Effects of panax notoginseng saponins on pneumocyte apoptosis and Fas/FasL expression in rabbits with lung ischemia/reperfusion injury

AIM: AIM: To explore the relationship between apoptosis in the lung tissues and lung ischemia/reperfusion injury, and observe effects of panax notoginseng saponins (PNS) on apoptosis in lung ischemia/reperfusion injury. METHODS: Single lung in situ ischemia/reperfusion animal model was used. Eighty four Japanese white rabbits were randomly divided into control group (control), ischemia/reperfusion 1 h group (IR1h), IR3h, IR5h, Panax Notoginseng Saponins 1 h group (PNS1h), PNS3h and PNS5h. TUNEL, immunocytochemistry and in situ hybridization techniques were used to observe apoptosis and Fas/FasL expression in various phases of lung ischemia/reperfusion. RESULTS: Cell apoptosis in lung tissues were significantly high, Fas/FasL mRNA and its protein were up-regulated in lung tissues of lung ischemia/reperfusion injury compared with control (all of P<0.01). The PNS suppressed apoptosis as well as expression of Fas/FasL mRNA and its protein (P<0.05 or P<0.01, respectively). There was a significant correlation between expression of Fas/FasL protein, Fas/FasL mRNA and cell apoptosis (r=0.540，0.658，0.668，0.686；all P<0.01). CONCLUSIONS: Activation of Fas/FasL system and its initiating cell apoptosis of lung tissues may contribute to the pathogenesis of lung ischemia/reperfusion injury. The protective effects of PNS include suppressing the activation of Fas/FasL system and blocking apoptosis in lung tissues in lung ischemia/reperfusion injury.     

Effects of Tertram ethylpyrazine on expression of Fas/FasL mRNA in lung tissue with ischemia-reperfusion injury in rabbits

AIM: To study the expression of Fas/FasL mRNA in lung tissue with ischemia-reperfusion lung injury in rabbits and the relationship with the apoptosis,and to observe the effects of Tertram ethylpyrazine on them.METHODS: The pulmonary ischemia-reperfusion models in rabbits with occlusion of left pulmonary hilum for 1 h and then reperfusion 1,3,5 h respectively were used in this experiment.In TMP group,Tertram ethylpyrazine was intravenously dropped at dose of 60 mg/kg at 1 h before ischemia.The TUNEL technique was used to explore apoptotsis of pulmonary cells.In situ hybridization was performed on the rabbit lung tissue to assay the expression of Fas/FasL mRNA.RESULTS: Apoptosis of pulmonary cells was found in both IR group and TMP group.Compared with group IR,the apoptosis index (AI) was decreased obviously in group TMP (P<0.01).There was a significant positive correlation between the expression of Fas/FasL mRNA and the apoptosis of pulmonary cells (r₁=0.900,r₂=0.869,P<0.01).CONCLUSION: The activation of Fas/Fas-L system may contribute significantly to induce pneumocyte apoptosis in pulmonary ischemic injury.Tertram ethylpyrazine inhibits the activation of Fas/FasL system to decrease apoptosis in pulmonary tissue,which may protect the pulmonary tissues in ischemia injury.     

Effect of Fas/FasL on late reperfusion of acute myocardial infarction and the potential oxidative stress mechanism in canine

AIM: To discuss the effect of Fas/FasL on the late reperfusion of acute myocardial infarction (AMI) and the potential oxidative stress mechanism. METHODS:Eighteen anesthetized dogs were randomly divided into three groups: late reperfusion group (n=6): ligated the coronary for 6 h, followed by reperfusion for 6 h; permanent ischemia group (n=6): after pericardium were opened for 6 h, ligated the coronary for 6 h, and did not reperfuse; control group (n=6): did not ligate the coronary but operation last for 12 h. Infarction brim myocardial Fas/FasL was detected by immunohistochemistry. Apoptosis index (AI) was detected by TUNEL. SOD and GR activity and MDA content were detected by colorimetry. RESULTS:The expression of Fas/FasL and apoptosis index were significantly higher in permanent ischemia group and late reperfusion group than those in control group (P<0.05, P<0.01), and the difference between them was also significant (P<0.05). SOD and GR activities were lower in permanent ischemia group and late reperfusion group than those in control group (P<0.05, P<0.01). The MDA contents in permanent ischemia group and late reperfusion group were higher than that in control group (P<0.05). CONCLUSION:The late reperfusion of AMI promotes the expression of Fas/FasL and myocardial apoptosis, and it may be due to oxidative stress mechanism.     

