In selection, proteins derived from these cells were analyzed by Western blotting using antibodies against NS5A to reflect HCV replication (upper panel), against cdN Madrasin web protein to determine the knockdown efficiency (middle panel) or against Erk-2 as a loading control (bottom panel). doi:10.1371/journal.pone.0068736.gAcknowledgmentsWe thank Dr. C.M. Rice for providing the plasmid p90/HCV FL-long pU, Dr. S.X. Skapek for the plasmid pBRII.hRBaK and Dr. J.-H. Ou for the HCV sub-genomic replicon cells. RNAi reagents were obtained from the National RNAi Core Facility located at the Institute of Molecular Biology/ Genomic Research Center, Academia Sinica.Author ContributionsConceived and designed the experiments: HCL SYL. Performed the experiments: CPF ZCL CHY THS JCC YJY. Analyzed the data: HCL SYL. Contributed reagents/materials/analysis tools: YLJ. Wrote the paper: SYL.
It has been demonstrated that activation of sympathetic nervous system (SNS) is one of the hallmarks of heart failure state, and the activation of SNS contributes to the worsening of mortality and left ventricular (LV) dysfunction [1?]. In heart failure, accumulating evidence suggests that inflammatory cascade in central nervous system is one of the important pathways in the activation of SNS [10?2]. In the inflammatory cascade, nuclear factorkappa B (NF-kB) is a predominant regulator for inflammatory cytokines. Thus, the signaling of NF-kB has been considered to be important in the central nervous system of heart failure [13,14]. Inflammatory response is usually associated with the activation of innate immunity [15]. Toll-like receptor (TLR) and interleukin 1 (IL-1) receptor share a common signaling pathway leading to NF-kB activation and proinflammatory cytokines. TLRs are considered to be family of conserved pattern-recognition receptors that are linking of immune and inflammatory process [15?7]. After the stimulation with a ligand, the TLRs relay a signal viamyeloid differentiation primary response protein 88 (MyD88) that is the common signal adaptor molecule, and trigger the 18055761 downstream stimulation of NF-kB and the induction of genes that encode proinflammatory cytokines. Recently we demonstrated that TLR4 and MyD88 were Hexaconazole web increased in brainstem of myocardial infarction (MI)-induced heart failure, and that intracerebroventricular (ICV) injection of angiotensin II type 1 receptor blocker prevented LV remodeling with sympathoinhibiton and reduction of TLR4 in brainstem [10]. These results suggest that TLR4 and MyD88-mediated inflammatory responses in brainstem would be involved in the mechanisms of LV remodeling associated with brain angiotensin II type 1 receptorevoked sympathoexcitation in MI-induced heart failure [10]. However, it has not been clarified whether direct inhibition of brain TLR4 could prevent LV remodeling with sympathoinhibition in MI-induced heart failure or not, because the no suitable direct inhibitor of TLR4 has been available.Brain TLR4-Mediated Sympathoexcitation in HFConsidering these backgrounds, in the present study, we examined whether silencing brain TLR4 by ICV injection of TLR4-SiRNA could prevent LV remodeling with sympathoinhibition in MI-induced heart failure or not.Results Effect of TLR4-SiRNA In Vitro and In VivoFigure 1 showed the expression of mRNA and protein of TLR4 in C6 cell line to identify the knockdown efficacy of different SiRNAs. These results suggested that SiRNA-2 was the most effective for knockdown of TLR4. Therefore, SiRNA-2 wa.In selection, proteins derived from these cells were analyzed by Western blotting using antibodies against NS5A to reflect HCV replication (upper panel), against cdN protein to determine the knockdown efficiency (middle panel) or against Erk-2 as a loading control (bottom panel). doi:10.1371/journal.pone.0068736.gAcknowledgmentsWe thank Dr. C.M. Rice for providing the plasmid p90/HCV FL-long pU, Dr. S.X. Skapek for the plasmid pBRII.hRBaK and Dr. J.-H. Ou for the HCV sub-genomic replicon cells. RNAi reagents were obtained from the National RNAi Core Facility located at the Institute of Molecular Biology/ Genomic Research Center, Academia Sinica.Author ContributionsConceived and designed the experiments: HCL SYL. Performed the experiments: CPF ZCL CHY THS JCC YJY. Analyzed the data: HCL SYL. Contributed reagents/materials/analysis tools: YLJ. Wrote the paper: SYL.
It has been demonstrated that activation of sympathetic nervous system (SNS) is one of the hallmarks of heart failure state, and the activation of SNS contributes to the worsening of mortality and left ventricular (LV) dysfunction [1?]. In heart failure, accumulating evidence suggests that inflammatory cascade in central nervous system is one of the important pathways in the activation of SNS [10?2]. In the inflammatory cascade, nuclear factorkappa B (NF-kB) is a predominant regulator for inflammatory cytokines. Thus, the signaling of NF-kB has been considered to be important in the central nervous system of heart failure [13,14]. Inflammatory response is usually associated with the activation of innate immunity [15]. Toll-like receptor (TLR) and interleukin 1 (IL-1) receptor share a common signaling pathway leading to NF-kB activation and proinflammatory cytokines. TLRs are considered to be family of conserved pattern-recognition receptors that are linking of immune and inflammatory process [15?7]. After the stimulation with a ligand, the TLRs relay a signal viamyeloid differentiation primary response protein 88 (MyD88) that is the common signal adaptor molecule, and trigger the 18055761 downstream stimulation of NF-kB and the induction of genes that encode proinflammatory cytokines. Recently we demonstrated that TLR4 and MyD88 were increased in brainstem of myocardial infarction (MI)-induced heart failure, and that intracerebroventricular (ICV) injection of angiotensin II type 1 receptor blocker prevented LV remodeling with sympathoinhibiton and reduction of TLR4 in brainstem [10]. These results suggest that TLR4 and MyD88-mediated inflammatory responses in brainstem would be involved in the mechanisms of LV remodeling associated with brain angiotensin II type 1 receptorevoked sympathoexcitation in MI-induced heart failure [10]. However, it has not been clarified whether direct inhibition of brain TLR4 could prevent LV remodeling with sympathoinhibition in MI-induced heart failure or not, because the no suitable direct inhibitor of TLR4 has been available.Brain TLR4-Mediated Sympathoexcitation in HFConsidering these backgrounds, in the present study, we examined whether silencing brain TLR4 by ICV injection of TLR4-SiRNA could prevent LV remodeling with sympathoinhibition in MI-induced heart failure or not.Results Effect of TLR4-SiRNA In Vitro and In VivoFigure 1 showed the expression of mRNA and protein of TLR4 in C6 cell line to identify the knockdown efficacy of different SiRNAs. These results suggested that SiRNA-2 was the most effective for knockdown of TLR4. Therefore, SiRNA-2 wa.
