- Email: [email protected]
Protective effect of crocetin against burn-induced intestinal injury
j o u r n a l o f s u r g i c a l r e s e a r c h 1 9 8 ( 2 0 1 5 ) 9 9 e1 0 7
Available online at www.sciencedirect.com
ScienceDirect journal homepage: www.JournalofSurgicalResearch.com
Protective effect of crocetin against burn-induced intestinal injury Chunxiang Zhou, PhD,a,b,c,1 Wei Bai, MD,d,e,1 Qiaohua Chen, MD,d Zhigang Xu, MD,d Xiongxiang Zhu, PhD,d Aidong Wen, PhD,a,b,c,** and Xuekang Yang, PhD, MDd,* a
Department of Pharmacy, Xijing Hospital, The Fourth Military Medical University, Xi’an, Shaanxi, PR China State Key Laboratory of Cancer Biology, The Fourth Military Medical University, Xi’an, Shaanxi, PR China c Department of Biochemistry and Molecular Biology, The Fourth Military Medical University, Xi’an, Shaanxi, PR China d Department of Burns and Cutaneous Surgery, Xijing Hospital, The Fourth Military Medical University, Xi’an, Shaanxi, PR China e Brigade of student, The Fourth Military Medical University, Xi’an, Shaanxi, PR China b
article info
abstract
Article history:
Background: Oxidative stress and inflammation exert central roles in burn-induced intes-
Received 18 February 2015
tinal injury. Crocetin, a natural carotenoid compound from gardenia fruits and saffron, has
Received in revised form
been shown to inhibit oxidative stress and inflammatory response. However, the possi-
22 April 2015
bility of crocetin to be used in the treatment of intestinal injury after burn injury has not
Accepted 27 May 2015
been investigated. The purpose of the present study was to investigate the effects and
Available online 3 June 2015
potential mechanisms of crocetin in burn-induced intestinal injury. Materials and methods: Several free radicalegenerating and lipid peroxidation models were
Keywords:
used to systematically assess the antioxidant activities of crocetin in vitro. A common burn
Crocetin
model was used to induce the intestinal injury in rats. Changes in the levels of malon-
Intestinal injury
dialdehyde, superoxidase dismutase, catalase, glutathione peroxidase, tumor necrosis
Oxidative stress
factor a, interleukin 6, polymorphonuclear neutrophil accumulation, intestinal perme-
Inflammation
ability, and intestinal histology were examined.
Burn injury
Results: In several models of antioxidant activity, crocetin exhibited marked inhibitory action against free radicals and lipid peroxidation. Crocetin increased levels of antioxidant enzymes and reduced intestinal oxidative injury in burn models. In addition, crocetin inhibited polymorphonuclear neutrophil accumulation, ameliorated tumor necrosis factor a and interleukin 6 levels, intestinal permeability, and histological changes. Conclusions: Crocetin treatment may protect against burn-induced small intestinal injury, possibly by inhibiting burn-induced oxidative stress and inflammatory response. ª 2015 Elsevier Inc. All rights reserved.
* Corresponding author. Department of Burns and Cutaneous Surgery, Xijing Hospital, The Fourth Military Medical University, Xi’an, Shaanxi, PR China. Tel./fax: þ86 2984775298. ** Corresponding author. Department of Pharmacy, Xijing Hospital, and The State Key Laboratory of Cancer Biology and The Department of Biochemistry and Molecular Biology, The Fourth Military Medical University,Xi’an, Shannxi 710032, P.R. China. Tel./fax: þ86 2984775298. E-mail addresses: [email protected] (A. Wen), [email protected] (X. Yang). 1 These authors contributed equally to this work and should be considered as co-first authors. 0022-4804/$ e see front matter ª 2015 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jss.2015.05.052
100
1.
j o u r n a l o f s u r g i c a l r e s e a r c h 1 9 8 ( 2 0 1 5 ) 9 9 e1 0 7
Introduction
Burn injury is one of the most common problems in China [1e6]. Individuals who survive the original insult may go on to develop systemic inflammatory response syndrome, sepsis, and multisystem organ failure, which all are major causes of late deaths after burns [7]. It is postulated that the gastrointestinal system is central to the development of systemic inflammatory response syndrome and multisystem organ failure after burn injury because mucosal injury leads to bacterial translocation and incites an inflammatory response [8]. These facts suggest that the preservation of intestinal mucosal barrier, therefore, has important clinical implications for the outcome of burn victims [7e9]. The mechanisms and treatments of intestinal injury after burn have been the topics with extensive research. Although the precise mechanisms involved have not been fully elucidated, oxidative stress and inflammatory pathways are thought to play important roles [10e12]. Oxidative stress is the imbalance between the production and elimination of reactive oxygen species (ROS). Under normal condition, the body has a potent antioxidant defense system, fighting against excessive generation of ROS. However, when either excessive ROS is generated or the antioxidant defense system is damaged, oxidative stress occurs [13]. ROS target cell membrane constituents cause lipid peroxidation, membrane disintegration, endothelial cell damage, and thus increase microvascular permeability, which in turn leads to the activation and adhesion of polymorphonuclear neutrophils (PMNs) and the release of proinflammatory substances [14]. The accumulation of PMNs further contributes to ROS formation and intestinal tissue damage, resulting in bacterial translocation, systemic complications, and eventually, multiple organ failure [15,16]. Treatments, such as antioxidants, free radical scavengers, and anti-inflammatory therapy, have been used successfully to attenuate intestinal injury in animal models of burn injury [7,10e12]. In recent years, there has been a global trend toward the use of natural substances as antioxidants and functional nutriments. Crocetin, a carotenoid compound, can be extracted from saffron (Crocus sativus L.) or gardenia jasminoides Ellis. This yellow compound has been used as an important spice and natural food colorant in various parts of the world [17]. In addition, saffron and gardenia fruits have been widely used as traditional medicines. In recent years, it has been suggested that crocetin might be an effective antioxidant to counter oxidative stress and inflammation in several models [18e21]. However, the specific roles of crocetins in the prevention of burn-induced intestinal injury remain unclear. Therefore, the aim of this study was to investigate whether crocetin could reduce oxidative damage and inflammatory response, and thus attenuate burn-induced intestinal injury.
2.
Materials and methods
2.1.
