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Transcriptome analysis of two structurally related flavonoids; Apigenin and Chrysin revealed hypocholesterolemic and ketogenic effects in mouse embryonic fibroblasts

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Abstract

There is no known single therapeutic drug for treating hypercholesterolemia that comes with negligible systemic side effects. In the current study, using next generation RNA sequencing approach in mouse embryonic fibroblasts we discovered that two structurally related flavonoid compounds. Apigenin and Chrysin exhibited moderate blocking ability of multiple transcripts that regulate rate limiting enzymes in the cholesterol biosynthesis pathway. The observed decrease in cholesterol biosynthesis pathway correlated well with an increase in transcripts involved in generation and trafficking of ketone bodies as evident by the upregulation of Bdh1 and Slc16a6 transcripts. The hypocholesterolemic potential of Apigenin and Chrysin at higher concentrations along with their ability to generate ketogenic substrate especially during embryonic stage is useful or detrimental for embryonic health is not clear and still debatable. Our study will serve as a steppingstone to further the investigation in whole animal studies and also in translating this knowledge to human studies.

Introduction

Increased cholesterol accumulation could lead to decreased cellular plasticity, inflammation and cell death (Spagnuolo et al., 2018; Tiscione et al., 2019). Currently in clinical practice, there are only limited FDA approved medications that could level the systemic increase of cholesterol. The drugs currently in clinical practice comes with the burden of adverse effects due to nonspecific effects (Hiatt and Smith, 2014; Miller et al., 2015). Regarding the available naturally derived agents for treating hypercholesterolemia, we don't have enough information about the systemic toxicological effects (Gao et al., 2013; Efentakis et al., 2015; Kammoun et al., 2018). In our current study, we have made a sincere attempt to understand the cellular effects of two naturally occurring flavonoid compounds which are structurally related; Apigenin and Chrysin.

Apigenin and Chrysin are flavones belong to the flavonoid super family having a keto group at C4 position. They are polyphenols by nature and synthesized and stored inside fruit or vegetable bearing plants, which are the major dietary sources (Fig. 1). These agents are endogenous plant metabolites that are passed on to humans mostly by dietary consumption.

Till date, these two agents have demonstrated substantial protection against cancer, cardiovascular and other metabolic diseases (Mantawy et al., 2017; Feng et al., 2018; Ren et al., 2018; Xu et al., 2018; Zhou et al., 2018; Dong et al., 2019; Hamadou et al., 2019; Qiu et al., 2019; Li et al., 2020) due to their anti-oxidant, anti-inflammatory and tumor-inhibitory property. In cardiovascular diseases, some of the major protective functions are due to inhibition of inflammatory mediators like tumor necrosis factor-alpha, intercellular adhesion molecule-1 (ICAM-1), interleukin - 1βeta (IL- 1β), and prevention of reactive oxygen species generation. Some of the reports also highlights their ability to inhibit transcription function of NF – Kappa B or upregulation of peroxisome proliferator activated receptor gamma function that is known to counteract the inflammatory gene induction. They have also shown to enhance the pro-survival growth factor signaling molecules like vascular endothelial growth factor (VEGF) and Akt signaling. These agents are also known to enhance the capacity of the cellular oxidative stress resistance by upregulating glutathione and super oxide dismutase levels. Regulating the levels of these secondary mediators or signaling molecules, Apigenin and Chrysin have exhibited their protective role in cardiovascular tissue. Most studies have only focused on the use of these flavonoids for a specific disease condition and focused only on their beneficial effects rather than their systemic impact. The conventional perspective has limited our understanding about the cellular or molecular mechanism and subsequent physiological effects of these agents. In this study, using next generation RNA Sequencing analysis, we have uncovered that Apigenin and Chrysin can inhibit multiple targets in the cholesterol biosynthesis pathway at transcript level. Maintaining systemic cholesterol homeostasis is very crucial during the embryonic development stage (Tadjuidje and Hollemann, 2006; Plosch et al., 2007; Sohi et al., 2011; Nikolova et al., 2017). Inhibiting cholesterol biosynthesis during embryonic stage can be deleterious to growth and function (Tadjuidje and Hollemann, 2006; Plosch et al., 2007; Sohi et al., 2011; Nikolova et al., 2017). These defects observed during embryonic stage include retarded myelin formation, impaired steroidogenesis, retarded growth, organ malformation and skeletal system deformation as observed in Smith-Lemli-Opitz syndrome, an autosomal recessive genetic defect accompanied by the inability of cells to synthesize cholesterol (Connor et al., 1995; Ramirez et al., 2003; Porter and Herman, 2011; Shackleton, 2012) (Nowaczyk and Wassif, 1993; Opitz and de la Cruz, 1994; Elias and Irons, 1995; Bick et al., 1999; Gaoua et al., 1999; Fitzky et al., 2001; Waage-Baudet et al., 2003; Wassif et al., 2003; Marcos et al., 2004; Merkens et al., 2004; Tadjuidje and Hollemann, 2006; Guizzetti and Costa, 2007; Marcos et al., 2007; Porter, 2008; Shackleton, 2012; Kanungo et al., 2013; Allen et al., 2019). Conversely, excessive cholesterol has also shown to be detrimental in both embryonic and adult stage. From all these findings, it becomes very crucial to determine when an agent with hypocholesterolemia potential can be administered, whether during embryonic (development) or adult (developed) stage. In our study, we demonstrate that both chrysin and apigenin downregulate multiple transcripts involved in cholesterol biosynthesis in mouse embryonic fibroblasts as observed by transcriptomic analysis. This inhibition of transcripts involved in cholesterol biosynthesis path was also accompanied by an increase in transcripts involved in an alternate metabolic path i.e., towards ketogenesis.

