Objective To investigate the effect and mechanism of Efferocytosis Relatived LncRNA (EFRL) on homocysteine-induced atherosclerosis in macrophage efferocytosis. Methods RAW264.7 cells were cultured in vitro, and the Control group (0 μmol/L Hcy) and Hcy intervention group (100 μmol/L Hcy) were set up. After GapmeR transfection of macrophages with Hcy intervention, EFRL knockdown negative control group (Hcy combined with LNA-NC) and EFRL knockdown group (Hcy combined with LNA-EFRL) were set up. High-throughput sequencing was applied for different expression of LncRNA MSTRG. 88917.16 (EFRL), UCSC was used to analyze its conservation, CPC and CPAT were used to analyze its ability to encode proteins, and GO and KEGG were used to analyze related biological functions. The localization of LncRNA EFRL in macrophages was analyzed by nucleoplasmic separation and RNA-FISH. Quantitative real-time PCR was used to detect the expression levels of LncRNA EFRL and its target gene SPAST in Hcy-treated macrophages. The apoptosis rate of Jurkat cells induced by UV was detected by flow cytometry. In vitro efferocytosis assay combined with immunofluorescence technique was used to analyze macrophage efferocytosis. ELISA was used to detect the levels of interleukin 1β(IL-1β) and IL-18. Results The new LncRNA MSTRG.88917.16 was identified and named EFRL(Efferocytosis Relatived LncRNA). UCSC, CPC and CPAT analyses showed that LncEFRL is highly conserved and does not have the ability to encode proteins. GO and KEGG analyses suggested that LncEFRL may be involved in macrophage efferocytosis. LncRNA EFRL was localized in the nucleus of macrophages as determined by nucleoplasmic separation and RNA-FISH. In comparison to the Control group, the expression levels of LncRNA EFRL and its target gene SPAST in the Hcy group were increased. In comparison to the Control group (0 min), the apoptosis rate of the experimental group (15, 30 min) Annexin V is more than 85%. Compared with Hcy combined with LNA-NC group, Hcy combined with LNA-EFRL group had enhanced macrophage efferocytosis and reduced levels of inflammatory factors. Compared with Hcy combined with LNA-NC group, the expression level of SPAST in Hcy combined with LNA-EFRL group was decreased. Conclusion Inhibition of EFRL expression can alleviate the process of Hcy inhibiting macrophage efferocytosis, and the mechanism is related to the regulation of the downstream target gene SPAST by EFRL.
RNA, Long Noncoding/physiology* ; Animals ; Homocysteine ; Mice ; Macrophages/drug effects* ; Humans ; RAW 264.7 Cells ; Atherosclerosis/chemically induced* ; Apoptosis/genetics* ; Phagocytosis/genetics* ; Jurkat Cells ; Interleukin-1beta/genetics* ; Efferocytosis

Objective:This study aimed to explore the molecular mechanisms of the maladaptation of H3N2 influenza virus in chicken embryos, provide a theoretical basis for the restoration of H3N2 influenza vaccine production in chicken embryos.Methods:Samples of respiratory secretions from patients with influenza-like symptoms (Influenza-like Illness, ILI) caused by H3N2 influenza virus were inoculated into chicken embryos and Madin-Darby Canine Kidney cells (MDCK), respectively. After isolating the virus, hemagglutination experiments were conducted to detect hemagglutination titers and hemagglutination inhibition experiments were used to compare antigenic differences; further, whole-genome sequencing of H3N2 influenza virus was performed using second-generation high-throughput gene sequencing (Next Generation High-Throughput Gene Sequencing, NGS), and key amino acid sites of mutations were identified through sequence alignment; combined with sialic acid receptor binding experiments, the differences in the binding of wild-type and mutant receptor binding sites (RBS) to sialic acid receptors were compared; finally, molecular docking and molecular dynamics simulation method were used to explore the specific molecular mechanisms of how mutation sites affect the differences in the affinity of the RBS pocket for sialic acid receptors.Results:The hemagglutination assay result indicated that both chicken embryos and MDCK cells could isolate the influenza virus, and the hemagglutination inhibition test showed that no antigenic differences were produced in the isolated strains. NGS analysis revealed that the H3N2 virus underwent an F195Y mutation in the (RBS) region of the hemagglutinin (HA) protein after adaptation through chicken embryo passages. Receptor-binding experiments demonstrated that the F195Y mutation enhanced the virus′s binding ability to α2, 3-linked sialic acid glycan (Neu5Acα2-3Galβ1-4GlcNAcβ-PAA, 3′SLN), while the mutation did not affect the affinity of the RBS pocket for α2, 6-linked sialic acid glycan (Neu5Acα2-6Galβ1-4GlcNAcβ-PAA, 6′SLN). Molecular docking and molecular dynamics simulation result indicate that the F195Y mutation, by replacing a hydrophobic amino acid with a hydrophilic one, leads to a significant decrease in the structure of the RBS pocket, enhancing the binding stability of the H3N2 influenza virus with α2, 3-sln. This is specifically manifested by an increase in binding time and an increase in the number of hydrogen bonds at the RBS site with the receptor. Furthermore, the F195Y mutation does not alter the binding of the virus to other receptors.Conclusions:The F195Y mutation in the RBS pocket of H3N2 influenza virus is a key site affecting the viral chicken embryo inadaptability.