Animals and burn protocol
Male SpragueeDawley rats (8e10 wk old, 200e250 g) were purchased from the Center of Experimental Animals of the
Fourth Military Medical University. Rats were housed for 6 d in standard cages and fed a standard chow diet with water ad libitum. All procedures were approved by the Institutional Animal Care and Use Committee of the Fourth Military Medical University, in accordance with the Guidelines for the Care and Use of Laboratory Animals published by the US National Institutes of Health. Unless otherwise noted, all the chemicals and reagents used in this study were procured from Sigma Chemical Co (St. Louis, MO), were of high purity, and were used without purification. For the burn protocol, animals were anesthetized with sodium pentobarbital, the dorsal fur was clipped using an electric clipper. Animals were placed into a template constructed to estimate a 30% total body surface area burn injury [11] and received immediately a subcutaneous injection of 1.5 mL normal saline containing buprenorphine for fluid resuscitation and pain control. Intraperitoneal injection of crocetin (100 and 200 mg/kg, Hong Kong Institute of Biotechnology, Shatin, Hong Kong) was performed immediately after burn injury. Animals in the control group were placed under general anesthesia, underwent dorsal fur clipping, and burn injury, received a subcutaneous injection of normal saline with buprenorphine and dimethyl sulfoxide, without crocetin treatment. Animals in basal group were subjected to the same procedures as the other group, except that they were not burned and received crocetin. All animals were killed 8 h after reperfusion for histologic examination of the bowel and for biochemical studies.
2.2. Intestinal permeability assay and histologic evaluation Animals were anesthetized, and a laparotomy incision was made. The distal ileum was identified, and a 5-cm segment was isolated with a silk suture. A 200-mL solution containing 25 mg of fluorescein isothiocyanate (FITC)e dextran in phosphate buffer saline was injected into the lumen of the isolated distal ileal loop. The laparotomy incision was closed with silk sutures, animals were euthanized 30 min later, and then the blood was collected via cardiac puncture. The serum was separated, and the fluorescence of FITCedextran was measured at 520 nm. The distal ileum was stored in 10% formalin and embedded in paraffin blocks by an automated processor. Sections of gut were cut at 7-mm thickness, placed onto glass slides, and stained with hematoxylineeosin. The stained slides were viewed with an Olympus IX70 light microscope (Olympus, Melville, NY) at 100 magnification. Images were obtained with the Q-imaging software package (Surrey, British Columbia, Canada).
2.3.
Free radical scavenging activity
Three models were used to assess the free radical scavenging activity of crocetin in vitro, as described previously [22]. The radical scavenging activity of 2,20 -diphenyl-1picrylhydrazyl (DPPH) was measured by DPPH assay. The hydroxyl radical scavenging activity was measured by Co(II)/ethylene diamine tetra acetic acid-induced luminol chemiluminescence measurements. The superoxide anion
j o u r n a l o f s u r g i c a l r e s e a r c h 1 9 8 ( 2 0 1 5 ) 9 9 e1 0 7
radical scavenging activity was measured by xanthine oxidase and xanthine-induced luminol chemiluminescence measurements.
2.4. model
Liver microsome preparation and lipid peroxidation
Rat liver microsomes were prepared by standard differential centrifugation techniques, as described by Satav and Katyare [23]. The protein concentration was measured by the Lowry method [24], with bovine serum albumin (fraction V, essentially fatty acid-free, low endotoxin) as the standard. Lipid peroxidation was induced in the rat liver microsomes by vitamin C/Fe2þ, cumine hydroperoxide (CHP), or CCl4/reduced form of nicotinamide-adenine dinucleotide phosphate (NADPH), as described previously [22]. The extent of lipid peroxidation was detected by the thiobarbituric acid (TBA; Merck, Darmsladt, Germany) method, by measuring the absorbance at 535 nm. TBA-reactive substances were calculated as malondialdehyde (MDA) equivalents [22,25]. Appropriate controls were performed to eliminate any possible interference with the assay.
2.5.
101
5-tetramethylbenzidine as a substrate. The MPO activity was expressed as units per gram of tissue.
2.6. Enzyme linked immunosorbent assay and Western blot analyses Enzyme immunoassay kits for interleukin 6 (IL-6) and tumor necrosis factor a (TNF-a; R&D Systems, Minneapolis, MN) were used to determine their concentrations in the plasma and in small intestine tissue specimens. For Western blot analysis, frozen small intestinal tissue samples were homogenized with radioimmune precipitation assay buffer (150 mM NaCl, 0.1% sodium lauryl sulfate, 1% Nonidet P40, 1% deoxycholate, and 50 mM TriseHCl) with protease inhibitor cocktail. They were kept on ice for 60 min with occasional mixing and centrifuged at 10,000 g for 5 min. The protein concentration was estimated with the Bradford assay, and the samples were analyzed by Western blotting. Antibodies against Nuclear Factor kB (NF-kB) p65 and phosphoeNF-kB p65 were used. Densitometric analysis of the protein levels was performed with the Quantity One software package (Bio-Rad, Shanghai, China).
Biochemical analyses
Superoxidase dismutase (SOD) activity was assayed based on the reduction of nitroblue tetrazolium (NBT) by superoxide anion produced by hydroxylamine hydrochloride autoxidation, as described previously [26]. Hydroxylamine hydrochloride was added to NBT, and the change in absorbance, A(x), was measured optically at 560 nm. The sample was added to this reaction mixture, and the change in absorbance A( y) was recorded at 560 nm. The difference in the rate was expressed as the percent inhibition (% inhibition) ¼ [A(x) A( y)]/A(x) 100. One unit (U) of SOD was defined as the amount of protein that inhibited 50% of the rate of NBT reduction. The catalase (CAT) activity was determined by a commercial kit (Nanjing Jiancheng Company, Nanjing, China). Ammonium molybdate can end the decomposition reaction of hydrogen peroxide (H2O2) catalyzed by CAT. Surplus H2O2 interacts with ammonium molybdate to generate a peroxomolybdic acid complex with a distinctive color. The absorbance was measured optically at 405 nm. One unit of enzyme was defined as the amount of enzyme required to break down 1 M H2O2 per second. The glutathione peroxidase (GPx) activity was assayed by spectrophotometry [26]. GPx can catalyze the reaction of glutathione (GSH) and hydroperoxides. The activity of the enzyme was evaluated by the consumption of GSH. The reaction was started by adding 400 mL of sample dilution. GSH reacts with 5,50 -dithiobis-(2-nitrobenzoic acid) to form a yellow product. The absorbance was measured optically at 422 nm. The MDA level was determined by the colorimetric absorption of the TBA-MDA chromophore used to determine the index of lipid peroxidation, as described previously [25e27]. As an index of PMN influx, the myeloperoxidase (MPO) activity was assessed by a spectrophotometric method (Victor-3 multilabel counter 1420; Perkin Elmer, Shanghai, China), with 3,3-5,
Fig. 1 e The antioxidant activity of different doses of crocetin. (A) DPPH, hydroxyl, and superoxide anion radicals were induced in different models. The inhibition ratios of crocetin in these free radicalegenerating models are shown. (B) Lipid peroxidation was rapidly induced by incubating rat liver microsomes with vitamin C/Fe2D, CHP, or CCl4/NADPH. The inhibition ratios of crocetin in these lipid peroxidation models are shown. Data are expressed as the mean ± standard deviation. CHP [ cumine hydroperoxide; DPPH [ 2,20 -diphenyl-1-picrylhydrazyl; NADPH [ nicotinamideadenine dinucleotide phosphate.
