| Objective: Irinotecan(CPT-11)is a kind of Chemotherapy drug commonly used for clinical large intestine cancer.Its adverse reaction named late-onset diarrhea is a dose-limiting toxic reaction,which seriously affects patient's quality of life and the tolerance to chemotherapy.The purpose of this study are(1)to establish the model of late-onset diarrhea induced by CPT-11 in mice;(2)to investigate whether the administration of Xiaochaihu Decoction(XD)has a preventive effect on the late-onset diarrhea model induced by irinotecan;(3)to determine the contents of irinotecan and its metabolites and active ingredients in XD by ultra-performance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS),and to explore the material basis of XD acting on delayed diarrhea to lay the foundation for mechanism research.Methods: 1.Establishing the model of late-onset diarrhea in mice: 20 healthy male C57BL6 mice were randomly divided into model group(n = 10)and normal group(n = 10)after fed with high-fat diet three weeks.The mice in the model group were given continuous intraperitoneal injection of CPT-11 once a day for 7 days.And the dose is 50 mg/kg.The mice in the normal group were given intraperitoneal injection of the same amount of solvent.During the experiment,we observed mice's anus,took pictures,collected feces for determining bloody stool by urine stool occult blood kit everyday.2.XD intervention reseach: 40 healthy male C57BL6 mice were randomly divided into 4 groups after fed with high-fat diet three weeks,and each group is 10 mice.They are normal control group(equal volume of physiological saline),late-onset model group(equal volume of physiological saline),XD group(1500 mg·kg-1·d-1)and loperamide group(0.5333 mg·kg-1·d-1),which was given intragastric administration once a day for 17 days.Mice except normal control group were given intraperitoneal injection of irinotecan for 7 days to induce delayed diarrhea.At the 0th,4th,6th,8th and 10 th days after injection of CPT-11,venous blood was collected from tail at 2 h after injection and was placed in the Pre-coated EP tube with heparin.Body weight changes in mice were observed.Feces were collected for determining bloody stool by urine stool occult blood kit everyday.On the 18 th day,all mice were anesthetized with diethyl ether.Their heart blood was taken and placed in the Pre-coated eppendorf(EP)tube with heparin to look for metabolites of Irinotecan and determine the content of effective ingredients of XD.And then,We executed mice to take their liver,kidney,large intestine,small intestine.Those samples are divided into two parts to reserve for different purpose.One part of them was frozen at-80 refrigerator for determining the content of the ?drug in tissues.Another was stored in formalin solution for making pathological sections,analyzing tissue injury.Results: 1.The establishment of the model of late-onset diarrhea in mice:(1)Compared with normal group mice,model group mice's fur is foul,damp and agglomeratic.Their bodies trembled and didn't struggle when they were caught.The anus is black,red and swollen.The weight of the mice(2)in normal group increased 0.57±041g,while the model group decreased by 5.98±2.08 g.(3)The diarrhea rate of mice in normal group was 0.After successive administration for 7 days,the diarrhea rate of mice in model group was 100%(P<0.01).2.XD intervention research:(1)Weight comparison: On the 1th day and the 4th day,there was no significant difference in body weight of mice(P>0.05).On the 10 th day,the mice in the normal control group were higher than other groups(P<0.05).It indicates that late-onset diarrhea model can significantly reduce the body weight of mice.On the 17 th day,the mice in the normal control group were still higher than other groups(P<0.05),but the body weight of XD group was higher than model control group(P<0.05),It suggests that XD can reduce the symptom of mice losing weight in a degree.(2)Bloody stool rate:The bloody stool rates of normal control group,model control group,XD group and loperamide group are 0%,100%,50% and 60% respectively.On the 8th day to 17 th day,The bloody stool rates of mice in XD group and loperamide group were lower than model control group(P<0.01),and the bloody stool rate of mice in XD group was lower than loperamide group(P<0.05);(3)Histopathological observation: The large intestine and small intestine were intact in the normal control group.Mucosal epithelial cells of large intestine and small intestine in model control group were accompanied with diffuse necrosis significantly and inflammatory cell infiltration.Mucosa tissues of mice intestine in XD group and loperamide group became normal.Continuous injection of CPT-11 for 7 days showed no obvious damage to the liver and kidney.3.Determination of drug concentration:(1)CPT-11 and its metabolites: After the mice in XD group were injected with CPT-11 for the 6th days,their plasma-drug concentration of CPT-11 was highest,it had no significant difference with the highest concentration of the control group on the 4th day(P<0.05);The maximum concentration of SN-38 in the model control group was 7 times higher than the experimental group;The plasma-drug concentration of SN-38 G in the model control group was the highest on the 4th day after injection of CPT-11,while the XD group was significantly higher than the model control group on sixth days when it's plasma-drug concentration was highest(P<0.05).Except for the 4th days,the plasma-drug concentration of the experimental group was lower than that of the control group,the plasma-drug concentration of the experimental group was higher than that of the control group on other days.In the model control group,the concentration of CPT-11 in the tissues according to the order is small intestine> liver> kidney> large intestine,the concentration of SN-38 and SN-38 G in the tissues according to the order is small intestine > kidney > large intestine > liver.In the control group,CPT-11,SN-38 and SN-38 G were the most distributed in the small intestine(P<0.05).In the XD group,the concentration of CPT-11 in the tissues according to the order is liver > kidney > small intestine > large intestine,the concentration of SN-38 and SN-38 G in the tissues according to the order is liver > small intestine > kidney > large intestine.In the XD group,CPT-11,SN-38 and SN-38 G were the most distributed in the liver(P<0.05),The content of each component in the XD group was significantly less than that in the model control group.(2)17 effective components in XD: after injection of irinotecan,only 14 components were detected in the blood on the 0th,4th,6th days.Baicalin,quercetin and baicalein were below detection limit in blood while they could be detected in all tissues.It suggested that these three components have organ targeting.The plasma-drug concentration of glycyrrhizin acid is much higher than other components,only a small amount exists in organizations,it maybe that glycyrrhizicn acid was mainly metabolized in the blood.In the liver,the concentration of baicalein was the highest(21700.7430 ng/mL),but the concentration of saikosaponin A is the lowest(12.4930 ng/mL).In the kidney,the concentration of ginsenoside Rd was the highest(21563.0884 ng/mL),and the content of saikosaponin A was the lowest(20.7965 ng/mL).In the large intestine,the concentration of baicalein was the highest(26370.0697 ng/mL),and the content of saikosaponin A was the lowest(3.7625 ng/mL).In the small intestine,the concentration of ginsenoside Rd was the highest(22622.3167 ng/mL),and the content of ginsenoside Rg1 was the lowest(45.0834 ng/mL).Conclusion: 1.After C57BL6 mice which were first used were fed with high-fat diet three weeks,the mice were given continuous intraperitoneal injection of CPT-11 once a day for 7 days,the dose is 50 mg/kg.The model of late-onset diarrhea induced by CPT-11 could be established successfully.2.XD can prevent bloody stool of mice caused by CPT-11,make weight of mice with delayed diarrhea grow normally,lower blood stool rate,improve intestinal tissue damage.3.XD contains 17 kinds of active ingredients.It mainly involved in the metabolism of SN-38,can inhibite bile efflux of CPT-11,SN-38,SN-38 G and speed up the elimination of these three substances.Enterohepatic circulation of CPT-11 can cause late-onset diarrhea.The prevention of late-onset diarrhea caused by irinotecan is a combinational interaction of multiple target organs,which may be related to the inhibition of enterohepatic circulation,and it can provide a reference for the follow-up mechanism. |
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