Risk Factors Analysis for the Development of Hypocomplementemia in Rheumatoid Arthritis Patients: A Single-Center Retrospective Study.

Objective: The purpose of the research was to explore the possible risk factors for the development of hypocomplementemia (HC) in rheumatoid arthritis (RA) patients by analyzing their clinical and laboratory features. Methods: This retrospective research contained 501 RA patients, divided into RA patients with HC (n=78) and RA patients without HC (n=423). Demographic characteristics and laboratory test results of RA patients were collected and analyzed, such as age, sex, anti-mutated citrullinated vimentin antibody (Anti-MCV), serum complements (C3, C4), immunoglobulins (IgA, IgG, IgM), hemoglobin (Hb), platelets (PLT) and erythrocyte sedimentation rate (ESR), etc. Spearman correlation was served as assessing the correlations of the levels of serum C3 and C4 with each index. Receiver operating characteristic (ROC) curves were served as assessing the diagnostic efficacy of each index for RA patients with HC. Furthermore, risk factors for the occurrence of HC in RA patients were analyzed by employing binary logistic regression of single and multiple factors. Results: Compared RA patients with HC to without HC, the former were older and had a longer disease duration with increased levels of Anti-MCV, IgM and DAS28 and lower levels of Hb, PLT and ESR; Spearman correlation analysis verified the level of serum Anti-MCV was a negative correlation with C3 (r=-0.156); the area under the ROC curve (AUC) of PLT in diagnosing RA patients with HC was the largest at 0.65 (95% CI: 0.60-0.69); binary logistic regression analysis indicated that advanced age (>66 years), long disease duration (>62 months), high DAS28 value (>6.13), the levels of Anti-MCV>107.68IU/mL, IgM>1.54g/L, ESR≤69.00mm/h, Hb≤99.00g/L and PLT≤305.00×109/L were probable risk factors for the occurrence of HC in RA patients. Conclusion: Age and disease duration, DAS28, Anti-MCV, IgM, ESR, Hb, and PLT are closely related to the development of HC in RA patients. Timely monitoring of these indicators can help to evaluate disease activity of RA patients and further improve their prognosis.

Standardized Treatment Strategy for Depressive Disorder.

The treatment strategies of depressive disorder include pharmacological treatment, psychotherapy, and physical therapy (electroconvulsive therapy [ECT], transcranial magnetic stimulation [TMS], etc.). The updated CANMAT guidelines recommended the most second-generation antidepressants as first-line treatments for patients with a major depressive disorder (MDD) of moderate or greater severity. Before antidepressant treatment, comprehensive assessment and safety monitoring are necessary. The application of measurement-based care in the diagnosis and treatment of depression would better ensure that enough dosage and response of antidepressant is achieved at each key point, and the final outcome of disease is improved. It is recommended that antidepressant is used with monotherapy in patients with depression. Antidepressants of different types and different mechanisms could be combined to improve the efficacy for patients with treatment-resistant depression (TRD). To prevent the relapse and recurrence of disease, the long-term treatment comprised of acute treatment, consolidation treatment, and maintenance treatment must be considered for all patients.

Salinomycin induces apoptosis and differentiation in human acute promyelocytic leukemia cells.

At present, acute promyelocytic leukemia (APL) is the most curable form of acute myeloid leukemia and can be treated using all-trans retinoic acid and arsenic trioxide. However, the current treatment of APL is associated with some issues such as drug toxicity, resistance and relapse. Therefore, other strategies are necessary for APL treatment. In the present study, we investigated the effects of salinomycin (SAL) on APL cell lines NB4 and HL-60 and determined its possible mechanisms. We observed that SAL inhibited cell proliferation, as determined by performing Cell Counting Kit-8 (CCK-8) assay, promoted cell apoptosis, as determined based on morphological changes, and increased Annexin V/propidium iodide (PI)-positive apoptotic cell percentage. Treatment with SAL increased Bax/Bcl-2 and cytochrome c expression and activated caspase-3 and -9, thus leading to poly(ADP-ribose) polymerase (PARP) cleavage and resulting in cell apoptosis. These results revealed that SAL induced cell apoptosis through activation of the intrinsic apoptosis pathway. The present study is the first to show that SAL induced the differentiation of APL cells, as determined based on mature morphological changes, increased NBT-positive cell and CD11b-positive cell percentages and increased CD11b and C/EBPß levels. Furthermore, SAL decreased the expression of ß-catenin and its targets cyclin D1 and C-myc. Results of immunofluorescence analysis revealed that SAL markedly decreased the ß-catenin level in both the nucleus and cytoplasm. Combination treatment with SAL and IWR-1, an inhibitor of Wnt signaling, synergistically triggered SAL-induced differentiation of APL cells. These findings demonstrated that SAL effectively inhibited cell proliferation accompanied by induction of apoptosis and promotion of cell differentiation by inhibiting Wnt/ß-catenin signaling. Collectively, these data revealed that SAL is a potential drug for treatment of APL.