102
j o u r n a l o f s u r g i c a l r e s e a r c h 1 9 8 ( 2 0 1 5 ) 9 9 e1 0 7
Fig. 2 e Crocetin administration reduced oxidative stress in serum. (A) The effect of crocetin administration on MDA content in serum; (B) the effect of crocetin administration on SOD activity in serum; (C) the effect of crocetin administration on CAT activity in serum; and (D) the effect of crocetin administration on GPx activity in serum. Data are expressed as the mean ± standard deviation, *P < 0.05 compared with the basal, **P < 0.05 compared with the control. CAT [ catalase; GPx [ glutathione peroxidase; MDA [ malondialdehyde; SOD [ superoxidase dismutase.
2.7.
Statistical analysis
All the results are expressed as the mean standard deviation of at least three experiments. Results were analyzed by one-way analysis of variance followed by an Student-Newman-Keuls
(SNK)-q test for multiple comparisons. The statistical significance of differences between two groups was determined by the Student t-test. All analyses were performed with the Statistical Package for the Social Sciences (SPSS Inc., Chicago, IL) software. Data were considered statistically significant for P < 0.05.
Fig. 3 e Crocetin administration reduced oxidative stress in small intestine tissue. (A) The effect of crocetin administration on MDA content in small intestine tissue; (B) the effect of crocetin administration on SOD activity in small intestine tissue; (C) the effect of crocetin administration on CAT activity in small intestine tissue; (D) the effect of crocetin administration on GPx activity in small intestine tissue. Data are expressed as the mean ± standard deviation, *P < 0.05 compared with the basal, **P < 0.05 compared with the control. CAT [ catalase; GPx [ glutathione peroxidase; MDA [ malondialdehyde; SOD [ superoxidase dismutase.
j o u r n a l o f s u r g i c a l r e s e a r c h 1 9 8 ( 2 0 1 5 ) 9 9 e1 0 7
103
the product of lipid peroxidation. Also, the administration of crocetin significantly inhibited the increase in MDA content. As shown in Figure 2B and D, burns markedly decreased the activity of serum SOD and GPx. Crocetin significantly increased the activity of serum SOD and GPx. Interestingly, burns remarkably increased the activity of serum CAT, and different doses of crocetin inhibited the increase in CAT activity (Fig. 2C). We next investigated the protective effect of crocetin in small intestine tissue. As shown in Figure 3A, the level of MDA was markedly increased after burns. Different doses of crocetin significantly decreased the content of MDA. In addition, burns significantly decreased the activities of SOD, CAT, and GPx, and different doses of crocetin significantly increased the enzymatic activities (Fig. 3BeD).
3.3. Crocetin administration reduced the levels of proinflammatory response
Fig. 4 e Crocetin administration reduced burn-induced inflammatory response in serum. Inflammatory cytokines IL-6 (A) and TNF-a (B) in serum were measured using an ELISA standardized procedure. Data are expressed as the mean ± standard deviation, *P < 0.05 compared with the basal, **P < 0.05 compared with the control. ELISA [ enzyme-linked immunosorbent assay; IL-6 [ interleukin 6; TNF-a [ tumor necrosis factor a.
3.
Results
To evaluate the inhibitory effect of crocetin on inflammatory reactions, the levels of two proinflammatory cytokines, IL-6 and TNF-a, were assessed. As shown in Figure 4, the serum levels of IL-6 and TNF-a were increased after burn injury. Treatment with crocetin significantly decreased the IL-6 and TNF-a levels. We next measured the levels of IL-6 and TNF-a in intestinal tissue, and found that they were significantly increased after burns. Additionally, a significant accumulation of neutrophils in intestinal tissues, as assessed by MPO activity, was also detected. Treatment with crocetin significantly reduced the tissue levels of IL-6 and TNF-a, and attenuated the neutrophil infiltration (Fig. 5AeC). To investigate the mechanism of crocetin on the inflammatory response, we further evaluated the activity of the transcription factor NF-kB. As shown in Figure 5D, nuclear NF-kB p65 phosphorylation was increased after burn injury; however, it was attenuated by crocetin administration.
3.1. Antioxidant activity levels of different doses of crocetin
3.4. Protective effect of crocetin on burn-induced intestinal injury
To evaluate the free radical scavenging activities of crocetin, several free radicalegenerating models were used. As shown in Figure 1A, crocetin remarkably inhibited DPPH free radical, hydroxyl radical, and superoxide anion radical. These data indicate that crocetin might be an effective free radical scavenger. Lipid peroxidation was induced rapidly by incubation of rat liver microsomes with vitamin C/Fe2þ, CHP, or CCl4/ NADPH. As shown in Figure 1B, different doses of crocetin remarkably inhibited lipid peroxidation in all three models. These data indicate that crocetin might be an effective lipid peroxidation inhibitor.
We first investigated the effect of crocetin on burn-induced intestinal permeability. As shown in Figure 6, the intestinal permeability to intraluminally injected FITCedextran was markedly increased after burn injury, whereas the systemic treatment with crocetin significantly attenuated the burninduced intestinal permeability. We next investigated burn-mediated histologic changes in the intestinal tissue. Compared with the sham group (Fig. 7A), the burn group (Fig. 7B) clearly showed mucosal ulceration and focal necrosis, and these changes were attenuated by treatment with different doses of crocetin (Fig. 7C and D). This result indicated that crocetin could significantly decrease the intestinal injury in burn injury model.
