Suppressive effect of isofraxidin on the overexpression of IL-6 and its molecular mechanism

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Recently, IL-6 has been widely recognized as an important pro-inflammatory cytokine involved in cytokine storm pathogenesis during severe inflammatory diseases, such as coronavirus disease 2019 (COVID-19). Therefore, IL-6 is considered to be a therapeutic target for inhibiting cytokine storm. In the present study, we investigated the suppressive effect of isofraxidin, a major coumarin compound of Acanthopanax senticosus, on the overexpression of IL-6 and its molecular mechanism. When human hepatocellular carcinoma cell lines, HuH-7 and HepG2, were treated with 12-O-tetradecanoylphorbol 13-acetate (TPA), a marked induction of IL-6 mRNA expression was observed in HuH-7 cells compared with HepG2 cells. Isofraxidin significantly suppressed TPA-induced IL-6 mRNA expression in HuH-7 cells in a dose-dependent manner. Furthermore, isofraxidin inhibited TPA-induced phosphorylation of ERK1/2 in a dose dependent manner. Similarly, the MAPK/ERK inhibitor U0126 suppressed TPA-induced IL-6 mRNA expression. However, isofraxidin had no effects on TPA-induced phosphorylation of SAPK/JNK, Akt (Ser473), and STAT3 (Tyr705), nuclear translocation of NF-κB p65, and degradation of IκB. Taken together, isofraxidin suppresses TPA-induced overexpression of IL-6 mRNA by selectively inhibiting the activation of the MAPK/ERK pathway in HuH-7 cells, indicating that isofraxidin may be an effective anti-inflammatory agent for treating cytokine storm. IL-6 Cytokine storm Suppressive effect Isofraxidin MAPK/ERK pathway Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Cytokine storm is a systemic inflammatory response that is commonly triggered by bacteria or viral infections and chimeric antigen receptor T cell therapy, and is one of the leading causes of morbidity and mortality in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-induced coronavirus disease 2019 (COVID-19) [ 1 – 4 ]. Cytokine storm is characterized by the overexpression and excessive production of pro-inflammatory cytokines, such as interleukin-1 (IL-1), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α), which may cause acute respiratory distress syndrome (ARDS) and subsequent severe tissue damage [ 1 – 4 ]. Among the pro-inflammatory cytokines, IL-6 is one of the most important cytokines involved in the initiation of cytokine storm, and induces other pro-inflammatory cytokines and the marker of endothelial injury plasminogen activator inhibitor-1 (PAI-1). Therefore, IL-6 is considered to be a therapeutic target for inhibiting cytokine storm. In fact, the blockade of IL-6 trans-signaling using a humanized monoclonal antibody against human IL-6 receptor (anti-IL-6R antibody) has been shown to be effective in patients with severe COVID-19 [ 1 ]. Moreover, it has been revealed that intracellular signaling pathways, such as MAPK/ERK, SAPK/JNK, PI3K/Akt, NFκ-B/IκB, and JAK/STAT3, are activated in host cells infected with SARS-CoV-2, resulting in the overexpression and excessive production of pro-inflammatory cytokines, such IL-6, in host cells [ 4 – 8 ]. Therefore, the inhibition of these signaling pathways using pharmacological agents may be an effective therapeutic strategy for counteracting cytokine storm in addition to the blockade of IL-6 trans-signaling using an anti-IL-6R antibody. However, only a limited number of pharmacological agents are available for the treatment of cytokine storm. Glucocorticoids have been used in the treatment of inflammation-related diseases, such as rheumatoid arthritis, sepsis, and COVID-19 [ 9 – 11 ]. Glucocorticoids bind intracellular glucocorticoid receptors (GRs) and subsequently downregulate the expression of pro-inflammatory cytokines by inhibiting the activation of intracellular signaling pathways and transcriptional factors [ 12 ]. However, the anti-inflammatory effects of glucocorticoids are often accompanied by several intolerable adverse effects, including indigestion, vomiting, weight gain, osteoporosis, hyperglycemia, cardiovascular diseases, and infections [ 13 , 14 ]. Thus, it is urgent to develop new anti-inflammatory agents that are effective for cytokine storm and exhibit tolerable adverse effects. Phytochemical compounds have become the primary sources for the discovery of new anti-inflammatory agents, due to their ability to alleviate inflammation with minimal side effects [ 15 ]. To date, the anti-inflammatory effects of various phytochemical compounds, including epigallocatechin-3-gallate (EGCG) and curcumin, have been reported [ 15 – 17 ]. Isofraxidin is a well-known coumarin compound that is naturally present plants, including Acanthopanax senticosus , and has been reported to exhibit several pharmacological activities, such as anti-inflammatory, anti-cancer, anti-depressive, anti-oxidant, and cardio-protective effects [ 18 – 27 ]. In 2007, we first reported the anti-inflammatory effects of isofraxidin using the SW982 human synovial sarcoma cell line [ 18 ]. In this study, we performed an examination of the effect of isofraxidin on IL-6 using ELISA and RT-PCR methods. Recently, reports have described the anti-inflammatory effects of isofraxidin using mouse peritoneal macrophages, human osteoarthritis chondrocytes, or LPS-injected mice [ 19 – 21 ]. However, these studies were not designed to elucidate the detailed molecular mechanisms underlying the effect of isofraxidin on the overexpression of IL-6. Therefore, the present study examined the effect of isofraxidin on TPA-induced IL-6 mRNA expression and activation of intracellular signaling pathways involved in the transcriptional regulation of the IL-6 gene using HuH-7 cells. The results of the present study may contribute to the development of effective treatments for cytokine storm. Materials and Methods Cell culture The human hepatocellular carcinoma cell lines, HuH-7 (RRID: CVCL_0336) and HepG2 (RRID: CVCL_0027) were obtained from JCRB (Osaka, Japan) and RIKEN BRC Cell Bank (Tsukuba, Japan), respectively. The cells were authenticated by STR profiling and mycoplasma DNA was not detected by PCR. These cells were routinely maintained in DMEM medium (Gibco, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS) at 37°C in a humidified atmosphere containing 5% CO 2 and 95% air. Treatment of cells with isofraxidin, dexamethasone, and signal transduction inhibitors Cells were plated onto 60-mm plastic dishes at a density of 3×10 4 cells/cm 2 in DMEM medium supplemented with 10% FBS. The next day, the medium was replaced with serum-free DMEM, and the day after that, cells were treated with 100 nM TPA (Sigma, St. Louis, MO, USA) and isofraxidin or dexamethasone (FUJIFILM Wako, Osaka, Japan) at the indicated concentrations in serum-free DMEM for 2, 8, and 24 h. Untreated cells were used as controls. For the inhibition of intracellular signaling pathways, individual signal transduction inhibitors (MAPK/ERK inhibitor U0126, PI3K inhibitor LY294002, JNK inhibitor SP600125, JAK2 inhibitor AZD1480, and NF-κB inhibitor PDTC (Cayman Chemicals, Ann Arbor, MI, USA)) were added in place of isofraxidin. MTT assay Cells were plated onto 96-well plates at a density of 3×10 4 cells/cm 2 in DMEM supplemented with 10% FBS. The next day, the medium was replaced with serum-free DMEM, and the day after that, cells were treated with or without isofraxidin at the indicated concentrations in serum-free DMEM for 2 and 24 h. After treatment with isofraxidin, cells were incubated with MTT (FUJIFILM Wako, 10 µL, 5 mg/mL) for 4 h. The formazan precipitates were dissolved in 100 µL DMSO, and the optical density (OD) values were read at 540 nm using a microplate reader. Total RNA extraction and quantitative real-time PCR Total RNA was extracted from cells using the RNeasy Mini Kit (Qiagen, Hilden, Germany). Quantitative real-time PCR was performed with the MyGo Mini Real Time PCR system (IT-IS Life Science, Dublin, Ireland) using Precision PLUS OneStep RT-qPCR Master Mix (Primerdesign Ltd, Chandler's Ford, UK), consisting of 10 µL of 2×OneStep RT-qPCR Master Mix, 0.5 µL of each 10 µM (0.25 µM final) forward and reverse primers, 2.5 µL (25 ng) of template RNA and 6.5 µL of RNase/DNase-free water was made to a total volume of 20 µL. The real-rime PCR cycling conditions were: 55°C for 10 min, 95°C for 2 min, followed by 40 cycles of 95°C for 10 s and 60°C for 1 min. The PCR primers used for amplification are as follows: IL-1β: sense 5’-ttcgacacatgggataacgagg-3’, antisense 5’-aggacatggagaacaccacttg-3; IL-6: sense 5’-gtagccgccccacacagacagcc-3’, antisense 5’-gccatctttggaaggttc-3’; TNF-α: sense 5’-tcttctcgaaccccgagtga-3’, antisense 5’-cctctgatggcaccaccag-3’; IL-8: sense 5’-ttggcagccttcctgatttc-3’, antisense 5’-atttctgtgttggcgcagtg-3’; MCP-1: sense 5’-tgctcatagcagccaccttc-3’, antisense 5’-tctccttggccacaatggtc-3’; GAPDH: sense 5’-ggtggtctcctctgacttcaaca- 3’, antisense 5’- gttgctgtagccaaattcgttgt-3’. GAPDH was used as an internal control. Western blot analysis Cell lysates and nuclear extracts were prepared using cell lysis buffer (Mammalian Cell PE LB, Biosciences, St. Louis, MO, USA) and nuclear extraction buffer (LysoPure Nuclear and Cytoplasmic Extraction Kit, FUJIFILM Wako), respectively. Protein concentrations were measured using a BCA Protein Assay Kit (Pierce, Rockford, IL, USA). Cell lysates (10–30 µg) and nuclear extracts (10 µg) were treated with 5×SDS sample buffer, separated by 10% SDS-polyacrylamide gels (SDS-PAGE), and then transferred to PVDF membranes. After the membranes were blocked with 2% blocking reagent in TBS-T buffer, they were incubated with 1:1000 dilution of anti-phospho (P)-ERK1/2, anti-ERK1/2, anti-P-SAPK/JNK, anti-SAPK/JNK, anti-P-Akt (Ser473, Thr308), anti-Akt, anti-P-STAT3 (Tyr705), anti-STAT3, anti-NF-κB p65, anti-IκB-α (Cell Signaling Technology, Danvers, MA, USA), anti-IL-6 (GeneTex, Taipei, Taiwan, ROC), and anti-actin (Sigma) in TBS buffer for 24 h at 4°C, and then