2’-Hydroxyflavanone prevents LPS-induced inflammatory response and cytotoxicity in murine macrophages

Abstract

2’-Hydroxyflavanone (2-HF) is a natural flavonoid isolated from citrus fruits. Multiple studies have demonstrated that 2-HF with its anti-proliferative and pro-apoptotic effects prevent the growth of various cancers. Although 2-HF is a well known anti-oxidative and chemopreventive agent, its role as an anti-inflammatory agent is not well established. In this study, we examined the effect of 2-HF on LPS-induced cytotoxicity and inflammatory response in murine RAW 264.7 macrophages. Flow cytometry analysis showed that pre-treatment of RAW 264.7 macrophages with 2-HF significantly prevented LPS-induced macrophage apoptosis. 2-HF also prevented LPS-induced reactive oxygen species (ROS) and nitric oxide (NO) production, lipid peroxidation, and loss of mitochondrial membrane potential in murine macrophages. Most importantly, the release of multiple inflammatory cytokines and chemokines such as eotaxin, IL-2, IL-10, IL-12p40, LIX, IL-15, IL-17, MCP-1, and TNF-α induced by LPS in the macrophages was inhibited by 2-HF. 2-HF also prevented LPS-induced activation of protein kinases p38MAPK and SAPK/JNK. Apart from this, LPS-induced phosphorylation, nuclear translocation, and DNA-binding of the redox transcription factor, NF-κB, was prevented by 2-HF. Our results demonstrate that 2-HF by regulating ROS/MAPK/NF-κB prevents LPS-induced inflammatory response and cytotoxicity in murine macrophages suggesting the potential development of 2-HF as an anti-inflammatory agent to ameliorate various inflammatory complications.

1. Introduction.

Lipopolysaccharides (LPS) present in the cell wall membranes of Gram-negative bacteria are well known to induce a potent inflammatory response in patients with bacterial infections. Even in patients treated with antibiotics, the LPS content in the bacterial debris can circulate in the blood, which maintains residual inflammation and promotes a sustained activation of monocytes and macrophages (Alexander and Rietschel, 2001; Erridge et al., 2002; Van Amersfoort et al., 2003). The activation of macrophages leads toincreased oxidative stress and expression of various inflammatory cytokines and chemokines, which by autocrine and paracrine manner induce an inflammatory response and organ toxicity (Fujiwara and Kobayashi, 2005; Tan et al., 2016). Generally, the inflammatory response is necessary to cope against pathogens, promoting tissue repair, and phagocytosis of apoptotic cells (Bennett et al., 2018). However, uncontrolled inflammatory response due to persistent production of various inflammatory cytokines and chemokines by activated macrophages leads to the alteration of various cell signaling pathways responsible for cell proliferation, differentiation, and apoptosis which lead to the tissue dysfunction and damage (Chen et al., 2018; Nathan and Ding, 2010). Multiple studies have provided evidence that a chronic inflammatory response plays a crucial role during the development and pathogenesis of complications such as sepsis, asthma, COPD, diabetes, atherosclerosis, neurological disorders, and multiple types of cancers (Coussens and Werb, 2002; Grivennikov et al., 2010). Recent studies have demonstrated that synthetic anti-oxidants, steroids, and nonsteroidal anti-inflammatory drugs (NSAIDs) that prevent macrophage activation and cytokine production could act as potential anti-inflammatory agents and prevent various inflammatory complications (Arulselvan et al., 2016; Dinarello, 2010; Maroon et al., 2010; Rayburn et al., 2009). However, these drugs are not effective in preventing certain chronic inflammatory diseases due to their off-target side effects, and their prolonged use results in the decreased patient’s quality of life (Fürst and Zündorf, 2014; Lanas and Hunt, 2006; Sostres et al., 2010). However, plant-based antioxidants, anti-inflammatory agents, and dietary phytochemicals have shown limited side effects and toxicity (Fürst and Zündorf, 2014; Islam et al., 2016). Therefore, it imperative that phytochemicals and natural plant-based anti-inflammatory agents have the potential for use as novel anti-inflammatory agents without significant side toxicities to treat various inflammatory complications.

