Rubi Fructus Water Extract Alleviates LPS-Stimulated Macrophage Activation via an ER Stress-Induced Calcium/CHOP Signaling Pathway

Despite the availability of antibiotics and vaccines, many intractable infectious diseases still threaten human health across the globe. Uncontrolled infections can lead to systemic inflammatory response syndrome and the excessive production of inflammatory cytokines, known as a cytokine storm. As cytokines also play necessary and positive roles in fighting infections, it is important to identify nontoxic and anti-inflammatory natural products that can modulate cytokine production caused by infections. Rubi Fructus, the unripe fruits of Rubus coreanus Miquel, are known to possess antioxidative properties. In this study, the effect of the water extract of Rubi Fructus (RF) on the lipopolysaccharide (LPS)-induced inflammatory response in RAW 264.7 macrophages was investigated using biochemical and cell biology techniques. Our data indicated that RF inhibits p38 phosphorylation, intracellular calcium release, and the production of nitric oxide (NO), interleukin (IL)-6, monocyte chemotactic activating factor (MCP)-1, tumor necrosis factor (TNF)-α, leukemia inhibitory factor (LIF), lipopolysaccharide-induced CXC chemokine (LIX), granulocyte-colony stimulating factor (G-CSF), granulocyte macrophage colony-stimulating factor (GM-CSF), vascular endothelial growth factor (VEGF), macrophage colony-stimulating factor (M-CSF), macrophage inflammatory protein (MIP)-1α, MIP-1β, MIP-2, and regulated on activation, normal T cell expressed and secreted (RANTES) in LPS-treated macrophages. In addition, we observed decreasing mRNA expression of Chop, Camk2a, Stat1, Stat3, Jak2, Fas, c-Jun, c-Fos, Nos2, and Ptgs2 without cytotoxic effects. We concluded that RF demonstrated immunoregulatory activity on LPS-stimulated macrophages via an endoplasmic reticulum (ER) stress-induced calcium/CCAAT-enhancer-binding protein homologous protein (CHOP) pathway and the Janus kinase (JAK)/signal transducers and activators of transcription (STAT) pathway.


Introduction
Inflammatory reactions in response to pathogenic infections are essential for human survival [1]. These inflammatory cascades are regulated by immune cells, such as neutrophils, monocytes, macrophages, dendritic cells, eosinophils, basophils, T-lymphocytes, and B-lymphocytes [2]. Among these cell types, macrophages are one of the major regulators of the innate immune system response to infectious pathogens [3]. Macrophages are well known to identify and destroy intrusive microorganisms

Effects of RF on NO Production in RAW 264.7 Macrophages Stimulated with LPS
The concentration of NO in culture medium was determined using a Griess reaction assay. Specifically, after incubation of cells in 96-well plates (1 × 10 4 cells/well) for 24 h with LPS (1 µg/mL) and RF, 100 µL of supernatant from each well was collected and mixed with 100 µL of Griess reagent in a new 96-well plate. After an incubation of 15 min at room temperature, OD was determined at 540 nm with a microplate reader (Bio-Rad) [15].

Effects of RF on Intracellular Calcium Release in RAW 264.7 Stimulated by LPS
After RAW 264.7 cells were seeded in 96-well plates (1 × 10 5 cells/well), LPS (1 µg/mL) and RF were added to the culture medium and incubated for 18 h at 37 • C. Thereafter, the medium was removed and cells were incubated with 100 µL of the Fluo-4 dye loading solution (Molecular Probes, Eugene, OR, USA) for 30 min at 37 • C. After 30 min incubation, cells were incubated for a further 30 min at room temperature. Then, the fluorescence intensity of each well was determined using a spectrofluorometer (Dynex, West Sussex, UK) at excitation and emission wavelengths of 485 nm and 535 nm, respectively [15].

