THE PROTECTIVE ROLE OF CURCUMIN IN ZYMOSAN-INDUCED MULTIPLE ORGAN DYSFUNCTION SYNDROME IN MICE

ABSTRACT

Aim:

Multiple Organ Dysfunction Syndrome (MODS) is characterized as progressive and uncontrolled inflammatory response which involves activation of inflammatory cascades, cytokines release, and endothelial dysfunction, leading to deterioration of several organ functions. Curcumin is a natural polyphenol related to the yellow color of turmeric and has been reported to exert an anti-inflammatory, anti-oxidative, and anti-tumor effect. We conducted the study to investigate the effects of curcumin in non-septic MODS caused by zymosan in mice model.

Method:

The mice were randomly allocated into five groups (six mice per group): control group (treated with physiological saline, 0.1 mL daily for 3 days before and 1 h after physiological saline treatment), DMSO group (treated with DMSO, 0.1 mL daily for 3 days before and 1 h after physiological saline treatment), Curcumin group (200 mg/kg, suspended in DMSO, in a final volume of 0.1 mL, used for 3 days daily before and 1 h after physiological saline treatment), Zymosan+DMSO group (treated with DMSO, 0.1 mL daily for 3 days before and 1 h after zymosan treatment) and Zymosan+ Curcumin group (treated with curcumin, suspended in DMSO at a dose of 0.1 mL daily for 3 days before and 1 h after zymosan treatment).

Mice in groups were sacrificed, and then the blood and tissues were collected to evaluate the severity of acute peritonitis, tissue histopathological changes, NO formation, oxidative stress, PMN infiltration, cytokines production, organ function, and NF-κB activation 18 h after when zymosan or physiological saline was injected. In another set of experiments, the mice were also grouped (20 mice per group) for monitoring the loss of body weight and mortality for 7 days after zymosan or physiological saline administration.

Results:

Curcumin induces a significant reduction of the volume exudate and the neutrophil infiltration. It also could exhibit an outstanding protective effect against histopathological injury by decreasing the NO formation, oxidative stress, cytokines production, and infiltration of inflammatory cells. The organ function is also improved by administration of curcumin. Moreover, the activation of NF-κB is attenuated by curcumin in the MODS mice model, suggesting that curcumin attenuated the zymosan-induced MODS via inhibiting the expression of NF-κB possibly. In addition, curcumin-treated mice were shown to alleviate the severity of MODS characterized by a minor systemic toxicity, less body weight loss, and lower mortality caused by zymosan administration.

Conclusion:

Curcumin attenuates zymosan-induced MODS.

INTRODUCTION

The sequential organ failure caused by various physiologic insults, including pancreatitis, empyrosis, shock, pyemia, trauma, etc., is known as “Multiple Organ Failure” (MOF) previously. Nowadays, MOF has been redefined as “Multiple Organ Dysfunction Syndrome” (MODS) (1). The MODS is characterized as the progressive deterioration of various organ functions. It has been a consensus that lung is the first organ affected by the MODS, subsequently followed by liver, intestine, kidney, hematological system, and eventually the cardiovascular system (2). Actually, the exact order of organ dysfunction depends on pre-existing disease or the sudden insult.

Previous studies indicated that MODS is the outcome of a generalized, highly amplified, progressive, and uncontrolled humoral and cellular response to the critical injury or stress, resulting in a generalized inflammatory state. The inflammatory cascades active the synthesis or release of complement, coagulation, kinin, and fibrinolytic. Moreover, phagocytes and endothelial cells are sensitized simultaneously by the inflammatory process of MODS (3). Vasodilatation, increased microvascular permeability, cellular adhesion, and coagulation activation are the four major events involved in the inflammatory cascades of MODS (4). Vasodilatation and increased microvascular permeability are related to the increase of local exploitable oxygen and nutrients, producing heat, swelling, and edema. Cellular adhesion and coagulation activation are associated with various molecules and cells, such as tumor necrosis factor (TNF-α), interleukins (IL), polymorphonucleocytes (PMNs), monocytes/macrophages, and endothelial cells (4, 5).

