Carvacrol protects mice against LPS-induced sepsis and attenuates inflammatory response in macrophages by modulating the ERK1/2 pathway

Macrophages play an important role in the development of life-threatening sepsis, which is characterized by multiorgan dysfunction, through their ability to produce inflammatory cytokines. Carvacrol is a phenolic compound that has been confirmed to possess strong anti‑inflammatory activity. In this study, we mainly investigated the effect of carvacrol on lipopolysaccharide (LPS)-induced macrophage proinflammatory responses and endotoxic shock. The results showed that carvacrol significantly reduced mouse body weight loss and ameliorated pathological damage to the liver, lung, and heart under LPS-induced sepsis. Carvacrol attenuated inflammatory responses by inhibiting the LPS-induced production of inflammatory cytokine interleukin-6 (IL-6) in vivo and in vitro. Mechanistically, carvacrol inhibited IL-6 production mainly through the ERK1/2 signalling pathway in macrophages. Furthermore, carvacrol improved the survival of septic mice. This study sheds light on the role of carvacrol in the pathogenesis of LPS-induced sepsis, and thus, its potential in treating sepsis patients may be considered.

protein-1 (MCP-1), and IL-6 14 .In addition, carvacrol significantly reduced the expression of immunoglobulin E (IgE), TNF-α and IL-4, while it significantly increased the expression of superoxide dismutase (SOD) and glutathione (GSH) in ovalbumin-induced inflammatory disease asthma 15 .Carvacrol prevents elevations in the levels of the inflammatory cytokines TNF-α and IL-6 and protects organs from damage in a murine model of caecal ligation and puncture-induced polymicrobial sepsis 16 .Besides, Carvacrol inhibits the production of proinflammatory cytokines such as IL-6 and IL-17 and increases the production of anti-inflammatory cytokines such as transforming growth factor-β (TGF-β) and IL-10 in EAE and exhibits anti-inflammatory properties 17 .Moreover, several studies have reported that carvacrol has protective effects against acute lung injury, inhibiting the production of inflammatory cytokines in serum and reducing oxidative stress in LPS-induced sepsis 18 .However, the effects of carvacrol on multiple organ damage and inhibition of macrophage activation remain unclear.Therefore, we first studied the role of carvacrol in LPS-induced multiple organ damage and macrophage activation and then explored its possible mechanism.
In this study, we observed that carvacrol reduced the mortality of septic mice, inhibited the LPS-induced production of proinflammatory cytokine IL-6 in serum, and ameliorated the pathological injury of liver, lung and heart.In addition, carvacrol significantly inhibited the production of the inflammatory cytokine Il-6 through the extracellular regulated protein kinase (ERK1/2) signalling pathway in macrophages.

