1.Arsenic trioxide inhibits KM3 cell growth and induces apoptosis and potentiated by bortezomib
Qing WANG ; Juan LI ; Jingli GU ; Junru LIU ; Lijin ZENG
Chinese Journal of Pathophysiology 2010;26(2):297-301
AIM: To observe if there is a synergistical effect on induction of apoptosis when arsenic trioxide alone or combination with bortezomib in KM3 cells. METHODS: KM3 cells were treated with arsenic trioxide alone or combined with bortezomib, the numbers of viable cells were determined by trypan blue exclusion. Cell growth inhibition was examined by MTT method. The cells were simultaneously stained with annexin V-FITC and PI and apoptosis was determined by bivariate flow cytometry using a FACScan. Reverse trascriptional-PCR (RT-PCR) method was used to examine the change of p65 mRNA and Western blotting to measure the expression of protein p65, p-p65, caspase-3, -8, -9, and poly ADP-ribose polymerase (PARP). RESULTS: Arsenic trioxide inhibited the cell growth and induced apoptosis. The mechanism was responsible for the activation of caspase-mediated induction of apoptosis. A synergistic effect of combination with bortezomib on apoptosis was observed. CONCLUSION: Arsenic trioxide inhibits KM3 cell growth and induces apoptosis with a synergistical effect when cotreated with bortezomib.
2.Protein expression of TIMP3 and RUNX3 in bone marrow mononuclear cells from acute leukemia patients
Ruifang FAN ; Zhigang FANG ; Xiangfu LIU ; Yongjiang ZHENG ; Binbin LIU ; Dongjun LIN
Chinese Journal of Pathophysiology 2010;26(2):293-296
AIM: To detect the protein expression of TIMP3 and RUNX3 in bone marrow mononuclear cells (BMMCs) from acute leukemia (AL) patients and to investigate the relationship between the methylation status of genes and their expressional levels. METHODS: Protein expression of TIMP3 and RUNX3 in 50 samples of BMMCs and 10 samples of peripheral blood mononuclear cells (PBMCs) from healthy volunteers was detected by Western blotting. The prognostic factors related to AL and data from methylation specific polymerase chain reaction were also analyzed. RESULTS: The expression level of RUNX3 with methylation was less than that without methylation in BMMCs from AL patients. The complete remission (CR) rate was related to RUNX3 expression and blasts in bone marrow (BM). BMMCs from patients with silencing of RUNX3 and higher blasts in BM had a lower CR rate. CONCLUSION: Absence of RUNX3 protein expression resulting from methylation of RUNX3 promoter probably plays a role in the pathogenesis of AL and is of value in prognosis. No relationship between methylation of TIMP3 promoter and the pathogenesis of AL is observed.
3.Identification of proteins related to proliferation and regulated by Epstein Barr virus encoded latent membrane protein 1 in nasopharyngeal epithelail cells
Zhiwei ZHANG ; Qiong ZHANG ; Jieqiong LIU ; Yanhui YU ; Zhimin HE
Chinese Journal of Pathophysiology 2010;26(2):287-292
AIM: To investigate the molecular mechanism of Epstein-Barr virus encoded latent membrane protein 1 regulated cellular proliferation in nasopharyngeal epithelial cells. METHODS: The nasopharyngeal epithelial cells NP69 were infected with RV-pLNSX (the empty vector) and RV-LMP1 retroviruses, respectively. Therefore, the NP69-pLNSX and NP69-LMP1 cell lines were established. Sequentially, cellular proliferation of NP69-pLNSX and NP69-LMP1 cells was compared to draw the cellular growth curve. The experiments of plate clone formation and forming of soft agar colony were conducted. Meanwhile, the differential expression of proteins were identified between NP69-pLNSX and NP69-LMP1 cell lines by proteomic methods, and the expression levels of partial identified proteins were verified. RESULTS: (1) LMP1 was able to accelerate cellular proliferation of nasopharyngeal epithelial cell NP69 (n=3, P<0.05). (2) Twenty two proteins (9 up-and 13 down-regulated) of LMP1 mediated regulation were identified from infected NP69 cell lines, and the differential expression of partial identified proteins was confirmed by Western blotting and fluorescent real-time quantitative RT-PCR. CONCLUSION: LMP1 probably mediates the regulation of vimentin protein and keratin 19 protein expression to promote cellular proliferation in NP69 cells.
