A Mouse Model Simulating The Effects Of Four Drinking Patterns On Colorectal Cancer Liver Metastasis

BackgroundThe proportion of people who consume alcohol and have colorectal liver metastasis(CRLM)is very high worldwide.To our knowledge,no detailed and well-recognized model of the relationship between alcohol consumption and CRLM has been established before.The lack of a mature mouse model of the effect of alcohol intake on CRLM has led to a lack of studies on the relevant mechanisms.There are only a few reports of the effect of chronic alcohol intake on CRLM.However,these studies not appear to take into account the fact that humans have different alcohol drinking patterns.ObjectiveMaintaining the same number calories in the feed,we innovatively simulated four categories of people:abstainer,binge drinker,chronic drinker,and chronic+binge drinker.To study the effect of four drinking patterns on CRLM,we establish the"ALD+CRLM dual model".It may be of great reference value for exploring the effects of different drinking patterns and alcohol intake on liver metastasis of colorectal cancer.MethodsThe "abstainer" was mimicked by CLD for 41 days plus gavage of 45%(wt/vol)maltose dextrin solution twice a week.The "binge drinker" was mimicked by CLD for 41 days plus gavage of 31.5%(vol/vol)ethanol solution twice a week.The"chronic drinker" was mimicked by 5%ELD for 41 days plus gavage of 45%(wt/vol)maltose dextrin solution twice a week.The "chronic+binge drinker" was mimicked by 5%ELD for 41 days plus gavage of 31.5%(vol/vol)ethanol solution twice a week.At day 30,the mice were anesthetized with pentobarbital,a 1-cm incision was made in the left subcostal region,the spleen was exteriorized,and MC38 cells(5 ×105)in 0.1 ml PBS were injected into the spleen.After 1 minute,we removed the spleen with an electric knife.Homoeostasis was assured by ligation with a suture.The four groups of mice were fed the control diet to facilitate wound healing for three days after the operation,and then CLD and 5%ELD were administered.Another gavage was performed eight days after the operation.On the 12th day after the surgery,the mice were sacrificed.The mice were weighed and their livers were weighed and photographed.Blood was collected by the retro-orbital blood vessel and processing it into a serum.Serum was stored at-80℃until further analysis.The left lobe livers of all mice were fixed in formalin and were embedded in paraffin.For the remaining liver,hepatic and tumor tissue samples were dissected and flash frozen in liquid nitrogen and stored at-80℃ for biochemical and histological analyses.ResultsWe established a new mouse model to study the effect of alcohol on CRLM.Visual examination of the liver and H&E staining demonstrated metastatic liver tumor formation in mice sacrificed 12 days after surgery.After 41 days of different diets,alcoholic liver injury was confirmed hepatic steatosis,hepatic TG,the levels of CYP2E1 in liver,hepatic oxidative stress,and the concentrations of ALT,AST,TG,and CHOL in serum.We found that the "chronic drinker" group and the "chronic+binge" drinker group had more steatosis and liver triglyceride accumulation than the"binge drinker" group and the "abstainer" group.CYP2E1 is an enzyme related to alcohol metabolism in the liver.The content of enzymes in the "abstainer" group and the "binge drinker" group is significantly lower than that in the "chronic drinker"group and the "chronic+binge drinker" group.Compared with the "abstainer" group,all other groups have more ROS expression.Compared with the concentrations of ALT,AST,and TG in the "abstainer" group,that in the "chronic+binge drinker"groups significantly increased.Compared with the "abstainer" group,the degree of liver metastasis in the "binge drinker" group increased but did not reach statistical significance.Nevertheless,both"chronic drinker" and "chronic+binge drinker" groups showed a statistically significant increase in liver metastasis.Compared with the "binge drinker" group,the liver metastasis of colorectal cancer in the "chronic+ binge drinker" group was significantly greater.Compared with the "chronic drinker" group,the degree of liver metastasis in the "chronic+binge drinker" group showed a mild increase but did not reach statistical significance.ConclusionsIn summary,this suggests that long-term sustained alcohol intake promotes CRLM more than binge drinking.The "chronic drinker" group and "chronic+binge drinker" group had more significant alcoholic liver injury and degree of liver metastasis than "binge drinker" group and "abstainer" group.It suggest that the degree of alcoholic liver injury may be associated with CRLM.The steps of the"ALD+CRLM dual model" were optimized several times and were recorded in detail.The four animal models designed in this study can be used as ideal animal models to study the risk of alcohol consumption on liver metastasis of colorectal cancer.