Justicia adhatoda induces megakaryocyte differentiation through mitochondrial ROS generation

Abstract

Background: Hepatoprotective activity along with improved survival percentage and hematological parameters prior to whole body irradiation were reported with Justicia adhatoda extracts. Purpose: To evaluate the thrombopoietic potential of Justicia adhatoda L. leaf extract in megakaryocyte differentiation Methods: Ethanol extracts were prepared using soxhlet extraction method, and IC50 value was determined. The effect of ethanol extracts obtained from Justicia adhatoda on megakaryocyte maturation and development in megakaryocytic Dami cell lines was tested. Expression of megakaryocyte specific markers, CD61 and CD41, were assessed using flow cytometry and fluorescence microscopy. In addition, cell cycle analysis and mitochondrial membrane potential were analyzed by flow cytometry. Gene expression analysis was performed using qRT-PCR. Results: At a concentration of 40 µg/ml, the leaf extracts of Justicia adhatoda for 72 h induced the megakaryocytic features in megakaryocytic Dami cell lines. The megakaryocyte specific markers, CD41 and CD61, were up-regulated (2.2 and 12.4 fold, respectively), and more number of cells entered into synthetic (S) and G2/M phase as compared with untreated cell (23.1% vs 16.6% and 70.2% vs 42.3%, respectively) showing maturation. RUNX1 (a transcription factor essential for embryonic hematopoiesis and adult megkaryocyte maturation) and c-Mpl (the receptor for TPO) were upregulated, and the suppressor of cytokine signaling (SOCS) 1 and SOCS3 were down-regulated upon treatment with Justicia adhatoda. Justicia adhatoda enhanced mitochondrial ROS generation by 28-fold, increased the permeability of mitochondrial membrane and showed an inverse correlation in superoxide dismutase levels. Conclusion: Justicia adhatoda could enhance mitochondrial ROS generation and increase the permeability of mitochondrial membrane, thereby inducing megakaryocytic maturation. Our findings suggest thrombopoietic potential of Justicia adhatoda leaf extract on megakaryocyte differentiation.