Abstract
Sepsis is a life-threatening disease caused by infection. Inflammation is a key pathogenic process in sepsis. Paeonol, an active ingredient in moutan cortex (a Chinese herb), has many pharmacological activities, such as anti-inflammatory and antitumour actions. Previous studies have indicated that paeonol inhibits the expression of HMGB1 and the transcriptional activity of NF-κB. However, its underlying mechanism is still unknown. In this study, microarray assay and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) results confirmed that paeonol could significantly up-regulate the expression of miR-339-5p in RAW264.7 cells stimulated by LPS. Dual-luciferase assays indicated that miR-339-5p interacted with the 3′ untranslated region (3′-UTR) of HMGB1. Western blot, immunofluorescence and enzyme-linked immunosorbent assay (ELISA) analyses indicated that miR-339-5p mimic and siHMGB1 both negatively regulated the expression and secretion of inflammatory cytokines (e.g., HMGB1, IL-1β and TNF-α) in LPS-induced RAW264.7 cells. Studies have confirmed that IKK-β is targeted by miR-339-5p, and we further found that paeonol could inhibit IKK-β expression. Positive mutual feedback between HMGB1 and IKK-β was observed when we silenced HMGB1 or IKK-β. These results indicated that paeonol could attenuate the inflammation mediated by HMGB1 and IKK-β by upregulating miR-339-5p expression. In addition, we constructed CLP model mice by cecal ligation and puncture. Paeonol was used to intervene to investigate its anti-inflammatory effect in vivo. The results showed that paeonol could improve the survival rate of sepsis mice and protect the kidney of sepsis mice.
Highlights
Sepsis is a life-threatening disease caused by infection
Immunofluorescence and reverse transcriptionquantitative polymerase chain reaction (RT-qPCR) analyses (P < 0.05; Fig. 1C,D) showed that miR-339-5p was successfully transfected into RAW264.7 cells
SiIKK-β and miR-339-5p were co-transfected into LPS-induced RAW264.7 cells, and the results showed that compared with the LPS group, the siIKK-β group showed downregulated production of High mobility group box 1 (HMGB1); co-transfection with the miR-339-5p mimic and siIKK-β inhibited HMGB1 more significantly than the transfection of siIKK-β alone (P < 0.05; Fig. 7D)
Summary
Sepsis is a life-threatening disease caused by infection. Inflammation is a key pathogenic process in sepsis. Microarray assay and reverse transcriptionquantitative polymerase chain reaction (RT-qPCR) results confirmed that paeonol could significantly up-regulate the expression of miR-339-5p in RAW264.7 cells stimulated by LPS. Immunofluorescence and enzyme-linked immunosorbent assay (ELISA) analyses indicated that miR339-5p mimic and siHMGB1 both negatively regulated the expression and secretion of inflammatory cytokines (e.g., HMGB1, IL-1β and TNF-α) in LPS-induced RAW264.7 cells. Studies have confirmed that IKK-β is targeted by miR-339-5p, and we further found that paeonol could inhibit IKK-β expression. Positive mutual feedback between HMGB1 and IKK-β was observed when we silenced HMGB1 or IKK-β These results indicated that paeonol could attenuate the inflammation mediated by HMGB1 and IKK-β by upregulating miR-339-5p expression. Numerous HMGB1-targeted therapies against inflammation have edu.cn www.nature.com/scientificreports/. MiRNAs (e.g., miR-142-3p, miR-181a, miR-126, and miR-155) have been reported to regulate inflammation by targeting HMGB123,24,26–28
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