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Aim To investigate the role of eelastrol in reactive oxygen species ( ROS) accumulation and DNA damage in hepatocellular carcinoma cells, and further investigate its effect on apoptosis induction in cancer cells.Methods Human liver cancer HepG2 and Huh7 cells were cultured with celastrol, then the morphological changes of cells were observed under microscope.MTT assay was employed to detect the proliferation of hepatocellular carcinoma cells, CM-H2DCFDA probe to detect intracellular ROS levels, and immunofluorescence to detect the expression level of -y-H2AX in celastrol-treated cells.Hoechst 33258 staining was used to observe nuclear condensation and fragmentation; Flow cytometry was used to evaluate cell death.J J Western blot was applied to measure the expression levels of -y-H2 AX, caspase-3, PARP and other proteins.Results Celastrol had a significant inhibitory effect on the proliferation of liver cancer cells in dose-dependent manner.Comparer] with the eontrol group, the eell viability was reduced and intracellular ROS level also increased significantly after celastrol treatment in a dose-dependent manner ( P < 0.05 ).With Hoechst staining, typical apoptotic characteristics such as nuclear chromatin condensation and fragmentation were observed in celastrol-treated cells.Western blot results showed that pro-form of caspase-3 significantly decreased, and the cleavage of PARP markedly increased by celastrol.After pretreatment with ROS inhibitor NAC, celastrol-mediated caspase-3 activation and PARP cleavage were significantly reversed ( P < 0.05 ).Conclusions Celastrol can induce apoptosis in hepatocellular carcinoma cells, and its anti-cancer effect is dependent on ROS-mediated DNA damage.
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Objective To investigate whether minicircle DNA-mediated shRNA can inhibit the replication and expression of HBV in HBV transgenic mice.Methods First,a universal plasmid shRNA,pU6-shRNA HBV and a minicircle shRNA vector pMC-U6-shRNA HBV targeting HBV gene was prepared by molecular cloning technique.Then,the HBV transgenic mice were divided into three groups and the pMC-U6-shRNA HBV,pU6-shRNA HBV and control vector pU6-control were transfected into them respectively by hydrodynamic injection (HDI).Sera were collected at different time points after injection.The changes of HBV DNA and HBsAg in serum of transgenic mice were detected by real-time PCR and chemiluminescence microparticle immunoassay (CMIA).Immunohistochemistry was used to analyze the expression of HBcAg in the liver of transgenic mice.Results Compared with pU6-control group,HBsAg and HBV DNA in serum of transgenic mice were significantly inhibited by pU6-shRNA HBV and pMC-U6-shRNA from day 7 to day 21 after HDI (P＜0.05).After that,the serum HBsAg and HBV DNA recovered and returned to the control level on the 35th day in pU6-shRNA HBV group.However,pMC-U6-shRNA HBV still maintained a strong inhibitory effect in vivo until 35 days post-injection compared with the other two groups (P＜0.05).Immunohistochemical results also suggest that pMC-U6-shRNA HBV could more significantly inhibit HBcAg expression than pU6-shRNA HBV in mouse liver tissue.Conclusion The minicircle DNA-based shRNA vector pMC-U6-shRNA HBV is of longer inhibitory effect on HBV replication and gene expression in HBV transgenic mice than pU6-shRNA HBV.Minicircle DNA is superior to pRNAT-U6.1/Neo for shRNA delivery in vivo.
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<p><b>OBJECTIVE</b>To observe the effect of dopamine receptor (DR2) activation on hypoxia/reperfusion injury (HRI) in the neonatal rat cardiomyocytes, and to explore its mechanism.</p><p><b>METHODS</b>The hypoxia/reperfusion (H/R) injury model was established in primarily cultured neonatal rat cardiomyocytes, and randomly assigned: control, H/R, bromocriptine (Bro) and haloperidol (Hal) groups. The cell apoptosis was detected using inverted microscope, transmission electron microscope and flow cytometry (FCM). The lactate dehydrogenase(LDH) and superoxide dismutase (SOD) activity and malondialdehyde (MDA) content in cell medium were analyzed. The expression of mRNA and protein of caspase-3, caspase-8, caspase-9, Fas, Fas-L, Cyt C and Bcl-2 were detected by RT-PCR and Western blot, respectively.</p><p><b>RESULTS</b>Compared with the control group, apoptosis rate, LDH activity, MDA content and the expression of pro-apoptotic factors and anti-apoptotic factors were increased, but SOD activity was decreased in H/R group. Compared with the H/R group, all index above-mentioned were down-regulated or reversed in Bro-group, and had no obvious differences in Hal-group.</p><p><b>CONCLUSION</b>The neonatal rat cardiomyocytes injury and apoptosis caused by hypoxia/reperfusion can be inhibited with DR2 activation, which mechanism is related to scavenging oxygen radical.</p>
Subject(s)
Animals , Rats , Animals, Newborn , Apoptosis , Cell Hypoxia , Myocardial Reperfusion Injury , Metabolism , Myocytes, Cardiac , Cell Biology , Metabolism , Oxidative Stress , Rats, Wistar , Receptors, Dopamine D2 , MetabolismABSTRACT
Objective To study the changes of soluble intercelluar adhesion molecule-1 (sICAM-1) in serum of neonates with hy-poxic-ischemic encephalopathy (HIE),and significance of changes of serum sICAM-1 in HIE pathogenesis. Methods There were 17 controls of neonates and their sICAM-1 concentrations in serum were detected with enzyme-linked immunosorbent assay (ELISA) at the critical stage and at the beginning of convalescent stage in 36 cases of HIE neonates and 17 normal neonates. The data were analyzed with analysis of variance, Newman-Keuls q test. Results The concentrations of sICAM-l[(216.64?85.32)?g/L] at the critical stage of 36 cases HIE neonates were significantly higher than those [(6. 16?4.05) ?g/L ] of control group (q = 17. 42 P