The Pharmacological Research Of Tek-1Relevance To Anti-neuroinflammation, A Candidate Compound Based On Telmisartan

Objective: Upon the important role of AT1receptor and PPARγ receptor inmodulating anti-oxidative damage and neuroprotection, we designed and acquired thecandidate compound Tek-1through the modification of biphenyl carboxylic acid unit.In this project, we identified the dual pharmacology characteristics based on AT1receptor and PPARγ receptor; furtherly, in vivo we evaluated the ameliorative effect ofTek-1on learning and memory dysfunction in AD mouse model induced bybeta-amyloid (Aβ); we investigated the role of Tek-1on regulating activated microgliaclosely related to the neuro-inflammation and signal transduction mechanism in order toobtain new structure AT1antagonist with better PPARγ receptor agonistic bioactivity.Methods: First, through radioligand binding assay on rat vascular smooth musclecells, calcium mobilization HTS assay and PPARγ responsive-element-luciferasereporter assay, we evaluated the AT1receptor affinity of Tek-1, and antagonistic activityas well as PPARγ agonistic activity respectively; Second, in Alzheimer’s disease mousemodel of C57BL/6J mice induced by introcerebroventricular injection of Aβ1-42,Telmisartan (1,3mg/kg) and Tek-1(1,3,10mg/kg) were continuously administered for4weeks. Then through the Morris water maze and the novel objects recognition test, weinvestigated the effect of Telmisartan and Tek-1on the spatial and nonspatial learningand memory abilities in AD mouse model. And the concentration of Interleukin (IL-6)and monocyte chemotactic protein-1(MCP-1) in brain were explored using ELISA;Third, we established BV-2microglia inflammatory model stimulated by bacteriallipopolysaccharide (LPS) in vitro, and then cells were treated with Telmisartan(0.1-10μM) and Tek-1(0.1-10μM), or in the presence of Telmisartan and Tek-1combined with specific PPARγ antagonist GW9662. Effects of Telmisartan and Tek-1on the release of tumor necrosis factor-alpha (TNF-α) in microglia were analyzed byELISA; the mRNA expression level of microglia marker CD11b, macrophages marker CD16and induced nitric oxide synthase (iNOS) were determined by real-timequantitative PCR; the mitogen-activated protein kinase (MAPKs) and nuclear factor-κB(NF-κB) signal transduction pathway in BV-2microglia stimulated by LPS weredetermined by Western Blot.Results: Through the radioassay of receptors, Tek-1had high affinity to AT1receptor, the Ki values for AT1receptor were1.1×10-9M. In the calcium mobilizationHTS assay, the effect of AT1agonist angiotensin II (ANGII) were inhibited by Tek-1ina concention-dependent manner with IC50values of1.02nM. Tek-1could significantlyactivate PPARγ in a concention-dependent manner ranging from0.1to10μM.Compared with rosiglitazone, a full agonist of PPARγ, Tek-1behaved as partial PPARγagonists.Telmisartan (1,3mg/kg) and Tek-1(1,3,10mg/kg) could significantly improve thenonspatial learning and memory abilities of AD mice induced by Aβ. Telmisartan (1,3mg/kg) and Tek-1(3,10mg/kg) could significantly improve the spatial learning andmemory ability on AD mice model induced by Aβ. ELISA results showed Telmisartan(1mg/kg) and Tek-1(1,3,10mg/kg) could significantly reduce the content of cytokineIL-6(P<0.001); Telmisartan (1mg/kg) and Tek-1(1,3mg/kg) could significantly reducethe content of chemokine MCP-1.In the inflammatory model of BV-2microglia cell induced by LPS,0.1-10μMTelmisartan and10μM Tek-1could significantly inhibit the release of TNF-α(P<0.001and P<0.05). Real time quantitative PCR results showed that10μM Telmisartan and1,10μM Tek-1could significantly inhibit the mRNA expression level of CD11b, CD16and iNOS. Specific PPARγ antagonist GW9662could partially reverse inhibiting theactivation of microglia and releasing of inflammatory mediator. Western blot resultsshowed that0.1-10μM telmisartan and0.1,1μM Tek-1decreased the phosphorylation ofERK1/2, JNK and p38or IκB-α degradation, up-regulated of NF-κB in BV-2microgliacells induced by LPS. Specific PPARγ antagonist GW9662could partially reverse theactivation effect of telmisartan and Tek-1on BV-2microglia cells.Conclusion:1. Tek-1had high affinity to AT1receptor and could significantlyinhibit the activation of intracellular calcium by ANGII, indicated that it was a anantagonist of AT1receptor;2. Compared with full agonist Rosiglitazone, Tek-1couldpartially activate PPARγ;3. Compared with telmisartan, chronic treatment with Tek–1could improve the ability of learning and memory ability on AD mice. And Tek-1could reduce the content of cytokine and chemokine in brain induced by Aβ. This implied theanti-inflammation effects of Tek-1related to ameliorative effect on learning andmemory dysfunction.4. Establishment of BV-2microglia inflammatory model inducedby LPS in vitro, Tek–1could inhibit the release of inflammatory mediator, themechanism of which mainly attributed to inhibiting the activation of mitogen-activatedprotein kinase (MAPKs) and NF-κB signal transduction pathway.