[Experimental study on the role of TRPM2 in regulating osteoblast differentiation through mitophagy in periodontitis].

Objective

To explore the regulatory mechanism of transient receptor potential cation channel subfamily M member 2 (TRPM2) on the osteogenic differentiation capacity of bone marrow mesenchymal stem cells (BMMSCs), and to clarify the role of TRPM2-regulated mitophagy in the progression of periodontitis.

Methods

Twelve TRPM2 gene knockout (TRPM2-/-) mice and twelve wild-type (WT) mice were used in this study. A periodontitis model was established by silk ligation on the maxillary second molars of mice, with sham operation performed in the control group, and the modeling lasted for 10 days. The mice were divided into four groups (n=6 per group): WT sham operation group, WT periodontitis group, TRPM2-/- sham operation group, and TRPM2-/- periodontitis group. In the periodontitis group, silk ligation was performed on the maxillary second molars of mice, while no silk ligation was conducted in the sham operation group. Micro-CT was employed to collect imaging data for three-dimensional reconstruction. CTvox and CTAn v1.15.4.0 software were used to quantify the distance from the cementoenamel junction to the alveolar bone crest (CEJ-ABC) and bone volume fraction (BV/TV) in maxillary bone tissues of the mice in four groups. Statistical analysis was performed subsequently. Immunohistochemical staining was conducted to detect the expression intensity of Runt-related transcription factor 2 (RUNX2) in periodontal tissues in maxillary bone tissues of the mice in four groups. BMMSCs were isolated from the femurs of the two types mice and cultured in vitro, followed by induction for osteogenic differentiation. Alizarin red S (ARS) staining and alkaline phosphatase (ALP) staining were used to evaluate the osteogenic differentiation potential of BMMSCs. Western blotting (WB) was performed to determine the expression levels of osteogenic-related markers [RUNX2, bone morphogenetic protein 2 (BMP2), ALP, osteopontin (OPN)]. Meanwhile, transmission electron microscopy (TEM) was used to observe intracellular mitophagy status. WB was applied to detect the expression of autophagy-related proteins [microtubule-associated proteins 1A/1B light chain 3 (LC3), PTEN-induced putative kinase 1 (PINK1), Parkin RBR E3 ubiquitin protein ligase (Parkin)], and immunofluorescence colocalization labeling was used to assess the fluorescence intensity of LC3, translocase of the outer mitochondrial membrane 20 (Tomm20), and Lysosomal-associated membrane protein (LAMP).

Results

No statistically significant differences were observed in bone volume fraction, trabecular number, and trabecular separation between TRPM2-/- and WT mice (all P>0.05). However, the buccal and palatal CEJ-ABC values in the TRPM2-/- periodontitis group [(265.40±21.72) μm and (273.30±17.56) μm, respectively] were significantly lower than those in the WT periodontitis group [(416.50±20.90) μm and (428.00±17.59) μm, respectively] (both P<0.001). In addition, the relative expression level of RUNX2 in periodontal tissues of the TRPM2-/- periodontitis group [(15.03±0.48) %] was significantly higher than that of the WT periodontitis group [(11.95±0.40) %] (P<0.001). In vitro experiments (ALP and ARS staining) demonstrated that the osteogenic differentiation capacity of BMMSCs derived from TRPM2-/- mice was significantly enhanced compared with that from WT mice. WB results showed that the expression levels of osteogenic-related markers (RUNX2, BMP2, ALP, OPN) in BMMSCs from TRPM2-/- mice were all upregulated compared with WT mice (all P<0.05, respectively), and so as the protein levels of mitophagy-related indicators (LC3 and BECLIN1)(both P<0.001). Furthermore, TRPM2 deficiency remarkably upregulated the expression of proteins related to the PINK1/Parkin pathway (all P<0.001).

Conclusions

TRPM2 regulates the osteogenic differentiation capacity of BMMSCs through mitophagy, thereby participating in the progression of periodontitis. Therefore, targeting TRPM2 is expected to serve as a novel and effective strategy for the treatment of periodontitis.