Objective To investigate the role and mechanism of neurotrophin-3(NT-3)in promoting neurological recovery after sep-sis-associated encephalopathy(SAE).Methods Twenty-four Sprague-Dawley(SD)rats were randomly divided into the Sham,SAE,and+NT-3 groups(n=8).Neurological deficits were evaluated using a behavioral scoring system.Western blotting was performed to assess the expression levels of NT-3,S100β,caspase-1,and microtubule-associated protein 1 light chain 3B(LC3B)in brain tissues.Immuno-histochemistry(IHC)was performed to assess interleukin-1 β(IL-1β)expression level.For cellular experiments,groups included normal,lipopolysaccharide(LPS),LPS+NT-3,LPS+NT-3+3-MA(autophagy inhibitor),and LPS+NT-3+compound C(AMPK inhibitor).Western blotting was used to analyze LC3B,NLRP3,ASC,AMPK,phosphorylated AMPK(p-AMPK),mTOR,and phosphorylated mTOR(p-mTOR)expression levels.Caspase-1 expression in cells was evaluated using immunofluorescence(IF).Results NT-3 expression in septic rat brain tissues increased progressively from d1 to d2(P＜0.05).Compared with the SAE group,the SAE+NT-3 group exhibited elevated neurological scores.The expression levels of IL-1β,S100β,and caspase-1 decreased from d1 to d2,while the aurtophagy level increased from d1 to d2(all P＜0.05).In cellular models,the LPS+NT-3 group showed decreased caspase-1 expression and increased autophagy activity compared to that in the LPS group from 1 h to 3 h(P＜0.05).Compared to the LPS+NT-3 group,the LPS+NT-3+3-MA group exhibited elevated NLRP3,ASC,and caspase-1-positive cells(P＜0.05),whereas the LPS+NT-3+Compound C group exhibited reduced p-AMPK,increased p-mTOR,and attenuated autophagy(P＜0.05).Conclusion NT-3 activates autophagy through the AMPK/mTOR signaling pathway during the early phase of SAE,thereby eliminating pyroptosis-associated proteins NLRP3 and ASC to promote neurolog-ical recovery.

Objective To investigate the role and mechanism of neurotrophin-3(NT-3)in promoting neurological recovery after sep-sis-associated encephalopathy(SAE).Methods Twenty-four Sprague-Dawley(SD)rats were randomly divided into the Sham,SAE,and+NT-3 groups(n=8).Neurological deficits were evaluated using a behavioral scoring system.Western blotting was performed to assess the expression levels of NT-3,S100β,caspase-1,and microtubule-associated protein 1 light chain 3B(LC3B)in brain tissues.Immuno-histochemistry(IHC)was performed to assess interleukin-1 β(IL-1β)expression level.For cellular experiments,groups included normal,lipopolysaccharide(LPS),LPS+NT-3,LPS+NT-3+3-MA(autophagy inhibitor),and LPS+NT-3+compound C(AMPK inhibitor).Western blotting was used to analyze LC3B,NLRP3,ASC,AMPK,phosphorylated AMPK(p-AMPK),mTOR,and phosphorylated mTOR(p-mTOR)expression levels.Caspase-1 expression in cells was evaluated using immunofluorescence(IF).Results NT-3 expression in septic rat brain tissues increased progressively from d1 to d2(P＜0.05).Compared with the SAE group,the SAE+NT-3 group exhibited elevated neurological scores.The expression levels of IL-1β,S100β,and caspase-1 decreased from d1 to d2,while the aurtophagy level increased from d1 to d2(all P＜0.05).In cellular models,the LPS+NT-3 group showed decreased caspase-1 expression and increased autophagy activity compared to that in the LPS group from 1 h to 3 h(P＜0.05).Compared to the LPS+NT-3 group,the LPS+NT-3+3-MA group exhibited elevated NLRP3,ASC,and caspase-1-positive cells(P＜0.05),whereas the LPS+NT-3+Compound C group exhibited reduced p-AMPK,increased p-mTOR,and attenuated autophagy(P＜0.05).Conclusion NT-3 activates autophagy through the AMPK/mTOR signaling pathway during the early phase of SAE,thereby eliminating pyroptosis-associated proteins NLRP3 and ASC to promote neurolog-ical recovery.

