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Objective To evaluate inhibitory effect of Chlamydia trachomatis plasmid-encoded protein pORF5 on HeLa cell apoptosis induced by tumor necrosis factor-alpha(TNF-α). Methods The recombinant lentiviral expression vector containing pORF5 gene and helper plasmids were co-transfected into 293T cells to prepare the recombinant lentivirus. Then, the lentivirus particles were collected and concentrated, and used to infect HeLa cells. Flow cytometric screening identified stable pORF5-expressing HeLa (pORF5-HeLa) cells. Meanwhile, the empty plasmid was transfected into HeLa cells to prepare control HeLa cells. The two cell lines were both divided into two subgroups to be treated with 20μg/L TNF-αand fresh culture medium respectively for 6 hours. Then, Hoechst 33258 staining was performed to observe morphological changes of apoptotic cells, flow cytometry to detect cell apoptosis, real-time PCR to measure the mRNA expression of Caspase3, Bax and Bcl-2, and Western blot analysis to determine the protein expression of Bax and Bcl-2. Results After 6-hour treatment with TNF-α, Hoechst 33258 staining showed variable degrees of karyopyknosis and karyorrhexis, and highly-refractive blue apoptotic bodies in the pORF5-HeLa cells and control HeLa cells. The pORF5-HeLa cells and control HeLa cells both showed significantly higher apoptosis rate in the treated subgroup than in the untreated subgroup (pORF5-HeLa cells:35.5%± 4.5%vs. 9.5%± 1.5%, t=13.53, P<0.01;control HeLa cells:63.6%± 5.8%vs. 7.9%± 0.9%, t=32.36, P<0.01). Compared with treated control HeLa cells, treated pORF5-HeLa cells showed significant decreases in mRNA expression of Bax(72.8%)and Caspase 3(84.5%)(t = 35.29, 42.25, respectively, both P < 0.01), as well as in Bax protein expression(t = 17.58,P < 0.01), but significant increases in Bcl-2 mRNA and protein(6.8 times)expression(t = 87.12, 18.93, respectively, both P <0.01). Conclusion pORF5 plasmid protein can inhibit TNF-α-induced HeLa cell apoptosis likely by increasing the expression of anti-apoptotic protein bcl-2 and decreasing the expression of pro-apoptotic proteins Caspase-3 and Bax.
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OBJECTIVE: To explore the effect of 1,2-dichloroethane(1,2-DCE) induced apoptosis on the expression of related proteins in human neuroblastoma cells(SH-SY5 Y cells). METHODS: SH-SY5 Y cells were cultured in complete medium with 1,2-DCE at final concentrations of 0,10,20,30,40,50,60,70 and 80 mmol/L. After being cultured for24 hours,the apoptosis of SH-SY5 Y cells was tested by flow cytometry using annexin Ⅴ-fluorescein isothiocyanate and propidium iodide. Western blot was used to detect the protein expression of P53,B cell lymphoma/leukmia-2(BCL-2)and BCL-2 associated X protein(BAX). RESULTS: At 1,2-DCE concentrations of 0-80 mmol/L,the total apoptosis rate of SH-SY5 Y cells increased with 1,2-DCE concentrations in a dose-dependent manner(P < 0. 01). At 1,2-DCE concentrations of 30-80 mmol/L,the early apoptosis rate and total apoptosis rate of SH-SY5 Y cells increased significantly than the control group(P < 0. 05). Compared with the other groups,the protein expression of P53 was the lowest when the1,2-DCE concentration was 20 mmol/L(P < 0. 05),and the protein expression of BCL-2 and the BCL-2/BAX ratio were the lowest when the 1,2-DCE concentration was 70 mmol/L(P < 0. 05). There is no dose-response relationship in the1,2-DCE concentrations and the protein expression levels of P53,BCL-2 and BAX,and BCL-2/BAX ratio. Linear multiple regression analysis revealed that the total apoptosis rate of SH-SY5 Y cells treated with 1,2-DCE was associated with the protein expression of P53 and BCL-2,and BCL-2/BAX ratio(P < 0. 05). CONCLUSION: 1,2-DCE could inhibit the apoptosis of SH-SY5 Y cells. The mechanisms may be related to the changes of P53 and BCL-2 protein expression,and BCL-2/BAX relative amount.
