Objective To investigate the protective effect of curcumin (Cur) against arsenic-induced neuroimmune toxicity and the underlying molecular mechanisms in vivo. Methods Eighty SPF female C57BL/6 mice were randomly assigned to four groups: a control group, an arsenic-treated group, a Cur-treated group and an arsenic+Cur group, with 20 mice in each group. The control group received distilled water; the arsenic-treated group was given 50 mg/L NaAsO₂ in the drinking water; the Cur-treated group was gavaged with 200 mg/kg of curcumin for 45 days; and the arsenic+Cur group received distilled water and was gavaged with 200 mg/kg of curcumin. Y-maze and Morris water maze experiments were conducted to assess the learning and memory ability of the mice. Western blot analysis was used to detect protein levels of blood-brain barrier tight junction proteins zonula occludens protein 1(ZO-1) and claudin 5, T lymphocyte subpopulation CD4 and CD8, Janus kinase 2/signal transducer and activator of transcription 3 (JAK2/STAT3) signaling pathway-related molecules JAK2 and STAT3. Real-time PCR was used to assess the mRNA levels of CD4⁺ T lymphocyte subsets type 1 T helper (Th1), Th2, Th17 and regulatory T cells (Treg) transcription factors and cytokines in hippocampus. Results Compared with the control group, the arsenic-treated group showed a significantly decreased correct rate, increased latency to reach the platform on the third and fifth days, and reduced times of crossing the platform. The expression of ZO-1 and claudin 5 protein decreased significantly, and the protein levels of CD4 and CD8 were up-regulated. The mRNA levels of Th1, Th17, and Treg transcription factor T-box expressed in T cell(T-bet), retinoid-related orphan receptor gamma t(RORγt), and forkhead box protein 3(FOXP3) in the arsenic-treated group were decreased. Th1 and Th17 cytokines interferon γ(IFN-γ) and interleukin 17(IL-17) were markedly decreased. In contrast, the mRNA levels of the Th2 transcription factor GATA binding protein 3(GATA3) and cytokine IL-4 in arsenic-treated group were higher than those in the control group. Furthermore, the protein levels of phosphorylated JAK2 (p-JAK2) and phosphorylated STAT3 (p-STAT3) increased. Compared with the arsenic-treated group, the arsenic+Cur group demonstrated a significantly increased correct rate, decreased latency to reach the platform on the third and fifth days, and increased times of crossing the platform. The protein expression levels of ZO-1 and claudin 5 increased significantly, and the protein levels of CD4 and CD8 were down-regulated. The mRNA levels of Th2 transcription factor GATA3 and cytokine IL-4 were decreased. The mRNA levels of Th17 transcription factor RORγt and cytokine IL-17 were markedly increased. Furthermore, the protein levels of p-JAK2 and p-STAT3 decreased. Conclusion Through inhibiting the JAK2/STAT3 signaling pathway, curcumin could improve arsenic-induced decline in learning and memory abilities in mice, reverse the destruction of blood-brain barrier permeability of innate immune system components in arsenic-exposed mice, and antagonize arsenic-induced increase in the number of renal CD4 and CD8 molecule as well as the imbalance of CD4⁺ T lymphocyte subsets (Th1, Th2, Th17 and Treg), ultimately counteracting arsenic-induced neurotoxicity.
Animals ; Janus Kinase 2/genetics* ; STAT3 Transcription Factor/genetics* ; Female ; Curcumin/pharmacology* ; Signal Transduction/drug effects* ; Mice, Inbred C57BL ; Mice ; Arsenic/toxicity*

Objective To investigate the immunomodulatory effect of mare's milk on dextran sulfate sodium (DSS)-induced ulcerative colitis (UC) mice. Methods Kunming mice were randomly divided into a blank group(0.8 mL/day saline by gavage) and a DSS modeling group. After modeling, the DSS modeling group was further divided into a control group (0.8 mL/day saline), a salazosulfapyridine (SASP) treated group(430 mg/(kg.d)) and a mare's milk group(0.8 mL/day), with 16 mice in each group. After 10 days of gavage administration, HE staining was performed to observe colonic inflammation, and the disease activity index (DAI) and colonic mucosal damage index (CMDI) were scored. ELISA was used to determine the levels of interleukin 1β (IL-1β), IL-6, and IL-10 in mouse colonic tissues, and flow cytometry was used to detect the percentages of CD4⁺ and CD8⁺ T lymphocytes in peripheral blood. Results Compared to the blank group, all indexes in mice of the control group indicated that DSS successfully induced UC. Compared to the control group, colon shortening in UC mice was attenuated in the mare's milk group; inflammation and ulcer formation in colonic tissues were inhibited; DAI and CMDI scores were lowere; IL-1β and IL-6 levels in mouse colonic tissues were significantly reduced; IL-10 levels were increased and the CD4⁺/CD8⁺ T cell ratio was reduced. Conclusion Mare's milk can inhibit the inflammation of DSS-induced UC mice through immune regulation.
Mice ; Animals ; Female ; Horses ; Colitis, Ulcerative/drug therapy* ; Interleukin-10 ; Dextran Sulfate ; Interleukin-6 ; Milk ; Signal Transduction ; Disease Models, Animal ; Inflammation ; Colon

