| Background:Methotrexate(MTX)is a typical chemotherapeutic drug that is widely used in the treatment of various malignant diseases as well as autoimmune diseases.Among them,gastrointestinal toxicity is a complication that commonly occurs when using chemical drugs and has a significant impact on the prognosis of patients,especially when used in a large doses.Therefore,Eliminating or reducing this complication will have an important clinical significance for the dose-limiting application of MTX.Aims:This study will explore that the effects of 30% dietary restriction(DR)on the survive rate and intestinal function of mice after lethal dose of MTX,compared with ad libitum(AL),as well as the changes and regulatory effects of gut microbiota.Methods:Two-month-old female C57BL/6J mice are fed with AL diet(Ad libitum)or DR diet(Dietary restriction)for 14 days,Then,mice were intraperitoneally injected with saline as control or MTX for 2 days at a dose of 120mg/kg(d-1)and 60mg/kg(d0).survival and percent change body weight change were monitored after MTX administration,and the intestinal tissues of the surviving mice on day 3 and day 6 after MTX administration were taken for section staining,histological analysis,crypt culture,qRT-PCR,and intestinal contents for 16 SrRNA sequencing etc.To analyze the Effect of DR on the survive rate and intestinal of mice after lethal doses of MTX chemotherapy.Two-month-old female C57BL/6J mice are fed with AL or DR for 14 days,mice were fed by gavage for 5 days and then in drinking water for the following days with broad-spectrum antibiotics or saline.After that,all mice were intraperitoneally injected with MTX for 2 days at a dose of 120mg/kg(d-1)and 60mg/kg,survival and percent change body weight change were monitored after MTX administration,and the intestinal tissues of the surviving mice on day 3 and day 6 after MTX administration were taken for section staining,histological analysis,crypt culture,qRT-PCR,and intestinal contents for 16 SrRNA sequencing etc.To analyze the role of gut microbiota in DR.Two-month-old female C57BL/6J mice are fed with AL,mice were fed by gavage for 5 day with lactobacillus rhammosus(LGG)at a dose of 5×10^9 cfu per mice,and then all mice were intraperitoneally injected with MTX for 2 days at a dose of 120mg/kg(d-1)and 60mg/kg,survival and percent change body weight change were monitored after MTX administration,and the intestinal tissues of the surviving mice on the day 3 and day 6 after MTX administration were taken for section staining,histological analysis,crypt culture,qRT-PCR,etc.To analyze the influences of LGG on survive rate and intestine function after lethal doses of MTX chemotherapy Results:The survival rate of mice in the AL group was only about 10% after lethal doses of MTX chemotherapy,while the survival rate of mice in the DR group was more than 90%,therefore,DR remarkably increases survival rate of mice exposed to lethal doses of MTX,when using broad-spectrum antibiotics to inhibit the gut microbiota of DR,the survival rate less than 10%,accordingly,DR protect the damage caused by the lethal dose of MTX is related to the gut microbiota.To detect the gut microbiota of AL and DR by 16 S rRNA sequencing,compared with AL group,we found that the genus of Lactobacillus that significantly increased after DR.When Lactobacillus rhamnosus in the genus of Lactobacillus by gastric gavage to AL mice,it was found that the survival rate is 52%,which can partially mimic the rescue effect of DR.These results indicated that supplementation of LGG can partially mimic the rescue effect by DR upon MTX.But the rescue effect was less effective compared to DR.Conclusion:DR significantly increases the survival rate of mice after lethal-dose MTX treatment by regulating gut microbiota and increasing the composition of "protective" flora in the intestinal flora,The mechanism of action is that DR can remarkably reduce the intestine damage and maintain the function of ISCs,... |
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