Effect of berberine on the adhesion between human umbilical vein endothelial cells and human pulmonary carcinoma cells

AIM: To study the mechanism of berberine on the adhesion between human pulmonary carcinoma cells (PG cells) and HUVECs. METHODS: The effect of berberine (2.5-40 mg/L) on the proliferation of HUVECs was detected by MTT method. Further, PG cells were treated with berberine at doses of 2.5, 5, 10 mg/L for 6, 12, 24 h. The adhesion between PG cells and HUVECs was determined by rose bengal staining. The expression of CD44s on PG cells were determined by fluorescence antibody staining. Fluorescence anisotropy imaging system was used to assay the fluidity of PG cell membrane. RESULTS: Berberine at concentrations of 2.5, 5, 10 mg/L were the safety doses to the proliferation of HUVECs treated for 6, 12, 24 h. Berberine inhibited the adhesion between PG cells and HUVECs significantly in a dose-dependent manner (P<0.05 or P<0.01). Meanwhile, berberine increased the expression of CD44s on PG cells (P<0.05 or P<0.01). Berberine decreased the fluidity of PG cell membrane in a dose-dependent manner after 24 h incubation. CONCLUSION: Berberine inhibits the adhesion between PG cells and HUVECs by regulating the expression of adhesion molecules and the fluidity of cell membrane on PG cells.     

中国园艺学会第七届青年学术讨论会

中国园艺学会第九届第8次常务理事扩大会决定,“中国园艺学会第七届青年学术讨论会”由山东农业大学园艺科学与工程学院和山东省园艺学会承办,将于2006年7月或8月在山东泰安举行。会议交流主题:(1)园艺作物种质资源、遗传育种与生物技术;(2)园艺作物有机、无公害及标准化安全生     

Effects of berberine on the apoptosis of NIT-1 cells induced by high glucose and saturated fatty acids

AIM: To investigate the effects of berberine on the apoptosis of NIT-1 cells induced by high glucose and saturated fatty acid. METHODS: The influence of berberine at different concentrations on NIT-1 cells cultured with or without high glucose and saturated fatty acid were determined and compared using MTT colorimeric assay. The cell apoptotic rate was also determined by flow cytometry assay and in situ TUNEL method. RESULTS: The effects of berberine at different concentrations on NIT-1 cells showed dose-dependent, low dose (≤5 μmol/L) had dispensable cytotoxicity; meanwhile, high dose showed distinct effects. On the other hand, low dose of berberine alleviated the apoptosis in NIT-1 cells induced by high glucose and saturated fatty acid, when adding berberine to cell medium. CONCLUSION: Berberine inhibited the apoptosis of NIT-1 cells induced by high glucose and saturated fatty acid.     

Effect of berberine on Helicobacter pylori-induced human gastric epithe-lial cells injury

AIM: To investigate the effect of berberine (Ber) on Helicobacter pylori (Hp)-induced human gastric epithelial cells (GES-1) injury and the underlying mechanism. METHODS: Berberine (5, 10 and 20 μmol/L) and PD98059 (20 μmol/L), a selective inhibitor of extracellular regulated protein kinases (ERK)1/2 signaling pathway, were added to Hp-infected GES-1 cells. The cell activity and apoptosis, the levels of interleukin (IL)-1β and IL-8, lactic dehydrogenase (LDH) activity and the protein levels of Bax, Bcl-2 and p-ERK1/2 in the GES-1 cells were determined by MTT assay, flow cytometry, ELISA, colorimetry and Western blot, respectively. RESULTS: Compared with control group, Hp significantly inhibited the cell activity, increased the apoptotic rate, LDH activity, IL-1β and IL-8 levels, the Bax and p-ERK1/2 protein levels but decreased the Bcl-2 protein level in GES-1 cells (P<0.05). However, these effects of Hp were reversed by berberine at medium-dose and high-dose, as compared with the Hp-infected GES-1 cells (P<0.05). Moreover, the protective effects of berberine were significantly enhanced by the co-incubation of berberine with PD98059, as compared with the berberine at higher dose (P<0.05). CONCLUSION: Berberine may attenuate Hp-induced human gastric epithelial GES-1 cells injury by anti-inflammation, promoting cell growth and anti-apoptosis via the inhibition of ERK1/2 signaling pathway.     

