Decreasing tumorigensis of mouse erthroleukemic cells by pre-infusion of adriamycin-treated erthroleukemic cells in mice

AIM: To investigate the effect of decreasing tumorigensis of mouse erthroleukemic (MEL) cells by pre-infusion of adriamycin(ADM)-treated MEL cells in mice.METHODS: The immungentic cell death of MEL cells was prepared by pre-treatment with ADM in vivo. The Kunming (KM) mice were divided into 3 group: MEL model group, ADM treatment group and control group. The mice in MEL model group were intravenously injected with PBS on the first day, and then with 1×10⁶ MEL cells on the 8th day. The mice in ADM treatment group were intravenously injected with 9×10⁶ MEL cells pretreated with ADM on the first day, and then with 1×10⁶ MEL cells without ADM pretreatment on the 8th day. The mice in control group didn't receive any intervention on the first day and 8th day. The survival time, the white cell count in peripheral blood and body weight were observed.RESULTS: All mice in control group were survived. All mice in MEL model group died from erythroleukemia. Only 1 mouse in ADM treatment group survived on the 80th day. The survival time in control group, MEL model group and ADM treatment group was 80 d, (28.00±8.43) d and (56.00±12.68) d, respectively. The survival time in ADM treatment group was longer than that in MEL model group (P<0.01). The peripheral blood white cell count in ADM treatment group was lower than that in ADM model group (P<0.01). The body weight of the mice in ADM treatment group on the 80th day was higher than that in MEL model group (P<0.01).CONCLUSION: The pre-infusion of adriamycin-treated MEL cells in erythroleukemic mice attenuates the tumorigenesis of MEL cells and improves survival time and life quality.     

Rapamycin enhances chemosensitivity of endometrial cancer cells to adria-mycin

AIM: To explore the therapeutic effect of adriamycin combined with rapamycin on endometrial cancer cells. METHODS: Two endometrial carcinoma cell lines with different PTEN gene states were chosen: HEC-1A (wild type) and Ishikawa (mutant type). Before adriamycin administration, the cells were pretreated with low concentration of rapamycin for 24 h. The cell viability and 50% inhibitory concentration (IC₅₀) of adriamycin at 24 h were determined by MTT assay. Multiple drug effect/combination index (CI) was used to evaluate the interaction between adriamycin and rapamycin. Apoptotic rate was measured by flow cytometry. The effects of the drugs on phosphorylation of PI3K/Akt and apoptosis protein caspase-3 were detected by Western blotting. RESULTS: Both adriamycin and rapamycin showed obvious growth inhibitory effects on the 2 endometrial cancer cell lines in a time- and dose-dependent manner. After pretreated with rapamycin, IC₅₀ of adriamycin decreased sharply. In Ishikawa cells, it decreased from (21.3±3.8) μmol/L to(11.9±1.2) μmol/L,P<0.05. In HEC-1A cells, it decreased from (14.3±2.8) μmol/L to (8.2±0.9) μmol/L,P<0.05. Combination index value of the 2 drugs was more than 1.15 in the 2 endometrial cancer cell lines, indicating synergistic effects. The combination therapy of adriamycin with rapamycin increased apoptotic rates in the 2 cell lines, and induced the down-regulation of phosphorylated Akt and over-expression of caspase-3 as compared with single drug treatment (P<0.05). CONCLUSION: Adriamycin combined with rapamycin significantly enhances the chemosensitivity of endometrial cancer cells and reduces drug resistance, which will become a new trend for treating endometrial cancer.     

Reverse effects of saikoside on multidrug resistance of human leukemic cell line K562/ADM in vitro

