Q M Zhai1, B Li2, Z W Wang1, L Liu1, Y Jin2, F Jin1. 1. Department of Orthodontics, School of Stomatology, The Fourth Military Medical University & State Key Laboratory of Military Stomatology & National Clinical Research Center for Oral Diseases & Shaanxi Clinical Research Center for Oral Diseases, Xi'an 710032, China. 2. Department of Histopathology, School of Stomatology, The Fourth Military Medical University & State Key Laboratory of Military Stomatology & National Clinical Research Center for Oral Diseases & Shaanxi International Joint Research Center for Oral Diseases, Xi'an 710032, China.
Abstract
Objective: To investigate the effect of endoplasmic reticulum (ER)-mitochondria coupling and the expression of mitofusion 2 (Mfn2) in periodontal ligament stem cells (PDLSC), so as to provide a theoretical basis and therapeutic target for promoting periodontal regeneration in treatment of periodontitis. Methods: The periodontal ligament tissue was scraped from extracted intact human teeth and teeth with periodontitis collected in the Department of Oral and Maxillofacial Surgery, the Fourth Military Medical University. The health PDLSC (H-PDLSC) and inflammatory PDLSC (P-PDLSC) were obtained respectively from the primary culture of the human teeth and cloned using 1imited diluted method. The level of ER-mitochondrial coupling was observed by transmission electron microscopy and organelle-specific fluorescence staining. Quantitative real-time PCR (qPCR) was used to detect the expression of Mfn2 in H-PDLSC and P-PDLSC. Tumor necrosis factor alpha (TNF-α) was used to simulate the inflammatory microenvironment. H-PDLSC was cultured in normal medium and media containing 5 and 10 mg/L TNF-α, named as H-PDLSC group, H-PDLSC+TNF-α (5 mg/L) group and H-PDLSC+TNF-α (10 mg/L) group, respectively. At the 7th day, qPCR was applied to detect the mRNA level of Mfn2. The expression of Mfn2 in P-PDLSC was down-regulated by small interfering RNA siMfn2. The osteogenic differentiation of P-PDLSC and P-PDLSC+siMfn2 were examined by qPCR at the 7th day, and alizarin red staining and cetyl pyridine chloride quantitative analysis at the 28th day after osteogenic induction. Results: The level of ER-mitochondrial coupling in the P-PDLSC group (the length of the coupling structure/mitochondrial perimeter was 0.55±0.10, the length of the coupling structure/endoplasmic reticulum perimeter was 0.44±0.08) was significantly higher than that in the H-PDLSC group (P<0.01). The co-localization of endoplasmic reticulum and mitochondria of P-PDLSC group was 0.71±0.09, which was significantly higher than that of H-PDLSC group (P<0.01). The expression level of Mfn2 in P-PDLSC (1.46±0.10) was higher than that in H-PDLSC (0.99±0.08). The expression levels of Mfn2 in H-PDLSC+TNF-α (5 mg/L) and H-PDLSC+TNF-α (10 mg/L) groups were 1.28±0.19, 1.54±0.43, respectively, which were both significantly higher than that in H-PDLSC (0.82±0.14) (P<0.01). P-PDLSC transfected with siMfn2 down-regulated the expression of Mfn2, and the osteogenic differentiation ability of P-PDLSC was restored. The results showed that the expression of alkaline phosphatase, Runt-related transcription factor-2 (RUNX2) and osteocalcin mRNA in P-PDLSC+siMfn2 group were significantly higher than that of the control group (P<0.01). The alizarin red staining and quantitative results of cetyl pyridinium chloride were consistent with the qPCR results. Conclusions: In the microenvironment of inflammation, ER-mitochondrial coupling and the expression of Mfn2 of PDLSC increased, which might lead to a decrease in osteogenic differentiation of PDLSC. The specific mechanism needs to be further studied.
Objective: To investigate the effect of endoplasmic reticulum (ER)-mitochondria coupling and the expression of mitofusion 2 (Mfn2) in periodontal ligament stem cells (PDLSC), so as to provide a theoretical basis and therapeutic target for promoting periodontal regeneration in treatment of periodontitis. Methods: The periodontal ligament tissue was scraped from extracted intact human teeth and teeth with periodontitis collected in the Department of Oral and Maxillofacial Surgery, the Fourth Military Medical University. The health PDLSC (H-PDLSC) and inflammatory PDLSC (P-PDLSC) were obtained respectively from the primary culture of the human teeth and cloned using 1imited diluted method. The level of ER-mitochondrial coupling was observed by transmission electron microscopy and organelle-specific fluorescence staining. Quantitative real-time PCR (qPCR) was used to detect the expression of Mfn2 in H-PDLSC and P-PDLSC. Tumor necrosis factor alpha (TNF-α) was used to simulate the inflammatory microenvironment. H-PDLSC was cultured in normal medium and media containing 5 and 10 mg/L TNF-α, named as H-PDLSC group, H-PDLSC+TNF-α (5 mg/L) group and H-PDLSC+TNF-α (10 mg/L) group, respectively. At the 7th day, qPCR was applied to detect the mRNA level of Mfn2. The expression of Mfn2 in P-PDLSC was down-regulated by small interfering RNA siMfn2. The osteogenic differentiation of P-PDLSC and P-PDLSC+siMfn2 were examined by qPCR at the 7th day, and alizarin red staining and cetyl pyridine chloride quantitative analysis at the 28th day after osteogenic induction. Results: The level of ER-mitochondrial coupling in the P-PDLSC group (the length of the coupling structure/mitochondrial perimeter was 0.55±0.10, the length of the coupling structure/endoplasmic reticulum perimeter was 0.44±0.08) was significantly higher than that in the H-PDLSC group (P<0.01). The co-localization of endoplasmic reticulum and mitochondria of P-PDLSC group was 0.71±0.09, which was significantly higher than that of H-PDLSC group (P<0.01). The expression level of Mfn2 in P-PDLSC (1.46±0.10) was higher than that in H-PDLSC (0.99±0.08). The expression levels of Mfn2 in H-PDLSC+TNF-α (5 mg/L) and H-PDLSC+TNF-α (10 mg/L) groups were 1.28±0.19, 1.54±0.43, respectively, which were both significantly higher than that in H-PDLSC (0.82±0.14) (P<0.01). P-PDLSC transfected with siMfn2 down-regulated the expression of Mfn2, and the osteogenic differentiation ability of P-PDLSC was restored. The results showed that the expression of alkaline phosphatase, Runt-related transcription factor-2 (RUNX2) and osteocalcin mRNA in P-PDLSC+siMfn2 group were significantly higher than that of the control group (P<0.01). The alizarin red staining and quantitative results of cetyl pyridinium chloride were consistent with the qPCR results. Conclusions: In the microenvironment of inflammation, ER-mitochondrial coupling and the expression of Mfn2 of PDLSC increased, which might lead to a decrease in osteogenic differentiation of PDLSC. The specific mechanism needs to be further studied.