| Monoacylglycerol lipase(MAGL)is the main hydrolase of endogenous cannabinoid 2-AG and belongs to serine hydrolase.2-AG is involved in a wide range of physiological and pathological activities,such as anti-inflammatory,by activating cannabinoid receptor 1 and cannabinoid receptor 2.Inhibition of MAGL can regulate the metabolism of 2-AG.Therefore,it is speculated that MAGL plays an important role in pig inflammatory response,and its mechanism of regulating inflammation was studied.The specific methods and main results are as follows:Firstly,the heterologous expression of porcine MAGL was carried out.The cDNA of porcine alveolar macrophage(PAM)was used as template to amplify the cDNA of MAGL.After sequencing and comparison,the sequence similarity with the MAGL gene sequence in Gen Bank was 99.78%,and the amino acid sequence was completely consistent.The cDNA of porcine MAGL was successfully cloned;pET15b-MAGL was successfully constructed by bienzyme cleavage of MAGL and ligation of expression vector pET15 b,then transferred to pre-prepared Origami-pGro7 competent cells to screen positive clones;L-Arabinose was added to induce the expression of the chaperone pGro7.1m M IPTG,24 ℃ induced 15 h,the recombinant MAGL expressed in soluble form,and highly efficient hydrolyzed serine hydrolases universal substrate p-NPA.LC-MS/MS results showed suggested that the recombinant MAGL can hydrolyze cannabinoid 2-AG(50.16 nmol/mg/min)with high activity.Secondly,LPS-induced PAM cell inflammation model was treated with different amounts of the recombinant MAGL for 18 hours,RT-qPCR and ELISA detected the expression levels of cytokines.The results demonstrated that in a dose-dependent way,the recombinant MAGL dramatically increased the expression levels of IL-1β、 IL-6and TNF-α.The specific inhibitor JZL184 significantly decreased the level of inflammation induced by MAGL,which proved that MAGL had pro-inflammatory effect.PAM treated with 2-AG showed anti-inflammatory effect,while cells treated with 2-AG and the recombinant MAGL showed pro-inflammatory effect,which proved that MAGL played pro-inflammatory role by hydrolyzing 2-AG to generate produced pro-inflammatory prostaglandin precursor arachidonic acid(AA).Finally,in order to prove that MAGL located in cells also has pro-inflammation effect,the MAGL eukaryotic expression plasmid was constructed and overexpressed in 293 T cells,which constituted a co-culture cell model with PAM.The co-cultured cell model once again proved that 2-AG could downregulate the level of inflammation,and MAGL hydrolyzed 2-AG to generate AA to exert a pro-inflammatory effect.The design and synthesis of 3 pairs of MAGL siRNAs,screening a pair of siRNAs with the highest silencing efficiency by Western Blot,using siRNA-3 silencing PAM,the silencing efficiency reached about 66%,which significantly downregulated the proinflammatory factors IL-1β、 IL-6 and TNF-α mRNA and protein induced by LPS,further demonstating that MAGL plays a pro-inflammatory role in inflammatory response,and inhibiting MAGL activity can play an anti-inflammatory effect.The above studies reveal that porcine MAGL can efficiently degrade endogenous cannabinoid 2-AG,generate pro-inflammatory mediator arachidonic acid,promote the production of pro-inflammatory factors,and thus up regulate the level of inflammation;Drug inhibition or silencing MAGL can reduce the release of proinflammatory factors and inhibit inflammation. |
