Protection Of Ferulic Acid On Lipopolysaccharide-induced Preterm Delivery And Glucuronidation And Sulfation Of Ferulic Acid

Lipopolysaccharide (LPS) is a toxic component of cell walls of Gram-negative bacteria and is widely present in the gastrointestinal tracts of humans and animals. In human, Gram-negative bacterial infections are recognized as the cause of preterm delivery and intrauterine fetal death (IUFD). In addition, LPS has been currently used for inducting preterm delivery and IUFD in animals. LPS induces generation of reactive oxygen species (ROS) and decline of reduced glutathione (GSH) in maternal liver, placenta as well as fetal liver, which are also responsible for the IUFD. Ferulic acid (FA) is widely distributed in plants and constitutes a bioactive ingredient of many staple foods such as grain bran, fruits and vegetables. Sodium ferulate (SF), a stable form of FA, is an effective anti-inflammatary drug. SF can significantly suppress the activation of NF-kB induced by LPS. Our previous study has proved that SF could significantly suppress the activation of NF-kB induced by IL-1β, which indicates that SF may have protective effect on LPS-induced preterm delivery and IUFD.FA biotransformation has been studied in rat. The studies showed that after oral administration of FA, the substance was quickly absorbed in stomach as the free form and then conjugated mainly in liver as sulfate and glucuronide conjugates. These reactions are catalyzed by UDP-glucuronosyltransferases (UGT) and sulfotransferases, respectively. Competition that between these two conjugation pathways for the same phenolic or amine substrate occurs frequently, for providing an efficient detoxication way to clear the substance. However, pharmacokinetics of FA in human has not been well documented.In this study, we evaluated the protective effect of SF on LPS-induced preterm delivery and IUFD. And we determined the anti-oxygen and anti-inflammation role of SF in those effects. On the other hand, we fingered out the characterization of the glucuronidation and sulfation of SF in human liver microsomes and cytosol. Our results pave the way for the further use of SF in clinic.PART ONE:Protective effects of sodium ferulate on LPS-induced preterm delivery and intrauterine fetal deathObjective:Study the effect of SF on LPS-induced preterm delivery and IUFD. Methods: Kunming mice were treated with SF during gestation day (GD) 10-15. After administration of LPS (150μg/kg, i.p.), the incidence of preterm delivery and the fetal death were calculated. Hematoxylin-eosin (HE) staining was used for histological evaluation. Malondialdehyde (MDA) levels were detected by thiobarbituric acid (TBA) method for assessing lipid oxidation. And the levers of GSH, glutathione S-transferase (GST) and glutathione peroxidase (GSH-Px) were detected in maternal liver, placenta and fetal liver. IL-1βand TNF-a level in amniotic fluid were evaluated by enzyme linked immunosorbent assay (ELISA). Subcellular localization of NF-kB was detected by immunohistochemistry. Results:For 150μg/kg LPS-treated group, incidence of preterm was 47.8%, delivery time was 17.5±1.3 gestational days, most of the pups were stillborn, and the pups'survival ratio was only 42.6%. Compared with LPS-treated group, SF-treated group showed a lower incidence of preterm (14.3%, P<0.01), a longer gestational days (18.4±0.5, P<0.05) and a higher pups'survival ratio (75.6%, P<0.01). In maternal liver, SF significantly suppress the reduce levels of GSH induced by LPS. And SF suppressed the NF-kB transfer to nucleus in uterus. TNF-a levels in amniotic·fluid induced by LPS was also suppressed by SF. Conclusion:SF has the protective effect on LPS-induced preterm delivery and IUFD. The suppression of NF-kB transfer and the improve of GSH levels might be responsible for the above effect.PART TWO:Identification of the human UDP-glucuronosyltransferase isoforms involved in the glucuronidation of the phytochemical ferulic acidObjective:Ferulic acid (FA), which may offer beneficial effects against cancer, cardiovascular disease, diabetes, osteoarthritis and Alzheimer's disease, is an abundant dietary antioxidant of the series of hydroxycinnamates. Methods:In this study, evidence for sulfation and glucuronidation of SF was investigated upon incubation of SF with human liver microsomes and cytosol. And optical pH levels were determined. HPLC-MS/MS was used for glucuronidation identification, while the S35 labeled thin layer chromatography method was used for sulfation identification. Identification of UDP-glucuronosyltransferases (UGT) isoforms involved in SF glucuronidation was applied with 12 human recombinant isoenzymes. Moreover, octyl gallate, a probe substrate of UGT1A1, competitively inhibited SF glucuronidation. Results:Two main glucuronides, ether O-glucuronide (M1) and ester acylglucuronide (M2), were formed with a similar affinity (apparent Km 3.53 and 5.15 mM, respectively) in pH 6.5. A phenol sulfoconjugate was also formed with a higher affinity (Km 0.53 mM) in pH 5.4. SF was mainly glucuronidated by UGT1A isoforms and UGT2B7. UGT1A4,2B4,2B15 and 2B17 failed to glucuronidate the substance. Kinetic constants examination revealed that SF was mainly glucuronidated by UGT1Al on the two nucleophilic groups. UGT1A3 had the same capability to glucuronidate the two positions, but efficiency was much lower than UGT1A1. UGT1A6 and 1A8 were strictly involved in the formation of the ether glucuronide, whereas UGT1A7,1A10 and 2B7 preferentially glucuronidated on the carboxyl group. Moreover, octyl gallate, competitively inhibited SF glucuronidation (Ml and M2) mediated by UGT1A1, with the Ki 472.6 and 42.0μM, respectively. Conclusion: Sulfation is the main metabolic pathway with the lower SF concentration, while glucuronidation is the main metabolic pathway with the higher SF concentration. UGT1A and UGT2B are responsible for the SF glucuronidation. SF glucuronidation is primarily mediated by UGTIAl.