The Study Of Toll-like Receptor 4-mediated Signal Transduction On The Pulmonary Microvascular Endothelial Cells Of Rats And The Inhibitory Effect Of Simvastatin On This Pathway

PART1 The study of the expression of Toll-like receptor 4 and its downstream signal transduction on the pulmonary microvascular endothelial cells of rats Objective: LPS, a major membrane constituent of gram-negative bacteria (GNB), plays a critical role in acute respiratory distress syndrome (ARDS) induced by infection of GNB. In classic theory LPS activates toll-like receptor 4 (TLR4) in immunity cells and mediates immunology response. However, giving evidences demonstrated that TLR4 is also expressed in non-immunity cells, modulates immunology response, and involves in pathological process. In this study we investigate whether TLR4 is expressed in the pulmonary microvascular endothelial cells (PMEC) of rats and its downstream signal pathway to understand the pathological mechanism of ARDS and provide the basis of therapy.Methods: The PMEC were established in vitro form the rat lung and characterized with the antibody against VII factor. The mRNA levels of the TLR family members were determined by RT-PCR. The protein level and cellular localization of TLR4 on the PMEC were assayed by Western blotting, immunofluroscence and flow cytometry. The NF-kB activation was investigated by immuofluroscence, luciferase reporter assay and Western blotting on the PMEC after LPS treatment. RT-PCR was performed to determine whether LPS could induce the mRNA expression of inflammatory factors TNF-α和iNOS. The blocking effects of TLR4 antibody and NF-kB inhibitor, Aspirin, on the LPS signal pathway were examined by immuofluroscence, luciferase reporter assay and Western blotting.Results: (1) Accordingly to the previous reports, we success in obtaining the PMEC in vitro from rats. These cells exhibited the regular cobblestone morphology and were constitutively cultured for 2-5 passages. These cells were positively stained with VI factor, indicated that they were microvascular endothelial cells. (2) PMEC generally expressed the mRNA of TLR1, TLR2, TLR3, TLR4, TLR6 and TLR7, but did not expressed TLR5 and TLR9．Among these TLRs the level of TLR4 was the highest. (3) Immuofluroscence showed that TLR4 protein was localized in the membrance in PMEC, but the cytosol was weakly stained with TLR4．Flow cytometry indicated that 95% PMEC exhibited the strong staining of TLR4 in their membrance. (4) LPS resulted in the increase of phosphoration of JNK and p38 (p<0．01) and the degradation of IkB-αupon 1h, 3h and 6h of treatment (P<0．01) . Furthermore, LPS treatment led to the entry of p65 into the nucleus and NF-kB dependent transcription activity. (5) LPS treatment obviously enhanced the mRNA levels of TNF-α和iNOS in a time-dependent manner and the peak was observed after 72 h (p<0．01) . (6) Pretreatment of anti-TLR4 antibody and NF-kB inhibitor, Aspirin, could almost completely block the degradation of IkB-α, the entry of p65 into the nucleus, NF-kB dependent transcription and enhancement of mRNA levels of TNF-α和iNOS induced by LPS.Conclusion: (1) TLR4 was highly expressed in the PMEC and located in the membrance. (2) LPS treatment results in the activation of TLR4-mediated signal and inflammatory response release of the inflammatory factors TNF-α和iNOS in PMEC. (3) The application of TLR4 antibody and NF-kB inhibitory can block NF-kB activation and release of the inflammatory factors TNF-α和iNOS , suggesting LPS increase the expression of inflammatory factors TNF-α和iNOS through activation of TLR4 and NF-kB. In conclusion, our study helps to understand of initiation and progression of GNB-induced diseases.PART2 The effect of simvastatin on TLR4 expression and downstream signalObjective: To investigate the effect of simvastatin on TLR4 expression and downstream signal to explore the possible usage of simvastatin on anti-inflammatory therapies in clinic.Method: RT-PCR and Western blotting were performed to analysis the mRNA and protein levels of TLR4 after simvastatin treatment. The effect of pretreatment of simvastatin on LPS-mediated NF-kB activation was assayed by immuofluroscence, luciferase reporter assay and Western blotting. The effect pf pretreatment of simvastatin on the expression of imflammatory factors triggered by TLR4 activation upon LPS application was determined by RT-PCR and ELISA.Results: (1) Simvastatin treatment led to the down-regulation of the mRNA and protein levels of TLR4 in a time-dependent manner (p<0．01) . The effect was obvious at 72 h after treatment. (2) Simvastatin significantly attenuated the degradation of IκB-αin a time-dependent manner, the entry of p65 into the nucleus and the NF-kB dependent transcription activity induced by TLR4 activation upon LPS treatment (p<0．01) . The effect preceded the down-regulation of TLR4．(3) Simvastatin significantly inhibits the expression of the imflammatory factors triggered by TLR4 activation upon LPS application (p<0．01) . Conclusion: Simvastatin harbors the ability to inhibit TLR4 expression as well as its downstream signal. Thus simvastatin is a potential inhibitors of TLR4 and used to treat TLR4-associated imflammatory diseases in clinic.