AIM: To study the reverse effects of saikoside (SS) on the multidrug resistance (MDR) of human leukemic cell line K562/ADM and to investigate the related mechanism. METHODS: K562 cells and K562/ADM cells in the culture were treated with SS at the concentrations of 1~100 mg/L. The inhibitory rate of the cell proliferation was measured by MTT assay. Non-cytotoxic dose of SS was determined. K562/ADM cells were treated with SS at non-cytotoxic doses of 1.25, 2.5 and 5.0 mg/L with different concentrations of adriamycin (ADM,0.05~100 mg/L). The 50% inhibitory concentration (IC₅₀) and the reversal index in all groups were determined. The cell morphology was observed after treated with SS+ADM. The effects of SS on ADM accumulation in K562/ADM cells, the cell cycle profile and apoptosis were examined by flow cytometry. RESULTS: The inhibitory rates were significantly increased in a dose-dependent manner when the cells were treated with different doses of SS (1~100 mg/L). The available reversal concentration of SS was 5.0 mg/L and the reversal index was 21.5 folds for K562/ADM cells. After treated with SS+ADM, the number of tumor cells was decreased and apoptotic cells were increased in a dose-response relationship. ADM accumulation in K562/ADM cells treated with SS was significantly higher than that in control cells (P<0.05). SS may significantly enhanced the apoptosis of K562/ADM cells treated with ADM (P<0.05). K562/ADM cells treated with SS were blocked in the stage of G₀/G₁. CONCLUSION: SS has effect on proliferation inhibition and MDR reversal in K562/ADM cell line. The reversal mechanisms of SS may be due to increasing the accumulation of chemo therapeutics in the cell, inducing the cell apoptosis and arresting the cells in G₀/G₁ phase.     

Effects of human urotensin II on ischemia/reperfusion injury in isolated perfused rat hearts

AIM: To investigate the effects of human urotensin II (hUII) on ischemia/reperfusion (I/R) injury in isolated rat hearts. METHODS: In the ischemia/reperfusion (I/R) model of isolated perfused rat hearts, the effects of hUII pretreatment on cardiac function was monitored with cardiac function software of MFL Lab200. ATP, total calcium, and malondialdehyde (MDA) content in myocardium were detected. The coronary perfusion flow (CPF) and lactate dehydrogenase (LDH) activity in coronary effluent were measured during reperfusion. RESULTS: In the hUII pretreated group, the release of LDH from myocardium was lower [(78.3±18.1)U/L] than I/R group [(109.3±23.9) U/L, P< 0.05], with decreased contents of MDA and calcium in myocardium (decreased by 24% and 27%, respectively, P< 0.05) and an increased myocardial ATP content [(3.8±0.4)μmol/g dw vs (2.2±0.4)μmol/g dw, P< 0.05)]. At the same time, hUII pretreatment increased CPF [(5.4±0.7) mL/min vs (3.8±0.8) mL/min in I/R group, P< 0.05], reduced left ventricular end-diastolic pressure (LVEDP) by 20% ( P< 0.05) with increased±d p /d t max [(217±38) kPa/s and (119±18) kPa/s vs (173±29) kPa/s and (82±25) kPa/s in I/R groups, respectively, P< 0.05]. hUII pretreatment also increased natrite/natrate (NO₂^-/NO₃^-) content in coronary effluent [(52.2±12.0)μmol/L vs (32.1±10.2)μmol/L in I/R group, P< 0.05)]. CONCLUSION: hUII pretreatment attenuated I/R injury in isolated perfused rat hearts. The protective mechanism might be associated with NO-mediated coronary vasodilation.     

Effects of leptin on BSEP protein expression and signaling pathway in HepG2 cells

AIM: To investigate the effects of leptin on the expression of bile salt export pump (BSEP) and signaling pathway in human hepatocellular carcinoma cell line HepG2. METHODS: HepG2 cells were cultured in vitro. Leptin at concentrations of 10^-8, 10^-7 and 10^-6 mol/L was used as a stimulating factor. The protein levels of adenosine monophosphate-activated protein kinase alpha subunit (AMPKa), phosphorylated AMPKa (p-AMPKa) and BSEP in the HepG2 cells at 24 h, 48 h and 72 h were detected by Western blotting. The optimal culture time and leptin concentration were selected, and compound C at concentration of 10 μmol/L was added to this group. The protein expression of BSEP was detected by Western blotting. RESULTS: Intervention of HepG2 cells with leptin for 72 h increased the protein expression of AMPKa gradually in a concentration-dependent manner, and leptin at concentration of 10^-6 mol/L induced the strongest AMPKa expression (P<0.01). Intervention of HepG2 cells with leptin for 24 h increased the phosphorylation level of AMPKa gradually in a dose-dependent manner (P<0.01). The effect of leptin on the increase in the protein expression of p-AMPKa was also in a time-dependent manner (P<0.01). After intervention with different concentrations of leptin for 24 h, the protein expression of BSEP in the HepG2 cells was gradually increased by the stimulation of leptin in a concentration- and time-dependent manner (P<0.01). Compared with NC group, the protein expression of BSEP in 10^-6 mol/L leptin group and 10^-6 mol/L leptin+10 μmol/L compound C group was increased at 72 h (P<0.01), and that in 10^-6 mol/L leptin+10 μmol/L compound C group was lower than that in 10^-6 mol/L leptin group at 72 h (P<0.01). CONCLUSION: Leptin promotes the protein expression of BSEP in HepG2 cells by leptin-AMPK-BSEP signaling pathway. Leptin promotes the increases in AMPKa protein and the level of phosphorylation of AMPKa in HepG2 cells.     

