Upregulation of endogenous erythropoietin expression by DLBS6747, a bioactive fraction of Ipomoea batatas L. leaves, via increasing HIF1α transcription factor in HEK293 kidney cells

Journal of Ethnopharmacology 235 (2019) 190–198

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Upregulation of endogenous erythropoietin expression by DLBS6747, a bioactive fraction of Ipomoea batatas L. leaves, via increasing HIF1α transcription factor in HEK293 kidney cells Guntur Berlian, Olivia Mayasari Tandrasasmita, Raymond Rubianto Tjandrawinata

T

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Dexa Laboratories of Biomolecular Sciences (DLBS), Industri Selatan V Block PP No. 7, Jababeka Industrial Estate II, Cikarang 17550, West Java, Indonesia

ARTICLE INFO

ABSTRACT

Keywords: Erythropoietin Erythropoietin stimulating agent Hypoxia inducible factor-1α Ipomoea batatas L. DLBS6747 Hypoxia

Ethnopharmacological relevance: Ipomoea batatas L., locally known as ubi jalar, is widely used in Indonesia and other countries as a folk remedy for various chronic diseases, including anemia-associated chronic kidney disease by increasing hematological parameters such as packed cell volume, white blood cells and platelet counts. Aim of the study: The aim of this study is to evaluate the effect of DLBS6747, a bioactive fraction of I. batatas L. leaves, on increasing EPO expression through the upregulation of HIF1α. Materials and methods: Effect of DLBS6747 on EPO expression and its transcription factor, HIFs, was evaluated in normoxia and hypoxia conditions. Effect of DLBS6747 on several genes involved in EPO expression were evaluated in a time-course manner using conventional and real-time PCR, while the protein level were revealed using western blot and ELISA. The involvement of HIF1α was also confirmed by HIF1α siRNA. Results: Administration of DLBS6747 increased transcriptional activity of EPO through the regulation of its transcriptional factors, which include HIF1α, HIF2α and NFᴋB. The effect was found to be dependent on oxygen availability, wherein DLBS6747-increased EPO expression was found to be more significant in hypoxic condition. In normoxia and hypoxia, 40 μg/mL DLBS6747 increased HIF1α and HIF2α expressions at mRNA level, wherein the peak appeared in 12 h treatment (up to 7.9- and 8.6-folds, respectively). On the other hand, increased protein level was only found in hypoxia, where the highest HIF1α expression was observed at 6 h (7.5folds increase) and started to decrease after the hours, while HIF2α was found to be increased time-dependently (up to 13.8-folds in 24 h). The mechanism of action of DLBS6747 as erythropoietin stimulating agent is more likely to affect the regulation of HIF1α, as confirmed by HIF1α siRNA which showed that DLBS6747 failed to increase EPO expression during co-incubation with HIF1α siRNA. DLBS6747 treatment also decreased NFᴋB time-dependently in normoxia, while no NFᴋB was detected in hypoxia, which revealed mimicking hypoxia activity of DLBS6747 to increase EPO expression. Conclusion: These findings showed convincing evidences that DLBS6747 increases endogenous EPO production primarily via upregulation of its transcription factors, especially HIF1α, in human embryonic kidney HEK293 cells. This is the first molecular report that reveals the mechanism of action of natural-based erythropenia drug in different oxygen availability.

1. Introduction It has widely been known that erythropoietin (EPO), which is mainly produced in kidney as the response to hypoxia, is the primary regulator of red blood cell production. Binding of EPO to its receptor induces phosphorylation of various signaling proteins that controls the synthesis of erythrocytes and survival of erythroid progenitor cells in bone marrow (Jelkmann, 2004). A low constitutive EPO production is

necessary to maintain the physiologic renewal of erythrocytes but EPO expression has been known to be increased dramatically during tissue hypoxia caused by several conditions such as high altitude and anemia (Jelkmann, 2011; Fandrey, 2004). Moreover, studies in various experimental models showed that injection of exogenous EPO can be used for treating anemia in critical illness, acute organ injuries and chemotherapy-induced anemia (Patel et al., 2011; Eckardt and Kurtz, 2005; Rossert and Eckardt, 2005).

Corresponding author. E-mail addresses: [email protected] (G. Berlian), [email protected] (O.M. Tandrasasmita), [email protected] (R.R. Tjandrawinata). ⁎

https://doi.org/10.1016/j.jep.2019.01.016 Received 19 October 2018; Received in revised form 22 January 2019; Accepted 23 January 2019 Available online 24 January 2019 0378-8741/ © 2019 Elsevier B.V. All rights reserved.