4.iASPP on apoptosis in breast cancer cells which expressed wild type p53
Changsong WANG ; Hong LI ; Chunfang GAO ; Yanping CHEN
Chinese Journal of Pathophysiology 2010;26(2):282-286
AIM: To investigate the RNAi effect of the inhibitory member of the ASPP family (iASPP) on the apoptosis of human breast cancer cell MCF-7 which expressed the wild type p53 gene. METHODS: The recombinant plasmid pAd-iASPP-RNAi was transfected into MCF-7 cells. The expression of iASPP mRNA and protein was analyzed by RT-PCR and Western blotting, respectively. The cell apoptosis was detected by FCM, and then the MCF-7 cells were transplanted into nude mice to set up transplantation model. The expression of iASPP RNA and protein in transplanted neoplasm were determined by RT-PCR and Western blotting, the apoptosis index was detected by FCM at the same time. RESULTS: The results showed that the expression of iASPP descended in MCF-7 cells (mRNA 95.4% and protein 96.8%, respectively, P<0.01) and the apoptosis rate and necrosis rate of MCF-7 cells increased (P<0.01) after transfection. As treated with pAd-iASPP-RNAi, the expression of iASPP in transplantation tumor cells descended 87.4% (mRNA) and 89.2% (protein), respectively (P<0.01), and the apoptosis rate and necrosis rate increased accordingly (P<0.01, P<0.05). CONCLUSION: The inhibition of iASPP may resume the ability of p53 to induce apoptosis in breast cancer cells which is able to express wild type p53.
5.JNK signal transduction pathway in anti-apoptotic effect of NGF on PC12 cells treated with 6-hydroxydopamine
Chinese Journal of Pathophysiology 2010;26(2):277-281
AIM: To investigate the anti-apoptotic effect of nerve growth factor (NGF) on PC12 cells and to observe the mechanism of signal transduction of JNK pathway. METHODS: PC12 cells were treated with 6-hydroxydopamine (6-OHDA) to induce cell apoptosis. NGF and SP600125, the c-Jun N-terminal kinase inhibitors, were added respectively in order to study the relationship between the activity of c-Jun N-terminal kinase and apoptosis of PC12 cells. The cells were divided into control group, 6- OHDA group, NGF group, 6-OHDA plus NGF group, NGF plus SP600125 group. The apoptotic rates of PC12 cells with different treatments were detected by flow cytometry and activities of JNK in PC12 cells were determined by Western blotting. RESULTS: 6-OHDA increased the apoptotic rate and activity of JNK1 in PC12 cells. Incubation with SP600125 or NGF for 15 min before adding 6-OHDA decreased the apoptotic rate of PC12 cells and activity of JNK1 in PC12 cells.CONCLUSION: Activity of JNK is involved in the pro-apoptotic effect of 6-OHDA on PC12 cells. Anti-apoptotic effect of NGF on PC12 cells affected by 6-OHDA is related to the decrease in the activity of JNK1.
6.Effects of sport fatigue and poverty of movement on neuroendocrine system in Wistar rats
Guoqiang YUAN ; Shizhen WU ; Haitao YANG ; Huailin GAO ; Junqing LIANG ; Zhenhua JIA ; Yiling WU
Chinese Journal of Pathophysiology 2010;26(2):272-276
AIM: To observe the different changes of neuroendocrine systems between the state of sport fatigue and poverty of movement. METHODS: 60 male Wistar rats were randomly divided into three groups: normal control group, sport fatigue model group and poverty of movement model group (20 rats in each group). The sport fatigue model was established by the method of combining basal diet and loaded swimming during 2 weeks, whereas the method of restricted activities was used to establish the poverty of movement model with total experimental time of 10 weeks. By the end of experiment, the climbing pole time was determined. The contents of hypothalamus thyrotropin releasing hormone (TRH), and serum norepinephrine (NE) and epinephrine (E) in rats with different treatments were determined by ELISA. In addition, the changes of hypothalamus corticotropin release hormone (CRH), pituitary adrenocorticotropic hormone (ACTH) and thyroid stimulating hormone (TSH), and serum corticosterone (CORT), triiodothyronine (T_3), tetraiodothyronine (T_4) were determined by radioimmunoassay to evaluate the functions of adrenergic nerve-adrenomedullin system, hypothalamo-pituitary-adrenal (HPA) axis and hypothalamo-pituitary-thyroid (HPT) axis. RESULTS: Compared to control group, the climbing pole time of the animals was obviously decreased in two model group. The adrenergic nerve-adrenomedullin system and HPA axis were inhibited in sport fatigue model rats, but HPT axis was unchanged. Interestingly, the HPA axis was hyperfunctional and HPT axis was inhibited in poverty of movement model rats. However, no change in the adrenergic nerve-adrenomedullin system was observed. CONCLUSION: Sport fatigue and poverty of movement all affect neuroendocrine system and lead to the adjustment mechanism imbalance, but the target and tendency are different.
