FTIR spectroscopic characterization reveals short-term macromolecular responses to photobiomodulation in mesenchymal stem cells

Abstract

Photobiomodulation (low-level laser therapy) was reported to promote tissue repair, stem cell differentiation and proliferation; however, the underlying cellular mechanisms remain unclear. Investigating early, subtle cellular responses may be the key to optimize therapeutic outcomes and understand the biological effects of photobiomodulation. This study aimed to assess short-term macromolecular alterations in adipose-derived mesenchymal stem cells (ADSCs) subjected to 671 nm low-level laser irradiation via Fourier transform infrared (FTIR) spectroscopy. ADSCs were divided into control, 1-hour (1 H post-L) and 3-hour (3 H post-L) post-irradiation groups. 671 nm low-level laser irradiation was applied at a fluency of 25 J/cm². After 1 and 3 h, cells were collected and placed on the ATR unit of the FTIR spectrophotometer, dried under mild nitrogen flow and analyzed. Photobiomodulation resulted in explicit differences in FTIR spectra of 1 H and 3 H post-L groups, including alterations in lipid composition, protein secondary structure, protein phosphorylation together with changes in metabolic turnover of carbohydrates over time. Additionally, the membrane order decreased and fluidity increased in the 1 H post-L group, suggesting a temporary lateral phase separation in ADSC membranes. Photobiomodulation induced short-term structural and compositional alterations in biomolecules of ADSCs, which is further remarkable to provide insights into its action mechanism.

Keywords: Adipose-derived mesenchymal stem cells; FTIR spectroscopy; Low-level laser therapy; Photobiomodulation; Short-term effects.

Fig. 1

(a) The schematic diagram of the timeline of the applied procedures. (b) The experimental setup for laser irradiation to the ADSCs. (c) the results of the MTT test demonstrating the changes in cell viability upon laser exposure at different energy densities of 0 (control), 1, 5, 10, 25 and 50 J/cm². (d) Western blot images of Hsp70 and GAPDH protein levels and (e) the fold change in Hsp70 protein expression levels compared to that of GAPDH in ADSCs of control, 1 H post-L and 3 H post-L groups. The full-length Western blots used for calculation are presented in Supplementary Figure S1. The data of MTT test and Hsp70 protein expression levels were compared by the use of one-way ANOVA with Dunn’s test as a post-hoc test. The degree of significance was denoted as *p < 0.05. The analysis of Hsp70 protein expression levels revealed non-significant alterations in between the groups.

Fig. 2

(a) The representative FTIR spectra of control ADSCs in 4000–850 cm⁻¹ region, the representative FTIR spectra of control, 1 H post-L and 3 H post-L groups (b) in 3050–2800 cm⁻¹, (c) in 1800–800 cm⁻¹, (d) in 1200–850 cm⁻¹ region. Spectra were baseline corrected and normalized with respect to Amide A band located at 3282 cm⁻¹ for visual demonstration.

Fig. 3

The bar graphs demonstrating the alterations in band area ratios of (a) Unsaturated lipid content, (b) Unsaturated/saturated lipids, (c) Carbonyl amount in lipids, (d) Aliphatic chain length, (e) Lipid/protein, (f) Protein concentration, (g) Protein phosphorylation, (h) Collagen integrity, (i) RNA/PO²⁻ antisym. + sym. stretching, (j) PO²⁻ antisym. + sym. stretching/protein, (k) Glucose/protein, (l) Glucose/phosphate for control 1 H post-L and 3 H post-L groups. The data was analyzed via one-way ANOVA with Tukey’s test as a post-hoc test. The degree of significance was denoted as *p < 0.05, **p < 0.01, ***p < 0.001.

Fig. 4

The representative absorbance (a) and second derivative (b) spectra of control, 1 H post-L and 3 H post-L groups in 1700–1600 cm⁻¹ region. The investigated protein secondary structural sub-bands under amide I band were labelled in the figure with numbers [1: antiparallel β-sheet structure; 2: α-helical structure; 3: native β-sheet structure; and 4: aggregated β-sheet structure].