereas both anti-Ku (n=2) samples exhibited positive reactions with consistent titers of 32. Following acid elution with subsequent heat elution, 8 of 10 anti-M samples showed positive results with titers ranging from 8 to 32, while 2 remained negative. Both anti-Ku samples demonstrated positive with titers of 4. Conclusion: Heat elution demonstrated superior efficiency for IgG anti-M compared to acid elution, whereas acid elution showed greater efficacy for IgG anti-Ku than heat elution.

Objective: To compare the efficacy of heat and acid elution methods for IgG anti-M and anti-Ku. Methods: Ten samples with IgG anti-M and two samples with IgG anti-Ku were selected and standardized to a titer of 64. These antibodies underwent overnight absorption at 4℃ with O-type MM and kk-type erythrocytes, and then heat and acid elution methods were used on the absorbed sensitized erythrocytes respectively by detecting the titer of anti-M and anti-Ku in the eluate to compare the differences in the elution efficiency of IgG anti-M and anti-Ku between the two elution methods. Results: In heat elution tests, all 10 anti-M samples showed positive results with titers ranging from 8 to 64, while 2 anti-Ku samples yielded negative results. In acid elution tests, all 10 anti-M samples demonstrated negative results, whereas both anti-Ku (n=2) samples exhibited positive reactions with consistent titers of 32. Following acid elution with subsequent heat elution, 8 of 10 anti-M samples showed positive results with titers ranging from 8 to 32, while 2 remained negative. Both anti-Ku samples demonstrated positive with titers of 4. Conclusion: Heat elution demonstrated superior efficiency for IgG anti-M compared to acid elution, whereas acid elution showed greater efficacy for IgG anti-Ku than heat elution.

Frontopsylla(Frontopsylla)ochotona sp.nov.is closely related to Frontopsylla(Fr.)adixsterna Liu,Shao et Liu,1976,but can be distinguished by the following characteristics:1.In males,the immutable processes exhibit a wide,finger-like shape with asymmetrical anterior and posterior edges at their ends.The posterior edge of st.VIII lacks a deep depression,and the distal arm of st.IX is split into two lobes.2.In females,the posterior edge of the st.VII abdominal plate lacks a deep sinus,but the posterior end has a truncated or arc-shaped broad leaf.The posterior edge of t.VIII has a wide sinus,distinguishing it from F.adixsterna and other species in this genus.The type specimen was collected in Linzhi City,Xizang Autonomous Region of China,in November 2018,from an agricultural area at an altitude of approximately 3 000 m.The host species include Ochotona sp.,Niviventer confucianus,and Pitymys leucurus.

Objective:To research the mechanism of the regulation of homeobox B8(HOXB8)for lysine demethylase 6B(KDM6B)-mediated CCAAT/enhancer binding protein α(C/EBPα)axis on the metastasis of high-grade serous ovarian cancer(HGSOC),so as to provide references for the study of the pathogenesis of HGSOC patients.Methods:The tumor tissue samples and corresponding adjacent normal tissue samples of HGSOC patients admitted to Wuhan Hospital of Traditional Chinese and Western Medicine from June to December 2024 were selected,and cell lines SKOV3 and A2780 of ovarian cancer were collected.Real-time fluorescence quantitative polymerase chain reaction(RT-qPCR)was adopted to detect the mRNA levels of KDM6B in HGSOC tumor tissues and its corresponding adjacent tissues.Western blot assay and immunohistochemistry were adopted to detect the expressions of KDM6B protein in the tissue.The A2780 cells of ovarian cancer were divided into the oe-HOXB8 group that was transfected by the HOXB8 overexpression vector,and the oe-NCHOXB8 group with the negative control(NC)vector.The SKOV3 cells of ovarian cancer were divided into the si-HOXB8 group that was transfected by the HOXB8 small interference sequence,and the si-NCHOXB8 group with negative control sequence.The transfected KDM6B was divided into the si-KDM6B group with small interference sequence and the oe-KDM6B group transfected with overexpression vector.The co-transfection HOXB8 and(or)KDM6B,C/EBPα were divided into si-HOXB8+si-KDM6B group and si-HOXB8+si-C/EBPα group of small interference sequence.The chromatin immunoprecipitation-qPCR(ChIP-qPCR)and dual-luciferase reporter gene assay were used to verify the mechanism that HOXB8 transcript and regulate KDM6B in SKOV3 and A2780 cells of ovarian cancer.The effects of overexpression or silencing of HOXB8 in A2780 and SKOV3 cells on the proliferation,invasion,migration and KDM6B expression of ovarian cancer cells were detected.The effects of overexpression or silencing of KDM6B in SKOV3 cells on the trimethylation modification of lysine 27 at histone H3(H3K27me3)and the expression of C/EBPα were detected.The effects of silencing KDM6B and C/EBPα on HOXB8-induced cell proliferation,invasion and migration were analyzed through functional rescue experiments.Results:In tumor tissues,the mRNA and protein expression levels of KDM6B were 1.02±0.03 and 1.02±0.04,respectively,which were significantly higher than those in the adjacent tissues,and the differences were statistically significant(t=62.440,38.737,P<0.01).The optical density value of proliferation,invasion rate and migration rate of A2780 cells in the oe-HOXB8 group that was transfected by the HOXB8 overexpression vector were respectively(1.74±0.15),(89.71±6.60)%and(85.33%±7.02)%,which were significantly higher than those in the oe-NCHOXB8 group,and the differences were statistically significant(t=7.778,7.353,4.759,P<0.01).The optical density value of proliferation,invasion rate and migration rate of SKOV3 cells in the si-HOXB8 group were respectively(0.54±0.06),(47.23±3.41)%and(43.20±3.12)%,all of which were significantly lower than those in the si-NCHOXB8 group,and the differences were statistically significant(t=9.400,8.615,9.040,P<0.01).The optical density value of proliferation,invasion rate and migration rate of SKOV3 cells in the si-HOXB8+si-KDM6B group were(1.04±0.09),(73.11±4.98)%and(68.65±4.45)%,respectively,which were significantly higher than those in the si-HOXB8 group,and the differences were all statistically significant(t=6.875,6.852,7.562,P<0.01).The optical density value of proliferation,invasion rate and migration rate of SKOV3 cells in the si-HOXB8+si-C/EBPα group were respectively(0.97±0.07),(75.87±5.12)%and(70.59±4.81)%,all of which were significantly higher than those in the si-HOXB8 group,and the differences were all statistically significant(t=6.355,7.500,7.884,P<0.01).Conclusion:HOXB8 can inhibit the C/EBPα expression and promote the HGSOC metastasis by regulating and controlling H3K27me3 modification of KDM6B-mediated C/EBPα histone.

