ASSESSMENT OF THE ROLE OF SOME PHYTOCHEMICALS FOR MAINTAINING IRON HOMEOSTASIS AND CURE OF CERTAIN RELATED DISEASES

Abstract

Iron homeostasis is a phenomenon of maintenance and regulation of iron in the storage and circulation in our body. An imbalance in iron regulation leads to the development of different diseased conditions, depending upon individuals' health. Inflammation is the primary initiator for several disorders in our body. Inflammation associated with an iron imbalance in body leads to the development of anemia of inflammation. Inflammation also leads to the development of cancer, cardiovascular disorders and kidney disorders. As the knowledge of heterogeneity of development of inflammation increases, there is a need to search for more effective and active molecules, with lesser side effects. Although the modern medicinal approach can mitigate inflammatory conditions in different diseased conditions, several unique complications must be addressed through a holistic method of therapy. Towards this end, a validation of our traditional knowledge of Ayurveda, and then combining it with the modern scientific approach may provide the best possible solution. Traditional plant-based medicines are safe, effective, economical, with lesser side effects and have easy accessibility than the other synthetic drugs. Hence, these herbal plants or active phyto-constituents are considered as the best possible alternative candidates in disease management. The present thesis entitled “Assessment of the role of some phytochemicals for maintaining iron homeostasis and cure of certain related diseases” deals with evaluating the anti-inflammatory and anti-cancer effects of dietary items through in vitro and in vivo approaches. Among different explored edible plants, black pepper and wheat were most potent in preventing the inflammatory condition. Thus, the detailed and mechanistic studies using in vitro and in vivo models for anemia of inflammation and chronic myeloid leukemia (cancer associated with the blood cells) were performed to establish the cross-talk among different diseased conditions. In the beginning, Chapter 1 introduces the present scenario of anemia of inflammation and a clinical approach for its treatment briefly. It also deals with a brief description of chronic myeloid leukemia and the therapeutic strategy for its treatment. Finally, the detailed objectives to be achieved in this study are specified in this chapter. Followed by this, Chapter 2 presents: (i) a detailed review of iron and iron homeostasis; (ii) a description of disorders associated with iron metabolism and hepcidin regulation; (iii) understanding the molecular mechanism involved in the pathophysiology of anemia of inflammation; (iv) the role of macrophages in inflammatory responses thus explaining the critical markers of inflammation such as cytokines, chemokines, main transcription factors and i inflammatory proteins; (v) a cross-talk of inflammation and cancer, cancer development, and the role of inflammation in hematopoiesis; (vi) a brief description of leukemia and its global burden, chronic myeloid leukemia and associated pathophysiology; (vii) current therapeutic approach for the treatment of chronic myeloid leukemia; (viii) hypothetical idea for the treatment of described disorders depending upon the approach of involvement of natural products for cancer prevention. Each of the hypotheses was further explored in the subsequent chapters of the thesis. The main objective of the present work is to decipher the anti-inflammatory and anti-cancer effects of various dietary items, followed by understanding their mode of action. There are different in vitro and in vivo assays, established to confirm a compound to be anti-inflammatory and anti-cancerous. Chapter 3 elaborates on the significant parameters for evaluation. The methodology starts with the preparation of fresh extracts from nutritional items, and phytochemical characterization of selected plants through the evaluation of phenolic and flavonoids content, evaluate the presence of various primary and secondary metabolites through gas chromatography. Further, we performed various biochemical and cell-based analyses such as cell proliferation and cytotoxicity evaluation, nuclear and cellular morphology characterizing, staining assays, flow cytometry assays (for ROS and apoptosis determination). More importantly, the biological activities were determined by analyzing the expression of target genes at the level of transcription and translation. Hepcidin, a 25- amino acid long peptide hormone, maintains the level of iron in our body and is encoded by the hepcidin gene in liver hepatocytes, macrophage cells, and duodenal enterocytes. Hepcidin maintains iron homeostasis in our body through three different modes. Firstly, it maintains the dietary absorption of iron in the duodenal region. Secondly, it regulates the release of iron from its storehouse, liver, and thirdly, it governs the iron recycling in macrophages. Hepcidin performs its iron regulation function by binding to the iron exporter channel, ferroportin. Ferroportin, a transmembrane protein, performs the export of iron moiety to the circulation. Iron is an essential element required in the body for accomplishing various functions, such as synthesis and repair of DNA, transport of oxygen, and ATP production in mitochondria. Iron also acts as a cofactor for several