| The prevention and treatment of liver cancer is one of the important issues which need be solved urgently.Rhubarb as a traditional Chinese medicine is often used in treatment of gastrointestinal diseases and chronic liver diseases in clinical,including liver cancer.Modern pharmacological research shows that Rhubarb has a variety of pharmacological activities.In particular,the free anthraquinones from rhubarb have a protective effect on the liver,such as anti-hepatitis,liver fibrosis,liver cirrhosis and liver cancer.However,many studies have shown that Rhubarb can cause hepatotoxicity in recent years.The"two-sided"effect of Rhubarb on the liver become a topic that cannot be ignored,the safety and effectiveness of Rhubarb in the treatment of liver cancer and further evaluation is needed.Objective:The DEN-induced rat liver cancer model is one of the commonly model that used to simulate the occurrence and development of human liver cancer.This study aims to explore the role and mechanism which Low-dose Rhubarb promote DEN-induced liver cancer in rats.Methods:1.The effect of Low-dose Rhubarb on the DEN-induced precancerous injury of liver cancer in ratsThirty-two SPF SD rats were randomly divided into four groups:normal control group,Low-dose Rhubarb treatment group,DEN treatment group,and Low-dose rhubarb-DEN treatment group.a.Normal control group:SD rats were given 1 m L normal saline,4 times a week for 8 weeks,and from the 6th week,1 m L of normal saline was added to SD rats once a day for 3 weeks;b.DEN treatment group:SD rats were given 1m L DEN(0.002 g/mouse)4 times a week for 8 weeks,and from the 6th week,1m L normal saline were added once a day for continuous 3 weeks;c.Low-dose Rhubarb treatment group:SD rats were given 1 m L normal saline,4 times a week for 8 weeks,and from the 6th week,SD rats were given Rhubarb(4g/Kg)every day,for 3 weeks;d.Low-dose Rhubarb-DEN treatment group:SD rats were gavaged with 1m L DEN(0.002 g/mouse),4 times a week for 8 consecutive weeks,and from the 6th week,with SD rats were given Rhubarb(4g/Kg)every day for 3 weeks.HE staining and Masson staining were used to observe the pathological changes of rat liver tissues in each group.The enzyme activity detection method was used to evaluate the liver function(serum ALT activity)of rats in each group.Western Blot method was used to detect the level of fibrosis marker molecule(α-SMA)in rat’s liver.2.The effect of Low-dose Rhubarb on the DEN-induced liver cancer in ratsThirty-two SPF SD rats were randomly divided into four groups:normal control group,Low-dose Rhubarb treatment group,DEN treatment group,and Low-dose Rhubarb-DEN treatment group.a.Normal control group:SD rats were given 1 m L normal saline,4 times a week for 17 weeks,and from the 12th week,1 m L normal saline was added to SD rats once a day for 6 weeks;b.DEN treatment group:SD rats were given 1 m L DEN(0.002 g/mouse),4 times a week for 17 weeks,and from the 12th week,1 m L of normal saline were added to SD rats once a day for 6 weeks.c.Low-dose Rhubarb treatment group:SD rats were gavaged with 1 m L normal saline,4 times a week for 17 weeks,and from the 12th week,SD rats were gavage with rhubarb(4g/Kg)every day for 6 weeks;d.Low-dose Rhubarb-DEN treatment group:SD rats were given1m L DEN(0.002 g/mouse)by gavage,4 times a week for 17 weeks,and from the 12th week,SD rats were gavage with Rhubarb(4g/Kg)every day for 6 weeks.HE staining and Sirius Scarlet staining were used to observe the pathological changes of rat liver tissues in each group.Immunohistochemical technique was used to detect the level of liver cancer marker molecules(CD34~+).The enzyme activity detection method was used to evaluate the liver function(serum ALT activity)of rats in each group.Western Blot method was used to detect the level of liver cancer marker molecules(AFP)in rat liver tissues.3.Metabonomics analyze that glucose metabolism in the rat liver tissue treated with Low-dose Rhubarb-DENIn the stage of precancerous injury,the livers of each group of rats were taken out,and the changes of glucose metabolism in the each group rat liver were analyzed by UHPLC-Q-TOF MS combined technology,and we screen different metabolites through comparing normal control group vs Low-dose Rhubarb-DEN treatment group,Low-dose Rhubarb-DEN treatment group vs Low-dose Rhubarb treatment group,and DEN treatment group vs Low-dose Rhubarb-DEN treatment group each group,and we use KEGG metabolic pathway to analyze metabolites.4.The role of glucose metabolism reprogramming in Low-dose rhubarb in promoting the occurrence of DEN-induced liver cancer in ratsThe enzyme activity detection method was used