7.Constitutive expression of HIF-1α in BMSCs reduces neuronal apoptosis and promotes neuronal proliferation after stroke in rats
Weibiao YE ; Yubin DENG ; Meihong YE ; Tuo CAI
Chinese Journal of Pathophysiology 2010;26(2):256-261
AIM: To explored the potential role of HIF-1α in reducing the neuronal apoptosis and promoting the neuronal proliferation after stroke in rats. METHODS: The bone marrow-derived mesenchymal stem cells (BMSCs) were lentivirally transduced to express the stable form of HIF-1α. Ischemic stroke was induced by permanent middle cerebral artery occlusion (MCAO) in Sprague-Dawley rats. Neurological function was evaluated by modified neurological severity score (mNSS). Cerebral infarct volume was measured by TTC staining. Immunohistochemistry and terminal deoxynucleotidyltransferase mediated dUTP nick end labeling (TUNEL) method were performed to detect neuronal proliferation and apoptosis. RESULTS: Significant improvement of neurological deficits was found in BMSCs-mHIF-1α rats as compared to the control animals at 14th d and 28th d after MCAO (P<0.05). Significant reduction of infarct volume was observed in rats in BMSCs-mHIF-1α group at 3rd day after MCAO (P<0.05). Histological evaluation showed that BMSCs-mHIF-1α treatment significantly promoted neuronal survival and proliferation in the ischemic boundary area. CONCLUSION: Constitutive expression of HIF-1α in BMSCs reduces the neuronal apoptosis and promotes the neuronal proliferation after stroke in rats.
8.Effects of hyperglycemia and cerebral ischemia on VEGF expression in different subfield of cerebral cortex in tree shrews
Chinese Journal of Pathophysiology 2010;26(2):251-255
AIM: To observe the changes of VEGF expression in different subfield of brain in tree shrews during hyperglycemia and focal cerebral ischemia, in order to explore the relationship between cerebral ischemia, hyperglycemia and VEGF. METHODS: High blood glucose in tree shrews was induced by intraperitoneal injection of streptozotoctin. Focal cortical thrombotic cerebral ischemia was induced by photochemical method in tree shrews. At 4 h, 24 h and 72 h after cerebral ischemia, the histopathological changes and hippocampal neuronal density were examined. VEGF expressions in the ischemic core, penumbra and contralateral cerebral cortex were detected by immunohistochemistry technique at different times after cerebral ischemia. RESULTS: The results of histopathological study showed that there was infarction zone in the exposured cerebral cortex at 4 h after photochemical reaction, and the damage was most severe at 24 h, subsequently accompanied with the glia multiplication and rehab reaction at 72 h. The animals in hyperglycemic ischemic group suffered from greater neurological lesion than the normoglycemic stroke animals, especially at 24 h (P<0.01) and 72 h (P<0.05) after cerebral ischemia. Immunohistochemical analyses of VEGF expression revealed that it started to increase at 4 h after brain ischemia in the penumbra, reached a peak at 24 h, and weakened at 72 h. The stimulated VEGF production was also observed in hyperglycemic only group. When hyperglycemia and brain ischemia were combined, the VEGF expression was higher than that in hyperglycemic only group (P<0.05). Compared to normoglycemic ischemic group, no additivity of the effects of hyperglycemia combined with brain ischemia was observed. CONCLUSION: (1) The model of experimental hyperglycemia and cerebral ischemia is replicated successfully by applying the method combined in vivo injection of streptozotocin in the lower primate tree shrew with thrombotic focal cerebral ischemia. (2) This study shows that hyperglycemia aggravates the focal cerebral ischemia damage. (3) Cerebral ischemia and hyperglycemia both can independently up-regulate VEGF expression, but there is no additional increase in VEGF expression when hyperglycemia combined with brain ischemia is applied.
