The Study On Regulation Mechanisn Of Proinflammatory Mediators On Pig Liver Carboxylesterase Expression

Pig liver carboxylesterase(PLE)belongs to the serine hydrolase,which has unique chiral hydrolysis characteristics and high hydrolysis activity,and it has become the main role of hydrolase in organic chemical synthesis.Our previous study had found that the two main subtypes of PLE could promote inflammation by hydrolyzing cannabinoid,which indicated that PLE was involved in regulation of inflammation.It is revealed that human and mouse carboxylesterase are the targets of inflammatory response regulation,plus the fact that PLE could regulate inflammatory response from our previous study,the author proposed that PLE could regulate inflammatory response while it could also be regulated by inflammatory response,and whether PLE can be regulated by pro-inflammatory mediators and its mechanism behind was studied.The contents and results of research were as follows:Firstly,primary porcine hepatocytes were treated with LPS at different concentrations(0.1?g/mL,1?g/mL,5?g/mL)and IL-1?/IL-6/TNF-? at different concentrations(0.1ng/mL,0.5ng/mL,5ng/mL).It was found that PLE mRNA and protein levels were significantly down-regulated by different concentrations of pro-inflammatory mediators.Secondly,the primary porcine hepatocytes were treated with 1 ?g/mL LPS and 0.5ng/mL IL-1?/IL-6/TNF-? for different time(3h,6h,12 h,24h).The results showed that the level of PLE mRNA was significantly decreased after 3 hours' treatment with four pro-inflammatory mediators,and the strongest effect was found after treatment for 12hours'.Besides,LPS,IL-1?,IL-6 and TNF-? down-regulated PLE mRNA level by 85%,77%,84%,75% respectively.It is known that LPS/IL-1?/IL-6/TNF-? can activate the NF-kappa B signal pathway.In this study,it was predicted by bioinformatics software that there were 15 binding sites of NF-kappa B in the PLE1 promoter-9916/+66 region.Therefore,it was speculated that inflammatory mediators could regulate the expression of PLE through the NF-kappa B signal pathway.The inhibition experiment of NF-kappa B on primary porcine hepatocytes was done in this study.By detecting the mRNA level of PLE,it was found that the inhibitor of NF-kappa B could completely impede the inhibition of LPS/IL-1?/IL-6/TNF-? on PLE.After that,the experiment concerning over-expressing NF-kappa B in the HepLi cells was done,it was found that NF-kappa B could decrease the activity of PLE promoter by about 50%.These results suggested that the down-regulation of PLE expression by pro-inflammatory mediators was indeed related tothe NF-kappa B signal pathway.Previous studies have shown that IL-6 can inhibit the expression of human cytochrome oxidase CYP3A4 through Pregnane X Receptor.Dexamethasone,a classical anti-inflammatory drug,can regulate the expression of human and rat CES through PXR and Glucocorticoid Receptor.In this study,four GR and four PXR binding sites of PLE1promoter-9916/+66 region were predicted with bioinformatics software.It is speculated that pro-inflammatory mediators may regulate the expression of PLE through GR and PXR.The mRNA levels and protein levels of PLE,GR and PXR in primary porcine hepatocytes which were treated with four pro-inflammatory mediators were detected.The results showed that the four pro-inflammatory mediators could significantly reduce the expression of GR and PXR,and such trend of GR and PXR was consistent with that of PLE.Next,the co-transfection of GR/PXR overexpression plasmids and PLE series reporter plasmids(truncated and uncut GR/PXR reaction elements)were carried out on HepLi cells.The activities of PLE reporter plasmids were detected and GR and PXR could indeed regulate the expression of PLE.These results suggested that pro-inflammatory mediators could indirectly regulate PLE expression by down-regulating GR and PXR expression.These results indicated that pro-inflammatory mediators could not only inhibit the expression of PLE through the NF-kappa B signal pathway,but also indirectly regulate the expression of PLE by down-regulating the expression of GR and PXR.Relevant studies have shown that NF-kappa B can inhibit the expression of PXR,which suggests that pro-inflammatory mediators may indirectly regulate the expression of PLE by inhibiting the expression of GR and PXR through NF-kappa B signal pathway.Therefore,the inhibition experiment of NF-kappa B on primary porcine hepatocytes was done in this study.The results showed that the inhibition of LPS/IL-1?/IL-6/TNF-?on GR and IL-6 on PXR could not be impeded by detecting the mRNA levels of PLE,PXR and GR.However,the inhibition of LPS/IL-1? on PXR could be partly impeded and the inhibition of TNF-? on PXR could be completely impeded.These results suggested that the mechanisms of down-regulation of GR and PXR expression by four pro-inflammatory mediators were different.LPS/IL-1?/IL-6/TNF-? couldn't down-regulate GR expression through NF-kappa B signal pathway,IL-6 couldn't down-regulate PXR expression through NF-kappa B,while LPS/IL-1?/IL-6/TNF-? could down-regulate PXR expression through NF-kappa B.