incubated with 1:2000-1:10000 dilution of anti-rabbit IgG, HRP-linked antibody (Cell Signaling Technology) in TBS-T buffer for 1 h at room temperature. Actin was used as an internal control. Detection of proteins was carried out using ECL Prime Western Blotting Detection System (GE Healthcare, Little Chalfont, UK). Protein band intensities were quantitated using Image J (NIH, Bethesda, MD, USA). Statistical analysis Statistical significance was determined by analysis of variance (ANOVA) followed by Tukey’s post hoc test for multiple comparison. Results Effect of isofraxidin on TPA-induced IL-6 mRNA expression in HuH-7 and HepG2 cells In order to elucidate whether isofraxidin suppresses the overexpression of IL-6, we first examined the effect of isofraxidin on TPA-induced IL-6 mRNA expression in HuH-7 and HepG2 cells using quantitative real-time PCR. When HuH-7 and HepG2 cells were treated with TPA for 2 h, a marked induction of IL-6 mRNA expression was observed in HuH-7 cells, whereas a modest induction was observed in HepG2 cells. In addition, isofraxidin significantly suppressed TPA-induced IL-6 mRNA expression in HuH-7 cells in a dose-dependent manner (Fig. 1 ). However, isofraxidin only slightly suppressed TPA-induced IL-6 mRNA expression in HepG2 cells. Time-course experiments of IL-6 mRNA expression in TPA-treated HuH-7 cells In order to determine the optimal treatment time in the suppression of IL-6 mRNA expression by isofraxidin, we performed time-course experiments. The effect of isofraxidin on TPA-induced IL-6 mRNA expression was examined in HuH-7 cells at 2, 8, and 24 h of treatment. The most marked induction by TPA and the most marked suppression by isofraxidin were observed at 2 h (Fig. 2 A). In addition, the suppression of IL-6 protein expression was also observed at 24 h of treatment (Fig. 2 B). Moreover, the effect of isofraxidin on cell viability of HuH-7 cells was evaluated using the MTT assay. The viability of HuH-7 cells was not significantly affected by treatment with isofraxidin at any concentration at 2 and 24 h (Fig. 2 C), indicating that the suppressive effect of isofraxidin on TPA-induced IL-6 mRNA expression is not due to cell toxicity. Based on these results, the following quantitative real-time PCR experiments were performed at 2 h of treatment. Effects of isofraxidin on TPA-induced mRNA expression of other pro-inflammatory cytokines and chemokines in HuH-7 cells In addition to IL-6, other pro-inflammatory cytokines (such as IL-1β and TNF-α) and chemokines (such as IL-8 and MCP-1) are also involved in cytokine storm pathogenesis. Therefore, we examined whether isofraxidin suppresses TPA-induced mRNA expression of other cytokines and chemokines. TPA treatment significantly induced the expression of IL-1β, TNF-α, and IL-8 mRNA. Isofraxidin tended to suppress IL-1β and TNF-α mRNA expression (Fig. 3 A). However, no suppression of IL-8 and MCP-1 mRNA expression was observed by treatment with isofraxidin at all concentrations tested (Fig. 3 B). Comparison of the suppressive effect of isofraxidin on TPA-induced IL-6 mRNA expression with dexamethasone in HuH-7 cells Dexamethasone is a well-known synthetic glucocorticoid that is commonly used in the treatment of inflammation-related diseases. Therefore, we compared the suppressive effect of isofraxidin on TPA-induced IL-6 mRNA expression with that of dexamethasone. At 50–200 µM, isofraxidin showed a dose-dependent suppression of TPA-induced IL-6 mRNA expression that was similar to dexamethasone (Fig. 4 ) Involvement of intracellular signaling pathways in TPA-induced IL-6 and IL-8 mRNA expression in HuH-7 cells Intracellular signaling pathways, such as MAPK/ERK, SAPK/JNK, PI3K/Akt, NF-κB/IκB, and JAK/STAT3, are known to be involved in the transcriptional regulation of IL-6 gene expression. Therefore, we confirmed the involvement of these signaling pathways in TPA-induced IL-6 mRNA expression in HuH-7 cells using signal transduction inhibitors. The addition of the MAPK/ERK inhibitor U0126, JNK inhibitor SP600125, Akt inhibitor LY294002, or JAK2 inhibitor AZD1480 resulted in a concentration-dependent suppression of IL-6 mRNA expression at 1–25 µM (Fig. 5 A, B, C, D). The NF-κB inhibitor PDTC did not suppress IL-6 mRNA expression at 1–25 µM, but suppressed at 50–200 µM (Fig. 5 E). As a reference, we also examined the effect of each inhibitor on TPA-induced IL-8 mRNA expression. U0126, SP600125, and PDTC suppressed IL-8 mRNA expression, but only weakly (Fig. 5 A, B, E), while LY294002 and AZD1480 did not suppress IL-8 mRNA expression (Fig. 5 C, D). These results indicate that the activation of the MAPK/ERK, SAPK/JNK, PI3K/Akt, NF-κB, and JAK/STAT3 pathways may be involved in TPA-induced IL-6 mRNA expression in HuH-7 cells. In addition, the present results indicate that the regulatory mechanism of IL-6 is different from that of IL-8 in TPA-treated HuH-7 cells. Time-course experiments for the detection of intracellular signaling molecules in TPA-treated HuH-7 cells In Fig. 5 , we showed that the activation of the MAPK/ERK, SAPK/JNK, PI3K/Akt, NF-κB/IκB, and JAK/STAT3 pathways may be involved in TPA-induced IL-6 mRNA expression in HuH-7 cells. For the detection of the phosphorylation of intracellular signaling molecules, it is important to examine their time-courses and to determine their inhibition kinetics. The phosphorylation of ERK1/2 was induced by TPA and inhibited by U0126 at all time points examined (15 min, 1 h, and 2 h), although U0126 inhibition was most prominent at 15 min (Fig. 6 A). The phosphorylation of SAPK/JNK was induced by TPA and inhibited by SP600125 only at 2 h (Fig. 6 B). The phosphorylation of Akt (Ser473) was induced by TPA and inhibited by LY294002 only at 15 min (Fig. 6 C). The phosphorylation of STAT3 (Tyr705) was not induced by TPA, but was inhibited by AZD1480 at all time points (15 min, 1 h, 2 h, Fig. 6 D). In contrast, IκB was degraded with TPA treatment, and PDTC clearly inhibited the degradation of IκB at 15 min (Fig. 6 E). The total protein levels of these signaling molecules and actin remained unchanged. Based on the results obtained in the present experiments, we next examined the effects of isofraxidin on intracellular signaling molecules. Effects of isofraxidin on intracellular signaling molecules in TPA-treated HuH-7 cells In order to elucidate the mechanisms responsible for the suppressive effect of isofraxidin on TPA-induced IL-6 mRNA expression, we examined the effects of isofraxidin on the phosphorylation of intracellular signaling molecules, nuclear translocation of NF-κB p65, and degradation of IκB using western blotting. As shown in Fig. 7 A and B, TPA significantly induced the nuclear translocation of NF-κB p65 and degradation of IκB. However, isofraxidin did not inhibit these effects at the concentration range studied (25–200 µM). Furthermore, isofraxidin did not inhibit TPA-induced phosphorylation of SAPK/JNK, Akt (Ser473), and STAT3 (Tyr705). On the other hand, isofraxidin inhibited TPA-induced ERK1/2 phosphorylation in a dose-dependent manner. The phosphorylation of Akt (Thr308) was not induced by TPA and was not inhibited by isofraxidin. The total protein levels of these signaling molecules and actin remained unchanged. These results indicate that isofraxidin selectively inhibits the MAPK/ERK pathway among several signaling pathways examined in TPA-treated HuH-7 cells. Discussion IL-6 is a pleiotropic cytokine with many biological activities, including inflammation, hematopoiesis, bone metabolism, embryonic development, and other fundamental processes [ 28 ]. Recently, IL-6 was considered to be a therapeutic target for inhibiting cytokine storm [ 1 ]. To date, although an anti-IL-6R antibody, dexamethasone, mRNA vaccines, and antiviral agents have been used for the treatment of COVID-19 [ 1 , 11 , 29 , 30 ], only a few of these agents (anti-IL-6R antibody [ 1 ] and dexamethasone [ 11 ]) have been shown to be effective for cytokine storm. In the present study, we investigated the suppressive effect of isofraxidin on the overexpression of IL-6 and its regulatory mechanism. The results of the present study suggest that isofraxidin may be an effective agent for the treatment of cytokine storm. Although TPA is one of the most utilized tumor promoters used to study the mechanisms of carcinogenesis [ 31 ], it is also frequently used as a stimulus for anti-inflammatory research because of its ability to induce inflammatory reactions [ 32 , 33 ]. Compared to viral infection experiments with SARS-CoV-2, in vitro experiments using TPA have the advantages of safety and simplicity. When HuH-7 and HepG2 cells were treated with TPA, a marked induction of IL-6 mRNA expression was observed in HuH-7 cells compared with HepG2 cells, although IL-6 induction was significantly elevated in both cell lines. Similarly, TPA was found to markedly activate several intracellular signaling pathways and induce the expression of IL-1β, TNF-α, and IL-8 mRNAs in HuH-7 cell, confirming that TPA is a useful stimulator for the present study. In the present study, the effect of isofraxidin on TPA-induced mRNA expression of several pro-inflammatory cytokines was evaluated using quantitative real-time PCR, and IL-6 mRNA expression was found to be significantly suppressed in a dose-dependent manner. In contrast, IL-1β and TNF-α mRNA expression were suppressed only at 200 µM. Moreover, the results also indicated that the suppressive effect of isofraxidin on TPA-induced IL-6 mRNA expression is more evident in HuH-7 cells than in HepG2 cells, suggesting that the effect of isofraxidin may exhibit the cell type differences. Thus, we have used TPA-treated HuH-7 cells as an in vitro model for further studies. Similarly, HuH-7 cells have been used in studies targeting the inhibition of cytokine storm in COVID-19 [ 4 ]. As described above, dexamethasone has been used as a therapeutic for treating cytokine storm [ 11 ]. Comparisons of isofraxidin with dexamethasone in the present study