OBJECTIVE: To explore the short-term efficacy and adverse reactions of orelabrutinib combined with high-dose methotrexate (HD-MTX) in the first-line treatment of elderly high-risk primary central nervous system lymphoma (PCNSL), as well as the survival of patients. METHODS: Twenty-five elderly patients with high-risk primary central nervous system diffuse large B-cell lymphoma admitted to Fujian Provincial Hospital from June 2016 to June 2022 were enrolled in this study, and complete clinical data from all patients were collected retrospectively, and the cut-off for follow-up was December 2022. 15 patients had received temmozolomide combined with HD-MTX regimen for at least four cycles, sequential lenalidomide maintenance therapy, while 10 patients had received orelabrutinib combined with HD-MTX regimen for at least four cycles, sequential orelabrutinib maintenance therapy. The short-term efficacy and adverse reactions of the two groups of patients after treatment were observed. Kaplan-Meier was used to analyze the progression-free survival (PFS) and time to progression (TTP). RESULTS: The objective response rate (ORR) and 2-year median FPS of orelabrutinib combined with HD-MTX regimen group were similar to the temozolomide combined with HD-MTX regimen group (ORR: 100% vs 66.7%; 2-year median PFS: 16 months vs 15 months, P>0.05). The 2-year median TTP of the orelabrutinib+HD-MTX regimen group was better than that of the temozolomide+HD-MTX regimen group (not reached vs 12 months, P<0.05). There were no significant differences in adverse reactions such as gastrointestinal reactions, bone marrow suppression, liver and kidney damage, cardiotoxicity, pneumonia and bleeding between these two groups (P>0.05). CONCLUSION For elderly patients with high-risk PCNSL, orelabrutinib combined with HD-MTX has reliable short-term efficacy, good safety, and tolerable adverse reactions, which is worthy of clinical promotion.
Humans ; Aged ; Methotrexate/adverse effects* ; Retrospective Studies ; Temozolomide/therapeutic use* ; Central Nervous System Neoplasms/drug therapy* ; Antineoplastic Combined Chemotherapy Protocols ; Lymphoma, Large B-Cell, Diffuse/drug therapy* ; Central Nervous System

OBJECTIVE: To investigate the significance of Tim-3 and Galectin-9 in Th1/Th2 imbalance in patients with multiple myeloma (MM). METHODS: 55 newly diagnosed MM patients and 20 healthy controls were included. Flow cytometry was used to detect the expression of Tim-3 on CD4⁺T cells, the proportion of Th1, Th2, Tim-3⁺Th1 and Tim-3⁺Th2 cells in peripheral blood. ELISA was used to detect the levels of cytokines IFN-γ and IL-4 in serum, and PCR was used to detect the level of Galectin-9 mRNA. Then the correlations between Galectin-9 mRNA expression and Th-cell subsets and related cytokine levels, as well as the relationship between Tim-3⁺Th1/Tim-3⁺Th2 ratio and corresponding clinical features were analyzed. RESULTS: Compared with the control group, the expression of Tim-3 on CD4⁺T cells in peripheral blood of MM patients was significantly increased (P<0.05), the proportions of Tim-3⁺Th1 cells, Tim-3⁺Th2 cells and Tim-3⁺Th1/Tim-3⁺Th2 ratio in MM patients were also increased (P<0.05), while the proportion of Th1 cells and Th1/Th2 ratio in MM patients were significantly decreased (P<0.05). The level of cytokine IFN-γ and IFN-γ/IL-4 ratio in MM patients were significantly decreased (P<0.05), while the level of cytokine IL-4 was increased (P<0.05). The mRNA levels of Galectin-9 in MM patients were significantly increased (P<0.05). The levels of Galectin-9 mRNA were positively correlated with Tim-3⁺CD4⁺T cells (r=0.663), Tim-3⁺Th2 cells (r=0.492) and IL-4 (r=0.470), while negatively correlated with IFN-γ (r=-0.593). The ratios of Tim-3⁺Th1/Tim-3⁺Th2 in MM patients were positively correlated with ISS stage (r=0.511), osteolytic damage (r=0.556) and chromosome abnormality (r=0.632). CONCLUSION These results suggest that Tim-3 and Galectin-9 are involved in Th1/Th2 imbalance in MM patients, and the high ratio of Tim-3⁺Th1/Tim-3⁺Th2 is associated with poor clinical prognosis.
Humans ; Cytokines/metabolism* ; Galectins/metabolism* ; Hepatitis A Virus Cellular Receptor 2/metabolism* ; Interleukin-4/metabolism* ; Ligands ; Multiple Myeloma/metabolism* ; RNA, Messenger/metabolism* ; Th1 Cells/metabolism* ; Th2 Cells/metabolism*

