Objective:
The aim of this study is to understand the effect of ROCK inhibitor on salispheres morphology and the expression of extracellular matrix proteins (ECM).
Methods:
Submandibular glands of female, ICR mice were used in this study. Salispheres were isolated by dissection and enzymatic digestion of the glands by collagenase II and hyaluronidase. The salispheres were then treated with Y-27632 (ROCK inhibitor) for 4 and 8 days. Next, the salispheres were imaged by phase-contrast microscopy and their relative size was determined by ImageJ. Later, the expression of the ECM proteins was analyzed by Western blotting. Statistical analysis was achieved by one-way ANOVA (SPSS program).
Results:
Treated salispheres with Y-27632 resulted in morphological changes at day 4 and day 8 of treatment compared to the untreated group. The spheres tend to adhere and form vacuoles, which are normally not observed in normal growing salispheres. In term of size, treated spheres at day 4 did not show significant difference compared to the untreated salispheres. However, there was a significant increase in the size of salispheres on day 8 treatment compared to day 4 treated and untreated groups (p<0.0005). In addition, there were differences in the expression of the ECM proteins such as; nidogen and laminin between treated and untreated salispheres.
Conclusion:
The morphological changes suggest that ROCK inhibitor plays a role in modulating the expression of the ECM proteins.