The Role Of Heme Oxygenase-1 In Indomethacin-Induced Small Intestinal Injury Using In Vitro Enteroids Technique

Background and objective: Non-steroidal anti-inflammatory drugs(NSIADs)may cause many side effects,most commonly in gastrointestinal tract.Recently,NSAIDsinduced small intestinal lesions have drawn particular attention as a result of the application of the capsule endoscope and double-balloon endoscope.However,the pathogenesis of NSAIDs-induced enteropathy remains unclear and the effective strategies for their treatment and prevention are urgent.Enteroids are considered to be the best tools for studies on intestinal epithelium in vitro and have a widely application prospects,however,there are few associated reports in China.This study aims to establish and optimize the culture technique for mouse enteroids and explore the role of heme oxygenase-1(HO-1)in NSAIDs-induced small intestinal injury using in vitro enteroids technique.Methods: The conditioned medium of L-WRN cells was collected,the crypts of mouse were digested with EDTA and then collected and embedded in Matrigel Matrix,after polymerization of Matrigel Matrix,the crypts were cultured in L-WRN conditioned medium at different concentration gradient.The viability of enteroids exposed to indomethacin were measured using MTT staining.The differentially expressed genes(DEGs)between the control group and the experimental group were identified using Illumia Hiseq sequencing systems.The functions and KEGG pathways of DEGs were mapped in Gene Ontology and KEGG pathway database,respectively.The viability of enteroids exposed to indomethacin at the indicated time points were measured using MTT staining,after pre-treated with the HO-1 inducer,hemin or the HO-1 inhibitor,Zn PP-IX.Results: Compared with L-WRN conditioned medium containing 20% FBS,the conditioned medium containing 10% FBS was more favorable for enteroids culture in vitro.When conditioned medium accounted for 10%,15%,20%,25% or 30% of the mixed medium,they all promoted the growth of enteroids and the 15% one seemed toyield better result.Indomethacin inhibited the growth of enteroids in a dose-dependent and time-dependent fashion.1079 differentially expressed genes between the two groups were uncovered.In the experiment group,726 m RNA were up-regulated and 353 m RNA were down-regulated.For cellular component,several GO terms were identified,especially those associated with microbody and peroxisome and the GO terms for biological process with DEGs were mostly in the category of organic acid metabolic process,oxoacid metabolic process and carboxylic acid metabolic process.Most of the molecular functional GO terms associated with DEGs fell into the category of oxidoreductase activity,glutathione transferase activity and carboxylic acid binding.The KEGG pathway enrichment analyses revealed that the DEGs were primarily associated with chemical carcinogenesis,metabolism of xenobiotics by cytochrome P450,drug metabolism-cytochrome P450,PPAR signaling pathway.Indomethacin induced an increase in HO-1 expression in enteroids.Pre-treated with hemin could enhance the viability of enteroids exposure to indomethacin.In contrast,Zn PP-IX had the opposite effect.Conclusions: We are one of the institutes which take the lead in establishing an enteroids culture technique in China.When the L-WRN conditioned medium containing 10% FBS accounting for 15% of the mixed medium,it might promote budding better than the others.Indomethacin inhibited the growth of enteroids and induced an increase in HO-1 expression in enteroids.Induction of HO-1 protected enteroids against indomethacin-induced injury.