Cloperastine Reduces IL-6 Expression via Akt/GSK3/Nrf2 Signaling in Monocytes/Macrophages and Ameliorates Symptoms in a Mouse Sepsis Model Induced by Lipopolysaccharide

Abstract

Cloperastine (CLP) is a drug with a central antitussive effect that is used to treat bronchitis. Therefore, we have attempted to examine the anti-inflammatory effects of CLP. CLP reduced the secretion of interleukin (IL)-6, a pro-inflammatory cytokine, from RAW264.7 monocyte/macrophage-linage cells treated with lipopolysaccharide (LPS). IL-6 is a biomarker of sepsis and has been suggested to exacerbate its symptoms. We found that the intraperitoneal administration of CLP reduced IL-6 levels in the lungs and also improved hypothermia in mice with LPS-induced sepsis. CLP ameliorated kidney pathologies such as congestion and increased the survival rate of mice administered with a lethal dose of LPS. To reveal the mechanisms underlying the anti-inflammatory function of CLP, we analysed the intracellular signaling in LPS-treated RAW264.7 cells. CLP induced the phosphorylation of protein kinase B (Akt) and glycogen synthase kinase 3 (GSK3) and also increased the amount of nuclear factor erythroid-2-related factor 2 (Nrf2) in RAW264.7 cells with/without LPS. Wortmannin, an inhibitor of phosphoinositide 3-kinase (PI3K), reduced the upregulated phosphorylation levels of Akt and GSK3 and the increased amount of Nrf2. It also halted the reduction of IL-6 secretion caused by CLP. These results suggest that CLP has an anti-inflammatory function via Akt/GSK3/Nrf2 signaling and could be a candidate drug for the treatment of inflammatory diseases, including sepsis.

INTRODUCTION

Sepsis is defined as a life-threatening organ dysfunction caused by a dysregulated host response to infection.¹⁾ The high incidence and mortality rate of severe sepsis means that it is a topic that requires attention.^2,3) The most common cause of sepsis is exposure to lipopolysaccharide (LPS), the main component of the cell walls of Gram-negative bacteria.⁴⁾ The symptoms of LPS-induced sepsis caused by LPS often involve an uncontrolled inflammatory response, organ failure, and death.⁵⁾ Although drugs that can alleviate the symptoms in patients with sepsis caused by Gram-negative bacteria, including LPS, are needed, specific drugs have not been developed.⁶⁾ Interleukin (IL)-6 is reported to be the most reliable biomarker among the pro-inflammatory cytokines in sepsis^7,8) and has been identified as a cytokine that exacerbates endotoxaemia in a model animal.⁹⁾ Compounds that reduce IL-6 levels are potential therapeutic agents.

We have previously established a T-cell activation-inhibitory assay for screening caloric restriction mimetics.¹⁰⁾ In the course of applying this screening assay, we found that this system can be used for screening drugs with anti-inflammatory functions.^11,12) Using this system, we were able to screen drugs and found that the antitussive drug cloperastine (CLP) (Fig. 1), which acts on the cough centre without acting on the respiratory centre,¹³⁾ reduces the secretion of pro-inflammatory cytokines, such as IL-6, in vitro. While the function of CLP in tumour growth and development has been reported,¹⁴⁾ the anti-inflammatory function of CLP has not been reported. Herein, we further attempted to ascertain the potential of CLP as a candidate drug for sepsis using an in vivo LPS-induced sepsis mouse model and to elucidate the mechanism of action of CLP on its anti-inflammatory function.

Fig. 1. Chemical Structure of CLP

MATERIALS AND METHODS

Reagents

CLP hydrochloride was purchased from Tokyo Chemical Industry Co., Ltd. (#C3038, Tokyo, Japan). LPS was obtained from Sigma-Aldrich (0111 E. coli B4, L2630; Sigma-Aldrich, St. Louis, MO, U.S.A.).

Animal Experiments

Male C57BL/6J mice were purchased from SLC (Shizuoka, Japan) at 6–7-weeks of age, maintained under controlled temperature and light (23 °C and 12-h light/dark, respectively), and used at 7–8-weeks of age; all of the animals received food and water ad libitum. All experimental procedures were in accordance with the guidelines for animal experimentation of the Animal Care and Use Committee of Matsuyama University (Matsuyama, Japan). The certification numbers verifying study approval were as follows: 24-005 on 2024.3.19., 23-008 on 2023.3.30., 22-007 on 2022.2.22., 21-009 on 2021.3.26.