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Objective Heart transplantation is an effective treatment of end-stage heart diseases and extending the time of donor heart preservation helps to make up for the shortage of donor hearts. This study was to investigate whether high-pressured mixed gas ( HPMG) of carbon monoxide and oxygen could prolong the time of donor heart preservation and its mechanisms. Methods Forty-eight C57BL/6 male mice aged 4-6 weeks were randomly divided in-to four groups of equal number:control ( the donor heart isolated but not transplanted) , immediate transplantation ( the donor heart transplanted right after isolated) , HTK-preservation ( the donor heart preserved in histidine-tryptophan-ketoglutarate solution for 24 hours after isolated, and HPMG preservation ( the donor heart preserved in an HPMG chamber with the oxygen partial pressure of 3200 hPa and carbon monoxide partial pressure of 800 hPa for 24 hours after isolated) .Another 36 recipient mice aged 6-8 weeks were randomly assigned to receive the donor heart immediately after harvested (n=12), preserved in HTK solution (n=12), or preserved in HPMG (n=12).At 2 hours after transplantation, the status of heart re-beating and cardiac function were compared among different groups of recipient mice.At 24 hours, tissues were taken from the transplanted hearts for examination of pathologic changes by HE stai-ning, detection of the apoptosis of cardiac cells by TUNEL, and determination of the expressions of microtubule-associated protein 1 light chain 3 -Ⅱ(LC3-Ⅱ) and B cell lymphoma/leukemia-2 (Bcl-2) by Western blot. Resul ts The re-beating rates of the imme-diately transplanted and HPMG-preserved hearts were significantly higher than that of the HTK-preserved ones (P<0.05).At 2 hours after transplantation, the cardiac function scores were 2.5 (2.0-2.9), 0.8 (0.5-1.0), and 4.5 (4.0-4.5) in the immediate implantation, HPMG-preservation and HTK-preservation groups respectively, with statistically significant differences between any two groups (P<0.05).The expressions of LC3-Ⅱand Bcl-2 were 2.06 ±0.29 and 0.87 ±0.18 in the HPMG-preserved heart recipients and 1.24 ±0.20 and 2.07 ±0.32 in the immediately transplanted heart recipients, both higher than 0.13 ±0.03 and 0.19 ±0.02 in the controls and 0.16 ±0.06 and 0.26 ±0.08 in the HTK-preserved heart recipients (P<0.05), the Bcl-2 higher in the HTK-pre-served heart recipients than in the controls (P<0.05), and the LC3-Ⅱ expression higher in the HPMG-preserved heart recipients than in the immediately transplanted heart recipients (P<0.05).HE staining showed that cell edema and inflammatory cell infiltration were more obvious in the HPMG-preserved heart recipients than in the controls and immediately transplanted heart recipients but less obvious than in the HTK-preserved heart recipients.The rate of cell apoptosis was dramatically increased in the HPMG-and HTK-pre-served heart recipients ([5.04 ±1.77]%and [26.72 ±5.23]%) in comparison with the controls ([1.08 ±0.56]%) (P<0.01) and immediately transplanted heart recipients ([2.13 ±1.71]%) (P<0.01) but decreased in the HPMG as compared with the HTK-preserved heart recipients (P<0.01). Conclusion High-pressured mixed gas preservation can reduce cold ischemia-reperfu-sion injury of the donor heart, which may be associated with its promotion of autophagy, provision of energy to cells, and apoptosis of cardiocytes in the donor heart.
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Granzyme B is an important effect factor of cytotoxic T lymphocytes in the immune killer function,can quickly induce cell apoptosis of the target cell.It is complicated for the reasons and the ways of the occurrence of the cell apoptosis,many genes are involved in gene regulation of cell apoptosis,and the B-cell lymphoma/leukemia-2 gene family members play a crucial role in the process of cell apoptosis.This paper mainly reviews the correlation of GrB and Bcl-2,Bid with cell apoptosis.
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Objective To explore the long-term effects of bubble baths on seizure-induced neurobehavior deficits and the expression of apoptotic/autophagic marker B cell lymphoma/leukemia-2 ( Bcl-2 ),Beclin-1,and plasticity-related gene-1 ( PRG-1 ) in newborn rats. MethodsSixty rats aged 6 days were randomly divided into 4 groups:a control group (CON),a control hydrotherapy group (HCON),a recurrent-seizure group (RS) and a recurrent-seizure hydrotherapy group (HRS),with 15 in each group.Flurothyl was used to induce 30 min of seizures daily for 6 consecutive days in the RS and HRS groups.Rats in the CON and HCON groups were placed in the same container for equal duration without exposure to flurothyl.Rats in the HCON and HRS groups were given bubble baths for 28 consecutive days after the end of the last seizure.Neurobehavioral damage was observed using open field behavior at postnatal day 26 (P26) and Morris water maze performance at postnatal day 43 to 49 (P43-P49) and a single-blind method.PRG-1,Bcl-2 and Beclin-1 protein levels in the hippocampus were detected by Western blotting at postnatal day 50 (P50). Results①The average open field test scores of the RS rats decreased significantly compared with those of the CON and HRS rats at P26.②In the Morris water maze test the average latencies of all rats decreased gradually from the 1st to 5th days (d1 to d5) after establishment of seizure model.The average escape latency was significantly longer for rats of the RS group than for CON group rats at the 4th and 5th days ( d4 and d5 ) after establishment of seizure model.The escape latency was significantly shorter for rats of the HRS group than for RS group rats at d4.③The level of Bcl-2 protein in the hippocampus was much lower in the RS group than in the HRS and control groups.In addition,the expression of PRG-1 in the RS group was significantly higher than in the CON group. ConclusionsRecurrent prolonged seizures cause long-term neurobehavior deficits,which might be associated with the down-regulated expression of Bcl-2 and up-regulated expression of PRG-1 in the hippocampus.Bubble baths can improve the neurobehavioral sequelae from seizures,perhaps through up-regulation of hippocampal Bcl-2 expression.