enzymes. Hepcidin regulates the body's iron level by binding to ferroportin, leading to its irreversible degradation, thus controlling its distribution. An altered level of hepcidin in circulation is an indication of the presence/ absence of active ferroportin in the body. The disease associated with increased hepcidin expression and less bioavailability of iron to the circulation from its storehouse, i.e., liver, has been termed as anemia of inflammation. In Chapter 4, a few of the phytochemical extracts were screened using in vitro models for establishing proof-of-concept for their role in prevention in anemia of ii inflammation. The selected phytochemicals were further analyzed for their potential to prevent anemia of inflammation, macrophage inflammatory state and chronic myeloid leukemia. Our results showed that among all-selected phytochemicals, black pepper and wheatgrass had potent anti-inflammatory activities as obtained from initial transcriptional analysis, and thus selected for further detailed studies. Chapter 5 depicts the molecular mechanisms involved in preventing the anemia of inflammation in the presence of black pepper and its major secondary metabolite, piperine. The initial evaluation confirms that the methanol extract is most potent in downregulating hepcidin's expression at transcriptional level compared to aqueous and ethanol extracts. The phytochemicals were further analyzed in HepG2 cells after inducing them with BMP-6 and IL-6 to mimic a condition of anemia of inflammation. The treatment resulted in the increased expression of hepcidin both at the transcriptional and the translational levels, which was through BMP/SMAD and JAK/STAT signalling pathways, respectively. The increased phosphorylation of SMAD/STAT proteins leads to the over-expression of hepcidin. However, treatment of BMP 6/IL-6 induced HepG2 cells with various concentrations of black pepper and piperine significantly downregulated the increased expression of phosphorylated SMAD/STAT proteins and ultimately inhibiting hepcidin expression. In the next phase, we analyzed the effect in vivo using anemia of inflammation-induced male Balb/c mice. Our data showed that both black pepper and piperine efficiently alleviated the anemia of inflammation in these animals. The transcriptional and translational studies confirmed that black pepper and piperine could regulate the expression of hepcidin. The molecular docking study also proposed the inhibitory potential of piperine on SMAD and STAT proteins by its physical interactions with two latter proteins and subsequently inhibiting their phosphorylation. Chapter 6 evaluated the anti-inflammatory property of wheatgrass extracts from different days of their germination in LPS-induced macrophage cells, RAW 264.7 cells. Initially, it was confirmed that wheatgrass extracts obtained from day 5 and day 7 old seedlings downregulated hepcidin mRNA expression, thus proposing that wheatgrass may be a potent anti inflammatory agent. In order to confirm this fact, RAW 264.7 cells were treated with lipopolysaccharide (LPS) to generate the inflammatory condition. A significant increase in the production of reactive oxygen species (ROS) and expression of pro-inflammatory cytokines (TNF-α, IL-6, IL-1β) and transcription factor (AP-1) confirmed the generation of inflammatory responses in these cells in response to LPS. Interestingly, the increased expressions of the above parameters were found to be downregulated in the presence of day 5 and day 7 old wheatgrass extracts. A similar pattern of inhibition of prominent inflammatory proteins, iNOS-2 and COX iii 2, were found in response to wheatgrass extract.It has been earlier reported that apigenin and inositol are the major secondary metabolites present in wheatgrass extract. The molecular docking study showed that, apigenin and inositol have the potentiality to inhibit COX-2 proteins, and the effect was comparable to its known inhibitor, naproxen. Chronic myeloid leukemia is a common form of leukemia, generated due to reciprocal translocation between chromosome 9 and 22, causing the Philadelphia chromosome (Ph+) in affected patients. In Chapter 7, we investigated the anti-cancer potential of black pepper and its major phytochemical, piperine, against chronic myeloid leukemia. The half inhibitory concentration (IC50) against the K-562 cell line was observed to be 116.6 µg/ml and 56.3 µM in the presence of black pepper methanol extract (BP-M) and piperine (PIP), respectively. Fluorescent microscopy and flow cytometry studies confirmed the induction of apoptosis and generation of ROS in K-562 cells in the presence of BP-M and PIP. The transcription and translational studies confirmed the stimulation of intrinsic apoptotic pathway. Both BP-M and PIP also generated the anti-proliferative effect against K-562 cells. Molecular docking studies showed the possibility of binding of PIP to Bcl2, PCNA and Apaf-1 proteins. These findings further confirmed the anti-cancer potential of both BP-M and PIP against chronic myeloid leukemia. Finally, Chapter 8 summarizes the current thesis's significant findings and provides a comprehensive suggestion for future work in the respective areas. The scientific conclusion dealt with in this thesis may be of potential use to the future researchers working in this area. Chapter 9 finally enlists the entire bibliography consulted in the course of the present work.