LIN28A alleviates inflammation, oxidative stress, osteogenic differentiation and mineralization in lipopolysaccharide (LPS)-treated human periodontal ligament stem cells

Abstract

Periodontitis is a complex dental condition that has a number of different etiologies. Lin-28 homolog A (LIN28A) has been previously reported to regulate inflammation, where its expression levels have been indicated to be lower in periodontal tissues following periodontitis. However, there is a lack of evidence to indicate the precise role of LIN28A in periodontitis. In the present study, LIN28A and Runt-related transcription factor 2 (RUNX2) expression were measured in human periodontal biopsy tissues using reverse transcription-quantitative PCR (RT-qPCR). RT-qPCR and western blot analyses were also used to measure LIN28A and RUNX2 expression in human periodontal ligament stem cells (hPDLSCs) following lipopolysaccharide (LPS) induction. Following construction of the LIN28A overexpression plasmid, the expression of LIN28A, RUNX2, osteopontin, osterix and osteocalcin were detected using RT-qPCR and western blotting. Additionally, RT-qPCR was used for the detection of proinflammatory biomarkers (IL-8, IL-1β and IL-6) and alkaline phosphatase (ALP) expression. Protein expression of intranuclear and cytoplasmic NF-κB p65 and NF-κB p65 phosphorylation were assessed using western blot analysis. The expression of antioxidant factors including SOD and GSH were determined using corresponding commercial assay kits. ALP and the mineralization capacity of hPDLSCs were detected by ALP activity assay and Alizarin red staining. The expression of LIN28A was found to be decreased in periodontal biopsy tissues from periodontitis patients compared with normal tissues and LPS-induced hPDLSCs compared with untreated hPDLSCs, which was positively correlated with RUNX2 expression. LIN28A overexpression was revealed to attenuate inflammatory damage and oxidative stress whilst improving ALP active damage, restoring RUNX2 expression and osteoblastic mineralization in LPS-induced hPDLSCs. In conclusion, the present study suggests that LIN28A serves a key role as a mediator of osteoblast differentiation and mineralization. In addition, LIN28A was able to alleviate inflammatory injury and oxidative stress in LPS-induced hPDLSCs.

Keywords: human periodontal ligament stem cells; lin-28 homolog A; lipopolysaccharide; oxidative stress; periodontitis.

Figure 1

LIN28A expression is reduced in periodontal tissue biopsies and LPS-induced hPDLSCs. The expression of (A) LIN28A and (B) RUNX2 in periodontal biopsy tissues was examined by RT-qPCR. (C) The correlation between the mRNA expression level of LIN28A and RUNX2 in periodontal biopsy tissues was examined. (D) Cell morphology and number of hPDLSCs after treatment with different concentrations of LPS were observed by microscope. Scale bars, 100 µm. LIN28A expression in LPS-induced hPDLSCs was measured by (E) western blotting and (F) RT-qPCR. RUNX2 expression in LPS-induced hPDLSCs was determined by (G) western blotting and (H) RT-qPCR. ^*P<0.05, ^**P<0.01 and ^***P<0.001 vs. Control. LIN28A, Lin-28 homeobox A; RUNX2, Runt-related transcription factor 2; RT-qPCR, reverse transcription-quantitative PCR; hPDSCs, human periodontal ligament stem cells; LPS, lipopolysaccharide.

Figure 2

LIN28A overexpression attenuates LPS-induced inflammatory damage in hPDLSCs. (A) A plasmid of LIN28A was constructed for the overexpression of LIN28A and transfection efficiency in hPDLSCs was tested by (A) western blotting and (B) RT-qPCR. The expression of LIN28A in transfected hPDLSCs induced by LPS was measured by (C) western blotting and (D) RT-qPCR. (E) The expression of IL-8, IL-1β and IL-6 in transfected hPDLSCs induced by LPS were quantified by RT-qPCR. (F) The protein expression levels of NF-κB p65 in the nucleus and cytoplasm in addition to NF-κB p65 phosphorylation in transfected hPDLSCs induced by LPS were measured by western blotting. ^*P<0.05, ^**P<0.01 and ^***P<0.001 vs. Control; ^#P<0.05, ^##P<0.01 and ^###P<0.001 vs. LPS + Ov-NC. LIN28A, Lin-28 homeobox A; LPS, lipopolysaccharide; hPDSCs, human periodontal ligament stem cells; RT-qPCR, reverse transcription-quantitative PCR; Ov, overexpression; NC, negative control.

Figure 3

LIN28A overexpression alleviates oxidative stress damage and upregulates RUNX2 expression in LPS-induced hPDLSCs. (A) Reactive oxygen species levels in transfected hPDLSCs induced by LPS were measured using a 2',7'-dichlorofluorescin diacetate measurement kit. Scale bars, 50 µm. The levels of (B) SOD and (C) GSH in transfected hPDLSCs induced by LPS were determined using their corresponding kits. (D) An ALP kit was used for detecting the activity of ALP in transfected hPDLSCs induced by LPS. (E) RT-qPCR was used to measure ALP expression in transfected hPDLSCs induced by LPS. RUNX2 expression in transfected hPDLSCs induced by LPS was tested by (F) western blotting and (G) RT-qPCR. ^**P<0.01 and ^***P<0.001 vs. control; ^##P<0.01 and ^###P<0.001 vs. LPS + Ov-NC. LIN28A, Lin-28 homeobox A; RUNX2, Runt-related transcription factor 2; LPS, lipopolysaccharide; hPDSCs, human periodontal ligament stem cells; Ov, overexpression; NC, negative control; SOD, superoxide dismutase; GSH, glutathione; ALP, alkaline phosphatase.

Figure 4

LIN28A facilitates osteoblast mineralization in LPS-induced hPDLSCs. (A) Alizarin red staining was used to evaluate the mineralization capacity of osteoblasts. Scale bars, 50 µm. The expression of (B) OPN, (C) OSX and (D) OCN in transfected hPDLSCs induced by LPS were assessed by reverse transcription-quantitative PCR. (E) The protein levels of OPN, OSX and OCN in transfected hPDLSCs induced by LPS were measured by western blotting. ^*P<0.05, ^**P<0.01 and ^***P<0.001 vs. Control; ^##P<0.01 and ^###P<0.001 vs. LPS + Ov-NC. Lin-28 homeobox A; LPS, lipopolysaccharide; hPDSCs, human periodontal ligament stem cells; OPN, osteopontin; OSX, osterix; OCN, osteocalcin; Ov, overexpression; NC, negative control.