Effects of lapatinib on cell proliferation and apoptosis in NB4 cells.

Acute promyelocytic leukemia (APL), characterized by the presence of the promyelocytic leukemia (PML)-retinoic acid α receptor (RARα) fusion protein, responds to treatment with all-trans retinoic acid (ATRA) and arsenic trioxide (ATO). However, drug resistance and side effects restrict the application of these reagents. Hence, the development of novel therapeutic drugs for APL treatment is critical. Lapatinib, a small-molecule tyrosine kinase inhibitor, has been used in the treatment of different tumors. However, it is unclear whether lapatinib exerts antitumor effects on APL. The present study investigated the antitumor effects and potential mechanisms of lapatinib on NB4 cells derived from APL. Cell Counting Kit-8 assay and colony forming analysis indicated that lapatinib inhibited NB4 cell proliferation in a dose-dependent manner. Flow cytometry analysis revealed that lapatinib induced cell cycle arrest at the S phase and promoted cell apoptosis. Furthermore, Liu's staining and Hoechst 33258 staining revelaed that lapatinib treatment induced an apoptotic nuclear phenomenon. Furthermore, lapatinib induced apoptosis by decreasing Bcl-2 and PML-RARα levels, and by increasing the levels of Bax, cleaved PARP, cleaved caspase-3 and cleaved caspase-9. In addition, lapatinib increased the levels of phospho-p38 MAPK and phospho-JNK, and decreased the levels of phospho-Akt. The p38 inhibitor PD169316 partially blocked lapatinib-induced proliferation inhibition and apoptosis, whereas the JNK inhibitor SP600125 had no such effects. Therefore, treatment with lapatinib may be a promising strategy for APL therapy.

[Mechanism underlying inhibition of proliferation and promotion of apoptosis by lapatinib in HL60 cells].

Objective To investigate the effect of lapatinib on cell proliferation and apoptosis in acute myeloid leukemia HL60 cells in vitro and the related molecular mechanisms. Methods The HL60 cells were treated with 5, 10, 15 µmol/L lapatinib for 24 hours, and then morphological changes of the cells were observed under optical microscope. CCK-8 assay was used to assess the cell viability. Colony formation assay was performed to detect the cell proliferation ability. Cell apoptosis labeled by annexinV-FITC/PI were analyzed by flow cytometry. Wright modified LIU's staining and Hoechst33342 fluorescent staining were used to observe the morphology of the nucleus. Western blotting was utilized to detect the expressions of Bax, Bcl2, caspase-3, caspase-9, cleaved caspase-3, cleaved caspase-9, cleaved poly-ADP-ribose polymerase (cleaved PARP), cell proliferation regulating inhibitor of protein phosphatase 2A (CIP2A), c-MYC, AKT and p-AKT. Results Compared with the control group, lapatinib inhibited cell proliferation and promoted apoptosis, induced nuclear fragmentation, chromatin condensation of HL60 cells in a dose-dependent manner. Meanwhile, it down-regulated the expression of Bcl2, up-regulated the levels of Bax, cleaved caspase-3, cleaved caspase-9 and cleaved PARP, and decreased the levels of CIP2A, p-AKT and c-MYC. Conclusion Lapatinib could inhibit cell proliferation and promote apoptosis in HL60 cells by inhibiting the CIP2A/AKT/c-MYC signal pathway.

Epigallocatechin-3-gallate induces apoptosis in acute promyelocytic leukemia cells via a SHP-1-p38α MAPK-Bax cascade.

