Binding Of Esculentoside A To Ribosomal Protein S3a Contributes Its Anti-inflammatory Effect

【Background and Objective】Esculentoside A (EsA), a saponin isolated from the root of Phytolacca esculenta, has been reported to exert anti-inflammatory effects in several animal models of acute and chronic inflammation by inhibiting the production and activity of pro-inflammatory cytokines in macrophages and epithelial cells. However, little is known about its intracellular mechanisms. We have previously identified ribosomal protein S3a(RPS3a), a component of the ribosomal small subunit (40S), as a specific intracellular binding protein of EsA. Studies have shown that RPS3a plays an important role in protein synthesis and also have unique extra-ribosomal functions. But it is not clear whether RPS3a is involved in regulation of signal transductions related to inflammatory and immune responses.This study aimed to elucidate the anti-inflammatory mechanism of EsA, and to investigate whether its specific binding protein RPS3a is involved in regulation of signal transductions related to inflammatory and immune responses.【Methods】We used LPS-stimulated RAW264.7cells and TNF-α-stimulated Jurkat cell and293cells as in vitro cell models. The expressions of pro-inflammatory cytokines were detected by RT-PCR and ELISA. The signaling cascades including the activation of NF-κB and MAPK (JNK and ERK, p38) pathways were detected by Western Blot and dual-luciferase reporter assay. Lentiviral RNAi and overexpression of RPS3a were conducted to investigate the possible role of RPS3a in regulation of signal transductions. Co-Immunoprecipitation was used to identify intracellular proteins that may interact with RPS3a.【Results and Conclusion】1. EsA suppressed the expression of pro--inflammatory cytokines. Within the range of5-40μM, EsA dose-dependently inhibited the expression of TNF-α in LPS-stimulated RAW264.7cells, and the expression of IL-8in TNF-a-stimulated Jurket cells and293cells.2. EsA inhibited the activation of NF-κB and MAPK pathways (1) The dual-luciferase reporter assay showed that EsA inhibited the expression of the NF-κB reporter gene in TNF-α-stimulated293cells in a dose-dependent manner in the range of5-40μM.(2) Western Blot assays showed that EsA remarkably blocked the IκB phosphorylation and degradation in LPS-stimulated RAW264.7cells and TNF-α-stimulated Jurket cells in a dose-dependent manner in the range of5-40μM.(3) Western Blot assays showed that EsA remarkably inhibited the phosphorylation of JNK and p38in LPS-stimulated RAW264.7cells and TNF-α-stimulated Jurket cells in a dose-dependent manner in the range of5-40μM.3. RPS3a knockdown by lentiviral RNAi inhibited the expression of pro-inflammatory cytokines in LPS-stimulated RAW264.7cells, blocked LPS-activated signaling cascades, and enhanced inhibitory effects of EsA.(1) RT-PCR and Western Blot assays showed that transfection of RAW264.7cells with RPS3a-specific lentiviral-shRNA resulted in the knockdown of RPS3a.(2) RNAi of RPS3a resulted in the decreased of expression of TNF-a and IL-6in LPS-stimulated RAW264.7cells.(3) Transfection of RAW264.7cells with RPS3a-specific lentiviral-shRNA resulted in the decrease of intracellular RPS3a, and simultaneously inhibited LPS-stimulated IκB phosphorylation and degradation, JNK and p38phosphorylation, and decrease of IRAK1, suggesting RPS3a is involved in LPS-activated signaling cascades.(4) EsA suppressed TNF-α production and NF-κB activation much more potently in RPS3a-knockdowned RAW264.7cells than in control cells, suggesting a synergistic inhibitory effect on immune cell activation between EsA and RNAi of RPS3a.4. In293cells overexpressed with RPS3a, TNF-a similarly enhanced the release of IL-8and stimulated the activation of signal pathways, but EsA lose its inhibitory effects.(1) Transfection of293cells with RPS3a-expressing plasmid pcDNA3.1-RPS3a resulted in the expression of His-RPS3a as detected by Western Blot48h after plasmid transfection.(2)293cells overexpressing His-RPS3a released the same level of IL-8after TNF-α stimulation as compared with control cells (transfected with pcDNA3.1vector). However, EsA no longer inhibited the release of IL-8in TNF-α-stimulated93cells overexpressing His-RPS3a. (3) TNF-a stimulation similarly led to IκB phosphorylation and degradation, as well as JNK and p38phosphorylation in293cells overexpressing His-RPS3a. However, EsA no longer inhibited these signaling cascades in RPS3a-overexpressing293cells. Combined with the results from RPS3a RNAi experiments, the present study suggested that the signal transduction of LPS or TNF-α stimulation require the participation of RPS3a, and EsA inhibited LPS or TNF-α-mediated signal transduction by binding to RPS3a and blocked its functions. Therefore, RPS3a might be a molecular target for EsA for its anti-inflammatory actions.5. searching for signal proteins that may interacted with RPS3a by co-immunoprecipitation to further explore the involvement of RPS3a in related signal transductions(1) Co-immunoprecipitation was conducted to identify intracellular proteins that may interact with RPS3a and several bands that might co-immunoprecipitated with were found.(2) Western Blot assays detected IκB and CK2in co-immunoprecipitated proteins.