Journal of Ethnopharmacology 235 (2019) 190–198

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Hypoxia inducible factors (HIFs) is a family of transcription factors which orchestrate signaling events to modulate a wide range of processes, including the process of erythrocyte synthesis which is known as erythropoiesis (Haase, 2006). HIF, consisting of an inducible HIFα subunit and a constitutive HIFβ subunit, is an oxygen sensor that plays a central role in the maintenance of oxygen homeostasis in body tissues (Ratcliffe, 2007). In the presence of oxygen, HIF is inactivated by prolyl hydroxylase domain (PHD) enzyme through post-transcriptional hydroxylation of specific amino acid residues within its α subunit. Upon hypoxic stimulation, the activated HIF binds onto the hypoxia response element (HRE) located on the promoter region of EPO gene which subsequently triggers the transcription of EPO gene (Haase, 2013). Recently, there has been growing interest in the use of natural products, especially those derived from plants and herbs. Our previous researches have been conducted in investigating the molecular and pharmacology mechanism of various herbals (Tandrasasmita et al., 2010, 2011, 2015; Tjandrawinata et al., 2010, 2013; Karsono et al., 2014; Berlian et al., 2016, 2018; Nailufar and Tjandrawinata, 2017), as well as other studies on various herbals (Tjandrawinata et al., 2012; Rahayu et al., 2016; Hiendrawan et al., 2017; Ramdani et al., 2017). Studies on various plants that possess the ability to promote hematopoietic function through the stimulation of EPO production, i.e. Astragalus propinquus and Angelica sinensis, have previously been done by other researchers. The studies reported that the flavonoids of these plants induced EPO expression through the upregulation of HIF1α expression in kidney (Zheng et al., 2011, 2010; Gao et al., 2008). Another study also reported that EPO expression was induced in a dose-dependent manner by the combination of Ligusticum striatum and A. sinensis extract in mice and Hep3B cells (Li et al., 2012; Bi et al., 2010). Ipomoea batatas L., locally known in Indonesia as ubi jalar, has widely been used in Indonesia and other countries as a folk remedy for various chronic diseases, including diabetes mellitus (Bachri et al., 2010), inflammatory disorders (Ayeleso et al., 2016; Konczak-Islam et al., 2003) and anemia associated with chronic kidney disease (Milind and Monika, 2015). The leaves of I. batatas have also been reported to possess hematinic effect by increasing hematological parameters such as packed cell volume, white blood cells and platelet counts in vivo in rabbit and mice (Montejo et al., 2015; Osime et al., 2008). In this study, we evaluated the effect of DLBS6747, a bioactive fraction of I. batatas leaves, on EPO expression and its transcription factors, HIFs, in normoxia and hypoxia conditions. Time series experiment was also conducted to further evaluate the effect of DLBS6747 to the feedback mechanism between EPO and HIFs in human embryonic kidney HEK293 cells. This is the first molecular evidence that demonstrates the mechanism of action of I. batatas L. bioactive fraction on increasing endogenous EPO as a potential drug to treat erythropoiesis dysfunctionrelated diseases caused by EPO deficiency.