Objective To investigate the effect of long intergenic non-coding RNA COX2 (lincRNA-COX2) on apoptosis and polarization of Listeria monocytogenes (Lm)-infected RAW264.7 cells. Methods RAW264.7 cells were cultured and divided into control group (uninfected cells), Lm infection group, negative control of small interfering RNA (si-NC) group, si-NC and Lm infection group, small interfering RNA of lincRNA-COX2 (si-lincRNA-COX2) group, si-lincRNA-COX2 and Lm infection group. RAW264.7 cells were infected with MOI=10 Lm for 6 hours, and then the inhibition efficiency of siRNA transfection was detected by fluorescence microscope and quantitative real-time PCR (qRT-PCR). The expression levels of cleaved-caspase-3(c-caspase-3), caspase-3, B-cell lymphoma-2 (Bcl2), Bcl2 associated X protein (BAX), arginase 1 (Arg1), inducible nitric oxide synthase (iNOS) were detected by Western blot analysis. Results c-caspase-3/caspase-3, BAX/Bcl2 and iNOS were significantly up-regulated, while the level of Arg1 was down-regulated in Lm-infected RAW264.7 cells compared with control group. LincRNA-COX2 knockdown inhibited the increase of protein levels for BAX/Bcl2, c-caspase-3/caspase-3 and iNOS in Lm-infected RAW264.7 cells, while the level of Arg1 in Lm-infected RAW264.7 cells was up-regulated. Conclusion Knockdown of lincRNA-COX2 can inhibit cell apoptosis and suppress the macrophage polarization into M1 type in Lm-infected RAW264.7 cells.
Apoptosis/genetics* ; bcl-2-Associated X Protein/metabolism* ; Caspase 3/metabolism* ; Cyclooxygenase 2/metabolism* ; Listeria monocytogenes/pathogenicity* ; Macrophages/microbiology* ; RNA, Long Noncoding/metabolism* ; RNA, Small Interfering/genetics* ; Animals ; Mice

OBJECTIVE: To investigate the effect and relative mechanism of Recombinant Human Thrombopoietin (rhTPO) on long-term hematopoietic recovery in mice with acute radiation sickness. METHODS: Mice were intramuscularly injected with rhTPO (100 μg/kg) 2 hours after total body irradiation with ⁶⁰Co γ-rays (6.5 Gy). Moreover, six months after irradiation, peripheral blood, hematopoietic stem cells (HSC) ratio, competitive transplantation survival rate and chimerization rate, senescence rate of c-kit⁺ HSC, and p16 and p38 mRNA expression of c-kit⁺ HSC were detected. RESULTS: Six months after 6.5 Gy γ-ray irradiation, there were no differences in peripheral blood white blood cells, red blood cells, platelets, neutrophils and bone marrow nucleated cells in normal group, irradiated group and rhTPO group (P>0.05). The proportion of hematopoietic stem cells and multipotent progenitor cells in mice of irradiated group was significantly decreased after irradiation (P<0.05), but there was no significant changes in rhTPO group (P>0.05). The counts of CFU-MK and BFU-E in irradiated group were significantly lower than that in normal group, and rhTPO group was higher than that of the irradiated group(P<0.05). The 70 day survival rate of recipient mice in normal group and rhTPO group was 100%, and all mice died in irradiation group. The senescence positive rates of c-kit⁺ HSC in normal group, irradiation group and rhTPO group were 6.11%, 9.54% and 6.01%, respectively (P<0.01). Compared with the normal group, the p16 and p38 mRNA expression of c-kit⁺ HSC in the irradiated mice were significantly increased (P<0.01), and it was markedly decreased after rhTPO administration (P<0.01). CONCLUSION The hematopoietic function of mice is still decreased 6 months after 6.5 Gy γ-ray irradiation, suggesting that there may be long-term damage. High-dose administration of rhTPO in the treatment of acute radiation sickness can reduce the senescence of HSC through p38-p16 pathway and improve the long-term damage of hematopoietic function in mice with acute radiation sickness.
Humans ; Mice ; Animals ; Thrombopoietin/metabolism* ; Hematopoietic Stem Cells ; Blood Platelets ; Recombinant Proteins/therapeutic use* ; Radiation Injuries ; RNA, Messenger/metabolism*

