Diospyrin Modulates Inflammation in Poly I:C-Induced Macrophages via ER Stress-Induced Calcium-CHOP Pathway

Diospyrin, plant-derived bisnaphthoquinonoid, is known to have anticancer activity. However, pharmacological activity of diospyrin on viral infection is not well known. We investigated effects of diospyrin on macrophages induced by polyinosinic-polycytidylic acid (poly I:C), a mimic of double-stranded viral RNA. Various cytokines, intracellular calcium, nitric oxide (NO), phosphorylated p38 MAPK, and phosphorylated ERK1/2 as well as mRNA expressions of transcription factors were evaluated. Diospyrin significantly reduced NO production, granulocyte-macrophage colony-stimulating factor production, and intracellular calcium release in poly I:C-induced RAW 264.7. The phosphorylation of p38 MAPK and ERK1/2 was also significantly suppressed. Additionally, diospyrin inhibited mRNA levels of nitric oxide synthase 2, C/EBP homologous protein (CHOP), calcium/calmodulin dependent protein kinase II alpha, signal transducers and activators of transcription 1 (STAT1), STAT3, STAT4, Janus kinase 2, first apoptosis signal receptor, c-Jun, and c-Fos in poly I:C-induced RAW 264.7. Taken together, this study represents that diospyrin might have the inhibitory activity against viral inflammation such as excessive production of inflammatory mediators in poly I:C-induced RAW 264.7 via ER stress-induced calcium-CHOP pathway.


Introduction
Innate immune activity is thought to be essential for overcoming infectious diseases [1]. Gilroy reported that immune reaction and inflammatory activity are important against infection [2]. Recently, Nahrendorf and Swirski reported that when stimuli persist and inflammation is not resolved, monocytes might cause a chronic inflammation [3]. Then, it is reasonable that the regulation of inflammatory responses is becoming more meaningful [4].
Macrophages are important in immunity. Zong et al. reported that various inflammatory diseases are related with a complex reaction generated by macrophages [5]. In inflammatory processes, macrophages and monocytes not only produce cytokines and nitric oxide (NO) but also release intracellular calcium. Cho et al. reported that viral infection can activate immune responses and trigger inflammatory diseases [6]. Alexopoulou et al. reported that double-stranded RNA (dsRNA) induces macrophages [7].

Cell Culture
RAW 264.7 was purchased from Korea Cell Line Bank (Seoul, Korea). In the previous study [9], we reported that diospyrin did not show any cytotoxicity up to a concentration of 10 μM, which was chosen for subsequent experiments.

NO Production
RAW 264.7 was seeded in a 96-well plate (1 × 10 4 cells/well) and treated with diospyrin and poly I:C for 24 h. After treatment, supernatants were collected, and NO content was measured using the modified Griess reagent assay kit (Millipore) [9,10]. In this experiment, we investigated inhibitory effects of diospyrin on RAW 264.7 induced by polyinosinic-polycytidylic acid (poly I:C), a mimic of double-stranded viral RNA. Diospyrin significantly reduced productions of GM-CSF and NO as well as calcium release in RAW 264.7 induced with poly I:C. The phosphorylation of p38 MAPK and ERK1/2 was also significantly suppressed. The mRNA expressions of nitric oxide synthase 2 (NOS2), C/EBP homologous protein (CHOP), calcium/calmodulin dependent protein kinase II alpha (Camk2a), signal transducers and activators of transcription 1 (STAT1), STAT3, STAT4, Janus kinase 2 (Jak2), first apoptosis signal receptor (Fas), c-Jun, and c-Fos were reduced. Data means diospyrin might have the inhibitory activity against viral inflammation such as excessive production of inflammatory mediators in dsRNA-stimulated macrophages via ER stress-induced calcium-CHOP pathway.

Cell Culture
RAW 264.7 was purchased from Korea Cell Line Bank (Seoul, Korea). In the previous study [9], we reported that diospyrin did not show any cytotoxicity up to a concentration of 10 µM, which was chosen for subsequent experiments.