Effect of berberine on endoplasmic reticulum stress-autophagy pathway in human ovarian cancer SKOV3 cells

AIM: To investigate the regulatory effect of berberine on the endoplasmic reticulum stress-auto-phagy pathway in human ovarian cancer SKOV3 cells. METHODS: Human ovarian cancer SKOV3 cells were cultured in vitro, and berberine at doses of 12.5, 25, 50, 100, 200 and 400 μmol/L were added. After exposure for 12 h, 24 h and 48 h, the viability of the SKOV3 cells was measured by MTT assay. The cells were divided into control group, berberine (50 μmol/L) group, berberine (100 μmol/L) group, and berberine (200 μmol/L) group. After treatment with berberine for 24 h, the effects of berberine on the morphological changes of SKOV3 cells were observed under inverted phase-contrast microscope. The protein expression of microtubule-associated protein 1 light chain 3 (LC3) and ubiquitin-binding protein p62 was observed by indirect immunofluorescence method under laser confocal microscope. The protein expression of beclin-1,LC3,p62, CCAAT/lenhancer binding protein homologous protein (CHOP) and glucose-regulated protein 78 (GRP78) was determined by Western blot. RESULTS: Berberine at 12.5, 25, 50, 100, 200 and 400 μmol/L significantly decreased the viability of SKOV3 cells at 12 h, 24 h and 48 h, and the IC₅₀ values of 12 h, 24 h and 48 h were (764.7±0.3) μmol/L, (231.6±0.1) μmol/L and (96.2±0.1) μmol/L, respectively. Laser confocal microscopy showed that the LC3 and p62 proteins were scattered and the fluorescence intensity was increased, while the point-like aggregation was also observed. Berberine at 200 μmol/L obviously enhanced the co-localization of LC3 and p62 proteins. Compared with control group, the expression of endoplasmic reticulum stress-related proteins GRP78 and CHOP, and autophagy-related proteins beclin-1, LC3 and p62 in berberine (200 μmol/L) group was increased significantly (P<0.05). CONCLUSION: Berberine may promote endoplasmic reticulum stress in SKOV3 cells by regulating autophagy.     

Effect of norcantharidin on proliferation and invasion of human breast cancer cell line SKBR3 in vitro

AIM: To investigate the effect of norcantharidin（NCTD）on proliferation and invasion of human breast cancer cell line SKBR3 in vitro and its anticancer mechanisms.METHODS: MTT assay was used to determine SKBR3 cell proliferation. Light and FACScan were used to detect apoptosis and cell cycle. The invasiveness of SKBR3 was evaluated by the adhesion test，Matrigel experiment and the crossing-river test.RESULTS: NCTD had inhibitive effects on growth of SKBR3 cells in a dose and time-dependent manner, with the IC₅₀ value of 12.5 mg/L at 24 h．The cells treated with 10 mg/L NCTD for 24 h and 48 h showed typical apoptotic morphology and hypodiploid peak before G₁ phase. The cell cycle was arrested at G₂/M phase. The apoptosis percentage was up to 3.44% and 6.17%, and the G₂/M percentage was up to 35.82% and 38.70%. NCTD also could inhibit obviously the adhesion, movement and invasive capability simulating human basement membrane of SKBR3. Its effect was also in a dose-dependent manner. In the NCTD-treated group, crossing-river time was prolonged significantly and passing-membrane cells markedly decreased. CONCLUSION: NCTD in vitro inhibits not only the proliferation and growth of human breast cancer cells but also invasion and metastasis of the cells at relatively low concentration. NCTD shows prominent anti-tumor effects.     

