OBJECTIVE: To investigate the effect and underlying mechanism of hispidulin on the proliferation and apoptosis of leukemia K562 cells. METHODS: K562 cells were cultured in vitro and treated with 0, 5, 25 or 100 μmol/L hispidulin for 24 h. Cell proliferation and apoptosis were detected by CCK-8 and flow cytometry, respectively. Western blot was used to assess the expression of Bax, Bcl-2 and interleukin (IL)-37 proteins. Bone marrow mononuclear cells were extracted from 17 chronic myeloid leukemia patients and 21 healthy individuals by Ficoll-Hypaque density gradient method, and the expression of IL-37 protein was measured by Western blot. K562 cells with IL-37 overexpression or knockdown were constructed, and then treated with 0 or 100 μmol/L hispidulin for 24 h. Cell proliferation, apoptosis and protein expression of Bax and Bcl-2 were determined in the same way as above. RESULTS: After K562 cells were treated with hispidulin, the cell inhibition rate, apoptosis rate, and the protein expression of Bax and IL-37 were significantly increased (P <0.05), but the cell proliferation and expression of Bcl-2 protein were decreased (P <0.05). The expression of IL-37 protein in bone marrow mononuclear cells of the leukemia patient was 0.24±0.03, which was significantly lower than 0.91±0.05 of healthy controls (P <0.05). Overexpression of IL-37 significantly promoted inhibition rate, apoptosis rate, and expression of Bax protein in K562 cells (P <0.05), but suppressed the expression of Bcl-2 protein (P <0.05). In addition, knockdown of IL-37 could reverse the effects of hispidulin on proliferation and apoptosis of K562 cells. CONCLUSION Hispidulin inhibits the proliferation and induces apoptosis of leukemia K562 cells, which may be related to the up-regulation of IL-37 protein in cells.
Humans ; K562 Cells ; bcl-2-Associated X Protein/pharmacology* ; Apoptosis ; Leukemia ; Proto-Oncogene Proteins c-bcl-2 ; Cell Proliferation

OBJECTIVE: To investigate the effects of knocking down nucleostemin ( NS) combined with rapamycin (RAPA) on autophagy and apoptosis in HL-60 cells , and to explore its role in HL-60 cells . METHODS: The expression of NS protein was detected using Western blot , after transfection of HL-60 cells was achieved by the recombinant lentviral vector NS -RNAi-GV248 . Flow cytometry was used to detect changes in cells apoptosis after NS silencing/ rapamycin for 24 , 48 hours , and the expressions of NS , LC3 , p62 , BCL-2 and Bax proteins in cells were detected by Western blot. RESULTS: The expression of NS in HL-60 cells was successfully down-regulated by recombinant lentiviral vector. After treatment with rapamycin for 24 and 48 h , the apoptosis rate of cells in each group increased (P < 0.05) , and the apoptosis was more obvious at 48 hours . Compared with the NS silencing group or rapamycin group , after treated with NS down-regulation combined with rapamycin for 48 hours , the apoptosis of HL-60 cells was significantly increased ( P < 0.05 ) , LC3 -II/LC3 -I ratio was significantly increased ( P < 0.05 ) , p62 protein expression was significantly decreased (P < 0.05) , and BCL-2/Bax ratio was significantly decreased ( P < 0.05) . CONCLUSION NS down-regulation combined with rapamycin can enhance the apoptosis and autophagy of HL-60 cells , and the induction of apoptosis of HL-60 cells may be related to the expression of BCL-2 and Bax proteins .
Humans ; HL-60 Cells ; Sirolimus/pharmacology* ; bcl-2-Associated X Protein ; Autophagy ; Apoptosis

This study aimed to explore the mechanism of albiflorin in the treatment of Alzheimer's disease(AD) based on network pharmacology, molecular docking, and in vitro experiments. Network pharmacology was used to predict the potential targets and pathways of albiflorin against AD, and molecular docking technology was used to verify the binding affinity of albiflorin to key target proteins. Finally, the AD cell model was induced by Aβ_(25-35) in rat pheochromocytoma(PC12) cells and intervened by albiflorin to validate core targets and pathways. The results of network pharmacological analysis showed that albiflorin acted on key targets such as mitogen-activated protein kinase-1(MAPK1 or ERK2), albumin(ALB), epidermal growth factor receptor(EGFR), caspase-3(CASP3), and sodium-dependent serotonin transporter(SLC6A4), and signaling pathways such as MAPK, cAMP, and cGMP-PKG. The results of molecular docking showed that albiflorin had strong binding affinity to MAPK1(ERK2). In vitro experiments showed that compared with the blank group, the model group showed decreased cell viability, decreased expression level of B-cell lymphoma 2(Bcl-2), increased Bcl-2-associated X protein(Bax), and reduced phosphorylation level of extracellular signal-regulated kinase 1/2(ERK1/2) and the relative expression ratio of p-ERK1/2 to ERK1/2. Compared with the model group, the albiflorin group showed potentiated cell viability, up-regulated expression of Bcl-2, down-regulated Bax, and increased phosphorylation level of ERK1/2 and the relative expression ratio of p-ERK1/2 to ERK1/2. These results suggest that the mechanism of albiflorin against AD may be related to its activation of the MAPK/ERK signaling pathway and its inhibition of neuronal apoptosis.
Animals ; Rats ; Alzheimer Disease/drug therapy* ; bcl-2-Associated X Protein ; Network Pharmacology ; Molecular Docking Simulation