Objective:This study aimed to explore the molecular mechanisms of the maladaptation of H3N2 influenza virus in chicken embryos, provide a theoretical basis for the restoration of H3N2 influenza vaccine production in chicken embryos.Methods:Samples of respiratory secretions from patients with influenza-like symptoms (Influenza-like Illness, ILI) caused by H3N2 influenza virus were inoculated into chicken embryos and Madin-Darby Canine Kidney cells (MDCK), respectively. After isolating the virus, hemagglutination experiments were conducted to detect hemagglutination titers and hemagglutination inhibition experiments were used to compare antigenic differences; further, whole-genome sequencing of H3N2 influenza virus was performed using second-generation high-throughput gene sequencing (Next Generation High-Throughput Gene Sequencing, NGS), and key amino acid sites of mutations were identified through sequence alignment; combined with sialic acid receptor binding experiments, the differences in the binding of wild-type and mutant receptor binding sites (RBS) to sialic acid receptors were compared; finally, molecular docking and molecular dynamics simulation method were used to explore the specific molecular mechanisms of how mutation sites affect the differences in the affinity of the RBS pocket for sialic acid receptors.Results:The hemagglutination assay result indicated that both chicken embryos and MDCK cells could isolate the influenza virus, and the hemagglutination inhibition test showed that no antigenic differences were produced in the isolated strains. NGS analysis revealed that the H3N2 virus underwent an F195Y mutation in the (RBS) region of the hemagglutinin (HA) protein after adaptation through chicken embryo passages. Receptor-binding experiments demonstrated that the F195Y mutation enhanced the virus′s binding ability to α2, 3-linked sialic acid glycan (Neu5Acα2-3Galβ1-4GlcNAcβ-PAA, 3′SLN), while the mutation did not affect the affinity of the RBS pocket for α2, 6-linked sialic acid glycan (Neu5Acα2-6Galβ1-4GlcNAcβ-PAA, 6′SLN). Molecular docking and molecular dynamics simulation result indicate that the F195Y mutation, by replacing a hydrophobic amino acid with a hydrophilic one, leads to a significant decrease in the structure of the RBS pocket, enhancing the binding stability of the H3N2 influenza virus with α2, 3-sln. This is specifically manifested by an increase in binding time and an increase in the number of hydrogen bonds at the RBS site with the receptor. Furthermore, the F195Y mutation does not alter the binding of the virus to other receptors.Conclusions:The F195Y mutation in the RBS pocket of H3N2 influenza virus is a key site affecting the viral chicken embryo inadaptability.