profile and microbiological tests. Physical description showed the fastest way to evaluate the stability of DLBS6747. Loss on drying was set to optimize the water (moisture) content in the DLBS6747 bioactive fraction. Solubility of DLBS6747 in water and ethanol was determined to ensure the consistency in the preparation of dosage form. Total ash content of DLBS6747 showed the total metal oxide from internal and external of raw material. TLC was able to show the marker compounds in its profile. TLC system was optimized to obtain the selected marker compound. Microbiological tests was performed to ensure that DLBS6747 comply to the regulation and safe to be consumed. The tests comprised of total aerobic microbial count, total yeasts and molds count and identification of microbial pathogens (Escherichia coli, Salmonella spp., Staphylococcus aureus, and Pseudomonas aeruginosa). 2.2. Cell culture and activity assay of DLBS6747 HEK293 cells (CRL-8065) were purchased from American Type Culture Collection (ATCC) (Manassas, VA, USA) and grown in Modified Eagle's Medium (MEM) (Gibco, CA, USA) supplemented with 10% New Born Calf Serum (NBCS) (Gibco, CA, USA), 100 μg/mL penicillinstreptomycin (Gibco, CA, USA) and 1 mM sodium pyruvate (Gibco, CA, USA) at 37 °C until it reached 80% of confluence. Cells were sub-cultured in 6-well plate in 2 mL medium and 10 cm petri dish in 10 mL medium. After 3–5 days, medium were aspirated and replenished with serum-free media for 4 h. In dose-response study, the cells were treated with 0–40 μg/mL of DLBS6747 for 24 h in normoxia condition to quantify the level of EPO and HIF1α. Using one concentration of DLBS6747 obtained from dose-response study, experiments were performed in normoxia and hypoxia conditions to measure the level of EPO, HIF1α, HIF2α, PHD-2 and NFκB gene expressions. 2.3. Hypoxia condition model Hypoxia condition was generated using Anaerocult® (Merck, Darmstadt, Germany) by incubating the cultures and Anaerocult® inside an anaerobic culture chamber to limit oxygen availability. Prior to the incubation, Anaerocult® was activated by the addition of 35 mL ddH2O. For time course study, cultures used for different incubation times (3, 6, 12 and 24 h) were placed in different anaerobic chamber to keep the hypoxia condition and prevent oxygen from the outside entering the chamber. 2.4. RNA interference HEK293 cells were grown in 24-well plate until it reached 90% of confluence. To validate the HIF1α siRNA study, increasing concentration of control GAPDH siRNA (silencer Select GAPDH control siRNA reference s815) was transfected to the cells. On the other hand, to further evaluate the effect of HIF1α siRNA on EPO expression, the cells were treated with 40 µg/mL DLBS6747 with or without 10 pmol HIF1α siRNA (Silencer Select HIF1α siRNA reference s6539) using Lipofectamine 3000 transfection reagent (Invitrogen, CA, USA) according to the manufacturer's instructions. At 72 h post-transfection, the cells RNA were isolated for further mRNA expression analysis. Cells without the addition of DLBS6747 were used as the negative control. Both HIF1α and GAPDH control siRNA were obtained from Ambion. The HIF1α siRNA sequence used were: sense 5’-CCAUAUAGAGAUAC UCAAATT-3’; antisense 5’-UUUGAGUAUCUCUAUAUGGTG-3’ and the control GAPDH siRNA sequence used were: sense 5’-GGUCAUCCAUG ACAACUUUTT-3’; antisense 5’-AAAGUUGUCAUGGAUGACCTT-3’.

2. Material and methods 2.1. Preparation of DLBS6747 DLBS6747 is a bioactive fraction from I. batatas L. leaves which were collected from Bogor, West Java, Indonesia. The plant was identified by Herbarium Bogoriense, Research Center of Biology, Indonesian Institute of Sciences with certificate No. 2153/IPH.1.01/If.07/X/2016. To obtain DLBS6747, 250 g of dried leaves of I. batatas L. were macerated for 2 h at 50 °C using 70% ethanol (raw material to ethanol ratio was 1:9 w/v). The obtained liquid was evaporated under reduced pressure at 60 °C to yield concentrated extract and then dried using conventional oven at 70 °C for 24 h. The resulted dried extract (about 14.7 g or 5.88% to the raw material) was packed and stored at room temperature (25–30 °C) for further analysis. DLBS6747 possessed several quality control parameters comprised of physical description, loss on drying, solubility in water, solubility in ethanol 96%, total ash content, thin layer chromatography (TLC)

2.5. Polymerase chain reaction Total RNA of control and treated-HEK293 cells with or without DLBS6747 was isolated by Trizol Reagent, and was reverse-transcribed using ReverTra Ace qPCR mastermix (Toyobo, Osaka, Japan) to obtain 191

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cDNA. cDNA was used as the template of gene fragment amplifications using conventional and real-time PCR techniques to measure the level of mRNA synthesis. The primers used are EPO forward, 5’-ACCAACAT TGCTTGTGCCAC-3’; EPO reverse, 5’-TCTGAATGCTTCCTGCTCTGG-3’; HIF1α forward, 5’-CGTCGCTTCGGCCAGTGTGT; HIF1α reverse, 5’TCCAGAGGTGGGGGTGCGAG-3’; HIF2α forward, 5’-TTGATGTGGAAA CGGATGAA-3’; HIF2α reverse, 5’-GGAACCTGCTCTTGCTGTTC-3’; GAPDH forward, 5’-ATGACAACAGCCTCAAGATCATCAG-3’; and GAPDH reverse, 5’-CTGGTGGTCCAGGGGTCTTACTCCT-3’. All primers were synthesized by First Base Laboratories (Singapore). Real-time PCR amplification was performed in a total volume of 20 µL, containing 2 µL cDNA solution, 10 µL 1X SsoFast PCR Mastermix (BioRad, Singapore), 2 µL of each primer of 10 µM and 4 µL nuclease-free water. PCR condition consisting of denaturation at 95 °C for 3 min, 35 cycles of denaturation at 95 °C for 5 s and annealing at 58 °C for 5 s, extension at 72 °C for 5 s and a final extension at 72 °C for 5 min. DNA amplification by conventional PCR was performed in a total volume of 25 µL, containing 3 µL cDNA solution, 12.5 µL 1X GoTaq Green Mastermix (Promega, WI, USA), 2.5 µL of each primer and 0.25 µL of GAPDH primer of 10 µM and 4 µL nuclease-free water. PCR condition consisting of denaturation at 95 °C for 3 min, 30–32 cycles of denaturation at 95 °C for 30 s and annealing at 58 °C for 30 s, extension at 72 °C for 1 min and a final extension at 72 °C for 5 min. The PCR products (10 µL) were run in a 2% agarose gel and DNA was visualized by EcoDye (SolGent, Daejeon, Korea) and photographed using GelDoc (BioRad). GAPDH was used to normalize the gene expression values.