showed that both agents suppress TPA-induced IL-6 mRNA expression in HuH-7 cells at 50–200 µM. However, a study using the A549 human lung cancer cell line, showed that dexamethasone suppressed IL-6 mRNA expression at 10–10,000 nM following IL-1β stimulation [ 34 ]. Determining whether isofraxidin suppresses IL-6 mRNA expression at lower concentrations (less than 50 µM) in HuH-7 cells under improved culture conditions is under investigation. It is known that intracellular signaling pathways, such as MAPK/ERK, SAPK/JNK, PI3K/Akt, NF-κB/IκB, and JAK/STAT3, are involved in the transcriptional regulation of the IL-6 gene [ 4 , 8 , 17 , 33 , 35 – 37 ]. Among these intercellular signaling molecules, isofraxidin only inhibited the TPA-induced phosphorylation of ERK1/2. We previously reported that isofraxidin suppresses TPA-induced MMP-7 expression in HuH-7 cells by inhibiting the phosphorylation of ERK1/2, but not of SAPK/JNK or p38 and activation of transcription factors AP-1 and NF-κB [ 22 ]. However, other investigators have reported that isofraxidin inhibits the PI3K/Akt, SAPK/JNK, and NF-κB/IκB pathways in studies using human lung cancer cells and others [ 20 , 21 , 23 , 24 , 25 , 26 ]. However, the present results shown in Fig. 7 indicated no inhibition of these signaling molecules by isofraxidin. These findings suggest that isofraxidin suppresses TPA-induced overexpression of IL-6 mRNA through selective inhibition of the MAPK/ERK pathway in HuH-7 cells. Recently, it has been reported that the MEK1/2 inhibitor ATR-002 alleviates SARS-CoV-2-induced expression of pro-inflammatory cytokines and chemokines [ 8 ], indicating that the inhibition of MAPK/ERK pathway may be useful in the treatment of cytokine storm. In addition, the PI3K/Akt, NF-κB/IκB, and JAK-STAT3 pathways were also reported to be important in the development of cytokine storm [ 4 , 5 , 7 ]. Taken together, use of isofraxidin alone or in combination with other agents that inhibit signaling pathways other than MAPK/ERK, may represent a more effective treatment of cytokine storm. Recently, the anti-inflammatory potential of phytochemical compounds, as well as their underlying mechanisms, have been actively studied. For example, luteolin, a flavonoid compound of celery or green pepper, suppresses IL-6 mRNA expression and protein production through the inhibition of phosphorylation of JNK and activity of AP-1 [ 37 ]. EGCG inhibits IL-6 synthesis and suppresses trans-signaling by inducing the production of soluble gp130 [ 16 ]. Curcumin, an active compound of turmeric, suppresses IL-6 expression by inhibiting the nuclear translocation of NF-κB p65 [ 17 ]. The present results suggest that the molecular mechanism by which isofraxidin suppresses IL-6 expression may be different from that of the aforementioned phytochemicals, although further studies are needed to verify this possibility. MicroRNAs (miRNAs) have been revealed to be involved in the regulation of activation and specificity of the MAPK/ERK pathway, with several miRNAs reported to decrease the activity of the MAPK/ERK pathway [ 38 ]. In gall bladder cancer cells, miR-29c-5p induces apoptosis through reducing the phosphorylation of MEK1/2, ERK1/2, and Akt (Ser473) [ 39 ]. MiR-101 is a down-regulated miRNA in hepatocellular carcinoma cells. The overexpression of miR-101 suppresses the proliferation of HepG2 cells through decreasing the activity of ERK1/2 [ 40 ]. MiR-148a inhibits the proliferation and invasion of esophagus squamous cell carcinoma cells through targeting MAP3K9 by the MAPK/ERK pathway [ 41 ]. Whether and how miRNAs are involved in the suppressing expression of IL-6 induced by isofraxidin is under investigation. In conclusion, isofraxidin suppresses the TPA-induced overexpression of IL-6 mRNA by selectively inhibiting the activation of the MAPK/ERK pathway in HuH-7 cells, indicating that isofraxidin may be an effective anti-inflammatory agent for treating cytokine storm. Declarations Author contributions TY and TT designed the study. TY performed the experiments and analyzed the data. All authors read and approved the final manuscript. Compliance with ethical standards Conflict of interest The authors declare that they have no conflict of interest. References Kang S, Tanaka T, Inoue H, et al. IL-6 trans-signaling induces plasminogen activator inhibitor-1 from vascular endothelial cells in cytokine release syndrome. PNAS. 2020; 117:22351-6. Del Valle-Mendoza J, Tarazona-Castro Y, Merino-Luna A, et al. Comparison of cytokines levels among COVID-19 patients living at sea level and high altitude. BMC Infect Dis. 2022; https:// doi.org/10.1186/s12879-022-07079-x Zhang F, Guo F, Zhang Y, et al. Huashibaidu formula attenuates sepsis-induced acute lung injury via suppressing cytokine storm: Implications for treatment of COVID-19. Phytomedicine. 2023; https:// doi.org/10.1016/j.phymed.2022.154549 Roshdy WH, Rashed HA, Kandeil A, et al. EGYVIR: An immunomodulatory herbal extract with potent antiviral activity against SARS-CoV-2. PLoS One. 2020; https:// doi.org/10.1371/journal.pone.0241739 Fakhri S, Nouri Z, Moradi SZ, et al. Targeting multiple signal transduction pathways of SARS-CoV-2: Approaches to COVID-19 therapeutic candidates. Molecules. 2021; https:// doi.org/10.3390/molecules26102917 Rex DAB, Dagamajalu S, Kandasamy RL, Raju R, Keshava Prasad TS. SARS-CoV-2 signaling pathway map: A functional landscape of molecular mechanisms in COVID-19. J Cell Commun Signal. 2021; 15:601-8. Fattahi S, Khalifehzadeh-Esfahani Z, Mohammad-Rezaei M, Mafi S, Jafarinia M. PI3K/Akt/mTOR pathway: A potential target for anti-SARS-CoV-2 therapy. Immunol Res. 2022; 70:269-75. Schreiber A, Viemann D, Schöning J, et al. The MEK1/2-inhibitor ATR-002 efficiently blocks SARS-CoV-2 propagation and alleviates pro-inflammatory cytokine/chemokine responses. Cell Mol Life Sci. 2022; https://doi.org/10.1007/s00018-021-04085-1 Prasad P, Verma S, Surbhi, et al. Rheumatoid arthritis: Advances in treatment strategies. Mol Cell Biochem. 2023; 478:69–88. Vandewalle J, Libert C. Glucocorticoids in sepsis: To be or not to be. Front Immunol. 2020; https://doi.org/10.3389/fimmu.2020.01318 Ripani U, Bisaccia M, Meccariello L. Dexamethasone and nutraceutical therapy can reduce the myalgia due to COVID-19 - a systemic review of the active substances that can reduce the expression of interlukin-6. Med Arch. 2022; 76:66-71. Hong H, Jang BC. Prednison inhibits the IL-1β-induced expression of COX-2 in HEI-OC1 murine auditory cells through the inhibition of ERK-1/2, JNK-1 and AP-1 activity. Int J Mol Med. 2014; 34: 1640-6. Timmermans S, Souffriau J, Libert C. A general introduction to glucocorticoid biology. Front Immunol. 2019; https://doi.org/10: 1545. 10.3389/fimmu.2019.01545 Wang Y, Wang X, Yang Y, et al. Comparison of the in vitro anti-inflammatory effect of cannabidiol to dexamethasone. Clin Cosmet Investig Dermatol. 2022; 15:1959-67 . Furst R, Zundorf I. Plant-derived anti-inflammatory compounds: Hopes and disappointments regarding the translation of preclinical knowledge into clinical progress. Mediators Inflamm. 2014; https://doi.org/10.1155/2014/146832 Ahmed S, Marotte H, Kwan K, et al. Epigallocatechin-3-gallate inhibits IL-6 synthesis and suppresses transsignaling by enhancing soluble gp130 production. PNAS. 2008; 105:14692-8. Jin CY, Lee JD, Park C, Choi YH, Kim GY. Curcumin attenuates the release of pro-inflammatory cytokines in lipopolysaccharide-stimulated BV2 microglia. Acta Pharmacol Sin. 2007; 28:1645-51. Yamazaki T, Shimosaka S, Sasaki H, Matsumura T, Tukiyama T, Tokiwa T. (+)-Syringaresinol-di-O-beta-D-glucoside, a phenolic compound from Acanthopanax senticosus Harms, suppresses proinflammatory mediators in SW982 human synovial sarcoma cells by inhibiting activating protein-1 and/or nuclear factor-kappaB activities. Toxicol In Vitro. 2007; 21:1530-7. Niu X, Xing W, Li W, Fan T, Hu H, Li Y. Isofraxidin exhibited anti-inflammatory effects in vivo and inhibited TNF-α production in LPS-induced mouse peritoneal macrophages in vitro via the MAPK pathway. Int Immunopharmacol. 2012; 14:164-71. Liu L, Mu Q, Li W, et al. Isofraxidin protects mice from LPS challenge by inhibiting pro-inflammatory cytokines and alleviating histopathological changes. Immunobiology. 2015; 220:406-13. Lin J, Li X, Qi W, et al. Isofraxidin inhibits interleukin-1β induced inflammatory response in human osteoarthritis chondrocytes. Int Immunopharmacol. 2018; 64:238-45. Yamazaki T, Tokiwa T. Isofraxidin, a coumarin component from Acanthopanax senticosus, inhibits matrix metalloproteinase-7 expression and cell invasion of human hepatoma cells. Biol Pharm Bull. 2010; 33:1716-22. Shen P, Wang HG, Li MM, et al. Isofraxidin inhibited proliferation and induced apoptosis via blockage of Akt pathway in human colorectal cancer cells. Biomed Pharmacother. 2017; 92:78-85. Zhang H, Feng QQ, Gong JH, Ma JP. Anticancer effects of isofraxidin against A549 human lung cancer cells via the EGFR signaling pathway. Mol Med Rep. 2018; 18:407-14. Li P, Zhao QL, Wu LH, et al. Isofraxidin, a potent reactive oxygen species (ROS) scavenger, protects human leukemia cells from radiation-induced apoptosis via ROS/mitochondria pathway in p53-independent manner. Apoptosis. 2014; 19:1043-53. Wu X, Gu J, Zou Z, et al. Suppressive effects of isofraxidin on depressive-like behaviors induced by chronic unpredictable mild stress in mice. Brain Sci. 2022; 12:1-12. Chen C, Song X, Lin D, Xu P. Isofraxidin alleviates myocardial infarction through NLRP3 inflammasome inhibition. Inflammation. 2020; 43:712-21. Hirano T. IL-6 in inflammation, autoimmunity and cancer. Int Immunol. 2021; 33:127-48. Szabó GT, Mahiny AJ, Vlatkovic I. COVID-19 mRNA vaccines: Platforms and current developments. Mol Ther. 2022; 30:1850-68. Lee TC, Murthy S, Del Corpo O, et al. Remdesivir for the treatment of COVID-19: A systematic review and meta-analysis. Clin Microbiol Infect. 