Male C57BL/6J mice were intraperitoneally (i.p.) administered CLP (0–10 mg/kg) in a sterilised 0.5% (w/v) methylcellulose 400 solution (WAKO 133-17815, FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan). The dose of CLP (0–10 mg/kg) was selected while referencing a literature reporting anti-inflammatory function of a low molecular compound, cynandione A, using LPS-induced mouse model of sepsis.¹⁵⁾ LPS (0, 5, or 12 mg/kg) in phosphate-buffered saline (PBS) was i.p. administered 4 h after the administration of CLP. Twenty-four hours later, rectal temperature was monitored using a thermometer (BAT-7001H THERMOMETER, PHYSITEMP INSTRUMENTS INC., Clifton, NJ, U.S.A.). Whole blood samples were collected from the heart for serum preparation and organs, including the lungs and kidneys, were harvested for extract preparation or histological analysis.

Mouse Monocyte/Macrophage RAW264.7 Cell Culture

Mouse monocyte/macrophage RAW264.7 cells were purchased from DS Pharma Biomedical (Osaka, Japan) and they were maintained in Dulbecco’s modified Eagle’s medium (DMEM) (#11960-044, GIBCO, Thermo Fisher Scientific, San Diego, CA, U.S.A.) supplemented with 10% Fetal Bovine Serum, qualified (#26140-079, GIBCO) and 1% penicillin/streptomycin/glutamine (#10378016, GIBCO). The RAW264.7 cells were treated with or without the samples at the indicated concentrations. One hour later, LPS was added to the culture at a concentration of 10 ng/mL, and the culture media were harvested for enzyme-linked immunosorbent assay (ELISA) at the indicated times.

Cytotoxicity Assay

The RAW264.7 cells were treated with or without the samples at the indicated concentrations. One hour later, LPS was added to the culture at a concentration of 10 ng/mL, and the cells were cultured for 24 h. In order to assess the cytotoxicity of the samples, we used a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cell proliferation kit (Roche Diagnostics, Basel, Switzerland), following the manufacturer’s instructions.

Western Blot Analysis

RAW264.7 cells were treated with or without the samples at the indicated concentrations. One hour later, LPS was added to the culture at a concentration of 10 ng/mL, and the cells were harvested using RIPA buffer or NE Nuclear and Cytoplasmic Extraction Reagents (#78833; Thermo Scientific, IL, U.S.A.) for Western blotting. The blots were probed with the indicated antibodies (Table 1). The membranes were cut horizontally to probe molecules with different molecular weights. For quantitative analysis, loading controls on the same blot such as glucose-3-phosphate dehydrogenase (G3PDH), α-tubulin, or proliferating cell nuclear antigen (PCNA) were reprobed and analysed. The primary antibodies were detected using horse radish peroxidase (HRP)-conjugated anti-rabbit or anti-mouse immunoglobulin G (IgG) antibodies (Cell Signaling Technology, Danvers, MA, U.S.A.). Data are presented as the fold-change of control mean ± standard error of the mean (S.E.M.).

Table 1. List of Antibodies Used for Western Blottig

Antibodies	Species	Manufacturer	Product No.	Molecular weight (kDa)
anti-Nrf2	Rabbit	Cell Signaling	#12721	95–110
anti-pGSK3α	Rabbit	Cell Signaling	#9327	51
anti-pGSK3β	Rabbit	Cell Signaling	#5558	46
anti-pAKT	Rabbit	Cell Signaling	#4060	60
anti-pAMPK	Rabbit	Cell Signaling	#2535	62
anti-NQO-1	Rabbit	Cell Signaling	#62262	29
anti-I-κB	Rabbit	Cell Signaling	#4812	39
anti-pERK	Rabbit	Cell Signaling	#9101	42, 44
anti-NF-κB	Mouse	Cell Signaling	#6956	65
anti-α-tubulin-HRP	—	Cell Signaling	#9099	52
anti-G3PDH-HRP	—	Sigma-Aldrich	#G9295	37
anti-PCNA	Rabbit	Proteintech	#10205-2-AP	30–40
anti-rabbit IgG-HRP	—	Cell Signaling	#7074	—
anti-mouse IgG-HRP	—	Cell Signaling	#7076	—

Protein Extraction from Lung Tissues for ELISA

The harvested mouse lungs were quickly frozen in liquid nitrogen and then stored in a deep freezer. Proteins in the lung tissue samples were extracted using a 50-fold volume of RIPA buffer without sodium dodecyl sulfate and used for ELISA.