2.8. Western blotting of NFᴋB Total of 35 μg proteins were boiled for 10 min in SDS buffer (1 M Tris pH 6.8, 87% glycerol, 10% SDS, 2-merkaptoetanol, Bromphenol Blue) and separated by 12% sodium dodecyl-sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), followed by electroblotting onto polyvinylidene difluoride (PVDF) membranes. The blots were incubated with NFᴋB and β-actin primary as well as secondary antibodies (Santa Cruz Biotechnology, Singapore), with dilution of 1:500 and 1:10,000, respectively. The protein band was visualized by the Amersham chemiluminescence kit (GE Life Sciences, Buckinghamshire, UK) and quantified using a ChemiDoc MP Imaging System (BioRad). β-actin was used to normalize the expression values of NFᴋB protein. 2.9. Statistical analysis Statistical differences between the test and control samples were determined by Student's t-test using StatView software package (Abacus Concepts, Piscataway, NJ, USA). Values were expressed as mean ± standard deviation from at least two independent experiments (p < 0.05). 3. Results 3.1. Dose response study of DLBS6747 on EPO and HIF1α expressions Cytotoxicity of DLBS6747 in human embryonic kidney HEK293 cell cultures was tested. Results showed that 0–40 µg/mL DLBS6747 did not significantly change the cell morphology and number, as revealed by microscopic examination and cell viability assay, respectively (data not shown), indicating that the doses were safe to the cells. DLBS6747-induced EPO mRNA expression were shown to be dose-dependent, with a peak response at 40 µg/mL DLBS6747, wherein 2.89-folds increment was found in the maximum induction compared to the control (Fig. 1A). Meanwhile, the effect of DLBS6747 on EPO was also evaluated at protein level. The same trend was found at protein level, as revealed by human EPO ELISA kit. Result showed that DLBS6747 induced EPO protein dose-dependently, with up to 2.5-folds increment compared to the control (Fig. 1B). Dose response of DLBS6747 on HIF1α, the transcription factor of EPO, was also evaluated. DLBS6747 started to significantly increase HIF1α mRNA by up to 2.9-folds at 30 and 40 µg/mL (Fig. 1C). As compared between the graphs, there is a clear correlation between DLBS6747 treatment and EPO expression and HIF1α in normoxia condition, which indicates that DLBS6747 increased EPO expression through the upregulation of HIF1α.

2.6. Protein preparation For EPO isolation, extracellular proteins were concentrated by centrifuging the media at 7000 × g for 15 min using a 50-kDa Amicon filter (Milipore, Carrightwohil, Ireland) in a 15 mL Falcon tube. To isolate HIF1α and HIF2α proteins, treated and untreated HEK293 cells (5 × 106 cells) were harvested and solubilized in 1 mL of extraction buffer. Cells were incubated in ice for 20 min and centrifuged at 16,000 × g at 4 °C for 20 min. The pellets were discarded and the supernatants were transferred into clean tubes. For PHD-2 protein isolation, HEK293 cells (5 × 106 cells) were harvested, resuspended in 1 mL of 1 × PBS and freeze-thawed at − 20 °C for three times to lyse the cells. The lysate was centrifuged at 1500 × g at 4 °C for 10 min to remove cellular debris. The pellets were then discarded, and the supernatants were transferred into clean tubes. To obtain NFκB protein, cells were washed with ice-cold phosphate-buffer saline (PBS) and lysed in a hypotonic buffer (150 mM NaCl, 20 mM Tris, 10% glycerol, 0.1% SDS, 1 mM EDTA) containing a protease inhibitor cocktail set I Calbiochem (Milipore, Carrightwohil, Ireland). Protein concentrations were determined according to Lowry et al. (1951).