At present,indocyanine green(ICG)is intraoperative imaging agent with the most of advantages,and the ICG fluorescence imaging technique that derived from it has been widely developed in surgery with laparoscope for colorectal cancer.This technique can effectively realize tumor localization,assessment for resection margin,imaging of lymph node and lymphatic vessel,which has important value in judging the blood supply of the anastomosis,and can further effectively improve surgical safety and reduce the risk of postoperative complications.In addition,ICG fluorescence imaging technique can also powerfully promote the development of minimally invasion and individualization of colorectal surgery.With the advancement of laparoscopic equipment,deepening of medical theory,progress of surgical techniques,and the integration of artificial intelligence and medicine,the application scenarios of ICG imaging technique are gradually expanding.This article reviewed the imaging principles,current status of application,and current existing issues of ICG fluorescence imaging technique in surgery for colorectal cancer,which conducted preliminarily exploration for its value in guiding the practice of the theory of membrane anatomy.

Objective To investigate the situation of unconformity of positive and reverse ABO blood typ-ing between outpatient and inpatient patients in the hospital,and explore the reasons for unconformity of posi-tive and reverse ABO blood typing detected by microcolumn gel method.Methods A retrospective analysis was conducted on the medical records of totally 425 patients with ABO blood type mismatch who underwent ORTHO VISION Max testing in the hospital from August 2023 to December 2024.Comprehensive analysis was conducted using various methods including the saline tube method,irregular antibody screening,increas-ing plasma volume,4 ℃ enhancement test,absorption elution,and gene sequencing,etc.Results Among 128 192 cases of ABO blood type tests,425 cases showed unconformity of positive and reverse ABO blood typ-ing,accounting for 0.33％.The causes were as follows:antibody weakening in 316 cases(74.35％),bone mar-row transplantation in 40 cases(9.41％),interference from irregular antibodies in 34 cases(8.00％),antigen weakening in 13 cases(3.06％),subgroups in 10 cases(2.35％),cold agglutinins in 6 cases(1.41％),inter-ference from irregular and autoantibodies in 4 cases(0.94％),auto-sensitization of red blood cells in 1 case(0.24％),and other reasons in 1 case(0.24％).Among 316 cases with antibody weakening,239 cases(75.63％)involved type A(anti-B weakening),61 cases(19.30％)involved type B(anti-A weakening),8 ca-ses(2.53％)involved type O(anti-A weakening),6 cases(1.91％)involved type O(anti-B weakening),and 2 cases(0.63％)involved type O(both anti-A and anti-B weakening).In patients with antibody weakening,the disease distribution was mainly cardiovascular disease in 127 cases(40.19％),a history of tumors in 67 ca-ses(21.20％),kidney diseases in 11 cases(3.48％),and orthopedic diseases in 10 cases(3.16％).Conclusion The main reason for the unconformity of positive and reverse ABO blood typing in the hospital is the weakening of the antibodies,especially the predominance of type A(anti-B weakening).Weakened anti-bodies are predominantly in the distribution of cardiovascular disease.For subtype-induced disturbances,a comprehensive analysis combining serology and gene sequencing is recommended.For disease-induced uncon-formity of positive and reverse ABO blood typing,monitoring the patients should be strengthened.

