Tanshinone IIA has anti-platelet effect and induces apoptosis in megakaryocytes via TNFR and Caspases

Abstract

Tanshinone IIA (TIIA) isolated from Danshen (Radix Salviae Miltiorrhiza Bge) is a derivative of phenanthrenequinone, which has been used for centuries to treat hypercoagulation related diseases. Our previous study showed that TIIA has neural protective effect (Xia et al, Pediatric Research, 2005). In this study, we investigated the effect of TIIA on platelets and megakaryocytes in a rabbit model, and its molecular mechanisms. Rabbit immune vasculitis model was established by intravenous injection of bovine serum albumin with TIIA treatment (5 mg/kg/day, iv). The platelet count, platelet aggregation, and inflammatory cytokine IL-1β level were significantly increased in vasculitis model compared with normal group at day 7. There were positive correlations between platelet count and IL-1β level. The number of megakaryocytes and CFU-MK formation in bone marrow were significantly increased in this model. Treatment with TIIA significantly decreased all these parameters, and also significantly reduced the injury vessel in immune vasculitis. Furthermore, the effect of IIA on in-vitro megakaryopoiesis was investigated. TIIA at 10-30 ug/ml significantly inhibited CFU-MK formation (p<0.05, n=6). TIIA also induced the apoptosis of megakaryocytic cell line CHRF. The cell viability was reduced to 81% after 72hrs of TIIA treatment. Using Annexin V/PI staining, TIIA induced apoptosis on CHRF cells in a dose dependent manner. This was verified by Caspase 3 assay which showed the activation of Caspase 3 increased from 5% to 16% after TIIA treatment. Using JC-1 assay, we found that the depolarized cells increased from 9% to 17% after TIIA treatment suggesting the involvement of intrinsic apoptotic pathway. To further determine the molecular mechanisms involved in the pro-apoptosis effect, Microarray studies using Affymetrix 133 plus genechips were conducted to identify the genes that were differentially expressed after TIIA treatment. Several groups of genes involved in apoptosis, calcium regulation and cell cycle checkpoints were found to be differentially expressed after the treatment. The differential expressions of these genes were validated using quantitative PCR. The most significantly upregulated gene (6.0±0.333 folds) was TNF Receptor Super-Family 9 (TNFRSF9). In addition, Receptor Interacting Protein Kinase (RIPK), a protein that likely interacts with TNFRSF9, was up-regulated to 2.0±0.167 folds. These results suggest that TIIA has an anti-platelet effect in this model. Its mechanism may be related to its inhibiting megakaryopoiesis and inducing apoptosis of megakaryocytes via TNFR and caspases. Disclosures:No relevant conflicts of interest to declare.