Study On The Mechanism And Correlation Of Quinone Oxidoreductase 1 And Sanguinarine Hepatic Cytotoxicity

Sanguinarine is a quaternary benzo[c]phenanthridine alkaloid and has been used as feed additives because of its ability of anti-bacterial, promote animal growth and insecticidal effect. But sanguinarine also has certain toxicity, for example, can induce hepatotoxicity. Sanguinarine metabolic pathways include oxidative metabolism and reduction metabolism. Research has found that phase I metabolism enzyme P450 enzymes (CYP1A1 and CYP1A2) can participate in the oxidation of sanguinarine; sanguinarine mainly metabolized to dihydrosanguinarine in cells that donot express cytochrome P450 enzymes. NQO1 can use NAD (P) H as the electron donor, and the reductive reaction of the compounds into the body is Ⅱ phase. It has not been reported that whether sanguinarine can be reduced to dihydrogensanguinarine by NQO1. Therefore, we speculated that NQO1 may be involved in this reaction.The rats BRL cells and rat hepatic stellate cells proliferation was determined by MTT test and apoptosis was determined by Hochest33342/PI staining method, the results showed that:sanguinarine and dihydrogen sanguinarine can both time and concentration dependent inhibition the proliferation of HSC-T6 and BRL cell, and the degree of proliferation inhibition of sanguinarine was significantly higher than that of the dihydrogen sanguinarine; Sanguinarine and dihydrogen sanguinarine can induce a time and concentration dependent apoptosis, cell apoptosis induced by sanguinarine role was significantly higher than that of the dihydrogen sanguinarine. The toxicity of sanguinarine on HSC-T6 cells and BRL cells was higher than that of the two hydrogen sanguinarine; the toxicity of sanguinarine on HSC-T6 cells was higher than on BRL cell.Application of fluorescence quantitative PCR technology. Western blotting analysis technology and flow cytometry analysis technology etc, to detect the mechanism of sanguinarine induced cell inhibition of proliferation and cell apoptosis. Results showed that: Sanguinarine induced BRL cells to apoptosis, ROS production, DNA ladder, cell cycle G1 arrest and decreased MMP; Sanguinarine concentration-dependently induced to the downregulation of BCL2 expression、the upregulation of Bax and increased Bax/Bcl-2 ratio; the decreased expression of BCL2 and increased with the promoter methylation of BCL2 gene and higher expression of miR-15a/16-1; Sanguinarine induced to caspase-dependent apoptosis, apoptosis can be impeded by caspase-8 inhibitor; Sanguinarine induced to ROS dependent caspase activation; NAC pretreatment or overexpression of BCL2 prevented sanguinarine induced downregulation of BCL2.Application of fluorescence quantitative PCR technology and western blotting analysis technology detect sanguinarine induced NQO1 and CYP1A1 expression, Adeno-associated virus mediated overexpression of NQO1 and CYP1A1 to research the poisonous of sanguinarine to BRL cells. The results showed that sanguinarine concentration dependently induced the down-regulation of NQO1 expression; lower concentration sanguinarine induced the upregulation of CYP1A1, yet higher concentration sanguinarine induced the downregulation of CYP1A1 expression; but at the same concentration of sanguinarine treatment, the downregulation of CYP1A1 was less obviously than NQO1. NQO1 and CYP1A1 were subcloned to pAAV-IRES-HrGFP vector; Three plasmids(pAAV-RC、 pAAV-Helper and pAAV-IRES-HrGFP carrying target gene) were co-transferred to Hek293 cells for the packaging of recombinant adeno-associated virus, electron microscopy and PCR detection results showed that rAAV-NQO1 and rAAV-CYP1A1 recombinant adeno-associated viruses were successfully packaged. Using the recombinant virus to infect the BRL cells, at the same concentration of sanguinarine treatment, rAAV-NQO1 infection group BRL cells activity were higher than the control group, rAAV-CYP1A1 infection group was significantly higher; at the same time, the apoptosis rate of rAAV-NQOl infection group BRL cells was significantly lower than rAAV-CYP1A1 infection group. Western-blotting results showed that:rAAV-CYP1A1 infection group showed the increase of BAX expression and the decrease of BCL2 expression, while rAAV-NQO1 infection group showed the increase of BAX expression and the decrease of BCL2 expression, but the decrease of BCL2 expression in rAAV-NQO1 group was not obviously than that of the rAAV-CYP1A1 infection group, the Bax/Bcl-2 ratio of rAAV-CYP1A1 infection group increase was more higher than that of rAAV-NQO1 infection group. These results showed that NQO1 has a protective effect to sanguinarine induced apoptosis and cell activity, while CYP1A1 is on the opposite.Applying the prokaryotic expression technology and cell free incubation technology to detect whether NQO1 is participate in the sanguinarine reduction reaction, the results showed that the rats of NQO1 gene was subcloned to pET28a (+) prokaryotic expression vector, and successfully expressed and purified the NQO1 protein; at the appearance of both NADPH and NQO1, sanguinarine can revert to dihydrogen sanguinarine; Without NADPH or NQO1 department, sanguinarine can not revert to dihydrogen sanguinarine. These results showed that NQO1 is the eduction reaction of metabolic enzymes of sanguinarine to dihydrogen sanguinarine, but require the participation of cofactor NADPH.