Effect And Mechanism Of IL-6 On Down-regulation Of Liver Carboxylesterases In Mice With Ulcerative Colitis

Aims:(1)To establish a LC-MS/MS analysis method for the metabolites CCAM and SN-38 of CESs in the mice hepatic microsome incubation system;(2)To evaluate the expression of hepatic CESs in the DSS-induced UC mice;(3)To study the effect and mechanism of IL-6 in down-regulating hepatic CESs in DSS-induced UC mice.Methods:(1)The first part: to establish the LC-MS/MS analysis methods of CCAM and SN-38 in the hepatic microsome incubation system.The in vitro culture conditions of clopidogrel and CPT-11 in mouse hepatic microsomes were optimized.The liquidliquid extraction method was used to process the liver microsome incubation system samples.CCAM was separated on a Ecosil ODS(2.1 mm×150 mm,5μm)column with an isocratic mobile phase consisted of 5 m M ammonium formate and 0.1% formic acid in water and acetonitrile(85:15,V:V)at a flow rate of 0.4m L/min.SN-38 was separated on a Waters symmetry C18(2.1 mm × 150 mm,3.5 μm)column;the mobile phase consisted of 5 m M ammonium formate and 0.1% formic acid in water(A)and acetonitrile(B)at a flow rate of 0.3m L/min and eluted with a gradient as follows: 0-1min,10%B;1-5min,90%B;5-7min,10%B.The injection volume is 5μL and the column temperature is 40°C.The mass spectrometry conditions are ESI,positive ion mode(+)and MRM.The MS parameters for analytes are follow: m/z 308.0→198.0 for CCAM;264.1 → 154.1 for ticlopidine;393.2 → 349.2 for SN-38;349.1 → 305.2 for camptothecin.(2)The second part: The mouse model of UC was established with 2%DSS solution.Its disease activity index(DAI),colon length,liver and spleen weight were measured;and histopathological examination was performed.Measuring the levels of ALT,AST,CRP and LPS of serum,the activity of MPO of colon,and detected the TNF-α,IL-1β and IL-6 levels of serum,hepatic and colonic.At the same time,the m RNA,protein expression and activity levels of CES1 and CES2 in the liver were detected respectively.(3)The three part: The mouse model of UC was established with 2%DSS solution,and In Vivo anti-mouse IL-6 antibody was injected through tail vein.Measuring DAI,liver weight,spleen weight and colon length;and conducting histopathological examination.Detecting the m RNA,protein expression level and metabolic activity of hepatic CES1 and CES2.And further detecting the protein expression of PXR,CAR,FXR,PPAR-α and NF-k B p65.Hepa RG cells were cultured in vitro to explore the optimal dose and time of down-regulation of CESs induced by IL-6 through different concentrations and times.After intervention with IL-6 and NF-κB inhibitor PDTC,the protein expression levels of CES1,CES2,PXR and CAR in cells were detected;the hydrolytic activity of CES1 and CES2 was determined by cytotoxicity assay.Results:(1)The first part: the established CCAM and SN-38 concentration test methods are stable and specific.The quantitative ranges are 100-20000ng/m L and 1-200 ng/m L,respectively.The intra-day and inter-day accuracy of CCAM were 91.00%-110.60%,and the RSD of intra-day and inter-day precision was 2.25% and 11.55%.The intra-day and inter-day accuracy of SN-38 was 92.75%-110.77%,and the RSD of intra-day and inter-day precision was 1.75%-8.97.The recovery rate,matrix effect and stability inspection results meet the requirements for quantitative analysis of biological samples in the 2020 edition of the Chinese Pharmacopoeia.(2)The second part: the DAI index,spleen weight and liver to body weight ratio were increased in DSS group,but the colon was shortened.Colonic MPO activity were significantly increased in the DSS group;at the same time,the levels of ALT,AST in the serum were increased in the DSS group;HE staining showed that the colon and liver of the DSS group showed obvious pathological damage.Meanwhile,CRP and LPS in the serum were increased in the model group.TNF-α,IL-1β and IL-6 were increased in the serum and colon;but Only IL-6 in the liver increased significantly,which was consistent with the results of immunohistochemistry.At the same time,the m RNA,protein expression levels and metabolic activity of CES1 and CES2 in the liver of the DSS group were significantly down-regulated.(3)The three part: IL-6 antibody intervention can significantly improve the weight loss of mice caused by DSS,increase the length of the colon and reduce the ratio of spleen weight and liver to body weight ratio;also improve the pathological changes of colon and liver tissues.IL-6 antibody intervention can effectively reverse the downregulation of m RNA,protein level and metabolic activity of hepatic CES1,CES2;as well as PXR,CAR protein levels and reduce NF-κB p65 nuclear translocation.In vitro experiments confirmed that NF-κB inhibitors can significantly reverse the IL-6 induced down-regulation of CES1,CES2,PXR and CAR protein levels;meanwhile,reverse the IL-6 induced decrease of hydrolysis activity of CES1 and CES2.Conclusions:(1)In this study,the concentration determination methods of CCAM and SN-38 in the mouse hepatic microsomal incubation system were established respectively.This analysis method is simple and sensitive,which meets the requirements of biological sample analysis and is suitable for the determination of CCAM and SN-38 concentration in the incubation system.At the same time,this study optimized the in vitro incubation conditions for CES1 and CES2 in mouse hepatic microsomes through a single factor experimental design,which provided a basis for studying the changes in the metabolic activity of CESs in mouse liver.(2)The liver of UC mice was significantly damaged,and the m RNA protein expressions of CES1 and CES2 and the hydrolytic activity in the liver were significantly down-regulated,which may be closely related to the abnormally elevated IL-6 in the liver.(3)IL-6 inhibited the expression of hepatic PXR/CAR by activating the NF-κB signaling pathway in DSS-induced colitis,and then down-regulated the expression and metabolic activity of CES1 and CES2.