Magnoliae flos Downregulated Lipopolysaccharide-Induced Inflammatory Responses via NF-κB/ERK-JNK MAPK/STAT3 Pathways

Background Magnoliae flos is the dried flower bud of Magnolia biondii and related plants. It has been used as a medicinal herb for the treatment of rhinitis, sinusitis, and sinus headaches. Nevertheless, the effects of Magnoliae flos in microbial infection or sepsis remain unclear. In this study, we investigated the anti-inflammatory effects of Magnoliae flos water extract (MF) in lipopolysaccharide- (LPS-) induced septic mice and LPS-stimulated RAW264.7 macrophages. Results We found that MF reduced the mortality of LPS-challenged mice. Enzyme immunoassays and reverse transcription polymerase chain reaction analysis revealed that MF administration attenuated mRNA expression and protein production of proinflammatory mediators, including cyclooxygenase 2, inducible nitric oxide synthase, tumor necrosis factor-α, and interleukin-6. In parallel to these results in mice, pretreatment with MF suppressed the LPS-induced production of proinflammatory mediators in RAW264.7 macrophages. In addition, we found that MF exerted its suppressive effects by inhibiting the activation of the mitogen-activated protein kinase, nuclear factor-κB, and signal transducer and activator of transcription pathways at the protein level. Conclusion MF could be a potential therapeutic agent for regulating excessive inflammatory responses in sepsis.


Introduction
Sepsis is a severe inflammatory condition characterized by organ dysfunction in severe cases, and it is mostly caused by bacterial infection [1]. Infectious diseases in humans have been documented dating as far back as 1000 BC, but pathogenic infections still remain the leading cause of morbidity and mortality worldwide [2]. Every year, sepsis accounts for one-third of all neonatal deaths worldwide [3]. According to the World Health Organization, neonatal sepsis is a major global health concern with the highest burden in low-and middle-income countries [4].
Despite the high mortality rate of sepsis, the etiology of the disease remains unclear. Pathogens invade cells, and their toxins trigger sepsis through two processes: massive burst of proinflammatory cytokines and dysregulation of host immune responses [5][6][7]. The core strategy for sepsis treatment is identifying and applying antibacterial and anti-inflammatory agents to the primary lesion. Corticosteroids, such as dexamethasone (Dexa), are a class of medications used in the treatment of severe acute inflammation [8]. Although steroidal agents are effective and provide quick relief, the severe adverse effects have caused reluctance to use corticosteroids even in high-risk individuals [9].
To investigate anti-inflammatory agents, researchers have used murine models of sepsis due to their advantages, such as ease of experimentation, availability of genetically engineered species, and the relative low cost [10]. Lipopolysaccharide-(LPS-) induced endotoxemia model is one such model that has been used for nearly 100 years in an effort to recapitulate human sepsis [11]. LPS, a major component of the outer membrane of gram-negative bacteria, activates innate immune cells, such as macrophages, to secrete proinflammatory cytokines and mediators through inflammatory pathways, such as nuclear factor-κB (NF-κB) and mitogenactivated protein kinase (MAPK) signaling pathways [12]. MAPK pathways include extracellular signal-regulated kinase (ERK) 1/2, c-Jun N-terminal kinase (JNK), and p38, all of which are crucial regulators of NF-κB and activator protein-(AP-) 1 activation [13]. LPS-activated pathways induce macrophages to produce various proinflammatory cytokines, such as tumor necrosis factor-(TNF-) α and interleukin-(IL-) 6, and proinflammatory mediators, such as nitric oxide (NO) and prostaglandins (PGs) [14]. Inducible NO synthase (iNOS) and cyclooxygenase-(COX-) 2 mediate inflammatory responses by synthesizing NO and prostaglandin E2 (PGE 2 ), respectively [15,16]. Therefore, the underlying molecular mechanism behind inflammatory responses may be correlated with the inhibition of NF-κB and/or MAPK signaling pathways [17]. In addition, IL-1β and IL-6 induce tyrosine-phosphorylation of signal transducer and activator of transcription (STAT) 3, which in return induces inflammatory responses [18,19].

