Abstract
BackgroundThe over-proliferation of fibroblasts is considered to be the main cause of scar adhesion after joint surgery. Hydroxycamptothecin (HCPT), though as a potent antineoplastic drug, shows preventive effects on scar adhesion. This study aimed to investigate the role of activating transcription factor 6 (ATF-6) in the HCPT-induced inhibition of fibroblast viability.MethodsThe cell counting kit-8 (CCK-8) assay, western blot analysis, lentivirus-mediated gene silencing, transmission electron microscopy (TEM) analysis, immunofluorescent staining for autophagy-related protein light chain 3 (LC3) were used to explore the effect of HCPT on triggering fibroblast apoptosis and inhibiting fibroblast proliferation, and the involvement of possible signaling pathways.ResultsIt was found that HCPT exacerbated fibroblast apoptosis and repressed its proliferation. Subsequently, endoplasmic reticulum stress (ERS)-related proteins were determined by western blot prior to ATF6 p50 was screened out and reexamined after it was silenced. As a result, ATF6-mediated ERS played a role in HCPT-induced fibroblast apoptosis. Autophagy-related proteins and autophagosomes were detected after the HCPT administration using western blot and TEM analyses, respectively. Autophagy was activated after the HCPT treatment. With the co-treatment of autophagy inhibitor 3-methyladenine (3-MA), both the western blot analysis and the CCK-8 assay showed inhibited autophagy, which indicated that the effect of HCPT on fibroblast proliferation was partially reversed. Besides, the LC3 immunofluorescence staining revealed suppressed autophagy after silencing ATF6 p50.ConclusionOur results demonstrate that HCPT acts as a facilitator of fibroblast apoptosis and inhibitor of fibroblast proliferation for curbing the postoperative scar adhesion, in which the ATF6-mediated ERS pathway and autophagy are involved.
Highlights
Intra-articular scar adhesion commonly occurs following knee arthroplasty, cruciate ligament reconstruction, internal fixation for tibial plateau fractures, and other operations, seriously affecting the postoperative efficacy and daily work of patients due to the limited flexion and extension and the stiffness of the joint [1, 2]
The effect of HCPT on fibroblasts Our previous studies have shown that HCPT triggered fibroblast apoptosis in a dose- and time-dependent manner
These results suggested that HCPT inhibited the viability of fibroblasts possibly by repressing its proliferation and triggering apoptosis
Summary
Intra-articular scar adhesion commonly occurs following knee arthroplasty, cruciate ligament reconstruction, internal fixation for tibial plateau fractures, and other operations, seriously affecting the postoperative efficacy and daily work of patients due to the limited flexion and extension and the stiffness of the joint [1, 2]. Effectively preventing postoperative scar adhesion is the priority to improve the success rate of the surgery. The exact mechanism of intra-articular adhesion still remains unclear, it is speculated that the excessive proliferation of fibroblasts in the surgical area is the main cause. Prohibiting fibroblast proliferation and inducing its apoptosis can be a feasible method against scar adhesion following joint surgery. A previous study has confirmed the preventive effect of HCPT on scar adhesion after knee surgery in rabbits [3]. In our study, HCPT has facilitated fibroblast apoptosis via the activation of ERS and thwarted joint adhesion through PERK and IRE1 signalings, and the role of ATF6 has been highlighted in our experiments. The over-proliferation of fibroblasts is considered to be the main cause of scar adhesion after joint surgery. This study aimed to investigate the role of activating transcription factor 6 (ATF-6) in the HCPT-induced inhibition of fibroblast viability
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