ELISA

Plasminogen activator inhibitor (PAI)-1, tumor necrosis factor (TNF)-α, monocyte chemotactic protein (MCP)-1 and IL-6 in the serum, lung extracts, or culture media were measured using mouse serpin E1/PAI-1 DuoSet ELISA (R&D Systems #DY3828-05, Minneapolis, MN, U.S.A.), Mouse TNF-α ELISA MAX Deluxe Set (Bio Legend #430904, CA, U.S.A.), Mouse MCP-1 ELISA MAX Deluxe Set (Bio Legend #432704), and Mouse IL-6 ELISA MAX Deluxe Set (Bio Legend #431304), respectively.

Histology

The kidneys of the mice were harvested and immersed in 4% paraformaldehyde solution in PBS for 2 d. Fixed organs were then embedded in paraffin, sectioned, and stained with haematoxylin–eosin (H&E).

Statistical Analyses

Data are expressed as the mean ± S.E.M. Data were analysed using one-factor ANOVA, followed by Tukey’s multiple comparison test. p < 0.05 was considered significant.

RESULTS

CLP Reduces LPS-Induced IL-6 Secretion from Monocyte/Macrophage Linage Cell Line in Vitro

We previously reported a practical approach, the T-cell activation-inhibitory assay, for screening caloric restriction mimetics based on inductive logic (Iura et al.). In our experience, we have noticed that most compounds screened in this assay show anti-inflammatory functions (Ishikawa et al.). Thus, we applied this method to screen anti-inflammatory drugs and found a candidate drug (CLP) (Fig. 1). We confirmed the anti-inflammatory functions of CLP using a monocyte/macrophage linage cell line, RAW264.7 cells. Secretion of inflammatory cytokines of IL-6 and TNF-α from RAW264.7 cells was induced by LPS treatment. CLP reduced the secretion of IL-6 induced by LPS, whereas that of TNF-α was conversely increased by CLP treatment. CLP did not affect the viability of RAW264.7 cells in the indicated range (Fig. 2).

Fig. 2. CLP Reduces IL-6 Secretion from RAW264.7 Cells Induced by LPS

RAW264.7 cells were cultured with CLP in the presence of LPS for 24 h. The cytotoxicity (A) was accessed using the MTT assay and IL-6 (B) and TNF-α (C) secreted from the RAW264.7 cells into culture media were measured with ELISA. * Significant difference (p < 0.05) between the control cultures treated with LPS only and the test cultures treated with LPS and the indicated concentrations of CLP. n = 4.

CLP Reduces the Levels of IL-6 in the Lung and Ameliorates Symptoms in a Mouse Model of LPS-Induced Sepsis in Vivo

To assess the anti-inflammatory function of CLP in vivo, we used a mouse model of LPS-induced sepsis. As reported by other groups,^9,16) administration of LPS (5 mg/kg) reduced the rectal temperature. We found that 1 mg/kg CLP ameliorated this reduction in regard to the rectal temperature. A high dose of CLP (10 mg/kg) impaired the recovery of the rectal temperature (Fig. 3A). The administration of LPS induced IL-6 upregulation in the lungs of mice, and CLP reduced this upregulated IL-6 levels (Fig. 3B). LPS slightly increased TNF-α levels in the lung of mice and CLP did not affect the TNF-α levels (Fig. 3C).

Fig. 3. CLP Ameliorates Hypothermia and Reduces IL-6 Contents in the Lung of Mice Treated with LPS

Mice were administered CLP 4 h before LPS challenge. (A) The rectal temperature was monitored 24 h after LPS challenge. The levels of IL-6 (B) and TNF-α (C) in the lung tissue of the LPS-challenged mice were measured by ELISA 24 h after the LPS challenge. * Significant difference (p < 0.05) between the indicated groups. n = 5.