NO Production
RAW 264.7 was seeded in a 96-well plate (1 × 10 4 cells/well) and treated with diospyrin and poly I:C for 24 h. After treatment, supernatants were collected, and NO content was measured using the modified Griess reagent assay kit (Millipore) [9,10].

Quantitative Polymerase Chain Reaction
RAW 264.7 was treated with diospyrin and poly I:C for 18 h in a six-well plate (1 × 10 6 cells/well). After 18 h treatment, RNA quantity of each well was measured using NucleoSpin RNA kit (Macherey-Nagel, Duren, Germany) and Experion RNA StdSens Analysis kit (Bio-Rad) with Experion Automatic Electrophoresis System (Bio-Rad). Each cDNA was produced using iScript cDNA Synthesis kit (Bio-Rad). Then, gene expressions were measured using quantitative polymerase chain reaction with iQ SYBR Green Supermix (Bio-Rad) [14,15]. The target genes are listed in Table 1. The β-actin was used as a housekeeping gene.

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

Discussion
Diospyrin, the medicinal plant (i.e., species of Diospyros such as Diospyros lotus and Diospyros montana)-derived bisnaphthoquinonoid, is known to suppress cancer [16], tuberculosis [17], and leishmaniasis [18,19]. In detail, Bailly reported in 2000 that diospyrin exerts the activity of a topoisomerase I suppressor, resulting in anti-tumor effect [16]. Lall et al. reported in 2005 that diospyrin exerted the inhibitory activity against Mycobacterium tuberculosis [20]. In 2013, Hazra et al. reported that diospyrin represented the anti-leishmanial activity [19]. But effects of diospyrin on viral inflammation have been rarely reported so far.
Although the innate immunity is essential for overcoming hazardous infections, the uncontrolled immuno-inflammatory reaction such as cytokine storm also might be fatal for human life. Thus, the resolution of excessive inflammation is being required continuously because there is no pertinent therapeutics against the newly emerged pandemic viral infection (such as Coronavirus Disease-19), which sometimes provokes cytokine storm.
Karpuzoglu and Ahmed reported that excessive NO production might be concerned with inflammatory diseases such as inflammatory autoimmune diseases [21]. Moreover, it is well reported that excessive production of NO contributes to septic shock [22]. Cooper et al. reported that viral infections might cause airway inflammation and increase the production of various cytokines excessively [23]. Interestingly, respiratory tract viral infections such as Influenza A and Parainfluenza virus were reported to show higher levels of GM-CSF, IL-17A, and IL-22 [24]. In this year, Crisci et al. reported that cytokines such as GM-CSF, VEGF, MCP-1, IL-6, and LIF were increased in severe patients with COVID-19 [25]. It is reasonable that excessive production of cytokines becomes a major target for relieving inflammatory illnesses [26]. In this study, we tried to find a material able to inhibit hyper-production of cytokines in macrophages induced with virus-like particles. During viral infection, dsRNA is produced in host cells and brings about viral inflammatory reaction in macrophages producing NO, ILs, TNF-α, and other cytokines [27]. Then, multiplex cytokine assay is regarded to be effective for checking anti-inflammatory activity of drug candidates [28][29][30][31][32]. For example, Kim et al. reported that baicalein showed inhibitory effects on productions of various cytokines in poly I:C-induced RAW 264.7 [14]. Meanwhile, Alexopoulou et al. reported in 2001 that dsRNA recognized by mammalian Toll-like receptor 3 might provoke inflammatory response via activation of p38 MAPK and ERK1/2 [7]. However, it is not yet fully reported for effects of diospyrin on viral inflammation.