Results
Carvacrol reduced LPS-induced body weight loss.In this study, we focused on elucidating the role of carvacrol in the proinflammatory response of macrophages and the pathogenesis of sepsis.To investigate the role of carvacrol during inflammation, we subjected the mice to sublethal doses of LPS.See the supplementary material for details regarding the groupings (Fig. S1).First, we investigated the body weight loss of mice treated with different doses of carvacrol under LPS-induced endotoxic shock.In accordance with our prediction, carvacrol reduced the body weight loss of the mice induced by LPS (Fig. 1A).To investigate whether carvacrol affects the survival of LPS-treated septic mice, mice were intraperitoneally administered different doses of carvacrol for 2 h before LPS injection, and the death of the mice was observed.As shown in Fig. 1B, LPS-treated mice given carvacrol had a significantly longer survival time and a better survival rate than LPS-treated mice given 0.2% Tween 80.The data revealed that carvacrol reduced the mortality of LPS-treated mice, further demonstrating that carvacrol protected the mice from endotoxic shock.
Carvacrol alleviated tissue injury under LPS-induced sepsis.LPS-induced endotoxic shock is a systemic inflammatory response syndrome, and excessive inflammation can lead to tissue damage 19,20 .To evaluate whether carvacrol influenced this process, we evaluated tissue damage in the carvacrol-treated mice subjected to LPS stimulation.The mice were pretreated with carvacrol for 2 h and stimulated with LPS for 12 h.Lung, liver, and heart samples were collected, and the pathological lesions were observed by HE staining.We observed much less damage in the liver, lung, and heart of carvacrol-treated mice (Fig. 2A-C); the levels of inflammatory cell infiltration, haemorrhage, and oedema were reduced in the lung tissue; the cytoplasmic colour fading, vacuolization, nuclear condensation, and nuclear fading of the liver cells were significantly reduced; and the levels of myocardial cell structural changes and inflammatory cell infiltration were also reduced in the heart.To further investigate whether carvacrol improved cardiac function in mice subjected to endotoxic shock, we assessed the ejection fraction (EF) and fractional shortening (FS) by echocardiography (Fig. 2D-F).The data showed that LPS-treated mice exhibited significantly lower EF and FS than mice in the control group, and those subjected to pretreatment with carvacrol had higher EF and FS than those in the LPS group.These results imply that carvacrol provides an indispensable function of organ protection under exposure to LPS.
Carvacrol inhibited the proinflammatory response of macrophages in septic mice.To explore whether the protective effect of carvacrol against sepsis occurred by reducing the number of LPS-exposed macrophages, we first measured the percentage and number of splenic macrophages.The results showed that mice administered carvacrol exhibited a significantly reduced percentage and number of splenic macrophages follow-  ing LPS exposure (Fig. 3A).To evaluate whether carvacrol inhibited the activation of macrophages, we examined CD86 and CD40 expression.The expression of CD40 in the macrophages of mice given carvacrol was significantly lower than that in mice given 0.2% Tween 80.In contrast, CD86 expression displayed no significant difference under the same conditions (Fig. 3B).To further elucidate the role of carvacrol in macrophage function, we next examined the levels of proinflammatory cytokines in mouse serum and peritoneal cells.It was interesting to observe that with carvacrol administration, the levels of serum IL-6 but not TNF-α were significantly reduced www.nature.com/scientificreports/after LPS injection for 6 h or 24 h (Fig. 3C).Notably, the same effect could be achieved by excessive peritoneal lavage (Fig. 3D).RT-qPCR analysis showed that carvacrol treatment significantly reduced IL-6 mRNA levels in LPS-stimulated mouse peritoneal cells (Fig. 3E).Neither the carvacrol-nor 0.2% Tween 80-treated groups displayed any significant difference in terms of the protein level and mRNA level of TNF-α.Generally, these observations suggest that carvacrol negatively regulates proinflammatory responses in macrophages under LPSinduced endotoxic shock.

Carvacrol inhibited the proinflammatory response of LPS-stimulated macrophages in vitro.
To further explore the protective effect of carvacrol on the LPS-induced macrophage proinflammatory response, we used BMDMs to evaluate the effect of carvacrol in vitro.Almost 100% of BMDMs differentiated from bone marrow were CD11b + F4/80 + positive cells (Figure S2).First, we assessed the viability of cells that had been treated with different doses of carvacrol for 24 h.As the concentration of carvacrol increased, the activity of cells was not affected (Fig. 4A), indicating that carvacrol within 20 µg/mL does not affect cell viability.Next, we selected 5 µg/mL and 10 µg/mL carvacrol to treat BMDMs for 2 h and then simulated them with LPS to examine the effect of carvacrol on CD86 and CD40 expression.After LPS stimulation, the cells treated with carvacrol exhibited significantly lower expression of CD40 than dimethyl sulfoxide (DMSO)-treated cells.At a carvacrol concentration of 10 µg/ml, CD86 expression changed significantly (Fig. 4B).Additionally, incubating BMDMs with carvacrol resulted in decreased expression of IL-6 at both the protein and mRNA levels.In addition, the mRNA but not the protein levels of TNF-α increased (Fig. 4C,D).In summary, all these data show that carvacrol negatively regulates LPS-induced proinflammatory responses primarily in macrophages.
Carvacrol inhibits the ERK1/2 signalling pathway activated by LPS.To further explore the mechanism by which carvacrol inhibits LPS-induced inflammation, we pre-treated BMDMs with different doses of carvacrol and then exposed these BMDMs to LPS for 10 min, 15 min or 24 h.We observed that carvac- rol only suppressed the phosphorylation of ERK1/2, but not that of p65, JNK, and p38, after LPS stimulation (Fig. 5A).We used specific inhibitor of MAPK (U0126) to pretreat BMDMs and evaluate the effect of carvacrol on LPS-induced inflammation.We found that U0126 inhibited the production of IL-6 and TNF-α in LPS-treated BMDMs.When cells were pretreated with U0126, the inhibitory effect of carvacrol on IL-6 production was significantly reduced (Fig. 5B).We also conducted in vivo experiments with the inhibitor and found that after treatment with U0126, the levels of IL-6 and TNF-α in the serum of septic mice were significantly reduced.Tubulin was used as a control.(B) BMDMs were pretreated with U0126 (10 μM) for 1 h, followed by carvacrol (10 μg/mL) for 2 h, and then administered LPS (1 μg/mL) for 24 h.IL-6 and TNF-α levels were measured in the culture medium.(C) Mice were pretreated with carvacrol (80 mg/kg) for 2 h and then stimulated with LPS (5 mg/kg of body weight).After 1 h, U0126 (20 μL of 20 mM) was injected intraperitoneally (n = 4 mice/group).The grouping of gels/blots cropped from different parts of the same gel (p-p65, p-p38 and p-JNK; ERK and Tubulin; p65 and p38 for 10 min samples) or different gels (p-ERK and JNK for 10 min samples).For 15 min and 24 h samples, the grouping of gels/blots cropped from different parts of the same gel (ERK and p65; p-JNK and p-p38) or different gels (p-ERK, p-p65, JNK, P38 and Tubulin).The full blots are shown in Supplementary Information S1.Data were analysed by two-way ANOVA with Bonferroni post-test (B-D).*P < 0.05; **P < 0.01, and ns not significant.www.nature.com/scientificreports/When mice were treated with U0126, the inhibitory effect of carvacrol on LPS-induced IL-6 production was significantly reduced (Fig. 5 C).These data indicate that carvacrol mainly negatively regulates ERK1/2 signalling and subsequently inhibits the production of inflammatory factors and tissue damage after LPS stimulation.