Next, we examined the serum levels of sepsis-related biomarkers including IL-6, MCP-1, and PAI-1. Administration of LPS upregulates serum levels of IL-6, MCP-1, and PAI-1 in mice. CLP treatment reduced the levels of these upregulated biomarkers in the serum, although no significant differences were observed between the LPS-treated and LPS plus CLP-treated groups (Fig. 4).

Fig. 4. Effect of CLP on IL-6, PAI-1, and MCP-1 Levels in the Serum of LPS-Challenged Mice

Mice were administered CLP 4 h before LPS challenge. The levels of IL-6 (A), PAI-1 (B), and MCP-1 (C) in the serum of mice were measured using ELISA 24 h after LPS challenge. n = 6–8.

CLP Ameliorates the Kidney Pathology and Increases the Survival Rate of a Mouse Model of LPS-Induced Sepsis

Based on the above finding that CLP has anti-inflammatory functions, we examined whether CLP could increase the survival rate of mice with lethal sepsis treated with high-dose LPS (12 mg/kg). Fifty-two hours after the LPS challenge, LPS-treated mice showed a survival rate of 10%. Administration of CLP (0.5 mg/kg) increased the survival rate of LPS-treated mice to 70% at 52 h after LPS challenge (Fig. 5A). Administration of a lower dose of CLP (0.2 mg/kg) had no significant effect (data not shown).

Fig. 5. CLP Enhances the Survival Rate of Mice Challenged with a Lethal Dose of LPS and Improves Histological Abnormalities in the Kidney of the LPS-Challenged Mice

Mice were administered CLP (0.5 mg/kg) 4 h before LPS challenge (12 mg/kg). (A) Kaplan–Meier survival curves were used to compare the LPS- and LPS plus CLP-treated groups. * Significant difference between groups (p < 0.05). n = 10. (B–D) Mouse kidneys were harvested 24 h after LPS challenge and then processed for histological examination. LPS-induced congestion (arrows) in the kidneys was protected by CLP treatment. (B) None, (C) LPS, and (D) LPS + CLP.

The kidneys of high-dose LPS-administered mice showed congestion in an extensive area of the organs in the HE sections. The pathology was completely prevented by treatment with 0.5 mg/kg CLP (Figs. 5B–D). Histological changes such as congestion or accumulation of mononuclear cells were not observed in the H&E section of the lung of high-dose LPS-administered mice (data not shown).

Anti-inflammatory Function of CLP Is Not Mediated by Obstructing Inhibitor of κB (I-κB)/Nuclear Factor-κB (NF-κB) Signaling or Extracellular Signal-Regulated Kinase (ERK) Signaling

In order to examine the mechanisms underlying the anti-inflammatory function of CLP, we analysed the intracellular signaling in LPS-treated RAW264.7 cells. First, we examined the involvement of I-κB/NF-κB signaling, because this signaling pathway is one of the most reported pathways concerning inflammatory signaling.¹⁷⁾ While the performed LPS treatment reduced I-κB levels in RAW264.7 cells, CLP did not halt the reduced I-κB levels but rather enhanced the reduction. While LPS treatment increased NF-κB levels in the nuclear fraction of RAW264.7 cells, CLP did not halt the increased NF-κB levels, rather increased the levels (Fig. 6). Thus, it cannot be suggested that the anti-inflammatory function of CLP is mediated by the downregulation of I-κB/NF-κB signaling.

Fig. 6. I-κB/NF-κB Signaling Induced by LPS Is Not Halted by Treatment with CLP

RAW264.7 cells were treated with CLP 1 h before LPS challenge. (A) Thirty minutes after the LPS challenge, the total cell lysates were harvested. The levels of I-κB in the cell lysates were analysed by Western blotting. (B) Fifteen minutes after LPS challenge, the cell lysates were harvested and nuclear fractions were prepared. The levels of NF-κB in the nuclear fraction were analysed by Western blotting. * Significant difference (p < 0.05) between the indicated cultures. n = 4.

