Efficacy of Photobiomodulation and Metformin on Diabetic Cell Line of Human Periodontal Ligament Stem Cells through Keap1/Nrf2/Ho-1 Pathway

Abstract

Background: Diabetes mellitus (DM) is a metabolic disorder resulting from hyperglycemia. Hyperglycemia contributes to oxidative stress, and the release of advanced glycation end products (AGEs) further promotes disease pathogenesis. Uncontrolled diabetes reflects great oral complications and affects human oral health. So, the present study aimed to assess the effects of photobiomodulation therapy (PBMT) and Metformin on proliferation and viability of human periodontal ligament stem cells (HPDLSCs) cultured in high glucose medium.

Methods: HPDLSCs were collected, isolated, and characterized and then divided into eight groups. Addition of extra glucose to diabetic groups 24 hours before cell irradiations. Metformin was added to half of the diabetic groups. Cells were irradiated with 808 nm diode laser 24, 48 hours. Cell viability was analyzed with MTT assay 24 hours post-irradiation to detect cell viability in each group. Real-time (PCR) was used to evaluate gene expression of Nrf2, Keap1, PIK3, and HO-1 and the effect of PBMT on Keap1/Nrf2/Ho-1 Pathway. ELISA reader was used to evaluating cell viability through (ROS, TNF-α, IL-10) protein levels after cell irradiation.

Results: Photobiomodulation at 1, 2, and 3 J/cm2 combined with metformin significantly promoted diabetic cell lines of HPDLSCs viability (in MTT assay and ELISA reader of ROS, TNF-α, IL-10 results) and gene expression of Nrf2, Keap1, PIK3, and HO-1 levels (p< 0.05).

Conclusion: photobiomodulation with 3 J/cm² combined with metformin enhanced proliferation and viability of diabetic cell lines of HPDLSCs and thus could improve differentiation and function of diabetic cell lines of HPDLSCs with minimum side effects.

Keywords: Diabetes Mellitus; Metformin; Periodontal Ligament Stem Cells; Photobiomodulation.

Fig. 1

A: show Group. 1 (normal cell) after 14 days, B: Group. 2 (diabetic cell) after 14 days, C: Group. 3 diabetic cells irradiated with 1 J, D: Group. 4 diabetic cells irradiated with 1 J & metformin, E: Group. 5 diabetic cells irradiated with 2 J, F: Group. 6 diabetic cells irradiated with 2 J & metformin, G: Group. 7 diabetic cells irradiated with 3 J, H: Group. 8 diabetic cells irradiated with 3 J & metformin.

Fig. 2

Picture of FACS analysis for immunological characterization of HPDLSCs where results +ve to CD 29, CD 90 and – ve to CD 45.

Fig. 3

Histogram of MTT assay at 24 h among different studied groups, showing significant between different groups of study (p value< 0.0001) Data was expressed as mean ± SD, p-value< 0.05 was significant.

Fig. 4

A: line graph for Nrf2 gene expression among different groups. highest value in group laser 3 J and metformin, B: line graph of Keap1 gene expression among studied groups with statistically significant, best results with laser 3 J and metformin against diabetic group, C: Line graph of PIK3 gene expression among all groups with statistically significant, best value in group irradiated with 3 J and metformin, D: Line graph of HO-1 gene expression among all studied groups, best value with laser 3 J and metformin against diabetic group. Data was expressed as mean ± SD, and p-value< 0.05 was significant.

Fig. 5

A: Histogram of ROS level among all studied groups, there is a statistical significant difference between different groups of study (p value< 0.0001), B: Histogram for TNF-α protein level in all studied groups with statistically significant between different groups against diabetic group (p value< 0.0001), C: Histogram for IL-10 protein level among all studied groups with statistically significant different groups of study (p value< 0.0001), All Data were expressed as mean ± SD, and p-value< 0.05 was significant.