Quercetin regulates Fas expression and induces apoptosis in hepatocellular carcinoma HepG2 cells

AIM: To investigate the role of 1, 4, 5- trisphosphate inositol (IP3) and Fas gene expression in apoptosis of HepG2 cells induced by quercetin. METHODS: HepG2 cells were treated with quercetin at different concentrations (including 20, 40, 60, 80 μmol/ L) for 72 h and treated with 60 μmol/ L quercetin for 6 h, 12 h, 24 h, 48 h and 72 h. IP3, Fas mRNA, Fas protein and apoptosis rate were assayed by IP3 - ［3H］ Birtrak assay, RT-PCR, Western blotting and flow cytometry, respectively. RESULTS: When HepG2 cells were incubated with different concentrations of quercetin for 72 h, the IP3 content was lower than those in control. Fas mRNA expression, Fas protein expression and the apoptosis rate were higher than those in control. When HepG2 cells were incubated with quercetin for 6 h, 12 h, 24 h, 48 h, 72 h, the IP3 contents were lower than those in control incubated with 60 μmol/L quercetin for 12 h. Fas mRNA expression was higher than that in control incubated with 60 μmol/L quercetin for 12 h . Fas protein expression was higher than that in control. The apoptosis rate was significantly higher than that in control incubated with 60 μmol/L quercetin for 24 h (P<0.01). CONCLUSION: Quercetin induces apoptosis of HepG2 cells by reducing IP3 production and upregulating Fas gene expression.     

Effects of leptin on hypoxia-induced apoptosis in cultured alveolar typeⅡ cells of fetal rat and its mechanism

AIM: To investigate the effects of leptin (LEP) on the alveolar type Ⅱ cells(AECⅡ) apoptosis induced by Na₂S₂O₄ and explore the molecular mechanisms. METHODS: Primary AECⅡ culture was prepared according to a specific immunosorption procedure with slight modification and the cells were identified by transmission electron microscope and immunocytochemistry. AECⅡ damage was induced by 5 mmol/L Na₂S₂O₄. LEP group cells were treated with LEP at concentrations from 100 μg/L to 1 600 μg/L. The cell survival rate was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays. Cell cycle and apoptosis were analyzed by flow cytometry and the level of caspase-3 was measured by Western blotting. RESULTS: Highly purified AECⅡ, obtained by the method of modified immunosorption, were identified with the positive expression of SP-A and intracellular lamellarbodies were found under electron micrography. The cells, exposed to 5 mmol/L Na₂S₂O₄, showed characteristic changes of apoptosis and activation of caspase 3. These damages were relieved by the treatment of LEP (100-1 600 μg/L), with survival increasing, apoptosis peak decreasing, cell morphology restoring and caspase 3 activation inhibiting.CONCLUSION: Leptin prevents AECⅡ from apoptosis induced by Na₂S₂O₄ or hypoxia. The potential mechanism of its action may be related to promoting cell cycle from G₁ phase to S phase and inhibiting the activating of caspase 3.     

Effects of Le Er Mai on apoptosis of hippocampus neuronal cells in anaphase of cerebral ischemic reperfusion injury in rats

AIM: To investigate the effects and mechanism of Le Er Mai (LEM) on the apoptosis of hippocampus neuronal cells in the anaphase of cerebral ischemic reperfusion injury in rats.METHODS: A rat model of middle cerebral artery occlusion reperfusion (MCAO) was produced with the intraluminal filament. During reperfusion for 30 d after 2 h of ischemia, the TUNEL staining methods were used to detect apoptosis of hippocampus neuronal cells, and immunohistochemical technique were employed to examine the protein expression of Fas, Bax, caspase-3 and caspase-9 in the hippocampial. The gene expressions of fas, bax, caspase-3 and caspase-9 in hippocampial were examined by RT-PCR. RESULTS: After 2 h ischemia and 30 d reperfusion, compared with sham-operated group, TUNEL-positive staining cells and expression levels of Fas, Bax as well as caspase-3 and caspase-9 obviously increased, and the mRNA expressions of fas, bax, caspase-3 and caspase-9 in hippocampial markedly up-regulated in model group. Compared with model group, LEM at dose of 2.00 g/kg or 0.87 g/kg, and flunarizinum significantly reduced apoptosis and decreased the protein expressions of Fas, Bax, caspase-3 and caspase-9 in hippocampial, and down-regulated the mRNA expressions of fas, bax, caspase-3 and caspase-9 (P<0.05), those action of LEM in 0.87 g/kg dosage group was lower than those in 2.00 g/kg dosage group.CONCLUSION: LEM obviously lower the injury of hippocampial in the anaphase of cerebral ischemia reperfusion through inhibiting the apoptosis of hippocampus neuronal cells. The mechanism of LEM may be related to regulate the expression of signal transduction pathway correlated gene of apoptosis in neuronal cells.     