Preparation of Magnoliae flos Water Extraction (MF).
The dried flower buds of M. biondii were obtained from HANHERB (Guri city, Gyeonggi province, Republic of Korea). The dried flower buds of the plants were processed and extracted with water at 100°C for 4 h with reflux condenser. The extract was filtered with Whatman filter paper and lyophilized. The percentage yield was 13.73% w/w. MF was diluted in saline prior to treat to the RAW264.7 cells or C57BL/6 mice.

Cell
Culture and Sample Treatment. The RAW 264.7 macrophages cell line was purchased from Korea Cell Line Bank (KCLB, Seoul, Republic of Korea). This cell line was cultured in DMEM supplemented with 10% FBS, penicillin (100 U/mL), and 1% streptomycin (100 μg/mL) in 37°C and 5% CO 2 incubator. MF was dissolved in distilled water, and the cells were treated with 50, 100, or 200 μg/mL MF. The cells (1 × 10 5 cells/mL) were stimulated with 1 μg/mL of LPS for the indicated time prior to treatment with MF for 1 h.

Experimental Animals and Sample Treatment.
Male C57BL/6 mice (6 weeks old) were obtained from Daehan Biolink Co. (Daejeon, Republic of Korea). All animals were housed in accordance with the guidelines for the care and use of laboratory animals. The guidelines were adopted and promulgated by Sangji University according to the requirements demonstrated by the National Institutes of Health. All the experimental protocols were approved based on the Institutional Animal Care and Use Committee (IACUC) of Sangji University before the beginning of the study (IACUC Animal approval protocol No.2020-10). The mice were housed in a cage and fed standard laboratory chow in the animal room with 12 h dark/light cycles and constant condition (20 ± 5°C temperature, 40-60% humidity) for a week. The mice randomly were assigned to one of five groups (n = 6 per group). The C57BL/6 mice were intraperitoneal injected with PBS or LPS (30 mg/kg dissolved in PBS). MF (50 and 100 mg/kg) was injected intraperitoneally 1 h before LPS injection. Survival was monitored for 108 h after LPS administration. Four hours after LPS 2 Mediators of Inflammation injection, peripheral blood and liver samples were obtained from each mouse.
2.5. Production Assay. We evaluated the production of NO, PGE2, and pro-inflammatory cytokines following the previous study [28].  Table 2).

Western Blot Analysis.
Protein samples were isolated from RAW264.7 macrophages using specific reagents (NE-PER Nuclear and Cytoplasmic Extraction Reagents provided Thermo; Pro-prepTM obtained from Intron biotechnology Inc.) and then separated using electrophoresis and evaluated following the previous analysis [28].

Statistical Analysis.
Results are expressed as the mean ± SD of triplicate experiments. Statistically significant differences were determined using ANOVA and Dunnett's post hoc test, and p values > 0.05 indicated statistical significance.
Since sepsis is a systemic inflammatory response induced by LPS, we investigated the effect of LPS injection on proinflammatory markers at the mRNA and protein level. As shown in Figure 1(b), the increase in iNOS and COX2 by LPS was suppressed after pretreatment with MF or Dexa the positive control. We also evaluated the levels of secreted protein and mRNA expression of proinflammatory cytokines, including TNF-α and IL-6 with enzyme immunoassay (EIA) and quantitative reverse transcription polymerase chain reaction (qRT-PCR), respectively (Figures 1(d)-1(g)).
LPS induced an increase in mRNA expression and secreted protein level of cytokines, and this was downregulated by pretreatment with MF.   Expression of COX-2 and iNOS was determined by western blot with specific antibodies. Densitometric analysis was performed using ImageJ software. β-Actin was used as an internal control. (d and f) Total RNA was isolated from the liver tissue samples, and then, mRNA expression of TNF-α (d) and IL-6 (f) was determined with qRT-PCR. (e and g) Peripheral blood samples were obtained from each mouse. Serum levels of TNF-α (e) and IL-6 (g) were determined using EIA kits. The data are presented as the mean ± SD.  (Figures 4(a) and 4(b)) and IL-6 (Figures 4(c) and 4(d)), respectively.

