| Objective: To observe the effect of diethylnitroamine(DEN)on the induction of hepatocellular carcinoma(HCC)in rats at different time,and to explore the susceptibility mechanism of the CYP2E1-ROS/NF-κB signaling pathway from the dynamic changes,so as to provide experimental basis for the prevention of primary HCC.Methods: 72 male SD rats weighing 165±15g were randomly divided into thre e groups:normal group,Maoshi group and Youshi group.DEN was injected intrap eritoneally at a dose of 50mg/kg twice a week in the two groups.After 4 week s,it was changed to once a week and stopped at 16 weeks.The normal group was injected intraperitoneally with normal saline at the same dose and time as the two groups.At the 4th,8th and 12 th weekend,3 rats in the normal group,6rats in the Maoshi group and 6 rats in the Youshi group were randomly select ed.At the 16 th weekend,the remaining rats were randomly selected.The serum and liver samples of the rats were collected for(1)ALT,AST,HA,γ-GT,SOD,GS H-Px,TGF-β1,MDA,AFP,8-OHd G,IL-6,TNF-α detection in the serum of the rats;(2)HE and Masson staining in the liver tissue;(3)immunohistochemistry The ex pression of PCNA,CYP2E1,NF-κB-p65 protein in liver tissue was detected by t he method of science;(4)the expression of CYP2E1,NF-κB-p65 m RNA in liver tissue was detected by real-time fluorescence quantitative PCR.Spss22.0 was us ed to analyze the results.Results:1.Effects of DEN on body weight and liver coefficient in rats From the end of the 4th week,the body weight of the two groups was signifi cantly lower than that of the normal group(P<0.05),With the increase of time,t he growth of body weight was slow and the change of body weight was not obvious(P>0.05),There was no significant difference between the two groups(P>0.05).The liver coefficient of the two groups increased significantly from the 4th we ekend(P<0.05),which was higher than that of the normal group(P<0.05).With the increase of time,there was a significant upward trend(P<0.05).The liver co efficient of the rats in Maoshi group was lower than that in Youshi group at f ourtime points,At the end of the 16 th week,the Maoshi group was significantly lower than the Youshi group,the difference was statistically significant(P<0.05).2.Effects of DEN on liver morphology and nodule in rats In the normal group,the liver tissue was intact and normal without obvious pat hological changes.At the end of the 4th and 8th week,liver congestion and swelling appeared in the two groups.The liver coefficient of the two groups was lighter than that of the Youshi group,and the difference was not statistically significant(P>0.05).At the end of the 12 th week,there were 3 liver nodules in Maoshi group,7of which were between 1-3 mm,and 4 in Youshi group,12 of which were betwee n1-3mm.At the end of the 16 th week,all the livers of the two groups had nod ules.Among them,32 liver nodules were in the range of 1-3mm and 14 were m ore than 3mm in the Maoshi group,51 were in the range of 1-3mm and 24 w ere more than 3mm in the Youshi group.3.Effect of DEN on HE staining of liver in rats Compared with the normal group,the liver of rats in Maoshi and Youshi groups gradually changed from hepatocyte injury,inflammatory infiltration(4/8weeks)-hep atic collagen deposition,pseudolobular formation,tuberous sclerosis(8/12 weeks)-cancer cell infiltration,and liver cancer formation(16weeks).At the end of the 12 th week,cirrhosis was found in 3 rats in Maoshi group,wit h a hardening rate of 50.0%;in Youshi group,cirrhosis was found in 4 rats,with a hardening rate of 66.7%(P>0.05).At the end of the 16 th week,4rats in Maoshi group were found canceration,the canceration rate was 33.3%,7 rats in Youshi group was found canceration,the c anceration rate was 58.6%,there was significant difference between the two gro ups(P<0.05).4.Effects of DEN on serum ALT,AST,HA and γ-GT in rats The serum ALT,AST,HA,γ-GT in Mao and You groups increased significantly a t the end of the 4th week,which were higher than those in the normal group(P<0.05);with the increase of time,there was a significant upward trend(P<0.05).Compared with Youshi group,ALT,AST,HA,γ-GT in Maoshi group were lower at four time points.At the end of the 16 th week,the serum γ-GT in Maoshi gro up was significantly lower than that in Youshi group(P<0.05).5.Effects of DEN on PCNA protein,serum TGF-β1 and AFP in rats At the end of the 4th,8th and 12 th week,the levels of PCNA protein,serum TG F-β1 and AFP in the two groups were significantly higher than those in the no rmal group(P<0.05).Compared with Youshi group,PCNA,TGF-β1 and AFP in liver tissue of Maoshi group were lower at four time points,but the difference was not statisticallysign ificant(P>0.05).The serum TGF-β1 in Maoshi group was significantly higheratt heend of the 8th week,and that in Youshi group was significantly higher at the end of the 4th week.6.Effect of DEN on the expression of CYP2E1 protein and m RNA inrat liver The expression of CYP2E1 protein and m RNA in the liver of the two groups increased significantly at the end of the 4th week,which was higher than that of the normal group at the same time(P<0.05).Compared with Youshi group,the expression of CYP2E1 protein and m RNA in Maoshi group was lower at four time points.The expression of CYP2E1 m RN Ain Maoshi group was significantly lower than that in Youshi group at the en d of the 12 th week(P<0.05);the expression of CYP2E1 protein in Maoshi group was significantly lower than that in Youshi group at the end of the 16 th w-eek(P<0.05).7.Effects of DEN on ROS,MDA,8-OHd G,SOD and GSH-Px in liver of rats ROS,MDA and 8-OHd G in liver of rats in Mao and You groups increased sig nificantly at the end of the 4th week,which were higher than those of rats in t-he same period(P<0.05).Compared with Youshi group,ROS,MDA and 8-OHd G in liver tissue of Maoshi group were lower at four time points,and ROS in liver tissue of Maoshi group was significantly lower than that of Youshi group at the end of 12 th and 16 th week(P<0.05).The serum SOD and GSH-Px in Mao and You groups decreased significantly a-t the end of the 4th week,which were significantly lower than those in the n-ormal group at the same time(P<0.05),but with the increase of time,the chan ges of SOD and GSH-Px were not significant(P>0.05).Compared with Youshi group,the serum SOD and GSH-Px of Maoshi group were slightly higher at four time points,but the difference was not statisticallysignificant(P>0.05).8.Effect of DEN on the expression of NF-κB-p65 protein and m RNA in rat li-ver The expression of NF-κB-p65 protein and m RNA in the liver of the two grou ps increased significantly at the end of the 4th week,both higher than that of t-he normal group(P<0.05),with the increase of time,there is a significant upwa rdtrend(P<0.05).Compared with Youshi group,the expression of NF-κB-p65 protein and m RNA in liver tissue of Maoshi group was lower at four time points,but the differen-ce was not statistically significant(P>.0.05).9.Effects of selective administration of den on serum IL-6 and TNF-α in rats The serum levels of IL-6 and TNF-α in the two groups increased significantly at the end of the 4th and 8th week,both higher than those in the normal group at the same time(P<0.05).Compared with Youshi group,serum IL-6 and TNF-α in Maoshi group were slightly lower at four time points,but the difference was not statistically significan-t(P>0.05).Conclusion:1.The susceptibility of diethylnitrosamine to hepatoma in rats was different at d-ifferent time.2.The difference of the susceptibility of DEN to hepatoma may be related to the activation of CYP2E1-ROS/NF-κB signaling pathway.3.It is suggested that in daily life,we can reduce the damage of nitrosamine to liver and prevent the occurrence of liver cancer by adjusting the time of eating foods containing nitrite or using antioxidants. |
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