AUTHOR=Iftikhar Fizza , Rahman Saeedur , Khan Muhammad Behroz Naeem , Khan Kanwal , Khan Muhammad Noman , Uddin Reaz , Musharraf Syed Ghulam 

TITLE=In Vitro and In Vivo Studies for the Investigation of γ-Globin Gene Induction by Adhatoda vasica: A Pre-Clinical Study of HbF Inducers for β-Thalassemia

JOURNAL=Frontiers in Pharmacology

VOLUME=13

YEAR=2022

URL=https://www.frontiersin.org/journals/pharmacology/articles/10.3389/fphar.2022.797853

DOI=10.3389/fphar.2022.797853

ISSN=1663-9812

ABSTRACT=<p>Fetal hemoglobin (HbF) is a potent genetic modifier, and the γ-globin gene induction has proven to be a sustainable therapeutic approach for the management of β-thalassemia. In this study, we have evaluated the HbF induction ability of <italic>A. vasica in vitro</italic> and <italic>in vivo</italic>, and the identification of potential therapeutic compounds through a bioassay-guided approach. <italic>In vitro</italic> benzidine-Hb assay demonstrated strong erythroid differentiation of K562 cells by <italic>A. vasica</italic> extracts. Subsequently, an <italic>in vivo</italic> study with an aqueous extract of <italic>A. vasica</italic> (100 mg/kg) showed significant induction of the γ-globin gene and HbF production. While in the acute study, the hematological and biochemical indices were found to be unaltered at the lower dose of <italic>A. vasica</italic>. Following the bioassay-guided approach, two isolated compounds, vasicinol (<bold>1</bold>) and vasicine (<bold>2</bold>) strongly enhanced HbF levels and showed prominent cellular growth kinetics with ample accumulation of total hemoglobin in K562 cultures. High HbF levels were examined by immunofluorescence and flow cytometry analysis, concomitant with the overexpression in the γ-globin gene level. Compound <bold>1</bold> (0.1 µM) and compound <bold>2</bold> (1 µM) resulted in a greater increase in F-cells (90 and 83%) with marked up (8-fold and 5.1-fold) expression of the γ-globin gene, respectively. Molecular docking studies indicated strong binding affinities of (<bold>1</bold>) and (<bold>2</bold>) with HDAC2 and KDM1 protein that predict the possible mechanism of compounds in inhibition of these epigenetic regulators in the γ-globin gene reactivation. Altogether, these observations demonstrated the therapeutic usefulness of <italic>A. vasica</italic> for fostering HbF production in clinical implications for blood disorders.</p>