3.2. Time course study of DLBS6747 on EPO and HIFs expression in different conditions of oxygen level

2.7. ELISA for quantitative measurement of proteins

Since HIF is a member of transcription factors family responsible for EPO gene as the response to oxygen availability in the environment, the effects of DLBS6747 in normoxia and hypoxia conditions were compared. A series of incubation time (0–24 h) was also applied in the treatment to evaluate the interaction profile between EPO and two members of HIF, which consist of HIF1α and HIF2α. Without DLBS6747 treatment, by comparing the control of normoxia and hypoxia groups, it was found that EPO protein in hypoxia (0.7 ng/mL) was lower than that in normoxia (2.3 ng/mL), while no significant difference was found at mRNA level (Fig. 2). On the other hand, the expressions of HIF1α protein in hypoxia (4.5 pg/mL) were significantly higher than that in normoxia (2.0 pg/mL), while no significant difference of HIF2α protein was found in normoxia and hypoxia (Figs. 3 and 4). The time course study revealed that 40 µg/mL of DLBS6747 increased the expression of EPO at mRNA level in a time-dependent

Effect of DLBS6747 on EPO, HIF1α, HIF2α and PHD-2 expressions at protein level were assayed using ELISA kits according to the manufacturer protocols (EPO kit: Abnova, CA, USA; HIF1α kit: Abnova, CA, USA; HIF2α kit: Mybiosource, CA, USA; PHD-2: Cloud-clone, TX, USA). According to the general protocol of the kits, each 50 µL of standard, sample and blank solution were loaded into the well-plate coated by immobilized specific antibody. After 1 h incubation at 37 °C, the wells were washed four times using washing solution. Biotin-conjugated secondary antibody was added into the wells and the mixture was incubated for 1 h at 37 °C. After re-washing the unbound secondary antibody, the streptavidin-HRP was added and the mixture was incubated for 30 min at 37 °C. TMB substrate (100 µL) was added into all wells and incubated for 15 min at room temperature. The reaction was stopped by adding 100 µL stop solution. The absorbance was then immediately measured at 450 nm. 192

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Fig. 1. Dose response study of DLBS6747 on EPO expression at (A) mRNA and (B) protein levels and (C) HIF1α expression at mRNA level. Gene expressions of EPO and HIF1α were analyzed using real-time PCR with GAPDH as the internal control, while the protein level was measured using human EPO ELISA kit. Figures represent mean ± SD of three independent experiments (*p < 0.05 compared to control).

manner (Fig. 2A); while a slight decrease was occurred at protein level in 24 h treatment (Fig. 2B). In addition, the effect of DLBS6747 on EPO was found to be more significant in hypoxia compared to normoxia. DLBS6747 40 µg/mL also increased the mRNA expression of HIF1α in both normoxia and hypoxia, wherein the peak appeared at 12–24 h

treatment (up to 8-folds), while no significant mRNA increase was found in HIF2α in normoxia (Figs. 3A and 4A). In the other hand, the increase of HIF1α and HIF2α at protein level only occurred in hypoxia condition. However, the effect of DLBS6747 on HIF1α and HIF2α in hypoxia condition was different. The highest protein level of HIF1α was Fig. 2. Effect of DLBS6747 on EPO expression at (A) mRNA and (B) protein levels. Gene expression of EPO was analyzed using real-time PCR with GAPDH as the internal control, while the protein level was measured using human EPO ELISA kit. Figures represent mean ± SD of three independent experiments (*p < 0.05 compared to control).

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Fig. 3. Effect of DLBS6747 on HIF1α expression at (A) mRNA and (B) protein levels. Gene expression of HIF1α was analyzed using realtime PCR with GAPDH as the internal control, while the protein level was measured using human HIF1α ELISA kit. Figures represent mean ± SD of three independent experiments (*p < 0.05 compared to control).

observed on 6 h of treatment (increased by 7.5-folds) and started to decrease after the hours, while HIF2α was found to be increased in a time-dependent manner (up to 13.8-folds increment in 24 h treatment) (Figs. 3B and 4B).

3.3. Effect of DLBS6747 on PHD-2 expression in different condition of oxygen level Due to the key function of PHD in the regulation HIF inactivation, in Fig. 4. Effect of DLBS6747 on HIF2α expression at (A) mRNA and (B) protein levels. Gene expression of HIF2α was analyzed using realtime PCR with GAPDH as the internal control, while the protein level was measured using human HIF2α ELISA kit. Figures represent mean ± SD of three independent experiments (*p < 0.05 compared to control).