Aim To investigate the effect of silencing circRNA ciRS-7 on cisplatin resistance of gastric cancer cells and its related mechanism. Methods Cisplatin-resistant gastric cancer cells HGC-27/DDP were constructed, ciRS-7 was silenced and HGC-27/DDP cells were treated with cisplatin. Real-time quantitative PCR (qRT-PCR) was used to detect the expression levels of ciRS-7 and microrNA-944 (miR-944). Cell counting kit-8 (CCK-8) was used to detect cell survival rate. Clone formation assay was used to detect the number of clones. Western blot was used to detect the protein expression levels of CyclinD1, proliferating cell nuclear antigen (PCNA), B-lymphocytoma-2-associated X protein (Bax), B-lymphocytoma-2 (Bcl-2) and cleaved aspartate-specific cysteine proteinase 3 (cleaved-caspase-3). Flow cytometry was used to detect cell apoptosis rate. In situ end labeling (TUNEL) was used to detect apoptosis index. Dual luciferase assay was used to verify the targeting relationship between ciRS-7 and miR-944. Results CiRS-7 was highly expressed in cisplatin-resistant gastric cancer tissues and cells, while miR-944 was low expressed in cisplatin-resistant gastric cancer tissues and cells. The survival rate, clonal formation number, protein expression levels of CyclinD1, PCNA and Bcl-2 in cisplatin-treated HGC-27/DDP cells were significantly reduced by silencing ciRS-7, while ciRS-7 expression level, apoptosis rate, apoptosis index, Bax, cleaved-caspase-3 protein expression levels of cisplatin-treated HGC-27/DDP cells were significantly raised. CiRS-7 targetedly inhibited the expression of miR-944. Conclusions Silencing ciRS-7 can reverse cisplatin resistance of gastric cancer cells, and the mechanism may be related to the promotion of miR-944 expression by silencing ciRS-7.

OBJECTIVE: To establish a based method flow cytometry to identify the antigen Jka in human red blood cells (RBCs) and verify its accuracy. METHODS: A total of 96 blood samples were enrolled in the study randomly from the voluntary blood donors in Shenzhen Blood Center. The RBCs were incubated with IgG anti-Jka primary antibody, and then labeled with the secondary antibody anti-IgG-Alexa Fluor 647. The fluorescence histograms of each sample were obtained by flow cytometry. Serological agglutination test was used to compare the accuracy of flow cytometry in the detecting of antigen Jka, while PCR-SSP and gene sequencing genotyping were used to verify the accuracy of flow cytometry in the detecting of the antigen in human RBCs. RESULTS: The results of flow cytometry for antigen Jka in human RBCs were consistent with those from serological tests. Samples that demonstrated higher serological agglutination intensity also showed higher fluorescence activity, which indicate more stronger of Jka antigen. The sensitivity of flow cytometry was higher than that of serological test; especially in distinguish Jka weak and negative samples. Flow cytometric results of all samples were consistent with the genotyping results, which confirmed the accuracy of flow cytometry. CONCLUSION The study established a new flow cytometry-based method successfully for the identification of Jka antigen of Kidd blood group in human RBCs. The Kidd blood group antigen Jka of different intensities can be accurately distinguished by the technique.
Blood Group Antigens ; Blood Grouping and Crossmatching ; Erythrocytes ; Flow Cytometry ; Humans ; Immunoglobulin G ; Kidd Blood-Group System

