W Qiu1,2,3, B Sun1,3, F He4, Y Zhang3,5. 1. Graduate School at Shenzhen, Tsinghua University, Shenzhen, China. 2. Department of Chemistry, Tsinghua University, Beijing, China. 3. Key Lab in Healthy Science and Technology, Division of Life Science, Graduate School at Shenzhen, Tsinghua University, Shenzhen, China. 4. Department of Stomatology, the Second Clinical Medical College, Shenzhen People's Hospital, Jinan University, Shenzhen, China. 5. Open FIESTA Center, Tsinghua University, Shenzhen, China.
Abstract
AIM: To determine the roles of autophagy and Notch signalling in mineral trioxide aggregate (MTA)-mediated proliferation and differentiation of human dental pulp cells (hDPCs). METHODOLOGY: hDPCs were separated from human healthy molar teeth using collagenase I/dispase II digestion and then cultured in α-MEM medium with 15% foetal bovine serum. hDPCs were seeded in 96-well plates, and cell counting kit assays were carried out to test their viability. Real-time quantitative polymerase chain reaction (qPCR) was used to evaluate ALP, Runx2, Notch1, Hes1 and Hey1 mRNA levels. Notch1, hes1, LC3 and p62 protein levels were quantified by Western blotting. Colocalization of LC3 and Notch1 was measured by immunofluorescence. Two-tailed Student's t-tests were used for statistical analysis. RESULTS: Autophagic flux was significantly (P < 0.05) inhibited by MTA extracts, causing Notch degradation arrest. This resulted in the promotion of cell proliferation and inhibition of differentiation during the logarithmic phase of cell growth. CONCLUSION: MTA extract promoted the proliferation of hDPCs in part by activating Notch signalling through inhibition of autophagic flux during the early stage and, thus, might potentially induce rapid restoration of injured pulps.
AIM: To determine the roles of autophagy and Notch signalling in mineral trioxide aggregate (MTA)-mediated proliferation and differentiation of human dental pulp cells (hDPCs). METHODOLOGY: hDPCs were separated from human healthy molar teeth using collagenase I/dispase II digestion and then cultured in α-MEM medium with 15% foetal bovine serum. hDPCs were seeded in 96-well plates, and cell counting kit assays were carried out to test their viability. Real-time quantitative polymerase chain reaction (qPCR) was used to evaluate ALP, Runx2, Notch1, Hes1 and Hey1 mRNA levels. Notch1, hes1, LC3 and p62 protein levels were quantified by Western blotting. Colocalization of LC3 and Notch1 was measured by immunofluorescence. Two-tailed Student's t-tests were used for statistical analysis. RESULTS: Autophagic flux was significantly (P < 0.05) inhibited by MTA extracts, causing Notch degradation arrest. This resulted in the promotion of cell proliferation and inhibition of differentiation during the logarithmic phase of cell growth. CONCLUSION: MTA extract promoted the proliferation of hDPCs in part by activating Notch signalling through inhibition of autophagic flux during the early stage and, thus, might potentially induce rapid restoration of injured pulps.
Authors: Li Fu; Bing Han; Yang Zhou; Jie Ren; Wenzhi Cao; Gopal Patel; Guoyin Kai; Jun Zhang Journal: Front Pharmacol Date: 2020-03-19 Impact factor: 5.810