Objective To explore the effect and mechanism of penehyclidine hydrochloride (PHCD) on vascular endothelial injury in septic rats. Methods Fifty male SD rats were randomly divided into control group, lipopolysaccharide (LPS) induced sepsis group (model group), low dose PHCD (0.3 mg/kg) group, medium dose PHCD (1.0 mg/kg) group and high dose PHCD (3.0 mg/kg) groups, ten mice for each group. Normal saline was injected into the tail vein of the control group, and 10 mg/kg lipopolysaccharide (LPS) was injected into the tail vein of the rats in other groups to prepare the sepsis rat models. After the models were successfully established, low, medium and high doses (0.3, 1.0, 3.0 mg/kg) of PHCD solution were injected into the tail vein of the rats of corresponding groups. Wet/dry mass ratio (W/D) of lung tissue of rats in each group was measured, and ELISA was used to assay interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), IL-6 content and rat plasma angiopoietin 2 (Ang2) content in bronchoalveolar lavage fluid (BALF). HE staining was used to observe the pathological changes of lung tissues. Immunohistochemical staining was used to observe the expression of Ang2 in the right lung tissues. Western blot analysis was performed to detect Ang2 and vascular endothelial cadherin (VE-cadherin) protein in lung tissues. Results Compared with the control group, the W/D ratio of the lung tissues of rats in the model group and the contents of IL-1β, IL-6 and TNF-α in BALF were significantly increased; the lung tissues showed obvious pathological damage, with up-regulation of Ang2 expression and down-regulation of VE-Cadherin expression. Compared with the model group, the W/D ratio of the lung tissues of rats in three PHCD treatment groups and the contents of IL-1β, IL-6 and TNF-α in BALF were significantly reduced; the pathological damage of lung tissue was significantly reduced, with down-regulation of Ang2 expression and up-regulation of VE-cadherin expression. Conclusion PHCD can reduce LPS-induced lung inflammation in rats with sepsis by regulating the Ang2/VE-Cadherin pathway, thereby improving vascular endothelial injury.
Rats ; Mice ; Animals ; Male ; Lipopolysaccharides/metabolism* ; Tumor Necrosis Factor-alpha/metabolism* ; Angiopoietin-2/pharmacology* ; Interleukin-6/metabolism* ; Rats, Sprague-Dawley ; Lung ; Acute Lung Injury/metabolism* ; Sepsis/metabolism*

OBJECTIVE: To calculate the pharmacokinetic parameters of recombinant human coagulation factor Ⅷ using myPKFiT in patients with severe hemophilia A, and provide an individualized treatment plan for patients. METHODS: A total of 42 patients with severe hemophilia A who were treated with recombinant human coagulation factor Ⅷ were included from January 2021 to December 2021. myPKFiT was used to calculate the pharmacokinetic parameters of FⅧ, and the individualized treatment plan for hemophilia A patients was formulated. RESULTS: The median age of 42 patients with severe hemophilia A was 31（16-50） years old, the average weight was 54.0±9.9 kg, the half-life of FⅧ was 12.05±1.6 h, the time to more than 1% of the baseline was 62.3±15.3 h, and the 0 bleeding rate after the guidance of myPKFiT was significantly increased from 39% to 49%, the Annual bleeding rate was reduced from 3.6±2.5 to 2.1±2.0, and the Annual joint bleeding rate was reduced from 3.2±2.2 to 1.9±0.9, all of which were statistically different (P<0.05). CONCLUSION Individualized therapy in patients with severe hemophilia A who were guided by myPKFiT assay of pharmacokinetics parameters can significantly reduce the annual bleeding rate and annual joint bleeding rate of patients.
Adult ; Humans ; Middle Aged ; Blood Coagulation Factors ; Factor VIII/pharmacokinetics* ; Hemophilia A ; Hemorrhage ; Recombinant Proteins/pharmacokinetics* ; Adolescent ; Young Adult

OBJECTIVE: To investigate the clinical characteristics, diagnosis, and treatment of one patient with TAFRO syndrome, and to strengthen the understanding of this rare type. METHODS: The clinical manifestations, diagnosis and treatment process, and prognosis of the patient admitted in Gansu Provincial People's Hospital were retrospectively analyzed. RESULTS: Combined with laboratory tests, bone marrow examination, imaging, pathology, etc, the patient was diagnosed with TAFRO syndrome. After three cycles of treatment with pomalidomide (2-3 mg/d, d1-21), cyclophosphamide (300 mg/m², 0.54 g once a week) and dexamethasone (20 mg/d, two days a week), platelet count, serum creatinine and procalcitonin returned to normal, the systemic edema disappeared, and the patient's condition was alleviated. The therapeutic effect was good. CONCLUSION TAFRO syndrome is rare, involves multiple systems, progresses rapidly, and has a worse prognosis. The choice of the "Pomalidomide+cyclophosphamide+dexamethasone" regimen is help to improve the survival prognosis of patient with TAFRO syndrome.
Humans ; Thrombocytopenia ; Retrospective Studies ; Castleman Disease/diagnosis* ; Dexamethasone ; Cyclophosphamide/therapeutic use*