Effect of IL-12 on the expression of Fas/FasL and TNFα

AIM:To study the effect of IL-12 on T lymphocytes apoptosis, the expression of Fas/FasL and TNFR/TNFα. METHODS:Terminal dUTP nick end labeling(TUNEL) and Annexin V assay were used. Anti-TNFR were labeled with FITC, anti-CD95 was labeled with PE and Anti-FasL with biotin. Three kinds of T cells (HTB176,TIB152 and human normal T cells) were analysed through flow cytometry. RESULTS:At 1st hour after being treated with IL-12, the expression of FasL protein and FasL mRNA in HTB176 and TIB152 began to increase and reached peak value in 24 hours. In the normal T cells, FasL just began to increase in 1 hour and maintained stability in 6, 12 and 24 hours through the later experiment period. All three kinds of T cells displayed no change in the expression of CD95 and TNFR/TNFα under the stimulation of IL-12. CONCLUSION:Expression of such apoptosis regulating factors were different in the apoptosis of T cells induced by IL-12.     

Effect of preconditioning with pioglitazone on ischemia reperfusion/hypoxia reoxygenation-induced mitochondrial structure and membrane potential in rats

AIM: To observe the effect of preconditioning with pioglitazone on ischemia reperfusion/hypoxia reoxygenation-induced mitochondrial ultramicro-structure and membrane potential in rats. METHODS: Sprague-Dawley rats were randomly divided into four groups: sham-operated (SO) group, ischemia reperfusion (IR) group, pioglitazone preconditioning group (Pio-P) and 5-HD+pioglitazone (5-HD+Pio) group. Apart from the SO group, IR, Pio-P and 5-HD+Pio groups were subjected to 30 min ischemia and 4 h reperfusion. The heart was quickly removed for observing the structure of mitochondria and measurement of the apoptosis index (AI) by TUNEL. Primary cultured cardiomyocytes of Sprague-Dawley rats were divided into control, hypoxic reoxygenation (HR) and different concentrations of Pio-P group. JC-1 staining flowcytometry was adopted to examine mitochondrial membrane potential (ΔΨm). RESULTS: The injury of mitochondrial structure in IR group was severer than that in Pio-P group, while the difference between 5-HD+Pio group and IR group was not evident. Flameng score in Pio-P group(1.62±0.60) was significantly lower than that in IR group (2.75±1.09), P<0.01. AI in Pio-P group (28.19%±4.93%) was lower than that in IR group (55.44%±6.63%),P<0.05. The rates of low ΔΨm cells in (5 μmol/L,10 μmol/L and 15 μmol/L) Pio-P group were (45.89±3.63)%, (17.13±1.37)% and (18.43±2.44)%, significantly lower than that in HR group (56.52%±2.87%),P<0.05, while the difference between 10 μmol/L group and 15 μmol/L group was not significant (P>0.05). CONCLUSION: Pioglitazone protects the heart from ischemia reperfusion/ hypoxia reoxygenation injury evidenced by improving mitochondrial ultrastructure and lessening the loss of mitochondrial membrane potential, and decreasing apoptosis. The cardioprotective effects can be inhibited by the blocker of mitochondrial ATP-sensitive potassium channels.     