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Fig. 5. Effect of DLBS6747 on PHD-2 protein. The protein level was measured using human PHD-2 ELISA kit. Figures represent mean ± SD of three independent experiments (*p < 0.05 compared to control).

this present experiment, we further evaluated the effect of DLBS6747 on PHD-2 expression in normoxia and hypoxia conditions. As depicted in Fig. 5, we found that PHD-2 expression in normoxia (109 pg/mL) was significantly higher compared to that in hypoxia (60 pg/mL). In both conditions, there was a clear trend that DLBS6747 decreased PHD-2 expression, wherein the lowest expression was found at 12 h (41 pg/ mL) and 6 h (45 pg/mL) treatment in normoxia and hypoxia conditions, respectively. 3.4. Effect of DLBS6747 on NFᴋB expression in different condition of oxygen level In addition to its positive regulators, we also evaluated the effect of DLBS6747 on NFᴋB, the transcriptional repressor of EPO promoter. The effect of DLBS6747 on NFᴋB at protein level was determined using western blot analysis. In normoxia, DLBS6747 treatment decreased NFᴋB time-dependently, while no NFᴋB was detected in hypoxia condition (Fig. 6). 3.5. Confirmation of the involvement of HIF1α pathway in DLBS6747induced EPO expression

Fig. 7. siRNA study of HIF1α and control GAPDH in HEK293 cells. (A) Increasing concentration of GAPDH siRNA did not affect HIF1α expression. (B) Effect of DLBS6747 on HIF1α and (C) EPO expressions at mRNA level. The mRNA level was measured using conventional and real-time PCR with GAPDH as the internal control. Figures represent mean ± SD of two independent experiments (*p < 0.05 compared to control).

Since HIF1α is generally considered as the primary mediator of hypoxia-induced EPO expression, it was suggested that DLBS6747 dominantly affects HIF1α. Prior to HIF1α siRNA study, we transfected the cells with increasing concentration of GAPDH siRNA (10–20 pmol) for 72 h to validate the study. The result showed that GAPDH siRNA treatment did not affect HIF1α mRNA expression (Fig. 7A). To confirm the involvement of HIF1α pathway in DLBS6747-induced EPO expression in HEK293 cells, we treated the cells with HIF1α siRNA. The result

showed that HIF1α siRNA significantly decreased HIF1α mRNA in previously DLBS6747-treated cells. DLBS6747 treatment (40 µg/mL) increased HIF1α up to 2-folds compared to control group, while administration of HIF1α siRNA together with DLBS6747 treatment

Fig. 6. Effect of DLBS6747 on NFᴋB protein. The protein level was measured using western blot with β-actin as the internal control. Figures represent three independent experiments.

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decreased the mRNA for 75% compared to DLBS6747 treatment group only (Fig. 7B). Subsequently, the level of EPO expression also decreased in line with that of HIF1α (Fig. 7C).

EPO expression is also induced not only by hypoxia but also by certain transition metal (cobalt and nickel) and iron chelation (Daghman et al., 1999; Ho and Bunn, 1996). Our literature study revealed that cobalt is one of mineral essentials found in I. batatas L. leaves (Taira et al., 2013), and this mineral could stimulate the production of HIF1α in normoxia condition in a dose-dependent manner (Befani et al., 2013; Huang et al., 1997). In this current study, significant increase of EPO by DLBS6747 through the upregulation of HIFα was found in normoxia, which was likely occurred due to cobalt contained in I. batatas L. leaves. In the presence of oxygen, HIFs are targeted for the inactivation and destruction by post-translational hydroxylation of specific amino acid residues within its α subunit. Prolyl hydroxylase domain (PHD) is an enzyme that regulates the balancing level of EPO in the cells. In normoxia condition, PHD promotes the interaction of ubiquitin protein into HIF complex and induces its proteolytic degradation (Souvenir et al., 2014; Schofield and Ratcliffe, 2004). PHD level has been found to be lower in hypoxia than normoxia, therefore the ubiquitination of HIF1α is decreased, then HIF1α will allow the occurrence of EPO transcription to induce EPO synthesis. Regarding to their function in regulating HIF, members of PHD inhibitor class drugs, i.e. molidustat, raxodustat and daprodustat, have generally been used to treat anemia related with EPO deficiency (Maxwell and Eckardt, 2016). Our findings showed that DLBS6747 decreased PHD expression, particularly in normoxia condition. These findings are likely correlated to the increased HIFα mRNA by DLBS6747 in normoxia, but not at protein level. It is suggested that there was a post-transcriptional regulation of HIFα to balance EPO level depending on oxygen availability. Effect of DLBS6747 in increasing EPO level is clearly shown in hypoxia condition. DLBS6747 also decreased NFᴋB protein level, which has been known as a suppressor of EPO promotor, in a dose-dependent manner in normoxia, while NFᴋB was almost undetectable in hypoxia. Our current result indicated that hypoxia repressed the expression of NFᴋB, which was correlated to the cell response to low oxygen level in environment. This result also indicated that DLBS6747 acted as a hypoxia mimetic agent in the presence of oxygen. NFᴋB is a family of transcription factors that is composed by RelA, RelB, cRel, NFᴋB1 (p105/p50) and NFᴋB2 (p100/p52). RelA, RelB and cRel contain a C-terminal transactivation domain, but no definable activation domains are found in p50 and p52. These transcription factors are usually held captive in the cytoplasm by a family of inhibitors known as Inhibitor of ᴋB (IᴋBs) (Jelkmann, 2004). Upon stimulation by different stresses, a number of pathways that lead to nuclear accumulation and DNA-binding of NFᴋB have been described (Gilmore, 2006). The role of NFᴋB in EPO gene expression has been reported by Figueroa et al. (2002) that found transient transfection of dominant negative of IᴋB prior to hypoxia treatment significantly decreased the expression of EPO mRNA, which revealed that indeed NFᴋB responds to oxygen level stimulus. Competition between NFᴋB and cofactor p300/CBP, which is known as one of HIF co-factors in EPO promoter, was occurred depending on the oxygen level and further defined the regulation of EPO synthesis (Ke et al., 2001; Gires et al., 2001; La Ferla et al., 2002; Jelkmann, 2004; Fandrey, 2004). In normoxia, NFᴋB level was higher than that in hypoxia. High level of NFᴋB inhibits p300 attached to the EPO promoter and suppresses EPO synthesis. On the other hand, decreased NFᴋB in hypoxia allows cofactor p300 and HIF complex to attach to the EPO promoter, therefore EPO synthesis will occur. In addition, NFᴋB has also been reported as one of several proteins that bind to HIF1 response element to increase HIF1 expression, however, NFᴋB activation in hypoxia seems to have a more complicated role in apoptosis depending on context (D’Ignazio and Rocha, 2016; Gordon et al., 2011). We suggested the suppression of NFᴋB by DLBS6747 in increasing EPO is more likely through suppressing the presence of NFᴋB in EPO promoter, and the

