Comparison of botulinum toxin type A and Rho-inhibitor on ATDC5 chondrocyte

Abstract

Knee osteoarthritis (OA) has an incidence rate of 25% per year and its responses to treatment are variable. The analgesic effect of intra-articular injection of botulinum toxin type A (BTX-A) has been observed in patients with knee OA. BTX-A might inhibit Rho GTPase by ADP-ribosylation of amino acid asparagine-41. Rho activity has a role in mediation of chondrocyte catabolic signaling pathways, and Rho/ROCK (Rho-associated coiled-coil containing kinase) activation induces articular cartilage degradation. In our unpublished data, chondroprotection of BTX-A was observed based on pathological findings in rat knee OA. ROCK I & II were found to be altered in chondrocytes treated with BTXA, with dose-dependent effect. This study aimed to compare the effect on chondrocyte of BTX-A and Rho-inhibitor. ATDC5 chondrocytes were compared based on cell phenotype and markers for chondrocyte differentiation and growth after administration of BTX-A, Rho-inhibitor (Y27632), and normal saline (control) to clarify the downstream signaling pathway. Reverse transcription-PCR (RT-PCR) showed that in ATDC5 chondrocyte cell culture, Y27632 and BTX-A elevated aggrecan on culture day 4 and day 7. On day 21, the level of collagen type X and MMP13 were lowered in Y27632 and BTX-A. ROCK I and II level were decreased on day 4 and 7 in all three groups, but were remarkably higher on day 14 and 21 in Y27632. On day 8, in ATDC5 cells not pretreated with insulin, decreased cell density, small and flat chondrocyte morphology were observed after addition of Y27632 and BTX-A, in comparison to control. BTX-A and Rho-inhibitor decreased ROCK I & II and increased aggrecan level during early stage, and decreased collagen type X and MMP13 during late stage. Potential dedifferentiation effect in BTX-A and Rho-inhibitor were observed as well. Rho-signaling pathway might play different roles during early and late stage in chondrocyte growth.