OBJECTIVE: To investigate the effects of Danmu Extract Syrup (DMS) on lipopolysaccharide (LPS)-induced acute lung injury (ALI) in mice and explore the mechanism. METHODS: Seventy-two male Balb/C mice were randomly divided into 6 groups according to a random number table (n=12), including control (normal saline), LPS (5 mg/kg), LPS+DMS 2.5 mL/kg, LPS+DMS 5 mL/kg, LPS+DMS 10 mL/kg, and LPS+Dexamethasone (DXM, 5 mg/kg) groups. After pretreatment with DMS and DXM, the ALI mice model was induced by LPS, and the bronchoalveolar lavage fluid (BALF) were collected to determine protein concentration, cell counts and inflammatory cytokines. The lung tissues of mice were stained with hematoxylin-eosin, and the wet/dry weight ratio (W/D) of lung tissue was calculated. The levels of tumor necrosis factor-α (TNF-α), interleukin (IL)-6 and IL-1 β in BALF of mice were detected by enzyme linked immunosorbent assay. The expression levels of Claudin-5, vascular endothelial (VE)-cadherin, vascular endothelial growth factor (VEGF), phospho-protein kinase B (p-Akt) and Akt were detected by Western blot analysis. RESULTS: DMS pre-treatment significantly ameliorated lung histopathological changes. Compared with the LPS group, the W/D ratio and protein contents in BALF were obviously reduced after DMS pretreatment (P<0.05 or P<0.01). The number of cells in BALF and myeloperoxidase (MPO) activity decreased significantly after DMS pretreatment (P<0.05 or P<0.01). DMS pre-treatment decreased the levels of TNF-α, IL-6 and IL-1 β (P<0.01). Meanwhile, DMS activated the phosphoinositide 3-kinase/protein kinase B (PI3K/Akt) pathway and reversed the expressions of Claudin-5, VE-cadherin and VEGF (P<0.01). CONCLUSIONS DMS attenuated LPS-induced ALI in mice through repairing endothelial barrier. It might be a potential therapeutic drug for LPS-induced lung injury.
Mice ; Male ; Animals ; Proto-Oncogene Proteins c-akt/metabolism* ; Lipopolysaccharides ; Phosphatidylinositol 3-Kinases/metabolism* ; Interleukin-1beta/metabolism* ; Vascular Endothelial Growth Factor A/metabolism* ; Tumor Necrosis Factor-alpha/metabolism* ; Claudin-5/metabolism* ; Acute Lung Injury/chemically induced* ; Lung/pathology* ; Interleukin-6/metabolism* ; Drugs, Chinese Herbal

Objective:To analyze the genes related to platelet activation in essential thrombocythemia (ET)based on transcriptome sequencing technology (RNA-seq ),and to explore the potential targets related to ET thrombosis. Methods:Blood samples from ET patients and healthy individuals were collected for RNA-seq,and differentially expressed lncRNAs,miRNAs,and mRNAs were selected to construct a lncRNA-miRNA-mRNA regulatory network. Differential mRNAs in the regulatory network were enriched and analyzed using Gene Ontology (GO ) and Kyoto Encyclopedia of Genes and Genomes (KEGG).The real-time PCR method was applied to validate differential mRNAs on crucial signaling pathways.Results:A total of 32 lncRNAs (3 up-regulated,29 down-regulated),16 miRNAs (8 up-regulated,8 down-regulated),and 35 mRNAs (27 up-regulated,8 down-regulated)were identified as differentially expressed.Among them,5 lncRNAs,12 miRNAs,and 19 mRNAs constituted the regulatory network.KEGG enrichment analysis showed that the differential mRNAs were related to the platelet activation signaling pathway,and there were 6 differential mRNAs related to platelet activation,namely F2R,ITGA2B,ITGB1,ITGB3,PTGS1,and GP1 BB,which were all up-regulated in their expression.RT-PCR results showed that the expression of five mRNAs including F2R,ITGA2B,ITGB1,ITGB3,and GP1BB were upregulated in ET patients compared with healthy subjects,and consistent with RNA-seq results,while PTGS1 expression was not significantly different.Conclusion:Differential mRNAs in ET patients are related to the platelet activation pathway,and F2R,ITGA2B,ITGB1,ITGB3,and GP1BB mRNAs may serve as novel targets associated with platelet activation in ET.

