Lipopolysaccharide-Induced Nitric Oxide, Prostaglandin E2, and Cytokine Production of Mouse and Human Macrophages Are Suppressed by Pheophytin-b.

Chun-Yu Lin,Yu-Wei Chang,Yen-Hsu Chen,Shin-Huei Chen,Chung-Yi Chen,Wen-Hung Wang,Ling-Chien Hung

doi:10.3390/ijms18122637

Abstract

Sepsis is an overwhelming systemic response to infection that frequently results in tissue damage, organ failure, and even death. Nitric oxide (NO), prostaglandin E2 (PGE2), and cytokine overproduction are thought to be associated with the immunostimulatory cascade in sepsis. In the present study, we analyzed the anti-inflammatory efficacy of the pheophytin-b on both RAW 264.7 murine macrophage and purified human CD14+ monocytes stimulated with lipopolysaccharide (LPS) and elucidated the mechanisms by analyzing the cell signaling pathways known to be activated in sepsis. Pheophytin-b suppressed the overexpression of NO, PGE2, and cytokines in LPS-stimulated macrophages without inducing cytotoxicity. It also reduced NOS2 and COX-2 mRNA and protein levels. The inhibitory effects on NO, PGE2, and cytokine overproduction arose from the suppression of STAT-1 and PI3K/Akt pathways; no changes in NF-κB, MAPK, and AP-1 signaling were detected. Thus, pheophytin-b may represent a potential candidate to beneficially modulate the inflammatory response in sepsis.

Highlights

Severe sepsis, and septic shock remain to be the main causes of death among critically ill patients [1], and the number of new sepsis cases are increasing [2,3] despite advances in understanding its etiology and the development of new therapeutic strategies [4]
We stimulated the purified human CD14+ monocytes to differentiate into macrophages by adding human GM-CSF (10 ng/mL) (PeproTech, Rocky Hill, NJ, USA) for 6 days at 37 ◦C in 5% CO2 [46] after which they were prepared for subsequent pheophytin-b treatment
Colorimetric analyses were obtained with an Enzyme-Linked Immunosorbent Assay (ELISA) plate reader at 570 and 600 nm, and the cytotoxicity of pheophytin-b treatment was evaluated relative to controls

Summary

Introduction

Severe sepsis, and septic shock remain to be the main causes of death among critically ill patients [1], and the number of new sepsis cases are increasing [2,3] despite advances in understanding its etiology and the development of new therapeutic strategies [4]. Effects of Pheophytin-b on NO and PGE2 Production in LPS-Stimulated Macrophages. Effects of Pheophytin-b on NO and PGE2 Production in LPS-Stimulated Macrophages In the present study, pre-treatment of RAW 264.7 cells with pheophytin-b for 30 min elicited significanInt, tdhoesper-edseepnet nsdtuednyt, spurpe-ptrreeastsmioenntoof fLRPAS-Wind26u4c.7edceNllsOwpirthodpuhcetoipohnyatisnd-betfeocrte3d0 mbyinaerleicdituecdtion in nistirginteifilceavnetl,sd(opse

Reagents and Antibodies

Cytotoxicity Analysis Following Pheophytin-b Treatment

Measurement of Nitrite Release Indicative of NO Production

Signal Transduction Pathway Analysis

4.10. Statistical Analysis