2-HF is a naturally isolated flavonoid from the citrus fruits, and it has been shown to be a good antioxidant with strong chemopreventive activities. Most of the recent studies have demonstrated that 2-HF exerts anti-cancer effects in various models of cancers such as lung (Awasthi et al., 2018; Hsiao et al., 2007) renal (Nagaprashantha et al., 2011) colon (Shin et al., 2012), prostate (Ofude et al., 2013; Wu et al., 2014; Wu et al., 2018) osteosarcoma (Lu et al., 2014), gastric (Zhang et al., 2015) and breast cancer (Nagaprashantha et al., 2018; Singhal et al., 2017; Singhal et al., 2018b; Singhal et al., 2019). Specifically, by inhibiting proliferation, metastasis, angiogenesis, epithelial-mesenchymal transition (EMT), and inducing apoptosis via regulation of MAPK, AKT, and PI3K, 2-HF has been demonstrated to exert anti-cancer effects (Hsiao et al., 2007; Nagaprashantha et al., 2011; Wu et al., 2014; Wu et al., 2018). Proteomic analysis of 2-HF treated ER⁺ breast cancer cells demonstrated changes in multiple regulators of cellular signaling pathways involved in breast cancer cell proliferation, DNA synthesis, antioxidant response, and transcriptional regulation (Nagaprashantha et al., 2019). 2-HF has been demonstrated to inhibit the activity of Ral interacting protein of 76 kDa (RLIP76); a multifunctional transporter protein involved in detoxification of xenobiotics including chemotherapeutic drugs (Awasthi et al., 2008; Vatsyayan et al., 2010). Both in vitro and in vivo results demonstrate that 2-HF by inhibiting RLIP76 exerted anti-proliferative effects on breast cancer cells (Nagaprashantha et al., 2018; Singhal et al., 2018a). 2-HF further inhibited RLIP76-mediated transport of chemotherapeutic drug doxorubicin from breast cancer cells, which may lead to an increase in the efficacy of the chemotherapeutic drug (Nagaprashantha et al., 2018) Combination of 2-HF with RLIP76 antibody prevented metastasis of breast cancer cells to the brain in xenograft models (Singhal et al., 2018b). Furthermore, using SENCAR mice (SENsitive to CARcinogen) model of breast cancer, Singhal et al., have demonstrated that combination treatment of tumor-bearing mice with 2-HF with either RLIP76 antibody or antisense RNA or both exert protective functions against 7,12-Dimethylbenz[a]anthracene (DMBA)-induced carcinogenesis (Singhal et al., 2019). Although these studies demonstrate the anti-cancer properties of 2-HF, the anti-inflammatory effects of 2-HF are not well known. It is important to analyze the anti-inflammatory properties of 2-HF as inflammation plays a central role in cancer progression and therapy and 2-HF might also exert anticancer effects through anti-inflammatory mechanisms. Furthermore, anti-inflammatory effects of flavonoids can be exploited for better anti-cancer effects in combination therapy for cancer including overcoming drug resistance in cancer therapy (Batra and Sharma, 2013). Apart from cancer, the anti-inflammatory effects of flavonoids can be useful for the treatment of multiple inflammatory complications (García-Lafuente et al., 2009; Ginwala et al., 2019; Pollack et al., 2016). Further, the mechanism through which 2-HF prevents inflammatory response is not known. Specifically, the effect of 2-HF in the prevention of LPS-induced inflammatory response and cytotoxicity is not reported.

Therefore, in this study, we have analyzed the anti-inflammatory roles of 2-HF using LPS-treated RAW 264.7 murine macrophages. Our results demonstrate that 2-HF significantly prevented LPS-induced inflammatory response in macrophages. 2-HF significantly prevented LPS-induced cytotoxicity, generation of ROS, production of NO, expression of various cytokines, and chemokines. Our study demonstrates that the anti-inflammatory effects of 2-HF were mediated by regulating the ROS/p38MAPK/SAPK/JNK/NF-κB pathway in murine macrophages. To the best of our knowledge, we demonstrate for the first time the anti-inflammatory effects of 2-HF in RAW 264.7 murine macrophages. These results suggest the potential scope for the development of 2-HF as an agent to prevent multiple human inflammatory complications apart from the reported chemoprevention and anticancer properties of 2-HF.

2. Materials and Methods:

2.1. Materials

2’-Hydroxyflavanone (#H3780) was purchased from Sigma Aldrich. DMEM (#11965092), Trypsin EDTA (#25200–056), Phosphate Buffered Saline (#21–030-CV), and Annexin-V/Propidium Iodide (#V13241) were obtained from Thermo Fisher Scientific (Invitrogen). Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) (#M2128), Griess reagent (#G4410), and lipopolysaccharides (LPS) (#L4130) were obtained from Sigma Aldrich. TMRE (#21426), nuclear extraction kit (#10009277), and NF-κB transcription factor assay kit (#10007889) were obtained from Cayman chemicals. Antibodies against NF-κB (p65) (#3033, dilution 1:1000), p-p38MAPK (#4511, dilution 1:1000), p38MAPK (#9212, dilution 1:1000), p-SAPK/JNK (#4668, dilution 1:1000), SAPK/JNK (#9252, dilution 1:1000), p-ERK1/2 (#4370, dilution 1:1000), ERK1/2 (#4695, dilution 1:1000), β-actin (#4970, dilution 1:5000), and GAPDH (#2118, dilution 1:5000) were obtained from Cell Signaling Technologies. Anti-rabbit secondary IgG antibodies (#170–6515, dilution 1:10000) and anti-mouse IgG (#170–6516, dilution 1:10000) were obtained from Bio-Rad. All other reagents and chemical used were of analytical grade and were obtained from either Fisher Scientific or Sigma Aldrich.

2.2. Cell culture

RAW 264.7 (# TIB-71) murine macrophages and human THP-1 monocytes (#TIB-202) were obtained from ATCC and were grown in Dulbecco’s modified eagle medium (DMEM high glucose #11965092) and RPMI-1640 (#11875093) respectively supplemented with 10% fetal calf serum and 1% penicillin/streptomycin. The cells were maintained at 37° C in a humidified atmosphere of 5% CO₂. The cells obtained are certified free of contaminants and mycoplasma by ATCC and no further confirmatory tests were performed in the laboratory. All experiments were carried out within 6–8 cell passage numbers after thawing.

2.3. Measurement of cytotoxicity

RAW 264.7 macrophages were seeded onto 96-well plates (5000 cells/cm²) and allowed to adhere overnight. The cells were then treated with different concentrations of 2-HF (10, 20, 30 & 50 μM) for 24 h and 48 h. For LPS-treatments, the cells were pre-treated overnight with the indicated concentrations of 2-HF followed by stimulation with LPS (1μg/mL) for 24 h and 48 h. At the end of the incubation period, 10 μL of 5 mg/mL MTT was added to the wells, incubated for 2 h and the formazan crystals were dissolved with DMSO and absorbance was recorded at 550 nm using a microplate reader (BioTek, Synergy2). For Annexin/Propidium Iodide (PI) staining and analysis by flow cytometry, after 24 h of treatment with LPS ± 2-HF, the cells were harvested by trypsinization and washed with Annexin V/PI binding buffer followed by incubation with Annexin V and PI for 30 min. The cells were then washed with Annexin-V/PI binding buffer and analyzed by BD LSRII flow cytometer. Data was analyzed by Flow Jo software.