ERK signaling is involved in IL-6 expression.^18,19) While LPS treatment induced the phosphorylation of ERK in RAW264.7, CLP did not halt the induction of ERK phosphorylation (Fig. 7). ERK signaling has not been suggested to mediate the CLP-induced reduction in IL-6 expression.

Fig. 7. ERK Signaling Induced by LPS Is Not Halted by Treatment with CLP

RAW264.7 cells were treated with CLP 1 h before LPS challenge. Thirty minutes after the LPS challenge, the total cell lysates were harvested. ERK phosphorylation levels in cell lysates were analysed by Western blotting. n = 4.

Protein Kinase B (Akt)/Glycogen Synthase Kinase 3β (GSK3β)/Nuclear Factor Erythroid-2-Related Factor 2 (Nrf2) Signaling Mediates the Anti-inflammatory Function of CLP

Next, we examined the relationship between the anti-inflammatory effects of CLP and Akt/GSK3/Nrf2 signaling. The two kinases, Akt and GSK3β, are well known players in the regulation of inflammation.²⁰⁾ Akt signaling downregulates the secretion of pro-inflammatory cytokines under LPS-induced conditions.^21,22) Activation of Akt signaling phosphorylates GSK3β and inactivates GSK3β, which promotes the stabilisation of Nrf2, a transcription factor.²³⁾ Nrf2 has been shown to interfere with the LPS-induced gene expression of pro-inflammatory cytokines, such as IL-6 and IL-1β.²⁴⁾ The phosphorylation of AMP-activated protein kinase (AMPK) is also suggested to IL-6 expression.²⁵⁾ In fact, CLP induced the phosphorylation of Akt, GSK3α/β, and AMPK and increased the amount of Nrf2 in RAW264.7 cells with or without LPS challenge (Figs. 8, 9A). We also observed enhanced expression of reduced nicotinamide adenine dinucleotide phosphate (NADPH)-quinone oxidoreductase-1 (NQO1) (Fig. 9B), a well-known downstream molecule of Nrf2.²⁶⁾ The role of NQO1 in mediating antioxidant effects has been well characterised, both in vitro and in vivo.²⁷⁾

Fig. 8. Akt/GSK3/AMPK Signaling Is Activated by Treatment with CLP

RAW264.7 cells were treated with CLP 1 h before LPS challenge. Twenty-four hours later, the total cell lysates were harvested. The phosphorylation levels of Akt (A), GSK3α/β (B) and AMPK (C) in cell lysates were analysed by Western blotting. * Significant difference (p < 0.05) between the indicated cultures. n = 4.

Fig. 9. Nrf2 and NQO1 Is Induced by Treatment with CLP

RAW264.7 cells were treated with CLP 1 h before LPS challenge. Twenty-four hours later, the total cell lysates were harvested. The levels of Nrf2 (A) and NQO1 (B) in cell lysates were analysed by Western blotting. * Significant difference (p < 0.05) between the indicated cultures. n = 4.

To confirm the involvement of Akt/GSK3/Nrf2/AMPK signaling in regard to the anti-inflammatory effects of CLP, we performed inhibition experiments using a potent and selective inhibitor of phosphoinositide 3-kinase (PI3K), wortmannin.²⁸⁾ Inhibition of PI3K by treatment with wortmannin clearly reduced the CLP-induced phosphorylation of Akt and GSK3 (Figs. 10A–D). The amount of Nrf2 was also reduced by the treatment with wortmannin (Figs. 10E, F). In contrast, the phosphorylation levels of AMPK were not affected by wortmannin (Figs. 10G, H). Wortmannin halted the reduction of IL-6 production induced by CLP (Fig. 11).

Fig. 10. Wortmannin Reduces Akt, GSK3, and Nrf2 Signaling, but Not AMPK Signaling

RAW264.7 cells were treated with wortmannin 2 h before LPS challenge and CLP 1h before LPS challenge. Thirty minutes after the LPS challenge, the total cell lysates were harvested. The phosphorylation levels of Akt (A, B), GSK3α/β (C, D) and AMPK (G, H) in cell lysates were analysed by Western blotting. The total levels of Nrf2 (E, F) were also analysed. * Significant difference (p < 0.05) between the indicated cultures. n = 3 for B and C, and 4 for A and D–H.