3.2.
Crocetin administration reduced oxidative stress
To evaluate the protective effect of crocetin against oxidative stress, several parameters in serum were detected, including MDA content, SOD, CAT, and GPx activity. As shown in Figure 2A, burns significantly increased the content of MDA,
4.
Discussion
The present study investigated the antioxidant activities of crocetin systematically and evaluated its role in burn-induced
104
j o u r n a l o f s u r g i c a l r e s e a r c h 1 9 8 ( 2 0 1 5 ) 9 9 e1 0 7
Fig. 5 e Crocetin administration reduced burn-induced inflammatory response in small intestine. IL-6 (A), TNF-a (B), MPO activities (C), and NF-kB activation (D) in intestinal tissues were measured. Data are expressed as the mean ± standard deviation, *P < 0.05 compared with the basal, **P < 0.05 compared with the control. IL-6 [ interleukin 6; MPO [ myeloperoxidase; NF-kB [ nuclear factor kB; TNF-a [ tumor necrosis factor a.
intestinal injury. Our findings suggest that crocetin is an effective free radical scavenger and lipid peroxidation inhibitor. Importantly, systemic treatment with crocetin effectively improved antioxidant biomarkers, attenuated inflammatory reaction, decreased intestinal permeability, and histologic changes in rats after burn injury. Some studies have demonstrated that crocetin has certain antioxidant ability [17e19]. However, studies on the mechanism of its antioxidant effects are currently limited.
Fig. 6 e Protective effect of crocetin on burn-induced intestinal permeability. Different doses of crocetin were administered immediately after burn injury. FITCedextran assay was used to detect the intestinal permeability. Data are expressed as the mean ± standard deviation, *P < 0.05 compared with the basal, **P < 0.05 compared with the control.
Therefore, in the first part of this study, the antioxidant activity of crocetin was evaluated systematically in vitro. Antioxidants function through several mechanisms, one of which is the scavenging of free radicals. Hydroxyl radical and superoxide anion radical are two kinds of the most important free radicals in organisms. Hydroxyl radical production plays a significant role in the initiation of lipid peroxidation [28]. The superoxide anion radical is a relatively low-energy radical, but it is responsible for the production of highly reactive and damaging hydroxyl radical [29]. DPPH is a stable free radical and used to evaluate the radical scavenging activity of natural substances [22,29]. Several free radicalegenerating models were used to evaluate the free radical scavenging activity of crocetin. Our results found that different doses of crocetin effectively inhibited the generation of DPPH, hydroxyl, and superoxide anion radicals. Consistent with our results, Yoshino et al. [18] have reported that crocetin significantly inhibited hydroxyl radical generation using in vitro X-band electron spin resonance and spin trapping. Taken together, these results indicate that crocetin could be an effective free radical scavenger. Membrane lipids are particularly susceptible to oxidation because of their high concentration of polyunsaturated fatty acids and their association with the enzymatic and nonenzymatic systems in the cell membrane, which are able to generate free radical species [30]. Crocetin was also tested for its antioxidant activity by measuring its ability to inhibit lipid peroxidation induced by vitamin C/Fe2þ, CHP, or CCl4/NADPH in rat liver microsomes [22,25]. The results of lipid peroxidation in rat liver microsomes suggested that crocetin exhibited antioxidant potential and may act as a lipid peroxidation inhibitor.
j o u r n a l o f s u r g i c a l r e s e a r c h 1 9 8 ( 2 0 1 5 ) 9 9 e1 0 7
105
Fig. 7 e Effects of crocetin on burn-induced intestinal injury. The intestinal microscopic appearances of four groups are shown (AeD). Each image was taken at 3100 magnification. Data shown are the typical results. The burn group (B) clearly shows mucosal ulceration and focal necrosis, and these changes were attenuated by treatment with different doses of crocetin (C and D). (Color version of figure is available online.)
Previously mentioned results are very significant findings from the point of view of clarifying the detailed antioxidant mechanism of crocetin. In the following experiments, we detected the antioxidant ability of crocetin in vivo. As we know, there is an intricate balance between the production and destruction of ROS in living organism. Excess ROS react with many biomolecules such as DNA [31], lipids [32], or proteins [33]. The body has developed an antioxidant defense system against harmful effects of ROS. There are several enzymes that play critical roles in the removal of excess ROS in living organism. Among them, SOD, CAT, and GPx are the three most crucial enzymes in the cellular antioxidant system [22,29]. Modulation of these antioxidant enzymes may be able to protect against oxidative stress. Therefore, we detected the effect of crocetin on these enzymes after burns. Results show that burns significantly decreased the activities of SOD, CAT, and GPx in intestine tissue, and different doses of crocetin increased the enzymatic activities (Fig. 3BeD). This could be responsible for the increased resistance to oxidative stress. Consistent with our results, Shen and Qian [34] and Shen et al. [35] have reported that crocetin markedly increased GPx and SOD activities in norepinephrine-induced cardiac injury. A similar trend was observed in the serum enzymatic activities of SOD and GPx, but not of CAT. As shown in Figure 2C, serum CAT activity was significantly increased after burns. It may be attributed to an instinct protective effect in response. Under normal conditions, there is no CAT in extracellular fluid of
human body, but in certain conditions, such as infection or stress, CAT would appear in serum. The higher activity of CAT could make cells capable of tolerating higher H2O2 concentrations. Tissue MDA content, the final product of lipid breakdown caused by oxidative stress, is considered to be a good indicator for radical-induced lipid peroxidation [26,27]. In our present study, MDA level was increased markedly in animals subjected to burns, and crocetin administration significantly decreased the MDA concentration in a dose-dependent manner. It indicated that crocetin may inhibit the lipid peroxidation induced by burns. Take together, previously mentioned results indicated that crocetin, which possesses both radical scavenging and antioxidant activities, could improve the antioxidant defense system and inhibit the oxidative damage of small intestine. In addition to radical scavenging and antioxidant activities, crocetin also exhibits anti-inflammation property [20,21]. There is increasing evidence showing that the inflammatory response plays a key role in burn-induced intestinal injury and suppression of inflammatory signaling can protect the injured intestinal tract [10e12]. In the present study, we found that crocetin treatment limited PMN accumulation in small intestine and decreased the levels of TNF-a and IL-6. Also, burn injury increases the level of NF-kB p65, whereas treatment with crocetin reduced burn-induced NF-kB activation. These results suggested that crocetin
106
j o u r n a l o f s u r g i c a l r e s e a r c h 1 9 8 ( 2 0 1 5 ) 9 9 e1 0 7
significantly inhibited the inflammatory response in rats after burn. On the basis of the antioxidative and anti-inflammatory properties of crocetin, we then investigated if crocetin could attenuate burn-induced intestinal injury. Consistent with others, the intestinal permeability to FITCedextran was strongly increased after burns [9]. Our treatment with different doses of crocetin attenuated intestinal permeability in a dose-dependent manner. We additionally observed changes in the intestinal tissue after burns microscopically. Damage to the crypt and shortening of the villi in the intestine are typical histologic changes after burns described previously [9,11]. Treatment with crocetin attenuated both of these in intestinal injury. Previously mentioned results indicated that crocetin could inhibit oxidative tissue damage and inflammatory response, resulting in the inhibition of burn-induced intestinal injury.