MF Inhibited Phosphorylation of STAT3 and Regulated
Activation of NF-κB/IκB-α Signaling Pathways in LPS-Stimulated RAW264.7 Macrophages. Given the results from LPS-challenged mice (Figures 1 and 2), we investigated 3 proinflammatory signaling pathways involved in the severe inflammatory responses. As shown in Figures 5(a) and 5(b), pretreatment with MF inhibited the phosphorylation of STAT3 induced by LPS. We also examined the effect of MF on the nuclear translocation of NF-κB p65. LPS stimulation showed notable nuclear translocation of p65 from cytosol to nucleus, and this was inhibited by pretreatment with MF though the tendency was not presented in dosedependent manner (Figures 5(c) and 5(d)). We also found that MF downregulated the phosphorylation of IκB-α stimulated by LPS; however, the effect of MF on the activation of IκB-α or restoration of degraded IκB-α was not dose dependent (Figures 5(c) and 5(e)). The protein levels of iNOS and COX2 were determined by western blot with specific antibodies. Densitometric analysis was performed using ImageJ software. The data are presented as the mean ± SD. ## p < 0:01 and ### p < 0:001 vs. the control group; * p < 0:05, * * p < 0:01, and * * * p < 0:001 vs. the LPS-treated group.

Discussion
Magnoliae flos, the dried flower bud of M. biondii and related plants, is an effective traditional remedy for rhinitis, sinusitis, and sinus headaches. The water extract of M. biondii has been studied previously, and compounds including magnosaline and magnolin, with antiallergic, antirheumatic, or antioxidant properties, have been identified [20][21][22][23][24]. In the present study, we investigated the effect of MF in LPSchallenged mice and LPS-stimulated RAW264.7 macrophages. LPS treatment in mice and murine macrophages induced excessive inflammatory responses through increased secretion of proinflammatory cytokines and mediators via activation of proinflammatory signaling pathways. Several previous reports showed anti-inflammatory activity of Magnoliae flos extracts in macrophage models. Different liquids for extract such as ethanol [30] or methanol [31] are available for keeping the anti-inflammation activity of Magnoliae flos.
Also, other previous studies have presented the various suppressing inflammatory signaling pathways accompanied by antioxidant downregulating NF-κB [30], ovariectomyinduced osteoporosis regulating osteoclastogenesis [32], and cancer-mediated bone destruction blocking the vicious cycle [33]. In addition, many active compounds of Magnoliae flos showed the anti-inflammatory effects such as fargesin [34], tetrahydrofurofuran-type lignans [33], or essential oil [35]. Though extraction solvent, accompanied disease or signaling pathways, or subjects on the experiments are different from this present work, all of them show the effects based on its anti-inflammatory effects. Inflammation is necessary to maintain homeostasis in the immune system, whereas it is needed to suppress the reactions when it comes to excessive situation causing chronic and undesirable phenomenon [36]. Prolonged inflammation can lead to chronic pathological conditions, such as chronic bronchitis, which are difficult to treat, with symptoms that worsen over time [37]. Therefore, it is important to investigate and develop both prevention and treatment strategies for excessive inflammation. Sepsis, characterized by its systemic inflammatory response syndrome, is recognized as a global public health issue with high mortality and economic burden [38]. To investigate preventative and/or therapeutic agents for sepsis, we used an in vivo model, LPS-challenged mice [10]. Although the LPSchallenged murine model of sepsis has been used for nearly 100 years and has various advantages, there are some inherent limitations in its ability to recapitulate sepsis in humans. The data are presented as mean ± SD. ### p < 0:001 vs. the control group; * p < 0:05, * * p < 0:01, and * * * p < 0:001 vs. the LPS-treated group.