Effects of RF on Cytokine Production in RAW 264.7 Cells Stimulated by LPS
RAW 264.7 cells were seeded in 96-well plates (1 × 10 4 cells/well) and treated with LPS (1 µg/mL) and RF [15]. After 24 h treatment, levels of the following cytokines in each well were analyzed: interleukin (IL)-6; monocyte chemoattractant protein (MCP)-1; tumor necrosis factor (TNF)-α; leukemia inhibitory factor (LIF); lipopolysaccharide-induced CXC chemokine (LIX; CXCL5); granulocyte colony-stimulating factor (G-CSF); granulocyte macrophage colony-stimulating factor (GM-CSF); macrophage colony-stimulating factor (M-CSF); vascular endothelial growth factor (VEGF); macrophage inflammatory proteins (MIP)-1α, MIP-1β, MIP-2; RANTES (CCL5; regulated on activation, normal T cell expressed and secreted); and interferon gamma-induced protein 10 (IP-10; CXCL10). Cytokines were measured using a Luminex assay based on xMAP technology with MILLIPLEX MAP Mouse Cytokine/Chemokine Magnetic Bead Panel kits (Millipore) and a Bio-Plex 200 suspension array system (Bio-Rad), as described previously [15]. The assay used in this experiment was designed for the multiplexed quantitative measurement of multiple cytokines in a single well, using as little Nutrients 2020, 12, 3577 4 of 14 as 25 µL of cell culture supernatant. Standard curves for each cytokine were generated using the kit-supplied reference cytokine samples. Briefly, the following procedure was performed: after pre-wetting the 96-well plate with Wash Buffer, Wash Buffer in each well was removed using a Handheld Magnetic Separation Block (HMSB). Next, cell culture supernatants from each well were incubated with antibody-conjugated beads on a plate shaker for 2 h at room temperature. After incubation, well contents were gently removed with a HMSB, and the 96-well plate was washed 2 times. Then, 25 µL of detection antibodies were added to each well and incubated with agitation on a plate shaker for 1 h at room temperature. Subsequently, 25 µL Streptavidin-Phycoerythrin was added to each well containing the detection antibodies and incubated for 30 min with agitation on a plate shaker at room temperature. After incubation, the well contents were gently removed and washed 2 times using a HMSB. Then, 150 µL of Sheath Fluid was added to all wells, and the beads bound to each cytokine were analyzed with a Bio-Plex 200 instrument (Bio-Rad). Raw data (fluorescence intensity) were analyzed using Bio-Plex Manager software (Bio-Rad). Baicalein (25 µM), a well-known anti-inflammatory flavonoid, was used as a positive control. RAW 264.7 cells were incubated with LPS (1 µg/mL) and RF for 18 h in 6-well plates (1 × 10 6 cells/well). After 18 h incubation, total RNA of each well was isolated using NucleoSpin RNA kit (Macherey-Nagel, Duren, Germany). Briefly, 350 µL Lysis Buffer RA1 and 3.5 µL β-mercaptoethanol was added to the cell pellet and vortexed vigorously to lyse cells. Lysate was cleared by filtration using a NucleoSpin ® Filter, and then 350 µL ethanol (70%) was added, and mixed by vortexing. The lysate was loaded into the NucleoSpin ® RNA Column, and 350 µL Membrane Desalting Buffer was added and centrifuged. A total of 95 µL DNase reaction mixture was applied directly to the center of the silica membrane of the column, followed by incubation at room temperature for 15 min. Samples were washed with Wash Buffer RA2 and Wash Buffer RA3, and silica membrane was dried. RNA was eluted in 60 µL RNase-free water and centrifuged [15].

Determination of RNA Concentration
RNA concentration was measured using Experion RNA StdSens Analysis kit (Bio-Rad) with the Experion Automatic Electrophoresis System (Bio-Rad). First, the electrodes were cleaned using a cleaning chip filled with 900 µL DEPC-treated water. Then, the Gel-Stain solution was prepared, 9 µL was added into labeled wells, and the chip was primed. Samples and RNA ladder were loaded into the chip, which was vortexed using the Experion vortex station for 1 min. Then the chip was loaded into the electrophoresis platform and the RNA StdSens Analysis program was run [15].

