Title

Anti-inflammatory and osteogenic effects of naringenin

Date of Award

5-2020

Degree Type

Thesis

Degree Name

Master of Science in Biomedical Sciences

First Advisor

Srujana Rayalam, PhD

Second Advisor

Shashidharamurthy Taval, PhD

Third Advisor

Harold Komiskey, PhD

Fourth Advisor

Richard White, PhD

Abstract

In the U.S., over half of the elderly population is afflicted with osteoporosis and low bone density (1). Osteoporosis can be defined as a bone disease that results in bone loss and fragility. It can be further characterized by accumulation of fat in bone marrow, excessive inflammation, and a decreased capability to form bone. Although there are several therapeutic options which attempts to quail the effects or occurrence of osteoporosis, there is continued discussion on safer and more natural treatments for the afflicted on a continued long-term basis. Naringenin as well as other phytochemicals have demonstrated promise for natural alternative therapies for their osteogenic, antioxidant, and anti-inflammatory properties (43). These two specific characteristics of naringenin bolster its potential benefits to target the underlying proinflammatory skewed remodeling present in osteoporosis.

Furthermore, phytochemicals have been demonstrated to activate energy sensing AMPK pathway (45). Phosphorylated AMPK has been associated with the same metabolic status illustrated by phytochemical administration. Essentially, this metabolic status consists of an increase in the catabolic ATP producing pathways and a decrease in anabolic energy consuming pathways (30). Some examples include, decreased fat accumulation, increased fat oxidation, increased osteogenic markers, mitochondrial biogenesis, an inhibition of inflammation, and much more. Although others have linked phytochemicals to the activation of AMPK, we aimed to demonstrate this particular activation in some way mediates naringenin’s anti-inflammatory and osteogenic characteristics.

We investigated osteogenic and anti-inflammatory effects of naringenin in two established in vitro models. MC3T3-E1 subclone 4, is a particular murine preosteoblast lineage that is capable of mineralization and has been extensively studied as an in vitro osteogenic model. RAW264.7, is a murine derived macrophage-like lineage commonly used as an in vitro immune response model. Western blotting, ELISA, Alizarin Red staining, alkaline phosphatase (ALP) assay, calcium assay and nitric oxide assay, were performed to demonstrate the osteogenic and anti-inflammatory effects of naringenin and determine whether those effects were mediated in part through AMPK-activation. Our data suggests that naringenin induces anti-inflammatory effects in RAW 264.7 cells by polarizing macrophages to M2 phenotype as evidenced by the increase in the secretion of IL10, a signature cytokine for M2 polarization. Further, naringenin increased catecholamine and suppressed TNFα and IL6 secretion in addition to suppressing the expression of lipopolysaccharide-induced expression of inducible nitric oxide synthase. In MCT3T3 cells, naringenin increased the ALP activity, calcium deposition and secretion of osteoprotegerin. Further, in the presence of an AMPK inhibitor, naringenin-induced increase in ALP activity, calcium deposition and OPG secretion were suppressed thus suggesting an important role of AMPK pathway in naringenin mediated osteogenic effects. Additional studies are required to establish naringenin as an AMPK activator and to confirm AMPK mediated osteogenic and anti-inflammatory effects of naringenin.

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