Tanshinone Iia Inhibits Megakaryopoiesis in Immune Vasculitis

Introduction Tanshinone IIA, an active component of Danshen (Salvia miltiorrhiza), has been used for centuries to treat hypercoagulation-related diseases, which attributed to its anti-platelet and anti-inflammatory effects. However, the role of Tanshinone IIA in megakaryocytes, the precursor of platelet within the bone marrow, remains unclear. Therefore, the present study established a rabbit model with immune vasculitis to examine the effect of Tanshinone IIA on megakaryopoiesis and to identify the underlying mechanism(s). Methods Immune vasculitis was established in rabbits (3-4 weeks old) by two intravenous injection of 10% bovine serum albumin (2.5 ml/kg) at two-week interval. Those rabbits were randomly treated with Tanshinone IIA (5 mg/kg/d, 7 d, iv) or aspirin (100 mg/kg/d, 7 d, ig). Megakaryocyte count and CFU-MK formation were measured by Wright's and AChE staining, respectively. Human megakaryotic cell lines Meg-01 and CHRF-288-11 were used to examine the effect of Tanshinone IIA on apoptosis by Annexin V-FITC/PI, mitochondrial membrane potential/JC-1 and Caspase-3 activity assays using flow cytometry. ResultsIn rabbits with immune vasculitis, the platelet count, platelet aggregation and the serum levels of inflammatory cytokines IL-1β, TNF-α and IL-6 were significantly increased when compared to their healthy controls. After 7 days of Tanshinone IIA treatment, all these parameters were significantly reduced, with the inhibitions comparable to those caused by aspirin. In addition, the number of megakaryocytes and the formation of CFU-MK were also statistically increased in rabbits with immune vasculitis, which could be significantly reduced by Tanshinone IIA. In vitro, Tanshinone IIA (1, 3, 10 and 30 μg/ml) also significantly inhibited the formation of CFU-MK of bone marrow cells of BALB/c mice (6-10 weeks) in a dose-dependent manner. In human megakaryocytic cell line Meg-01, Tanshinone ⅡA (10 μg/ml, 72 h) induced apoptosis; both early and late apoptotic rates were significantly increased. In another human megakaryocytic cell line CHRF-288-11, Tanshinone ⅡA (10 μg/ml, 72 h) statistically increased the proportion of depolarized cells, from 9.70% to 14.13%, according to mitochondrial membrane potential using JC-1 assay. The expression of active Caspase-3 in CHRF-288-11 was also significantly increased by Tanshinone ⅡA (10 μg/ml, 72 h) from 5.25% to 15.86%. Conclusion The present study shows that Tanshinone IIA ameliorates immune vasculitis by inhibiting megakaryopoiesis and inducing apoptosis of megakaryocytes, which might explain the anti-platelet and anti-inflammatory effects of Tanshinone IIA. Disclosures No relevant conflicts of interest to declare.

An extraordinary pulmonary artery involvement in granulomatosis with polyangiitis

Granulomatosis with polyangiitis (GPA) is an autoimmune mediated systemic disease and characterized by pauci-immune vasculitis mainly of small and medium vessels with typical necrotizing granulomatous lesions in the affected tissues. A 44 year old lady who was known to have GPA presented with unusual presentation of its category with extraordinary large vessels involvement including pulmonary arteries and her condition improved with immunosuppressive therapy. We reported this case, to improve the awareness about other overlapping categories of vessels vasculitis that may involve large vessels and main pulmonary arteries and to avoid misdiagnosing these patients with category of classical large vessels vasculitis.