Acute promyelocytic leukemia (APL) is characterized by a specific chromosomal translation, resulting in a fusion gene that affects the differentiation, proliferation and apoptosis of APL cells. Epigallocatechin-3-gallate (EGCG), a catechin, exhibits numerous biological functions, including antitumor activities. Previous studies have reported that EGCG induces apoptosis in NB4 cells. However, the molecular mechanism underlying EGCG-induced apoptosis remains unclear. The present study aimed to determine the molecular basis of EGCG-induced apoptosis in NB4 cells. EGCG treatment significantly inhibited the viability of NB4 cells in a dose-dependent manner. In addition, EGCG treatment induced apoptosis and increased the levels of (Bcl-2-like protein 4) Bax protein expression. Moreover, EGCG treatment was able to increase phosphorylated (p)-p38α mitogen-activated protein kinase (MAPK) and Src homology 1 domain-containing protein tyrosine phosphatase (SHP-1) expression. Pretreatment with PD169316 (a p38 MAPK inhibitor) partially blocked EGCG-induced apoptosis and inhibited EGCG-mediated Bax expression. Similarly, pretreatment with NSC87877, an inhibitor of SHP-1, partially blocked EGCG-induced apoptosis and inhibited EGCG-mediated increases in p-p38α MAPK and Bax expression. Therefore, the results of the present study indicate that EGCG is able to induce apoptosis in NB4 cells via the SHP-1-p38αMAPK-Bax cascade.

Verteporfin Inhibits Cell Proliferation and Induces Apoptosis in Human Leukemia NB4 Cells without Light Activation.

Background and Aims: Verteporfin (VP), clinically used in photodynamic therapy for neovascular macular degeneration, has recently been proven a suppressor of yes-associated protein (YAP) and has shown potential in anticancer treatment. However, its anti-human leukemia effects in NB4 cells remain unclear. In this study, we investigated the effects of VP on proliferation and apoptosis in human leukemia NB4 cells. Methods: NB4 cells were treated with VP for 24 h. The effects of VP on cell proliferation were determined using a Cell-Counting Kit-8 assay (CCK-8) assay and colony forming assay. Apoptosis and cell cycle were evaluated by flow cytometry (FCM). The protein levels were detected by western blot. Results: We found that VP inhibited the proliferation of NB4 cells in a concentration and time-dependent manner. FCM analysis showed that VP induced apoptosis in a concentration dependent manner and that VP treatment led to cell cycle arrest at G0/G1 phase. Moreover, VP significantly decreased the protein expression of YAP, p-YAP, Survivin, c-Myc, cyclinD1, p-ERK, and p-AKT. In addition, VP increased the protein expression of cleaved caspase3, cleaved PARP, Bax, and p-p38 MAPK. Conclusions: VP inhibited the proliferation and induced apoptosis in NB4 cells.

Effect of YAP Inhibition on Human Leukemia HL-60 Cells.

Background: Yes-associated protein (YAP), the nuclear effector of the Hippo pathway, is a candidate oncoprotein and participates in the progression of various malignancies. However, few reports have examined the effect of YAP inhibition in human leukemia HL-60 cells. Methods: We examined the effects of YAP knockdown or inhibition using short hairpin RNA (shRNA) or verteporfin (VP), respectively. Western blot assays were used to determine the expression levels of YAP, Survivin, cyclinD1, PARP, Bcl-2, and Bax. Cell proliferation was assessed using the cell counting kit (CCK-8) assay. Cell cycle progression and apoptosis were evaluated by flow cytometry, and apoptotic cell morphology was observed by Hoechst 33342 staining. Results: Knockdown or inhibition of YAP led to cell cycle arrest at the G0/G1 phase and increased apoptosis, inhibited cell proliferation, increased levels of Bax and cleaved PARP, and decreased levels of PARP, Bcl-2, Survivin, and cyclinD1. Moreover, Hoechst 33342 staining revealed increased cell nuclear fragmentation. Conclusion: Collectively, these results show that inhibition of YAP inhibits proliferation and induces apoptosis in HL-60 cells. Therefore, a novel treatment regime involving genetic or pharmacological inhibition of YAP could be established for acute promyelocytic leukemia.