2.4. Measurement of oxidative stress

RAW 264.7 murine macrophages were seeded onto 6-well plates (5000 cells/cm²) and allowed to adhere overnight. The cells were then pre-treated with different concentrations of 2-HF overnight in 0.1% serum-containing media followed by stimulation with LPS (1μg/mL) for 24 h. After 24 h incubation, the cells were harvested by trypsinization and used to assay ROS or lipid peroxidation. Briefly, for ROS, the cells were stained with CMH2DCFDA dye (5 μM) for 30 min and analyzed with a flow cytometer.

For assay of lipid peroxidation, the cells were stained with 10 μM lipid peroxidation senor dye provided with Image–iT Lipid peroxidation kit (Invitrogen) and analyzed by BD LSRII flow cytometer. Fluorescence was recorded using 581/591nm (Ex/Em) (Texas red) and 488/510 nm (Ex/Em) (FITC) filters and the ratio of fluorescence intensities at 590/510 was determined using Flow Jo software which provides a readout for lipid peroxidation. The lipid peroxidation sensor dye BODIPY 581/591 C11 reagent upon oxidation in live cells shifts fluorescence from red (590) to green (510) and the ratio of 590/510 is inversely proportional to lipid peroxidation in cells.

For nitric oxide (NO) estimation, media obtained from the treatment groups were used for NO assay using Griess reagent (Sigma Aldrich) as reported earlier (Shukla et al., 2018). Briefly, equal amounts of freshly prepared Griess reagent (40 mg/mL) and media were incubated for 15 min at RT followed by recording the absorbance at 540 nm using a Synergy 2 microplate reader.

2.5. Analysis of mitochondrial membrane potential

Mitochondrial membrane potential was analyzed with Tetramethylrhodamine, Ethyl Ester, Percolate dye (TMRE) (Cayman chemicals). Cells were pre-treated with the indicated concentrations of 2-HF overnight in 0.1% serum-containing media followed by stimulation with LPS (1 μg/mL) for 24 h. After 24 h incubation, the cells were harvested by centrifugation and incubated with 100 nM TMRE dye for 30 min. After 30 min, TMRE fluorescence was recorded using a flow cytometer (Ex/Em: 544/590). The percentage of TMRE positive and negative cells in each condition was analyzed by Flow Jo software.

2.6. Western blot analysis

After the indicated treatments, the cells were collected using a scrapper, centrifuged, washed with ice-cold PBS, and lysed with RIPA buffer containing phosphatase and protease inhibitors. Equal amounts of cell lysates were loaded and separated by 12% SDS-PAGE, followed by the transfer of proteins on to PVDF membranes. The membranes were then blocked with 5% non-fat dried milk and incubated with the specific antibodies overnight. The next day, the membranes were washed, incubated with specific secondary antibodies. Immunolabeling was detected using a SuperSignal West Pico chemiluminescent substrate (#34078, Thermo Scientific). For re-probing with different antibodies or loading control, the membranes were stripped with Restore Plus stripping buffer (#46430, Thermo Scientific).

2.7. Analysis of inflammatory cytokines

RAW 264.7 murine macrophages or human THP-1 monocyteswere pre-treated with 2-HF overnight in 0.1% serum-containing media. The next day, the cells were stimulated with 1μg/mL LPS in 0.1% serum-containing media for 24 h. After 24 h treatment, an equal amount of media was collected, cleared by centrifugation to remove debris and stored in −80°C. The media was then concentrated 10x using a vacuum evaporator (Savant SC210A, Thermo scientific) and 25 μL media from each condition was used in the assay. The media was incubated with the labeled magnetic beads provided with the mouse multiplex (#MCYTOMAG-70K-PMX) or human multiplex (#HCYTOMAG-60K) cytokine chemokine kit from Millipore Sigma. After incubating overnight at 4°C, the beads were washed twice with wash buffer, incubated with detection antibodies, and counterstained with Streptavidin PE. Acquisition and analysis were carried out using a Luminex analyzer from Millipore and data are presented as pg/mL based on the standard curve generated using the standards provided with the kit.

2.8. Statistical analysis

Data are presented as mean±SD and p-values were determined by student’s t-test using Microsoft office excel or GraphPad Prism software. p < 0.05 was considered as statistically significant.

3. Results:

3.2. Effect of 2’-Hydroxyflavanone on LPS-induced macrophage cytotoxicity

Since the effect of 2-HF on the LPS-induced macrophage cytotoxicity is not known, therefore, we first examined the effect of 2-HF on the viability of LPS treated RAW 264.7 macrophages. Macrophages were treated with different concentrations of 2-HF for 24 h and 48 h and the cell viabilities were determined by MTT assay. Results shown in Fig. 1A, demonstrate that RAW 264.7 macrophages treated with different concentration of 2-HF for 48 h did not induce any observable changes in cell viability. Similarly, no significant differences in the cell viability were observed after 24 h when macrophages were incubated with different concentrations of 2-HF (data not shown). Further, incubation of RAW 264.7 macrophages with LPS for 24 h and 48 h induced a significant decrease in the cell viability (Fig. 1B&1C). MTT assay did not show any observable difference in exerting protection by 2-HF against LPS-induced decrease in cell viability at 24 h time-period. However, at 48 h incubation with 30 μM and 50 μM 2-HF prevented LPS-induced decrease in the cell viability (Fig. 1C). We further confirmed the effect of 2-HF on RAW 264.7 cell death by Annexin-V/PI staining by flow cytometry. Exposure to LPS induced a significant increase in PI-positive cells and pre-treatment with 2-HF prevented it (Fig. 1D&E). These results demonstrate that 2-HF exerted protective functions against LPS-induced cytotoxicity in RAW 264.7 macrophages.