OBJECTIVE: To study the effect of interleukin-6 (IL-6) gene deletion on radiation-induced hematopoietic injury in mice and relative mechanism. METHODS: Before and after whole body ⁶⁰Co γ-ray irradiation, it was analyzed and compared that the difference of peripheral hemogram, bone marrow hematopoietic stem and progenitor cells conts in IL-6 gene knockout (IL-6^-/-) and wild-type (IL-6^+/+) mice and serum IL-6 and G-CSF expression levels in above- mentioned mouse were detected. Moreover, 30 days survival rate of IL-6^-/- and IL-6^+/+ mice after 8.0 Gy γ-ray irradiation were analyzed. RESULTS: IL-6 levels in serum of IL-6^+/+ and IL-6^-/- mice were respectively (98.95±3.85) pg/ml and (18.36±5.61) pg/ml, which showed a significant statistical differences (P<0.001). There were no significant differences of peripheral blood cell counts and G-CSF level in serum between IL-6^+/+ and IL-6^-/- mice before irradiation (P>0.05). However, the number of leukocytes, neutrophils, lymphocytes, monocytes, platelets in peripheral blood and G-CSF level in serum of IL-6^-/- mice were significantly decreased at 6 h after 8.0 Gy γ-ray irradiation compared with that of IL-6^+/+ mice. On days 30 after 8.0 Gy γ-ray irradiation, the survival rate of IL-6^+/+ and IL-6^-/- mice was 62.5% and 12.5%, and the mean survival time of dead mice was 16.0±1.0 and 10.6±5.3 days, respectively. On days 14 after 6.5 Gy γ-ray irradiation, bone marrow nucleated cells in IL-6^+/+ and IL-6^-/- mice were respectively (10.0±1.2)×10⁶ and (8.3±2.2)×10⁶ per femur. Compared with IL-6^+/+ mice, the proportion of Lin^-Sca-1^-c-kit⁺ (LK) in bone marrow of IL-6^-/- mice had no significant change (P>0.05), but the proportion of Lin^-Sca-1⁺c-kit⁺ (LSK) was significantly decreased (P<0.05). CONCLUSION IL-6 plays an obvious role in regulating hematopoietic radiation injury, and IL-6 deficiency can inhibit the radiation-induced increase of endogenous G-CSF level in serum, aggravates the damage of mouse hematopoietic stem cells（HSC） and the reduction of mature blood cells in peripheral blood caused by ionizing irradiation, resulting in the shortening of the survival time and significant decrease of the survival rate of mice exposed to lethal dose radiation.
Animals ; Gene Deletion ; Granulocyte Colony-Stimulating Factor/pharmacology* ; Interleukin-6/metabolism* ; Mice ; Radiation Injuries ; Whole-Body Irradiation

OBJECTIVE: To explore the extrinsic regulation mechanism of bone marrow microenvironment in leukemia cells, and investigate the promoting effect of osteoblast niche on the proliferation and self-renewal of leukemia stem cell by up-regulating the expression of interleukin-1 (IL-1) in leukemia cell. METHODS: The gene expression profiles on leukemia cells derived from AE9a mouse bone marrow endosteum and central bone marrow were determined by RNA sequencing and gene set enrichment analysis (GSEA). Quantitative real-time PCR (qRT-PCR) was used to detect the expression of IL-1 in AE9a mouse leukemia cells co-cultured with or without osteoblasts in vitro. In addition, qRT-PCR was also used to determine the expression of IL-1 in bone marrow mononuclear cell (BMMNC) from 43 patients with acute myeloid leukemia (AML). For leukemia cells co-cultured with osteoblasts or treated with IL-1β, colony forming ability of AE9a leukemia cells was determined by colony formation assay. RESULTS: In AE9a leukemia mouse, RNA-seq data and GSEA showed that the enrichment of the upregulated genes in leukemia cells located in endosteum fell into inflammatory response gene set, among them, IL-1α and IL-1β were significantly higher expressed in AE9a leukemia cells that located osteoblast niche (IL-1α: P<0.001, IL-1β:P<0.001). After AE9a leukemia cells were co-cultured with osteoblasts in vitro, the expression of IL-1α and IL-1β in leukemia cells were increased by 2.5 and 3.5 times respectively. In colony formation assay, the number of colonies was increased significantly after leukemia cells were co-cultured with osteoblasts (P<0.001). In addition, when AE9a leukemia cells were treated with IL-1β, the number of colonies was also increased significantly (P<0.01). In AML patients, BMMNC with high percentage of CD34 positive cells exhibited higher level of IL-1 expression. CONCLUSION Osteoblast niche can promote leukemia cell proliferation and self-renewal through up-regulating the expression of IL-1 in leukemia cells. In AML patients, the expression level of IL-1 was correlated to the percentage of CD34 positive cells in BMMNC.
Animals ; Antigens, CD34/metabolism* ; Bone Marrow/metabolism* ; Cell Proliferation ; Leukemia, Myeloid, Acute/metabolism* ; Mice ; Osteoblasts/metabolism* ; Stem Cells ; Tumor Microenvironment