Correlation of Blimp1 with ATF4/CHOP Signaling Pathway in Multiple Myeloma U266 Cells / 中国实验血液学杂志

OBJECTIVE@#To investigate whether Blimp1 plays an anti-apoptosis role in myeloma by interfering with ATF4/CHOP cell apoptosis pathway induced by endoplasmic reticulum stress, and to explore the anti-myeloma mechanism of aspirin.@*METHODS@#The bone marrow fluid of 40 newly diagnosed multiple myeloma patients without treatment and 30 control people with relatively normal bone marrow was collected. Flow cytometry was used to separated the normal and abnormal plasma cells, LV-Blimp1-RNAi (40051-2) recombinant lentivirus down-regulates the expression of Blimp-1 in U266 cell line and detected the changes of the expression of ATF4 and CHOP gene. U266 cells were stimulated by aspirin at different concentrations (0, 0.5, 2.5, 5.0 mmol/L) in vitro. Then the effect of aspirin on proliferation of U266 cells was measured by CCK-8 assay, the mRNA expression levels of Blimp1, ATF4 and CHOP in four groups were detected by real-time PCR.@*RESULTS@#The expression level of Blimp1 in phenotype abnormal plasma cells was significantly increased as compared with normal cells, while the expression of ATF4 and CHOP in phenotype abnormal plasma cells was significantly decreased as compared with normal cells (P＜0.05). In the case of MOI=100, the transfection efficiency of U266 cells was beyond 80% as detected by fluorescence microscopy. Compared with blank conrol and negatine control groups, Blimp1 mRNA expression level in positive group was significantly reduced while ATF4 and CHOP expression significantly increased. CCK-8 showed that the proliferation activity of U266 cells could be inhibited by aspirin, which showed a time-and dose-dependent manner; at the same time, the expression level of Blimp1 in U266 cells were decreased with the increasing of aspirin concentration, while the expression level of ATF4 and CHOP was increased with the increasing of aspirin concentration.@*CONCLUSIONS@#Blimp1 may display the anti-apoptosis of myeloma cells through interfering with ATF4/CHOP signaling pathway; low dose of aspirin may play anti-myeloma effect by inhibiting the expression of Blimp1 in myeloma cells.

Expression of Blimp1、ATF4 and CHOP in Multiple Myeloma Cells and Effect of Aspirin on Their Expression / 中国实验血液学杂志

OBJECTIVE@#To explore the expression of Blimp1, ATF4 and CHOP in bone marrow mononuclear cells from patients with multiple myeloma as well as the effect of aspirin on their expression.@*METHODS@#Sixty untreated patients with multiple myeloma and 30 patients with relatively normal bone marrow were selected. Mononuclear cells from the bone marrow fluid were separated using Ficoll separation solution. CD138 plasma cells were sorted by immunomagnetic beads method. RT-PCR was used to detect the expression levels of Blimp1, ATF4 and CHOP mRNA in U266 cells cultured in vitro. The cells were divided into blank control group, negative control group (no-loaded virus transfection) and positive experimental group [LV-Blimp1-RNAi (40051-2) transfection] by lentivirus transfection. RT-PCR was used to detect the expression of Blimp1, ATF4 and CHOP mRNA in cells of different groups. U266 cells were stimulated in vitro with different concentrations of aspirin solution (0, 0.5 mmol/L, 2.5 mmol/L, 5.0 mmol/L) for 24, 48 h and 72 h, respectively. The ability of cell proliferation in different groups was measured by CCK-8. U266 cells were stimulated with different concentrations of aspirin for 48 hours. And the mRNA expression of Blimp1, ATF4 and CHOP was detected by RT-PCR.@*RESULTS@#Compared with plasma cells in normal group, the expression of Blimp1 mRNA in CD138 plasma cells of MM patients significantly increased (8.040±1.878), and the mRNA expression levels of ATF4 and CHOP significantly decreased (0.735±0.089; 0.837±0.062) (P＜0.05). U266 cells were cultured in vitro. Compared with the blank control group and the negative control group, the mRNA expression level of Blimp in the positive experimental group was significantly down-regulated after infection with LV-Blimp1-RNAi (40051-2) lentiviral expression vector (0.637±0.021). ATF4 and CHOP mRNA expression levels were significantly increased (1.452 ± 0.027; 1.721 ± 0.038) (P＜0.05). The proliferation of U266 cells decreased after stimulation with aspirin. In the range of (0.5-5) mmol/L, aspirin could significantly inhibit the proliferation of U266 cells. The inhibition effect of aspirin was increased along with prolongation of time and increase of concentrations. After aspirin stimulation of different concentrations for 48 hours, the expression level of Blimp1 in U266 cells decreased with increasing of drug concentration, while the expression levels of ATF4 and CHOP increased with increasing of drug concentration.@*CONCLUSION@#Inhibition of Blimp1 expression in multiple myeloma cells can promote the expression of ATF4 and CHOP. Aspirin can inhibit the proliferation activity of myeloma cells by down-regulating Blimp1 expression in myeloma cells and up-regulating ATF4 and CHOP expression, therefore plays an anti-tumor rote.

