Anti-inflammatory effect of green photobiomodulation in human adipose-derived mesenchymal stem cells

Abstract

Photo biomodulation (PBM) as a non-invasive and safe treatment has been demonstrated the anti-inflammatory potential in a variety of cell types, including stem cells. However, further investigations using different laser parameters combined with more accurate methods such as quantitative measurement of inflammatory gene expression at the mRNA level are still necessary. The aim of this study was to evaluate the effect of 532 nm green laser on cell proliferation as well as expression of inflammatory genes in human adipose-derived mesenchymal stem cells (hADMSCs) using RNA sequencing (RNA-seq) technique and confirmatory RT-PCR. hADMSCs were cultured in DMEM low glocuse medium with 10% fetal bovine serum until the fourth passage. Cultured cells were divided in two groups: control group (no laser irradiation) and laser group, irradiated with 532 nm laser at 44 m J/cm² with an output power of 50 mW and a density of 6 mW/cm², every other day, 7 s each time. The cell viability was assessed using MTT assay 24 h after each irradiation on days 3, 5, and 7 after cell seeding, followed by performing RNA-seq and RT-PCR. The MTT assay showed that PBM increased cell proliferation on day 5 after irradiation compared to day 3 and decreased on day 7 compared to day 5. In addition, gene expression analysis in hADMSCs using RNA-seq revealed down-regulation of inflammatory genes including CSF2, CXCL2, 3, 5, 6, 8, and CCL2, 7. These results indicate that 532 nm PBM with the parameters used in this study has a time-dependent effect on hADMSCs proliferation as well as anti-inflammatory potential.

Keywords: Inflammation; Low-level light therapy; Mesenchymal stem cells; RNA; Sequence analysis.

Fig. 1

The time frame of our experimental design. Following seeding of human adipose-derived mesenchymal stem cells, laser therapy was conducted at days D2, D4, and D6. To explore the effect of laser on the cells, MTT assay, RNA-seq, and RT-PCR were subsequently conducted

Fig. 2

C1-3: control MSCs; L1-3: laser-treated MSCs at days D2, D4, and D6

Fig. 3

A MTT assay shows a significant increase in optical density between the control and laser groups on day 3 and day 5 after treatment (*p-value < 0.05) (**p-value < 0.001). B Cell viability shows a significant increase)***p-value < 0.0005 ( from day 3 to 5 after irradiation and a significant decrease)**p-value < 0.001) from day 5 to day 7 after treatment. The values are expressed as means (± SEM; n = 4)

Fig. 4

The expression levels for the candidate genes relative to β-actin as control (see Table 2). The values are expressed as means (± SEM; n = 3), (**p < 0.001); (*p < 0.05)