OBJECTIVE: To understand the biological characteristics of polycythemia vera (PV) patients with myeloid fibroplasia, and further analyze the risk factors affecting myeloid fibroplasia in PV patients, so as to provide ideas for predicting the occurrence of myeloid fibroplasia in PV patients. METHODS: Forty patients with PV in the Department of Hematology, Xiyuan Hospital of China Academy of Chinese Medical Sciences were collected and divided into two groups, with (hyperplasia group) and without (Non-proliferative group) hyperplasia of bone marrow fibers. The differences of basic clinical characteristics, blood routine, biochemistry, bone marrow cells, coagulation function and other indicators between the two groups were compared, and the independent risk factors affecting the proliferation of bone marrow fibrous tissue in PV patients were further analyzed by multivariate regression. RESULTS: Compared with Non-proliferative group, the JAK2 mutation rate (95% vs 70%，P=0.037), eosinophilic cell count (0.19 vs 0.11, P=0.047) and eosinophilic percentage (1.84 vs 1.27, P=0.001) in PV patients with hyperplasia were significantly increased, triglycerides (1.55 vs 1.91, P=0.038) and low-density lipoprotein (1.50 vs 3.08, P=0.000) were significantly reduced, bone marrow hematopoietic volume (0.85 vs 0.6, P=0.001), granulocyte/erythrocyte ratio (3.40 vs 1.89, P=0.033), lymphocyte/erythrocyte ratio (0.60 vs 0.42, P=0.033), and granulocyte+lymphocyte/erythrocyte ratio (3.72 vs 2.37, P=0.026) were significantly increased, thrombin time (18.84 vs 18.12, P=0.043) was significantly prolonged. Multivariate regression analysis results showed that peripheral blood eosinophil ≥2% and low-density lipoprotein ≤2 mmol/L were independent risk factors for bone marrow fibrous tissue hyperplasia in PV patients (P<0.05). CONCLUSION Increased proportion of peripheral blood eosinophils and decreased low density lipoprotein are risk factors for bone marrow fibrous tissue hyperplasia in PV patients.
Humans ; Bone Marrow/pathology* ; Polycythemia Vera ; Hyperplasia/pathology* ; Granulocytes/pathology* ; Janus Kinase 2/genetics* ; Risk Factors ; Lipoproteins, LDL ; Polycythemia/pathology*