Figure.1: Effect of 2’-Hydroxyflavanone (2-HF) on cell viability:

Serum starved RAW 264.7 murine macrophages were treated with different concentrations of 2-HF (10, 20, 30, 50 μM) for 24 h and 48 h either alone or in combination with LPS (1 μg/mL) in 0.1% serum-containing media and cell viability was determined by MTT assay. Bars showing MTT absorbance values after (A) 48 h treatment with different concentrations of 2-HF. MTT assay showing the effect of 2-HF on LPS induced a decrease in cell viability after (B) 24 h and (C) 48 h of the indicated treatments. RAW 264.7 macrophages were pre-treated with the indicated concentrations of 2-HF (20, 30, 50 μM) overnight in 0.1% serum-containing media followed by incubation with LPS (1 μg/mL) for 24 h. Flow cytometry analysis of Annexin-V/PI stained RAW 264.7 cells after 24 h of indicated treatments was done. (D) Percentage of propidium iodide positive cells in the indicated treatments (E) Representative dot plots showing Annexin-V/PI positive stained RAW 264.7 cells after the indicated treatments. Representative data from 3-independent experiments is shown. Bars represent mean±SD (n=3). **p<0.005 vs control; #p<0.05, ##p<0.005 vs LPS treated.

3.3. Effect of 2’-Hydroxyflavanone on LPS-induced oxidative stress

Since LPS induced oxidative stress plays an important role in mediating cellular signaling responses, we next examined the effect of 2-HF on LPS-induced ROS production. Our results shown in Fig. 2A and 2B demonstrate that LPS-induced ROS production in RAW 264.7 macrophages was significantly prevented by 2-HF pretreatment in a dose-dependent manner. Since ROS initiate lipid peroxidation and increased lipid peroxidation is a marker for oxidative stress, we next measured the effect of 2-HF on LPS-induced lipid peroxidation in murine macrophages. Results shown in Fig. 3A indicate that LPS induces lipid peroxidation in macrophages and 2-HF prevents it. Since NO has been shown to cause macrophage cytotoxicity, we next examined the effect of 2-HF on LPS-induced NO levels in macrophages. Treatment of macrophages with LPS-induced NO release in the culture media and 2-HF pretreatment prevented LPS-induced release of NO in a dose-dependent manner (Fig. 3B).

Figure.2: Effect of 2’-Hydroxyflavanone (2-HF) on LPS-induced ROS production:

RAW 264.7 murine macrophages were pre-treated with different concentrations of 2-HF (20, 30, 50 μM) overnight in 0.1% serum-containing media followed by incubation with LPS (1 μg/mL) for 24 h. To determine ROS levels, the cells were stained with CMH2DCFDA dye (5 μM) and analysis was done by flow cytometry. (A) Histograms showing CMH2DCFDA fluorescence intensity in RAW 264.7 macrophages treated with LPS alone or in combination with 2-HF. (B) Bars showing the geometric mean of fluorescence intensity (MFI) of the indicated treatments shown in Fig. 2A. Bars are mean±SD (n=3). Representative data is shown. **p<0.005 vs control; #p<0.05, ##p<0.005vs LPS treated.

Figure.3: Effect of 2’-Hydroxyflavanone (2-HF) on LPS-induced lipid peroxidation, NO production, and mitochondrial membrane potential:

RAW 264.7 murine macrophages were pre-treated with different concentrations of 2-HF (20, 30, 50 μM) overnight in 0.1% serum-containing media followed by incubation with LPS (1 μg/mL) for 24 h. (A) Bars showing the ratio of red (590)/green (510) fluorescence ratios in RAW 264.7 macrophages after the indicated treatments assayed using Image-iT lipid peroxidation assay kit from Invitrogen. The ratio of red (590)/green (510) is inversely proportional to lipid peroxidation. (B) Nitric oxide levels in RAW 264.7 cell culture media assayed by Griess reagent after 24 h of indicated treatments. To determine mitochondrial membrane potential, the cells were stained with TMRE dye, and analysis was done by flow cytometry. (C) Histograms showing TMRE positive and negative cells after the indicated treatments. Bars showing quantification of TMRE positive and TMRE negative cells as determined in C. Bars represent mean±SD. Representative data from 3-independent experiments is shown. *p<0.05 **p<0.005 vs control; #p<0.05, ##p<0.005vs LPS treated.

3.4. Effect of 2’-Hydroxyflavanone on LPS-induced mitochondrial membrane potential.

An increase in ROS and NO induces damaging effects in multiple cellular organelles including mitochondria in the cells. Therefore, we examined the effect of 2-HF in the prevention of LPS-induced mitochondrial membrane potential in RAW 264.7 macrophages. Flow cytometry analysis of TMRE stained cells demonstrated that LPS exposure leads to a loss of mitochondrial membrane potential and pre-treatment with 2-HF prevented it. Exposure to LPS caused a significant decrease in TMRE retention capacity as evidenced by the decrease in TMRE positive cells, which is an indicator of loss in mitochondrial membrane potential and pre-incubation with different concentrations of 2-HF significantly prevented it (Fig. 3C).