RT-qPCR Analysis
Gene expression was measured using quantitative polymerase chain reaction with iQ SYBR Green Supermix (Bio-Rad) using the CFX96 Real-Time PCR Detection System (Bio-Rad) [15]. Briefly, a master mix was prepared for all reactions by adding iQ SYBR Green Supermix and Forward/Reverse primers for each target gene. This master mix was thoroughly mixed to ensure homogeneity, and 7 µL was dispensed into the wells of a qPCR plate. A total of 3 µL of cDNA was added to each well; any air bubbles in the vessel bottom were removed, and the PCR plate was loaded into the real-time PCR instrument. PCR was performed using the following protocol: denaturation of DNA at 95 • C for 3 min, followed by 40 cycles of 95 • C for 10 sec and 55 • C for 30 sec. The 2 -∆∆Ct cycle threshold method was used to normalize the relative mRNA expression levels to the internal control, β-actin. The primers used in this assay are listed in Table 1.

Effects of RF on Phosphorylation of p38 MAPK in RAW 264.7 Cells Stimulated by LPS
Flow cytometry was performed to detect phosphorylated p38 MAPK in RAW 264.7 cells using an Attune NxT flow cytometer (Thermo Fisher Scientific) [17]. Briefly, RAW 264.7 macrophages were seeded in 6-well plates (1 × 10 6 cells/well) and incubated with LPS (0.1 µg/mL) and RF for 30 min. After incubation, cells were harvested and washed with Flow Cytometry Staining Buffer (SB). Prior to antibody staining, cells were fixed with the pre-warmed Fix Buffer I for 10 min. Then, cells were washed with SB and permeabilized with Perm Buffer III on the ice for 30 min. Then, cells were stained with 5 µg/mL of phospho-p38 MAPK Antibody (T180/Y182) (eBioscience 17-9078-42), or 1.2 µg/mL of Mouse IgG2b kappa Isotype Control (eBioscience 12-4732-81), and analyzed on the Attune NxT flow cytometer (Thermo Fisher Scientific) using Attune NxT software (Thermo Fisher Scientific).

Statistics
Data are presented as means ± SD. All data were analyzed by one-way analysis of variance (ANOVA) test followed by Tukey's multiple comparison test using GraphPad Prism (version 4; GraphPad Software, San Diego, CA, USA).

Determination of the Total Flavonoid Content of RF
We found that the total flavonoid content of RF was 9.15 mg RE/g extract.

Effects of RF on Cell Viability
In this study, RF at concentrations of 25, 50, 100, or 200 µg/mL did not decrease cell viability of RAW 264.7 cells after 24 h (108.00 ± 7.84%, 103.84 ± 3.71%, 104.47 ± 4.77%, and 103.19 ± 0.83% of the normal group (Nor) treated with media only, respectively). These results indicated that RF does not exert any cytotoxic effect on macrophages at concentrations of up to 200 µg/mL, which were used in all subsequent experiments ( Figure 1A). Nutrients 2020, 12, x FOR PEER REVIEW 6 of 14 Values represent means ± SD of three independent experiments (n = 3). Statistical significance was calculated by oneway ANOVA and a Tukey multiple comparison test. ** p < 0.01 vs. Con; *** p < 0.001 vs. Con.

NO Production in RAW 264.7 Cells
Data showed that RF significantly inhibited NO production from RAW 264.7 cells stimulated by 24 h treatment with LPS. NO production in RAW 264.7 cells incubated with RF at concentrations of 25, 50, 100, and 200 µg/mL were 88.29 ± 7.33%, 87.6 ± 2.22%, 87.22 ± 8.5%, and 86.26 ± 1.98% of that treated with LPS alone ( Figure 1B). These data indicated that RF might modulate excessive NOinduced inflammatory signaling.

Calcium Release in RAW 264.7 Cells
Data showed that RF significantly inhibited calcium release in RAW 264.7 cells stimulated by 18 h LPS treatment. Calcium release in RAW 264.7 cells incubated with RF at concentrations of 25, 50, 100, and 200 µg/mL were 35.25 ± 1.86%, 34.91 ± 1.12%, 34.72 ± 2.6%, and 34.94 ± 1.08% of that induced by LPS treatment alone ( Figure 1C). Our results indicated that RF might exert a regulatory effect over the calcium-related ER stress response pathway.