Objective To explore the role of autophagy, apoptosis of neutrophils and neutrophils extracellular traps (NET) formation in systemic lupus erythematosus (SLE). Methods Thirty-six patients with SLE were recruited as research subjects, and 32 healthy controls matched accordingly were enrolled as control subjects. The expression levels of microtubule associated protein 1 light chain 3B (LC3B), autophagy-related gene5(ATG5), P62, B-cell lymphoma 2(Bcl2), Bcl2-related X protein (BAX) in neutrophils were detected by Western blot analysis. Flow cytometry was employed to analyze the expression of LC3B on neutrophils. The expression level of myeloperoxidase(MPO) in plasma was estimated by ELISA. Furthermore, neutrophils were cultured in vitro and stimulated by 100 nmol/L rapamycin and 10 μg/mL lipopolysaccharide (LPS) for 6 hours, respectively. And then, the expression levels of LC3B, ATG5, P62, Bcl2 and BAX in neutrophils were detected by Western blot analysis. The level of MPO in culture supernatant was detected by ELISA. The change of fluorescence intensity of NET in culture supernatant was assayed by Sytox^TM Green staining combined with fluorescence spectrophotometry. Results Compared with healthy controls, the levels of autophagy and apoptosis of neutrophils and NET formation in SLE patients were increased. The level of apoptosis and NET formation was positively associated with neutrophil autophagy. The level of autophagy showed an increase but had no effect on apoptosis and NET formation for neutrophil stimulated by rapamycin. The levels of autophagy and NET formation also increased with no significant effect on apoptosis for neutrophil induced by LPS. Conclusion The autophagy, apoptosis and NET formation of neutrophils increase in SLE patients. The activation of autophagy and NET in neutrophils possibly result from the inflammatory internal environment in SLE patients.
Humans ; Neutrophils ; Extracellular Traps/metabolism* ; Lipopolysaccharides/pharmacology* ; bcl-2-Associated X Protein/metabolism* ; Sirolimus/pharmacology* ; Lupus Erythematosus, Systemic ; Autophagy

In order to investigate the role of melatonin in inhibiting the proliferation of murine gastric cancer and the underlying molecular mechanism, we performed an in vivo study by inoculating murine foregastric carcinoma (MFC) cells in mice, and then tumor-bearing mice were treated with different concentrations of melatonin (i.p.). The changes of Bcl-2, Bax, p21 and p53 expressions in tumor tissue were detected by using real-time fluorescence quantitative RT-PCR and Western blot. We found that: (1) melatonin resulted in reductions of tumor's volume and weight in the gastric cancer-bearing mice and thus showed anti-cancer effect; (2) melatonin reduced Bcl-2 expression, but increased the expression of Bax, p53 and p21 in tumor tissue. Our results suggest that melatonin could inhibit the growth of tumors in gastric cancer-bearing mice through accelerating the apoptosis of tumor cells.
Animals ; Apoptosis ; Melatonin ; pharmacology ; Mice ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Stomach Neoplasms ; drug therapy ; metabolism ; Tumor Suppressor Protein p53 ; metabolism ; bcl-2-Associated X Protein ; metabolism

This study was aimed to explore the apoptotic effect of Resveratrol (RES) on KG-1 cells and the expression of bcl-2/bax in vitro, and to clarify the possible mechanism of apoptotic effect of RES on leukemia cells. After treating with different concentrations of RES, the suppressive effect of RES on proliferation of KG-1 cells was analyzed by MTT method. Transmission electron microscope technique were used to detect the apoptosis status of KG-1 cells. The cell cycle and apoptosis percentage of KG-1 cells treated with RES were detected by flow cytometry. The expressions of bcl-2, bax mRNA and protein were assessed by semiquantitative RT-PCR and flow cytometry. The results showed that RES could obviously inhibit proliferation of KG-1 cells (p < 0.05). Compared with the control group, the cell number in S phase of KG-1 cells treated with RES increased (p < 0.05), the apoptosis rate enhanced significantly (p < 0.01) and the expression level was down-regulated, while expression level of bax was obviously up-regulated (p < 0.01). It is concluded that RES significantly induces apoptosis of KG-1 cells in vitro, which is probably related to the down-regulation of bcl-2 expression and up-regulation of bax expression.
Antineoplastic Agents, Phytogenic ; pharmacology ; Apoptosis ; drug effects ; Cell Line, Tumor ; Humans ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Stilbenes ; pharmacology ; bcl-2-Associated X Protein ; metabolism