Discussion
Sepsis is a complex and rapidly developing inflammatory disease, and the molecular mechanism underlying the immune response to this condition warrants further exploration 21 .Sepsis results in many deaths every year and represents a major global health problem.Therefore, it is urgent to identify new therapeutic drugs for effective sepsis treatment.Macrophages play key roles in causing excessive inflammatory responses and organ failure and have important functions during all stages of sepsis 22,23 .Therefore, modulation of macrophage immune function is of great importance for the treatment of sepsis 24,25 .Many studies have revealed that natural products, such as astragalus polysaccharide 26 , mushroom polysaccharides 27 , and curcumin 28 exert protective and therapeutic effects on sepsis.We identified that carvacrol attenuates the proinflammatory responses of macrophages and protects against septic shock in mice.Carvacrol has been proven by numerous studies to exert anti-inflammatory activities 14,17,29 .Interestingly, we found that carvacrol prevents elevations in the levels of the inflammatory cytokines IL-6 not TNF-α in serum and peritoneal lavage and protects organs from damage in a murine model of LPS-induced sepsis.Besides, carvacrol inhibits the production of IL-6 not TNF-α in LPS stimulated bone marrow derived-macrophages in vitro.However, there are no reports on the effect of carvacrol on LPS-induced macrophage function.Whether carvacrol can attenuate LPS-induced inflammatory responses in macrophages in sepsis and its underlying mechanisms still need to be further explored.
Macrophages are one of the key types of cells involved in the pathogenesis of sepsis and exert effects by secreting inflammatory cytokines 30 .Carvacrol inhibits dendritic cell (DC) activation in vitro by decreasing the expression of CD40, which is also important for macrophage activation 31 .These studies indicate that carvacrol may protect mice from injury by inhibiting macrophage activation in the endotoxic shock model.We pre-treated cells with a certain concentration of carvacrol prior to LPS exposure and found that carvacrol effectively reduced the expression of CD40 and IL-6, which suggests that carvacrol can inhibit macrophage activation and proinflammatory cytokine production in vivo.The in vitro experimental results also showed that pre-treatment with carvacrol before LPS stimulation limited the activation and function of BMDMs.
Increasing evidence has shown that LPS-induced macrophage activation is mainly mediated by the ERK1/2 and NF-κB signalling pathways 32,33 .LPS activates the phosphorylation of the mitogen-activated protein kinases ERK1/2, p38, JNK, and p65.Consistent with previous research showing that carvacrol suppressed LPS-induced RAW264.7 cell activation through the ERK1/2 and NF-κB pathways 34,35 , we observed that carvacrol only significantly decreased p-ERK1/2 expression in BMDMs.Whether carvacrol interferes with LPS binding with TLR4 and the receptors for carvacrol warrants further study.In addition, both in vitro and in vivo results demonstrated that carvacrol reduced IL-6 expression but had no effect on TNF-α expression.Similar research found that javamide-II inhibits IL-6 production via suppressing the p38 signal pathway, without significant effects on the productions of TNF-alpha and IL-1beta in macrophage-like THP-1 cells, suggesting that carvacrol may regulate molecules that only affect IL-6 expression, and the specific molecular mechanism still needs further exploration.
In conclusion, the results of this study suggest that targeted modulation of the inflammatory cytokine storm with carvacrol could open new avenues for sepsis treatment strategies and demonstrate the potential application of carvacrol in pharmacological studies.
Animals.Female C57BL/6 mice (6-8 weeks old) were purchased from the Hunan Laboratory Animal Center.All mice were housed in specific pathogen-free animal facilities at Jiangxi University of Traditional Chinese Medicine, and the mice experiments used protocols that were approved by the Institutional Animal Care and Use Committee.