OBJECTIVE: To establish an intestinal organoid model that mimic acute graft versus host disease (aGVHD) caused intestinal injuries by using aGVHD murine model serum and organoid culture system, and explore the changes of aGVHD intestine in vitro by advantage of organoid technology. METHODS: 20-22 g female C57BL/6 mice and 20-22 g female BALB/c mice were used as donors and recipients for bone marrow transplantation, respectively. Within 4-6 h after receiving a lethal dose (8.0 Gy) of γ ray total body irradiation, a total of 0.25 ml of murine derived bone marrow cells (1×10⁷/mice, n=20) and spleen nucleated cells (5×10⁶/mice, n=20) was infused to establish a mouse model of aGVHD (n=20). The aGVHD mice were anesthetized at the 7th day after transplantation, and the veinal blood was harvested by removing the eyeballs, and the serum was collected by centrifugation. The small intestinal crypts of healthy C57BL/6 mice were harvested and cultivated in 3D culture system that maintaining the growth and proliferation of intestinal stem cells in vitro. In our experiment, 5%, 10%, 20% proportions of aGVHD serum were respectively added into the organoid culture system for 3 days. The formation of small intestinal organoids were observed under an inverted microscope and the morphological characteristics of intestinal organoids in each groups were analyzed. For further evaluation, the aGVHD intestinal organoids were harvested and their pathological changes were observed. Combined with HE staining, intestinal organ morphology evaluation was performed. Combined with Alcian Blue staining, the secretion function of aGVHD intestinal organoids was observed. The distribution and changes of Lgr5⁺ and Clu⁺ intestinal stem cells in intestinal organoids were analyzed under the conditions of 5%, 10% and 20% serum concentrations by immunohistochemical stainings. RESULTS: The results of HE staining showed that the integrity of intestinal organoids in the 5% concentration serum group was better than that in the 10% and 20% groups. The 5% concentration serum group showed the highest number of organoids, the highest germination rate and the lowest pathological score among experimental groups, while the 20% group exhibited severe morphological destruction and almost no germination was observed, and the pathological score was the highest among all groups(t=3.668, 4.334,5.309,P<0.05). The results of Alican blue staining showed that the secretion function of intestinal organoids in serum culture of aGVHD in the 20% group was weaker than that of the 5% group and 10% of the organoids, and there was almost no goblet cells, and mucus was stainned in the 20% aGVHD serum group. The immunohistochemical results showed that the number of Lgr5⁺ cells of intestinal organoids in the 5% group was more than that of the intestinal organoids in the 10% aGVHD serum group and 20% aGVHD serum group. Almost no Clu⁺ cells were observed in the 5% group. The Lgr5⁺ cells in the 20% group were seriously injuried and can not be observed. The proportion of Clu⁺ cells in the 20% group significantly increased. CONCLUSION The concentration of aGVHD serum in the culture system can affect the number and secretion function of intestinal organoids as well as the number of intestinal stem cells in organoids. The higher the serum concentration, the greater the risk of organoid injury, which reveal the characteristics of the formation and functional change of aGVHD intestinal organoids, and provide a novel tool for the study of intestinal injury in aGVHD.
Mice ; Female ; Animals ; Mice, Inbred C57BL ; Bone Marrow Transplantation ; Graft vs Host Disease ; Stem Cells ; Organoids

BACKGROUND: Pneumonia-like primary pulmonary lymphoma (PPL) was commonly misdiagnosed as infectious pneumonia, leading to delayed treatment. The purpose of this study was to establish a computed tomography (CT)-based radiomics model to differentiate pneumonia-like PPL from infectious pneumonia. METHODS: In this retrospective study, 79 patients with pneumonia-like PPL and 176 patients with infectious pneumonia from 12 medical centers were enrolled. Patients from center 1 to center 7 were assigned to the training or validation cohort, and the remaining patients from other centers were used as the external test cohort. Radiomics features were extracted from CT images. A three-step procedure was applied for radiomics feature selection and radiomics signature building, including the inter- and intra-class correlation coefficients (ICCs), a one-way analysis of variance (ANOVA), and least absolute shrinkage and selection operator (LASSO). Univariate and multivariate analyses were used to identify the significant clinicoradiological variables and construct a clinical factor model. Two radiologists reviewed the CT images for the external test set. Performance of the radiomics model, clinical factor model, and each radiologist were assessed by receiver operating characteristic, and area under the curve (AUC) was compared. RESULTS: A total of 144 patients (44 with pneumonia-like PPL and 100 infectious pneumonia) were in the training cohort, 38 patients (12 with pneumonia-like PPL and 26 infectious pneumonia) were in the validation cohort, and 73 patients (23 with pneumonia-like PPL and 50 infectious pneumonia) were in the external test cohort. Twenty-three radiomics features were selected to build the radiomics model, which yielded AUCs of 0.95 (95% confidence interval [CI]: 0.94-0.99), 0.93 (95% CI: 0.85-0.98), and 0.94 (95% CI: 0.87-0.99) in the training, validation, and external test cohort, respectively. The AUCs for the two readers and clinical factor model were 0.74 (95% CI: 0.63-0.83), 0.72 (95% CI: 0.62-0.82), and 0.73 (95% CI: 0.62-0.84) in the external test cohort, respectively. The radiomics model outperformed both the readers' interpretation and clinical factor model ( P <0.05). CONCLUSIONS The CT-based radiomics model may provide an effective and non-invasive tool to differentiate pneumonia-like PPL from infectious pneumonia, which might provide assistance for clinicians in tailoring precise therapy.
Humans ; Retrospective Studies ; Pneumonia/diagnostic imaging* ; Analysis of Variance ; Tomography, X-Ray Computed ; Lymphoma/diagnostic imaging*

