Objective To investigate the protective effects of antitumor necrosis factor-α antibody （TNF-αAb） on lung injury after cardiopulmonary bypass （CPB） and their mechanisms. Methods Forty healthy New Zealand white rabbits，weighting 2.0-2.5 kg，male or female，were randomly divided into 4 groups with 10 rabbits in each group. In groupⅠ，the rabbits received CPB and pulmonary arterial perfusion. In group Ⅱ，the rabbits received CPB and pulmonary arterial perfusion with TNF-αAb. In group Ⅲ，the rabbits received CPB only. In group Ⅳ，the rabbits only received sham surgery. Neutrophils count，TNF-α and malondialdehyde （MDA） concentrations of the blood samples from the left and right atrium as well as oxygenation index were examined before and after CPB in the 4 groups. Pathological and ultrastructural changes of the lung tissues were observed under light and electron microscopes. Lung water content，TNF-α mRNA and apoptoticindex of the lung tissues were measured at different time points. Results Compared with group Ⅳ，after CPB，the rabbitsin group Ⅰ to group Ⅲ showed significantly higher blood levels of neutrophils count，TNF-α and MDA（P＜0.05），higherTNF-α mRNA expression，apoptosis index and water content of the lung tissues （P＜0.05），and significantly lower oxyg-enation index （P＜0.05） as well as considerable pathomorphological changes in the lung tissues. Compared with group Ⅱ，after CPB，the rabbits in groups Ⅰ and Ⅲ had significantly higher blood concentrations of TNF-α （5 minutes after aortic declamping，220.43±16.44 pg/ml vs.185.27±11.78 pg/ml，P＜0.05；249.99±14.09 pg/ml vs.185.27±11.78 pg/ml，P＜0.05），significantly higher apoptosis index （at the time of CPB termination，60.7‰±13.09‰ vs. 37.9‰±7.78‰，P＜0.05；59.6‰±7.74‰ vs. 37.9‰±7.78‰，P＜0.05），significantly higher blood levels of neutrophils count and MDA （P＜0.05），significantly higher TNF-α mRNA expression and water content of the lung tissues （P＜0.05），and significantly loweroxygenation index （P＜0.05） as well as considerable pathomorphological changes in the lung tissues. Compared with groupⅠ，rabbits in group Ⅲ had significantly higher above parameters （P＜0.05） but lower oxygenation index （P＜0.05） only at 30 minutes after the start of CPB. Conclusion Pulmonary artery perfusion with TNF-αAb can significantly attenuate inflammatory lung injury and apoptosis of the lung tissues during CPB.
【 Abstract 】 Objective To probe into the role of inositol 1, 4, 5-trisphosphate (IP3) and bax gene expression in apoptosis of HepG2 cells induced by genistein (Gen). Methods HepG2 cells were treated with different concentrations including 20, 40, 60 and 80 μ mol/L Gen as HepG2 cells cultured with 0 μmol/L Gen for 72 h was control; HepG2 cells were treated with 60 μmol/L Gen for 6, 12, 24, 48 and 72 h as HepG2 cells treated with 60 μmol/L Gen for 0 h was control. IP3 content, bax mRNA expression and apoptosis rate were assayed by IP3- ［ 3H ］ Birtrak assay, RT-PCR and flow cytometry, respectively. ResultsHepG2 cells incubated with each concentration of Gen for 72 h ， IP3 content was lower than that of control 〔 (17.7 ± 1.3), (11.2 ± 0.9), (4.9 ± 0.5), (4.8 ± 0.3) pmol/106 cells vs (29.4 ± 0.5) pmol/106 cells 〕 , P ＜ 0.01 ； bax mRNA expression (RI which was the gray degree multiply area of bax/the gray degree multiply area of β -actin) was higher than that of control (0.26 ± 0.02, 0.33 ± 0.05, 0.35 ± 0.06, 0.38 ± 0.05 vs 0.09 ± 0.01), P ＜ 0.01 ； The apoptosis rate was higher than that of control 〔 (10.1 ± 0.9)%, (18.7 ± 1.6)%, (28.7 ± 2.5)%, (27.9 ± 2.0)% vs (2.6 ± 0.1)% 〕 , P ＜ 0.01. HepG2 cells were incubated with 60 μ mol/L Gen for 6, 12, 24, 48 and 72 h ， IP3 content was lower than that of control 〔 (22.6 ± 0.9), (12.0 ± 1.4), (7.5 ± 0.8), (5.6 ± 0.5), (4.3 ± 0.6) pmol/106 cells vs (29.2 ± 0.6) pmol/106 cells 〕 , P ＜ 0.01 ； bax mRNA expression was higher than that of control incubated with 60 μ mol/L Gen for above 12 h (0.25 ± 0.06, 0.29 ± 0.02, 0.30 ± 0.02, 0.35 ± 0.04 vs 0.09 ± 0.01), P ＜ 0.01 ； The apoptosis rate in groups incubated with 60 μ mol/L Gen for 24, 48 and 72 h was significantly higher than that in control 〔 (7.4 ± 0.5)%, (20.5 ± 2.0)%, (30.7 ± 1.6)% vs (2.6 ± 0.1)% 〕 , P ＜ 0.01. ConclusionGen induces apoptosis of HepG2 cells by reducing IP3 production and increasing bax gene expression.