Effects of obstructive jaundice on enterocyte chloridion secretion in rats

AIM: To investigate the effects of obstructive jaundice (OJ) on enterocyte chloridion secretion in rats. METHODS: The OJ model of rats was set up. The rats were divided into OJ1 group and OJ2 group randomly. The animals in OJ1 group were sacrificed by exsanguination 7 days after operation and the rats in OJ2 group were sacrificed 14 days after operation. The Cl^- concentration in peripheral blood was detected. The epithelial cells in the terminal ileum mucosas were cultured in vitro. The concentration of intracellular Cl^- was detected by fluorospectrophotometry. The Cl^- current was measured by whole-cell patch clamp experiments. Western blotting was used to examine the change of voltage-gated Cl^- channel 2 protein (ClC-2) and the images were analyzed by image analysis system and statistical software quantitatively. RESULTS: The serum Cl^- concentration in OJ1 group and OJ2 group were obviously lower than that in control group . The intracellular Cl^- relative concentrations in OJ1 group and OJ2 group were higher than that in control group (3.14±0.38 and 3.55±0.47 vs 2.69±0.41,both P<0.05). The Cl^- current of normal epithelial cells displayed outward rectification, and transformed negative value into positive value following cell membrane depolarization. The Cl^- current was (-15.45±7.56) pA/pF and (5.85±0.81) pA/pF when voltage was -80 mV and 80 mV. The ranges of upgrade of absolute values of average electric current density in OJ groups were lower than those in control group (all P<0.05). Western blotting analysis showed that the ClC-2 protein generated a broad band about 90 kD. The average gradations of Western blotting images were lower in OJ groups than that in control group (0.20±0.04 and 0.19±0.06 vs 0.27±0.06, both P<0.05). However, no difference between the 2 OJ groups was observed (P>0.05). CONCLUSION: The chloridion secretion of intestinal epithelium is restrained in rats with OJ. The concentration gradient between exterior and interior of epithelial cells is decreased, and the Cl^- outward current is reduced. All of above are concerned with downregulation of ClC-2 protein in cell membrane.     

Effect of microRNA-100 on proliferation activity and cell cycle of hepatocarcinoma cells

AIM：To investigate the effect of microRNA-100 (miR-100) on the proliferation activity and cell cycle of hepatocarcinoma cells. METHODS：Synthetic miR-100 mimic and its negative control were transfected into human hepatocarcinoma HepG2 cells by liposome method. After transfection, the cell counting kit-8 (CCK-8) was used to measure the cell proliferation activity. The cell cycle distribution was determined by flow cytometry. The expression of Polo-like kinase 1 (Plk1) at mRNA and protein levels was detected by quantitative real-time PCR (qRT-PCR) and Western blotting. RESULTS：The transfection efficiency mediated by cationic liposome was greater than 85%. The inhibitory rates of cell proliferation in HepG2 cells were (43.5±12.2)%, (46.5±3.7)% and (52.1±0.2)% at 24 h, 48 h and 72 h after transfected with miR-100 mimic, respectively, which were significantly increased as compared with the control cells. Moreover, the cell proliferation index in experimental group (35.8 ± 1.4) was higher than that in negative control group (39.2 ± 1.0) and simple liposome group (40.7 ± 2.0) at 72 h. At the same time, the mRNA and protein expression levels of Plk1 obviously decreased in HepG2 cells transfected with miR-100 at 72 h after transfection. CONCLUSION：miR-100 suppresses the proliferation activity of hepatocarcinoma cells by down-regulating Plk1 gene expression.     