Zymosan, obtained from the cell wall of the yeast Saccharomyces cerevisiae, is a nonbacterial, nonendotoxic substance that induces acute peritonitis and MODS characterized by functional and structural organ changes that concur with human beings (6). The inflammatory cascades of MODS caused by zymosan in mice within 18 h are associated with systemic hypotension, high level of nitric oxide (NO) in peritoneum and plasma, maximal cellular infiltration, exudate formation, and cyclooxygenase activity (7). Moreover, the severity of MODS is dependent on the dosage of zymosan used in the experiment.

Curcumin is a natural polyphenol related to the yellow color of turmeric. Turmeric is a spice produced from the root of Curcuma longa, a member of the ginger family, Zingiberaceae. Because of the medicinal properties of turmeric, it has been used for a long time in India. Medically curcumin is mainly used for its anti-inflammatory, anti-oxidative, and anti-tumor effect. Curcumin harbors these effects because it can regulate certain molecular targets including pro-inflammatory cytokines, transcription factors, etc. (8–10). Previous studies have indicated curcumin inhibits the LPS-induced activation of nuclear NF-κB. Moreover, curcumin has the ability to inhibit the production of inflammatory cytokines in both rat vascular smooth muscle cell and RAW264.7 cell which was administered with LPS (11, 12).

However, whether curcumin can attenuate non-septic shock remains unclear. In this study, we investigated the effects of curcumin in non-septic MODS caused by zymosan in mice model.

MATERIALS AND METHODS

Experimental animals

Male wild-type C57BL/6 mice (4–5 weeks old, 20–25 g) were purchased from Animal Feeding Center of Xi’an Jiaotong University Health Science Center. All mice were housed in pathogen-free polycarbonate cages (three per cage) in a controlled environment and temperature (24 ± 1°C) with 12h light–dark cycles. All of the mice were fed standard rodent chow and water ad libitum and were adapted to the environment for 7 days before use. Animal care was in compliance with criteria outlined in the Guide for the Care and Use of Laboratory Animals established by the US National Institutes of Health. The study was approved by the Animal Research Committee of Xi’an Jiaotong University Health Science Center.

Reagents

Curcumin and Zymosan were obtained from Sigma-Aldrich (St. Louis, MO, USA). Curcumin was dissolved in DMSO and stored at 4°C. Zymosan was dissolved in 1 × PBS and stored at 4°C. The Nitrite/Nitrate assay kit was purchased from the Beyotime Institute of Biotechnology of Nantong, Jiangsu, China. Total Superoxide Dismutase (T-SOD) assay kit, Malondialdehyde (MDA) assay kit, and Myeloperoxidase (MPO) assay kit were provided by the Jiancheng Bioengineering Institute of Nanjing, Jiangsu. Mouse TNF-α, IL-6, and IL-1β ELISA kits were obtained from Dakewe Biotech Company, Shenzhen, China. Rabbit monoclonal antibodies including phosphorylated and non-phosphorylated forms of NF-κB were obtained from Cell Signaling Technology Inc, Beverly, MA, USA.

Zymosan-induced non-septic shock

The mice were randomly allocated into five groups as follows (six mice per group): Control group (Con): the mice were treated with physiological saline (0.9% NaCl solution) via intraperitoneal injection (i.p.). DMSO group (DMSO): the mice were treated with DMSO instead of physiological saline via i.p. which is identical to the control group (at a dose of 0.1 mL daily for 3 days before and 1 h after physiological saline treatment). Curcumin group (Cur): the mice were treated with curcumin via i.p. (200 mg/kg, suspended in DMSO, in a final volume of 0.1 mL, used for 3 days daily before and 1 h after physiological saline treatment). Zymosan+DMSO group (Zym+DMSO): the mice were both treated with zymosan (500 mg/kg, solubilized in physiological saline) and DMSO (at a dose of 0.1 mL daily for 3 days before and 1 h after zymosan treatment). Zymosan+Curcumin group (Zym+Cur): the mice were both treated with zymosan and curcumin via intraperitoneal injection (at a dose of 200 mg/kg, suspended in DMSO, used for 3 days daily before and 1 h after zymosan treatment). Eighteen hours after the administration of zymosan or saline, the animals were sacrificed after anesthetizing for estimating the non-septic shock as described below (13–15). In another set of experiments, the mice were grouped (20 mice per group) as mentioned above for monitoring the loss of body weight and mortality for 7 days after zymosan or physiological saline administration (13–15). During the observation period, new added mice were banned. The mortality of mice was calculated at the end of the observation period. The procedure of the experiment is shown as follows (Fig. 1).