Application of quality by design in granulation process for ginkgo leaf tablet (Ⅳ)： influence and control of raw materials' quality variation / 中国中药杂志

Raw materials' quality variation could affect the quality consistency of product and the clinical efficacy. In this paper, the high shear wet granulation (HSWG) process of the ginkgo leaf tablet was taken as the research object. Ginkgo biloba extracts and excipients microcrystalline cellulose collected from various sources and batches were used to simulate raw materials' quality variation. Real-time torque was recorded to analyze the viscosity of the wetting mass, and then by combining with physical fingerprint, the impact of physical quality variation of powders on granule properties could be investigated. Based on regime map thesis, whether the granules' nucleation mode was in mechanical dispersion regime was determined by calculating dimensionless parameters, which would lead to the unstable output in considerations of granule yield ratio and particle size distribution (PSD) curve. The orthogonal partial least square (OPLS) model was adopted to build the relationship between the micromeritic properties and the mediangranule size (D50) of Ginkgo biloba granules and then the critical material attributes (CMAs) were screened by variable importance in the projection (VIP) indexes. The results demonstrated that the properties of powders including hygroscopicity, angle of repose, Hausner ratio, Carr index, D10 and loss on drying affected the granule size. Besides, Ginkgo biloba granules were compressed into tablets. In view of tensile strength analysis, the raw materials' quality variation did not result in decrease of tensile strength of the ginkgo leaf tablets. The design space of critical quality attributes (CQAs) and the process design space which could cope with raw materials' quality variation were proved to be robust..

Expression of Blimp-1 mRNA in Bone Marrow Mononuclear Cells in Multiple Myeloma Patients and Its Clinical Significance / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To investigate the expression level of B lymphocyte-induced maturation protein-1(Blimp-1) mRNA in bone marrow mononuclear cells(BMMNC) of multiple myeloma(MM) patients and its clinical significance.</p><p><b>METHODS</b>Fluorescent quantitative real-time PCR(qRT-PCR) was used to measure Blimp-1 mRNA expression in BMMNC and flow cytometry(FCM) was performed to detect the number of malignant plasma cells in bone marrow of MM group (39 newly-diagnosed and untreated patients) and IDA group (5 IDA patients). The clinical data of all the patients' were collected, and the 39 patients in MM group were divided into 2 subgroups: in BD group 20 cases were treated with bortezomib-based regimen and in VOD group 19 patients were treated with VAD regimen. The age, sex, clinseal stage and type between the 2 subgroups were not statistically different. Blimp-1 mRNA expression level in BMMNC of MM patients was detected by qRT-PCR after 3 treatment cycles.</p><p><b>RESULTS</b>The expression levels of Blimp-1 mRNA in BMMNC of IDA patients and MM patients divided into 3 groups according to ISS were (0.00047±0.00027), ISS I(0.09543±0.02800), Ⅱ(0.13606±0.04162),Ⅲ (0.21202±0.03940), separately. There was statistical difference among the 4 groups(F=56.929,P<0.05) and there was significant difference between any 2 groups of these 4 groups(P<0.05). Significant positive correlation was found between Blimp-1 mRNA expression level and the number of malignant plasma cells, serum monoclonal proteins (M protein), β2-microglobulin(β2-MG), lactic dehydrogenase(LDH), C-reactive protein(CRP)(P<0.05). There was significant negative correlation between Blimp-1 mRNA and hemoglobin (Hb) level (P<0.05). After 3 cycles of chemotherapy, Blimp-1 mRNA level of patients with a >50% reduction of M protein was significantly lower than that of patients whose M protein did not decrease significantly(P<0.05). After 3 treatment cycles, Blimp-1 mRNA expression in BMMNC in BD group was significantly lower than that in VAD group [(0.02388±0.00871) vs (0.04823±0.00219), P<0.05].</p><p><b>CONCLUSION</b>The Blimp-1 mRNA expression level in BMMNC may reflect the tumor burden in MM patients, which related with ISS, and positively correlated with the malignant plasma cell number, M protein, β2-MG, LDH, CRP level, and negatively correlated with Hb. The change of Blimp-1 mRNA expression level in BMMNC relates with the extent of M protein reduction, suggesting it may be used as a marker for response to therapy. Bortezomib may have effect on malignant plasma cells by suppressing Blimp-1 mRNA expression.</p>

Effect of Krüppel-like factor 4 overexpression on heat stress-induced apoptosis of Raw264.7 macrophages / 中南大学学报(医学版)

OBJECTIVE@#To observe the effect of Krüppel-like factor 4 (KLF4) overexpression on heat stress-induced apoptosis of Raw264.7 macrophages.@*METHODS@#The fragment containing full length mouse KLF4 cDNA coding sequence was inserted into the pcDNA3.1 vector and Raw264.7 macrophages were transfected with pcDNA3.1-KLF4 plasmids using lipofectamine.The expression of KLF4 was examined by Western blot in the Raw264.7 macrophages stably transfected with pcDNA3.1- KLF4 plasmids. Raw264.7 cells transfected with pcDNA3.1 and pcDNA3.1-KLF4 were exposed to heat stress (42 degrees C) for 1h and recovered at 37 degrees C for 12h. Flow cytometry, Hoechest 33258 staining assay, and DNA ladder assays were performed to assess the apoptosis.@*RESULTS@#The KLF4 overexpressed Raw264.7 macrophages were established. After the heat stress, flow cytometry showed that apoptotic cells increased significantly in KLF4 overexpressed cells compared with the vector control; Hoechest 33258 staining was characterized with classical changes including apoptotic body, and nuclear condensation. Agarose gel electrophoresis showed that "DNA ladder" could be observed clearly.@*CONCLUSION@#KLF4 overexpression can increase heat stress-induced apoptosis of Raw264.7 macrophages.