OBJECTIVE: To explore the clinical characteristics and prognosis of multiple myeloma(MM) patients with secondary primary malignancies. METHODS: The clinical data of newly diagnosed MM patients admitted to the First Affiliated Hospital of Zhengzhou University from January 2011 to December 2019 were retrospectively analyzed. The patients with secondary primary malignancies were retrieved, and their clinical features and prognosis were evaluated. RESULTS: A total of 1 935 patients with newly diagnosed MM were admitted in this period, with a median age of 62 (18-94) years old, of which 1 049 cases were hospitalized twice or more. There were eleven cases with secondary primary malignancies (the incidence rate was 1.05%), including three cases of hematological malignancies (2 cases of acute myelomonocytic leukemia and 1 case of acute promyelocytic leukemia) and eight cases of solid tumors (2 cases of lung adenocarcinoma, and 1 case each of endometrial cancer, esophageal squamous cell carcinoma, primary liver cancer, bladder cancer, cervical squamous cell carcinoma, and meningioma). The median age of onset was 57 years old. The median time between diagnosis of secondary primary malignancies and diagnosis of MM was 39.4 months. There were seven cases with primary or secondary plasma cell leukemia, the incidence rate was 0.67%, and the median age of onset was 52 years old. Compared with the randomized control group, the β2-microglobulin level in the secondary primary malignancies group was lower (P=0.028), and more patients were in stage I/II of ISS (P=0.029). Among the 11 patients with secondary primary malignancies, one survived, ten died, and the median survival time was 40 months. The median survival time of MM patients after the secondary primary malignancies was only seven months. All seven patients with primary or secondary plasma cell leukemia died, with a median survival time of 14 months. The median overall survival time of MM patients with secondary primary malignancies was longer than that of the patients with plasma cell leukemia (P=0.027). CONCLUSION The incidence rate of MM with secondary primary malignancies is 1.05%. MM patients with secondary primary malignancies have poor prognosis and short median survival time, but the median survival time is longer than that of patients with plasma cell leukemia.
Humans ; Middle Aged ; Aged ; Aged, 80 and over ; Multiple Myeloma/complications* ; Leukemia, Plasma Cell ; Retrospective Studies ; Esophageal Neoplasms/complications* ; Esophageal Squamous Cell Carcinoma/complications* ; Prognosis ; Neoplasms, Second Primary

OBJECTIVE: To investigate the expression of CSF3R mutation in acute myeloid leukemia (AML) and analyze its clinical characteristics and prognosis. METHODS: A retrospective study was conducted in 212 patients with AML who were newly diagnosed in the Second Hospital of Shanxi Medical University from January 1th 2018 to June 30th 2021, including 22 patients with CSF3R mutations as mutation group and 190 patients with CSF3R wild type ［66 cases of them were screened by propensity score matching (PSM), as control group］. The early efficacy and survival between the two groups were compared. RESULTS: The median age of patients in the mutation group was 50(17-73) years old, and the ratio of male to female was 1.2:1 The main types were AML with maturation (11 cases) and acute myelomonocytic leukemia (9 cases). Prognostic stratification was carried out according to the risk stratification system of the European leukemia network in 2017, with 16 cases (72.73%) in the middle and high-risk group. At the initial diagnosis, the median count of white blood cell (WBC) was 44.75(1.30-368.71)×10⁹/L, among which 15 cases (68.18%) were >10×10⁹/L, and the median count of platelet (PLT) was 24(4-55)×10⁹/L. CSF3R T618I (68.18%) was a common mutation site, which had concomitant gene mutations, in which CEBPA mutation was the most common (10 cases, 45.45%), but only existed in CSF3R T618I mutation. The CR/CRi rate was 68.18% and 71.21% in the mutant group and the control group (P >0.05), the median over all survival time was 15 months and 9 months (P >0.05), and the median disease-free survival time was 8 months and 4 months (P >0.05), respectively. CONCLUSION Most AML patients with CSF3R mutation are middle-aged patients, the main types are AML with maturation and acute myelomonocytic leukemia, and most of them have middle and high-risk prognosis. CSF3R mutation may not be an independent prognostic marker for newly diagnosed AML patients.
Middle Aged ; Humans ; Male ; Female ; Aged ; Leukemia, Myelomonocytic, Acute ; Retrospective Studies ; Leukemia, Myeloid, Acute/diagnosis* ; Prognosis ; Mutation ; Receptors, Colony-Stimulating Factor/genetics*

