Effects of PLAB on apoptosis and Smad signal pathway of hypertrophic scar fibroblasts

Abstract

Pseudolaric acid-B (PLAB), a diterpene acid, was isolated from the root and trunk barks of Pseudolarix kaempferi. It has shown antifungal and anti-fertility effects and cytotoxic activities in previous studies. Our goals are to study the effects of PLAB on cell proliferation and Smad signal pathway of hypertrophic scar fibroblasts. Our results showed that PLAB induced apoptosis in hypertrophic scar fibroblasts and inhibited cell proliferation of hypertrophic scar fibroblast. MTT assay showed that its IC50 value toward hypertrophic scar fibroblasts was 12.9 ± 1.20 μmol/L. Furthermore, the results of cell growth curve matched with the above results. Inducing apoptosis by PLAB in hypertrophic scar fibroblast was assessed by various morphological and biochemical characteristics, including cell shrinkage, chromatin condensation, membrane blebbing, formation of apoptotic bodies, and DNA ladder formation. A typical “Sub-G1 peak” was also checked through flow cytometry. The Smad2 and Smad7 mRNA levels of 48-h PLAB treatment were determined by reverse transcription–polymerase chain reaction (RT-PCR) 48 h later. RT-PCR showed that Smad7 mRNA level increased and significant differences were observed between control group and experimental group (P < 0.05); While there is no significant difference in Smad2 mRNA between the two groups. Our results showed that PLAB interfered with the microtubule dynamics of tubulin polymerisation and depolymerisation, which results in the inhibition of chromosomal segregation in mitosis and consequently the inhibition of cell division. These results suggest that PLAB inhibits hypertrophic scar fibroblast growth through apoptosis and Smad signal pathway.