Section snippets

Mammalian cell line used for the experiment

In this work, we have used Mouse Embryonic Fibroblasts (MEFs) from passages 2–5. The MEFs were purchased from Lonza Walkersville, Inc. (MD, USA) with Cat#M-FB-481. The MEFs were isolated from CD-1 mouse embryos and shipped in cryopreserved vials.

Cell culture

MEFs were cultured in Dulbecco's Modification of Eagle's Medium (DMEM), Corning® containing high glucose (4.5 g/l) in 10% Fetal Bovine Serum (A3840201) from Life Technologies – Thermo Fisher Scientific, USA), 1% penicillin/Streptomycin and amphotericin

RNA-seq analysis of chrysin and apigenin reveal their cholesterol biosynthesis inhibitory property

We selected a higher dose of 50 μM for both Apigenin and Chrysin for treating mouse embryonic fibroblasts for 24 h. It is deemed necessary to understand the toxicological properties along with their beneficial pharmacological properties for both these compounds at this concentration (Polier et al., 2011). Treatment of mouse embryonic fibroblasts (MEFs) with either 50 μM of Chrysin (Fig. 1 A) or 50 μM of Apigenin (Fig. 1 A) in 5 mM glucose medium for 24 h in comparison to control group (only

Discussion

Our study unveils a new mechanism for these natural agents of flavonoid origin as ideal therapeutics for controlling systemic cholesterol levels, by downregulating cellular cholesterol biosynthesis. They bring about this function through inhibition of multiple transcripts involved in cholesterol biosynthesis pathway. Our study also reveals that these two flavonoids could be bringing about the desired effect partly by inhibiting SREBP transcriptional targets especially the ones involved in

Data Availability

The RNA sequencing data can be found at NIH-Gene Expression Omnibus - public genomic data repository with the accession number GSE155852.

Ethics approval

N/A. We have not used data from whole animal models or human subjects, so there were no ethical issues involved in this study.

Funding

The funding source for this study is from the Midwestern University and Department of Pharmacology new faculty startup funds and the outsourcing funds provided by the Midwestern University core committee to Dr. Puthanveetil.

Author contributions

PP conceived the idea, performed the experiments, collected the data, wrote, and edited the manuscript. PP wrote and edited the manuscript and was primarily involved in plotting the figures and making diagrams. SB and WAP have contributed to the figures and edited the manuscript. XK helped with the figures and edited the manuscript. SB, GV and WAP edited the manuscript and provided their inputs and critics that has improved the quality of the manuscript. All authors have read and approved the

Declaration of competing interest

All the authors declare no conflict of interest.

Acknowledgements

I would like thank Dr. Kyle Ramsey (Vice President and Chief Academic Officer), Dr. Gloria Yeuh (Dean - CGS), Dr. Mike Fay (Associate Dean -CGS), and Dr. Philip Kopf (Chair- Department of Pharmacology) for their continued support. Dr. Walt Prozialeck and Dr. Joshua Edwards and lab members (Mr. Ameir Barakat and Mr. Pete Lamar) for sharing space and facilities and being wonderful colleagues. Dr. Kathleen O'Hagan (Chair, Department of Physiology) for the scientific inputs, collaborations and for

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