NO Production in RAW 264.7 Cells
Data showed that RF significantly inhibited NO production from RAW 264.7 cells stimulated by 24 h treatment with LPS. NO production in RAW 264.7 cells incubated with RF at concentrations of 25, 50, 100, and 200 µg/mL were 88.29 ± 7.33%, 87.6 ± 2.22%, 87.22 ± 8.5%, and 86.26 ± 1.98% of that treated with LPS alone ( Figure 1B). These data indicated that RF might modulate excessive NO-induced inflammatory signaling.

Calcium Release in RAW 264.7 Cells
Data showed that RF significantly inhibited calcium release in RAW 264.7 cells stimulated by 18 h LPS treatment. Calcium release in RAW 264.7 cells incubated with RF at concentrations of 25, 50, 100, and 200 µg/mL were 35.25 ± 1.86%, 34.91 ± 1.12%, 34.72 ± 2.6%, and 34.94 ± 1.08% of that induced by LPS treatment alone ( Figure 1C). Our results indicated that RF might exert a regulatory effect over the calcium-related ER stress response pathway.

Cytokine Production in RAW 264.7 Cells
RF significantly reduced the production of cytokines in RAW 264.7 cells stimulated by LPS for 24 h. In particular, RF reduced productions of IL-6, MCP-1, G-CSF, LIF, LIX, MIP-1α, MIP-1β, MIP-2, VEGF, and RANTES in a dose-dependent manner. Production of IL-6 from RAW 264. 7 Figure 2). These data indicated that RF might alleviate hyper-inflammation leading to excessive production of cytokines, chemokines, and growth factors in LPS-stimulated macrophages. Values represent means ± SD of three independent experiments (n = 3). Statistical significance was calculated by one-way ANOVA and a Tukey multiple comparison test. * p < 0.05 vs. Con; ** p < 0.01 vs. Con; *** p < 0.001 vs. Con.

Phosphorylation of p38 MAPK in RAW 264.7
Phosphorylation of p38 MAPK was significantly inhibited by RF (Figure 4). p38 MAPK phosphorylation in RAW 264.7 cells incubated with RF (25, 50, 100, and 200 µg/mL) for 30 min was 48.76 ± 3.79%, 39.64 ± 14.53%, 26.11 ± 3.87%, and 38.29 ± 8.46% of that induced by LPS. No non-specific staining was observed with Mouse IgG2b kappa Isotype Control. These data indicated that RF might exert its anti-inflammatory effects via suppressing the activation of p38 MAPK signaling pathway. Values represent means ± SD of three independent experiments (n = 3). Statistical significance was calculated by one-way ANOVA and a Tukey multiple comparison test. * p < 0.05 vs. Con; ** p < 0.01 vs. Con.

Discussion
SIRS caused by gram-negative bacteria is known to be a host response to lipopolysaccharide [18,19] and accompanied by increased production of inflammatory mediators such as NO, IL-1, IL-6, and TNF-α resulting in vascular leakage and multiple organ dysfunction syndrome (MODS) [20,21]. Increased endothelial permeability is central to SIRS, leading to MODS and death [22][23][24][25]. Indeed, in addition to the cytopathic role in host defense and innate immune functions, NO plays a major role in vasodilation in SIRS [26]. Despite a definitive link between cytokine levels and morbidity/mortality following infection, no effective therapeutic modalities have been developed to subdue the pathology associated with cytokine storm [27]. However, because of the necessary and positive function of cytokines against pathogenic infections, global blunting, rather than ablation, of inflammatory mediators will likely be required to ameliorate pathology associated with cytokine storm [27]. Among immune cells, macrophages are well known to produce many kinds of inflammatory mediators, such as NO and cytokines. Therefore, a drug candidate able to modulate the excessive activation of macrophages stimulated by LPS, including the excessive production of NO and cytokines, might be a beneficial for the regulation of endotoxemia-related inflammatory cascades.
Until recently, many studies have reported on the anti-inflammatory effects of natural products. In the previous study, we reported that Angelica sinensis root water extract has an anti-inflammatory effect on LPS-stimulated RAW 264.7 via calcium-mediated JAK-STAT pathway [15]. Since Rubi Fructus has also antioxidative properties like Angelica sinensis root, we set the hypothesis that Rubi Fructus inhibits inflammatory reactions in LPS-stimulated macrophages via calcium-STAT signaling pathway. Actually, Rubi Fructus has traditionally been used as a medical drug in East Asia, including Korea and China [8,9]. While the use of Rubi Fructus is indicated in textbooks of traditional medicine Values represent means ± SD of three independent experiments (n = 3). Statistical significance was calculated by one-way ANOVA and a Tukey multiple comparison test. * p < 0.05 vs. Con; ** p < 0.01 vs. Con.

