Inducing cathepsin L expression/production, lysosomal activation, and autophagy of human dental pulp cells by dentin bonding agents, camphorquinone and BisGMA and the related mechanisms

Camphorquinone (CQ) and resin monomers are included in dentin bonding agents (DBAs) and composite resin to restore tooth defects due to abrasion, crown fracture, or dental caries. DBAs, CQ, and bisphenol A-glycidyl methacrylate (BisGMA) applications influence the biological activities of the dental pulp. The current investigation aimed to delineate the effect of DBAs, CQ, and BisGMA on cathepsin L production/expression, lysosomal activity, and autophagy induction in human dental pulp cells (HDPCs). HDPCs were exposed to DBAs, CQ, or BisGMA with/without inhibitors for 24 h. Enzyme-linked immunosorbent assay was employed to determine the cathepsin L level in culture medium. The cell layer was utilized to measure cell viability by 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl -tetrazolium bromide (MTT) assay. Real-time PCR was used to evaluate the mRNA expression. Western blotting or immunofluorescent staining was used to study protein expression. Lysosomal density was evaluated by lysotracker red staining. We found that DBAs, CQ, and BisGMA stimulated cathepsin L mRNA, protein expression, and production in HDPCs. In addition, CQ and BisGMA induced lysosomal activity, Beclin1, ATG12, LC3B, Bax, and p53 expression in HDPCs, indicating the stimulation of autophagy. Glutathione (GSH) prevented CQ- and BisGMA-induced cytotoxicity. Moreover, E64d, cathepsin L inhibitor (two cathepsin inhibitors), and Pifithrin-α (a p53 inhibitor) showed little preventive effect toward CQ- and BisGMA-induced cytotoxicity. Autophagy inhibitors (NH4Cl, Lys05) mildly enhanced the CQ- and BisGMA-induced cytotoxicity. These results indicate that DBAs stimulated cathepsin L, possibly due to their content of CQ and BisGMA that may induce cathepsin L in HDPCs. CQ and BisGMA stimulated lysosomal activity, autophagy, and apoptosis, possibly via induction of Beclin 1, ATG12, LC-3B, Bax, and p53 expression. In addition, CQ and BisGMA cytotoxicity was related to redox change and autophagy. These events are important role in pulpal changes after the restoration of tooth decay using CQ- and BisGMA-containing DBAs and resin composite.

 

Comments:

The investigation described aimed to examine the effects of dentin bonding agents (DBAs), camphorquinone (CQ), and bisphenol A-glycidyl methacrylate (BisGMA) on human dental pulp cells (HDPCs). The study investigated the production/expression of cathepsin L, lysosomal activity, and autophagy induction in HDPCs following exposure to DBAs, CQ, and BisGMA.

The researchers conducted various assays and analyses to evaluate the effects of these substances on HDPCs. They used enzyme-linked immunosorbent assay (ELISA) to measure the level of cathepsin L in the culture medium. Cell viability was assessed using the 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay. Real-time PCR was employed to evaluate mRNA expression, while Western blotting and immunofluorescent staining were used to study protein expression. Lysosomal density was evaluated using lysotracker red staining.

The results of the investigation showed that DBAs, CQ, and BisGMA stimulated the production, mRNA expression, and protein expression of cathepsin L in HDPCs. Furthermore, CQ and BisGMA induced lysosomal activity and the expression of Beclin1, ATG12, LC3B, Bax, and p53, indicating the stimulation of autophagy. The researchers also found that CQ and BisGMA exhibited cytotoxic effects on HDPCs. However, the cytotoxicity was prevented by glutathione (GSH), a cellular antioxidant.

In terms of preventive effects, inhibitors of cathepsin L (E64d), p53 (Pifithrin-α), and autophagy (NH4Cl, Lys05) showed limited effectiveness in preventing the cytotoxicity induced by CQ and BisGMA.

The study suggests that DBAs stimulate the production of cathepsin L in HDPCs, potentially due to the presence of CQ and BisGMA in these agents. Moreover, CQ and BisGMA were found to induce lysosomal activity, autophagy, and apoptosis, possibly through the upregulation of Beclin 1, ATG12, LC-3B, Bax, and p53 expression. The cytotoxic effects of CQ and BisGMA were associated with changes in redox balance and autophagy.

Overall, these findings indicate that the use of CQ- and BisGMA-containing DBAs and resin composites may lead to pulpal changes after tooth restoration, involving cathepsin L production, lysosomal activity, autophagy, and apoptosis.