2022; 28:1203-10. Zhao Z, Sun YS, Chen W, Lv LX, Li YQ. Hispolon inhibits breast cancer cell migration by reversal of epithelial-to-mesenchymal transition via suppressing the ROS/ERK/Slug/E-cadherin pathway. Oncol Rep. 2016; 35:896-904. Khan AQ, Khan R, Rehman MU, et al. Soy isoflavones (daidzein & genistein) inhibit 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced cutaneous inflammation via modulation of COX-2 and NF-κB in Swiss albino mice. Toxicology. 2012; 302:66-74. Balkrishna A, Nain P, Chauhan A, et al. Super critical fluid extracted fatty acids from Withania somnifera seeds repair psoriasis-like skin lesions and attenuate pro-inflammatory cytokines (TNF-α and IL-6) release. Biomolecules. 2020; https:// doi.org/10.3390/biom10020185 Chen Y, Zhang C, Xiao CX, et al. Dexamethasone can attenuate the pulmonary inflammatory response via regulation of the lncH19/miR-324-3p cascade. J Inflamm (Lond). 2021; https://doi.org/10.1186/s12950-020-00266-0 Liu T Zheng S, Guo P. Effect of torin1 on suppressing inflammation in mice with dextran sodium sulfate-induced colitis. Int Clin Exp Med. 2017; 10:4723-31. Kim JH, Choi HS, Kim SL, Lee DS. The PAK1-Stat3 signaling pathway activates IL-6 gene transcription and human breast cancer stem cell formation. Cancers (Basel). 2019; https://doi.org/10.3390/cancers11101527 Jang S, Lelley KW, Johnson RW. Luteolin reduces IL-6 production in microglia by inhibiting JNK phosphorylation and activation of AP-1. PNAS. 2008; 105:7534-9. Safa A, Abak A, Shoorei H, Taheri M, Ghafouri-Fard S. MicroRNAs as regulators of ERK/MAPK pathway: A comprehensive review. Biomed Pharmacother. 2020; https://doi.org/10.1016/j.biopha.2020.110853 Shu YJ, Bao RF, Jiang L, et al. MicroRNA-29c-5p suppresses gallbladder carcinoma progression by directly targeting CPEB4 and inhibiting the MAPK pathway. Cell Death Differ. 2017; 24:445-57. Meng X, Shi Y, Xiang X, et al. Influence of miR-101 on proliferation of liver cancer cells through the MAPK/ERK signaling pathway. Oncol Lett. 2020; 19:1310-6. Zhang BX, Yu T, Yu Z, Yang XG. MicroRNA-148a regulates the MAPK/ERK signaling pathway and suppresses the development of esophagus squamous cell carcinoma via targeting MAP3K9. Eur Rev Med Pharmacol Sci. 2019; 23:6497-504. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3935003","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":271558445,"identity":"f7a54c79-30d9-4479-841a-44d1d5405869","order_by":0,"name":"Taisuke Yamazaki","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA2klEQVRIiWNgGAWjYDADCWbmwyAKzDHAp5IHoYUtmVQtDDzGxLnHnoHH8NONmjp5yXaezwY/cywY+KUPMBQX4LWFx1g65xib4Wxm3s2JvdskGCT7EhiMZ+DXYiCdw8bDOA+o5TAjUIvBGQYGYx78Wox/5/yTsJ/HzPOYaC1m0rltBomzmXmYk4nTcpitzDq3LyF5ZjObsSHQLzySPYwNeP3C3t68+XbOtzrbGecPP5b4ua1Ojp+H+ZgxvhBjYOZAjTegkxjbCMQQ+wNMYx7j1zIKRsEoGAUjDAAAv7851jsosVkAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0001-7669-0341","institution":"Kohno Clinical Medicine Research Institute","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Taisuke","middleName":"","lastName":"Yamazaki","suffix":""},{"id":271558446,"identity":"a981b8ba-02c6-4729-a5eb-b707a6f4808c","order_by":1,"name":"Takayoshi Tokiwa","email":"","orcid":"","institution":"Kohno Clinical Medicine Research Institute","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Takayoshi","middleName":"","lastName":"Tokiwa","suffix":""}],"badges":[],"createdAt":"2024-02-06 21:54:37","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3935003/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3935003/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":50921870,"identity":"4c107cd4-3f9e-480b-8912-7b626607038d","added_by":"auto","created_at":"2024-02-09 16:06:12","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":360869,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of isofraxidin on TPA-induced IL-6 mRNA expression. HuH-7 and HepG2 cells were treated with 100 nM TPA and various concentrations of isofraxidin for 2 h. IL-6 mRNA expression was determined by quantitative real-time PCR. Levels are expressed relative to those of untreated cells, which were arbitrarily set at 1. Values represent the mean±standard deviation of three independent experiments. Statistical significance was determined by analysis of variance (ANOVA) followed by Tukey’s post hoc test for multiple comparison (*\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, **\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01 compared with the TPA treatment only).\u003c/p\u003e","description":"","filename":"Iso1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3935003/v1/680811049a7bf9a2e04d4afe.jpg"},{"id":50921332,"identity":"1361044b-560c-49d1-b26b-d36fcfac2c1f","added_by":"auto","created_at":"2024-02-09 15:58:12","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":484279,"visible":true,"origin":"","legend":"\u003cp\u003eTime-course experiments of IL-6 mRNA expression.\u003cstrong\u003e A \u003c/strong\u003eQuantitative real time-PCR analysis. HuH-7 cells were treated with 100 nM TPA and various concentrations of isofraxidin for 2, 8, and 24 h. IL-6 mRNA expression was determined by quantitative real-time PCR. Relative expression levels were calculated as describe in the legend to Fig. 1. \u003cstrong\u003eB \u003c/strong\u003eWestern blot analysis of IL-6. HuH-7 cells were treated with 100 nM TPA and various concentrations of isofraxidin for 24 h. IL-6 protein expression was determined by western blotting. Results are representative of two independent experiments. \u003cstrong\u003eC\u003c/strong\u003e MTT assay. The cell viability of HuH-7 cells was evaluated using MTT assay.\u003c/p\u003e","description":"","filename":"Iso2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3935003/v1/bf7ebef79cc07639f2faf83f.jpg"},{"id":50921334,"identity":"fc1a0b27-4678-4f40-b19a-8526ea56b996","added_by":"auto","created_at":"2024-02-09 15:58:12","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":411675,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of isofraxidin on the TPA-induced mRNA expression of other pro-inflammatory cytokines and chemokaines. HuH-7 cells were treated with 100 nM TPA and various concentrations of isofraxidin for 2 h. The mRNA expression of pro-inflammatory cytokines and chemokines was determined by quantitative real-time PCR. Relative expression levels were calculated as describe in the legend to Fig. 1.\u003c/p\u003e","description":"","filename":"Iso3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3935003/v1/1839755dbbe6049f38791395.jpg"},{"id":50921330,"identity":"748a18a4-c8f7-4a56-91b9-e395ca1c9fde","added_by":"auto","created_at":"2024-02-09 15:58:11","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":328032,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of the suppressive effect of isofraxidin on TPA-induced IL-6 mRNA expression with dexamethasone. HuH-7 cells were treated with 100 nM TPA and various concentrations of isofraxidin or dexamethasone for 2 h. IL-6 mRNA expression was determined by quantitative real-time PCR. Relative expression levels were calculated as describe in the legend to Fig. 1.\u003c/p\u003e","description":"","filename":"Iso4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3935003/v1/762d34a1acbc13476fed4913.jpg"},{"id":50921331,"identity":"bf73ee07-f9fe-4d18-a163-6184a0bae5ed","added_by":"auto","created_at":"2024-02-09 15:58:12","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":600068,"visible":true,"origin":"","legend":"\u003cp\u003eInvolvement of intracellular signaling pathways in TPA-induced IL-6 and IL-8 mRNA expression. HuH-7 cells were treated with 100 nM TPA and various concentrations of each signal transduction inhibitors for 2 h. IL-6 and IL-8 mRNA expression were determined by quantitative real-time PCR. Relative expression levels were calculated as describe in the legend to Fig. 1.\u003c/p\u003e","description":"","filename":"Iso5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3935003/v1/2530fde44f469086bfb9959f.jpg"},{"id":50921336,"identity":"7416266a-253c-4483-90ca-6f5d8ea0c2a5","added_by":"auto","created_at":"2024-02-09 15:58:12","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":798700,"visible":true,"origin":"","legend":"\u003cp\u003eTime-course experiments for the detection of intracellular signaling molecules. HuH-7 cells were treated with 100 nM TPA and various concentrations of each signal transduction inhibitors for 15 min, 1h, and 2 h. The phosphorylation of intracellular signaling molecules and degradation of IκB were determined by western blotting. Results are representative of two independent experiments.\u003c/p\u003e","description":"","filename":"Iso6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3935003/v1/895708d843419fda1838789b.jpg"},{"id":50921335,"identity":"0b70c73c-644a-414b-baee-576c6f67889e","added_by":"auto","created_at":"2024-02-09 15:58:12","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":2348060,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of isofraxidin on intracellular signaling molecules. \u003cstrong\u003eA\u003c/strong\u003e Western blot analysis of intracellular signaling molecules.\u003cstrong\u003e B\u003c/strong\u003e Densitometric analysis of western blotting results. HuH-7 cells were treated with 100 nM TPA and various concentrations of isofraxidin for 15 min (ERK1/2, Akt, STAT3, NF-κB p65, and IκB) and 2 h (SAPK/JNK). The phosphorylation of intracellular signaling molecules, nuclear translocation of NF-κB p65, and degradation of IκB were determined by western blotting. Protein band intensities were quantitated by Image J. Levels are expressed relative to those of untreated cells, which were arbitrarily set at 1. Values represent the mean±standard deviation of three independent experiments. Statistical significance was determined by analysis of variance (ANOVA) followed by Tukey’s post hoc test for multiple comparison (*\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, **\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01 compared with the TPA treatment only).