Effects of artesunate on proliferation,apoptosis and chemotherapeutic drug sensitivity of human hepatocellular carcinoma HepG2 cells

AIM: To investigate the effects of artesunate on proliferation and apoptosis in human hepatocelluar carcinoma cell line HepG2 and to study the sensitizing effect of artesunate on HepG2 cells to chemotherapeutic drugs. METHODS: The proliferation of HepG2 cells was determined by the assay of cell counting kit-8 (CCK-8) and the colony formation test. The morphology of HepG2 cells with Hoechst 33258 staining was observed under fluorescent microscope. Annexin V/propidium iodide (PI) was used to analyze the apoptosis and the cell cycle. The sensitizing effects of artesunate on HepG2 cells to chemotherapeutic drugs were determined by CCK-8 assay. RESULTS: When treated with artesunate for 48 h, the proliferation of HepG2 cells was significantly inhibited in a dose-dependent manner. The IC₅₀ was 19.2 μmol/L. Compared with the control cells, the colony formation of HepG2 cells treated with artesunate for 7 days was significantly inhibited. The nuclear fragmentation, karyopyknosis, chromosomal condensation, cell shrinkage, and attachment loss in HepG2 cells treated with artesunate were observed. The cells in G₂ phase increased obviously, and the percentages of hypodiploid cells and early apoptotic rates were significantly higher in artesunate treatment groups than those in control group. The IC₅₀ of 5-FU, carboplatin and epirubicin combined with artesunate was 3.33, 2.02 and 1.71 times sensitized as compared with control group, respectively. CONCLUSION: Artesunate effectively inhibits proliferation and induces apoptosis of HepG2 cells. Aresunate also sensitizes HepG2 cells to chemotherapeutic drugs.     

Effect of antisense locked nucleic acid blocking translation initiation region of c-myc exon 2 on apoptosis of hepatocellular carcinoma cells

AIM: To observe the effect of antisense locked nucleic acid (anti-LNA) blocking the translation initiation region of c-myc exon 2 on the viability and apoptosis of hepatocellular carcinoma cells.METHODS: The anti-LNA that was complementary to the translation initiation region of c-myc exon 2 was designed, synthesized, and introduced into the HepG2 cells by cationic liposome-mediated transfection. The mRNA and protein levels of c-Myc in the cells were determined by RT-PCR and Western blot. The change of cell apoptosis was analyzed by flow cytometry, and the toxicity of anti-LNA to the cells was detected by MTT assay.RESULTS: Five days after transfection, the mRNA level of c-Myc in anti-LNA group was 0.335±0.016, and the protein level was 0.448±0.037, significantly lower than those in control group (both P<0.05). The ratio of apoptotic cells in anti-LNA group was 32%±6%, which was higher than that in control group (P<0.05).CONCLUSION: Antisense locked nucleic acid targeting at the translation initiation region of c-myc exon 2 shows strong inhibitory effects on the apoptosis of hepatocellular carcinoma cells.     

Effects of exogenous ADM on HPA axis in early stage of acute mechanical renal trauma

AIM: To explore the effects of exogenous adrenomedullin(ADM) on hypothalamus-pituitary-adrenal cortex (HPA)axis in the early stage of acute mechanical renal trauma.METHODS: Healthy adult Wistar rats were randomly divided into 4 groups: 8 in control group, 32 in trauma group, 32 in the group injected with ADM before trauma and 32 in the group injected with ADM after trauma. To induce renal trauma, the rats in the latter 3 groups were subjected to mechanical impact directly on the skin of renal region by a free-fall iron hammer. The rats in 2 treatment groups were injected with ADM (0.1 nmol/kg) intraperitoneally 10 min just before and after trauma,respectively. The rats in the 3 groups with kidney injury were executed in batches by drawing all the blood quickly in the hearts at the time points of 1 h, 6 h, 12 h and 24 h after trauma. The hypothalamus tissues were also collected. The expression of corticotropin-releasing hormone(CRH) in hypothalamus and the concentrations of adrenocorticotropic hormone(ACTH) and cortisol(CORT) in plasma were detected by immunohistochemical method and radioimmunoassay. RESULTS: The expression of CRH in hypothalamus and the concentrations of ACTH and CORT in plasma in trauma group,but were slightly higher than those in control group,but without statistical significance. The expression of CRH in hypothalamus at 1 h and 24 h, the concentration of ACTH in plasma at 12 h and CORT at 6 h,12 h and 24 h in the group injected with ADM before trauma significantly higher than those in trauma group and control group (P<0.05). The expression of CRH in hypothalamus at 1 h, 6 h and 12 h and the concentration of CORT in plasma at 12 h and 24 h in the group injected with ADM after trauma were obviously higher than those in trauma group and control group (P<0.05). CONCLUSION: Exogenous ADM stimulates HPA axis and activates the function of HPA axis markedly. However, the different layers of HPA axis have different responses to exogenous ADM. Injection of ADM before or after trauma has different effects on HPA axis.     