The Effect of Tanshinone-IIa on IL-1β Induced-Thrombocytosis in an Immune Vasculitis Model

Inflammation cytokines may be involved in the pathogenesis of thrombocytosis with vasculitis. Our previous study showed that inflammation cytokine IL-1β plays an important role on in-vitro megakaryopoiesis (Yang M et al, Br J Haematol 2000). The role of IL-1β and Tanshinone IIA (TIIA) (Isolated from Danshen, Radix Salviae Miltiorrhiza Bge) on platelets and megakaryocytes (MKs) in immune vasculitis model was investigated in this study. Rabbit model with immune vasculitis was established by injection (iv) of BSA. After treatment with BSA for 7 days, the platelet count, platelet aggregation and the expression of AnnexinⅤ were significantly increased in this vasculitis model group compared with normal control group (n=7). IL-1β levels was also significantly higher in vasculitis model. There were positive correlations between platelet count and IL-1β levels (R=0.65), platelet aggregation and IL-1β levels (R=0.60). Treatment with TIIA (5 mg/kg/day, iv) and aspirin significantly decreased all these parameters. MKs and CFU-MK number were also significantly increased in vasculitis group as compared to normal group. Treatment with TIIA and aspirin significantly reduced the number of MKs and CFU-MK in this model. Study further demonstrated that IL-1β alone or in combination with TPO induced in-vitro CFU-MK formation. The mRNA of of IL-1 type I and type II receptors (IL-1 RI and RII) were detected in cultured MK (CD61+ CD41+) cells. The expression of IL-1 RI and RII was also confirmed by immunofluorescence staining in bone marrow MKs. Moreover, the IL-1R bloker can reduced IL-1β induced megakaryopoiesis. TIIA on in-vitro megakaryopoiesis was also investigated. TIIA at 10-30 ug/ml significantly inhibited CFU-MK formation. TIIA also induced the apoptosis of MKs in a dose dependent manner by Anexin V assay. Caspase 3 assay showed the activation of Caspase 3 increased from 5% to 16%. Using JC-1 assay found that the depolarized cells increased from 9% to 17% suggesting the involvement of intrinsic apoptotic pathway. IL-1β may play an important role in the thrombocytosis of immune vasculitis by inducing megakaryopoiesis. TIIA has anti-platelet effect in this model which may be mediated via inhibiting IL-1β induced-megakaryopoiesis. Disclosures No relevant conflicts of interest to declare.

IL-1β Plays An Important Role On Thrombocytosis In An Immune Vasculitis Model Via Promoting Megakaryopoiesis

Thrombocytosis and inflammation cytokines may be involved in the pathogenesis of vasculitis. Our previous study have showed that major inflammation cytokine IL-1β play an important role on in-vitro megakaryopoiesis (Yang M et al, Br J Haematol 2000). In this study, we investigated the changes of IL-1β and megakaryopoiesis and the effect of aspirin in an immune vasculitis model. Rabbit immune vasculitis model was established by intravenous injection of bovine serum albumin. In this model, platelet number and function of periphery blood, megakaryocyte number and the CFU-MK formation of the bone marrow, and serum levels of inflammatory cytokines were investigated. After treatment with BSA for 7 days, the platelet count, platelet aggregation and the expression of AnnexinⅤ were significantly increased in this vasculitis model group compared with normal control group (n=6). The serum levels of inflammatory cytokine IL-1β was also significantly higher in vasculitis model. There were positive correlations between platelet count and IL-1β levels (R=0.55), platelet aggregation and IL-1β levels (R=0.603). Treatment with aspirin (100 mg/kg/d) significantly decreased all these parameters, showing aspirin had anti-platelets and anti-inflammation effects. Our results also demonstrated that megakaryocyte number and the formation of CFU-MK were significantly increased in vasculitis group as compared to those in normal group. Treatment with aspirin significantly reduced the number of megakaryocytes and the formations of CFU-MK in bone marrow in this immune vasculitis model. Our study further demonstrated that IL-1β alone or in combination with TPO induced in-vitro CFU-MK formation. Using RT-PCR techniques, the mRNA of of IL-1 type I and type II receptors (IL-1 RI and RII) were detected in cultured CD61+ CD41+ cells and four megakaryocytic cell lines. The expression of IL-1 RI and RII was also confirmed by flow cytometry and immunofluorescence staining in bone marrow megakaryocytes. Moreover, the IL-1R bloker can reduced IL-1β induced megakaryopoiesis. This sudy showed that IL-1β may play an important role in the pathogenesis of immune vasculitis. Aspirin has anti-inflammation effects in this model which may be mediated via inhibiting megakaryopoiesis and platelet formation. Disclosures: No relevant conflicts of interest to declare.

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

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.