Objective:To observe the effect of moxibustion on the protein and mRNA expressions of corticotropin-releasing factor (CRF)and corticotropin-releasing factor receptor 1 (CRFR1) in hypothalamus of trinitrobenzene sulfonic acid (TNBS)-induced experimental colitis rats,and to explore the central mechanisms of moxibustion in improving visceral pain and the pain-related emotions in experimental colitis rats.Methods:Thirty-six Sprague-Dawley (SD) rats were randomly divided into a normal group (NG),a model group (MG),a herb-partitioned moxibustion group (HPMG) and a sham herb-partitioned moxibustion group (SHPMG).Except the NG,rats in the remaining three groups all received TNBS enema to establish experimental colitis models.The HPMG received herb-partitioned moxibustion (HPM) at bilateral Tianshu (ST 25) and Qihai (CV 6) for intervention;for the SHPMG,the herbal cakes and moxa cones were only placed on the acupoints but not ignited;rats in the MG and NG were only fixed in the same way as those in the HPMG but did not receive any treatment.At the end of the intervention,the abdominal withdrawal reflex (AWR) score,the open field test (OFT) score and the elevated plus maze (EPM) score were observed to measure the changes in visceral pain and pain-related emotions of the rats.The enzyme-linked immunosorbent assay (ELISA) was used to examine the expressions of CRF and CRFR1 proteins in hypothalamus;the fluorescence-based quantitative polymerase chain reaction (PCR) was used to detect the expressions of CRF and CRFR1 mRNAs in hypothalamus.Results:Compared with the NG,the AWR score increased significantly and the OFT and EPM scores dropped significantly in the MG (all P＜0.05),and the expressions of hypothalamic CRF and CRFR1 proteins and mRNAs increased significantly (all P＜0.01).Compared with the MG and SHPMG,the AWR score dropped significantly and the OFT and EPM scores increased significantly in the HPMG (all P＜0.01),and the expressions of hypothalamic CRF and CRFR1 proteins and mRNAs decreased significantly (all P＜0.05).There were no significant differences between the MG and the SHPMG (all P＞0.05).Conclusion:HPM can down-regulate the abnormally increased expressions of CRF and CRFR1 proteins and mRNAs in hypothalamus of the TNBS-induced experimental colitis rats,which is plausibly one of its action mechanisms in mitigating visceral pain and the pain-related emotions in the experimental colitis rats.

Aged ; Central Nervous System Neoplasms ; pathology ; Humans ; Lymphoma, Non-Hodgkin ; pathology ; Magnetic Resonance Imaging ; Male ; Middle Aged

OBJECTIVETo prepare cytochrome (CYP)2E1-silenced hepatocytes by lentivirus-mediated RNA interference technology and to investigate the hepatotoxicity of trichloroethylene (TCE) in CYP2E1-silenced hepatocytes.

METHODSShort 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.

RESULTSThe 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).

CONCLUSIONCYP2E1-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.

Apoptosis ; drug effects ; genetics ; Cell Line ; Cell Survival ; drug effects ; genetics ; Cytochrome P-450 CYP2E1 ; genetics ; metabolism ; Genetic Vectors ; Hepatocytes ; drug effects ; metabolism ; Humans ; Lentivirus ; genetics ; RNA Interference ; Trichloroethylene ; toxicity