Fig. 11. Wortmannin Halts the Reduced Secretion of IL-6 by CLP Treatment

RAW264.7 cells were treated with wortmannin 2 h before LPS challenge and CLP 1h before LPS challenge. 4 h after LPS challenge, the levels of IL-6 secreted from the cells into the culture media were measured by ELISA. * Significant difference (p < 0.05) between the indicated cultures. n = 5–6.

DISCUSSION

IL-6 has been reported to be upregulated in the serum of patients with sepsis.^7,29) A positive correlation between IL-6 serum levels and symptom severity in patients with sepsis has been repeatedly reported.^30–33) In the present study, we found IL-6 modulating functions of CLP such as the reduction of IL-6 secretion from LPS-treated RAW 264.7 cells, the reduction of IL-6 levels in the lung of LPS-challenged mice, and the decline of IL-6 levels in the serum of LPS-challenged mice. Therefore, the IL-6-reducing effect of CLP may ameliorate sepsis symptoms.

While measuring IL-6 in the serum of mice, we found that IL-6 levels in the serum show drastic changes after the LPS administration, as reported by Higa et al.³⁴⁾ When LPS was challenged by i.p. injection, serum IL-6 levels increased until 2 h and declined sharply after that (data not shown). We presume that the drastic changes of IL-6 levels in the time course evoke severe dispersion at the time point of measurement and disturb the verification of significant differences between groups. Thus, CLP dose dependently reduces the average of IL-6 levels despite the circumstances, which is the most important statistical indicator, although no statistically significant differences between groups (Fig. 4).

A high dose of CLP administration (10 mg/kg) to a mouse model of LPS-induced sepsis showed less effect for ameliorating hypothermia than a low dose of CLP administration (1 mg/kg) (Fig. 3). To probe the cause of the unexpected results, we preliminary measured IL-6 levels in the mouse serum. The average of IL-6 levels in the serum of the model was reduced by the low dose of CLP administration (1 mg/kg). The high dose of CLP administration (10 mg/kg) dampened the IL-6 reducing effect of the low dose of CLP (1 mg/kg). Although the differences in the average of serum IL-6 levels between groups did not accompany with the statistical significance (as described in the above paragraph), we presume that cancellation of the IL-6 reducing effect by the high dose of CLP evoke the change of phenotypes of the sepsis model such as the rectal temperature. The reason why the high dose of CLP administration (10 mg/kg) induce the cancellation of the IL-6 reducing effect remains unclear.

Hypothermia is a symptom of sepsis¹⁾ and is indicative of LPS-induced sepsis.¹⁶⁾ Marginal zone B cells in the spleen produce IL-6 in response to LPS and thus exacerbate endotoxic shock, including hypothermia.⁹⁾ Other cells, including alveolar and ascites macrophages, are also known to produce IL-6.^35,36) We found that CLP prevented LPS-induced hypothermia in mice (Fig. 3) and reduced IL-6 levels in the lungs of LPS-challenged mice, suggesting the possibility that CLP alleviated hypothermia by reducing IL-6 expression in critical cells, including alveolar macrophages. Further experiments must be performed to arrive at definitive conclusions.

CLP reduced the IL-6 and MCP-1 levels and rescued congestion in the kidneys of mice with LPS-induced sepsis (Fig. 5). CLP might ameliorate the symptoms of sepsis, such as kidney congestion, by decreasing IL-6 and MCP-1 levels. MCP-1 is induced by IL-6 in myeloid lineage cells³⁷⁾ and plays a key role in the recruitment of inflammatory cells, such as monocytes and lymphocytes, into inflamed tissue.^38,39) Acute inflammation induced by MCP-1 results in oedema. Sepsis is associated with congestion caused by vascular leakage through damaged endothelial cells.^40–42)

In the in vitro experiments, CLP showed a contrasting effect on the levels of IL-6 and TNF-α in LPS-treated RAW264.7-cell culture, where IL-6 expression was found to be reduced and TNF-α expression was increased (Fig. 2). We suspect that CLP has the IL-6 reducing function and the reduction of IL-6 levels augmented up-regulation of TNF-α in response to LPS stimulation, because Yimin et al. have reported that the production of IL-6 and TNF-α is negatively regulated by each other.^43,44) In fact, TNF-α in the lung of LPS-challenged mice was not induced by CLP. Furthermore, the levels of TNF-α in the lung of LPS-treated mice were approximately a hundredth of those of IL-6 (Fig. 3) and the changes in TNF-α levels would be negligible.