Objective To evaluate the phenomena of apoptosis and its relevant mechanism during ischemia-reperfusion period. Methods The published papers to explore the apoptotic phenomena and its mechanism in organs or tissues which experienced ischemia-reperfusion injury were reviewed. Results Apoptosis was common in ischemia-reperfusioned organ or tissue. The severity of apoptosis was influenced by many factors such as ischemia, hypoxia, oxygen free radials, intracellular free calcium ion overloading, various cytokines, et al; and also was regulated by bcl-2 family, caspase family and NF-κB，et al. Conclusion Apoptosis is a common phenomenum in ischemiareperfusioned organ or tissue which is affected and regulated by various factors.
Objective To explore the molecular mechanism of miR-515-5p in inhibiting chondrocyte apoptosis and alleviating inflammatory response in osteoarthritis (OA). Methods Human cartilage cell line C28/I2 was cultured in vitro and treated with 10 ng/mL interleukin 1β (IL-1β) for 24 hours to construct an in vitro OA model. C28/I2 cells were transfected with miR mimics, mimics negative control (NC), over expression (oe)-NC, and oe-Toll-like receptor 4 (TLR4), respectively, and then treated with 10 ng/mL IL-1β for 24 hours to establish OA model. Cell proliferation capacity was detected by cell counting kit 8 and 5-Ethynyl-2’-deoxyuridine, cell apoptosis and cell cycle were detected by flow cytometry, and B-cell lymphoma 2 protion (Bcl-2), Bcl-2-associated X protein (Bax), cleaved-Caspase-3, TLR4, myeloid differentiation primary response gene 88 (MyD88), p65 and phosphorylated p65 (p-p65) protein expression levels were detected by Western blot. Real-time fluorescence quantitative PCR was used to detect mRNA expression levels of miR-515-5p and TLR4, and ELISA was used to detect pro-inflammatory factor prostaglandin E2 (PGE2), tumor necrosis factor α (TNF -α), and IL-6 levels in cell supernatant. The potential binding sites between miR-515-5p and TLR4 were predicted by BiBiServ2 database, and the targeting relationship between miR-515-5p and TLR4 was verified by dual luciferase reporting assay. Results After the treatment of C28/I2 cells with IL-1β, the expressions of miR-515-5p and Bcl-2 protein and the proliferation ability of C28/I2 cells significantly reduced. The expression levels of Bax and cleaved-Caspase-3 protein, the levels of pro-inflammatory factors (PGE2, TNF-α, IL-6) in the supernatant of C28/I2 cells, and the apoptosis of C28/I2 cells significantly increased. In addition, the proportion of the cells at S phase and G2 phase decreased significantly, and the proportion of cells at G1 phase increased significantly, suggesting that the cell cycle was blocked after IL-1β treatment. After transfection with miR mimics, the expression level of miR-515-5p in the cells significantly up-regulated, partially reversing the apoptosis of OA chondrocytes induced by IL-1β, and alleviating the cycle arrest and inflammatory response of OA chondrocytes. After treating C28/I2 cells with IL-1β, the mRNA and protein levels of TLR4 significantly increased. Overexpression of miR-515-5p targeted inhibition of TLR4 expression and blocked activation of MyD88/nuclear factor κB (NF-κB) pathway. Overexpression of TLR4 could partially reverse the effect of miR mimics on IL-1β-induced apoptosis and inflammation of OA chondrocytes. ConclusionmiR-515-5p negatively regulates the expression of TLR4, inhibits the activation of MyD88/NF-κB pathway and apoptosis of OA chondrocytes, and effectively alleviates the inflammatory response of the cells.