Relation of Fas/FasL-mediated caspase-3 activation with acinar cell apoptosis in rats with acute pancreatitis

AIM: To observe the expression and interrelationship of apoptosis controlling genes and proteins Fas, FasL, caspase-3 in rats with acute pancreatitis (AP). METHODS: Forty male Sprague Dawley rats were randomly divided into four groups, 10 rats each group. Acute pancreatitis with different inflammatory degree was induced by retroinjecting 2.0%, 3.5% or 5.0% sodium taurocholate at dose of 1 mL/kg body weight into the pancreaticobiliary duct. All the rats were sacrificed 6 h after operation. The pathologic changes of pancreas were observed under optical microscope. The protein and mRNA expressions of Fas, FasL, caspase-3 in pancreatic tissue were measured by Western blotting and RT-PCR. The apoptosis of acinar cells was measured by the methods of in situ end labeling. RESULTS: In normal pancreatic tissue, there appeared the protein and mRNA expression of Fas, FasL, caspase-3. In acute pancreatitis with different inflammatory degree, with the degree of inflammation worsen, the apoptosis cells tapered, the expression of above protein and mRNA also descended gradually. Furthermore, the variation tendency of caspase-3 among the four groups was in coincidence with Fas or FasL. CONCLUSION: Fas/FasL -mediated apoptotic pathway participates in the regulation of acinar cell apoptosis in acute pancreatitis.     

Effect of puerarin on expression of Fas/FasL mRNA in lung tissue with pulmonary injury induced by ischemia-reperfusion in rabbits

AIM：To investigate the effect of puerarin (Pur) on expression of Fas/FasL mRNA in lung tissue during pulmonary ischemia and reperfusion injury (PIRI) in rabbits.METHODS：Single lung ischemia and reperfusion animal model was used.The rabbits were randomly divided into three groups,sham operated group (sham,n=10),PIR group (I-R,n=30) and PIR+ Pur group (Pur,n=30).Changes of several parameters included apoptotic index (AI),wet to dry ratio of lung tissue weight (W/D) and index of quantitative assessment of histologic lung injury (IQA) were measured at 60,180 and 300 minutes after reperfusion in lung tissue.Meanwhile,the location and expression of Fas/FasL mRNA were observed.Lung tissue was prepared for light microscopic and electron microscopic observation at 60,180,300 minutes after reperfusion.RESULTS：As compared with group I-R,Fas/FasL mRNA slightly expressed in intima and extima of small pulmonary artery,alveoli,and bronchiole epithelia in group Pur.The values of AI,W/D and IQA showed significantly lower than that in group I-R at 60,180,300 minutes after reperfusion in lung tissue (P<0.01 and P<0.05).Meanwhile,abnormal changes of the lung tissue in morphologically were lessen markedly in group Pur.CONCLUSION：Puerarin produces a notable protective effects on PIRI in rabbits by inhibiting Fas/FasL mRNA expression and decreasing apoptosis.     

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.     

Dual-direction effect of crenulatin on apoptosis of cerebral microvascular endothelial cells and it's mechanism

) ［ABSTRACT］AIM: To study the effect and the mechanism of crenulatin, an effective constituent of Chinese traditional medicine, on apoptosis of cerebral microvascular endothelial cells. METHODS: The following terminal concentrations of crenulatin were used in the study: 25 mg/L and 100 mg/L. Apoptosis of mouse cerebral microvascular endothelial cells (bEnd.3 cell line) was evaluated by flow cytometer, immunocytochemical assay (Fas, Bcl-2) and Western blotting (caspase-3) after culture for 24 h. RESULTS: Compared with control group, apoptosis of bEnd.3 cells in 25 mg/L group was significantly inhibited (P<0.05), but apoptosis in the 100 mg/L group was significantly increased (P<0.05). In apoptosis inhibited group, the Fas immunocytochemical staining was weaker, the positive cells were significantly decreased (P<0.05) and caspase-3 expression was decreased compared with control group; however, the Bcl-2 staining was stronger and the positive cells were significantly increased (P<0.05). On the other hand, in apoptosis increased group (100 mg/L group), the changes were just opposite. CONCLUSIONS: The effect of crenulatin on apoptosis of mouse cerebral microvascular endothelial cells possesses a dual-direction change, inhibitive effect in 25 mg/L and stimulative effect in 100 mg/L group, respectively. The mechanism is related to the alterations of Fas/Bcl-2 expression and caspase-3 activity.     