Fig. 1.

The procedure of the experiment.

Clinical scoring of systemic toxicity

The clinical severity of systemic toxicity in administrated mice was assessed depending on a subjective scale created by Di Paola et al. throughout the period of the experiment (7 days) (16). The scale ranging from 0 to 3 (0 = absence, 1 = mild, 2 = moderate, 3 = serious) was applied for assessing each of the toxic signs, including conjunctivitis, ruffled fur, diarrhea, and lethargy, which were observed in the mice models. The final score of the systemic toxicity was the summation of the four single evaluations and the maximum value of score was 12. Two independent investigators, who had no knowledge of the administration received by respective animals, assessed the clinical score via discussion and consensus on all formulary criteria of the clinical scoring of systemic toxicity.

Assessment of acute peritonitis

After the mice were administrated with zymosan or physiological saline for 18 h, all mice were sacrificed, and sequentially the parameters of acute inflammation in peritoneum were measured. Through an incision in abdominal linea alba, a total of 5 mL phosphate-buffer-saline (PBS) was injected into the abdominal cavity. After abdominal massage for a while, the washing buffer was drawn out carefully through the incision in abdominal linea alba. Then the washing buffer was collected in a 10-mL centrifuge tube by a plastic pipette. The volume of exudate was calculated by subtracting the volume injected (5 mL) from the total volume collected. The peritoneal exudate was centrifuged at 7,000 × g for 10 min at room temperature. The precipitates of cell were counted by Turk's solution which was composed of 0.01% crystal violet and 3% acetic acid. The absorption value of the supernatant was measured by a Beckmann 520 spectrophotometer at OD650.

Histological study and evaluation

Samples of liver, lung, kidney, and distal intestine were excised and fixed in 10% formalin solution to assess morphological changes after 18 h when zymosan or physiological saline was injected. Then all the samples were embedded in paraffin and sliced into a serial of 5-μm thicknesses stained with Hematoxylin & Eosin (H&E) to evaluation. Evaluation of the histologic changes was conducted by two independent pathologists who were blinded to the administration received by respective animals. The tissue injury scores were also assessed by the two independent pathologists (17, 18).

Measurement of nitrite/nitrate concentrations

The production of nitrite/nitrate (NO₂/NO₃) was an indicator of the synthesis of NO. Plasma samples of mice were obtained 18 h after administration of zymosan or physiological saline to assess the concentration of nitrite/nitrate by a nitrite/nitrate colorimetric commercial kit obtained from Beyotime. The optical density at 550 nm (OD550) was measured by a spectrophotometer. The concentration of nitrate was calculated by comparison with OD550 of standard solutions of sodium nitrate prepared in saline solution.

Measurement of total superoxide dismutase (T-SOD), myeloperoxidase (MDA), and malondialdehyde (MPO) concentrations

Total superoxide dismutase (T-SOD) activity, used as an indicator of clearance of superoxide was measured by a total superoxide dismutase (T-SOD) assay kit according to the manufacturer's instruction. The concentration of Malondialdehyde (MDA) was an indicator of the lipid peroxide, which was involved in lipid peroxidation and production of oxyradical. It was measured by Malondialdehyde (MDA) assay kit according to the manufacturer's instruction which was similar to T-SOD measurement. Myeloperoxidase (MPO) activity, an indicator of PMN infiltration in lung, liver, and intestine, was also measured by Myeloperoxidase (MPO) assay kit.

Measurement of cytokines

The levels of indicators of inflammation including TNF-α, IL-6, and IL-1β were measured 18 h after the administration of zymosan or physiological saline by using plasma samples. The mouse TNF-α, IL-6, and IL-1β ELISA kits, purchased from Dakewe Biotech Company, were used for measurement.