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Objective To study the effects of He-Ne laser irradiation on histological changes and the expression of proliferating cell nuclear antigen ( PCNA ) and B-cell lymphoma/leukemia-2 ( Bcl-2 ) in the gastric mucosa of rats with chronic atrophic gastritis (CAG) so as to elucidate the relationship of He-Ne laser irradiation with precancerous lesions and apoptosis in the gastric mucosa. Methods The rats were divided into a normal group,a model group and a laser group.A model of CAG was established by gastric perfusion with a mixture of sodium salicylate and alcohol combined with irregular fasting and forced exercise.A He-Ne laser was used to irradiate the rats at 3.36 J/cm2 for 7 min daily for 20 d.Histopathological changes including the severity of inflammation in the gastric mucosa and the morphology and structure of the parietal cells were observed with a light microscope,and the expression of PCNA and Bcl-2 was detected with immunohistochemical methods. Results The pathologic morphological changes in the gastric mucosa of the model group were atrophy of the glands of the gastric mucosa and notable inflammatory infiltration.But in the laser group inflamed cells decreased,and the morphology,structure and volume of the cells all recovered close to normal.The immunohistochemistry results showed that during the atrophy of the gastric mucosa the expression of PCNA and Bcl-2 was elevated,and it was significantly higher in the model group than in the normal group.After irradiation the expression of PCNA and Bcl-2 was significantly lower. Conclusions There was hyper-proliferation in the gastric mucosa of the CAG model rats,with high expression of apoptosis suppressor PCNA and Bcl-2 proteins.Laser irradiation can reduce the expression of PCNA and Bcl-2,enhance cell proliferation and induce apoptosis,preventing the development of cancer.Laser irradiation has a good adjuvant therapeutic effect for all the pathological changes observed.
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Objective To study the effect of electroacupuncture (EA) combined with transcranial magnetic stimulation (TMS) on the expression of the B-cell lymphoma/leukemia-2 gene (Bcl-2) and brain derived neurotrophic factor (BDNF) after cerebral infarction. Methods One hundred Sprague-Dawley rats were randomly and equally divided into a normal group, a sham-operated control group, a model group and an EA plus TMS group. A cerebral infarction model was established in the latter two groups using left middle cerebral artery occlusion (MCAO). Five-member subgroups of the EA plus TMS group were then treated at 6, 12, 24,48 and 72 hours after reperfusion. Sham EA plus TMS was given to similar sub-groups from the other groups at the same time points. The expression of Bcl-2 mRNA and BDNF mRNA were measured using a RT-PCR at the 14th day. Results Positive expression of Bcl-2 mRNA and BDNF mRNA was detected around the infarction in all groups. The average expression of both was significantly higher in the EA plus TMS group than in the model group. Bcl-2 mRNA peaked when the therapy was administered at 24 hours and BDNF mRNA at 48 hours.Conclusions The expression of Bcl-2 mRNA and BDNF mRNA is maximized when EA plus TMS is administered 24-48 hours after cerebral infarction. EA plus TMS does have protective and rehabilitative effects on rats after cerebral infarction.
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Objective To study the effect of transcranial magnetic stimulation (TMS) on the rehabilitation of rats with cerebral infarction. Methods One hundred Sprague-Dawley rats were randomly divided into a normal group, a sham-operated control group, a model group and a TMS group with 25 rats in each group. A cerebral infarction model was established in the latter two groups by left middle cerebral artery occlusion (MCAO). TMS was started at either 12 or 24 hours after reperfusion, and sham-TMS was given to the first two groups at the same time points. The expression of Bcl-2 mRNA and BDNF mRNA were measured by RT-PCR after 14 days. Results Bcl-2 mRNA and BDNF mRNA were detected in all groups. The expression of Bcl-2 mRNA in the TMS-12 h group, and that of BDNF mRNA in the TMS-24 h group were significantly higher than in the other groups. Conclusions The expression of Bcl-2 mRNA and BDNF mRNA in the brains of rats after cerebral infarction peak when TMS is administered 12 h and 24 h after reperfusion, respectively. TMS might have protective and rehabilitative effects on rats after cere-bral infarct.