5.
Conclusions
The present study, for the first time, investigated the antioxidant activity of crocetin systematically by using a variety of in vitro models and examined its protective effect against burn-induced intestinal injury. We found that crocetin, which showed both radical scavenging and antioxidant activities, could inhibit oxidative tissue damage, improve the antioxidant defense system, decrease the inflammatory response, and exhibit a protective effect on small intestine insulted by burn injury. These results suggested that crocetin would be a promising candidate against intestine injury after burn. However, the potential mechanisms of crocetin on signal transduction pathways, cellular homeostasis, and biologic effects as a potential clinical agent still remain to be clarified.
Acknowledgment This work was in part supported by grants from the National Natural Science Foundation of China (No. 81201463) and the Shanxi Province Natural Science Foundation of China (No. 2014JQ4153). We thank all other members of our laboratory for their insight and technical support. All authors have contributed significantly. Chunxiang Zhou and Wei Bai performed the experiment and collected the data; Qiaohua Chen and Zhigang Xu performed data analysis; Xiongxiang Zhu did critical revision; Aidong Wen contributed toward conception and design; Xuekang Yang contributed toward analysis, interpretation, funding, and writing. Each author certifies that they have made a direct and substantial contribution to the work reported in the manuscript by participating in each of the following three areas: (1) conceiving and designing the study, collecting the data, or analyzing and interpreting the data; (2) writing the manuscript or providing critical revisions that are important for the intellectual content; and (3) approving the final version of the manuscript.
Disclosure The authors declare that they have no conflicts of interest.
references
[1] Zhou B, Zhou X, Ouyang LZ, et al. An epidemiological analysis of paediatric burns in urban and rural areas in south central China. Burns 2014;40:150. [2] Zhu L, Zhang Y, Liu L, et al. Hospitalized pediatric burns in North China: a 10-year epidemiologic review. Burns 2013;39: 1004. [3] Wang C, Zhao R, Du WL, Ning FG, Zhang GA. Firework injuries at a major trauma and burn center: a five-year prospective study. Burns 2014;40:305. [4] Zhang LJ, Cao J, Feng P, et al. Influencing factors of the quality of life in Chinese burn patients: investigation with adapted Chinese version of the BSHS-B. Burns 2014;40:731. [5] Xu JH, Qiu J, Zhou JH, et al. Pediatric burns in military hospitals of China from 2001 to 2007: a retrospective study. Burns 2014;40:1780. [6] Zhu L, Zhang H, Shi F, Yi D, Zhu G. Epidemiology and outcome analysis of scalds in children caused by “guo lian kang”: an 11-year review in a burn center in China. Burns 2015;41:289. [7] Magnotti LJ, Deitch EA. Burns, bacterial translocation, gut barrier function, and failure. J Burn Care Rehabil 2005;26:383. [8] Gosain A, Gamelli RL. Role of the gastrointestinal tract in burn sepsis. J Burn Care Rehabil 2005;26:85. [9] Costantini TW, Peterson CY, Kroll L, et al. Role of p38 MAPK in burn-induced intestinal barrier breakdown. J Surg Res 2009;156:64. [10] Liu DM, Sun BW, Sun ZW, Jin Q, Sun Y, Chen X. Suppression of inflammatory cytokine production and oxidative stress by CO-releasing molecules-liberated CO in the small intestine of thermally-injured mice. Acta Pharmacol Sin 2008;29:838. [11] Yang XK, Chen J, Bai H, et al. Inhibition of Naþ/Hþ exchanger 1 by cariporide reduces burn-induced intestinal barrier breakdown. Burns 2013;39:1557. [12] Schwacha MG. Macrophages and post-burn immune dysfunction. Burn 2003;29:1. [13] Ha H, Park J, Kim Y, Endou H. Oxidative stress and chronic allograft nephropathy. Yonsei Med J 2004;45:1049. [14] Flessas II, Papalois AE, Toutouzas K, Zagouri F, Zografos GC. Effects of lazaroids on intestinal ischemia and reperfusion injury in experimental models. J Surg Res 2011; 166:265. [15] Hernandez LA, Grisham MB, Twohig B, Arfors KE, Harlan JM, Granger DN. Role of neutrophils in ischemia reperfusion-induced microvascular injury. Am J Phys 1987; 253:H699. [16] Katada K, Bihari A, Mizuguchi S, et al. Carbon monoxide liberated from CO-releasing molecule (CORM-2) attenuates ischemia/reperfusion (I/R)-induced inflammation in the small intestine. Inflammation 2010;33:92. [17] Selim K, Tsimidou M, Biliaderis CG. Kinetic studies of degradation of saffron carotenoids encapsulated in amorphous polymer matrices. Food Chem 2000;71:199. [18] Yoshino F, Yoshida A, Umigai N, Kubo K, Lee MC. Crocetin reduces the oxidative stress induced reactive oxygen species in the stroke-prone spontaneously hypertensive rats (SHRSPs) brain. J Clin Biochem Nutr 2011;49:182.