Evaluation of organ function

In order to evaluate the function and injury of organs in the macroscopic scale, the blood samples of mice were collected using cardiac puncture 18 h after the administration of zymosan or physiological saline. Then the blood samples were centrifuged (1,610× g for 3 min at room temperature) to obtain plasma. All plasma samples were analyzed via standard laboratory techniques in a veterinary clinical laboratory. The indexes used as biochemical indicators of MODS were as follows: liver injury was assessed by measuring the level of aspartate aminotransferase (AST), which is a non-specific marker for hepatic injury, and alanine aminotransferase (ALT), which is a specific marker for hepatic parenchymal injury in plasma. Lung injury was assessed by blood gas analysis. The pO₂, pCO₂, HCO₃⁻, and pH were measured using the arterial blood samples via a Blood-gas Analyzer. Renal injury was evaluated by the level of creatinine, which is an indicator of glomerular filtration rate. Moreover, lower glomerular filtration rate means more serious renal failure. The levels of amylase and lipase in plasma samples were measured to assess the pancreatic injury.

Western blot

Proteins were extracted from the tissue samples according to the manufacturer's instruction. Then BCA protein assay kit was used to measure the concentration of extracted proteins. Equal amounts of protein were loaded and separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Then the gel electrophoresis was transferred onto polyvinylidene difluoride (PVDF) membranes, which were immunoblotted with the appropriate primary antibody at 4°C overnight. Then the membranes were incubated with HRP conjugated anti-goat or anti-rabbit antibody. The signals were generated using an enhanced chemiluminescence (ECL) Western blotting kit, finally.

Statistical analysis

The survival and mortality rates were expressed as percentage. The data were expressed as mean ± standard deviation (SD). The differences between the respective groups were evaluated by either analysis of variance (ANOVA) or nonparametric test, as applicable, using SPSS v20 (SPSS Inc, Chicago, IL,

IBM Inc). A P value less than 0.05 was considered statistically significant.

RESULTS

Effect of curcumin on weight change, mortality, and systemic toxicity

Administration of zymosan caused a severe peritonitis in mice characterized as systemic toxicity and distinct weight loss. Obviously, the body weight decreased. However, on the third to fifth day, the rate of weight loss slowed down (Fig. 2A). An upward trend appeared at the seventh day in the Zym+Cur group, but there was no statistical significance compared with the level at the fifth day (P = 0.47) (Fig. 2A). Moreover, the mortality also persistently decreased throughout the observation period (7 days), and the mortality of the mice treated with zymosan was 75% at the end of the observation period (Fig. 2B). In addition, the clinical scoring of systemic toxicity significantly rose over time and reached the maximum at the end of the seventh day (Fig. 2C). The maximum of systemic toxicity score was 8.6 ± 0.51 (mean ± SD). Curcumin (200 mg/kg, i.p.) protected the mice against toxicity and decreased the loss of body weight and mortality caused by zymosan. Moreover, curcumin treatment did not cause significant changes in these parameters in control mice and could attenuate the toxicity of DMSO (Fig. 2, A and C).

Fig. 2.

Curcumin reduces the loss of body weight, mortality, and systemic toxicity induced by zymosan (n = 20).

A, The loss of body weight was measured at −3, 0, 3, 5, 7 days. B, The survival curves of mice during the 7 days after administration of zymosan or saline. C, Systemic toxicity score was measured at 0, 3, 5, 7 days to evaluate the severity of MODS caused by zymosan. #P < 0.01, ^∗P < 0.05, ^∗1P < 0.05 Cur vs. DMSO group, #₁P < 0.01 Cur vs. DMSO group.

Effect of curcumin on acute peritonitis

At 18 h after the administration of zymosan or saline, the exudates and peritoneal exudate cells that both stood for severity of acute peritonitis were detected. The Zym+DMSO group showed a significant increase in the volume exudate and the polymorphonuclear leukocyte number synchronously compared with the control, DMSO, or Zym+Cur group (Fig. 3, A and B). Moreover, the nitrite/nitrate levels, indicating the NO formation, also increased in the exudate in the Zym+DMSO group (Fig. 3C). The mice in Zym+Cur group showed an obvious reduction in volume exudate, the polymorphonuclear leukocyte number, and NO formation (Fig. 3). Curcumin treatment did not cause significant changes in the sham group.

Fig. 3.

Curcumin reduces the severity of acute peritonitis (n = 6).