3.5. Effect of 2’-Hydroxyflavanone on LPS-induced expression of inflammatory cytokines and chemokines

Our results described in the previous section demonstrate that 2-HF inhibited LPS induced ROS production and lipid peroxidation. These results indicate that 2-HF exerts anti-oxidative and anti-inflammatory functions. Therefore, we next analyzed the expression of multiple pro-inflammatory cytokines in culture media obtained from LPS-treated RAW 264.7 macrophages either alone or in combination with 2-HF using a multiplex kit from Millipore. Results shown in Table-1 demonstrate that 2-HF significantly prevented LPS induced release of inflammatory cytokines and chemokines in RAW 264.7 macrophages. Exposure to LPS induced a significant increase in the expression of multiple pro-inflammatory cytokines as observed in Table-1. Pre-treatment with 2-HF led to a significant decrease in the levels of eotaxin, IL-2, IL-10, IL-12p40, LIX, IL-15, IL-17, MCP-1, and TNF-α in LPS treated cells (Table-1). These results demonstrate that 2-HF exert potent-anti-inflammatory properties. To examine, if 2-HF also demonstrates anti-inflammatory actions in human cell lines, we have also examined the effect of 2-HF on LPS - induced release of various inflammatory cytokines and chemokines in human THP-1 monocytes. Our data shown in Table-2 indicate that similar to the results observed with the murine macrophages, 2-HF also prevented the LPS-induced inflammatory response in human cells (Table- 2).

Table-1. Effect of 2’-Hydroxyflavanone (2-HF) on LPS-induced activation of inflammatory cytokines and chemokines in RAW 264.7 murine macrophages:

RAW 264.7 murine macrophages were pre-treated with 2-HF (30 μM) overnight in 0.1% serum-containing media followed by incubation with LPS (1 μg/mL) for 24 h. The culture media was collected and centrifuged to remove debris and stored in −80°C. Thereafter, the media was concentrated 10x using a vacuum evaporator and was used to assay the levels of cytokines using a multiplex mouse cytokine/chemokine kit from Millipore Sigma. Table showing the effect of 2-HF on LPS-induced release of cytokines in RAW 264.7 culture media after the indicated treatments (n=3).

	Control	2-HF	LPS	LPS+2-HF
G-CSF	83.6±15.06	50.3±4.8	26413.3±904.9**	26846.3±585.9
Eotaxin	5.1±0.4	3.6±0.5	30.1±1.2**	13.2±2.8#
GM-CSF	63.2±7.5	39.7±12.4	257.2±10.9**	339.7±62.3
IFNγ	8.5±0.3	4.0±1.7	27.2±2.2**	28.5±3.6
IL-1α	67.3±1.5	35.1±5.8	865.7±48.2**	2610.7±215.8
IL-β	43.4±2.3	81.5±13.4	1271.6±39.8**	1540.9±266.6
IL-2	8.9±0.9	7.5±1.3	163.1±12.8**	39.6±7.5#
IL-4	1.1±0.1	0.7±0.1	3.4±0.3**	2.5±0.2
IL-3	0.9±0.2	1.1±0.5	4.4±2.3**	4.8±0.1
IL-5	10.2±0.1	3.1±1.5	29.6±3.5*	30.0±4.9
IL-6	9.04±1.1	6.7±2.2	21679.3±75.5**	21277.3±385.7
IL-9	0.4±0.06	2.2±1.3	5.7±0.9**	7.6±0.5
IL-10	1380.7±41.2	1249.8±90.7	65044±275.1**	2156.8±460.9#
IL-12 (p40)	20.8±1.7	9.3±3.1	219.3±16.8**	146.4±9.1#
IL-12 (p70)	8.04±1.4	2.5±1.2	56.6±6.6**	40.6±11.7
LIF	6.2±1.6	3.5±1.6	36.4±2.3**	31.7±2.6
IL-13	7.2±0.3	3.5±0.4	2229.3±215.3**	2339.1±594.8
LIX	7.3±9.2	8.1±0.9	91.5±0.4**	35.3±5.6#
IL-15	3320.1±1247.3	840.5±367.4	>10000**	8465.7±610.6#
IL-17	23.2±9.9	13.5±10.5	251.5±5.06**	139.8±25.2#
IP-10	10.6±1.1	3.8±0.9	21.7±1.1*	17.1±0.9
MCP-1	25.7±0.7	21.2±1.8	1030.1±34.8**	768.6±122.9#
MIP-2	60.7±12.2	29.2±10.6	196.7±15.4**	293.0±107.2
RANTES	10.0±5.0	9.6±7.1	258.2±17.1**	199.4±46.6
TNF-α	11.9±1.0	10.9±0.8	136.7±30.3**	28.2±5.4#

^**p<0.005 vs untreated control;

^#p<0.05 vs LPS treated.

The values show the levels of cytokines in pg/mL.

ND=not detected.

Table- 2. Effect of 2’-Hydroxyflavanone (2-HF) on LPS-induced activation of inflammatory cytokines and chemokines in THP1 monocytes:

Serum starved THP-1 monocytes were stimulated with LPS (1 μg/mL) alone or in combination with 2-HF (30 μM) overnight. After treatment, the cell culture media was collected, centrifuged to remove debris, and stored at −80°C. The media was concentrated 10x using a vacuum evaporator and 25 μL was used to assay the levels of cytokines and chemokines using a human magnetic multiplex cytokine/chemokine kit (#HCYTOMAG-60K) from Millipore Sigma. Table showing the concentration of various cytokines (pg/mL) in THP-1 cell culture media after the indicated treatments (n=3). Values are mean±SD.