Epigallocatechin-3-gallate promotes all-trans retinoic acid-induced maturation of acute promyelocytic leukemia cells via PTEN.

Acute promyelocytic leukemia (APL) is a distinctive subtype of acute myeloid leukemia (AML) in which the hybrid protein promyelocytic leukemia protein/retinoic acid receptor α (PML/RARα) acts as a transcriptional repressor impairing the expression of genes that are critical to myeloid cell mutation. We aimed at explaining the molecular mechanism of green tea polyphenol epigallocatechin-3-gallate (EGCG) enhancement of ATRA-induced APL cell line differentiation. Tumor suppressor phosphatase and tensin homolog (PTEN) was found downregulated in NB4 cells and rescued by proteases inhibitor MG132. A significant increase of PTEN levels was found in NB4, HL-60 and THP-1 cells upon ATRA combined with EGCG treatment, paralleled by increased myeloid differentiation marker CD11b. EGCG in synergy with ATRA promote degradation of PML/RARα and restores PML expression, and increase the level of nuclear PTEN. Pretreatment of PTEN inhibitor SF1670 enhances the PI3K signaling pathway and represses NB4 cell differentiation. Moreover, the induction of PTEN attenuated the Akt phosphorylation levels, pretreatment of PI3K inhibitor LY294002 in NB4 cells, significantly augmented the cell differentiation and increased the expression of PTEN. These results therefore indicate that EGCG targets PML/RARα oncoprotein for degradation and potentiates differentiation of promyelocytic leukemia cells in combination with ATRA via PTEN.

Expression, purification, and characterization of mouse nesfatin-1 in Escherichia coli.

Nesfatin-1 is a newly discovered satiety molecule expressed mainly in the hypothalamic nuclei. It suppresses both short-term and long-term appetite. Six synthetic deoxyoligonucleotides overlapped by PCR encoding nesfatin-1 were cloned into a pET28a vector after the hexa-histidine-tagged multiple cloning sites sequence with an enterokinase recognition site incorporated in-between. The recombinant plasmid was transformed into Escherichia coli strain Rosetta to express the fusion protein, which constituted 27% of the total cell proteins. After purified by Ni-sepharose affinity chromatography, the fusion protein was treated with enterokinase to release nesfatin-1. The nesfatin-1 sample was further purified with reverse-phase high performance liquid chromatography (HPLC), and its molecular weight was determined by mass spectrometry. The biological activities of recombinant nesfatin-1 were also assessed using in vivo animal models. The method described here promises to produce about 8 mg biologically active nesfatin-1 with homogeneity over 98% from 1-L shaking flask culture of E. coli, which can be considered as an easy and cost-effective way to synthesize nesfatin-1.

NLS-RARα Inhibits the Effects of All-trans Retinoic Acid on NB4 Cells by Interacting with P38α MAPK.

Nuclear localization signal retinoic acid receptor alpha(NLS-RARα), which forms from the cleavage of promyelocytic leukemia-retinoic acid receptor alpha(PML-RARα) protein by neutrophil elastase(NE), possesses an important role in the occurrence and development of acute promyelocytic leukemia(APL). However, the potential mechanism underlying the effects of NLS-RARα on APL is still not entirely clear. Here, we investigated the effects of NLS-RARα on APL NB4 cells and its mechanism. We found that all-trans retinoic acid(ATRA) could promote differentiation while inhibit proliferation of APL NB4 cells via upregulating the expression of phosphorylated p38α mitogen-activated protein kinase(p-p38α MAPK). We also found that NLS-RARα could inhibit differentiation while accelerate proliferation of NB4 cells via downregulating the expression of p-p38α protein in the presence of ATRA. Furthermore, immunofluorescence and co-immunoprecipitation assays confirmed NLS-RARα interacted with p38α protein directly. Finally, application of PD169316, an inhibitor of p38α protein, suggested that recruitment p38α-combinded NLS-RARα by ATRA eventually caused activation of p38α protein. In summary, our study demonstrated that ATRA cound promote differentiation while inhibit proliferation of APL NB4 cells via activating p38α protein after recruiting p38α-combinded NLS-RARα, while NLS-RARα could inhibit the effects of ATRA in the process.