9.Effects of metformin on pressure overload-induced cardiac hypertrophy in rats
Chengxi ZHANG ; Sinian PAN ; Rongsen MENG ; Zhaojun XIONG ; Baolin CHEN ; Chaoquan PENG ; Yugang DONG
Chinese Journal of Pathophysiology 2010;26(2):238-244
AIM: To study the effects of metformin on the pressure overload-induced cardiac hypertrophy in rats. METHODS: Transverse aortic constriction (TAC) model of rat was made through laparotomy. One week after TAC surgery, the rats were randomly divided into 5 groups (n=8 in each group) and were administered with the corresponding drugs orally every day for 8 weeks: sham group (sham surgery, administered with 2 mL distilled water);TAC group (TAC rats, administered with 2 mL distilled water);metformin(MET) group (TAC rats, administered with MET at dose of 300 mg·kg~(-1)·d~(-1));MN group [TAC rats, administered with MET at dose of 300 mg·kg~(-1)·d~(-1) plus NOS inhibitor, NG-nitro-L-arginine methyl ester (L-NAME) 50 mg·kg~(-1)·d~(-1)] and L-NAME group (TAC rats, administered with L-NAME at dose of 50 mg·kg~(-1)·d~(-1)). After treated for 8 weeks, the echocardiography, hemodynamics, the ratio of heart weight to body weight (HW/BW) and histological examination of the heart were performed. The levels of myocardial AMP-activated protein kinase subunit α (AMPKα), p-AMPKα~(Thr172), endothelial nitric oxide synthase (eNOS) and p-eNOS~(Ser1177) were detected by Western blotting. Plasma and myocardial nitric oxide (NO) were detected biochemically. RESULTS: After 8 weeks treatment, the wall thickness of left ventricle, the heart weight/body weight ratio (HW/BW), and the left ventricular myocardial perivascular fibrosis and myocardial interstitial fibrosis of the animals in TAC group were significantly increased as compared to those in sham rats. Treatment with MET for 8 weeks significantly attenuated left ventricular hypertrophy and improved cardiac function in TAC rats. These effects of MET were mostly abolished by L-NAME. Molecular biology and biochemical testing revealed that the levels of left ventricular myocardial p-AMPKα~(Thr172) and p-eNOS~(Ser1177), as well as the levels of myocardial and serum NO were significantly increased in MET group. CONCLUSION: Long-term MET treatment significantly inhibits the cardiac hypertrophy and the myocardial fibrosis and improves the cardiac functions in pressure-overload rats. The anti-hypertrophic effects of MET may be mediated via activation of AMPK-eNOS signaling pathway.
10.Effect of pressure phase plane derived τ and K on evaluation of left ventricular diastolic function in isolated rat heart during ischemia/reperfusion
Ying OU ; Mingzhi ZHENG ; Jianping JIANG ; Jiali BAO ; Yingying CHEN ; Yueliang SHEN
Chinese Journal of Pathophysiology 2010;26(2):216-221
AIM: To analyze and compare the changes of pressure phase plane(PPP) derived τ and K on isolated rat heart during ischemia/reperfusion, and to explore the value of PPP derived τ and K for evaluation of left ventricular diastolic dysfunction. METHODS: LVEDP, -d(p/dt)_(max), τ and K were measured and calculated during ischemia/reperfusion in Sprague-Dawley rat hearts. Meanwhile, the level of lactate dehydrogenase (LDH) in the coronary effluent was measured, and the ultrastructure changes in myocardium were observed under electron microscope. RESULTS: Compared with control group, τ increased and K reduced significantly in each ischemic group in a time dependent manner (P<0.05). With prolonged ischemia, τ was even higher and K was even lower (P<0.05). Compared with control group, except ischemia 15 min, LDH in other groups increased significantly at 10 min and 20 min after reperfusion (P<0.05). Compared with ischemia 30 min, LDH of ischemia 45 min and ischemia 60 min were even higher at 10 min and 20 min after reperfusion (P<0.05). With prolonged ischemia, the abnormal changes of the myocardial ultrastructure were observed. CONCLUSION: PPP derived τ and K may be promising indexes for quantitative assessment of left ventricular diastolic function on isolated) rat heart during ischemia/reperfusion, and indication of the severity of ischemia/reperfusion injury.