	Control	2-HF	LPS	LPS+2-HF
EGF	9.9±0.4	10.4±0.9	31.1±1.01**	20.1±0.8#
FGF-2	46.7±3.8	52.8±4.9	135.1±8.1**	102.7±4.3#
Eotaxin	ND	ND	22.8±1.4**	19.8±0.9
TGF-α	3.8±0.2	3.4±0.2	13.5±1.1**	8.7±0.4#
G-CSF	ND	ND	201.3±11.2**	78.3±5.7##
Flt-3L	4.3±0.8	4.8±0.6	27.1±2.9**	22.1±3.2
GM-CSF	ND	5.1±0.4	142.0±4.4**	223.5±10.7#
Fractalkine	19.2±6.1	42.0±1.5	688.6±57.2**	374.5±41.7#
IFNα2	13.7±2.8	21.3±1.0	188.9±14.6**	130.5±8.1#
IFNγ	1.5±0.5	2.7±1.3	43.5±1.5**	28.4±3.7
IL-10	3.7±0.2	3.7±0.6	1871.2±76.9**	457.5±39.9##
MCP-3	7.1±2.7	5.8±2.4	2549.1±92.6**	224.3±6.3##
IL-12p40	2.9±0.3	4.1±1.0	36343.5±12756.4*	2113.9±76.6
MDC	349.8±11.8	204.5±6.0	14437.7±970.4**	1116.5±5.5##
IL-12p70	ND	ND	43.8±2.3**	14.1±2.7##
PDGF-AA	228.5±17.1	245.6±6.9	5665.6±573.1**	5885.7±651.5
IL-13	ND	ND	9.2±1.8*	5.03±1.04
PDGF-AA/BB	11.2±1.2	15.1±2.8	44.1±7.8*	32.07±6.1
IL-15	0.8±0.2	1.2±0.5	11.2±1.5*	5.8±0.6
sCD40L	ND	ND	3.4±0.1	2.5±0.9
IL-17α	ND	ND	4.2±0.9	2.03±0.3
IL-1RA	75.06±1.8	84.6±3.8	2435.8±138.7**	736.7±68.2##
IL-la	ND	ND	36.8±2.4**	30.8±2.5
IL-9	ND	ND	6.8±1.02*	4.7±1.2
IL-1β	ND	ND	2342.4±120.7**	200.5±18.2##
IL-6	ND	ND	2932.5±258.8**	218.5±21.2##
IL-7	7.7±0.5	9.6±1.6	57.4±4.07**	43.1±6.2
IL-8	31.3±2.3	81.2±4.2	10531.2±510.7**	10398.8±253.2
IP-10	508.1±10.7	98.8±5.5	11463.5±281.7**	10910.5±104.4
MCP-1	139.4±7.7	743.9±21.4	10018.2±521.6**	10212.7±402.6
MIP-lα	66.1±4.5	75.1±1.3	4700.2±254.3**	4724.5±235.1
MIP-1β	63.5±5.2	41.4±3.5	52978.3±9225.4**	18781.3±2434.3#
TNF-α	100.5±2.9	133.0±6.8	7267.4±502.6**	2575.7±211.1##
TNF-β	ND	ND	2.9±1.2	1.06±0.5
VEGF	2833.9±331.6	4096.3±208.9	9389.1±1214.2*	7435.2±213.5

^*p<0.05,

^**p<0.005 vs untreated control;

^#p<0.05 #p<0.005 vs LPS-treated.

ND=Not detected

3.6. Effect of 2’-Hydroxyflavanone (2-HF) on LPS-induced activation of signaling pathways

To decipher the signaling pathways which are modulated by 2-HF that lead to anti-inflammatory effects of 2-HF, we next examined if 2-HF prevented LPS-induced activation of NF-κB. Western blot analysis of nuclear extracts obtained from RAW 264.7 macrophages stimulated with LPS-alone or in combination with 2-HF (30 μM) demonstrate that LPS-induced nuclear translocation of phosphorylated NF-κB was prevented by 2-HF (Fig. 4A). Further analysis of the DNA-binding activity of nuclear extracts using an NF-κB transcription factor binding assay suggest that LPS-induced NF-κB-DNA binding was inhibited by 2-HF treatment (Fig. 4B).

Figure.4: Effect of 2’-Hydroxyflavanone (2-HF) on LPS-induced activation of signaling pathways:

RAW 264.7 murine macrophages were pre-treated with 30 μM 2-HF overnight in 0.1% serum-containing media followed by incubation with LPS (1 μg/mL) for the indicated periods (0, 15, 30, 60, 120 min).To determine the effect of 2-HF on NF-κB, after the indicated treatments, nuclear extracts were prepared using a kit from Cayman chemicals and nuclear lysates were subjected to western blotting or ELISA to quantify NF-κB activity. (A) Western blotting of nuclear extracts showing the effect of 2-HF on LPS-induced nuclear translocation of phosphorylated NF-κB. (B) NF-κB–DNA binding activity was measured by using an NF-κB-transcription factor assay kit. (C-E) Western blot showing expression of phosphorylated and total p38MAPK, ERK and SAPK/JNK in whole-cell lysate treated with LPS (1 μg/mL) alone or in combination with 2-HF for the indicated time. β-actin was used as a loading control. Bars represent the quantification of data. Values are mean±SEM. Representative data from 3-independent experiments is shown. *p<0.05 **p<0.005 vs control; #p<0.05 vs LPS treated.

Since protein kinases such as MAPK activates NF-κB, we next analyzed the expression of p38MAPK, ERK, and SAPK/JNK in RAW 264.7 macrophages treated with LPS ± 2-HF. Our results shown in Fig. 4C–D demonstrate that LPS-induced activation of p38MAPK and SAPK/JNK was prevented by 2-HF. Although LPS induces a significant increase in phosphorylation of ERK1/2, 2-HF did not inhibit ERK1/2 phosphorylation. Thus, our results suggest that 2-HF protects macrophages from LPS-induced cytotoxicity by regulating MAPK/NF-κB signaling pathways.