The dosage of CLP (0.5 mg/kg) used in the survival experiment is estimated to translate to approximately 2.3 mg for a 60 kg human according to human equivalent dose conversion based on body surface area.⁴⁵⁾ As the clinical dose of CLP is 30–60 mg per day for antitussive adult patients, CLP could be a possible candidate drug for patients with sepsis through drug repositioning.

We showed that CLP suppressed the symptom of a mouse model of LPS-induced sepsis via the reduction of IL-6 secretion. These results suggest that CLP has a preventive effect against sepsis. Although preventive treatment is better than cure in general, curative medicine is more desirable than preventive medicine in the case of sepsis. Thus, we should examine the effect of CLP when CLP is administered posterior to LPS or at the same time as LPS, in future studies.

CLP treatment did not obstruct inflammatory signaling, such as I-κB/NF-κB and ERK signaling. CLP increased the amount of NF-κB induced by LPS in the nuclear fraction of RAW264.7 cells. Also, CLP showed a tendency to reduce the amount of I-κB and increase the phosphorylation levels of ERK with/without LPS (Figs. 6, 7). On the other hand, CLP treatment with/without LPS induced the expression of Nrf2 and NQO1 which have anti-inflammatory and/or antioxidant effects (Fig. 9). Thus, it is difficult to draw a conclusive definition whether CLP treatment causes positive or negative consequences under the circumstances. Further investigation is required for beneficial utilization of CLP.

Experiments using a selective inhibitor of PI3K, wortmannin, suggested the involvement of GSK3 signaling on the regulation of IL-6 expression by CLP treatment. While LPS clearly induced the phosphorylation of Akt (Fig. 10B), the phosphorylation level of GSK3 was not changed by LPS treatment (Fig. 10D). The treatment of wortmannin reduced the phosphorylation level of GSK3. Similar degree of reduction in the phosphorylation level of GSK3 was induced by wortmannin under 10 or 20 µM CLP treatment. However, as the treatment of 20 µM CLP slightly upregulated the phosphorylation level of GSK3, the level of GSK3 phosphorylation under 20 µM CLP plus wortmannin treatment was not reduced below the control level without CLP and wortmannin. In the case of 10 µM CLP plus wortmannin treatment, the level of GSK3 phosphorylation was reduced below the control level, comparable to that with wortmannin without CLP. The reduction in the GSK3 phosphorylation level indicates the increase in the amount of non-phosphorylated active GSK3. It is presumed that the increase in the amount of non-phosphorylated active GSK3 induced the degradation and reduction of Nrf2²³⁾ (Fig. 10F). Consequently, the wortmannin treatment under 10 µM CLP reduced the levels of Nrf2 which interferes with the gene expression of IL-6²⁴⁾ and caused the rebound in the IL-6 secretion (Fig. 11).

In the present study, we have proposed a mechanism by which CLP reduces IL-6 secretion in LPS-challenged monocytes/macrophages. CLP does not reduce the LPS-induced IL-6 expression by interfering the inflammation-promoting mechanism such as I-κB/NF-κB or ERK signaling but does by promoting the signaling of Nrf2, the master regulator of endogenous antioxidant responses and inflammations⁴⁶⁾: CLP is suggested to activate PI3K, which phosphorylates Akt.²⁰⁾ The activated Akt phosphorylates and inactivates GSK3, which promotes the stabilisation of Nrf2.²³⁾ The stabilised Nrf2 interferes with IL-6 gene expression.²⁴⁾ CLP is also suggested to activate AMPK, which is independent of the Akt/GSK3/Nrf2 signaling. In future studies, the precise mechanism of CLP action, for example, the molecular target of CLP, must be clarified.

Acknowledgments

This study was supported by JSPS KAKENHI (Grant No. JP22K11819).

Author Contributions

AK: Investigation; AS: Investigation, Methodology; SO: Methodology, Writing—original draft; MN: Conceptualization, Methodology, Writing—original draft, Writing—review & editing.

Conflict of Interest

The authors declare no conflict of interest.

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