Discussion
SIRS caused by gram-negative bacteria is known to be a host response to lipopolysaccharide [18,19] and accompanied by increased production of inflammatory mediators such as NO, IL-1, IL-6, and TNF-α resulting in vascular leakage and multiple organ dysfunction syndrome (MODS) [20,21]. Increased endothelial permeability is central to SIRS, leading to MODS and death [22][23][24][25]. Indeed, in addition to the cytopathic role in host defense and innate immune functions, NO plays a major role in vasodilation in SIRS [26]. Despite a definitive link between cytokine levels and morbidity/mortality following infection, no effective therapeutic modalities have been developed to subdue the pathology associated with cytokine storm [27]. However, because of the necessary and positive function of cytokines against pathogenic infections, global blunting, rather than ablation, of inflammatory mediators will likely be required to ameliorate pathology associated with cytokine storm [27]. Among immune cells, macrophages are well known to produce many kinds of inflammatory mediators, such as NO and cytokines. Therefore, a drug candidate able to modulate the excessive activation of macrophages stimulated by LPS, including the excessive production of NO and cytokines, might be a beneficial for the regulation of endotoxemia-related inflammatory cascades.
Until recently, many studies have reported on the anti-inflammatory effects of natural products. In the previous study, we reported that Angelica sinensis root water extract has an anti-inflammatory effect on LPS-stimulated RAW 264.7 via calcium-mediated JAK-STAT pathway [15]. Since Rubi Fructus has also antioxidative properties like Angelica sinensis root, we set the hypothesis that Rubi Fructus inhibits inflammatory reactions in LPS-stimulated macrophages via calcium-STAT signaling pathway. Actually, Rubi Fructus has traditionally been used as a medical drug in East Asia, including Korea and China [8,9]. While the use of Rubi Fructus is indicated in textbooks of traditional medicine such as "Donguibogam (Principles and Practice of Eastern Medicine)" and "Ben Cao Gang Mu (Compendium of Materia Medica)", many researchers are interested in investigating the pharmacological activities of Rubi Fructus [8,9]. A number of studies have reported on the biomedical efficacy of Rubi Fructus, such as its anti-inflammatory effects [12], antioxidative effects [10], improvements in visual sensitivity [28], acetylcholinesterase inhibitory activity [29], improvements in diabetic osteoporosis by simultaneous regulation of osteoblasts and osteoclasts [30], hepatoprotective effects [31], anti-fatigue effects [32], increased hypocholesterolemic activity [33], chemopreventive effects in prostate cancer [34], increased anti-anaphylactic activity [35], and enhanced spermatogenesis [36]. In detail, Seo et al. reported in 2019 that ERF inhibited the production of NO, IL-1β, and IL-6 as well as the activation of inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX-2) via inhibition of the NF-κB signaling pathway in LPS-stimulated RAW 264.7 [12]. Lee et al. reported in 2014 that ERF reduced production of NO, PGE2, TNF-α, IL-1β, and IL-6, and reduced expression of iNOS and COX-2 through suppression of NF-κB activation, as well as phosphorylation of JNK and p38 MAPKs [13]. Kim et al. reported in 2013 that the WRF suppressed NF-κB activation, ROS production, and inflammatory and phase II gene expression in LPS-treated RAW 264.7 cells [14]. Additionally, Park et al. reported in 2006 that ERF exerts anti-inflammatory effects in macrophages via activation of the heme oxygenase-1 signaling pathway [37].
In these experiments, RF significantly inhibited excessive production of NO, IL Our data indicate that RF may be useful to relieve NO-aggravated vasodilation and cytokine storm in SIRS due to gram-negative bacterial infection. Additionally, RF significantly decreased the release of intracellular calcium, mRNA expression of Chop, and phosphorylation of p38 MAPK in LPS-stimulated RAW 264.7 cells, which led to the hypothesis that RF-mediated regulation of inflammatory mediators in LPS-treated RAW 264.7 macrophages might be achieved through ER stress-related CHOP activation.
