Effects of trichloroethylene on hepatotoxicity in cytochrome 2E1-silenced hepatocytes / 中华劳动卫生职业病杂志

<p><b>OBJECTIVE</b>To prepare cytochrome (CYP)2E1-silenced hepatocytes by lentivirus-mediated RNA interference technology and to investigate the hepatotoxicity of trichloroethylene (TCE) in CYP2E1-silenced hepatocytes.</p><p><b>METHODS</b>Short hairpin RNA fragments were designed and synthesized and were then ligated into the lentiviral vector; single colonies were screened; the plasmid was extracted after PCR and sequence identification and then transferred into L02 hepatocytes; the CYP2E1-silenced hepatocytes were selected; real-time quantitative PCR and Western blot were used to evaluate the interference effects. The obtained CYP2E1-silenced hepatocytes, as well as normal L02 hepatocytes, were treated with TCE (0, 0.25, 0.50, 1.00, 2.00, and 4.00 mmol/L). The cell viability and half maximal inhibitory concentration (IC50) of TCE were measured; the apoptotic rate of cells was measured by flow cytometry; the mRNA expression levels of apoptosis genes and oncogenes were measured by real-time quantitative PCR.</p><p><b>RESULTS</b>The IC50s of TCE for L02 hepatocytes and CYP2E1-silenced hepatocytes were 15.1 mmol/L and 23.6 mmol/L, respectively. The apoptotic rate increased as the dose of TCE rose in the two types of cells; the CYP2E1-silenced hepatocytes hada significantly lower apoptotic rate than L02 hepatocytes when they were exposed to 2.0 and 4.0 mmol/L TCE (P < 0.05 or P < 0.01). The mRNA expression level of bcl-2 (anti-apoptosis gene) in CYP2E1-silenced hepatocytes was 15% ∼ 60% higher than that in L02 hepatocytes (P < 0.01), while the mRNA expression levels of caspase-3 and caspase-9 (apoptosis genes) in CYP2E1-silenced hepatocytes were 30% ∼ 60% lower than those in L02 hepatocytes (P < 0.01). The mRNA expression level of p53 (cancer suppressor gene) in CYP2E1-silenced hepatocytes was 81 - 278% higher than that in L02 hepatocytes (P < 0.01), while the mRNA expression levels of c-fos and k-ras (oncogenes) in CYP2E1-silenced hepatocytes were 20-68% lower than those in L02 hepatocytes (P < 0.01).</p><p><b>CONCLUSION</b>CYP2E1-silenced cells can be successfully prepared by lentivirus-mediated RNA interference technology. Silencing CYP2E1 gene can reduce the hepatotoxicity of TCE and inhibit the expression of some apoptosis genes and oncogenes, suggesting that CYP2E1 gene plays an important role in TCE metabolism and is related to the hepatotoxicity of TCE.</p>

Apoptosis and expression of iNOS after spinal cord injury in rats / 中国康复理论与实践

@# ObjectiveTo study the characteristic of apoptosis and the expression of inducible nitric oxide synthase(iNOS),as well as relation between them after spinal cord injury (SCI) in rats.Methods84 adult SD rats were randomly divided into three groups,and made into mild,moderate and severe acute SCI models.The rats were killed at 4h,8h,1d,3d,7d,14d and 21d after surgery.The histopathology changes in spinal cord tissue were observed with HE staining microscopic examination.The expression of iNOS was determined by immunohistochemistry and the apoptosis was labeled by terminal deoxynucleotidyl transferase mediated dUTP nick end labeling(TUNEL).ResultsThe histopathology changes in spinal cord tissue deteriorated with increasing in injury degree.A little iNOS immunoreactivity (iNOS-IR) was detected in nerve cells,gliocytes,ependymocytes and blood vessel endotheliocytes of normal and vertebra shelf operated controls,increasing at 8 h, and in flood tide in 7 d. The expression of Bax and the rates of TUNEL positive cells were similar with that of iNOS after SCI. There was positive correlation between expression of iNOS and degree of primary SCI ,apoptosis index(r=0.414,P<0.01;r=0.854,P<0.01).Conclusions Expression of iNOS and Bax increase and a large number of TUNEL positive cells present after SCI in rats. There is positive correlation between expression of iNOS and degree of primary SCI,apoptosis index.