A, The volume of exudate was less in curcumin-treated group. B, The peritoneal exudate cell count was reduced in Zym+Cur group compared with Zym+DMSO group. C, The level of nitrate/nitrite in exudate was markedly reduced in Zym+Cur group compared with Zym+DMSO group. #P < 0.01, ^∗P < 0.05.

Effect of curcumin on tissue histopathological change

Eighteen hours after zymosan or saline injection, the samples of liver, lung, kidney, and intestine were stained with H&E and revealed several distinct pathological changes. The examination of lung biopsies demonstrated an obvious infiltration of neutrophils, macrophages, and plasma cells (Fig. 4A). The intestine samples stained with H&E revealed a significant edema in the space bounded besides infiltration of inflammatory cells (Fig. 4A). In liver and kidney, there were inflammatory infiltration and damages of normal structures (Fig. 4A). However, the mice treated with curcumin showed a marked reduction of histological injury, especially the inflammatory infiltration in lung, intestine, liver, and kidney. Moreover, tissue injury scores for each organ were assessed by two independent pathologists (Fig. 4B). None of these changes in histopathology was observed in the control group (Fig. 4).

Fig. 4.

Curcumin improves the pathological changes of tissue samples.

A, 18 h after zymosan was injected, the animals were sacrificed and tissue sections were stained by H&E. Liver, lung, kidney, and intestine samples from curcumin-treated group exhibited a markedly reduction of histological injury, especially the inflammatory infiltration. Original magnification ×200. Figures were representative of at least three experiments conducted at different days. B, Tissue injury scores for the H&E staining of each organ. #P < 0.01, ^∗P < 0.05.

Effect of curcumin on NO formation and oxidative stress

The NO formation was associated with inflammation in MODS and the levels of nitrate/nitrite were the indicators of the formation of NO. The level of nitrate/nitrite in plasma, an indicator of NO formation, was markedly elevated in zymosan-treated mice in comparison with control group (Fig. 5A). Moreover, the nitrate/nitrite level in exudate also rose significantly in the Zym+DMSO group (Fig. 3C). In addition, the level of SOD, which can protect tissue against oxidative damage, was sharply decreased in zymosan-treated mice while it was retrieved by curcumin (Fig. 5B). Similarly, the MDA level, an indicator of the lipid peroxide which was associated with lipid peroxidation and production of oxyradical, was elevated in Zym+DMSO group in comparison with control group. The increase of the level of MDA was attenuated by curcumin compared with Zym+DMSO group (Fig. 5C). Altogether, curcumin showed a protective effect against NO formation and oxidative stress (Figs. 3C and 5).

Fig. 5.

Curcumin reduces the NO formation and oxidative stress (n = 6).

A, The level of nitrate/nitrite in plasma, an indicator of NO formation, was markedly reduced in Zym+Cur group compared with Zym+DMSO group. B, The activity of SOD retrieved in Zym+Cur group compared with Zym+DMSO group. C, The level of MDA, an indicator of the lipid peroxide which was associated with lipid peroxidation and production of oxyradical, was reduced by zymosan significantly compared with zymosan-treated group. #P < 0.01, ^∗P < 0.05.

Effect of curcumin on PMN infiltration

It is generally known that the increase of MPO activity indicated accumulation and infiltration of neutrophils in certain tissues and augmented tissue damages. Therefore, the level of MPO, an enzyme contained in PMN lysosome, was measured in tissue samples to reveal the severity of inflammation. As a result, the MPO activity was significantly increased in lung, intestine, liver, and kidney after administration of zymosan (Fig. 6). The MPO activity was decreased in tissue samples of zymosan-treated mice that have been administrated with curcumin (Fig. 6).

Fig. 6.

Curcumin attenuates PMN infiltration (n = 6).

The MPO activity, indicating accumulation and infiltration of neutrophils in certain tissues, was significantly increased in liver, lung, kidney, and intestine after 18 h when zymosan was administrated. Curcumin can reduce the elevated level of MPO. #P < 0.01, ^∗P < 0.05.

