Effect And Possible Mechanism Of Maotai Liquor On Hepatocellular Carcinoma Formation In Diethylnitrosamine-initiated Mice

Objective To investigate the relationship and the possible mechanisms of long-term Maotai consumption on hepatocarcinogenesis in a DEN-initiated HCC animal model. Methods 192 male C57BL/6J mice were randomly divided into six groups, included:Control group(C), Maotai group(M), Ethanol group(E), Maotai+DEN group(MD), Ethanol+ DEN group(ED) and DEN group(D). DEN was given to mice at a dose of 100 mg/kg, ip, and 50 mg/kg, ip in the following week. Mice were simultaneously given Maotai or an equal amount of ethanol (53%,5ml/kg/day,5days/week for up to 35weeks). Body weight and general condition were monitored. At 3-week,13-week and 35-week of the experiment, serum and livers were collected respectively for biochemical and histopathological examination of liver injury and incidence of HCC. The liver index, serum alanine transaminase(ALT)and aspartate transaminase(AST) were examined. Malondialdehyde(MDA)of liver homogenate were determined. All liver tissue samples were detected the liver histopathology, such as the lesions of hepatocytes, fibrogenesis and the evaluation of liver structural change by using hematoxylin and eosin (H&E), Masson and Reticular fiber stain and transmission electron microscope. Expression level of glypican-3 (GPC3) were analyzed using immunohistochemical assays. Real-time RT-PCR, immunohistochemistry and Western blotting were used to examine the expression of liver metallothionein-1/2 (MT-1/2), NF-E2-related factor 2 (Nrf2), glutamate-cysteine ligase catalytic subunit (GCLC) and modified subunit (GCLM). Custom-builded SABioscience PCR array and Real-time RT-PCR analysis were used to profile changes in liver gene expression, relevant to liver fibrosis, ethanol responsive, apoptosis, tumor suppressor and hepatoprotective. Further analysis were done on gene having significant mRNA level change by using immunohistochemistry and western blotting assays. The apoptosis index and the early apoptosis rate of liver tissue cell was detected by using POD and AnnexinV/PI flow cytometry analysis methods. Results We identified tissue damage and dysfunction of liver in ethanol+DEN-treated mice, whereas the extent of injury was reduced in Maotai+DEN-treated mice. Significant Glypican-3(GPC3) expression and precancerous injury or HCC could be seen in mice with ethanol+DEN, but barely be seen in Maotai+DEN-treated mice. A higher expression of MT-1/2, Nrf2 and GCLC could be seen in Maotai+DEN-treated mice. Real-time RT-PCR analysis demonstrated Maotai+DEN treatment could significantly activate the MT-1 transcription at all the time points, and the specific up-regulation of MT-1 was confirmed through immunohistochemistry and western blotting assay. Ethanol+DEN group showed transient activation in MT-1 transcription, but the protein levels of MT in this group are generally lower than control group. Similarly, Maotai+DEN treatment slightly enhanced the mRNA and protein levels of Nrf2 in all time points, but ethanol+DEN treatment only transiently increased the transcriptional level of Nrf2 within 3-week treatment. The expression of Nrf2 in this group showed no difference with the control level at 35-week time point. In addition, Maotai+DEN group always showed significantly increased GCLC expression level than control group, but the ethanol+DEN group always showed lower GCLC expression level than control group. Maotai+DEN could upregulate the transcriptional level and protein level of GCLM only at 3-week, but no any increased expression of GCLM could be seen either in Maotai+DEN group or ethanol+DEN group at 35-week. The current study demonstrated ethanol induced more pronounced gene expression alterations than an equal amount of Maotai. Ethanol+DEN treatment significantly increased gene expressions relevant to liver fibrosis, carcinogenesis, and the ethanol responsive that were distinctly less pronounced in the Maotai+DEN. For example, Atm,Fzd7, NQOl,Irsl, Lefl,Tgfbr2,CK8 and CK18. Maotai+DEN treatment significantly or lightly increased gene expressions relevant to apoptosis, tumor suppressor and hepatoprotective, such asTAp63, Wtl, P53, Pten,Rbl, GST-Pi, especially TAp63.But all these increases were less pronounced in ethanol+DEN mouse livers. The Maotai+DEN treatment increased the expression of caspase9 7-fold and 5-fold compared with ethanol+DEN and DEN alone. The significantly induction of p21 also could be seen in Maotai+DEN treatment group. In addition to these, a significant increased liver tissue cell apoptosis index and early apoptosis rate could be seen in Maotai+DEN group not as done with ethanol+DEN treatment. Conclusion Maotai liquor ameliorates the formation of DEN-induced HCC in mice even though equal amounts of ethanol were given. The protection mechanisms are possibly related with the activation of antioxidation factors, such as MTs, Nrf2 and GCLC, and the dramatic induction of TAp63, caspase9 and p21 with Maotai liquor also could be important adaptive responses to reduce liver injury resulted by the synergy between alcohol and DEN.