Protective effect of ethyl pyruvate on brain tissues in neonatal rats with hypoxic-ischemic brain damage

AIM：To study the effects of ethyl pyruvate (EP) on brain tissues in neonatal rats with hypoxic-ischemic brain damage (HIBD) and its underlying mechanisms. METHODS：A total of 165 seven-day-old Sprague-Dawley (SD) rats were randomly divided into 3 groups：sham operation group (n=43), HIBD group (n=61) and HIBD+EP group (n=61). The rats in HIBD+EP group were intraperitoneally injected with EP (50 mg/kg) 30 min before operation, and once a day after surgery. Superoxide dismutase (SOD) activity and malondialdehyde (MDA) content in brain homogenate, water content of brain and apoptotic cells in cortex were detected 3 days later. Ischemic and non-ischemic brain tissues were weighed to assess the extent of brain atrophy 14 days later. RESULTS：Higher level of SOD ［(125.78±18.35)×103 U/(g protein)］ and lower level of MDA ［(4.42±1.04) μmol/(g protein)］ in HIBD+EP group than that in HIBD group ［(97.84±15.50)×103 U/(g protein) and (6.02±0.89) μmol/(g protein), respectively］ was observed (P<0.05)．In addition, the water content of ischemic hemisphere was significantly higher than that of non-ischemic one in HIBD group (P<0.05), and was indistinguishable from that of non-ischemic one after EP treatment (P>0.05), indicating the protective effect of EP against brain edema. The apoptotic cells in cortex and hippocampus in HIBD+EP group ［(96.63±10.08)/field and (41.91±9.96)/field, respectively］ were obviously decreased compared with HIBD group ［(111.54±1.64)/field and (51.73±1.77)/field, respectively］, but still higher than those in sham operation group (P<0.05). The atrophy ratio of ischemic hemisphere in HIBD+EP group was (13.25±5.19)%, significantly lower than that in HIBD group ［(20.32±5.10)%, P<0.05］. CONCLUSION：Ethyl pyruvate is neuroprotective against HIBD in neonatal rats via increasing SOD level, decreasing MDA level, attenuating brain edema, decreasing apoptotic cells in cortex and alleviating atrophy of hypoxic-ischemic hemisphere.     

Anti-lung cancer efficacy of magnetic nanoscale carrier with camptothecin

AIM: To study the preparation, drug release, cell absorption and anti-lung cancer efficacy of magnetic nanoscale carrier with camptothecin (MNC-Camp). METHODS: The drug release ability of MNC-Camp was detected by ultraviolet spectrophotometric method. Absorption of magnetic nanoparticles by A549 cells was measured by pullulan staining method. The growth inhibition ability of Camp and MNC-Camp on the A549 cells and white blood cells was analyzed by MTT assay. The metastasis ability of A549 cells treated with Camp and MNC-Cmap was evaluated by cell invasion method. The activity of apoptosis protein caspase-3 was detected by ELISA. RESULTS: A549 cells had a good absorption ability on MNC, and the drug release of MNC-Camp increased with time increasing. Compared with MNC group, Camp and MNC-Camp induced the apoptosis of A549 cells, and the half inhibition concentration (IC₅₀) were (35.14±3.21) and (7.16±2.54) mg/L, respectively.Camp and MNC-Camp also decreased the A549 cell transfer number and increased the activity of apoptosis protein caspase-3. All the effects of MNC-Camp were obviously significant than those of free Camp (P<0.05). CONCLUSION: MNC-Camp has a better inhibitory effect on the growth of A549 cells than free Camp, and finally plays an important role in anti-lung cancer effect.     

Effect of Aurora protein kinase inhibitor VX-680 on homogeneity adhesion and migration in human hepatoma HepG2 cell

