Cycloastragenol reduces inflammation in CLP-induced septic MICE by suppressing TLR4 signaling pathways.

BACKGROUND

Aggressive systemic inflammation due to activation of macrophage-derived excessive immune responses is a critical cause of sepsis leading to clinical death. The effect of cycloastragenol (CAG) on cecal ligation and puncture (CLP)-induced systemic inflammation in mice with sepsis and the underlying mechanism are still unknown.

PURPOSE: Here, we firstly investigated the ameliorative functions of CAG in CLP-induced systemic inflammation in sepsis and LPS-mediated inflammatory response, and the impact of Toll-like receptor 4 (TLR4) pathway on the anti-inflammatory effects of CAG.

METHODS

The in vitro effect of CAG on RAW264.7 cells and THP-1-derived macrophages induced by LPS was detected with quantitative polymerase chain reaction (qPCR), enzyme-linked immunosorbent assay (ELISA), and Western blotting (WB) assays. In addition, the association of TLR4-MD2 complex with CAG was measured through molecular docking, molecular dynamics (MD) simulation, surface plasmon resonance imaging (SPRi), cellular thermal shift assay (CETSA), immunofluorescence and WB. A specific inhibitor of TLR4 receptor TAK-242 and a TLR4-encoding adenovirus were adopted for verifying the functions of CAG. Meanwhile, the in vivo effects of CAG on cardiopulmonary structure, inflammatory factors and survival of CLP-induced septic mice were analyzed through hematoxylin and eosin staining, qPCR, ELISA, and survival analysis.

RESULTS

CAG hindered the LPS-induced production of inflammatory mediators like TNF-α, IL-6 and IL-1β within macrophages in vitro. It also inhibited MAPK and NF-κB pathway activation induced by binding of LPS to TLR4 receptor. As suggested by molecular docking results, the MD2-CAG binding energy was -9.53 kcal/mol. During the MD simulation, CAG could tightly bind to the binding pocket of MD2. SPRi revealed that the equilibrium dissociation constant (KD) value for CAG and TLR4 was 5.24× 10-9 M. Moreover, CAG enhanced the thermal stability of TLR4 by approximately 2.68 °C. It further inhibited the binding between LPS-488 and cell membrane receptors. These inhibitory effects of CAG could be partly reversed by TLR4 overexpression and could not increase by specifically blocking TLR4. In vivo, CAG attenuated cardiopulmonary injury and inflammation and improved survival in septic mice dose-dependently.

CONCLUSION

CAG exerts its anti-inflammatory activity through suppressing MAPK and NF-κB pathway activation caused by TLR4 activation and inhibiting inflammatory factor production dose-dependently.