Discussion
Diospyrin, the medicinal plant (i.e., species of Diospyros such as Diospyros lotus and Diospyros montana)-derived bisnaphthoquinonoid, is known to suppress cancer [16], tuberculosis [17], and leishmaniasis [18,19]. In detail, Bailly reported in 2000 that diospyrin exerts the activity of a topoisomerase I suppressor, resulting in anti-tumor effect [16]. Lall et al. reported in 2005 that diospyrin exerted the inhibitory activity against Mycobacterium tuberculosis [20]. In 2013, Hazra et al. reported that diospyrin represented the anti-leishmanial activity [19]. But effects of diospyrin on viral inflammation have been rarely reported so far.
Although the innate immunity is essential for overcoming hazardous infections, the uncontrolled immuno-inflammatory reaction such as cytokine storm also might be fatal for human life. Thus, the resolution of excessive inflammation is being required continuously because there is no pertinent therapeutics against the newly emerged pandemic viral infection (such as Coronavirus Disease-19), which sometimes provokes cytokine storm.
Karpuzoglu and Ahmed reported that excessive NO production might be concerned with inflammatory diseases such as inflammatory autoimmune diseases [21]. Moreover, it is well reported that excessive production of NO contributes to septic shock [22]. Cooper et al. reported that viral infections might cause airway inflammation and increase the production of various cytokines excessively [23]. Interestingly, respiratory tract viral infections such as Influenza A and Parainfluenza virus were reported to show higher levels of GM-CSF, IL-17A, and IL-22 [24]. In this year, Crisci et al. reported that cytokines such as GM-CSF, VEGF, MCP-1, IL-6, and LIF were increased in severe patients with COVID-19 [25]. It is reasonable that excessive production of cytokines becomes a major target for relieving inflammatory illnesses [26]. In this study, we tried to find a material able to inhibit hyper-production of cytokines in macrophages induced with virus-like particles. During viral infection, dsRNA is produced in host cells and brings about viral inflammatory reaction in macrophages producing NO, ILs, TNF-α, and other cytokines [27]. Then, multiplex cytokine assay is regarded to be effective for checking anti-inflammatory activity of drug candidates [28][29][30][31][32]. For example, Kim et al. reported that baicalein showed inhibitory effects on productions of various cytokines in poly I:C-induced RAW 264.7 [14]. Meanwhile, Alexopoulou et al. reported in 2001 that dsRNA recognized by mammalian Toll-like receptor 3 might provoke inflammatory response via activation of p38 MAPK and ERK1/2 [7]. However, it is not yet fully reported for effects of diospyrin on viral inflammation.
In this experiment, bio-activity of diospyrin in poly I:C-induced RAW 264.7 was evaluated using multiplex cytokine assay, flow cytometry assay, etc. Experimental data means that diospyrin reduces the production of NO and GM-CSF as well as phosphorylation of p38 MAPK and ERK1/2 in poly I:C-induced RAW 264.7. As NO is related with septic shock, diospyrin could be tested for relieving cytokine storm with viral sepsis.
Recently, pandemic viral infection COVID-19 has produced a global threat. Interestingly, TNF-α-converting enzyme (TACE) was reported to be involved in the cell entry of SARS-CoV [33,34]. Xiao et al. reported that SARS-CoV-2 binds angiotensin-converting enzyme 2 (ACE2) for cell entry [35]. ACE2 is known to be shed by TACE and Palau et al. suggested that TACE inhibition may be important for protecting COVID-19 [36]. Additionally, Scott et al. reported in 2011 that TACE activity is upregulated in LPS-induced human monocytes through p38 activation [37]. In this study, the authors presented TACE inhibitory potential of diospyrin predicted by structure-based virtual screening in Figure 6. However, more studies are needed to ascertain whether diospyrin exerts a meaningful effect on TACE activity. In this experiment, bio-activity of diospyrin in poly I:C-induced RAW 264.7 was evaluated using multiplex cytokine assay, flow cytometry assay, etc. Experimental data means that diospyrin reduces the production of NO and GM-CSF as well as phosphorylation of p38 MAPK and ERK1/2 in poly I:Cinduced RAW 264.7. As NO is related with septic shock, diospyrin could be tested for relieving cytokine storm with viral sepsis.