j o u r n a l o f s u r g i c a l r e s e a r c h 1 9 8 ( 2 0 1 5 ) 9 9 e1 0 7
[19] Venkatraman M, Konga D, Peramaiyan R, Ganapathy E, Dhanapal S. Reduction of mitochondrial oxidative damage and improved mitochondrial efficiency by administration of crocetin against benzo[a]pyrene induced experimental animals. Biol Pharm Bull 2008;31:1639. [20] Yang R, Yang L, Shen X, et al. Suppression of NF-kB pathway by crocetin contributes to attenuation of lipopolysaccharide-induced acute lung injury in mice. Eur J Pharmacol 2012;674:391. [21] Nam KN, Park YM, Jung HJ, et al. Anti-inflammatory effects of crocin and crocetin in rat brain microglial cells. Eur J Pharmacol 2010;648:110. [22] Wang X, Ye XL, Liu R, et al. Antioxidant activities of oleanolic acid in vitro: possible role of Nrf2 and MAP kinases. Chem Biol Interact 2010;184:328. [23] Satav JG, Katyare SS. Effect of experimental thyrotoxicosis on oxidative phosphorylation in rat liver, kidney and brain mitochondria. Mol Cell Endocrinol 1982;28:173. [24] Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem 1951;193:265. [25] Xi MM, Hai CX, Tang H, Chen M, Fang K, Liang X. Antioxidant and antiglycation properties of total saponins extracted from traditional Chinese medicine used to treat diabetes mellitus. Phytother Res 2008;22:228. [26] Wang X, Hai CX, Liang X, Yu SX, Zhang W, Li YL. The protective effects of Acanthopanax senticosus harms aqueous extracts against oxidative stress: role of Nrf2
[27]
[28]
[29]
[30]
[31] [32] [33] [34]
[35]
107
and antioxidant enzymes. J Ethnopharmacol 2010;127: 424. Sato Y, Itagaki S, Oikawa S, et al. Protective effect of soy isoflavone genistein on ischemia-reperfusion in the rat small intestine. Biol Pharm Bull 2011;34: 1448. Mallick IH, Yang W, Winslet MC, Seifalian AM. Ischemiarepefusion injury of the intestine and protective strategies against injury. Dig Dis Sci 2004;49:1359. Yang XK, Bai H, Cai WX, et al. Lycium barbarum polysaccharides reduce intestinal ischemia/reperfusion injuries in rats. Chem Biol Interact 2013;204:166. Aruoma OI. Free radicals, oxidative stress, and antioxidants in human health and disease. J Am Oil Chem Soc 1998;75:199. Marnett LJ. Oxyradicals and DNA damage. Carcinogenesis 2000;21:361. Yla-Herttuala S. Oxidized LDL and atherogenesis. Ann N Y Acad Sci 1999;874:134. Stadtman ER, Levine RL. Protein oxidation. Ann N Y Acad Sci 2000;899:191. Shen XC, Qian ZY. Effects of crocetin on antioxidant enzymatic activities in cardiac hypertrophy induced by norepinephrine in rats. Pharmazie 2006;61:348. Shen XC, Qian ZY, Wang YJ, Duan JA. Crocetin attenuates norepinephrine-induced cytotoxicity in primary cultured rat cardiac myocytes by antioxidant in vitro. J Asian Nat Prod Res 2009;11:417.
Available online at www.sciencedirect.com
ScienceDirect journal homepage: www.JournalofSurgicalResearch.com
Protective effect of crocetin against burn-induced intestinal injury Chunxiang Zhou, PhD,a,b,c,1 Wei Bai, MD,d,e,1 Qiaohua Chen, MD,d Zhigang Xu, MD,d Xiongxiang Zhu, PhD,d Aidong Wen, PhD,a,b,c,** and Xuekang Yang, PhD, MDd,* a
Department of Pharmacy, Xijing Hospital, The Fourth Military Medical University, Xi’an, Shaanxi, PR China State Key Laboratory of Cancer Biology, The Fourth Military Medical University, Xi’an, Shaanxi, PR China c Department of Biochemistry and Molecular Biology, The Fourth Military Medical University, Xi’an, Shaanxi, PR China d Department of Burns and Cutaneous Surgery, Xijing Hospital, The Fourth Military Medical University, Xi’an, Shaanxi, PR China e Brigade of student, The Fourth Military Medical University, Xi’an, Shaanxi, PR China b
article info
abstract
Article history:
Background: Oxidative stress and inflammation exert central roles in burn-induced intes-
Received 18 February 2015
tinal injury. Crocetin, a natural carotenoid compound from gardenia fruits and saffron, has
Received in revised form
been shown to inhibit oxidative stress and inflammatory response. However, the possi-
22 April 2015
bility of crocetin to be used in the treatment of intestinal injury after burn injury has not
Accepted 27 May 2015
been investigated. The purpose of the present study was to investigate the effects and
Available online 3 June 2015
potential mechanisms of crocetin in burn-induced intestinal injury. Materials and methods: Several free radicalegenerating and lipid peroxidation models were
Keywords:
used to systematically assess the antioxidant activities of crocetin in vitro. A common burn
Crocetin
model was used to induce the intestinal injury in rats. Changes in the levels of malon-
Intestinal injury
dialdehyde, superoxidase dismutase, catalase, glutathione peroxidase, tumor necrosis
Oxidative stress
factor a, interleukin 6, polymorphonuclear neutrophil accumulation, intestinal perme-
Inflammation
ability, and intestinal histology were examined.
Burn injury
Results: In several models of antioxidant activity, crocetin exhibited marked inhibitory action against free radicals and lipid peroxidation. Crocetin increased levels of antioxidant enzymes and reduced intestinal oxidative injury in burn models. In addition, crocetin inhibited polymorphonuclear neutrophil accumulation, ameliorated tumor necrosis factor a and interleukin 6 levels, intestinal permeability, and histological changes. Conclusions: Crocetin treatment may protect against burn-induced small intestinal injury, possibly by inhibiting burn-induced oxidative stress and inflammatory response. ª 2015 Elsevier Inc. All rights reserved.