\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-3935003/v1/3aa60798a16e186c29fc5965.png"},{"id":51322247,"identity":"c00262e4-8b55-4415-8af3-9b823c22851b","added_by":"auto","created_at":"2024-02-19 14:51:27","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":994761,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3935003/v1/53dd3ccf-1bdb-4d5e-a06b-68026f10e183.pdf"}],"financialInterests":"","formattedTitle":"Suppressive effect of isofraxidin on the overexpression of IL-6 and its molecular mechanism","fulltext":[{"header":"Introduction","content":"\u003cp\u003eCytokine storm is a systemic inflammatory response that is commonly triggered by bacteria or viral infections and chimeric antigen receptor T cell therapy, and is one of the leading causes of morbidity and mortality in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-induced coronavirus disease 2019 (COVID-19) [\u003cspan additionalcitationids=\"CR2 CR3\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Cytokine storm is characterized by the overexpression and excessive production of pro-inflammatory cytokines, such as interleukin-1 (IL-1), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α), which may cause acute respiratory distress syndrome (ARDS) and subsequent severe tissue damage [\u003cspan additionalcitationids=\"CR2 CR3\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Among the pro-inflammatory cytokines, IL-6 is one of the most important cytokines involved in the initiation of cytokine storm, and induces other pro-inflammatory cytokines and the marker of endothelial injury plasminogen activator inhibitor-1 (PAI-1). Therefore, IL-6 is considered to be a therapeutic target for inhibiting cytokine storm. In fact, the blockade of IL-6 trans-signaling using a humanized monoclonal antibody against human IL-6 receptor (anti-IL-6R antibody) has been shown to be effective in patients with severe COVID-19 [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eMoreover, it has been revealed that intracellular signaling pathways, such as MAPK/ERK, SAPK/JNK, PI3K/Akt, NFκ-B/IκB, and JAK/STAT3, are activated in host cells infected with SARS-CoV-2, resulting in the overexpression and excessive production of pro-inflammatory cytokines, such IL-6, in host cells [\u003cspan additionalcitationids=\"CR5 CR6 CR7\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Therefore, the inhibition of these signaling pathways using pharmacological agents may be an effective therapeutic strategy for counteracting cytokine storm in addition to the blockade of IL-6 trans-signaling using an anti-IL-6R antibody. However, only a limited number of pharmacological agents are available for the treatment of cytokine storm.\u003c/p\u003e \u003cp\u003eGlucocorticoids have been used in the treatment of inflammation-related diseases, such as rheumatoid arthritis, sepsis, and COVID-19 [\u003cspan additionalcitationids=\"CR10\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Glucocorticoids bind intracellular glucocorticoid receptors (GRs) and subsequently downregulate the expression of pro-inflammatory cytokines by inhibiting the activation of intracellular signaling pathways and transcriptional factors [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. However, the anti-inflammatory effects of glucocorticoids are often accompanied by several intolerable adverse effects, including indigestion, vomiting, weight gain, osteoporosis, hyperglycemia, cardiovascular diseases, and infections [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Thus, it is urgent to develop new anti-inflammatory agents that are effective for cytokine storm and exhibit tolerable adverse effects. Phytochemical compounds have become the primary sources for the discovery of new anti-inflammatory agents, due to their ability to alleviate inflammation with minimal side effects [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. To date, the anti-inflammatory effects of various phytochemical compounds, including epigallocatechin-3-gallate (EGCG) and curcumin, have been reported [\u003cspan additionalcitationids=\"CR16\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIsofraxidin is a well-known coumarin compound that is naturally present plants, including \u003cem\u003eAcanthopanax senticosus\u003c/em\u003e, and has been reported to exhibit several pharmacological activities, such as anti-inflammatory, anti-cancer, anti-depressive, anti-oxidant, and cardio-protective effects [\u003cspan additionalcitationids=\"CR19 CR20 CR21 CR22 CR23 CR24 CR25 CR26\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. In 2007, we first reported the anti-inflammatory effects of isofraxidin using the SW982 human synovial sarcoma cell line [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. In this study, we performed an examination of the effect of isofraxidin on IL-6 using ELISA and RT-PCR methods. Recently, reports have described the anti-inflammatory effects of isofraxidin using mouse peritoneal macrophages, human osteoarthritis chondrocytes, or LPS-injected mice [\u003cspan additionalcitationids=\"CR20\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. However, these studies were not designed to elucidate the detailed molecular mechanisms underlying the effect of isofraxidin on the overexpression of IL-6. Therefore, the present study examined the effect of isofraxidin on TPA-induced IL-6 mRNA expression and activation of intracellular signaling pathways involved in the transcriptional regulation of the IL-6 gene using HuH-7 cells. The results of the present study may contribute to the development of effective treatments for cytokine storm.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eCell culture\u003c/h2\u003e \u003cp\u003eThe human hepatocellular carcinoma cell lines, HuH-7 (RRID: CVCL_0336) and HepG2 (RRID: CVCL_0027) were obtained from JCRB (Osaka, Japan) and RIKEN BRC Cell Bank (Tsukuba, Japan), respectively. The cells were authenticated by STR profiling and mycoplasma DNA was not detected by PCR. These cells were routinely maintained in DMEM medium (Gibco, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS) at 37\u0026deg;C in a humidified atmosphere containing 5% CO\u003csub\u003e2\u003c/sub\u003e and 95% air.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eTreatment of cells with isofraxidin, dexamethasone, and signal transduction inhibitors\u003c/h2\u003e \u003cp\u003eCells were plated onto 60-mm plastic dishes at a density of 3\u0026times;10\u003csup\u003e4\u003c/sup\u003e cells/cm\u003csup\u003e2\u003c/sup\u003e in DMEM medium supplemented with 10% FBS. The next day, the medium was replaced with serum-free DMEM, and the day after that, cells were treated with 100 nM TPA (Sigma, St. Louis, MO, USA) and isofraxidin or dexamethasone (FUJIFILM Wako, Osaka, Japan) at the indicated concentrations in serum-free DMEM for 2, 8, and 24 h. Untreated cells were used as controls. For the inhibition of intracellular signaling pathways, individual signal transduction inhibitors (MAPK/ERK inhibitor U0126, PI3K inhibitor LY294002, JNK inhibitor SP600125, JAK2 inhibitor AZD1480, and NF-κB inhibitor PDTC (Cayman Chemicals, Ann Arbor, MI, USA)) were added in place of isofraxidin.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eMTT assay\u003c/h2\u003e \u003cp\u003eCells were plated onto 96-well plates at a density of 3\u0026times;10\u003csup\u003e4\u003c/sup\u003e cells/cm\u003csup\u003e2\u003c/sup\u003e in DMEM supplemented with 10% FBS. The next day, the medium was replaced with serum-free DMEM, and the day after that, cells were treated with or without isofraxidin at the indicated concentrations in serum-free DMEM for 2 and 24 h. After treatment with isofraxidin, cells were incubated with MTT (FUJIFILM Wako, 10 \u0026micro;L, 5 mg/mL) for 4 h. The formazan precipitates were dissolved in 100 \u0026micro;L DMSO, and the optical density (OD) values were read at 540 nm using a microplate reader.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eTotal RNA extraction and quantitative real-time PCR\u003c/h2\u003e \u003cp\u003eTotal RNA was extracted from cells using the RNeasy Mini Kit (Qiagen, Hilden, Germany). Quantitative real-time PCR was performed with the MyGo Mini Real Time PCR system (IT-IS Life Science, Dublin, Ireland) using Precision PLUS OneStep RT-qPCR Master Mix (Primerdesign Ltd, Chandler's Ford, UK), consisting of 10 \u0026micro;L of 2\u0026times;OneStep RT-qPCR Master Mix, 0.5 \u0026micro;L of each 10 \u0026micro;M (0.25 \u0026micro;M final) forward and reverse primers, 2.5 \u0026micro;L (25 ng) of template RNA and 6.5 \u0026micro;L of RNase/DNase-free water was made to a total volume of 20 \u0026micro;L. The real-rime PCR cycling conditions were: 55\u0026deg;C for 10 min, 95\u0026deg;C for 2 min, followed by 40 cycles of 95\u0026deg;C for 10 s and 60\u0026deg;C for 1 min. The PCR primers used for amplification are as follows: IL-1β: sense 5\u0026rsquo;-ttcgacacatgggataacgagg-3\u0026rsquo;, antisense 5\u0026rsquo;-aggacatggagaacaccacttg-3; IL-6: sense 5\u0026rsquo;-gtagccgccccacacagacagcc-3\u0026rsquo;, antisense 5\u0026rsquo;-gccatctttggaaggttc-3\u0026rsquo;; TNF-α: sense 5\u0026rsquo;-tcttctcgaaccccgagtga-3\u0026rsquo;, antisense 5\u0026rsquo;-cctctgatggcaccaccag-3\u0026rsquo;; IL-8: sense 5\u0026rsquo;-ttggcagccttcctgatttc-3\u0026rsquo;, antisense 5\u0026rsquo;-atttctgtgttggcgcagtg-3\u0026rsquo;; MCP-1: sense 5\u0026rsquo;-tgctcatagcagccaccttc-3\u0026rsquo;, antisense 5\u0026rsquo;-tctccttggccacaatggtc-3\u0026rsquo;; GAPDH: sense 5\u0026rsquo;-ggtggtctcctctgacttcaaca- 3\u0026rsquo;, antisense 5\u0026rsquo;- gttgctgtagccaaattcgttgt-3\u0026rsquo;. GAPDH was used as an internal control.