AIM:To study the effect of Aurora protein kinase inhibitor VX-680 on homogeneous adhesion and migration ability in human hepatocellular carcinoma cell line HepG2. METHODS:The HepG2 cell were divided into experimental group and control group, respectively. VX-680 was used in experimental groups at 3 concentrations (3.125 μmol/L group, 6.25 μmol/L group and 12.5 μmol/L group). DMSO was used in the control group. The effects of VX-680 at different concentrations on the adhesion ability of human hepatocellular carcinoma HepG2 cells were observed by cell slow aggregation test and separation experiment. The effects of VX-680 at different concentrations on the migration ability of HepG2 cells was detected by wound healing assay. The expression of E-cadherin in HepG2 cells was detected by Western blot. RESULTS:The results of the slow aggregation test showed that compared with the control group, the number of cell clumps formed in experimental groups was significantly decreased (P<0.01). The results of separation experiment showed that the ratio of N_TC/N_TE gradually decreased with the increased concentration of VX-680. The results of wound healing assay showed that as the concentration of VX-680 increased, the cell scratch healing ability gradually weakened compared with control group. The results of Western blot showed that the protein expression of E-cadherin in the HepG2 cells increased with the increased concentration of VX-680 (P<0.05). CONCLUSION:VX-680 increases the homogeneous adhesion and inhibits the migration of HepG2 cells.     

Effects of MEG3 and adenosine on autophagy and proliferation of HepG2 cells

AIM:To study the effects of maternal expressed gene 3 (MEG3) and adenosine on the autophagy and proliferation of human hepatocellular carcinoma HepG2 cells, and to explore the possible mechanisms of autophagy and the effect on the proliferation of HepG2 cells induced by MEG3 and adenosine. METHODS:HepG2 cells were cultured according to the conventional cultural method, and divided into control group, MEG3 group (the cells were transfected with MEG3 lentivirus), adenosine group (the cells were treated with 1 mmol/L adenosine) and MEG3+adenosine group (the cells were treated with 1 mmol/L adenosine and MEG3 lentivirus). The protein expression of LC3-Ⅱ, LC3-Ⅰ and mammalian target of rapamycin (mTOR) was determined by Western blot. MDC staining was used to observe the number of autophagosomes. The cell viability was measured by CCK-8 assay, and the number of viable cells were counted by automated cell counter. RESULTS:Compared with control group, LC3-Ⅱ expression and the ratio of LC3-Ⅱ/LC3-Ⅰ were decreased, mTOR expression was increased (P<0.05), the viability of HepG2 cells was decreased, and the number of autophagosomes were reduced in MEG3 group and adenosine group. In MEG3+adenosine group, LC3-Ⅱ expression and the ratio of LC3-Ⅱ/LC3-Ⅰ were decreased significantly (P<0.01), mTOR expression was increased significantly (P<0.01), and the viability and autophagosomes of HepG2 cells were reduced markedly as compared with MEG3 group and adenosine group. After treated with MEG3 and adenosine for 24~72 h, the viable HepG2 cells reduced significantly in MEG3 group and adenosine group (P<0.01), especially in MEG3+adenosine group (P<0.01). CONCLUSION:MEG3 overexpression and low concentration of adenosine activate the mTOR pathway, and inhibit the autophagy and proli-feration of HepG2 cells. MEG3 enhances the effect of adenosine on HepG2 cells.     

Ethyl acetate extract of Pleione bulbocodioides (Franch.) Rolfe induces apoptosis of human leukemia K562 and HL-60 cells through intrinsic mitochondrial apoptosis pathway

AIM:To investigate the effects of ethyl acetate (EtOAc) extract of Pleione bulbocodioides (Franch.) Rolfe on proliferation and apoptosis of human leukemia K562 and HL-60 cells and the possible apoptosis pathway. METHODS:Human leukemia cell lines were treated with EtOAc extract of Pleione bulbocodioides at different concentrations. XTT method was used to evaluate the viability of K562 cells and HL-60 cells. The cell growth inhibition was calculated by Trypan blue exclusion test. The percentage of apoptotic cells was determined by flow cytometry, and 4',6-diamidino-2-phenylindole (DAPI) was used to observe morphological changes of the cells. The cell cycle was observed by propidium iodide (PI) staining. The protein expression of Bcl-2, Bax, cleaved poly(ADP-ribose) polymerase (PARP), cleaved caspase-3, cytochrome C and apoptosis-inducing factor (AIF) wase determined by Western blot. RESULTS:The cell viability and proliferation were inhibited by EtOAc extract of Pleione bulbocodioides with IC₅₀ of (42.14±2.54) mg/L for HL-60 cells and (51.28±3.12) mg/L for K562 cells at 24 h. The results of Annexin V-FITC/PI and DAPI staining showed that EtOAc extract of Pleione bulbocodioides induced cell apoptosis in a dose-dependent manner. The apoptotic rate was increased compared with control group (P<0.05). The G₂ phase increased with typical cell apoptosis-induced morphological changes. The levels of pro-apoptotic proteins Bax, cleaved PARP and cleaved caspase-3 were increased, while Bcl-2 was down-regulated (P<0.05). Cytochrome C and AIF in cytosol, characteristic proteins of intrinsic mitochondrial apoptosis pathway, also increased with the concentration of EtOAc extract of Pleione bulbocodioides increasing (P<0.05). CONCLUSION:EtOAc extract of Pleione bulbocodioides significantly inhibits cell proliferation and induces cell apoptosis in human leukemia cell lines HL-60 and K562 through intrinsic mitochondrial apoptosis pathway.     

