Studies On The Regulation Mechanism Of Pig Liver Esterases On β-lactam Drug Effects And Inflammation Response

Mammalian carboxylesterases,belonging to serine hydrolase family,are widely distributed in various animal tissues and a series of immune cells.CES could highly hydrolyze the endogenous/exogenous compounds containing ester bond,thioester bond and amide bond,which play an important role in prodrug activation,drug metabolism,signal transduction,inflammatory response,nutrient absorption and cholesterol homeostasis in vivo.PLEs are the most complex member of mammalian carboxylesterase family,which contain a variety of isoenzymes.PLE possess properties such as wide substrate speificity,catalytic flexibility,and excellent chemo-,stereo-,and regioselectivity.Inspired by the efficiently hydrolysis activity of PLE,and the important role of human and mouse carboxyesterase played in drug metabolism and inflammatory response,it is speculated that PLE may involved in veterinary drug metabolism and inflammatory response in pigs by virtue of its highly hydrolytic activity,and act as a regulator of drug metabolism and inflammatory level.This study carried out a serious of experiments in vitro and piglet inflammation model.The main methods and results are as follows:1. PLE highly hydrolyze AMO/AMP and decreased the antibacterial activityFirstly,LC-MS/MS analysis found that both AMO and AMP were rapidly hydrolyzed by recombinant PLE1 and PLE6 to produce AMA and APA,respectively.PLE1 and PLE6 were more active toward AMO（25.94 U/mg,25.32 U/mg）than AMP（8.34 U/mg,10.44 U/mg）.PLE1 and PLE6 showed a comparable V_max value toward AMO,but PLE6 exhibited a higher V_maxvalues toward AMP than PLE1.PLE1 and PLE6 had the lower K_m value for AMO than AMP.These results suggested that PLE6was kinetically favorable than PLE1 for both AMO and AMP.Between two antibiotics,AMO was kinetically favorable.In addition,antibacterial tests in vitro confirmed that incubations with recombinant PLE1 or PLE6 sharply decreased or even eliminated the antibacterial activities of AMO and AMP for Staphylococcus aureus,Salmonella typhimurium and E.coli K88ac.Secondly,western blot and LC-MS/MS detection revealed that Large white and Tongcheng pig liver S9 fractions showed comparable hydrolytic activities toward AMO and AMP,respectively.AMO and AMP could enter into cells and were effectively hydrolyzed by PLE.Additionally,a very low level of PLE existed in pig serum and could not hydrolyze AMO and AMP.These results indicated that AMO and AMP in the body need to enter into cells to be metabolized by PLE.But unlike the recombinant PLE,liver S9 fractions and eukaryotic expression PLE hydrolyzed AMO to produce DIKETO instead of AMA.This study revealed that PLE can openβ-lactam ring and efficiently hydrolyze AMO and AMP to reduce their antibacterial activity,which are likely to be involved in the development of drug resistance.In particular,the expression profiles and activity of PLE in vivo are likely to reduce the bioavailability and therapeutic effect of these antibiotics.Comprehensively,the expression level and hydrolytic activity difference of PLE isoenzymes most probably were the foundation of clinical rationale administration of antibiotics and improving therapeutic effect.2. PLE participate in endocannabinoid metabolism to promote inflammationThe endocannabinoid 2-AG and AEA regulate inflammation in the body by activating the cannabinoid receptor.Based on the molecular structural characteristics,we hypothesized that PLEs can regulate inflammation in pigs through hydrolyzing2-AG and AEA.Firstly,LC-MS/MS results suggested that the recombinant PLEs can efficiently hydrolyzed 2-AG and AEA to produce AA.The hydrolytic activities of PLE1 and PLE6 for 2-AG were 1.56 nmol/mg/min and 62.70 nmol/mg/min,which were about 3 times and 41 times for AEA,respectively.In addition,PLE1 and PLE6reflected extremely high activities of 17.90μmol/mg/min,101.11μmol/mg/min,respectively,forto produce.PLEs in the pig liver S9 fractions are responsible for most of the hydrolysis of 2-AG（53.4%）,AEA（75%）and（95.4%）.Meanwhile,2-AG was also efficiently hydrolyzed by PLE in live cells.Secondly,western blot and LC-MS/MS results demonstrated that PLEs were present in PAM in much lower levels than in PHC,and the homogenates of PAM and PHC could effectively hydrolyze 2-AG with hydrolytic activities of 4.47nmol/min/mg and 3.25 nmol/min/mg.BNPP inhibited 77.3%and 88.4%of the total2-AG hydrolysis in PAM and PHC homogenates,respectively,making PLEs the rate-limiting enzymes for 2-AG inactivation.In the cell culture models,2-AG inhibited inflammation while PLE-hydrolyzed 2-AG enhanced LPS-induced pro-inflammatory cytokines,which positively correlated with PLEs level.BNPP inhibited the hydrolytic activity of PLEs for 2-AG and reduced the inflammatory response.Finally,LPS were injected into crossbred healthy pigs.The results revealed that LPS induced local necrosis in the liver,disintegration of hepatocytes,thickening of alveolar septa,shedding,necrosis of intestinal villi,neutrophil infiltration.BNPP provided substantial protection against tissue injury and attenuated the neutrophil infiltration.Moreover,LC-MS/MS results showed that LPS induced a robust increase in the levels of AA,PGD2 and PGE2 in tissues,which were markedly blunted by BNPP in almost all the tissues tested.These data demonstrated that PLE contributed to the AA precursor pool for PGs in vivo.It was worth mentioning that,in vivo,LPS significantly down-regulated the levels of PLE in the tissue S9 fractions,and decreased the hydrolytic activities of the tissue homogenates for p-NPA and 2-AG.These suggest that the pro-inflammatory function of PLE makes it the negative-feedback regulation target of inflammation and that down-regulation of PLE levels is likely to be the crucial mechanism in maintaining inflammation homeostasis.In conclusion,we systematically elucidated that PLE participate in endocannabinoid metabolism and promote inflammation,which may be one of the crucial mechanisms of non-specific immune response.These findings identify PLE as a distinct metabolic node that couples endocannabinoid to prostaglandins signaling networks.Inhibition of PLE might be a potentially safe way to suppress the pro-inflammatory cascades that underlie inflammation disorders in pigs.Comprehensively,the investigation of PLE inhibitors may lay a solid foundation for the development of new anti-inflammatory drugs for 2-AG metabolism-related inflammatory diseases in pigs,which provides a significant resource for human medicine.