Effect of lentinan on apoptosis and PI3K/AKT signaling pathway in leukemic HL-60 cells in vitro

AIM:To study of the regulatory effect of lentinan on human leukemic HL-60 cell apoptosis and its effect on PI3K/AKT signaling pathway in HL-60 cells in vitro.METHODS:Lentinan at concentrations of 0 mg/L, 15 mg/L, 30 mg/L and 45 mg/L was applied to HL-60 cells cultured to the logarithmic phase in vitro, and the inhibitory effect of lentinan on the viability of HL-60 cells was measured by MTT assay after 24 h, 48 h and 72 h. The apoptosis induced by lentinan was analyzed by flow cytometry. The protein levels of cleaved PARP, cleaved caspase-9, cleaved caspase-3, cleaved caspase-8, cytochrome C, PI3K, AKT and p-AKT were determined by Western blot. After treatment with PI3K inhibitor LY294002 at 5 mg/L for 72 h, the apoptosis of HL-60 cells was analyzed by flow cytometry. RESULTS:The viability of HL-60 cells was inhibited after treatment with lentinan at concentrations of 15 mg/L, 30 mg/L and 45 mg/L for 24 h, 48 h and 72 h in concentration-dependent and time-dependent manners (P<0.05). The apoptosis of HL-60 cells was promoted after treatment with lentinan (15 mg/L, 30 mg/L and 45 mg/L) for 72 h in a concentration-dependent manner (P<0.05). The protein levels of cleaved PARP, cleaved caspase-9, cleaved caspase-3 and cytoplasmic cytochrome C in the HL-60 cells induced by 30 mg/L lentinan were increased significantly with the increase in the treatment time (P<0.05), but caspase-8 did not show any change. The protein levels of PI3K, AKT and p-AKT were decreased obviously with the increase in the lentinan concentration (P<0.05). Treatment of HL-60 cells with LY294002, a PI3K pathway inhibitor, produced apoptosis-inducing effect similar to lentinan (P<0.05). CONCLUSION:Lentinan induces HL-60 cell apoptosis by inhibiting PI3K/AKT signaling pathway.     

Effects of azathioprine on proliferation,cell cycles and apoptosis of me-senchymal stem cells from rat bone marrow in vitro

AIM: To investigate the effects of azathioprine (AZA) on the proliferation, cell cycle and apoptosis of mesenchymal stem cells (MSCs) from the bone marrow of Sprague-Dawley rats in vitro. METHODS: MSCs were cultured in low-glucose DMEM containing 10% FBS,and treated with AZA at concentrations of 50 mg/L, 100 mg/L, 200 mg/L and 300 mg/L for 24 h, 48 h and 72 h. The effects of AZA on the growth curve and proliferation of MSCs were tested by cell counter under microscope. The apoptosis and cell cycle was determined by flow cytometry. RESULTS: Pure MSCs were gained by 3 times of passages. No significant effect of AZA at concentration of less than 100 mg/L on the proliferation, cell cycle and apoptosis of MSCs was observed (P>0.05). Under the condition of more than 200 mg/L for 72 h, AZA inhibited the growth of MSCs.The inhibitory rate was more than 66%, and the rate of apoptosis was increased (P<0.05). However, at the concentration of 300 mg/L for 72 h, AZA decreased the apoptotic rate and the necrotic rate of MSCs was obviously increased (P<0.05). Using AZA at concentration of more than 200 mg/L, as the action time prolonged, the MSCs in G₀/G₁ phase were increased, and those in S phase were decreased (P<0.05). CONCLUSION: At some concentrations, AZA significantly affects the proliferation, apoptosis and cell cycle of MSCs. Large dose of AZA may cause MSCs to death.     

