Title:Anti-inflammatory Effect of Novel 2-Phenylphthalazin-2-ium Bromides on LPS-induced RAW264.7 Cells and their Mechanism
Volume: 21
Issue: 12
Author(s): Fang-Jun Cao*, Jian Shen, Hui Zhang and Lu Wang
Affiliation:
- Shaanxi Key Laboratory of Qinling Ecological Security, Shaanxi Institute of Zoology, Xi’an, Shaanxi, 710072, P. R.
China
Keywords:
Isoquinolinebenzo[c]phenanthridine alkaloids, 2-phenylphthalazin-2-ium bromides, Anti-inflammatory effect, Inflammatory cytokines, TLR4-NF-κB/MAPK signal pathway.
Abstract:
Background: Inspired by natural anti-inflammatory quaternary benzo[C]phenanthridine
alkaloids, novel 2-phenylphthalazin-2-ium bromides were previously designed and synthesized.
Objective: The anti-inflammatory effect of 2-phenylphthalazin-2-ium bromides was evaluated based
on inflammatory cytokines, and their possible mechanism was explored through the NF-κB, TLR4
and MAPK signaling pathways.
Methods: The tested concentrations of two compounds were assessed using MTT assay in vitro.
Griess assay was used to determine the changes in nitric oxide (NO) in the cell culture supernatant.
qRT‒PCR was used to detect the mRNA levels of inflammatory cytokines, such as IL-6, IL-1ß, IL-10,
TNF-α, TLR4 and iNOS. The secretion levels of TNF-α and IL-1β were detected by ELISA. Western
blot test was used to detect the protein expression of IL-6, IL-10, TLR4, iNOS, NF-κB, p-P38/P38, p-
ERK/ERK and p-JNK/JNK.
Results: 2-(3,5-Dichlorophenyl)phthalazin-2-ium bromide (2) with a concentration below 1 μg/mL
showed no significant effect on the growth inhibition of RAW264.7 cells, so the concentrations of
compound 2 used for experiments were set to 0, 0.25, 0.5 and 1 μg/mL. Compared with the blank
control group, the model group showed increased release of NO, transcription levels of IL-6, IL-1ß,
IL-10, TNF-α, TLR4 and iNOS (p<0.05), and ratios of p-P38/P38, p-ERK/ERK, p-JNK/JNK
(p<0.05). Compared with the model group, the sample groups displayed decreased NO release and
reduced transcriptional levels of IL-6, IL-1ß, IL-10, TNF-α, TLR4, and iNOS and reduced protein
expression ratios of IL-6, IL-1ß, IL-10, TNF-α, NF-κB, TLR4, iNOS, p-P38/P38, p-ERK/ERK and p-
JNK/JNK (p<0.05).
Conclusion: This study showed that 2-phenylphthalazin-2-ium bromides partially protected macrophages
from the LPS-induced inflammatory response by suppressing TLR4-NF-κB/MAPK signaling
and reducing NO production.
