Study On LPS Suppresses The Expression And Activity Of CES1 And CES2

Carboxylesterases areα/β-hydrolase-fold protein and comprise a multigene superfamily.The expression of Carboxylesterases is ubiquitous with high levels in various tissues.Among them,the liver expresses two major carboxylesterases, including CES1 and CES2,whereas the gastrointestinal tract expresses predominantly CES2.These enzymes efficiently catalyze the hydrolysis of a variety of ester- and amide-containing chemicals as well as drugs (including prodrugs) to the respective free acids.In some pathological states,such as infection,it is apparent that inflammation can decrease metabolic clearance of DMEs substrates by 20%-70%. Obviously,the potential for clinically relevant consequences of these changes will be highest when large changes in clearance occur,and with drugs that have a low safety margin.Lipopolysaccharide (LPS) is a toxic component of cell walls of gram-negative bacteria and is widely present in the digestive tracts of humans and animals.Humans are constantly exposed to low levels of LPS through infection. LPS has been used extensively as a model of sepsis to study the inhibitory effects of inflammation and infection on CYP activity and expression.In this study,we investigated the role of LPS on the expression and activity of CES1 and CES2 both in vivo and vitro.Then,we examined the pharmacological/toxicological consequences of the reduced expression of HCE1 and HCE2 in HepG2 cells by antithrombogenic agent clopidogrel and anticancer agent irinotecan.Through the above experiment,we found that inflammation can reduce the expression and activity of CES1 and CES2.In addition,pretreatment with LPS altered the cellular responsiveness toward various ester therapeutic agents,indicating inflammation can significantly influence the efficacy of the drug.In a word,the study on LPS of the modulation and effect of CES1 and CES2 gives a theortical basis of reasonable clinical drug administration window.It is not only money-saving,socially and economically profitable but also improves effect as well as avoids oversdosage and decreases toxicity of drugs.PartⅠ: Study on LPS suppresses the expression and activity of CES1 and CES2 in mice livers and intestinesTo study the effects of inflammation on the expression and activity of CES1 and CES2,we treated mice with LPS.Mice livers and intestines CES1 and CES2 mRNA levels were determined using qRT-PCR.Results showed that in mice treated with LPS,the liver CES1 and CES2 mRNA levels were relatively lower than the control group by 96% and 92%, the differences were statistically significant(P < 0.05).Similarly,mice treated with LPS ,the intestinal CES1 and CES2 mRNA levels were relatively lower than the saline group by 75% and 87%, the differences were also statistically significant(P < 0.05).To investigate whether the decreases of CES1 and CES2 mRNA levels in mice livers and intestines translate into decreases in the hydrolytic activity,the overall hydrolytic activity by the homogenate were determined with standard substrate para-nitrophenylacetate.Consistent with the decreases in CES1 and CES2 mRNA,the hydrolysis of PNPA was markedly decreased.The value of LPS treated mice livers carboxylesterase total activity was 1.572±0.319,significantly lower than the control group value of 3.269±0.228(P < 0.05). The total activity of intestinal carboxylesterase of LPS treated group was 0.413±0.082,also significantly lower than the control group value of 0.885±0.102.Finally, mice livers and intestines carboxylesterases protein levels were determined using Western blotting.Results showed that LPS significantly down-regulated the expressions of CES1 and CES2 in mice livers and intestines.Compared with control group,the levels of both CES1 and CES2 proteins in LPS treated mice livers were decreased by 62% and 58%, the differences were statistically significant(P < 0.05).While the levels of CES1 and CES2 proteins in LPS treated mice intestines were decreased by 35% and 42%. the differences were also statistically significant(P < 0.05).Our results showed that inflammation not only downregulate the expression of CES1 and CES2 in mice livers and intestines,but also can reduce the total hydrolytic activity.PartⅡ: Study on LPS suppresses the expression and activity of HCE1 and HCE2 in HepG2 cellsThrough the study of the first part,we found that in both mice livers and intestines,treatment with LPS not only decreased the expression of CES1 and CES2,but also reduced its activity.So, will CES1 and CES2 downregulation have any effect on clinical drug delivery?We took human hepatoma cell line HepG2 as research object and studied the expression of HCE1 and HCE2 at cellular level. Treatment of human hepatoma cell line HepG2 with LPS could significantly reduce the HCE1 and HCE2 protein expression.In addition,to determine whether the decreased protein level of HCE1 and HCE2 reduce its hydrolysis activity. The hydrolysis of para-nitrophenylacetate was determined spectrophotometrically as described under Materials and Methods.The results show that the total hydrolytic activity of protein samples was significantly reduced.Since LPS not only inhibit HCE1 and HCE2 protein levels but also reduce its total hydrolytic activity,for further observation whether the decreased level of HCE1 and HCE2 protein expression will changes its metabolism and the toxicity of drugs reaction,we treated HepG2 cells with different concentrations of clopidogrel and irinotecan for 48h (drug medium was replaced with fresh drug-containing medium at 24 h),then determined cell viability and morphologic changes using MTT and take photos.When exposed to clopidogrel,cells pretreated with LPS exhibited profound changes,whereas cells without LPS pretreatment showed normal appearance.The results of irinotecan was contrast with clopidogrel.Cell viability results was consistent with morphological changes.Through the above study on LPS influence the expression and activity of CES1 and CES2,we found that inflammation can downregulation the expression and activity of carboxylesterase.By MTT and morphological experiments we demonstrated the reduced expression of carboxylesterase by inflammation may change its metabolic effects and toxicity of drugs.In a word,the study on LPS of the effect of CES1 and CES2 gives a theortical basis of reasonable clinical drug administration window.