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eWestern blot analysis\u003c/h2\u003e \u003cp\u003eCell lysates and nuclear extracts were prepared using cell lysis buffer (Mammalian Cell PE LB, Biosciences, St. Louis, MO, USA) and nuclear extraction buffer (LysoPure Nuclear and Cytoplasmic Extraction Kit, FUJIFILM Wako), respectively. Protein concentrations were measured using a BCA Protein Assay Kit (Pierce, Rockford, IL, USA). Cell lysates (10\u0026ndash;30 \u0026micro;g) and nuclear extracts (10 \u0026micro;g) were treated with 5\u0026times;SDS sample buffer, separated by 10% SDS-polyacrylamide gels (SDS-PAGE), and then transferred to PVDF membranes. After the membranes were blocked with 2% blocking reagent in TBS-T buffer, they were incubated with 1:1000 dilution of anti-phospho (P)-ERK1/2, anti-ERK1/2, anti-P-SAPK/JNK, anti-SAPK/JNK, anti-P-Akt (Ser473, Thr308), anti-Akt, anti-P-STAT3 (Tyr705), anti-STAT3, anti-NF-κB p65, anti-IκB-α (Cell Signaling Technology, Danvers, MA, USA), anti-IL-6 (GeneTex, Taipei, Taiwan, ROC), and anti-actin (Sigma) in TBS buffer for 24 h at 4\u0026deg;C, and then incubated with 1:2000-1:10000 dilution of anti-rabbit IgG, HRP-linked antibody (Cell Signaling Technology) in TBS-T buffer for 1 h at room temperature. Actin was used as an internal control. Detection of proteins was carried out using ECL Prime Western Blotting Detection System (GE Healthcare, Little Chalfont, UK). Protein band intensities were quantitated using Image J (NIH, Bethesda, MD, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eStatistical significance was determined by analysis of variance (ANOVA) followed by Tukey\u0026rsquo;s post hoc test for multiple comparison.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eEffect of isofraxidin on TPA-induced IL-6 mRNA expression in HuH-7 and HepG2 cells\u003c/h2\u003e \u003cp\u003eIn order to elucidate whether isofraxidin suppresses the overexpression of IL-6, we first examined the effect of isofraxidin on TPA-induced IL-6 mRNA expression in HuH-7 and HepG2 cells using quantitative real-time PCR. When HuH-7 and HepG2 cells were treated with TPA for 2 h, a marked induction of IL-6 mRNA expression was observed in HuH-7 cells, whereas a modest induction was observed in HepG2 cells. In addition, isofraxidin significantly suppressed TPA-induced IL-6 mRNA expression in HuH-7 cells in a dose-dependent manner (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). However, isofraxidin only slightly suppressed TPA-induced IL-6 mRNA expression in HepG2 cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eTime-course experiments of IL-6 mRNA expression in TPA-treated HuH-7 cells\u003c/h2\u003e \u003cp\u003eIn order to determine the optimal treatment time in the suppression of IL-6 mRNA expression by isofraxidin, we performed time-course experiments. The effect of isofraxidin on TPA-induced IL-6 mRNA expression was examined in HuH-7 cells at 2, 8, and 24 h of treatment. The most marked induction by TPA and the most marked suppression by isofraxidin were observed at 2 h (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). In addition, the suppression of IL-6 protein expression was also observed at 24 h of treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Moreover, the effect of isofraxidin on cell viability of HuH-7 cells was evaluated using the MTT assay. The viability of HuH-7 cells was not significantly affected by treatment with isofraxidin at any concentration at 2 and 24 h (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC), indicating that the suppressive effect of isofraxidin on TPA-induced IL-6 mRNA expression is not due to cell toxicity. Based on these results, the following quantitative real-time PCR experiments were performed at 2 h of treatment.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eEffects of isofraxidin on TPA-induced mRNA expression of other pro-inflammatory cytokines and chemokines in HuH-7 cells\u003c/b\u003e \u003c/p\u003e \u003cp\u003eIn addition to IL-6, other pro-inflammatory cytokines (such as IL-1β and TNF-α) and chemokines (such as IL-8 and MCP-1) are also involved in cytokine storm pathogenesis. Therefore, we examined whether isofraxidin suppresses TPA-induced mRNA expression of other cytokines and chemokines. TPA treatment significantly induced the expression of IL-1β, TNF-α, and IL-8 mRNA. Isofraxidin tended to suppress IL-1β and TNF-α mRNA expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). However, no suppression of IL-8 and MCP-1 mRNA expression was observed by treatment with isofraxidin at all concentrations tested (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eComparison of the suppressive effect of isofraxidin on TPA-induced IL-6 mRNA expression with dexamethasone in HuH-7 cells\u003c/b\u003e \u003c/p\u003e \u003cp\u003eDexamethasone is a well-known synthetic glucocorticoid that is commonly used in the treatment of inflammation-related diseases. Therefore, we compared the suppressive effect of isofraxidin on TPA-induced IL-6 mRNA expression with that of dexamethasone. At 50\u0026ndash;200 \u0026micro;M, isofraxidin showed a dose-dependent suppression of TPA-induced IL-6 mRNA expression that was similar to dexamethasone (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e)\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eInvolvement of intracellular signaling pathways in TPA-induced IL-6 and IL-8 mRNA expression in HuH-7 cells\u003c/h2\u003e \u003cp\u003eIntracellular signaling pathways, such as MAPK/ERK, SAPK/JNK, PI3K/Akt, NF-κB/IκB, and JAK/STAT3, are known to be involved in the transcriptional regulation of IL-6 gene expression. Therefore, we confirmed the involvement of these signaling pathways in TPA-induced IL-6 mRNA expression in HuH-7 cells using signal transduction inhibitors. The addition of the MAPK/ERK inhibitor U0126, JNK inhibitor SP600125, Akt inhibitor LY294002, or JAK2 inhibitor AZD1480 resulted in a concentration-dependent suppression of IL-6 mRNA expression at 1\u0026ndash;25 \u0026micro;M (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, B, C, D). The NF-κB inhibitor PDTC did not suppress IL-6 mRNA expression at 1\u0026ndash;25 \u0026micro;M, but suppressed at 50\u0026ndash;200 \u0026micro;M (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE). As a reference, we also examined the effect of each inhibitor on TPA-induced IL-8 mRNA expression. U0126, SP600125, and PDTC suppressed IL-8 mRNA expression, but only weakly (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, B, E), while LY294002 and AZD1480 did not suppress IL-8 mRNA expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC, D). These results indicate that the activation of the MAPK/ERK, SAPK/JNK, PI3K/Akt, NF-κB, and JAK/STAT3 pathways may be involved in TPA-induced IL-6 mRNA expression in HuH-7 cells. In addition, the present results indicate that the regulatory mechanism of IL-6 is different from that of IL-8 in TPA-treated HuH-7 cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eTime-course experiments for the detection of intracellular signaling molecules in TPA-treated HuH-7 cells\u003c/h2\u003e \u003cp\u003eIn Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, we showed that the activation of the MAPK/ERK, SAPK/JNK, PI3K/Akt, NF-κB/IκB, and JAK/STAT3 pathways may be involved in TPA-induced IL-6 mRNA expression in HuH-7 cells. For the detection of the phosphorylation of intracellular signaling molecules, it is important to examine their time-courses and to determine their inhibition kinetics. The phosphorylation of ERK1/2 was induced by TPA and inhibited by U0126 at all time points examined (15 min, 1 h, and 2 h), although U0126 inhibition was most prominent at 15 min (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). The phosphorylation of SAPK/JNK was induced by TPA and inhibited by SP600125 only at 2 h (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). The phosphorylation of Akt (Ser473) was induced by TPA and inhibited by LY294002 only at 15 min (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). The phosphorylation of STAT3 (Tyr705) was not induced by TPA, but was inhibited by AZD1480 at all time points (15 min, 1 h, 2 h, Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD). In contrast, IκB was degraded with TPA treatment, and PDTC clearly inhibited the degradation of IκB at 15 min (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE). The total protein levels of these signaling molecules and actin remained unchanged. Based on the results obtained in the present experiments, we next examined the effects of isofraxidin on intracellular signaling molecules.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eEffects of isofraxidin on intracellular signaling molecules in TPA-treated HuH-7 cells\u003c/h2\u003e \u003cp\u003eIn order to elucidate the mechanisms responsible for the suppressive effect of isofraxidin on TPA-induced IL-6 mRNA expression, we examined the effects of isofraxidin on the phosphorylation of intracellular signaling molecules, nuclear translocation of NF-κB p65, and degradation of IκB using western blotting. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA and B, TPA significantly induced the nuclear translocation of NF-κB p65 and degradation of IκB. However, isofraxidin did not inhibit these effects at the concentration range studied (25\u0026ndash;200 \u0026micro;M). Furthermore, isofraxidin did not inhibit TPA-induced phosphorylation of SAPK/JNK, Akt (Ser473), and STAT3 (Tyr705). On the other hand, isofraxidin inhibited TPA-induced ERK1/2 phosphorylation in a dose-dependent manner. The phosphorylation of Akt (Thr308) was not induced by TPA and was not inhibited by isofraxidin. The total protein levels of these signaling molecules and actin remained unchanged. These results indicate that isofraxidin selectively inhibits the MAPK/ERK pathway among several signaling pathways examined in TPA-treated HuH-7 cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIL-6 is a pleiotropic cytokine with many biological activities, including inflammation, hematopoiesis, bone metabolism, embryonic development, and other fundamental processes [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Recently, IL-6 was considered to be a therapeutic target for inhibiting cytokine storm [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. To date, although an anti-IL-6R antibody, dexamethasone, mRNA vaccines, and antiviral agents have been used for the treatment of COVID-19 [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e], only a few of these agents (anti-IL-6R antibody [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e] and dexamethasone [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]) have been shown to be effective for cytokine storm. In the present study, we investigated the suppressive effect of isofraxidin on the overexpression of IL-6 and its regulatory mechanism. The results of the present study suggest that isofraxidin may be an effective agent for the treatment of cytokine storm.