Effect of Tianshengyuan-1 (TSY-1) on telomerase activity and hematopoietic recovery - in vitro, ex vivo, and in vivo studies

Abstract

Aplastic anemia is a heterogeneous disorder of bone marrow failure syndrome. Accumulating evidence indicates that both acquired and congenital aplastic anemia is linked to telomerase activity and telomere length. Chinese herbal medicine Tianshengyuan-1 (TSY-1), a liquid extraction of multiple Chinese herbs, appears to stimulate hematopoiesis in patients with bone marrow deficiencies; however, the exact mechanism of action remains unclear. In this study, we investigated the effect of TSY-1 on telomere length and telomerase activity. We first investigated the effects of TSY on in vitro cultured cell lines including CD34+ hepatic stem cells and CD4+/CD8- Jurkat cells. An immune-mediated murine aplastic anemia model and human samples, including peripheral blood samples of 4 healthy donors and bone marrow hematopoietic cells from 4 patients with hypocellular myelodysplastic syndrome (MDS), were also used to test the efficacy of TSY on hematopoiesis, telomerase activity and telomere length. Our results indicated that TSY-1 increased the telomerase activity and telomere length in a dose-response manner in vitro, in vivo, and in human samples including 3 of 4 healthy individuals and 3 of 4 bone marrow samples from MDS patients. In immune-mediated murine aplastic anemia model, TSY-1 activity on Telomere length was parallel to the significant increasing of the RBC, hemoglobin, hematocrit, and platelet count in peripheral blood, increasing of CD34+ cell count and hematopoiesis, and decreasing of fatty infiltration in bone marrow samples. Our study demonstrated that TSY-1 may exert its effects by modulating telomerase activity of hematopoietic cells. Further studies are warranted to explore the precise molecular mechanisms of how TSY-1 regulates telomerase activity and telomere length, and also to test the TSY-1 in randomized control trials.

Keywords: TSY-1; hematopoietic cells; telomerase; telomere.

Figure 1

The effect of TSY on telomerase activity, telomere length and cell count in CD34+ stem cells (A) and Jurkat cells (B). The left panel shows telomerase activity and telomere length of cells after 24 h of treatment with TSY with various dilutions (0, 20, 40, 80 μg/ml). The right panel shows quantification of telomerase activity, telomere length and cell count after 24 h of treatment with TSY with various dilutions (0, 20, 40, 80 μg/ml). Detail methods of determining telomerase activity, telomere length, and cell count were described in Materials and Method section.Telomerase activity and length plotted the ratio of each TSY treated sample relative to the untreated control, whereas cell count was the average of cell counts in 4 fields selected randomly under 10x objective using Leica inverted microscope. Each experiment repeated three times. The differences of telomerase activity, telomere length, and cell counts among control and various dosage groups all reached significance level by ANOVA test (P<0.05).

Figure 2

The effect of TSY on bone marrow CD34+ cells count, bone marrow histology and telomere length in immune mediate AA mouse model. A & C: Bone marrow CD34+ cell population analyzed by flow cytometry. Note significant increase of CD34 positive (**P<0.01 and ***P<0.001 by Student t-test) in TSY-1 treated group relative to the model control group. B: Histopathological study of bone marrow after treatment with TSY. D: Telomere length in peripheral blood sample, plotted the ratio of each TSY treated sample relative to the untreated control. The differences of telomere length among control and various dosage groups reached significance level by ANOVA test (P<0.05).

Figure 3

The effect of TSY on telomerase activity and telomere length in normal T-cells isolated from peripheral blood of 4 healthy donors. Isolated T lymphocytes were incubated with concentration of 0, 20, 40 μg/ml of TSY-1 for 24 hours and harvested for determination of telomerase activity and telomere length according to the methods described in Materials and Methods section. (A) Telomerase activity; (B) Telomere length. Telomerase activity (A) and telomere length (B) were determined according to the methods described in Materials and Methods section. The values plotted were the ratio of treated cells relative to untreated control.

Figure 4

The effect of TSY on telomerase activity in hematopoietic cells isolated from bone marrow of MDS patients. Hematopoietic cells were isolated from bone marrow of four MDS patients and cultured in presence of TSY with various concentrations (0, 20, 40 μg/ml) for 24 hours before the analysis of telomerase length. Telomere length was determined according to the methods described in Materials and Methods section. The values plotted were the ratio of treated cells relative to untreated control.