Effect of curcumin on cytokines production

The pro-inflammatory cytokines TNF-α, IL-6, and IL-1β, which modulated the process of inflammation via regulation of cytokines secretion, were measured in plasma. A substantial increase of TNF-α, IL-6, and IL-1β was observed in the zymosan-treated mice, while a significant inhibition of TNF-α, IL-6, and IL-1β was observed in the curcumin-treated group (Fig. 7, A–C). In addition, curcumin did not cause significant changes in the cytokines in the control mice (Fig. 7, A–C). Moreover, curcumin attenuated the increase of IL-1β induced by DMSO in comparison with curcumin group (Fig. 7C).

Fig. 7.

Curcumin reduces the production of pro-inflammatory cytokines (n = 6).

A, TNF-α was significantly attenuated in curcumin-treated mice after zymosan injection. B, IL-6 was significantly attenuated in curcumin-treated mice after zymosan injection. C, IL-1β was significantly attenuated in curcumin-treated mice after zymosan injection. Curcumin attenuated DMSO induced increase of IL-1β. #P < 0.01, ^∗P < 0.05.

Effect of curcumin on organ function and injury

The mice treated with saline, DMSO, or curcumin did not show any significant alterations in the indicators of liver function. However, the zymosan-treated mice demonstrated a sharp rise of the AST, ALT, and ALP level, suggesting the injury of hepatocyte and dysfunction of liver (Fig. 8A). Curcumin had the ability to attenuate the liver injury induced by zymosan (Fig. 8A).

Fig. 8.

Curcumin improves organ function after administration of zymosan (n = 6).

Liver (A), lung (B), kidney and pancreas (C) were protected against injury or dysfunction by curcumin. #P < 0.01, ^∗P < 0.05.

After administration of zymosan, lung function was impaired. Compared with zymosan-treated mice, administration of curcumin could improve the level of HCO₃⁻ and pH value, but did not have the same effect of pO₂ or pCO₂ (Fig. 8B).

In addition, the zymosan-treated mice demonstrated a significant increase of the level of creatinine, amylase, and lipase, suggesting the development of renal dysfunction and injury of pancreas. Contrarily, treatment with curcumin sharply reduced the level of creatinine, amylase, and lipase, suggesting a protective effect against injury of kidney and pancreas (Fig. 8C).

Effect of curcumin on NF-κB activation

The expression of NF-κB was detected by Western blot. The results showed that the expression of phosphorylation of NF-κB p65 was markedly elevated in zymosan-treated mice (Fig. 9). However, administration of curcumin reduced the expression of phosphorylation of NF-κB p65, suggesting that curcumin could attenuate the zymosan-induced MODS via inhibiting the NF-κB expression (Fig. 9).

Fig. 9.

Curcumin attenuates the expression of NFκB.

A, The expression of phosphorylation of NF-κB was elevated by administration of zymosan. Contrary, curcumin-treated mice expressed less phosphorylation of NF-κB. B, Densitometry analysis of (A).

DISCUSSION

MODS is characterized as a progressive and uncontrolled inflammatory response that involves activation of inflammatory cascades, cytokines release, and endothelial dysfunction (11), leading to deterioration of several organ functions, starting mostly with lung failure and followed by failures of the liver, intestine, and kidney. Nowadays, MODS is the leading cause of morbidity and mortality in ICU practice (12). During the process of MODS, sustained hypermetabolism and negative nitrogen balance are the foremost events promoting the disease progression. Clinically, lung injury is the most common cause of single-organ failure and the percentage of direct cause of death in patients is 19% (19). Moreover, renal failure and progressive severity of liver failure herald a later stage of MODS, and the mortality risk is almost 100% (19).

In 1986, Goris et al. established a model known as zymosan-induced generalized inflammation (ZIGI) model. ZIGI model shared a lot of characteristics with human MODS and became the only well-tested experiment model of human MODS.

Zymosan, derived from the cell wall of the yeast Saccharomyces cerevisiae, is a nonbacterial, nonendotoxic substance. As zymosan is not degradable, phagocytosis by macrophages results in a prolonged inflammatory response. The mechanisms of MODS induced by zymosan might be the activation of a wide range of inflammatory mediators including components of the complement system, prostaglandins and leukotrienes, platelet aggregation factor, oxygen radicals, lysosomal enzymes, and activated macrophages (3).