\u003c/p\u003e \u003cp\u003eAlthough TPA is one of the most utilized tumor promoters used to study the mechanisms of carcinogenesis [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], it is also frequently used as a stimulus for anti-inflammatory research because of its ability to induce inflammatory reactions [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Compared to viral infection experiments with SARS-CoV-2, \u003cem\u003ein vitro\u003c/em\u003e experiments using TPA have the advantages of safety and simplicity. When HuH-7 and HepG2 cells were treated with TPA, a marked induction of IL-6 mRNA expression was observed in HuH-7 cells compared with HepG2 cells, although IL-6 induction was significantly elevated in both cell lines. Similarly, TPA was found to markedly activate several intracellular signaling pathways and induce the expression of IL-1β, TNF-α, and IL-8 mRNAs in HuH-7 cell, confirming that TPA is a useful stimulator for the present study.\u003c/p\u003e \u003cp\u003eIn the present study, the effect of isofraxidin on TPA-induced mRNA expression of several pro-inflammatory cytokines was evaluated using quantitative real-time PCR, and IL-6 mRNA expression was found to be significantly suppressed in a dose-dependent manner. In contrast, IL-1β and TNF-α mRNA expression were suppressed only at 200 \u0026micro;M. Moreover, the results also indicated that the suppressive effect of isofraxidin on TPA-induced IL-6 mRNA expression is more evident in HuH-7 cells than in HepG2 cells, suggesting that the effect of isofraxidin may exhibit the cell type differences. Thus, we have used TPA-treated HuH-7 cells as an \u003cem\u003ein vitro\u003c/em\u003e model for further studies. Similarly, HuH-7 cells have been used in studies targeting the inhibition of cytokine storm in COVID-19 [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAs described above, dexamethasone has been used as a therapeutic for treating cytokine storm [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Comparisons of isofraxidin with dexamethasone in the present study showed that both agents suppress TPA-induced IL-6 mRNA expression in HuH-7 cells at 50\u0026ndash;200 \u0026micro;M. However, a study using the A549 human lung cancer cell line, showed that dexamethasone suppressed IL-6 mRNA expression at 10\u0026ndash;10,000 nM following IL-1β stimulation [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Determining whether isofraxidin suppresses IL-6 mRNA expression at lower concentrations (less than 50 \u0026micro;M) in HuH-7 cells under improved culture conditions is under investigation.\u003c/p\u003e \u003cp\u003eIt is known that intracellular signaling pathways, such as MAPK/ERK, SAPK/JNK, PI3K/Akt, NF-κB/IκB, and JAK/STAT3, are involved in the transcriptional regulation of the IL-6 gene [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan additionalcitationids=\"CR36\" citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Among these intercellular signaling molecules, isofraxidin only inhibited the TPA-induced phosphorylation of ERK1/2. We previously reported that isofraxidin suppresses TPA-induced MMP-7 expression in HuH-7 cells by inhibiting the phosphorylation of ERK1/2, but not of SAPK/JNK or p38 and activation of transcription factors AP-1 and NF-κB [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. However, other investigators have reported that isofraxidin inhibits the PI3K/Akt, SAPK/JNK, and NF-κB/IκB pathways in studies using human lung cancer cells and others [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. However, the present results shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e indicated no inhibition of these signaling molecules by isofraxidin. These findings suggest that isofraxidin suppresses TPA-induced overexpression of IL-6 mRNA through selective inhibition of the MAPK/ERK pathway in HuH-7 cells. Recently, it has been reported that the MEK1/2 inhibitor ATR-002 alleviates SARS-CoV-2-induced expression of pro-inflammatory cytokines and chemokines [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], indicating that the inhibition of MAPK/ERK pathway may be useful in the treatment of cytokine storm. In addition, the PI3K/Akt, NF-κB/IκB, and JAK-STAT3 pathways were also reported to be important in the development of cytokine storm [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Taken together, use of isofraxidin alone or in combination with other agents that inhibit signaling pathways other than MAPK/ERK, may represent a more effective treatment of cytokine storm.\u003c/p\u003e \u003cp\u003eRecently, the anti-inflammatory potential of phytochemical compounds, as well as their underlying mechanisms, have been actively studied. For example, luteolin, a flavonoid compound of celery or green pepper, suppresses IL-6 mRNA expression and protein production through the inhibition of phosphorylation of JNK and activity of AP-1 [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. EGCG inhibits IL-6 synthesis and suppresses trans-signaling by inducing the production of soluble gp130 [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Curcumin, an active compound of turmeric, suppresses IL-6 expression by inhibiting the nuclear translocation of NF-κB p65 [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. The present results suggest that the molecular mechanism by which isofraxidin suppresses IL-6 expression may be different from that of the aforementioned phytochemicals, although further studies are needed to verify this possibility.\u003c/p\u003e \u003cp\u003eMicroRNAs (miRNAs) have been revealed to be involved in the regulation of activation and specificity of the MAPK/ERK pathway, with several miRNAs reported to decrease the activity of the MAPK/ERK pathway [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. In gall bladder cancer cells, miR-29c-5p induces apoptosis through reducing the phosphorylation of MEK1/2, ERK1/2, and Akt (Ser473) [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. MiR-101 is a down-regulated miRNA in hepatocellular carcinoma cells. The overexpression of miR-101 suppresses the proliferation of HepG2 cells through decreasing the activity of ERK1/2 [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. MiR-148a inhibits the proliferation and invasion of esophagus squamous cell carcinoma cells through targeting MAP3K9 by the MAPK/ERK pathway [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Whether and how miRNAs are involved in the suppressing expression of IL-6 induced by isofraxidin is under investigation.\u003c/p\u003e \u003cp\u003eIn conclusion, isofraxidin suppresses the TPA-induced overexpression of IL-6 mRNA by selectively inhibiting the activation of the MAPK/ERK pathway in HuH-7 cells, indicating that isofraxidin may be an effective anti-inflammatory agent for treating cytokine storm.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor contributions\u0026nbsp;\u003c/strong\u003eTY and TT designed the study. TY performed the experiments and analyzed the data. All authors read and approved the final manuscript. \u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompliance with ethical standards\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eConflict of interest\u0026nbsp;\u003c/strong\u003eThe authors declare that they have no conflict of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eKang S, Tanaka T, Inoue H, et al. IL-6 trans-signaling induces plasminogen activator inhibitor-1 from vascular endothelial cells in cytokine release syndrome. PNAS. 2020; 117:22351-6.\u003c/li\u003e\n\u003cli\u003eDel Valle-Mendoza J, Tarazona-Castro Y, Merino-Luna A, et al. Comparison of cytokines levels among COVID-19 patients living at sea level and high altitude. BMC Infect Dis. 2022; https:// doi.org/10.1186/s12879-022-07079-x\u003c/li\u003e\n\u003cli\u003eZhang F, Guo F, Zhang Y, et al. Huashibaidu formula attenuates sepsis-induced acute lung injury via suppressing cytokine storm: Implications for treatment of COVID-19. Phytomedicine. 2023; https:// doi.org/10.1016/j.phymed.2022.154549\u003c/li\u003e\n\u003cli\u003eRoshdy WH, Rashed HA, Kandeil A, et al. EGYVIR: An immunomodulatory herbal extract with potent antiviral activity against SARS-CoV-2. PLoS One. 2020; https:// doi.org/10.1371/journal.pone.0241739\u003c/li\u003e\n\u003cli\u003eFakhri S, Nouri Z, Moradi SZ, et al. Targeting multiple signal transduction pathways of SARS-CoV-2: Approaches to COVID-19 therapeutic candidates. Molecules. 2021; https:// doi.org/10.3390/molecules26102917\u003c/li\u003e\n\u003cli\u003eRex DAB, Dagamajalu S, Kandasamy RL, Raju R, Keshava Prasad TS. SARS-CoV-2 signaling pathway map: A functional landscape of molecular mechanisms in COVID-19. J Cell Commun Signal. 2021; 15:601-8.\u003c/li\u003e\n\u003cli\u003eFattahi S, Khalifehzadeh-Esfahani Z, Mohammad-Rezaei M, Mafi S, Jafarinia M. PI3K/Akt/mTOR pathway: A potential target for anti-SARS-CoV-2 therapy. Immunol Res. 2022; 70:269-75.\u003c/li\u003e\n\u003cli\u003eSchreiber A, Viemann D, Sch\u0026ouml;ning J, et al. The MEK1/2-inhibitor ATR-002 efficiently blocks SARS-CoV-2 propagation and alleviates pro-inflammatory cytokine/chemokine responses. Cell Mol Life Sci. 2022; https://doi.org/10.1007/s00018-021-04085-1 \u003c/li\u003e\n\u003cli\u003ePrasad P, Verma S, Surbhi, et al. Rheumatoid arthritis: Advances in treatment strategies. Mol Cell Biochem. 