This study was purposed to observe the changes of caspase-3 activity during apoptosis of HL-60 cells induced by an iron chelator, DFO (deferoxamine), and to explore the mechanism underlying apoptosis in HL-60 cells. The HL-60 cells treated with DFO were examined by light microscopy, flow cytometry (FCM) and DNA agarose gel electrophoresis; the activity of caspase-3 was determined by cellular immunohistochemistry; the transcription of the apoptotic gene of bax was detected by hybridization in situ. The results showed that the typical morphological character of apoptosis cells, DNA ladder and FCM assay confirmed that DFO could induce the apoptosis in HL-60 cells. The apoptotic rate increased in dose-and time-dependent manner. When cells had been cultivated with 100 micromol DFO for 12 hours, a few caspase-3 positive cells were found. In the process of time, the rate of caspase-3 positive cells was progressively higher than that in control (P < 0.05), while the level of bax transcription was also higher than that in the control. It is concluded the activation of caspase-3 and gene bax may be involved in the apoptosis of HL-60 cells induced by DFO.
Apoptosis ; drug effects ; Caspase 3 ; metabolism ; Deferoxamine ; pharmacology ; HL-60 Cells ; Humans ; bcl-2-Associated X Protein ; biosynthesis ; genetics

Rhus chinensis is an important resource plant. The aqueous extract of R. chinensis roots or stems was to produce Shuguantong Syrup, which is mainly used for the treatment of coronary heart disease and angina pectoris with definite curative effect. On this basis, the crude phenolic part of R. chinensis prepared by macroporous resin was evaluated for the cardio protective effect against myocardial ischemia in mice. The results showed that the phenolic part group with oral administration at the dosages of 190.8-381.6 mg·kg~(-1), compared with the model group, reduced the values of left ventricular end systolic diameter(LVEDs) and the left ventricular end diastolic diameter(LVEDd), and increased the cardiac ejection fraction(EF) and left ventricular fractional shortening(FS) rate, which could effectively improve cardiac function and exert its anti-myocardial ischemia effect, and reduce the rising levels of creatine kinase isoenzyme(CK-MB) and lactate dehydrogenase(LDH) in serum. HE staining showed that the phenolic part group reduced the infiltration of myocardial inflammatory cells and alleviated the degree of myocardial fibrosis and collagen deposition. TUNEL staining showed that the blue-green fluorescence of the phenolic part group decreased successively, and the degree of myocardial cell apoptosis was reduced. Immunohistochemical staining suggested that it could reduce the number of positive cells for p53 protein expression and significantly improve myocardial cell damage. All above data suggested that the phenolic part group had an anti-mycardial ischemis effect. Related mechanism studies revealed that the crude phenolic part could regulate the expressions of the p53 gene(p53), Bcl-2-associated X protein(Bax), B lymphoma-2 gene(Bcl-2), and caspase-3 protein(caspase-3) in myocardial tissue, suggesting that it could reduce cardiac remodeling and myocardial ischemic damage, and improve cardiac function by inhibiting myocardial apoptosis.This research laid a foundation for the elucidation of the pharmacological ingredients R. chinensis.
Animals ; Apoptosis ; Mice ; Myocardial Ischemia/drug therapy* ; Myocardium ; Myocytes, Cardiac ; Plant Extracts/pharmacology* ; Rhus ; bcl-2-Associated X Protein

Animals ; Captopril ; pharmacology ; Heart Failure ; metabolism ; Male ; Myocytes, Cardiac ; metabolism ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Rats ; Rats, Sprague-Dawley ; bcl-2-Associated X Protein ; metabolism

The study was aimed to explore the apoptosis effect of ouabain on Jurkat cells and its mechanism. MTT method was used to observe the inhibitory effect of ouabain on Jurkat cell proliferation. Apoptosis was detected by using flow cytometry (FCM) and the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling reaction method (TUNEL). The protein expressions of Bax, Bcl-2 and active subunits of caspase-3 were measured by Western blot. Activities of caspase-3 were determined by colorimetry method. The results showed that ouabain could induce apoptosis of Jurkat cells, the expression of Bax protein in process of cell apoptosis, caspase-3 activity of Jurkat cells were remarkably enhanced after ouabain treatment. It is concluded that ouabain may induce apoptosis of Jurkat cells due to the activation of caspase-3 resulting from regulation of Bax protein and Bcl-2 gene expressions.
Apoptosis ; drug effects ; Caspase 3 ; metabolism ; Cell Proliferation ; drug effects ; Genes, bcl-2 ; genetics ; Humans ; Jurkat Cells ; Ouabain ; pharmacology ; bcl-2-Associated X Protein ; biosynthesis ; genetics