Protective effect of procyanidins on PC12 cells exposed to Aβ25-35

AIM: To investigate the protective effect of procyanidins on the PC12 cells exposed to Aβ_25-35 and the mechanisms.METHODS: Aβ_25-35 at 25 μmol/L was used to treat the PC12 cells for 48 h, and the PC12 cells were pretreated with procyanidins at 25, 50 and 100 mg/L for 24 h. The cell vitality was measured by MTT assay. The content of reactive oxygen species (ROS) was detected by DCFH-DA staining. The change of mitochondrial membrane potential was examined by JC-10 staining. The apoptosis was analyzed by flow cytometry with Annexin V/PI double staining. The protein levels of activated caspase-3 was determined by Western blot.RESULTS: Under the exposure of the PC12 cells to Aβ_25-35, procyanidins increased the cell viability, reduced intracellular ROS level, prevented mitochondrial membrane potential decline, attenuated the caspase-3 activation and inhibited the apoptosis of PC12 cells (P<0.05 or P<0.01).CONCLUSION: Procyanidins have a significant protective effect on the PC12 cells exposed to Aβ_25-35. Its mechanism may be related to removing intracellular ROS induced by Aβ_25-35, relieving the damage to the mitochondrial membrane, and thereby inhibiting cell apoptosis.     

Effect of salidroside on behaviors of primary mouse T-lymphocytes in vitro

AIM: To observe the effect of salidroside on behaviors of primary mouse T-lymphocytes in vitro. METHODS: The lymphocytes from the lymphoid nodes of BALB/c mice were isolated and primarily cultured. The viability of T cells was assessed by MTT assay. Fluorescence-conjugated monoclonal antibody and flow cytometry (FCM) were used to analyze the expression of T-cell activation marker CD69 in response to concanavalin A (Con A) in vitro. Carboxyfluorescein diacetate succinimidyl ester (CFDA-SE) staining was used to detect the proliferation of T cells in vitro. FCM analysis was used to determine the production of reactive oxygen species (ROS) in the T cells by staining with 2',7'-dichlorodihydrofluorescein diacetate (H₂DCFDA). The mean fluorescence intensity of DiOC₆(3) staining in the T cells was detected by FCM in order to analyze the effects of salidroside on the activity of the mitochondrial and the mitochondrial membrane potential in the T cells induced by dexamethasone (DEX). The thymus T cells from BALB/c mice were isolated and primarily cultured, and then FCM was also used to analyze the apoptosis of the thymus T cells treated with DEX. RESULTS: Salidroside increased the expression of T-cell activation marker CD69 at the final concentration of 80, 160 and 320 μmol/L (P<0.05). Salidroside promoted the proliferation of T cells induced by Con A for 72 h in vitro (P<0.01). Salidroside reduced the production of ROS (P<0.05) and protected the mitochondrial membrane potential of T cells from the injury of DEX (P<0.01). Salidroside also decreased the apoptosis rate of the thymus T cells induced by DEX in vitro (P<0.01). CONCLUSION: Salidroside promotes the activation and proliferation of T cells induced by Con A, reduces the production of ROS, maintains the mitochondrial membrane potential and protects thymus T cells against apoptosis induced by DEX in vitro.     

Mechanisms underlying the protection of berberine against liver injury induced by lipopolysaccharide in mice

AIM: To investigate the protective effects of berberine against liver injury induced by lipopolysaccharide in mice and the mechanisms underlying its protective effect. METHODS: The male mice were divided randomly into control, berberine group, LPS group and berberine treatment group. Mice were administered intragastrically with distilled water (0.01 mL/g) or 5 g/L neutral sulfate berberine (0.01 mL/g) once a day for 5 days and injected intraperitoneally with normal saline or LPS （0.02 mL/g，28 mg/kg）at 1 h after gavage on day 5. Blood was collected for determining alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities, the content of tumor necrosis factors-α (TNF-α) at 10 h and 2 h after LPS or normal saline injection, respectively. Furthermore, the liver tissue was processed, and histological changes and ultrastructure in liver were observed with light and electron microscopy, malondialdehyde (MDA) content and superoxide dismutase (SOD) activity in liver were also detected. RESULTS: Both ALT and AST activities in serum in LPS group were higher than those in control and berberine treatment group. LPS increased the serum TNF-ɑ content at 2 h after injection, which was reversed by berberine pretreatment. The histological examination showed that LPS caused severe hepatic cell edema, degeneration, apoptosis and even necrosis, and ultrastructure observation demonstrated that LPS induced mitochondrial swelling, condensation and margination of chromatin, irregular nuclear envelope in hepatocytes. The above pathological changes produced by LPS were attenuated by berberine pretreatment. Moreover, MDA contents in liver tissue were higher in LPS group than control and berberine treatment group, but there were no significant difference in SOD activity between berberine treatment and LPS group. CONCLUSION: Berberine has a protective effect on LPS-induced liver injury in mice, the mechanisms may be related to its decreasing the production of TNF-α, inhibiting lipid peroxidation and protecting mitochondria.     