Curcumin is a natural polyphenol related to the yellow color of turmeric. Previous studies showed that curcumin could inhibit inflammation in a number of disease models. It was reported that curcumin inhibited the production of TNF-α and IL-1 in the human monocytic macrophages induced by LPS, probably (20). Curcumin was also reported to inhibit the LPS-induced activation of nuclear NF-κB. In addition, some studies exhibited that curcumin most likely attenuate the sepsis and septic shock via decreasing oxidative stress, production of cytokines, and infiltration of PMNs (21, 22). Another study concluded that curcumin may attenuate the MODS caused by endotoxemia (23). Moreover, a study conducted by Fu et al. indicated that curcumin could attenuate inflammatory responses by suppressing TLR4-mediated NF-κB signaling pathway (24). Similarly, inhibition of activation of NF-κB induced by curcumin is mediated by IκBα kinase and ATK, resulting in the suppression of NF-κB dependent gene production (25, 26). In the present study, we found that curcumin could exhibit an obvious protective effect against MODS induced by zymosan by reducing the severity of peritonitis, the release of cytokines and oxygen-free radicals, the infiltration of PMNs, the injury and dysfunction of liver, lung, kidney and intestine, the mortality of animals, and the clinical symptoms of models, potentially. The course of MODS is composed of three major phases as previously reported. The first phase on which we focused in this study starts right after the administration of zymosan. During this phase, animals develop an acute peritonitis. Moreover, the level of oxygen consumption, MPO activity, concentration of MDA, permeability of endothelial cell and recruitment of neutrophil could increase, and SOD activity could decrease, synchronously. Among all the mechanisms, the recruitment and infiltration of neutrophils in certain tissues may play a crucial role in clearance of foreign antigens, destruction and remodeling of injured tissue which promotes the inflammatory processes. In our study, the histological examination indicated a gradual development and generalized inflammatory response in various organs and also exhibited that the inflammatory response with PMNs infiltration might be improved in treatment group compared with the model group (Fig. 4). It is generally known that MPO activity stands for the relative number of neutrophils. Therefore, the activity of MPO, an important index of tissue damage, can be used to evaluate the protective effects of curcumin against zymosan-induced MODS. In our study, the activity of MPO reduced markedly in the treatment group compared with the model group (Fig. 6). Moreover, the level of MPO in intestine in DMSO group was decreased by curcumin, suggesting the protective effect of curcumin against DMSO toxicity, potentially. Previous studies also obtained the similar results that curcumin could attenuate the accumulation and infiltration of neutrophils. In 2000, a study conducted by Jones et al. indicated that curcumin might inhibit neutrophils infiltration and inflammatory cytokines activation, and also could enhance the activity of SOD and reduced superoxide generation in the renal ischemia reperfusion injury (27). In addition, a water-soluble curcumin complex was proved to attenuate numerous markers of inflammation and injury, including pulmonary edema and neutrophil infiltration in an acute lung injury model (28). Similarly, Hu et al. found that curcumin could promote apoptosis of neutrophils by activation of p38 MAPK or an increased activity of caspase-3 (29).

The level of oxidants, released by activated phagocytes, coincides with the occurrence of severe cell damage. The peroxidation of cell membrane lipids is a noticeable damage. In addition, the production of superoxide is involved in the organ damage. However, the SOD activity and concentration of MDA are biomarkers of superoxide clearance and lipid peroxide level, respectively. Thus, increased MDA level and decreased SOD activity are considered to be associated with a more serious peroxidation. Several studies have shown that the level of SOD decreased and MDA could increase after zymosan administration (3, 7, 30). In the present study, we confirmed that the activity of SOD could increase and the concentration of MDA could decrease in the treatment group, implying less oxidative damage in cells and organs, probably (Fig. 5, B and C). Therefore, oxidant activity might be increased in the zymosan-induced MODS model, but curcumin could cause a substantial decrease of toxicity from oxygen metabolites.

