Ribosomal Protein S3a Inhibits Endotoxin-Induced Imflammation And Synergizes With Anti-imflammatory Effect Of Esculentoside A

Objective:To investigate effect of Ribosomal Protein S3a (RPS3a), which is the cellular binding protein of anti-inflammatory agent esculentoside A, on LPS-induced inflammation for further exploring the anti-inflammatory mechanism of esculentoside A and providing new strategies to treat inflammation-related diseases.Methods:The RPS3a gene was obtained by RT-PCR from cDNA reversely transcribed from total mRNA of human HEK.293cell. The RPS3a gene fragments were subcloned into the shuttle vector pShuttle-CMV. Recombinant pShuttle-CMV-RPS3a was then identified by enzyme digestion and DNA sequence analysis, lineared by enzyme Pmel and transformed into competent E.Coli BJ5183carrying backbone plasmid pAdEasy-1for homologous recombination; The recombinant adenoviral vector was lineared by enzyme PacI and transfected into293cells by Lipofectamine2000to package adenovirus. The packaging efficiency was evaluated with green fluorescence and the virus titre was measured by morphology of293cells after adenovirus infection. In order to verify the efficacy of AdRPS3a, different cell types were infected with AdRPS3a or AdGFP control at the same MOI and western blotting was used to measure RPS3a protein expression level. Cell inflammatory model was established using murine RAW264.7stimulated with LPS. RAW264.7cells were infected with AdRPS3a or AdGFP for48h and then treated with or without EsA prior to LPS(1μg/ml) stimulation for24h. Levels ofNO, TNF-a and IL-10in the cell culture supernatants were collected and determined by Griess method and ELISA assay. Meanwhile, intracellular NF-κB, MAPK (ERK, JNK and P38), AKT and STAT3signaling pathways were detected by Western blot analysis after infected cells sitmulated with LPS for30min. Endotoxin shock model was established in mouse by injecting intraperitoneally with lethal dose of LPS (15mg/Kg). To evaluate effect of RPS3a on endotoxin shock, mice were injected via tail vein with AdRPS3a or AdGFP.3d later, mice were challenged with LPS and monitored over a10-day period for survival.Results:The RPS3a gene was obtained by RT-PCR using total RNA extracted from human HEK293cell as the template, then cloned and inserted into the Sol Ⅰ/Hind Ⅲ sites of shuttle vector pShuttle-CMV to successfully form transfer vector pShuttle-CMV-RPS3a. The construct was verified by enzyme digestion with Sal Ⅰ/Hind Ⅲ and DNA sequence determination. Recombinant adenovirus vector pAdRPS3a was fruitfully obtained through homologous recombination of Pmel-lineared pShuttle-CMV-RPS3a and pAdEasy-1in E.Coli BJ5183and confirmed by digestion with Pacl. The lineared recombinant adenoviral vector was transfected to293cells and effectively packaged as AdRPS3a adenovirus. The Titre of amplied recombinant adenovirus could reach to1011pfu/ml. Western Bloting showed that RPS3a protein was markedly increased inNIH3T3cells、HSC-T6cells and293cells infected with AdRPS3a compared with that of AdGFP control. RPS3a overexpression in RAW264.7cells resulted in the decreased release of TNF-a and NO in cell supernatant after LPS stimulation, but elevated the level of IL-10; Western Blot analysis showed that RPS3a overexpression inhibited LPS-stimulated ERK,JNK,p38and IκB phosphorylation. but enhanced STAT phosphorylation. However, RPS3a overexpression rendered no evident influence on the IκB degradation and AKT phosphorylation. Overexpression RPS3a conferred a survival rate of70%in LPS-induced lethality, compared with0%for the control. ESA enhanced inhibitory effects of RPS3a on NO production in LPS-stimulated RAW264.7.Conclusion:Ribosomal Protein S3a exhibits anti-inflammatory activities in vivo and vitro, at least in part, through inhibiting the activation of MAPK and NF-κB signaling pathways and enhancing STAT3phosphorylation. Synergistic effect of EsA with RPS3a on inflammation inhibition suggest that binding of EsA to RPS3a may contribute to its anti-inflammatory effects.