Recently, pandemic viral infection COVID-19 has produced a global threat. Interestingly, TNFα-converting enzyme (TACE) was reported to be involved in the cell entry of SARS-CoV [33,34]. Xiao et al. reported that SARS-CoV-2 binds angiotensin-converting enzyme 2 (ACE2) for cell entry [35]. ACE2 is known to be shed by TACE and Palau et al. suggested that TACE inhibition may be important for protecting COVID-19 [36]. Additionally, Scott et al. reported in 2011 that TACE activity is upregulated in LPS-induced human monocytes through p38 activation [37]. In this study, the authors presented TACE inhibitory potential of diospyrin predicted by structure-based virtual screening in Figure 6. However, more studies are needed to ascertain whether diospyrin exerts a meaningful effect on TACE activity. With an infection of microorganisms, macrophages show a distinct reaction, called pyroptosis, which is different from both apoptosis and necrosis. In pyroptosis, various inflammatory mediators are produced from infected macrophages [38]. In 2016, Broz and Dixit also reported about pyroptotic cells to produce many kinds of cytokines [39]. In 2019, Goddard et al. reported that microbial infections can stimulate macrophages [40]. However, it is not yet fully reported for virus-induced pyroptosis accompanying massive production of inflammatory mediators with the calcium signaling pathway.
With ER stress, NO is well known to increase CHOP expression with releasing calcium from ER calcium store and leads to macrophage apoptosis [41][42][43][44]. In 2009, Timmins et al. suggested in their impressive report that ER stress-induced Camk2a activation might enable macrophage apoptosis through Fas induction and/or activation of STAT1 in macrophages [43]. In 2006, Endo et al. reported that LPS causes the overexpression of CHOP, which mediates apoptosis in macrophages and ER stress [45]. Interestingly, Stout et al. reported in 2007 that a brief activation of STAT1 induces ER stress and calcium release from ER calcium store in IFN-gamma-induced airway epithelial cell death with increase of STAT1 protein level [46]. In 2008, Lim et al. reported atheromata-related macrophage apoptosis might be provoked by ER stress through a rise in cytosolic calcium [47].
In the current study, poly I:C-induced RAW 264.7 represents propyrototic ER stress with enhancing mRNA expression of NOS2, CHOP, Camk2a, STAT1, STAT3, STAT4, Jak2, Fas, c-Jun, and c-Fos. Additionally, diospyrin modulates calcium release and mRNA expression of NOS2, CHOP, Camk2a, STAT1, STAT3, STAT4, Jak2, Fas, c-Jun, and c-Fos in poly I:C-induced RAW 264.7. This data With an infection of microorganisms, macrophages show a distinct reaction, called pyroptosis, which is different from both apoptosis and necrosis. In pyroptosis, various inflammatory mediators are produced from infected macrophages [38]. In 2016, Broz and Dixit also reported about pyroptotic cells to produce many kinds of cytokines [39]. In 2019, Goddard et al. reported that microbial infections can stimulate macrophages [40]. However, it is not yet fully reported for virus-induced pyroptosis accompanying massive production of inflammatory mediators with the calcium signaling pathway.
With ER stress, NO is well known to increase CHOP expression with releasing calcium from ER calcium store and leads to macrophage apoptosis [41][42][43][44]. In 2009, Timmins et al. suggested in their impressive report that ER stress-induced Camk2a activation might enable macrophage apoptosis through Fas induction and/or activation of STAT1 in macrophages [43]. In 2006, Endo et al. reported that LPS causes the overexpression of CHOP, which mediates apoptosis in macrophages and ER stress [45]. Interestingly, Stout et al. reported in 2007 that a brief activation of STAT1 induces ER stress and calcium release from ER calcium store in IFN-gamma-induced airway epithelial cell death with increase of STAT1 protein level [46]. In 2008, Lim et al. reported atheromata-related macrophage apoptosis might be provoked by ER stress through a rise in cytosolic calcium [47].
However, this study could not elucidate whether intracellular calcium level is increased through influx of extracellular calcium or ER calcium store depletion in poly I:C-induced RAW 264.7. Further