Effect of Nao-re-qing oral liquid on cAMP content in hypothalamus and CSF,and AVP content in ventral septal area of endotoxin-induced febrile rabbits

AIM: To explore the mechanism of Nao-re-qing oral liquid (NRQ) decreasing endotoxin (ET)-induced fever in rabbits. METHODS: (1) The ET-induced fever model was established in rabbits. Febrile response of rabbits was observed. (2) The arginine vasopressin (AVP) content in the ventral septal area (VSA), and cAMP content in hypothalarmus (HP) and CSF were determined by radioimmunoassay.RESULTS: (1) In ET group, the maximal increment in body temperature (ΔT) [(1.80±0.16) ℃], 6 h thermal respone index (TRI₆)(11.31±0.20), the cAMP content in the HP [(1.35±0.21)nmol/g], the cAMP content in CSF [(66.69±1.82) nmol/L] and AVP content in the VSA [(30.80±9.59)ng/g ] were significantly higher than those in NRQ+ET group[ΔT(0.82±0.08) ℃, TRI₆(5.73±0.09), HP: cAMP(0.70±0.50)nmol/g, CSF: cAMP(56.86±1.34), AVP:(11.91±3.47)ng/g]( P<0.01). (2) The AVP content in VSA, and cAMP content in HP and CSF were separately paralleled with the fluctuation of body temperature (AVP: r=0.972, P<0.01; HP: cAMP r=0.899, P<0.05; CSF: cAMP r=0.991, P<0.01). CONCLUSION: The antipyretic action of NRQ may be due to inhibiting the increase in cAMP in HP and meanwhile promoting the release of AVP in VSA.     

Effect of lipopolysaccharide on viability and secretion function of human umbilical vein endothelial cells

AIM: To observe the direct effect of lipopolysaccharide (LPS) on secretion of endothelin-1 (ET-1) and nitric oxide by human umbilical vein endothelial cell and cell viability of the secretor. METHODS: The third passage of human umbilical vein endothelial cells were incubated with different concentrations of LPS (1 g/L, 100 mg/L, 10 mg/L, 1 mg/L, 100 μg/L, 10 μg/L, 1 μg/L) for 6 hours, and the culture supernatants were collected. The concentrations of ET-1 were determined by radioimmunoassay, the concentrations of nitric oxide were determined using Greiss's method. The viabilities of cells were measured by MTT method. RESULTS: The concentration of ET-1 (pg/L) of normal control group was 251.64±10.90. The concentrations of ET-1 (pg/L) of LPS treated groups were 220.85±19.14, 278.67±15.45, 306.40±11.60, 312.87±33.50, 324.38±17.02, 291.49±14.30, 282.11±13.38, respectively (each group compared with normal control group, P<0.05 or P<0.01). The concentration of NO_x (μmol/L) of normal control group was 629.46±13.36. The concentrations of NO_x (μmol/L) of LPS treated groups were 732.58±23.21, 669.87±9.32, 661.24±16.80, 650.33±13.24, 606.59±12.94, 626.75±9.83, 627.61±5.61, respectively (each group compared with normal control group, P<0.05 or P<0.01). The viabilities of endothelial cells of LPS treated groups were 74%, 81%, 86%, 88%,91%, 93%, 93%, respectively. CONCLUSION: LPS of lower concentrations had no significantly lethal effect on human umbilical vein endothelial cells, but enhanced secretion of ET-1 and inhibited NO production. LPS in higher concentrations showed significant lethal effect on human umbilical vein endothelial cells, inhibited secretion of ET-1 and enhanced NO production.     