Effect of activated protein C on apoptosis in human umbilical vein endothelial cells induced by lipopolysaccharide

AIM:To study the effect of activated protein C (APC) at different concentrations on apoptosis of human umbilical vein endothelial cells (HUVECs) induced by lipopolysaccharide (LPS).METHODS:The HUVECs were induced by LPS (1.0 mg/L) as apoptotic model that was administered by different concentration of APC (10 μg/L or 50 μg/L). Meanwhile, the control group and induced apoptosis group induced by LPS (1.0 mg/L) stimulation were also set up. The changes of cellular ultrastructures were observed under electron microscope. The DNA ladder and TUNEL fluorescent staining were measured in cells. Annexin-Ⅴ/PI double staining was used to measure the cell apoptosis rate by flow cytometry. Cell survival rate was measured by MTT assay. The proliferating cell nuclear antigen (PCNA) expression levels in cells were also measured by Western blotting to reflect the proliferation of the cells.RESULTS:There were significant apoptotic changes in the cells induced by LPS, but the apoptotic changes were reduced and apoptosis rates were decreased in the cells treated with APC. Meanwhile, cell survival rate and the protein levels of PCNA were increased after APC treatment, particularly at the concentration of 50 μg/L, which showed difference when compared with those induced apoptosis group by LPS (P<0.05).CONCLUSION:APC can inhibit HUVECs apoptosis induced by LPS and promote cell proliferation, thus protect the cells from injury.     

Effects of sinapic acid on proliferation and apoptosis of rat vascular smooth muscle cells induced by high glucose

AIM: To investigate the effects of sinapic acid(SA) on the proliferation and apoptosis of rat vascular smooth muscle cells(VSMCs) induced by high glucose(HG). METHODS: Cultured A7r5 cells were randomly divided and treated as indicated. The cell viability was determined by MTT assay. DNA synthesis was measured by BrdU assay. Cell cycle progression and cell apoptotic rate were determined by flow cytometry analysis. The levels of reactive oxygen species(ROS) were detected by ELISA. The protein levels of cyclin D1, P21, P27, phosphorylated protein kinase C(p-PKC), p-P38 and β-actin were evaluated by Western blot. RESULTS: Compared with control group, the viability of A7r5 cells was significantly enhanced, the DNA synthesis was increased, the cell cycle progression was promoted, the levels of ROS were elevated, the cell apoptotic rate was reduced, the protein expression of P21 and P27 was decreased, and the protein levels of cyclin D1, p-PKC and p-P38 were increased in HG group(all P<0.05). These effects were reversed by SA(0.1, 1 and 10 μmol/L) treatment in a dose-dependent manner(all P<0.05). Both P38 inhibitor SB203580 and PKC inhibitor chelerythrine significantly inhibit HG-induced PKC/P38 activation and cell viability(P<0.05).CONCLUSION: SA inhibits HG-induced VSMCs proliferation and promotes cell apoptosis via reducing PKC/P38 activation.     