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Objective To observe the effects of passive movement on the functional outcome after occlusion of the middle artery in the brain and reperfusion, and to explore the molecular mechanisms involved. Methods Cerebral infarction models were established in rats using left middle cerebral artery occlusion ( MCAO). The survivors were randomly divided into a passive movement group and a natural recovery group. There was also a sham-operated group and a normal group. Passive movement treatment (twice a day, twenty min per time) was started at different times after reperfusion. The expression of brain-derived neurotrophic factor (BDNF) and B cell lymphoma/leukemia-2 gene (Bcl-2) were determined using real-time PCRs. Results Expression of BDNF and Bcl-2 was detected a-round the infarction area in both groups. The expression of BDNF and Bcl-2 was highest in the sub-groups where passive movement was begun 24 or 48 h after the operation. Conclusions The expression of BDNF and Bcl-2 in the brain peaks when daily, moderate intensity passive movement is administered beginning 24 to 48 h after reperfusion. Passive movement might have a protective and rehabilitative effect after cerebral infarction.
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Objective To study the effect of manganese chloride on apoptosis,p53 and Bcl-2 expression in the spermatogenic cells of rats.Methods 24 healthy male SD rats were randomly divided into three groups.Two groups were treated with MnCl2?4H2O through intraperitoneal injection at 15 mg/kg and 30 mg/kg respectively,once a day and 5 times in a week for four weeks,the third group served as the control and given normal saline.The spermatogenic cell apoptosis was examined by transmission electronic microscope and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling(TUNEL)technique.Expression of p53 and Bcl-2 were measured with immunohistochemistry.Results The apoptosis index(AI)and p53-positive-cell rate in manganese exposed group were significantly higher than those in the control group(P
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Objective To explore the expression of Bcl-2 and Bax in embryonic neuroepithelial cells in Golden hamster exposured to the teratogenic dose of alcohol. Methods The experimental pregnant golden hamsters were given ethanol through intraperitoneally injected, 5.8 g/kg body weight. The Bcl-2 and Bax expressions in neuroepithelial cells of experimental and control animals were detected with immunohistochemical and image analysis at 4,8,16,24,48 and 72 h time points after treatment. Results Bcl-2 and Bax proteins distributed in nuclear membranes and cytoplasm of the neuroepithelial cells as well as mesenchymal cells. In control groups, the expressions of these two kind proteins increased as the time passed at beginning, 16 hours after, came to the summit and then descended gradually. Compared with the control, the expressions of Bcl-2 in experimental groups were down-regulated, expressions of Bax were up-regulated except at 8 h time point. Conclusion Alcohol can induce down-regulation of the expression of Bcl-2 protein and up-regulation of the expression of bax protein, which may play a an important role in regulation of apoptosis in alcohol-induced neural tube malformation.
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Objective To study the effect of alcohol on apoptosis and apoptosis-associated genes-Bcl-2 and Bax of cardiac muscle cell.Methods We established an animal experimental model by giving different concentration of alcohol to the healthy mice.Myocardial apoptosis and the expression of apoptosis-associated gene Bcl-2 and Bax were quantitatively analysed by the nuclear fast red-crystal violet staining and immunohistochemical method.Results Some nuclears of the experimental groups were stained purple and agglutinated.Counting under the high power microscope,it was found that the amount of abnormal nuclears significantly increased in alcohol-treated groups compared with the control group (P
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Objective To explore the apoptosis of alveolar epithelial cell(AEC)and the dynamic changes of Caspase-3 mRNA and Bax and Bcl-2 expression in premature rat with hyperoxia-induced chronic lung disease(CLD).Methods Sixty premature rats within 1 day after birth were randomly divided into 2 groups:hyperoxia group(n=30)and control group(n=30).Hyperoxia-induced premature rat CLD models were prepared,and situ nick end-labeling(TUNEL),reverse transcription polymerasechain reaction(RT-PCR)and immunohistochemical technique were used to determine apoptosis index(AI)of AEC and the expressions of Caspase-3 mRNA and Bax and Bcl-2 proteins in lung tissues at 1,3,7,14 and 21 d after birth.Results Compared with control group,in the hyperoxia group,on the third day after exposured to hyperoxia,the AI of AEC and the expressions of Caspase-3 mRNA and Bax began to increase,and the expression of Caspase-3 mRNA was kept at high level on 7-21 d.The expression of Bcl-2 began to decrease on 7 d,and significantly decreased on 7-21 d.AI of AEC was positively correlated with the expression of Caspase-3 mRNA and Bax,and negatively with the expression of Bcl-2.Conclusions Hyperoxia may induce the increased expression of Caspase-3 mRNA,which might result in the abnormal expression of Bax and Bcl-2 in lung tissues and their imbalance,which might be one of the underlying mechanisms of apoptosis of AEC in premature rats with CLD.