| Prostaglandin E2 (PGE2) is the major products generated by the action of cyclooxygenase (COX) on arachidonic acid and it is widely distributed in the body. PGE2 plays important roles in regulating the biological functions and development of many diseases through four G-protein-coupled receptors. The distribution of EP4 in the body is extensive. PGE2 can participate in keeping the normal physiological function of a living organism, such as immunoregulation, blood pressure regulation, reproduction and gastrointestinal hcmeostasis, and also can participate in the development process of many diseases, such as cardiovascular disease and cancers. However, till now, the comprehension of the physiological functions of EP4 is incomplete. The pioneer contributors studied the functions of EP4 in some diseases by employing the agonist, antagonist or transgenic animals. However, to our knowledges, report on the metabolic responses of a living organism to these kinds of treatments is rare. To investigate the endogenous metabolic responses of organisms to EP4 antagonist, we carried out the following studies:First, we studied the effects of DMSO exposure for 4 weeks on the systemic metabolism of mice. The results showed that DMSO intevention induced significant metabolic changes in urine, including the alterations of intestinal flora, the acceleration of choline metabolism and tricarboxylic acid cycle. Besides, the significant decrease of glucose and pyruvate in serum suggested that the energy metabolism was accelerated. Then the endogenous metabolism of multiple organs of mice, including spleen, kidney, myocardium and lung, were also affected by the treatment of DMSO. The metabolic responses of myocaridium was much more obvious than other organs, including the alterations of choline and amino acids metabolism. This research will provide metabonomics basic data for the studies of employing DMSO as drug carrier.Secend, the changes of metabolic profiles of mice biofluids (serum and urine) and liver to three dosages of EP4 antagonist were systematic investigated using NMR and GC-FID based metabonomics. In addition, transcriptomics profile was also used to investigate the variance of liver of high-dosed mice on transcriptome level. The results of high-dosed group showed that:1) The effects of three dosages of EP4 antagonist on serous and urinary metabolism were weak, and not dose-dependent and successive.2) The metabolic responses to moderate-and high-dose of EP4 antagonist were much stronger. The results of high-dose group showed that:(A) The results of GC-FID showed that the fatty acids in liver of mice treated with EP4 antagonist decreased dramaticlly. The significant changes of a series of genes related to fatty acids metabolism (INSIG2, SCD1, CD36, ACACB, RGS16 et al.) suggested that the acceleration of fatty acid oxidation together with the inhibition of fatty acid synthesis caused the deletion of fatty acids in liver. (B) Oxidative stress was incuded by the fatty acid oxidation in liver, which was verified by the changes of GSH and GSTM3 and SCAD in liver. (C) The choline metabolism was influenced by EP4 antagonist. The increased level of choline in liver and serum together with the up-regulated level of CHKA in liver indecated that the synthesis of phosphatidylcholine was inhibited and the catabolism of choline was enhanced. (D) The increased levels of uracil, xanthine and hypoxanthine together with the up-regulated expression level of UPP2 suggested that nucleotide catabolism was accelerated. The increased levels of AMP, ADP and UMP together with the down-regulated exprssion levels of CTPS2 and ADK indicated that the nucleotide anabolism was inhibited. (E) The expression levels of genes (RETSAT, MTHFR, NAMPT and NNMT) related to vitamin A, vitamin B3 and folate metabolism also changed in EP4 antagonsit treated group. (F) The metabolism of amino acids also changed; (G) The changs of enzymes (ANG and CYP2B9) related to the metabolism of arachidonate indicated that arachidonate pathway was also influenced.Third, metabolic responses of mice multiple organs, including kidney, spleen, myocardium and testicel to three dosages of EP4 antagonist were investigated. The metabolism of these organs were all affected by these three dosage, among which the moderate-dose of EP4 antagonist had much stronger influence. The EP4 antagonist intervention caused inhibition of glycolysis, cell membrane damage and distrubance of osmotic pressure to kideny, spleen and myocardium. Many amino acids in spleen and kidney decreased dramaticlly. These results showed that systemic metabolic changes taken place in multiple organs, induing energy, amino acids, choline and nucleotide metabolism. These changes of metabolism were probable due to the interference of EP4 to exert its physiological functions normally.In summary, by combination of NMR and GC-FID based metabonomics and transcriptomics methods, we comprehensively investigated the metabolic changes of mice fluid and multiple orgens to EP4 antagonist intervention, which can deepen our understanding of the biological functions of EP4 and provide clues for the discovery of new functions associated with EP4. |
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