Role of 17-AAG in inducing apoptosis and cell cycle arrest of HCT-15 cells

AIM: To investigate the effects of 17-AAG on apoptosis and cell cycle of HCT-15 cells and to clarify the related mechanisms. METHODS: MTT method was employed to evaluate the inhibitory effects of 17-AAG with Aifferent time and different doses on the proliferation of HCT-15 cells. The cells were stained with Annexin V-FITC/propidiumiodide and measured by flow cytometry. The expression of STAT3, cyclin D1, Cyt C, caspase 9 and caspase 3 at mRNA and protein levels was determined by RT-PCR and Western blotting. RESULTS: Treatment with 17-AAG at concentration of 1.25~20 mg/L for 24 h and 48 h significantly inhibited the activity of HCT-15 cells at both time-and concentration-dependent manners. Treatment with 17-AAG at concentrations of 0.425, 0.85 and 1.7 mg/L for 48 h significantly induced apoptosis and cell cycle arrest of HCT-15 cells. The exposure of 17-AAG at concentrations of 0.425, 0.85 and 1.7 mg/L for 48 h to the HCT-15 cells significantly down-regulated the expression of STAT3 and cyclin D1 at mRNA and protein levels, but up-regulated Cyt C, caspase 9 and caspase 3 mRNA and protein in a concentration-dependent manner. CONCLUSION: 17-AAG inhibits the cell activity, induces apoptosis and G₁ arrest by down-regulating the expression of cyclin D1, and promoting the mitochondria apoptosis through STAT3 pathway.     

Berberine promotes apoptosis of human breast cancer cells by regulating miRNA-146a

ATM: To observe the effect of berberine on apoptosis of MCF-7 cells and its potential mechanism. METHODS: The MCF-7 cells were divided into control group and the groups with 3 different doses of berberine. The cell viability was detected by MTT assay, while the cell apoptosis was measured by Hoechst 33258 staining and flow cytometry assay. The protein levels of p-P65, Bax and Bcl-2 were Western blot. The levels of microRNA-146a(miRNA-146a) in the MCF-7 cells were detected by RT-qPCR. The miRNA-146a siRNA was transfected to the MCF-7 cells after an evaluation of transfection efficacy, which was co-incubated with berberine to observe its effects on the mRNA levels of Bax and Bcl-2. RESULTS: Compared with control group, the cell viabilities were decreased significantly in medium and high doses of berberine treatment groups with a dose-dependent manner (P<0.01). The cell apoptosis was increased significantly in medium and high doses of berberine treatment groups dose-dependently (P<0.05). The protein levels of Bax were up-regulated, while those of Bcl-2 and p-P65 were down-regulated significantly by the treatment of berberine (P<0.05). In addition, the miRNA-146a levels were increased significantly in medium and high doses of berberine treatment groups (P<0.05) and showed a dose-dependent manner. The mRNA levels of Bax were decreased, while the mRNA levels of Bcl-2 were increased after transfection with miRNA-146a siRNA and co-incubated with berberine.CONCLUSION: Berberine promotes apoptosis of MCF-7 cells. The mechanism may be related to inhibit the activity of NF-κB by incresing the levels of miRNA-146a.     

Effect of NF-κB inhibitor pyrrolidine dithiocarbamate on proliferation and apoptosis of human multiple myeloma U266 cells