Mediators of Inflammation
The bacterial infection model, bacteria and fibrin clot implantation, cecal slurry injection, and colon ascendens stent peritonitis model are some other models for studying sepsis in vivo [39,40].
In the present study, we used LPS-induced murine sepsis model to investigate the anti-inflammatory properties of MF. Considering the wide spectrum of LPS dependent on doses or species, we would be able to perform other model of sepsis for further study. In mice, LPS dose of 30 mg/kg (intraperitoneal) had 100% mortality 60 h postinjection. While 67% of mice in the Dexa group and 50.0% of mice in the high-dose MF (100 mg/kg) group survived 96 h postinjection. Although it was not reflected in the survival rate (Figure 1), analysis of proteins extracted from liver tissue and serum suggests that MF can induce an antiinflammatory effect.
As an endotoxemia model, LPS-challenged sepsis model starts with binding to Toll-like receptor-(TRL-) 4 [10]. TLR4 is an important receptor that mediates the phosphorylation of NF-κB pathways through signal transduction, which involves the innate immune system. LPS-induced TLR4-mediated NF-κB signaling pathway has been related to improper immune responses [41]. The pathway activates immune cells to produce proinflammatory cytokines or mediators, such as TNF-α, IL-6, NO, or PGE 2 . NO is distinctly produced in the inflammatory response of LPSstimulated macrophages. As a small diffusible molecules playing a variety of physiological activities, NO is produced by activated macrophages with iNOS [42]. In addition, it seems to be produced by other proinflammatory mediators such as interferone-γ [43] or MF itself which resulted in significantly higher production at 50 μg/mL of MF. PGE 2 is an  Figure 5: Effect of MF on phosphorylation of STAT3 and activation of NF-κB/IκB-α in LPS-stimulated RAW264.7 macrophages. The cells were pretreated with MF for 1 h prior to the addition of LPS (1 μg/mL) for either 2 h (a and b) or 15-30 min (c-e). (a and b) Total proteins extracted from the cells were evaluated with specific antibodies against STAT3 and pSTAT3 by western blot. (c-e) Nuclear and cytosolic extracts were isolated, and the level of p65 in each fraction, i.e. p65 (N) and p65 (C), and LPS-induced degradation of IκB-α was examined by western blot. The data are presented as mean ± SD. ### p < 0:001 vs. the control group; * p < 0:05 and * * * p < 0:001 vs. the LPS-treated group. 8 Mediators of Inflammation important mediator that increases during trauma and sepsis and enhances neutrophilic inflammation [44]. In the present study, we evaluated the induction of proinflammatory cytokines and mediators by LPS stimulation in vivo and in vitro.
We found that MF suppressed the effects of LPS in the pro-duction of proinflammatory markers in LPS-challenged mice and LPS-stimulated macrophages (Figures 1, 3, and  4). These effects were confirmed with downregulation of mRNA expression of TNF-α and IL-6 by MF treatment (Figures 1(d), 1(f), 4(a), and 4(c)). In addition, we evaluated   Mediators of Inflammation the effects of MF on RA264.7 macrophages depending on the noncytotoxicity concentration. In Figure 3(a), cell viability of MF treatment at 200-1000 μg/mL showed proliferation effect which is assumed to be noncytotoxic condition. Based on the nontoxic and anti-inflammatory range of MF, we investigated the effects of MF on murine cell line as well as LPS-propelled sepsis animal model. MAPK activation by LPS is a crucial signal transduction event that regulates the production of proinflammatory cytokines and mediators and the activation of NF-κB [13]. MF showed inhibitory effects on the phosphorylation of MAPK or nuclear translocation of p65. While effects of MF were presented on the MAPKs, the effects of MF on nuclear translocation of p65 showed discrepancy and increased nuclear p65 level in mouse liver whereas suppressed nuclear translocation of p65 in murine cell line. We assumed that it is caused by lack of time let the subjects under the stimuli (Figure 2(d) and 5(c)) [5]. Meanwhile, the representative endotoxin, LPS, triggers excessive production of the STAT protein family, specifically STAT3, which is associated with the upregulation of LPS-binding protein, causing an amplification of inflammation in sepsis [45]. We also found that MF suppressed signaling pathways in the nucleus and the cytosol in LPS-stimulated cells and mice (Figures 2,  5, and 6). However, further work should be conducted to investigate the effect of MF on the translocation of STAT3 or LPS-stimulated inflammatory responses in other organs.

Conclusion
In conclusion, we reported that MF decreased mortality in LPS-challenged mice and suppressed the expression of proinflammatory cytokines and related proteins. MF also decreased production and expression of proinflammatory markers in LPS-stimulated macrophages. The suppressive effects of MF on the inflammatory responses seem to be regulated by the NF-κB/JNK-ERK MAPK/STAT3 pathways. Therefore, we suggest the possibility of MF as an antiinflammatory agent (Figure 7).

Data Availability
The data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest
The authors declare that there is no conflict of interest regarding the publication of this paper.