Nec-1 attenuates inflammation and cytotoxicity induced by high glucose on THP-1 derived macrophages through RIP1.

OBJECTIVES

This research aimed to study whether necrostain-1 (Nec-1) could alleviate inflammatory injury induced by high glucose upon THP-1 derived macrophages through RIP1.

DESIGN

Firstly, THP-1 derived macrophages were incubated with 5.5 mM glucose (normal glucose, NG), 25 mM glucose (high glucose, HG), and mannitol as the high osmotic pressure group (5.5 mM glucose+19.5 mM mannitol) for 24, 48, and 72 h respectively. TNF-α, IL-1β, IL-6, and IL-8 levels were measured by ELISA. Secondly, macrophages were exposed to NG, HG, or HG plus 5 μM necrostatin-1 (Nec-1) for 72 h. mRNA expression of inflammatory cytokine was measured by RT-PCR, and protein levels of inflammatory cytokines and LDH leakage were determined by ELISA. RIP1 expression was determined by RT-PCR and WB. Thirdly, macrophages were transfected with si-RIP1 or negative control (si-NC). Wild type and RIP1-silenced macrophages were incubated with NG or HG, and TNF-α, IL-1β, IL-6, IL-8, and LDH levels were measured again by ELISA.

RESULTS

1. TNF-α, IL-1β, IL-6, and IL-8 levels were elevated in the HG group, as compared with that the NG group. Inflammation remained unchanged in the mannitol group. 2) Inflammatory response and LDH levels in the HG plus Nec-1 group were remarkably lower than in the HG group. 3) Inflammatory injury in the si-NC group was more severe than in the si-RIP1 group.

CONCLUSIONS

Current results indicated that Nec-1 could alleviate HG-caused inflammatory injury on THP-1 derived macrophages by regulating RIP1. These findings could help cast light on the relationships between diabetes and periodontitis.