OBJECTIVE: To investigate the inflammatory effects of Cinobufotalin on monocytes in resting state and macrophages in activated state and its molecular mechanism. METHODS: THP-1 cells were stimulated with Phorbol 12-myristate 13-acetate to induce differentiation into macrophages. Lipopolysaccharides was added to activate macrophages in order to establish macrophage activation model. Cinobufotalin was added to the inflammatory cell model for 24 h as a treatment. CCK-8 was used to detect cell proliferation, Annexin V /PI double staining flow cytometry was used to detect cell apoptosis, flow cytometry was used to detect macrophage activation, and cytometric bead array was used to detect cytokines. Transcriptome sequencing was used to explore the gene expression profile regulated by Cinobufotalin. Changes in the significantly regulated molecules were verified by real-time quantitative polymerase chain reaction and Western blot. RESULTS: 1∶25 concentration of Cinobufotalin significantly inhibited the proliferation of resting monocytes(P<0.01), and induced apoptosis(P<0.01), especially the activated macrophages(P<0.001, P<0.001). Cinobufotalin significantly inhibited the activation of macrophages, and significantly down-regulated the inflammatory cytokines(IL-6, TNF-α, IL-1β, IL-8) released by activated macrophages(P＜0.001). Its mechanism was achieved by inhibiting TLR4/MYD88/P-IκBa signaling pathway. CONCLUSION Cinobufotalin can inhibit the inflammatory factors produced by the over-activation of macrophages through TLR4/MYD88/P-IκBa pathway, which is expected to be applied to the treatment and research of diseases related to the over-release of inflammatory factors.
Humans ; Toll-Like Receptor 4/metabolism* ; Myeloid Differentiation Factor 88/genetics* ; Macrophages/metabolism* ; Cytokines/metabolism* ; Lipopolysaccharides/pharmacology* ; NF-kappa B

OBJECTIVE: To explore the anti-inflammatory effects of ethyl lithospermate in lipopolysaccharide (LPS)-stimulated RAW 264.7 murine-derived macrophages and zebrafish, and its underlying mechanisms. METHODS: 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazoliumbromide (MTT) assays were performed to investigate the toxicity of ethyl lithospermate at different concentrations (12.5-100 µ mol/L) in RAW 264.7 cells. The cells were stimulated with LPS (100 ng/mL) for 12 h to establish an inflammation model in vitro, the production of pro-inflammatory cytokines interleukin (IL)-6 and tumor necrosis factor α (TNF-α) were assessed by enzyme linked immunosorbent assay (ELISA). Western blot was used to ascertain the protein expressions of signal transducer and activator of transcription 3 (STAT3), nuclear factor kappa B (NF-κB) p65, phospho-STAT3 (p-STAT3, Tyr705), inhibitor of NF-κB (IκB) α, and phospho-I κB α (p-IκB α, Ser32), and confocal imaging was used to identify the nuclear translocation of NF-κB p65 and p-STAT3 (Tyr705). Additionally, the yolk sacs of zebrafish (3 days post fertilization) were injected with 2 nL LPS (0.5 mg/mL) to induce an inflammation model in vivo. Survival analysis, hematoxylin-eosin (HE) staining, observation of neutrophil migration, and quantitative real-time polymerase chain reaction (qRT-PCR) were used to further study the anti-inflammatory effects of ethyl lithospermate and its probable mechanisms in vivo. RESULTS: The non-toxic concentrations of ethyl lithospermate have been found to range from 12.5 to 100 µ mol/L. Ethyl lithospermate inhibited the release of IL-6 and TNF-α(P<0.05 or P<0.01), decreased IκBα degradation and phosphorylation (P<0.05) as well as the nuclear translocation of NF-κB p65 and p-STAT3 (Tyr705) in LPS-induced RAW 264.7 cells (P<0.01). Ethyl lithospermate also decreased inflammatory cells infiltration and neutrophil migration while increasing the survival rate of LPS-stimulated zebrafish (P<0.05 or P<0.01). In addition, ethyl lithospermate also inhibited the mRNA expression levels of of IL-6, TNF-α, IκBα, STAT3, and NF-κB in LPS-stimulated zebrafish (P<0.01). CONCLUSION Ethyl lithospermate exerts anti-Inflammatory effected by inhibiting the NF-κB and STAT3 signal pathways in RAW 264.7 macrophages and zebrafish.
Animals ; Mice ; NF-kappa B/metabolism* ; Lipopolysaccharides ; RAW 264.7 Cells ; Zebrafish ; NF-KappaB Inhibitor alpha/metabolism* ; Interleukin-6/metabolism* ; Tumor Necrosis Factor-alpha/metabolism* ; STAT3 Transcription Factor/metabolism* ; Inflammation/metabolism* ; Anti-Inflammatory Agents/therapeutic use*