Effects of LG268 on Cell Proliferation and Apoptosis of NB4 Cells.

AIMS: To investigate the effect of LG100268 (LG268) on cell proliferation and apoptosis in NB4 cells. METHODS: NB4 cells were treated with LG268 for 24 h or 48 h. The effect of LG268 on cell proliferation was assessed by the CCK-8 assay and colony-forming assay. Apoptosis and cell cycle were evaluated by flow cytometry. The protein expression levels of Survivin, PARP, c-Myc, cyclin D1, ERK, p-ERK, p38 MAPK, and p- p38 MAPK were detected by western blot. RESULTS: We found that LG268 inhibited the proliferation of NB4 cells in a dose-dependent manner. Flow cytometry analysis showed that LG268 accelerated apoptosis in NB4 cells in a time- dependent manner and that LG268 treatment led to cell cycle arrest at G0/G1 phase. Moreover, LG268 significantly decreased the protein levels of Survivin, c-Myc, and cyclinD1. Cleaved PARP was observed in the LG268 treatment group but not in the control group. In addition, LG268 increased the phosphorylation level of p38 MAPK and decreased the phosphorylation level of ERK. CONCLUSIONS: LG268 inhibited cell proliferation and promoted cell apoptosis in NB4 cells.

Serum DHEAS levels are associated with the development of depression.

The aim of study was to evaluate the association between serum DHEAS levels and depression with a case-control study together with a meta-analysis. Radioimmunoassay (RIA) was performed to measure the serum DHEAS levels of all participants before and after treatment. Depression Patients were divided into mild depression and severe depression based on Hamilton depression scale (HAMD24) and received 5-hydroxytryptamine (5-HT) and citalopram (20mg/d) for 8 weeks. Case-control studies related to our study theme were enrolled for meta-analysis and Comprehensive Meta-analysis 2.0 (CMA 2.0) was used for statistical analysis. After treatment, DHEAS levels in depression patients were significantly increased, while before and after treatment, DHEAS levels were all lower in depression patients than in controls (all P<0.001); further analysis on age revealed that DHEAS levels were decreased with the rising of age. Meta-analysis results suggested that serum DHEAS levels (ng/mL) were significantly higher in healthy controls compared to depression patients (SMD=0.777, 95%CI=0.156-1.399, P=0.014). In conclusion, our study suggests that serum DHEAS levels are associated with the development of depression and it decreased with the rising of age.

The Kringle-2 domain of tissue plasminogen activator significantly reduces mortality and brain infarction in middle cerebral artery occlusion rats.

Tissue plasminogen activator (TPA) showed brain-protective activity within the first 15 min after cerebral ischemia in rats. To understand its molecular mechanism, TPA derivates were intracerebroventricularly administered at 15 min before, and 15, 90, 120 min after middle cerebral artery occlusion (MCAO) in rats. The reduction in mortality and cerebral infarction at 24 h was seen only with TPA administered at 15 min after MCAO. The down-regulation of endogenous TPA by the intracerebroventricular injection of TPA was found to be responsible for the protective effect on the integrity of blood-brain barrier after MCAO, as well as for the reduction in mortality and cerebral infarction. Moreover, for the first time we have found that the Kringle-2 domain is essential for the brain-protective activity of TPA.

Selective trans-cinnamic acid uptake impairs [Ca2+]cyt homeostasis and growth in Cucumis sativus L.

To obtain insight into interspecies interactions mediated by allelochemicals, the response of cucumber (Cucumis sativus L. cv Jinyan No.4) and figleaf gourd (Cucurbita ficifolia Bouché) seedlings to trans-cinnamic acid (CA) (1) was investigated. While trans-CA is an autotoxin in cucumber root exudates, figleaf gourd is resistant to it. Cucumber, however, had a high rate of trans-CA uptake by the roots, leading to reduced root growth. The trans-CA treatment also resulted in an intracellular release of Ca(2+) from the vacuole to the cytoplasm, and, thus, an increased [Ca(2+)](cyt) level accompanied by gradual loss of cell viability in cucumber roots. Taken together, these results suggest that [Ca(2+)](cyt) homeostatic disturbance is one of the primary triggers for trans-CA phytotoxicity in cucumber.