Effect of vitamin E on ox-LDL-induced monocyte/endothelium adhesion

AIM:To observe the role of ox-LDL in rabbit endothelium/circulating monocyte adhesion in vitro and the effect of vitamin E.METHODS:Cultured rabbit's endothelial cells were incubated with different concentrations of ox-LDL, then incubated with rabbit's monocytes to observe cell's adhesion. With Northern blotting, endothelial VCAM-1 mRNA expression was measured. By using vitamin E incubation before ox-LDL, above steps were repeated to observe the effect of vitamin E.RESULTS:When ox-LDL concentrations were 2.5 mg/L,5 mg/L,10 mg/L,monocytes adhesive to endothelium per microscope field were 132.8±20.2,350.0±37.2,502.6±78.8,respectively They were al significantly higher than that of control group(51.2±7.7,P<0.01)Addition of vitamin Eat comcentration of 10 mol/L,20 mol/L,40 mol/L respect ively before ox-LDL incubation yield fewer adhesive monocytes as follow:422.3±32.2,296.0±21.7,205.2±36.6,respectively,but only the last two concentrations had significant effects(P<0.01)Endothelial VCAM-1 mRNA expression in ox-LDL group was higher than that in vitamin E group(0.49±0.09 vs 0,33±0.10,P<0.05).CONCLUSIONS:ox-LDL directly enhances rabbit's endothelium/monocyte adhesion by inducing expression of VCAM-1 in endothelium. Vitamin E inhibites these effects of ox-LDL.     

Salidroside increases release of intracellular free Ca2+ from sarcoplasmic reticulum in cultured rat cardiomyocytes

AIM: To observe the effects of salidroside on intracellular free Ca²⁺ concentration in cultured rat cardiomyocytes. METHODS: Primarily cultured cardiomyocytes of neonatal rats were divided into control group, different concentrations of salidroside groups and verapamil pretreatment+different concentrations of salidroside groups. The fluorescent intensity of intercellular free calcium concentration ([Ca²⁺]_i) in cultured cardiomyocytes of newborn rats loaded with fluo-3/AM(5 μmol/L) was measured by laser scanning confocal microscopy. RESULTS: Salidroside at concentrations of 15 mg/L, 30 mg/L and 60 mg/L elevated [Ca²⁺]_i in cultured rat cardiomyocytes with the peak values of 574.08±4.65, 591.86±3.64 and 618.66±4.27, respectively (all P<0.01), indicating that the effect of salidroside on the level of [Ca²⁺]_i was dose-dependent. In the presence of verapamil in D-Hanks solution, salidroside also elevated the fluorescent intensity of [Ca²⁺]_i in cardiomyocytes from 357.74±3.13, 387.17±2.37 and 391.43±1.34 to 480.86±3.98, 496.70±3.08 and 522.18±3.19, respectively (all P<0.01). CONCLUSION: Salidroside increases the release of [Ca²⁺]_i from sarcoplasmic reticulum in cultured rat cardiomyocytes.     

Stable reversal of multidrug resistance in A549/DDP cells by shRNA targeting MRP1 gene

AIM: To reverse multidrug resistance (MDR) of A549/DDP cells with short hairpin RNA (shRNA) expression vectors. METHODS: Two multidrug resistance-associated protein 1( MRP1 ) gene-specific shRNA expression plasmids pSilencer 2.1-U6 neo-MRP1 were constructed and introduced into A549/DDP cells. MRP1 mRNA was assayed by real-time fluorescent quantitative PCR. The MRP1 function was determined by rhodamine 123(Rho123) retention and the protein expression of MRP1 was detected by immunofluorescent staining. The viability of A549/DDP cells was evaluated by MTT method. RESULTS: MRP1 shRNA expression plasmids were successfully constructed. The expression of MRP1 at mRNA and protein levels was significantly decreased after sh-MRP1-2.1-1 and sh-MRP1-2.1-2 were transfected into A549/DDP cells. The intracellular accumulation of Rho123 significantly increased from(16.93±0.58)% to (89.02±0.59)% and (82.56±1.37)%. IC₅₀ of cisplatin were decreased from (101.45±0.64) μmol/L to (38.06±0.05) μmol/L and (53.72±0.36) μmol/L. IC₅₀ of 5-fluorouracil were decreased from (263.20±2.00) μmol/L to (98.82±1.16) μmol/L and (141.81±0.49) μmol/L. CONCLUSION: The shRNA expression plasmid pSilencer 2.1-U6 neo-MRP1 can stably and permanently inhibit MRP1 gene. The sensitivity of A549/DDP cells to drug is reversed.