Zymosan-induced MODS in mice also produces a variety of pro-inflammatory cytokines such as TNF-α, IL-6, and IL-1β, which play a critical role in inflammation, similarly. TNF-α is the earliest and primary endogenous mediator of inflammatory reaction (24). IL-6, a pleiotropic cytokine, is produced by monocytes and macrophages and is involved in vascular inflammation and immune reaction. IL-1β has the ability to increase the level of TNF-α in inflammatory cascades. Previous studies demonstrated that TNF-α, IL-6, and IL-1β may be involved in promoting the local or systemic inflammatory process (24, 31, 32). In the present study, the level of TNF-α, IL-6, and IL-1β increased significantly in the plasma samples after administration of zymosan compared with treatment group (Fig. 7). Moreover, curcumin could attenuate the increase of IL-1β induced by DMSO in comparison with curcumin group. Therefore, curcumin may decrease the concentration of these pro-inflammatory cytokines induced by zymosan.

NO is a reactive nitrogen species which has the ability to damage tissues via directly and indirectly ways during numerous serious illnesses (7, 30). The nitrite/nitrate levels in plasma and exudate, an indicator of NO formation, indicated that curcumin could attenuate the NO formation induced by zymosan (Figs. 5A and 3C). However, the DMSO group and curcumin group demonstrate a slight increase in the parameters compared with the control group which might be caused by the toxicity of DMSO and curcumin did not have the ability to reduce the toxicity induced by DMSO (Figs. 5A and 3C). The P values of volume exudate, peritoneal exudate cell, and nitrite/nitrate levels in exudate were 0.063, 0.189, and 0.632 severally compared between DMSO group and curcumin group (Fig. 3). Therefore, curcumin treatment did not cause significant changes in the control group.

NF-κB is a critical factor which plays an essential role in regulating immune responses. Normally, NF-κB locates in the cytoplasm and translocates into the nucleus when it is activated. Then nucleus-located NF-κB will trigger the transcription of cytokines’ gene, such as TNF-α, IL-6, and IL-1β. In this study, NF-κB was activated and resulted in the release of pro-inflammation cytokines in the model group which was treated with zymosan (Fig. 9). However, curcumin could antagonize the activation of NF-κB induced by zymosan (24, 33–35).

To further study the effect of curcumin in zymosan-induced MODS, we measured the indicators of organ function and found that curcumin could improve the levels of HCO₃⁻ and pH. However, the levels of pO₂ and pCO₂ could not be improved by curcumin. Lung was the first affected organ by zymosan-induced MODS and curcumin might play a limited role in the improvement of respiratory function. On the contrary, the histopathological changes, SOD, MDA, and MPO, were all improved by curcumin, potentially. Therefore, curcumin could attenuate the injury of lung partly. Moreover, curcumin may prevent liver dysfunction, supported by the data that curcumin could decrease the level of AST, ALT, and ALP in the treatment group. The levels of creatinine, amylase, and lipase which stand for the severity of MODS were all also reduced by curcumin administration. In addition, the functional changes in organs could be also proved by the H&E staining of sample tissues. However, there are some limitations to our study. Because of restrictions of us, we did not explore the up-regulation or down-regulation of NF-κB in this study. As a result, we could not judge the importance of NF-κB in the zymosan-induced MODS. Based on the experimental data, the NF-κB may affect the proceeding of MODS partly. Additionally, we just explored the protective effect of curcumin in the present study. As a result, all mice were pretreated with curcumin. However, the curative effect of curcumin also should be examined. Therefore, mice will be administrated with curcumin following injury to explore the clinical relevancy by us in the future.

Nowadays, curcumin has been used in clinical trials and obtained favorable results. Storka et al. indicated that short-term intravenous dosing of liposomal curcumin appeared to be safe and had laid the foundation for the clinical application of curcumin (36). A randomized controlled trial conducted by Lang demonstrated that curcumin was a safe and promising agent for the treatment of ulcerative colitis (37). Moreover, curcumin was proved to attenuate the severity of premenstrual syndrome symptoms and reduce IL-22 serum level in patients with psoriasis vulgaris (38, 39).

In conclusion, the MODS induced by zymosan in animal models could be remarkably attenuated by administration of curcumin. Based on the data of this study, the protective effect of curcumin may function by attenuating the oxidative damage and neutrophils infiltration through inhibiting the NF-κB expression, potentially and partially. Furthermore, our findings might demonstrate broad applicable prospects of curcumin that used in the therapy of MODS or other inflammatory diseases.