Effect of naoluo xintong on expression of Fas,Fas L in hippocampus CA1 area and Fas mRNA in the cortex of frontal or parietal lobe after local cerebral ischemia/reperfusion in MCAO rats

AIM: To investigate the effets of naoluo xintong on the expression of Fas, FasL protein in hippocampus CA1 area and Fas mRNA in the cortex of frontal or parietal lobe after local cerebral ischemia/reperfusion in MCAO rats. METHODS: The local cerebral ischemia /reperfusion model was established by intraluminal thread occlusion of the middle cerebral arteries (MCAO), the middle cerebral arteries of rats were occluded for 2 hours and reperfused for 1, 3 and 7 days. The animals were divided into pseudo surgery group(sham group), model group, Yiqi group, Huoxue group and naoluo xintong group. Using the techniques of immuno-histochemical staining and in situ hybridization, the expression of Fas and FasL was observed in hippocampus CA1 area, the expression of Fas mRNA was also observed in the cortex of frontal and parietal lobe. RESULTS: A value of Fas and FasL protein expression or A value and positive unit of Fas mRNA expression in control group were higher than those in sham in hippocampus CA1 area, the cortex of frontal or parietal lobe after local cerebral ischemia/reperfusion in MCAO rats (P<0.01). A value and/or positive unit of their expression in naoluo xintong group were lower than those in control group (P<0.05 or P<0.01). A value and/or positive unit of their expression in Yiqi and Huoxue groups were higher than those in naoluo xintong group for 3 and/or 7 days (P<0.05 or P<0.01). CONCLUSION: naoluo xintong could resist neuron apoptosis, alleviate pathologic injury after local cerebral ischemia/reperfusion in MCAO rats by inhibiting the expression of Fas, FasL protein and Fas mRNA.     

Expression of IGFBP2 and IGFBP4 in hepatocytes with experimental injury

AIM:To explore the mechanism of IGFBP2 and IGFBP4 in hepatocytes with injury induced by TNF-α and TGF-β1. METHODS:Human hepatocyte line (HL-7702) was cultured in vitro and treated with TNF-α or TGF-β1 for 24 h. The expression of IGFBP2 and IGFBP4 was detected by immunochemistry staining. The inhibition ratio of hepatocytes was detected by MTT assay. HL-7702 was treated with TNF-α at concentration of 20 μg/L for 48 h, then the apoptosis of hepatocytes was detected by both Annexin-V /PI and TUNEL assay. RESULTS:The expression of IGFBP2 and IGFBP4 in TNF-α or TGF-β1 treated groups was significantly higher than that in control group (P<0.05). The positive staining of IGFBP2 and IGFBP4 in TNF-α (20 μg/L) treated groups or TGF-β1 (4 μg/L) treated groups was the strongest among all groups. A positive correlation was found between IGFBP2 or IGFBP4 and inhibitory ratio of hepatocytes (P<0.05 or P<0.01). Compared with normal control group, the percentage of apoptosis markedly increased in TNF-α (20 μg/L) treated group (P<0.01). CONCLUSION:IGFBP2 and IGFBP4 involved in the injury process in hepatocytes, indicating an important role in injury of hepatocytes induced by TNF-α or TGF-β1.     

Modulation of bacterial redox protein azurin induces human osteosarcoma cell apoptosis by Fas antigen and caspase-8

AIM: To determine the role of Fas antigen and caspase-8 in modulating apoptosis of osteosarcoma cells induced by bacterial redox protein azurin. METHODS: The human osteosarcoma cell line U2OS was treated with bacterial redox protein azurin (150 mg/L) for 6, 12, 24 and 48 h, respectively. Cell immunohistochemistry and quantitative image pattern analysis were applied for detecting the expression of Fas antigen. Caspase-8 activity was detected using caspase-8 fluorescent assay kit. The apoptotic rate was measured by FCM. RESULTS: Compared with the control group, the expression of Fas antigen and activity of caspase-8 significantly increased in U2OS cells treated with 150 mg/L azurin (P<0.01). There was positive correlation between the expression of Fas antigen and activity of caspase-8 (r=0.873, P<0.01). The increased Fas antigen expression had the function to transfer apoptotic signals and the anti-Fas antibody promoted azurin induced apoptosis through increasing Fas antigen expression. IETD-FMK blocked the activation of procaspase-8 and reduced apoptosis of U2OS cells in the presence of azurin or anti-Fas antibody. The apoptotic rates in azurin group and anti-Fas antibody group were significantly different from the inhibitor group (P<0.01). CONCLUSION: The Fas-dependent signalling is the important pathway of azurin inducing apoptosis in U2OS cells. The up-regulation of csepase-8 may play an important role.     