to evaluate the levels of lactic acid and isocitrate dehydrogenase in the liver tissues of the normal control group,Low-dose rhubarb treatment group,DEN treatment group,and Low-dose rhubarb-DEN treatment group.Western Blot The method was used to detect the levels of glycometabolism reprogramming marker molecules(Erk1/2 and HIF-1α)in the liver tissues of 4 groups of rats.Intervention with glycometabolism reprogramming inhibitor(resveratrol)to detect the effect of Low-dose rhubarb on DEN-induced precancerous injury of liver cancer in rats(HE staining,liver function assessment andα-SMA level detection).Results:1.Precancerous injury stage of liver cancer:HE staining results show that compared with DEN treatment group,liver tissue of Low-dose Rhubarb-DEN treatment group has more serious pathological changes;Masson staining andα-SMA level detection results show that the degree of Low-dose Rhubarb-DEN treatment group liver fibrosis of the rats was higher;the results of serum ALT levels showed that the liver function damage of the low-dose Rhubarb-DEN treatment group rats was more significant.These findings indicate that Low-dose Rhubarb promotes the precancerous injury of DEN-induced liver cancer in rats.2.Liver cancer stage:HE staining results showed that compared with DEN treatment group,liver tissue of Low-dose Rhubarb-DEN treatment group showed more serious pathological changes;Sirius scarlet staining results showed that the liver fibrosis symptom of Low-dose Rhubarb-DEN treatment group rat was higher;the results of serum ALT levels showed that the liver function damage of rats in the Low-dose Rhubarb-DEN treatment group was more significant;the results of immunohistochemistry and Western Blot showed that the level of CD34+and AFP in Low-dose Rhubarb-DEN treatment group rat liver tissue also increased significantly.These findings indicate that Low-dose Rhubarb promotes the occurrence of DEN-induced liver cancer in rats.3.The analysis of non-targeted metabolomics technology showed that:(1)Between the normal control group and the Low-dose Rhubarb-DEN treatment group,97 different metabolites were screened out,which 15 different metabolites were related to glucose metabolism.The results show that these differential metabolites are involved in the TCA cycle,pentose and glucuronate interconversions,HIF-1 signaling pathway,glycolysis,cancer center carbon metabolism,carbohydrate digestion and absorption,and pentose phosphate pathway based on the analysis of the KEEG metabolic pathway;(2)65different metabolites were screened between the Low-dose Rhubarb-DEN treatment group and the Low-dose Rhubarb-DEN treatment group,which 8 different metabolites were related to glucose metabolism;these differential metabolisms are involved in the TCA cycle,pentose and glucuronate interconversions,HIF-1 signal transduction pathway,glycolysis,cancer center carbon generation metabolism based on the KEEG metabolic pathway analysis;(3)43 different metabolites were screened out among the DEN treatment group and the Low-dose Rhubarb-DEN treatment group,which 9 different metabolites are related to glucose metabolism,these differential metabolites involve in pentose and glucuronate interconversions,HIF-1 signal transduction pathway,glycolysis,cancer center carbon metabolism and based on the results of KEEG metabolic pathway analysis.These results indicate that,glucose metabolism reprogramming(enhanced glycolysis and reduced aerobic oxidation of glucose)was significantly enhanced in the liver tissues of rats treated with Low-dose Rhubarb-DEN.4.We further explore the role of glucose metabolism reprogramming in Low-dose Rhubarb promote DEN-induced precancerous injury of liver cancer in rats.Biochemical experiments(enzyme activity detection and Western Blot)once again proved that Low-dose Rhubarb-DEN treatment reprograms glucose metabolism of rat liver tissue.The inhibitor of glycometabolism reprogramming(resveratrol)can significantly weaken the promoting effect of Low-dose Rhubarb on the precancerous injury of DEN-induced liver cancer in rats.Conclusion:Low-dose Rhubarb can aggravate DEN-induced precancerous injury and liver cancer in rats;Low-dose Rhubarb can promote DEN-induced glucose metabolism reprogramming in rat liver tissues(enhanced glycolysis and reduced aerobic oxidation of glucose);and glucose metabolism reprogramming is closely related to Low-dose Rhubarb promote the DEN-induced liver cancer.Our research shows that the hepatotoxicity of Rhubarb cannot be ignored,and its effectiveness and safety in the treatment of liver cancer require more in-depth research. |
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