Studies On The Regulation Mechanism Of Pig Liver Carboxylesterase On The Inflammation In Pig

Pig liver carboxylesterase is the most complex enzyme family in mammalian enzymes.At present,our research team has cloned 108 kinds of PLE isoenzymes,including 7 reported subtypes,of which 55 are of high frequently occurring.Mammalian carboxylesterase has high enzyme activity and substrate flexibility.It can hydrolyze a variety of endogenous and exogenous substances and exert irreplaceable pharmacological and toxicological effects.However,little is known about its physiological effects,that is not quite proportional with the abundance of pig liver esterases in the body.Other studies have shown that human CES can hydrolyze endocannabinoids,which provides ideas for the physiological role of PLE.The common endocannabinoids are 2-AG and AEA.They are synthesized on demand and have anti-inflammatory effects in the body.They are mainly hydrolyzed by classical cannabinoid degrading enzymes MAGL and FAAH.PLE and classical cannabinoid degrading enzymes belong to the family of serine hydrolases,and 2-AG and AEA contain ester and amide bonds respectively for PLE hydrolysis.It is reasonable to assume that PLE may regulate inflammation levels by hydrolyzing cannabinoids.To demonstrate the role of PLE in the inflammatory response and elucidate its mechanism of function,this study first prokaryotic functional expressed subtypes PLE1 and PLE6 to hydrolysis of endocannabinoids in vitro.It was found that both PLE1 and PLE6 can hydrolyze cannabinoid 2-AG,and enzyme activity levels about PLE6 hydrolysis of 2-AG as high as 62.7nmol/min/mg.This provides a direct basis for PLE to hydrolyze endocannabinoids and regulate inflammatory responses in the body.Our research team found that PLE was mainly distributed in the liver.Therefore,in this study,the pig liver S9 containing PLE was used for hydrolysis of cannabinoid2-AG and AEA in vitro.Experiments have shown that liver S9 can not only hydrolyze cannabinoids 2-AG and AEA,but that after treatment with the specific inhibitor BNPP,the level of enzyme activity of adult pig liver S9 is down-regulated,suggesting that PLE in liver S9 can hydrolyze endocannabinoids.PLE is located in the endoplasmic reticulum of the cell and can only exert hydrolysis of substances entering the cell.Therefore,in this study,the 293 T cell line was overexpressed the PLE6 containing the signal peptide.At the level of living cells,it was tested whether the cannabinoid 2-AG could enter the cell and be hydrolyzed by PLE.The 2-AG hydrolyzate arachidonic acid was detected by LC-MS/MS.As a result,the product AA was significantly higher in the cell culture medium than in the control group,as well as in the mixture of cell culture medium and the cell.That indicating that the cannabinoid 2-AG is able to enters the cell and is hydrolyzed by PLE that located in the lumen of the endoplasmic reticulum,which also suggests that the endogenous cannabinoid produced by the body can enter the cell and is hydrolyzed by PLE.This laid a good foundation for the study of regulation of inflammatory mechanisms by PLE.PLE hydrolyzes endocannabinoids to produce arachidonic acid,which is also a precursor of prostaglandins.Therefore,PLE hydrolysis of cannabinoids involved in the regulation of inflammation is more complicated.For this reason,we established a co-cultured model of primary liver cells,primary Kupffer cells,and primary lymphocytes in this study to investigate the regulation mechanism of PLE on inflammation in the body.First,added 2-AG treatment of LPS-induced co-cultured cell inflammation model,protein chip results demonstrated that 2-AG exerts anti-inflammatory effects.Secondly,the co-cultured LPS-induced inflammation model treated with 2-AG hydrolyzed by recombinant PLE6,and the protein chip test results showed that 2-AG hydrolyzed by PLE6 played a proinflammatory role.Then,LPS-induced co-cultured cell inflammation model was treated with BNPP,a specific inhibitor of carboxylesterase,and the results of protein chip showed that BNPP exerted anti-inflammatory effect after inhibiting PLE,showing that PLE played a pro-inflammatory role.After the above studies proved that 2-AG has anti-inflammatory effects,and PLE hydrolysis of cannabinoids played a pro-inflammatory role.Finally,the co-cultured cell model was treated with the carboxylesterase inhibitor BNPP for 3 h,and then the cannabinoid 2-AG was added to the LPS-induced co-cultured cell inflammation model,Protein chip results show that BNPP significantly down-regulates LPS-induced inflammation after 2-AG treatment,further proving that PLE hydrolysis of cannabinoids to play a pro-inflammatory role.Due to the limited number of isolated primary Kupffer cells,MoDC and alveolar macrophage cell lines were added in this cell model.The model were co-cultured with primary porcine hepatocytes,MoDC cells,KCs cells,and alveolar macrophage cell lines.First,the RT-qPCR results showed that 2-AG down-regulated LPS-induced inflammation in co-cultured cells,demonstrating that 2-AG exerts anti-inflammatory effects in a co-cultured cell model.Next,this study was to add PLE1 and PLE6 in a co-cultured cell inflammatory model,Protein chip results showed that PLE1 andPLE6 play a pro-inflammatory role.Finally,BNPP was used to treat co-cultured cell inflammation model induced by LPS.Protein chip results showed that BNPP significantly inhibited the level of inflammation,demonstrating that PLE plays a pro-inflammatory role in co-cultured cell inflammation model.The above experiments are consistent with the three cell co-cultured model test results.Finally,using the classical cannabinoid degrading enzyme MAGL inhibitor JZL184,compared with the effect of MAGL and PLE in co-cultured cell inflammatory model,the protein chip results showed that JZL184 down-regulated LPS-induced inflammation level,indicating that PLE and MAGL have the same tendency to modulate inflammatory responses.