Resveratrol Enhances The Protective Effect Of JBP485 Against Indomethacin-induced Small Intestine Injury In Vivo And In Vitro By Up-regulating Pept1

Part Ⅰ Development of LC-MS/MS methods for simultaneous determination of JBP485 and resveratrol in biological samplesObjective: To develop a sensitive,quick,specific and high performance LC-MS/MS method for simultaneous determination of JBP485 and resveratrol(Res).Methods: After protein precipitation,the analytes were separated on a Hypersil ODS-C18 column.The mobile phase consisted of 60%(v/v)acetonitrile,40%(v/v)water with 0.1% formic acid.The ionization was conducted using a TurboIonspray interface in positive ion mode for JBP485 and resveratrol.The selected transitions of m/z were m/z 201.1→86.1 for JBP485,m/z 229.1→107 for resveratrol,and m/z 309.2→120.0 for bestatin(IS).Results: The quantitative result was linear in the concentration range of 1.00 to 1000.00 ng/ml for JBP485 and resveratrol.This method had good precision and accuracy.Conclusions: We developed and validated a LC/MS/MS method to detect the concentration of JBP485 and resveratrol in biological samples.Part Ⅱ Resveratrol enhances the protective effect of JBP485 against indomethacin-induced small intestine injury in vivo and in vitro by up-regulating Pept1Objective: The purpose of present study was to assess the effect of resveratrol(Res)on the protective effect of JBP485 against indomethacin-induced intestinal injury in rats and in IEC-6 cells.Methods: Intestinal injury model was established by intragastric administration of indomethacin(14 days,1.5 mg / kg,twice a day).We recorded the diarrhea of each group of rats.We anesthetized rats,taken the jejunum to observe the ulcer,conducted HE staining and measured the concentrations of PGE2 and HDL-c.Concentrations or activities of GSH,SOD,MDA and CAT were measured to identify the effect of resveratrol on antioxidant capacity of JBP485 in rats and IEC-6 cells.The MPO activity and the mRNA levels of IL-6,TNF-α and IL-1β in small intestine were measured to evaluated the effect of resveratrol on the anti-inflammatory ability of JBP485.TUNEL assay and hoechst 33342 staining was used to identify the effect of resveratrol on the anti-apoptotic ability of JBP485.Western blotting assay was used to investigate the expression of Bax,Bcl-2 COX1/2 and Pept1.In vivo absorption studies,in situ jejunal perfusion technique and uptake assay in IEC-6 cells were used to investigate the effect of resveratrol on intestinal absorption and intracellular accumulation of JBP485.Results: JBP485 treatment ameliorated indomethacin-induced enteropathy characterized by severe histopathological changes and a large number of TUNEL-positive cells in rat intestinal epithelium,elevated levels of MDA,MPO,TNF-α,IL-6,IL-1β and increased expression of COX-2,and reduced levels of SOD,GSH,CAT,HDL-c,PGE2,expression of COX-1 and Bcl-2/Bax ratio in rat intestinal tissues.Co-treatment with Res enhanced activity of JBP485 against indomethacin-induced intestinal injury in rats and in IEC-6 cells.Furthermore,Res increased the plasma concentration of JBP485 in both indomethacin-treated and healthy rats,as well as the intracellular accumulation of JBP485 in IEC-6 cells,through upregulation of Pept1.Conclusions: JBP485 alleviated indomethacin-induced intestinal injury through restoring the redox balance,suppressing inflammation,reducing apoptosis,which was further enhanced by Res via upregulation of Pept1 and improvement of bioavailability of JBP485,at least in part.