4. Discussion:

Inflammation plays a significant role in maintaining normal physiological functions including conferring protection against infections, pathogens, getting rid of damaged cells, and in tissue repair and regeneration (Oishi and Manabe, 2018; Prame Kumar et al., 2018). Apart from normal physiological functions, inflammation has a major role in aging and age-associated complications and multiple otherhuman pathological complications (Rea et al., 2018). However, excess or uncontrolled inflammation has been recognized as a driver of pathological complications associated with diseases such as obesity, autoimmune disorders, cardiovascular complication, colitis and different types of cancers (Ferrucci and Fabbri, 2018; Hunter, 2012; Rajendran et al., 2018; Rea et al., 2018). In the present study, we have investigated the anti-inflammatory effects of 2’-Hydroxyflavanone (2-HF) in LPS stimulated RAW 264.7 macrophages. Our results demonstrate that 2-HF prevents LPS-induced ROS production, loss of mitochondrial membrane potential and activation of kinases and transcription factors such as p38 MAPK, SAPK/JNK and NF-κB, thus preventing the release of inflammatory cytokines and chemokines and apoptosis of macrophages. The anticancer effects of 2-HF has been detailed in multiple studies (Nagaprashantha et al., 2019; Nagaprashantha et al., 2018; Nagaprashantha et al., 2011; Singhal et al., 2018a; Singhal et al., 2017; Singhal et al., 2018b; Wu et al., 2018) but the anti-inflammatory properties of 2-HF is not known. Although in a preliminary report by Patel et al. experimental and molecular docking studies on 2-HF derivatives demonstrated the inhibitory effects of multiple derivatives of 2-HF on NO production, IL-1β and TNF-α secretion in macrophages (Patel et al., 2015), our study is the first report providing a detailed description of the anti-inflammatory effects of 2-HF.

Amongst the cells of the immune system, macrophages are important players in mediating inflammatory response in the body (Oishi and Manabe, 2018). Apart from the macrophages, other cells of the immune system such as monocytes, Tregs, MDSC, T and B-cells are also activated in response to pathogenic stimuli and play a central role in regulating inflammation (Ong et al., 2018). Moreover, studies have provided evidence that activated or inflamed monocytes can migrate to the sites of inflammation, which further potentiates the inflammatory response. The activated monocytes have the potential to differentiate into macrophages and modulate multiple cellularregulatory processes in the sites of inflammation (Evans et al., 2009; Parisi et al., 2018; Yang et al., 2014).

The interplay of multiple cytokines, chemokines, and growth factors induces activation of macrophages to a pro-inflammatory or anti-inflammatory phenotype and each subset has been demonstrated to exert distinct and specific roles in multiple human pathologies (Parisi et al., 2018; Turner et al., 2014). The activated or inflamed macrophages play a critical role in regulating chronic low-grade inflammation during obesity, which has important implications in obesity-associated insulin resistance and diabetic progression (Lauterbach and Wunderlich, 2017). Further, macrophages play an important role in atherosclerosis and the development of atherosclerotic plaque. In atherosclerotic plaques, an abundance of classically activated or pro-inflammatory macrophages have been reported, which leads to the development of an inflammatory niche and regulate plaque stability and macrophage foam cell formation. These cells are also an abundant source of inflammatory cytokines such as IL-1β, TNF-α, IL-12, and IL-18, which further contributes to enhancing inflammation(Dickhout et al., 2008; Moore et al., 2013; Tang et al., 2015). Inhibiting macrophage proliferation and preventing pro-inflammatory macrophage accumulation has been demonstrated to suppress atherosclerotic plaque formation (Tang et al., 2015). In fact, in regressing atherosclerotic plaques, an abundance of alternatively activated or anti-inflammatory macrophages has been reported, which promotes tissue repair and by secreting anti-inflammatory cytokines such as IL-10 and TGF-β (Peled and Fisher, 2014; Rahman et al., 2017). Apart from this, in cancer, the inflammatory or activated macrophages in the tumor microenvironment have been reported to play pro-tumoral roles and promote cancer cell proliferation, migration and metastasis, angiogenesis and modulate response to chemotherapy (Aras and Zaidi, 2017; Mantovani et al., 2017). Recent studies also provide evidence that macrophages play an important role during immunotherapy for cancer (Cassetta and Kitamura, 2018; DeNardo and Ruffell, 2019). Multiple strategies to target macrophages have been highlighted in the recent past which may help modulate disease progression and potentiate therapeutic response. Targeting inflammatory monocytes and macrophages have provided evidence that inhibiting inflammation is a key step in ameliorating disease (Ponzoni et al., 2018). Owing to their limited toxic side effects, various dietary and plant-based polyphenols and flavonoids including natural anti-oxidants have attained attention in the recent past as preferred agents to target inflammatory macrophages in ameliorating human pathologies (Pandey and Rizvi, 2009; Santhakumar et al., 2018; Saqib et al., 2018). However, some concerns with potential toxicity and unwanted side-effects remain (Galati and O’Brien, 2004), and hence proper understanding of the mechanisms of action and toxicity of these compounds are warranted. In this study, we observed that 2-HF did not affect the viability of RAW 264.7 macrophages and 2-HF prevented LPS-induced decrease in cell viability to a significant extent. Thus 2-HF can be deemed safe. Moreover, prior studies described earlier using 2-HF in vivo, reported no toxic effects of 2-HF in vivo.