AIM:To explore the effect of pyrrolidine dithiocarbamate (PDTC),an NF-κB inhibitor,on the proliferation and apoptosis of human multiple myeloma U266 cells and its mechanisms.METHODS:The U266 cells were treated with PDTC at different concentrations (0,25,50,100 and 200 μmol/L)in vitro.The growth inhibitory rate of the U266 cells was detected by CCK-8 assay and cell counting.The cell cycle of the U266 cells was determined by flow cyto-metry,and the apoptosis was examined by flow cytometry with Annexin V-FITC/PI staining.The effect of PDTC on the expression of DNA methyltransferase 1(DNMT1) at mRNA and protein levels was measured by RT-qPCR and Western blot,respectively.The effects of PDTC on the protein levels of NF-κB (P65),DNMT1,Bcl-2,cyclin D1,cleaved caspase-3 and cleaved caspase-8 were determined by Western blot.RESULTS:The protein level of NF-κB (P65) was decreased after treatment with PDTC for 48 h or 72 h.PDTC inhibited the proliferation of U266 cells in both dose-and time-dependent manners.After treatment with PDTC for 48 h,the percentage of U266 cells in G₂ phase increased compared with control group (P<0.05).PDTC induced the apoptosis of U266 cells in a dose-dependent manner.The expression of DNMT1 at mRNA and protein levels decreased (P<0.05).The results of Western blot showed that the expression of Bcl-2 in PDTC groups decreased,while the protein levels of cyclin D1,cleaved caspase-3 and cleaved caspase-8 were higher than those in control group (P<0.05).CONCLUSION:The NF-κB inhibitor PDTC inhibits the proliferation of U266 cells by inducing cell apoptosis.It may be related to the down-regulated expression of DNMT1,cell cycle arrest and activation of the apoptotic pathways.     

Recombinant human defensin α1 induces proliferation of rat glomerular mesangial cells in vitro

AIM: To study the effects and mechanism of recombinant human defensin α₁ on cell proliferation in cultured rat glomerular mesangial cells.METHODS: The influences of defensin α₁ at various concentrations on rat 1097 mesangial cell line cultured in vitro were evaluated with MTT assay.The different concentrations of U0126,signal-regulated protein kinase (MEK) inhibitor,were added into the culture mediums of mesangial cells to do blocking test.Incubated with a final concentration of 3 mg/L defensin α₁,the phosphorylation of extracellular signal regulated kinase (ERK)1/2 and type IV collagen of mesangial cells in different times were evaluated by Western blotting.RESULTS: Defensin α₁ at 3-20 mg/L enhanced proliferation of rat glomerular mesangial cells.The incubation times for the maximum effect on proliferation was 12 h (P<0.01),whereas defensin α₁ concentration >20 mg/L decreased cell proliferation.The cell proliferation induced by defensin α₁ was inhibited by U0126.Stimulation of the cells with defensin α₁ at concentration of 3 mg/L for 5 minutes induced a maximum effect on a ratio of phosphorylation of ERK1/2 to total ERK.After 12 h incubation with defensin α₁,an increase in type IV collagen was observed by Western blotting and continued to increase at 24 h and 48 h (P<0.01).CONCLUSION: Defensin α₁ enhances rat glomerular mesangial cell proliferation and induces type IV collagen production by MAPK signaling pathway.     

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.     

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 L-carnitine on apoptosis and oxidant damage of cardiomyocytes induced by hypoxia/reoxygenation in vitro

AIM: To examine the effects of L-carnitine on apoptosis and oxidant injury in cultured neonatal rat cardiomyocytes induced by hypoxia/reoxygenation and its possible mechanism. METHODS: The cultured cardiomyocytes were divided into three groups， control， A/R group （anoxia for 120 min, reoxygenation for 240 min） and L-carnitine treatment group, in which cells were exposed to 20 mg/L, 50 mg/L, 100 mg/L, 200 mg/L L-carnitine respectively at 2 h before anoxia. The superoxide dismutase (SOD), succinate dehydrogenase (SDH) activities and malondialdehyde (MDA) content were examined, and the apoptosis was determined by flow of cytometry (FCM). In addition, the ultrastructure was observed by transmission electron microscopy. RESULTS: In A/R group, SOD and SDH activities were lower, the apoptosis rate and MDA content were higher than those in control group (P<0.05). In L-carnitine treatment group, SOD and SDH activities were higher, the apoptosis rate and MDA content were lower than those in A/R group, a L-carnitine concentration-dependent effect was found. Moreover, impairment of myocardial ultrastructure was more severe in A/R group than L-carnitine treatment group. CONCLUSION: L-carnitine can protect cardiomyocytes against hypoxia/reoxygenation-induced injury in a dose-dependent manner.