Mechanism of Innate Immune Response Induced by Saponin Active Fraction Using C2C12 Myoblasts.

saponin active fraction (AJSAF), is a prospective adjuvant with dual Th1/Th2 and Tc1/Tc2 potentiating activity. Its adjuvant activity has previously been proven to be strictly dependent on its spatial co-localization with antigens, highlighting the role of local innate immunity in its mechanisms. However, its potential targets and pathways remain unclear. Here, its intracellular molecular mechanisms of innate immune response were explored using mouse C2C12 myoblast by integrative analysis of the in vivo and in vitro transcriptome in combination with experimental validations. AJSAF elicited a temporary cytotoxicity and inflammation towards C2C12 cells. Gene set enrichment analysis demonstrated that AJSAF regulated similar cell death- and inflammatory response-related genes in vitro and in vivo through activating second messenger-MAPK-CREB pathways. AJSAF markedly enhanced the Ca, cAMP, and reactive oxygen species levels and accelerated MAPK and CREB phosphorylation in C2C12 cells. Furthermore, Ca chelator, CREB inhibitor, and MAPK inhibitors dramatically blocked the up-regulation of IL-6, CXCL1, and COX2 in AJSAF-treated C2C12 cells. Collectively, these results demonstrated that AJSAF induced innate immunity via Ca-MAPK-CREB pathways. This study is beneficial for insights into the molecular mechanisms of saponin adjuvants.