Humans ; Mycobacterium tuberculosis/genetics* ; Microspheres ; Antitubercular Agents/pharmacology* ; Mutation ; Microbial Sensitivity Tests ; Fluoroquinolones ; Drug Resistance, Bacterial/genetics* ; Tuberculosis, Multidrug-Resistant/microbiology*

Aim To investigate the effect of prodigiosin on the proliferation, migration, cell cycle and apopto- sis of adriamycin-resistant breast cancer cell line MCF- 7/ADR.Methods CCK-8, colony formation assay, scratch test and Transwell were used to detect the pro¬liferation and migration of MCF-7/ADR after treatment with different concentrations of prodigiosin.How cy¬tometry and DAP1 staining were used to detect the cell cycle and apoptosis of MCF-7/ADR cell line after treatment with different concentrations of prodigiosin.Western blot was used to detect the effect of prodigiosin on the expression of caspase-3, Bax and Bcl-2 in MCF- 7/ADR cells.Results Prodigiosin inhibited the pro¬liferation and migration of MCF-7/ADR cell line in a concentration- and time-dependent manner ( P <0.05 ).In addition, prodigiosin enhanced the in¬hibitor}' effect of adriamycin on MCF-7/ADR cell line.Prodigiosin arrested MCF-7/ADR cell line cycle in the S phase and induced cell apoptosis in a time- and dose- dependent manner.The percentage of S phase cells treated with 2 mg • L 1 prodigiosin for 48 hours in¬creased to 35.3% , and the apoptotic rate was as high as 64.83% , which were statistically significant com¬pared with the control group ( P < 0.05 ).Prodigiosin could up-regulate the expression of apoptosis protein caspase-3 and Bax and inhibit the expression of Bcl-2 protein in MCF-7/ADK cell.Conclusions Prodigio¬sin can effectively inhibit the proliferation, migration and promote cell apoptosis and regulate cell cycle in adriamvcin-resistant breast cancer cell line MCF-7/ ADR.Prodigiosin can enhance the sensitivity of MCF- 7/ADR cell line to adriamycin.

Objective To observe the distribution of mesencephalic astrocyte derived neurotrophic factor (MANF) within the brain in human, mice and rats and the comparison across the species. Methods An immunohistochemical method was applied to investigate the expression of MANF and to compare the differences among species in the brain of human specimens ( n = 5) , mice ( n = 6) , and rats ( n = 6). Results The expression of MANF varied in different brain regions in human, mice and rats. In human, in the cortex, the expression of MANF was the highest among all brain regions. In the subcortical areas or the nucleus, brainstem and cerebellum expression level of MANF were relatively lower. The distribution of MANF in mouse and rat brain were slightly different from that in human brain, but generally consistent. Conclusion The distribution pattern of MANF is similar across the species, however, within a species, MANF expression levels varies in different brain regions.