4. Discussion EPO is the hormone responsible for the production of red blood cell in the bone marrow. It is primarily produced by the kidney when a drop in blood oxygen level is perceived, though 10–15% of the total EPO is derived from the liver (Jelkmann, 2011; Rodriguez et al., 2000). The abnormalities of these organs may cause anemia due to EPO deficiency (Schwenger et al., 2007; Eckardt and Kurtz, 2005). Since most EPO is produced by kidney, a kidney cell line, HEK293, was used in this study as the in vitro model to investigate the effect of DLBS6747 as a promising compound to stimulate red blood cells production, or generally known as erythropoietin-stimulating agent (ESA), and revealed its mechanism of action. The regulation of EPO gene expression occurs primarily at mRNA level (Wenger, 2002). Failure to increase circulating EPO as the response to hypoxic stress may result in anemia (Jelkmann, 2011; Schwenger et al., 2007). In the present study, we reported that DLBS6747 positively increased EPO production at both mRNA and protein levels in HEK293 cells. Thus, the regulation of EPO by DLBS6747 may serve as one of the molecular bases for its hematopoietic effect. Transcriptional regulation of EPO expression is not solely regulated by one transcription factor. Instead there are numerous potential binding sites for different transcription factors in the promoter and 3’enhancer region. EPO expression is a result of the interplay between many different transcription factors of partly opposite functions, where the balance of these transcription factors determines the extent of EPO expression (Batmunkh et al., 2006). One of the most important transcription factors of EPO is HIFs, which consist of a constitutive β-subunit and one of the two alternative oxygen-regulated HIFα subunits that possess a C-terminal transactivation domain, which are HIF1α and HIF2α, where HIF1α is generally considered as the primary mediator of hypoxia-induced EPO gene expression (Jelkmann, 2011; Stockmann and Fandrey, 2006). Nevertheless, in vitro study in HEK293 and knockout studies in mice demonstrate that HIF1α and HIF2α play nonredundant roles, and inactivation of each one results in a distinctly different target and phenotypes depending on the cell type and oxygen concentrations (Hu et al., 2003; Wiesener et al., 2003; Rosenberger et al., 2002). Accordingly, HIF1α has been suggested to represent the response to acute hypoxia, whereas HIF2α is the predominant subunit to chronic exposure to low oxygen that occurs at high altitude (van Patot and Gassmann, 2011). In this study, we found that DLBS6747 induced the expression of HIF1α and HIF2α, where the increment were only found in hypoxia at protein level; however, their optimum time was different: HIF1α at 6 h while HIF2α at 24 h. It seemed that HIF1α might govern the response to hypoxia at the beginning as a response to acute hypoxia caused by Anaerocult® treatment, and further passing to the increment of HIF2α as the cells adapt to the low oxygen condition. On the other hand, HIF1β is continuously expressed, with their mRNA level being unaltered by the induction of hypoxia (Jelkmann, 2011). It was found that the level of constitutive β-subunit was also sufficient to form HIF complex with the increased α-subunit, since the increased HIF1α and HIF2α by DLBS6747 successfully promoted EPO transcription. We also found that the increment was depending on oxygen availability in the environment, where DLBS6747-increased EPO expression was found to be more significant in hypoxic condition. These findings were in accordance to the theory that HIFα expressions are greatly affected by oxygen level (Haase, 2013; Jelkmann, 2011; Maxwel, 2003). However,

