Low level laser (LLL) attenuate LPS-induced inflammatory responses in mesenchymal stem cells via the suppression of NF-κB signaling pathway in vitro

by Kan Yin, Rongjia Zhu, Shihua Wang, Robert Chunhua Zhao

Considering promising results in animal models and patients, therapeutic use of MSCs for immune disease is likely to undergo continued evaluation. Low-lever laser (LLL) has been widely applied to retard the inflammatory reaction. LLL treatment can potentially be applied in anti-inflammatory therapy followed by stem cell therapy.

The purpose of this study was to investigate the effect of LLL (660 nm) on the inflammatory reaction induced by LPS in human adipose derived mesenchymal stem cells (hADSCs) and pertinent mechanism.

Anti-inflammatory activity of LLL was investigated by LPS-induced mesenchymal stem cells. The production and expression of pro-inflammatory cytokines were evaluated by ELISA kits and RT-qPCR. Nuclear translocation of NF-κB was indicated by immunofluorescent staining. Phosphorylation status of NF-κB p65 and IκBα were illustrated by western blot assay. ROS generation was measured with CM-H2DCFDA, and NO secretion was determined by DAF-FM. We studied surface expression of lymphocyte activation markers when Purified peripheral blood mononuclear cell (PBMC) were activated by phytohaemagglutinin (PHA) in the presence of 3 types of treated MSCs.

LLL reduced the secretion of IL-1β, IL-6, IL8, ROS and NO in LPS treated MSCs. Immunofluorescent assay demonstrated the nuclear translocation decrease of NF-κB in LLL treated LPS induced MSCs. Western blot analysis also suggested that LLL suppressed NF-κB activation via regulating the phosphorylation of p65 and IκBα. MSC significantly reduced the expression of activation markers CD25 and CD69 on PHA-stimulated lymphocytes.

The results indicate that LLL suppressed the activation of NF-κB signaling pathway in LPS treated MSCs through inhibiting phosphorylation of p65 and IκBα, which results in good anti-inflammatory effect. In addition, LLL attenuated activation-associated markers CD25 and CD69 in co-cultures of PBMC and 3 types of treated MSCs.