Expression of apoptosis signal protein induced by Fas in labial salivary gland of patients with primary Sjgren’s syndrome

AIM:To study the expression of apoptosis signal proteins induced by Fas in labial salivary gland of patients with primary Sjgren’s syndrome (pSS), and investigate the possible pathway of the cell signaling transduction in pSS. METHODS:Biopsies of minor submucosal labial salivary gland were obtained from 32 patients with pSS and 8 normal controls. Fas, FasL and FADD were detected by Western blotting. Caspase-3 was measured by immunohistochemistry and CAD mRNA expression was determined by RT-PCR. RESULTS:The expressions of Fas, FasL, FADD, caspase-3 and CAD mRNA in labial salivary glands of patients with pSS were significantly higher than those in control group (P＜0.05). CONCLUSION:The apoptosis signalling transduction in labial salivary glands of patients with pSS may be that the product of FasL combined with Fas activates caspase-8 through FADD, then ICE family is cascaded, and finally CAD is splited by caspase-3 from ICAD, which catalyzes the degradation of DNA.      

Effect of low molecular weight heparin on hypoxia-induced apoptosis in primary cultured neonate rat cerebral cortical neurons

AIM:To investigate the mechanism by which low molecular weight heparin (LMWH) inhibits apoptosis of primary cultured cerebral cortical neurons caused by hypoxia. METHODS:The anti-apoptosis effect of LMWH on primary cultured neurons was observed by methods of the primary culture of cerebral neurons of postnatal rats in free-serum with neurobasal medium supplied with 2% B27 supplement, hypoxic culture of neurons, Hoechst 33342 staining and immunocytochemistry. RESULTS:At concentrations of 250-500 U/L, LMWH reduced apoptosis rate of cerebral cortical neurons induced by hypoxia (P<0.05) and apoptosis rate was lower in LMWH groups than that in BDNF (50 μg/L) group (P<0.05). LMWH (500 U/L) increased Bcl-2 protein expression (P<0.05) and decreased Bax protein expression (P<0.01) in the cerebral cortical neurons induced by hypoxia, the ratio of Bcl-2/Bax was improved (P<0.01). The ratio of Bcl-2/Bax in LMWH (500 U/L) group was higher than that in normal control group, BDNF group and apoptosis positive group (P<0.05). CONCLUSION:LMWH at concentrations of 250-500 U/L is able to prevent cerebral cortical neurons from apoptosis in primary culture under hypoxia. The effect of anti-apoptosis may be related to up-regulation of Bcl-2 protein expression, down-regulation of Bax-2 protein expression, and increase in the ratio of Bcl-2/Bax.     

Proapoptotic mechanism and changes of endogenous TGF-β1 in NB4 cells induced by exogenous TGF-β1

AIM: To study the effects of transforming growth factor-β1 (TGF-β1) on cell apoptosis, cell cycle, production of endogenous TGF-β1, expressions of P27Kip1, cyclin E and bcl-2 mRNA levels in NB4 cells. METHODS: Apoptotic morphological changes were observed by Wright-Giemsa staining. Cell cycle and apoptosis were detected with flow cytometry. Semiquantitative RT-PCR was used to examine the mRNA levels of endogenous TGF-β1, P27Kip1, cyclin E and bcl-2. RESULTS: TGF-β1 significantly restrained the growth and promoted the apoptosis of NB4 cells. The blockage of NB4 cells treated by TGF-β1 at concentration of 5 μg/L was in G1 phase. Endogenous TGF-β1 mRNA expression in NB4 cells was up-regulated when the concentration of exogenous TGF-β1 was <5 μg/L. Meanwhile, the expression of endogenous TGF-β1 mRNA was down-regulated when the concentration of exogenous TGF-β1 was 10 μg/L. After treated with TGF-β1 at concentration of 5 μg/L, P27Kip1 mRNA expression in NB4 cells was up-regulated, cyclin E and bcl-2 were reduced. CONCLUSION: TGF-β1 is able to induce apoptosis and cell cycle distribution abnormally in NB4 cells by (1) Up-regulation of endogenous TGF-β1, so that NB4 cells was induced into apoptosis through consequently high expression of P27Kip1. (2) TGF-β1 may lead to cell cycle arrest by inhibiting the expression of cyclin E directly, or by inhibiting the activity of cyclin E through the increased expression of P27Kip1. (3) Down-regulation of bcl-2 induces apoptosis of NB4 cells.