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Fig. 8. Proposed mechanism of action of DLBS6747 in increasing EPO in human kidney HEK293 cells.

involved pathways was different from the activity of DLBS6747 in increasing HIF1α. Study reported that mimicking hypoxia is one of the novel treatment concepts for restoring endogenous EPO to treat anemia, i.e. BAY85-3934 (molidustat), a member of PHD inhibitor class drugs, that has been reported to stimulate EPO production through mimicking hypoxia by inhibiting HIF-PHDs, thereby stabilizing HIF (Flamme et al., 2014). However, as reported by Pediatric Committee of the European Medicines Agency (2014), molidustat has been associated with potential unwanted adverse effects in children. In case of DLBS6747, there is no study yet regarding its potential side effect. Flavonoids, the major compound found in DLBS6747, are associated with a broad spectrum of health-promoting effects and has been proposed as a possible therapy for treating anemia (Panche et al., 2016; Zhang et al., 2016). However, excessive flavonoid intake may lead to pro-oxidants that generate free radicals and inhibitor of enzymes involved in hormone metabolism (Skibola and Smith, 2000). Cellular regulatory mechanism of DLBS6747 in increasing endogenous EPO expression through the regulation of its transcription factors in both normoxia and hypoxia conditions are depicted in Fig. 8. The mechanism of action of DLBS6747 as ESA is more likely to affect the regulation of HIF1α, as confirmed by HIF1α siRNA which showed that DLBS6747 did not increase EPO expression when it was co-incubated with HIF1α siRNA. These findings showed convincing evidences that DLBS6747 increases endogenous EPO production primarily through the upregulation of its transcription factors, especially HIF1α, in human embryonic kidney HEK293 cells. Although clinical studies are still required, erythropoietin-stimulating activity of DLBS6747 may be considered as one of the promising treatments for erythropenia-related diseases. This current study is the first report that provides molecular evidences of a potential bioactive fraction to treat erythropenia condition and its molecular action in different oxygen availability.

CRediT authorship contribution statement Guntur Berlian: Conceptualization, Data curation, Formal analysis, Validation, Visualization, Writing - original draft. Olivia Mayasari Tandrasasmita: Conceptualization, Data curation, Methodology, Supervision, Writing - review & editing. Raymond Rubianto Tjandrawinata: Conceptualization, Methodology, Writing review & editing. Acknowledgements The authors would like to thank PT Dexa Medica for the financial support. The authors would also like to thank Irfan Agustian Darfiansyah for preparing DLBS6747, Tia Mariana for supporting the laboratory works and Debbie Retnoningrum and Isabela Anjani for editing this manuscript. Conflict of interest/Declarations of interest None. References Ayeleso, T.W., Ramachela, K., Mukwevho, E., 2016. A review of therapeutic potentials of sweet potato: pharmacological activities and influence of the cultivar. Trop. J. Pharm. Res. 15, 2751–2761. Bachri, M.S., Jang, H.W., Choi, J., Park, J.O., 2010. Protective effect of white-skinned sweet potato (Ipomoea batatas L.) from Indonesia on streptozotocin-induced oxidative stress in Rats. J. Life Sci. 20, 1569–1576. Batmunkh, C., Krajewski, J., Jelkmann, W., Hellwig-Burgel, T., 2006. Erythropoietin production: molecular mechanisms of the antagonistic action of cyclic adenosine monophosphate and interleukin-1. FEBS Lett. 580, 3151–3160. Befani, C., Mylonis, I., Gkotinakou, I.M., Georgoulias, P., Hu, C.J., Simos, G., et al., 2013. Cobalt stimulates HIF-1-dependent but inhibits HIF-2-dependent gene expression in liver cancer cells. Int. J. Biochem. Cell Biol. 45 (11). Berlian, G., Tandrasasmita, O.M., Tjandrawinata, R.R., 2016. Standardized bioactive fraction of Phaleria macrocarpa (Proliverenol) prevents ethanol-induced

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