| BackgroundThe Nrf2/Keap1 signaling pathway plays a key role in the cell's ability to defend itself from oxidative stress. Nrf2 is a nuclear transcription factor that controls the expression and coordinated induction of a battery of defensive genes encoding detoxifying enzymes and antioxidant proteins.Under homeostasis, Nrf2 is retained in the cytoplasm by an inhibitor, Keap1. When a cell is exposed to oxidative stress Nrf2 dissociates from the Keap1 complex, stabilizes, and translocates into the nucleus, forms heterodimers with unknown proteins, and binds the antioxidant response element, which leads to coordinated activation of gene expression. In our study,we found oridonin activate transcription of Nrf2 target genes and protect mice from acute liver injury.Normally Keap1 targets Nrf2 for ubiquitin-mediated degradation. The loss of Keap1 function has been shown to lead to nuclear accumulation of Nrf2, activation of metabolizing enzymes, and drug resistance. Studies have reported mutations resulting in dysfunctional Keap1 in lung, breast, and bladder cancers. An increasing body of evidence indicates that certain types of tumour might constitutively activate Nrf2 through genetic and epigenetic mechanisms as a means of increasing their survival or proliferation. Initial reports uncovered Keap1 mis-sense mutations in the H1184 and H1648 lung cancer cell lines that resulted in loss of Nrf2 repression. Shortly thereafter, mis-sense mutations, insertions and deletions were identified in Keap1 from the tumours of ten of 54 non-small cell lung cancer (NSCLC) patients and in six of 12 cell lines. Importantly, the mutations were reported to result in permanent Nrf2 activation. Furthermore, Keap1 mutation was frequently accompanied by loss of heterozygosity at the gene locus, thereby indicating that biallelic Keap1 loss is common in NSCLC. Further Keap1 mutations were later identified in additional lung cancer patients; one of eight with small cell lung carcinoma (SCLC), three of 29 with adenocarcinoma and one of two with large cell carcinoma were affected. Mutations have also been reported in patients with neoplastic disease of the biliary tract; four of 13 patients with gall bladder cancer harboured Keap1 mutations. A single study reported that the human Q293 breast cancer cell line contains an inactive Keap1 protein. Taken together, these results indicate that Keap1 can be rendered inactive through somatic mutations in several tumour types besides lung cancer. We found a same sense Keap1 Exon4 mutation in esophageal carcinoma through amplification and sequencing, but it is uncertain that the spot can lead to loss of Keap1 function and there is no reports that mutation in other exon in esophageal carcinoma exists.ObjectiveTo confirm that Nrf2/Keap1 can protect mice from liver acute injury. To prove that somatic Keap1 mutations occur in esophageal carcinoma by detecting exon4 of Keap1 mutations.MethodsThe 40 C57BL/6 mice were randomly divided into four groups: normal control group, small-dose oridonin group, middle-dose oridonin group, large-dose oridonin group, and build models of different protection of liver. Oridonin small-, middle-, large-dose groups were given oridonin at different doses(at dose of 1,5,10mg/kg)for 4 weeks,but the control group were administered with PEG400 at 0.05ml. After that,acute liver injury of mice was induced by irrigation of alcohol at dose 0.1ml/10g for one week. Then, the mice were put to death, the level of serum ALT, AST were detected. Pathological changes were observed by HE. We extracted DNA from surgically resected esophageal squmous cancer tissues ( n = 36) and matched adjacent cancer tissues and amplified Keap1 exon4.After we got objective fragment, PCR products were directly sequenced after purification. The senquence data were analyzed to identify potential genetic alteration. ResultsOridonin can decrease acute liver injury of mice treated by alcohol though Nrf2/Keap1 signaling pathway. We found a same sense mutation in Keap1 Exon4 in esophageal carcinoma.ConclusionOridonin can reduce the acute liver injury of mice through activation of the transcription factor Nrf2. We found a same sense mutation in Keap1 Exon4 in esophageal carcinoma. It is possible that the Nrf2/Keap1 pathway is abnormal in esophageal carcinoma. |
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