[Objective] To explore the effects of different methods on antibody detection through investigating the causes of cross-matching incompatible in a patient with gastric malignant tumor, and to establish flow cytometry protocol for confirming hemolytic transfusion reaction (HTR). [Methods] Antibodies in the patient's serum were identified by red blood cells (RBCs) blood grouping, antibody screening and identification, acid elution test and PEG enhancement test. To confirm HTR, patient RBCs, proximal and distal ends RBCs, separated by capillary centrifugation, were tested by direct antiglobulin test (DAT) and Jk^a antigen single label and double label flow cytometry. [Results] Routine serological technology revealed the presence of anti-C, e (titer:2) and anti-Jka (titer >1) in the patient’s serum. After separation using capillary centrifugation technology, both the proximal and distal DAT and Jk^a antigen tests were negative. Both DAT and Jk^a antigen positive red blood cells (0.21%, 6/6 327) were found in the patient's blood samples by flow cytometry. After separation of blood samples by capillary centrifugation, there were significantly more DAT and Jk^a antigen double-positive RBCs in the distal end (0.43%, 33/7 707) than in the proximal end (0.09%, 15/7 225). Two blood samples were screened from over 100 donor blood samples that are compatible with the patient's cross-matching, and the transfusion effect was favorable. [Conclusion] Serological methods combined with flow cytometry could improve the sensitivity of antibody detection, provide a more accurate basis for the diagnosis of HTRs, and guarantee the safety of blood transfusion.

Objective:To investigate the effect of transforming growth factor beta 1 (TGF-β1) on the biological activity of infantile hemangioma (IH) -derived endothelial cells (HemECs) .Methods:Three proliferating IH tissues and three involuting IH tissues were collected from IH patients receiving surgical resection at the Department of Pediatric Surgery, West China Hospital, Sichuan University from February to August 2021. Primary HemECs were isolated from proliferating IH tissues, and human umbilical vein endothelial cells (HUVECs) served as the control. The TGF-β1 expression levels in tissues and cells were detected by immunohistochemical study and Western blot analysis. Cell counting kit-8 (CCK8) assay was performed to assess the effect of 0 (control group) - 100 ng/ml TGF-β1 on HemEC proliferation. HemECs were treated with 5 ng/ml TGF-β1 or without (control group), and after several hours of treatment, Transwell assay was performed to evaluate cell migration ability, and immunofluorescence assay to assess the changes in the expression of endothelial markers (platelet-endothelial cell adhesion molecule-1 [CD31], vascular endothelial cadherin [VE-cadherin]) and mesenchymal markers (α-smooth muscle actin [α-SMA], collagen type Ⅰ α 1 [COL1A1]). Comparisons between groups were conducted by t test or one-way analysis of variance. Results:Immunohistochemical study showed that proliferating IH tissues were stained positively for TGF-β1, which was expressed relatively abundantly; the percentages of TGF-β1-positive signal area were higher in the proliferating IH tissues (24.68% ± 3.74%) than in the involuting IH tissues (almost no expression). Western blot analysis revealed that the relative expression level of TGF-β1 was significantly higher in HemECs (1.08 ± 0.13) than in HUVECs (0.30 ± 0.04, t = 9.93, P < 0.001). CCK8 assay showed increased proliferative activity of HemECs in the 3.125-, 6.25-, 12.5-, 25-, 50- and 75-ng/ml TGF-β1 groups compared with the control group (all P < 0.05), and no significant difference was found between the 100-ng/ml TGF-β1 group and the control group ( P > 0.05). Transwell assay revealed an increased number of migratory HemECs in the 5-ng/ml TGF-β1 group (127 ± 6) compared with the control group (103 ± 9; t = 5.32, P < 0.01). Immunofluorescence assay showed significantly decreased fluorescence intensity of endothelial markers CD31 and VE-cadherin in the 5-ng/ml TGF-β1 group (5.441 ± 1.254, 5.073 ± 0.412, respectively) compared with the control group (9.518 ± 1.728,7.671 ± 0.921, t = 3.31, 4.46, P = 0.030, 0.011, respectively), and significantly increased fluorescence intensity of mesenchymal markers α-SMA and COL1A1 in the 5-ng/ml TGF-β1 group (8.074 ± 0.846, 5.885 ± 0.216, respectively) compared with the control group (0.393 ± 0.342, 0.295 ± 0.125, t = 14.58, 38.76, P < 0.001, < 0.000 1, respectively) . Conclusion:TGF-β1 was relatively highly expressed in the proliferating IH tissues and HemECs, and could promote the proliferation, migration and endothelial-to-mesenchymal transition of HemECs.