The Anti-inflammation Strategies Based On Nuclear Factor PPARγ And FXR

Inflammation is a protective host response to external challenge or cellular injury thatleads to the activation of a complex array of inflammatory mediators, finalizing therestoration of tissue structure and function[1]. An important general mechanism responsible forthis activity is referred to as transrepression, in which nuclear receptors interfere withsignal-dependent activation of inflammatory response genes through protein-proteininteractions with coregulatory proteins and promoter-bound transcription factors, rather thandirect, sequence-specific interactions with DNA. Nuclear receptors comprise a superfamily ofstructurally conserved, ligand-dependent transcription factors that regulate diverse aspects ofdevelopment and homeostasis by both positively and negatively regulating gene expression.Our studies foucused on two classical NRs, PPARγ and FXR, employing the LPS-inducedAcute lung injury mice （wild-type and A2AR KO mice）, LPS-induced murine macrophagesRaw264.7cells and HUVECs, discussed the underlying mechaniasms of regulation of thepotential inhibitor A2AR and TM respectively in anti-inflammation effection.In order to investigate the interaction between PPARγ and A2AR, we carried out ourstudies as followed:1. PPARγ activation attenuate lung damages of mice with ALI in an A2AR-dependentmanner.2. A2AR deficiency blocks the inhibitory effect of PPARγ activation on inflammatorycytokine expression.3. PPARγ agonist upregulate A2AR expression in lung tissues of mice with ALI andLPS-stimulated murine macrophages.4. A2AR activation positively regulates PPARγ mRNA and protein expressions in aPKA-dependent manner.In above parties, comparisons between histopathological evaluation, immunofluorescence analysis and test of lung water content of lung tissues of wild-type mice and A2AR geneknockout mice, which were treated with or without agonist and antagonist of A2AR orPPARγ showed that PPARγ activation attenuate lung damages of mice with ALI in anA2AR-dependent manner, while activation of A2AR could obviously prevent the inhibitionof LPS effect on the expression of PPARγ. Real-time, Western blot assays were employedto demonstrate the positive expression loop between PPARγ and A2AR in vivo and in vitro.5. PPARγ activation enhances the transcriptional activation of A2AR promoter.6. PPARγ directly targets A2AR in the A2AR promoter region.7. CREB, the downstream of PKA, directly mediates the A2AR-induced upregulationof PPARγ.In above parties, Luciferase reporter assays, point mutation technology, EMSA andChIP assays were showed that PPARγ activation enhance the transcriptional activation ofA2AR via a DR10PPRE in-218to-197sites in A2AR promoter region in murinemacrophages. And CREB, phosphorylated in A2AR-PKA-CREB pathway as a downstreamtranscription factor, directly mediated the A2AR-induced upregulation of PPARγ by bindingto a CRE-like site locating in+4to+11sites in PPARγ promoter region.8. Combined treatment of PPARγ agonist ROSI and A2AR agonist CGS21680exertsbetter protective effect than the single administration of them.In the above part, we used immunofluorescence to detecte the infliltration ofneutrophil in the lung tissues of LPS-induced ALI mice. And the combined treatement ofROSI and CGS21680achieved a better protective effect than the single administration ofthem.Taken together, our findings suggest PPARγ and A2AR form a positive feedback loopto inhibit inflammation and attenuate lung damages in ALI. This transcriptional regulationof PPARγ and A2AR appears to be critical in controlling their endogenous ligand’s (such as15d-PGJ2to PPARγ while adenosine to A2AR) response under pathophysiologicalconditions. In addition, it provides novel evidence for understanding the molecular basis ofPPARγ and A2AR expressions and their functions in ALI, as well as the action of the tworeceptors-related drugs for ALI.For another, we focused our studies on the regulation of FXR effect onThrombomdulin (TM), which is a significant mediator molecules expressed on vascular endothelial cells. Endothelial cells (ECs) dysfunction induced a variety of diseases such asinflammation, vascular diseases, fibrosis, thrombosis and even spontaneous metastasis[21-22].In the studies of Li. et al., we have already demonstrated that activation of FXR by itsnatural ligand CDCA or synthetic ligand GW4064lead to both a significant up-regulationof TM expression and an increase of TM activity. Furthermore, FXR induces TMexpression maybe though directly binding to the IR8FXRE sites in the promoter region ofTM. Then we performed directly sites mutant, electrophoretic mobility shift assay (EMSA)and ChIP assays to inquire into the underlying mechanisms of the regulation and clearifiedthis transcriptional regulation of TM appears to be achieved via a novel IR8FXRE in thepromoter region of TM. Additionally, it provides novel angel to investigate FXR and TMfunctions in the inflammation, fibrosis, and other vascular diseases.