Many studies have reported ER stress-related calcium release and CHOP expression in stressed cells. In 1996, Wang and Ron reported that CHOP was known to be activated by p38 MAPK in stressed cells [38]. In 2006, Endo et al. reported that LPS stimulation of macrophages causes ER stress and CHOP expression, which initiated inflammasome activation and subsequent macrophage pyroptosis [39]. In 2007, Stout et al. reported that ER calcium stores were reduced and intracellular calcium concentration was initially increased during an inflammatory signaling cascade [40]. Mori also reported that NO depletes ER calcium, causes ER stress-induced CHOP expression, and leads to apoptosis [41]. In 2009, Timmins et al. reported that ER stress-induced calcium release from the ER lumen activates CAMK2a, which might enable macrophage apoptosis via Fas induction and/or activation of Stat1 in macrophages [42]. Tabas et al. also reported that ER-stress-induced CHOP activation, resulting in a release of ER calcium stores into the cytoplasm, and that cytoplasmic calcium activates CAMK2a, which in turn activates a number of pro-apoptotic processes [5]. In 2011, Cho et al. reported that COX-2 is an important mediator of the inflammatory response related to ER stress [43]. In 2017, Lee et al. reported that LPS induces expression of iNOS, COX-2, NO, TNF-α, and IL-6 via p38 MAPK and JAK/STAT signaling pathways in RAW 264.7 macrophages [44]. Guha and Mackman reported that p38 MAPK signaling in LPS-stimulated macrophages can activate a variety of transcription factors, including AP-1 (c-Fos/c-Jun) [45]. In the current study, RF was shown to reduce mRNA expression of Camk2a, Stat1, Stat3, Jak2, Fas, c-Jun, c-Fos, Nos2, and Ptgs2 in LPS-stimulated RAW 264.7 macrophages. These results suggest that RF alleviates LPS-stimulated macrophage activation via ER stress-induced calcium/CHOP pathway and the JAK/STAT pathway, resulting in reduced production of inflammatory mediators such as NO, cytokines, chemokines, and growth factors ( Figure 5). However, this study could not determine the exact source from which intracellular calcium is released. It remains for future studies to elucidate whether the intracellular calcium level is increased through the influx of extracellular calcium or through depletion of ER calcium stores in LPSstimulated RAW 264.7 macrophages. Moreover, we could not evaluate effects of RF on IL-1β production, phosphorylation of JNK, NF-κB activation, and ROS production in LPS-stimulated RAW 264.7. More detailed research will clarify the efficacy of RF in treating bacterial infectious diseases.

Conflicts of Interest:
The authors declare no conflict of interest. The funders had no role in the design of the study, in the collection, analyses, or interpretation of data, in the writing of the manuscript, or in the decision to publish the results.  However, this study could not determine the exact source from which intracellular calcium is released. It remains for future studies to elucidate whether the intracellular calcium level is increased through the influx of extracellular calcium or through depletion of ER calcium stores in LPS-stimulated RAW 264.7 macrophages. Moreover, we could not evaluate effects of RF on IL-1β production, phosphorylation of JNK, NF-κB activation, and ROS production in LPS-stimulated RAW 264.7. More detailed research will clarify the efficacy of RF in treating bacterial infectious diseases.

Conflicts of Interest:
The authors declare no conflict of interest. The funders had no role in the design of the study, in the collection, analyses, or interpretation of data, in the writing of the manuscript, or in the decision to publish the results.