2023; 478:69\u0026ndash;88.\u003c/li\u003e\n\u003cli\u003eVandewalle J, Libert C. Glucocorticoids in sepsis: To be or not to be. Front Immunol. 2020; https://doi.org/10.3389/fimmu.2020.01318\u003c/li\u003e\n\u003cli\u003eRipani U, Bisaccia M, Meccariello L. Dexamethasone and nutraceutical therapy can reduce the myalgia due to COVID-19 - a systemic review of the active substances that can reduce the expression of interlukin-6. Med Arch. 2022; 76:66-71.\u003c/li\u003e\n\u003cli\u003eHong H, Jang BC. Prednison inhibits the IL-1\u0026beta;-induced expression of COX-2 in HEI-OC1 murine auditory cells through the inhibition of ERK-1/2, JNK-1 and AP-1 activity. Int J Mol Med. 2014; 34: 1640-6.\u003c/li\u003e\n\u003cli\u003eTimmermans S, Souffriau J, Libert C. A general introduction to glucocorticoid biology. Front Immunol. 2019; https://doi.org/10: 1545. 10.3389/fimmu.2019.01545\u003c/li\u003e\n\u003cli\u003eWang Y, Wang X, Yang Y, et al. Comparison of the in vitro anti-inflammatory effect of cannabidiol to dexamethasone. Clin Cosmet Investig Dermatol. 2022; 15:1959-67 .\u003c/li\u003e\n\u003cli\u003eFurst R, Zundorf I. Plant-derived anti-inflammatory compounds: Hopes and disappointments regarding the translation of preclinical knowledge into clinical progress. Mediators Inflamm. 2014; https://doi.org/10.1155/2014/146832\u003c/li\u003e\n\u003cli\u003eAhmed S, Marotte H, Kwan K, et al. Epigallocatechin-3-gallate inhibits IL-6 synthesis and suppresses transsignaling by enhancing soluble gp130 production. PNAS. 2008; 105:14692-8.\u003c/li\u003e\n\u003cli\u003eJin CY, Lee JD, Park C, Choi YH, Kim GY. Curcumin attenuates the release of pro-inflammatory cytokines in lipopolysaccharide-stimulated BV2 microglia. Acta Pharmacol Sin. 2007; 28:1645-51.\u003c/li\u003e\n\u003cli\u003eYamazaki T, Shimosaka S, Sasaki H, Matsumura T, Tukiyama T, Tokiwa T. (+)-Syringaresinol-di-O-beta-D-glucoside, a phenolic compound from Acanthopanax senticosus Harms, suppresses proinflammatory mediators in SW982 human synovial sarcoma cells by inhibiting activating protein-1 and/or nuclear factor-kappaB activities. Toxicol In Vitro. 2007; 21:1530-7.\u003c/li\u003e\n\u003cli\u003eNiu X, Xing W, Li W, Fan T, Hu H, Li Y. Isofraxidin exhibited anti-inflammatory effects in vivo and inhibited TNF-\u0026alpha; production in LPS-induced mouse peritoneal macrophages in vitro via the MAPK pathway. Int Immunopharmacol. 2012; 14:164-71. \u003c/li\u003e\n\u003cli\u003eLiu L, Mu Q, Li W, et al. Isofraxidin protects mice from LPS challenge by inhibiting pro-inflammatory cytokines and alleviating histopathological changes. Immunobiology. 2015; 220:406-13.\u003c/li\u003e\n\u003cli\u003eLin J, Li X, Qi W, et al. Isofraxidin inhibits interleukin-1\u0026beta; induced inflammatory response in human osteoarthritis chondrocytes. Int Immunopharmacol. 2018; 64:238-45. \u003c/li\u003e\n\u003cli\u003eYamazaki T, Tokiwa T. Isofraxidin, a coumarin component from Acanthopanax senticosus, inhibits matrix metalloproteinase-7 expression and cell invasion of human hepatoma cells. Biol Pharm Bull. 2010; 33:1716-22.\u003c/li\u003e\n\u003cli\u003eShen P, Wang HG, Li MM, et al. Isofraxidin inhibited proliferation and induced apoptosis via blockage of Akt pathway in human colorectal cancer cells. Biomed Pharmacother. 2017; 92:78-85.\u003c/li\u003e\n\u003cli\u003eZhang H, Feng QQ, Gong JH, Ma JP. Anticancer effects of isofraxidin against A549 human lung cancer cells via the EGFR signaling pathway. Mol Med Rep. 2018; 18:407-14.\u003c/li\u003e\n\u003cli\u003eLi P, Zhao QL, Wu LH, et al. Isofraxidin, a potent reactive oxygen species (ROS) scavenger, protects human leukemia cells from radiation-induced apoptosis via ROS/mitochondria pathway in p53-independent manner. Apoptosis. 2014; 19:1043-53.\u003c/li\u003e\n\u003cli\u003eWu X, Gu J, Zou Z, et al. Suppressive effects of isofraxidin on depressive-like behaviors induced by chronic unpredictable mild stress in mice. Brain Sci. 2022; 12:1-12.\u003c/li\u003e\n\u003cli\u003eChen C, Song X, Lin D, Xu P. Isofraxidin alleviates myocardial infarction through NLRP3 inflammasome inhibition. Inflammation. 2020; 43:712-21.\u003c/li\u003e\n\u003cli\u003eHirano T. IL-6 in inflammation, autoimmunity and cancer. Int Immunol. 2021; 33:127-48.\u003c/li\u003e\n\u003cli\u003eSzab\u0026oacute; GT, Mahiny AJ, Vlatkovic I. COVID-19 mRNA vaccines: Platforms and current developments. Mol Ther. 2022; 30:1850-68. \u003c/li\u003e\n\u003cli\u003eLee TC, Murthy S, Del Corpo O, et al. Remdesivir for the treatment of COVID-19: A systematic review and meta-analysis. Clin Microbiol Infect. 2022; 28:1203-10.\u003c/li\u003e\n\u003cli\u003eZhao Z, Sun YS, Chen W, Lv LX, Li YQ. Hispolon inhibits breast cancer cell migration by reversal of epithelial-to-mesenchymal transition via suppressing the ROS/ERK/Slug/E-cadherin pathway. Oncol Rep. 2016; 35:896-904. \u003c/li\u003e\n\u003cli\u003eKhan AQ, Khan R, Rehman MU, et al. Soy isoflavones (daidzein \u0026amp; genistein) inhibit 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced cutaneous inflammation via modulation of COX-2 and NF-\u0026kappa;B in Swiss albino mice. Toxicology. 2012; 302:66-74.\u003c/li\u003e\n\u003cli\u003eBalkrishna A, Nain P, Chauhan A, et al. Super critical fluid extracted fatty acids from \u003cem\u003eWithania somnifera\u003c/em\u003e seeds repair psoriasis-like skin lesions and attenuate pro-inflammatory cytokines (TNF-\u0026alpha; and IL-6) release. Biomolecules. 2020; https:// doi.org/10.3390/biom10020185\u003c/li\u003e\n\u003cli\u003eChen Y, Zhang C, Xiao CX, et al. Dexamethasone can attenuate the pulmonary inflammatory response via regulation of the lncH19/miR-324-3p cascade. J Inflamm (Lond). 2021; https://doi.org/10.1186/s12950-020-00266-0\u003c/li\u003e\n\u003cli\u003eLiu T Zheng S, Guo P. Effect of torin1 on suppressing inflammation in mice with dextran sodium sulfate-induced colitis. Int Clin Exp Med. 2017; 10:4723-31.\u003c/li\u003e\n\u003cli\u003eKim JH, Choi HS, Kim SL, Lee DS. The PAK1-Stat3 signaling pathway activates IL-6 gene transcription and human breast cancer stem cell formation. Cancers (Basel). 2019; https://doi.org/10.3390/cancers11101527\u003c/li\u003e\n\u003cli\u003eJang S, Lelley KW, Johnson RW. Luteolin reduces IL-6 production in microglia by inhibiting JNK phosphorylation and activation of AP-1. PNAS. 2008; 105:7534-9.\u003c/li\u003e\n\u003cli\u003eSafa A, Abak A, Shoorei H, Taheri M, Ghafouri-Fard S. MicroRNAs as regulators of ERK/MAPK pathway: A comprehensive review. Biomed Pharmacother. 2020; https://doi.org/10.1016/j.biopha.2020.110853\u003c/li\u003e\n\u003cli\u003eShu YJ, Bao RF, Jiang L, et al. MicroRNA-29c-5p suppresses gallbladder carcinoma progression by directly targeting CPEB4 and inhibiting the MAPK pathway. Cell Death Differ. 2017; 24:445-57. \u003c/li\u003e\n\u003cli\u003eMeng X, Shi Y, Xiang X, et al. Influence of miR-101 on proliferation of liver cancer cells through the MAPK/ERK signaling pathway. Oncol Lett. 2020; 19:1310-6. \u003c/li\u003e\n\u003cli\u003eZhang BX, Yu T, Yu Z, Yang XG. MicroRNA-148a regulates the MAPK/ERK signaling pathway and suppresses the development of esophagus squamous cell carcinoma via targeting MAP3K9. Eur Rev Med Pharmacol Sci. 2019; 23:6497-504. \u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"IL-6, Cytokine storm, Suppressive effect, Isofraxidin, MAPK/ERK pathway","lastPublishedDoi":"10.21203/rs.3.rs-3935003/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3935003/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eInterleukin-6 (IL-6) is a pleiotropic cytokine that has many biological activities, including inflammation, hematopoiesis, bone metabolism, embryonic development, and other fundamental processes. Recently, IL-6 has been widely recognized as an important pro-inflammatory cytokine involved in cytokine storm pathogenesis during severe inflammatory diseases, such as coronavirus disease 2019 (COVID-19). Therefore, IL-6 is considered to be a therapeutic target for inhibiting cytokine storm. In the present study, we investigated the suppressive effect of isofraxidin, a major coumarin compound of Acanthopanax senticosus, on the overexpression of IL-6 and its molecular mechanism. When human hepatocellular carcinoma cell lines, HuH-7 and HepG2, were treated with 12-O-tetradecanoylphorbol 13-acetate (TPA), a marked induction of IL-6 mRNA expression was observed in HuH-7 cells compared with HepG2 cells. Isofraxidin significantly suppressed TPA-induced IL-6 mRNA expression in HuH-7 cells in a dose-dependent manner. Furthermore, isofraxidin inhibited TPA-induced phosphorylation of ERK1/2 in a dose dependent manner. Similarly, the MAPK/ERK inhibitor U0126 suppressed TPA-induced IL-6 mRNA expression. However, isofraxidin had no effects on TPA-induced phosphorylation of SAPK/JNK, Akt (Ser473), and STAT3 (Tyr705), nuclear translocation of NF-κB p65, and degradation of IκB. Taken together, isofraxidin suppresses TPA-induced overexpression of IL-6 mRNA by selectively inhibiting the activation of the MAPK/ERK pathway in HuH-7 cells, indicating that isofraxidin may be an effective anti-inflammatory agent for treating cytokine storm.\u003c/p\u003e","manuscriptTitle":"Suppressive effect of isofraxidin on the overexpression of IL-6 and its molecular mechanism","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-02-09 15:58:07","doi":"10.21203/rs.3.rs-3935003/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"b12e4f6c-8c1a-4ced-ac7d-8d75edb23fd2","owner":[],"postedDate":"February 9th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-02-19T14:43:19+00:00","versionOfRecord":[],"versionCreatedAt":"2024-02-09 15:58:07","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3935003","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3935003","identity":"rs-3935003","version":["v1"]},"buildId":"cTy_lsJlmDsVRNrSptgXS","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}