* Corresponding author. Department of Burns and Cutaneous Surgery, Xijing Hospital, The Fourth Military Medical University, Xi’an, Shaanxi, PR China. Tel./fax: þ86 2984775298. ** Corresponding author. Department of Pharmacy, Xijing Hospital, and The State Key Laboratory of Cancer Biology and The Department of Biochemistry and Molecular Biology, The Fourth Military Medical University,Xi’an, Shannxi 710032, P.R. China. Tel./fax: þ86 2984775298. E-mail addresses: [email protected] (A. Wen), [email protected] (X. Yang). 1 These authors contributed equally to this work and should be considered as co-first authors. 0022-4804/$ e see front matter ª 2015 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jss.2015.05.052
100
1.
j o u r n a l o f s u r g i c a l r e s e a r c h 1 9 8 ( 2 0 1 5 ) 9 9 e1 0 7
Introduction
Burn injury is one of the most common problems in China [1e6]. Individuals who survive the original insult may go on to develop systemic inflammatory response syndrome, sepsis, and multisystem organ failure, which all are major causes of late deaths after burns [7]. It is postulated that the gastrointestinal system is central to the development of systemic inflammatory response syndrome and multisystem organ failure after burn injury because mucosal injury leads to bacterial translocation and incites an inflammatory response [8]. These facts suggest that the preservation of intestinal mucosal barrier, therefore, has important clinical implications for the outcome of burn victims [7e9]. The mechanisms and treatments of intestinal injury after burn have been the topics with extensive research. Although the precise mechanisms involved have not been fully elucidated, oxidative stress and inflammatory pathways are thought to play important roles [10e12]. Oxidative stress is the imbalance between the production and elimination of reactive oxygen species (ROS). Under normal condition, the body has a potent antioxidant defense system, fighting against excessive generation of ROS. However, when either excessive ROS is generated or the antioxidant defense system is damaged, oxidative stress occurs [13]. ROS target cell membrane constituents cause lipid peroxidation, membrane disintegration, endothelial cell damage, and thus increase microvascular permeability, which in turn leads to the activation and adhesion of polymorphonuclear neutrophils (PMNs) and the release of proinflammatory substances [14]. The accumulation of PMNs further contributes to ROS formation and intestinal tissue damage, resulting in bacterial translocation, systemic complications, and eventually, multiple organ failure [15,16]. Treatments, such as antioxidants, free radical scavengers, and anti-inflammatory therapy, have been used successfully to attenuate intestinal injury in animal models of burn injury [7,10e12]. In recent years, there has been a global trend toward the use of natural substances as antioxidants and functional nutriments. Crocetin, a carotenoid compound, can be extracted from saffron (Crocus sativus L.) or gardenia jasminoides Ellis. This yellow compound has been used as an important spice and natural food colorant in various parts of the world [17]. In addition, saffron and gardenia fruits have been widely used as traditional medicines. In recent years, it has been suggested that crocetin might be an effective antioxidant to counter oxidative stress and inflammation in several models [18e21]. However, the specific roles of crocetins in the prevention of burn-induced intestinal injury remain unclear. Therefore, the aim of this study was to investigate whether crocetin could reduce oxidative damage and inflammatory response, and thus attenuate burn-induced intestinal injury.
2.
Materials and methods
2.1.
Animals and burn protocol
Male SpragueeDawley rats (8e10 wk old, 200e250 g) were purchased from the Center of Experimental Animals of the
Fourth Military Medical University. Rats were housed for 6 d in standard cages and fed a standard chow diet with water ad libitum. All procedures were approved by the Institutional Animal Care and Use Committee of the Fourth Military Medical University, in accordance with the Guidelines for the Care and Use of Laboratory Animals published by the US National Institutes of Health. Unless otherwise noted, all the chemicals and reagents used in this study were procured from Sigma Chemical Co (St. Louis, MO), were of high purity, and were used without purification. For the burn protocol, animals were anesthetized with sodium pentobarbital, the dorsal fur was clipped using an electric clipper. Animals were placed into a template constructed to estimate a 30% total body surface area burn injury [11] and received immediately a subcutaneous injection of 1.5 mL normal saline containing buprenorphine for fluid resuscitation and pain control. Intraperitoneal injection of crocetin (100 and 200 mg/kg, Hong Kong Institute of Biotechnology, Shatin, Hong Kong) was performed immediately after burn injury. Animals in the control group were placed under general anesthesia, underwent dorsal fur clipping, and burn injury, received a subcutaneous injection of normal saline with buprenorphine and dimethyl sulfoxide, without crocetin treatment. Animals in basal group were subjected to the same procedures as the other group, except that they were not burned and received crocetin. All animals were killed 8 h after reperfusion for histologic examination of the bowel and for biochemical studies.
2.2. Intestinal permeability assay and histologic evaluation Animals were anesthetized, and a laparotomy incision was made. The distal ileum was identified, and a 5-cm segment was isolated with a silk suture. A 200-mL solution containing 25 mg of fluorescein isothiocyanate (FITC)e dextran in phosphate buffer saline was injected into the lumen of the isolated distal ileal loop. The laparotomy incision was closed with silk sutures, animals were euthanized 30 min later, and then the blood was collected via cardiac puncture. The serum was separated, and the fluorescence of FITCedextran was measured at 520 nm. The distal ileum was stored in 10% formalin and embedded in paraffin blocks by an automated processor. Sections of gut were cut at 7-mm thickness, placed onto glass slides, and stained with hematoxylineeosin. The stained slides were viewed with an Olympus IX70 light microscope (Olympus, Melville, NY) at 100 magnification. Images were obtained with the Q-imaging software package (Surrey, British Columbia, Canada).
2.3.
Free radical scavenging activity
Three models were used to assess the free radical scavenging activity of crocetin in vitro, as described previously [22]. The radical scavenging activity of 2,20 -diphenyl-1picrylhydrazyl (DPPH) was measured by DPPH assay. The hydroxyl radical scavenging activity was measured by Co(II)/ethylene diamine tetra acetic acid-induced luminol chemiluminescence measurements. The superoxide anion
j o u r n a l o f s u r g i c a l r e s e a r c h 1 9 8 ( 2 0 1 5 ) 9 9 e1 0 7
radical scavenging activity was measured by xanthine oxidase and xanthine-induced luminol chemiluminescence measurements.