Objective To study the effect of low-dose cyclophosphamide (CY) on apoptosis of lung parenchyma cells in the early severe burn stage in rats. Methods Ninety clean SD male rats were randomly divided into 3 groups: the normal group (n=10), the experimental group (n=40) and the burn group (n=40). The model of degree III with 30% burn area was made in the experimental group and the burn group. CY (2 mg/kg) was injected into the abdominal cavity right after burn in the experimental group. No treatment was done in the normal group and burn group. Lung tissues were obtained at 3, 6, 12and 24 hours, respectively, after burn, and were observed by HE staining. Apoptosis of lung parenchyma cells was observed by TUNEL. Results Lung tissues were observed under the opticalmicroscopy in the normal group: the pulmonary structure was clear, and there were no inflammatory cells and exudation in the alveolar space and bronchial lumen. Besides, a few RBCs were seen. Pathological changes of lung tissues were observed under the opticalmicroscopy in the burn group: alveolar septum was obviously widened; alveolar wall was destroyed; interstitial edema and atelectasis occurred; and pathological lesion was gradually aggravated as time passed by. The pathological lesion of lung tissues mentioned above in the experimental group was better than those in the burn group. Compared with the normal group, the apoptosis ratio of lung parenchyma cells continuously increased in the burn group from the 3 hour after burn, and reached the peak at 12 hours. There were significant differences between the two groups (P lt; 0.05). However, in the experimental group, the apoptosis ratio of lung parenchyma cells increased at 3 hours after burn, cut down to normal at 6 and 12 hours, respectively, and notably decreased at 24 hours. There were significant differences between the experimental group and the normal group (P lt; 0.05). Compared with the burn group, the apoptosisrate of lung parenchyma cells in the experimental group began to decrease strikingly from the 6 hours after burn, and there were significant differences between the two groups (P lt; 0.05). Conclusion Low-dose CY can restrain the apoptosis of lung parenchyma cells in the early severe burn stage in rats and alleviate the injury of the lung.
To evaluate the significance of bcl-2 protein, estrogen receptor (ER), progesterone receptor (PR) and p53 oncogene in invasive breast cancer of ductal and lobular type. Tumor tissues were examined immunohistochemically in paraffin embedded tissues from 125 patients. Results: The invasive ductal breast carcinomas expressed bcl-2 protein significantly less frequently than the lobular type (Ｐ＜0.001). And the expression of bcl-2 protein was significantly correlated with ER, PR (Ｐ＜0.001) and p53 (Ｐ＜0.001), also correlated with primary tumor size and grade. No statistical evidence was found to indicate the relationship between bcl-2 protein expression and anxillary lymph node metastasis. Conclusions: The expression of bcl-2 protein may be regarded as a biological index and may play important role in evaluating the biological characteristics of breast cancer.
目的研究依达拉奉影响肝脏缺血再灌注过程中TNF-α的表达情况，探讨依达拉奉对肝脏缺血再灌注损伤的逆转作用。 方法将80只Wistar大鼠编号，根据计算机产生随机数字，前40为一组，后40为一组，分为实验组和对照组2组，建立常温下部分肝缺血再灌注损伤动物模型。 在肝脏缺血再灌注损伤开始前1 h和开始时对实验组大鼠给予依达拉奉注射液10 ml，对照组则给予同等容量的生理盐水。分别于再灌注后0、1、2及4 h测定肝脏脂质过氧化物酶（LPO）和肝脏谷草转氨酶(AST) 浓度； 应用RT-PCR法检测肝组织TNF-α mRNA含量，并测定肝组织和血清中TNF-α水平； 应用TUNEL染色法检测缺血肝组织的细胞凋亡情况。结果再灌注后1、2及4 h，实验组大鼠肝脏LPO及AST浓度均明显低于对照组（Plt;0.001）； 实验组再灌注后1 h时肝组织TNF-α mRNA表达量、肝组织和血清TNF-α含量均明显升高且达峰值，但均明显低于对照组（Plt;0.05）； 再灌注后各时相实验组肝细胞凋亡率明显升高，但均明显低于对照组（Plt;0.05）。 结论依达拉奉能抑制氧化应激反应，从而降低肝缺血再灌注损伤； 并显著减少炎性细胞因子TNF-α的产生，抑制炎性反应的发生，减少肝细胞的凋亡。
Objective To investigate the effect of ginkgolide B (GB) on cysteinyl aspartate specific proteinase-3 (Caspase-3)/chromosome 10 deletion phosphatase-tension protein homologue (PTEN)/protein kinase B (Akt) pathway and cell proliferation and apoptosis in hypoxia/reoxygenation cardiomyocytes. Methods H9C2 cells were cultured in vitro. A control group was cultured in serum-free DMEM high glucose medium at 37°C and 5% CO2 for 28 hours. The remaining groups were prepared with hypoxia/reoxygenation models. A GB low-dose group and a GB high-dose group were treated with GB pretreatment with final concentration of 50 μmol/L and 200 μmol/L respectively at 1 h before hypoxia/reoxygenation. A carvedilol group was treated with carvedilol of a final concentration of 10 μmol/L at 1 h before hypoxia/reoxygenation. The proliferation and apoptosis of H9C2 cells were detected, and the levels of lactate dehydrogenase (LDH), malondialdehyde (MDA), reactive oxygen species (ROS), PTEN, Akt, phosphorylated Akt (p-Akt) and Caspase-3 in H9C2 cells were also detected. Results Compared with the control group, the proliferation rate of H9C2 cell, and the levels of PTEN, Akt and p-Akt in other groups decreased, and the apoptosis rate, and the levels of LDH, MDA, ROS and Caspase-3 increased (P<0.05). Compared with the hypoxia/reoxygenation group, the proliferation rate of H9C2 cell, and the levels of PTEN, Akt and p-Akt in all GB dose groups and the carvedilol group increased; the apoptosis rate, and the levels of LDH, MDA, ROS and Caspase-3 decreased, and the effect of GB was in a dose dependent manner; however, the effect of GB was not as strong as carvedilol (P<0.05). Conclusion GB can inhibit H9C2 cell apoptosis and promote H9C2 cell proliferation by activating Caspase-3/PTEN/Akt pathway.