Oxidative stress is a driver of pathological complications and multiple signaling pathways are activated by ROS, including toxicity to macromolecules such as mitochondria (Ježek et al., 2018). ROS activate multiple signaling cascades leading to the generation of inflammatory cytokines and growth factors, and pro-inflammatory programming of cells. We have observed that 2-HF exert potent anti-oxidative effects in response to LPS-induced oxidative stress. Hence, the ability of 2-HF to inhibit LPS-induced ROS production could be a major step in mediating the anti-inflammatory activities of 2-HF. In consistence with this study, several other studies using plant-based flavonoids and phytochemicals have also demonstrated that inhibition of ROS and NO exerts cytoprotective functions (Hussain et al., 2016; Kuo et al., 2020; Nijveldt et al., 2001). Further, we observed that 2-HF prevents LPS-induced lipid peroxidation and loss of mitochondrial membrane potential. Lipid peroxidation -derived products such as HNE, 4-HHE, MDA, and acrolein generated by oxidative damage to lipids have been shown to be more stable than ROS and propagate the toxic effects of ROS in cells (Negre-Salvayre et al., 2008). Inhibition of lipid peroxidation end-products and their detoxification is a major step in preventing oxidative stress and inflammation-induced side effects in multiple human inflammatory pathologies (Barrera, 2012). Loss of mitochondrial membrane potential is also a major event in ROS-induced toxicity in cells. An increase in mitochondrial ROS along-with loss of mitochondrial membrane potential and cytochrome C release initiates apoptotic signaling pathways in the cells (Yue and Yao, 2016). Thus, by inhibiting ROS, NO, and preventing loss of mitochondrial membrane potential, 2-HF could exert protective functions during LPS-induced cytotoxicity in RAW 264.7 macrophages.

NF-κB is a master regulator of the inflammatory response and transcribes multiple genes responsible for regulating inflammation in various human diseases (Lawrence, 2009; Liu et al., 2017). Multiple studies have shown that inflammatory response in macrophages including various other immune cells is regulated by NF-κB (Dorrington and Fraser, 2019; Liu et al., 2017). Inhibition of NF-κB is a target for the anti-inflammatory effects of antioxidants and plant based-polyphenols (Gasparrini et al., 2018). NF-κB further plays an important role in the polarization of macrophages to pro-inflammatory or anti-inflammatory phenotype, which has important implications in regulating inflammation in pathologies such as cancer, arthritis, obesity, and atherosclerosis (Biswas and Lewis, 2010; Liu et al., 2017; Tugal et al., 2013). Furthermore, ROS or external stimuli-induced activation of NF-κB also leads to nitric oxide (NO) production, which induces oxidative stress (Jones et al., 2007). Our results demonstrate that LPS induced activation and nuclear translocation of NF-κB including DNA binding is prevented by 2-HF. Various protein kinases such as MAPK, Src, AKT, and JNK are activated upon oxidative stress which act as upstream signaling events in the activation of transcription factors such as NF-κB and AP1. Further activation of MAPK and AKT has been demonstrated to regulate multiple cellular signaling pathways playing a significant role in pathological complications (Kim and Choi, 2010; Lu and Xu, 2006). Several plant-derived antioxidants have been shown to prevent the activation of MAPKs and preventcellular toxicity (Qi et al., 2018; Upadhyay and Dixit, 2015). Similarly, we have observed that LPS-induced activation of p38MAPK and SAPK/JNK in macrophages. Thus, by preventing upstream protein kinases, 2-HF could prevent LPS-induced NF-κB activation and expression of various inflammatory cytokines, which are involved in the immune response as well as cytotoxicity. In summary, our study suggests that 2-HF prevents LPS-induced inflammatory response and cytotoxicity by inhibiting signaling through ROS/MAPK/NF-κB in RAW 264.7 macrophages. Our study demonstrates a novel anti-inflammatory role for the potent anticancer flavonoid, 2-Hydroxyflavaonone, which will open new avenues to investigate the immunomodulatory potential of this compound in multiple pathologies.

Highlights:

1. 2-hydroxyflavanone (2-HF) prevents LPS –induced apoptosis of murine macrophages.

2. 2-HF prevents LPS-induced generation of ROS, NO and lipid peroxidation.

3. 2-HF reverses the LPS-induced decrease in mitochondrial membrane potential.

4. 2-HF prevents LPS-induced activation of protein kinases: p38MAPK and SAPK/JNK.

5. 2-HF prevents LPS-induced activation of NF-κB and inflammatory response.

Abbreviations:

2-HF

2’-Hydroxyflavanone

4-HHE

4-hydroxy Hexenal

DMBA

7,12-Dimethylbenz[a]anthracene

DMEM

Dulbecco’s Modified Eagle’s medium

DNA

Deoxyribonucleic acid

EMT

Epithelial-mesenchymal transition

ERK

Extracellular signal-regulated kinase

FITC

Fluorescein isothiocyanate

HNE

4-Hydroxynonenal

IL

Interleukin

LPS

Lipopolysaccharides

MAPK

Mitogen-activated protein kinase

MCP-1

Monocyte Chemoattractant Protein −1

MDA

Malondialdehyde

MDSC

Myeloid-derived suppressor cells

MTT

Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide

NF-κB

Nuclear Factor kappa-light-chain-enhancer of activated B cells

NO

Nitric Oxide

NSAID

Nonsteroidal anti-inflammatory drugs

PI3K

Phosphoinositide 3-kinases

PVDF

Polyvinylidene fluoride or polyvinylidene difluoride

RLIP76

Ral interacting protein of 76 kDa

RNA

Ribonucleic acid

ROS

Reactive Oxygen Species

SAPK/JNK

Stress-activated protein kinases /Jun amino-terminal kinases

TGF-β

Transforming growth factor β

TMRE

Tetramethylrhodamine, Ethyl Ester, Percolate dye

TNFα

Tumor necrosis factor α