LPS-induced sepsis.
We randomly divided mice into different groups, pretreated them for 2 h with either carvacrol or 0.2% Tween 80, and finally administered LPS by intraperitoneal injection.Mice were intraperitoneally injected with 10 mg/kg body weight LPS, peritoneal cells and serum were collected after 6 h and 24 h, and the liver and lung were collected after 12 h for haematoxylin and eosin staining.The survival after administration of LPS (25 mg/kg of body weight) was also monitored.
Bone marrow-derived macrophage preparation.Bone marrow (BM) cells were isolated from the tibia and femur of C57BL/6 mice and cultured in complete Dulbecco's Modified Eagle Medium (DMEM) supplemented with M-CSF (10 ng/mL).Half of the medium was replaced with fresh DMEM supplemented with

Figure 2 .
Figure 2. Carvacrol alleviated mouse liver, lung and heart injury in LPS-induced sepsis.(A,B) We treated C57BL/6 mice with carvacrol (80 mg/kg of body weight) or 0.2% Tween 80 for 2 h and then used LPS (10 mg/kg of body weight) for stimulation.Lung, liver, and heart samples were harvested and fixed with 4% paraformaldehyde at 12 h after LPS injection.Staining of paraffin-embedded liver (A), lung (B), and heart (C) sections with haematoxylin-eosin staining (H&E) and histological analysis of acute liver injury (the arrow marks cytoplasmic colour fading, vacuolization, nuclear condensation, nuclear fading), lung injury (the arrow marks haemorrhage, lung oedema, inflammatory cell infiltration) and heart injury (the arrow marks myocardial cell structure and inflammatory cell infiltration) (n = 3 mice/group).(D) Representative echocardiographic images from each group.(E,F) The ejection fraction and fraction shortening of the heart were measured using the images in (D).Data were analysed by one-way ANOVA with Tukey's multiple comparisons test.The error bars show the means ± SEMs.*P < 0.05; **P < 0.01; ***P < 0.001.

Figure 3 .
Figure 3. Carvacrol inhibited LPS-induced macrophage activation and the proinflammatory response in vivo.We treated C57BL/6 mice with either carvacrol (80 mg/kg of body weight) or 0.2% Tween 80 for 2 h, followed by LPS (10 mg/kg of body weight) stimulation.(A) Flow cytometry analysis of cell numbers and percentages of CD11b + F4/80 + macrophages in the spleen after stimulation with LPS or PBS for 12 h (n = 4 mice/group).(B) Representative flow cytometry analysis of the levels of CD86 and CD40 and the mean fluorescence intensity of CD86 and CD40 in spleen macrophages as in (A).ELISA analysis of IL-6 and TNF-α in the serum (C) or peritoneal lavage (D) from carvacrol-or 0.2% Tween 80-treated mice 6 or 24 h after LPS exposure (n ≥ 3 mice/group).(E) IL-6 and TNF-α mRNA expression in mouse peritoneal cells was measured by RT-qPCR (n = 3 mice/group).Data were analysed by two-way ANOVA with Bonferroni posttest (A,B) or one-way ANOVA with Tukey's multiple comparisons test (C-E).The error bars show the means ± SEMs.*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 and ns not significant.

Figure 4 .
Figure 4. Carvacrol inhibited the LPS-induced macrophage proinflammatory response in vitro.(A) BMDMs were pretreated with different concentrations of carvacrol (0-20 μg/mL) and then treated with PBS or LPS for 24 h, and cell viability was assessed by CCK8 assay.(B-D) BMDMs were pretreated with different doses of carvacrol (0, 5, and 10 μg/mL) for 2 h and then treated with LPS (1 μg/mL).(B) FACS analysis of CD86 and CD40 expression in BMDMs after stimulation with LPS for 24 h.(C) After LPS stimulation for 24 h, the protein levels of IL-6 and TNF-α in the supernatant were measured by ELISA.(D) After 6 h of LPS stimulation, the mRNA levels of IL-6 and TNF-α were measured by RT-qPCR.Data were analysed by two-way ANOVA with Bonferroni posttest (A,B) or one-way ANOVA with Tukey's multiple comparisons test (C,D).The error bars show the means ± SEMs.*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 and ns: not significant.