The Effect Of Bile Acid Malabsorption Associated With Ileal Asbt On The Delay-onset Diarrhea Induced By Irinotecan In Mice

Aims(1)To investigate the mechanism of delay-onset diarrhea induced by Irinotecan(CPT-11)based on bile acid malabsorption related to ileal apical sodium-dependent bile acid transporter(Asbt)in mice.(2)To investigate the regulatory effect and the underlying mechanism of CPT-11 on the expression or function of Asbt via Vitamin D Receptor(VDR)/ retinoic X receptor-?(RXR-?)and retinoic acid receptor-?(Rar-?)/ Rxr-? signaling pathway.Methods(1)Mice were randomly divided into control group and CPT-11 group.CPT-11(40mg/kg/d)or vehicles were administered to mice intravenously once a day for one day,two days,three days and four days.The total bile acid(TBA)levels in the small intestine,colon,feces,liver,serum and gallbladder were evaluated by automatic biochemical analyzer,and the individual bile acids were measured by LC-MS/MS.The intestinal reabsorption ratio of bile acids was evaluated by the concentrations of representive bile acids in the ileum or the colon to that in the bile.(2)The protein or m RNA expression of hepatic cholesterol 7-alpha hydroxylase(Cyp7a1)or sterol 27-hydroxylase(Cyp27a1),ileal Asbt/ASBT or organic solute transporters ?/?(Ost?/?)were evaluated by Western Blot or RT-q PCR techniques in mice or Caco-2 cells.In situ loop method was also carried out to evaluate the absorption ability of ileum.In situ loop experiment was conduced to evaluate the function of Asbt,and the transporting rate was calculated by the ratio of ileal T-CDCA concentration to the Asbt relative expression.The tauro-cholic acid(T-CA)uptake was also determined in Caco-2 cells to evaluate the function of ASBT.(3)Mice were randomly divided into control group,CPT-11 group,and CPT-11+Seq group.Mice in CPT-11+Seq group were co-administrated i.g.with bile acid sequestrant,cholestyramine(0.8g/kg/d)for four days since the third experimental day.The body weight and severity of diarrhea were monitored and calculated daily,and the colon tissue was harvested for pathological evaluation.(4)The effects of CPT-11 or SN-38 on protein or m RNA expression of Vdr/VDR,Rar-?/RAR-?,and Rxr-?/RXR-? were evaluated by Western Blot or RT-q PCR techniques in mice or Caco-2 cells.Mice were co-administrated with Vdr agonist 1,25-dihydroxyvitamin D or Rar-? agonist 9-cis retinoic acid in order to investigate the effect of co-administration with agonists on the body weight and severity of diarrhea.Results(1)Compared with control group,the bile acid pool size and ileal TBA levels were decreased at 2,3,and 4 days post CPT-11 treatment in mice(P<0.05,P<0.05,P<0.01);whereas the TBA levels in the ileum were decreased at 2,3,and 4 days(P<0.05,P<0.01,P<0.01);the TBA levels in the colon were increased at 3 and 4 days(P<0.05,P<0.05);the TBA levels in the feces were increased at 3 and 4 days(P<0.05,P<0.05);the TBA levels in the liver and the bile were also increased.In the ileum,the concentrations of T-CA,tauro-?-muricholic acid(T-?-MCA),chenodeoxycholic acid(CDCA),and cholic acid(CA)were significantly decreased after CPT-11 treatment when compared to that of the control group.In the colon,the concentrations of deoxycholic acid(DCA)were increased at 2,3,and 4 days post CPT-11 treatment.In the feces,the concentrations of DCA and T-CA were increased at 3 and 4 days post CPT-11 treatment.(2)The expression of hepatic Cyp7a1 showed no difference in mice between control group and CPT-11 group,whereas the the expression of hepatic Cyp27a1 was increased after CPT-11 treatment.The protein expression of ileal Asbt was decreased at 4 days post CPT-11 treatment(P<0.05),and the m RNA levels were decreased at 3 and 4 days post CPT-11 treatment(P<0.05,P<0.05).In Caco-2 cells,the protein expression of mature ASBT was decreased whereas the immature ASBT was increased after incubation with CPT-11.In situ loop experiment,the tauro-chenodeoxycholic acid(T-CDCA)concentration in CPT-11-treated group was reduced when compared to that of control group(P<0.05),and the transporting rates of Asbt were also decreased at 3 and 4 days post CPT-11 treatment(P<0.05,P<0.05).Data from T-CA uptake test also showed that the T-CA uptake was reduced after CPT-11 or SN-38 treatment in Caco-2 cells.(3)CPT-11 treatment significantly induced body weight loss in mice,and this alteration could not be reversed by cholestyramine co-administration.However,cholestyramine co-administration significantly ameliorated the colonic pathology,severity of diarrhea,and the mortalityin mice.(4)The expression of VDR,RAR-? and RXR-? were decreased after 12 h and 24 h incubation with CPT-11 in Caco-2 cells.The expression of VDR didn't show any difference after SN-38 treatment,but the expression of RAR-? in 5.0 ?M group(P<0.05,P<0.01),10.0 ?M group(P<0.05,P<0.05),20.0 ?M group(P<0.05,P<0.01)were decreased after 12 h and 24 h incubation with SN-38,and the expression of RXR-? in 2.5 ?M group(P<0.05),5.0 ?M group(P<0.01),10.0 ?M group(P<0.01),20.0 ?M group(P<0.01)were decreased after 24 h incubation with SN-38 in Caco-2 cells.The ileal expression of Vdr were decreased at 2(P<0.05),3(P<0.01),and 4(P<0.01)days post CPT-11 treatment,and the expression of Rxr-? were decreased at 4 days post CPT-11 treatment in mice(P<0.01).However,the expression of Rar-? in mice were increased at 2(P<0.05)and 3(P<0.01)days post CPT-11 treatment.The m RNA expression of nuclear receptors were consistent well with protein expression.Co-administration with Vdr or Rar-? agonists in mice reversed CPT-11-induced body weight loss and the severity of diarrhea.Conclusion(1)This study showed that the reduced expression or function of Asbt attributed to CPT-11-induced bile acid malabsorption,which is one of the main mechanisms of CPT-11-induced diarrhea.(2)CPT-11 and/or SN-38 decreased the expression or function of Asbt via regulating VDR/RXR-?(Vdr/Rxr-?)or RAR-?/RXR-?(Rar-?/Rxr-?)signaling pathway,and the decreased expression or function of Asbt could lead to bile acid malabsorption and diarrhea.