ObjectiveTo introduce the relationship between the apoptosis hepatocyte and its genic mediation and the ischemia of portal vein. MethodsThe combination of related literatures and our research findings were made.ResultsPortal vein ischemia may induced hepatocyte apoptosis, p53 and bcl2 gene alternatively adjust hepatocyte apoptosis. Expression of p53 gene is enhanced in hepatic tissue when hepatocyte apoptosis is not obvious, but after 24-72 h of portal vein ischemia, when hepatocyte apoptosis is obvious, enhanced expression of p53 gene or reduced expression of bcl2 gene occur. There exists close relationship between portal vein ischemia and hepatocyte apoptosis. Conclusion Apoptosis hepatocyte is involved in organic atrophy after ischemia of portal vein, and p53 and bcl2 gene alternatively adjust hepatocyte apoptosis. At present, the mechanism of apoptosis of hepatocyte induced by ischemia of portal vein is not clear, which needs further study.
Objective To investigate the effect of heme oxygenase 1 (HO-1) on the apoptosis of human degenerated nucleus pulposus (NP) cells induced by tumor necrosis factor α (TNF-α), and explore its possible molecular mechanism. Methods The intervertebral disc tissues were derived from patients with lumbar intervertebral disc herniation. Then, the NP cells were cultured in vitro and the third generation of NP cells were used for subsequent experiments. Cell counting kit 8 (CCK-8) method was used to observe the proliferative effect of TNF-α on the NP cells in vitro at the concentration of 10, 20, 50, 100, and 200 ng/mL. The most apropriate concentration was selected according to the result of CCK-8. The NP cells were cultured with basal medium (control group), TNF-α (TNF-α group), TNF-α and CoPP 10 μmol/L (CoPP group), and TNF-α and ZnPP 15 μmol/L (ZnPP group), respectively. After cultured, the cell poptosis was detected by Hoechst staining and flow cytometry; the expression of cleaved Caspase-3, epithelial membrane protein 1 (EMP-1), HO-1, and p-P65 proteins were detected by Western blot. In order to further explore the potential molecular mechanisms of HO-1 for cell apoptosis, the NP cells were cultured with TNF-α (TNF-α stimulated group), TNF-α and pyrrolidine dithiocarbamate (PDTC) 5 μmol/L (TNF-α+PDTC stimulated group), respectively. Then the cell apoptosis rate was measured by flow cytometry at 24 hours after cultured. Results The optimal concentration of TNF-α was 100 ng/mL. Hoechst staining showed that a few apoptotic cells could be observed in control group and CoPP group; the apoptosis-like nucleis were observed in TNF-α group and ZnPP group, which was the most significant in ZnPP group. Flow cytometry showed that the cell apoptosis rates of TNF-α group, CoPP group, and ZnPP group were significantly increased when compared with the control group (P<0.05). Compared with TNF-α group, the cell apoptosis rate in CoPP group decreased (P<0.05), while in ZnPP group it increased (P<0.05). Western blot showed that the expression of HO-1 protein in TNF-α group was decreased, and the expressions of cleaved Caspase-3, EMP-1, and p-P65 proteins were increased when compared with the control group (P<0.05). Compared with TNF-α group, the expression of HO-1 protein in CoPP group increased, and the expressions of cleaved Caspase-3, EMP-1, and p-P65 proteins were reduced (P<0.05); the expression of HO-1 protein in ZnPP group decreased (P<0.05), the expressions of cleaved Caspase-3 and EMP-1 proteins increased (P<0.05), and the expression of p-P65 protein was not significantly changed (P>0.05). Compared with TNF-α stimulated group, the cell apoptosis rate in TNF-α+PDTC stimulated group was significantly reduced (t=3.076, P=0.031). Conclusion HO-1 can inhibit the apoptosis of degerated NP cells induced by TNF-α, and its mechanism effect is by inhibiting the nuclear factor кB signaling pathway.