2.4. model
Liver microsome preparation and lipid peroxidation
Rat liver microsomes were prepared by standard differential centrifugation techniques, as described by Satav and Katyare [23]. The protein concentration was measured by the Lowry method [24], with bovine serum albumin (fraction V, essentially fatty acid-free, low endotoxin) as the standard. Lipid peroxidation was induced in the rat liver microsomes by vitamin C/Fe2þ, cumine hydroperoxide (CHP), or CCl4/reduced form of nicotinamide-adenine dinucleotide phosphate (NADPH), as described previously [22]. The extent of lipid peroxidation was detected by the thiobarbituric acid (TBA; Merck, Darmsladt, Germany) method, by measuring the absorbance at 535 nm. TBA-reactive substances were calculated as malondialdehyde (MDA) equivalents [22,25]. Appropriate controls were performed to eliminate any possible interference with the assay.
2.5.
101
5-tetramethylbenzidine as a substrate. The MPO activity was expressed as units per gram of tissue.
2.6. Enzyme linked immunosorbent assay and Western blot analyses Enzyme immunoassay kits for interleukin 6 (IL-6) and tumor necrosis factor a (TNF-a; R&D Systems, Minneapolis, MN) were used to determine their concentrations in the plasma and in small intestine tissue specimens. For Western blot analysis, frozen small intestinal tissue samples were homogenized with radioimmune precipitation assay buffer (150 mM NaCl, 0.1% sodium lauryl sulfate, 1% Nonidet P40, 1% deoxycholate, and 50 mM TriseHCl) with protease inhibitor cocktail. They were kept on ice for 60 min with occasional mixing and centrifuged at 10,000 g for 5 min. The protein concentration was estimated with the Bradford assay, and the samples were analyzed by Western blotting. Antibodies against Nuclear Factor kB (NF-kB) p65 and phosphoeNF-kB p65 were used. Densitometric analysis of the protein levels was performed with the Quantity One software package (Bio-Rad, Shanghai, China).
Biochemical analyses
Superoxidase dismutase (SOD) activity was assayed based on the reduction of nitroblue tetrazolium (NBT) by superoxide anion produced by hydroxylamine hydrochloride autoxidation, as described previously [26]. Hydroxylamine hydrochloride was added to NBT, and the change in absorbance, A(x), was measured optically at 560 nm. The sample was added to this reaction mixture, and the change in absorbance A( y) was recorded at 560 nm. The difference in the rate was expressed as the percent inhibition (% inhibition) ¼ [A(x) A( y)]/A(x) 100. One unit (U) of SOD was defined as the amount of protein that inhibited 50% of the rate of NBT reduction. The catalase (CAT) activity was determined by a commercial kit (Nanjing Jiancheng Company, Nanjing, China). Ammonium molybdate can end the decomposition reaction of hydrogen peroxide (H2O2) catalyzed by CAT. Surplus H2O2 interacts with ammonium molybdate to generate a peroxomolybdic acid complex with a distinctive color. The absorbance was measured optically at 405 nm. One unit of enzyme was defined as the amount of enzyme required to break down 1 M H2O2 per second. The glutathione peroxidase (GPx) activity was assayed by spectrophotometry [26]. GPx can catalyze the reaction of glutathione (GSH) and hydroperoxides. The activity of the enzyme was evaluated by the consumption of GSH. The reaction was started by adding 400 mL of sample dilution. GSH reacts with 5,50 -dithiobis-(2-nitrobenzoic acid) to form a yellow product. The absorbance was measured optically at 422 nm. The MDA level was determined by the colorimetric absorption of the TBA-MDA chromophore used to determine the index of lipid peroxidation, as described previously [25e27]. As an index of PMN influx, the myeloperoxidase (MPO) activity was assessed by a spectrophotometric method (Victor-3 multilabel counter 1420; Perkin Elmer, Shanghai, China), with 3,3-5,
Fig. 1 e The antioxidant activity of different doses of crocetin. (A) DPPH, hydroxyl, and superoxide anion radicals were induced in different models. The inhibition ratios of crocetin in these free radicalegenerating models are shown. (B) Lipid peroxidation was rapidly induced by incubating rat liver microsomes with vitamin C/Fe2D, CHP, or CCl4/NADPH. The inhibition ratios of crocetin in these lipid peroxidation models are shown. Data are expressed as the mean ± standard deviation. CHP [ cumine hydroperoxide; DPPH [ 2,20 -diphenyl-1-picrylhydrazyl; NADPH [ nicotinamideadenine dinucleotide phosphate.
102
j o u r n a l o f s u r g i c a l r e s e a r c h 1 9 8 ( 2 0 1 5 ) 9 9 e1 0 7
Fig. 2 e Crocetin administration reduced oxidative stress in serum. (A) The effect of crocetin administration on MDA content in serum; (B) the effect of crocetin administration on SOD activity in serum; (C) the effect of crocetin administration on CAT activity in serum; and (D) the effect of crocetin administration on GPx activity in serum. Data are expressed as the mean ± standard deviation, *P < 0.05 compared with the basal, **P < 0.05 compared with the control. CAT [ catalase; GPx [ glutathione peroxidase; MDA [ malondialdehyde; SOD [ superoxidase dismutase.
2.7.
Statistical analysis
All the results are expressed as the mean standard deviation of at least three experiments. Results were analyzed by one-way analysis of variance followed by an Student-Newman-Keuls
(SNK)-q test for multiple comparisons. The statistical significance of differences between two groups was determined by the Student t-test. All analyses were performed with the Statistical Package for the Social Sciences (SPSS Inc., Chicago, IL) software. Data were considered statistically significant for P < 0.05.
Fig. 3 e Crocetin administration reduced oxidative stress in small intestine tissue. (A) The effect of crocetin administration on MDA content in small intestine tissue; (B) the effect of crocetin administration on SOD activity in small intestine tissue; (C) the effect of crocetin administration on CAT activity in small intestine tissue; (D) the effect of crocetin administration on GPx activity in small intestine tissue. Data are expressed as the mean ± standard deviation, *P < 0.05 compared with the basal, **P < 0.05 compared with the control. CAT [ catalase; GPx [ glutathione peroxidase; MDA [ malondialdehyde; SOD [ superoxidase dismutase.
j o u r n a l o f s u r g i c a l r e s e a r c h 1 9 8 ( 2 0 1 5 ) 9 9 e1 0 7
103
the product of lipid peroxidation. Also, the administration of crocetin significantly inhibited the increase in MDA content. As shown in Figure 2B and D, burns markedly decreased the activity of serum SOD and GPx. Crocetin significantly increased the activity of serum SOD and GPx. Interestingly, burns remarkably increased the activity of serum CAT, and different doses of crocetin inhibited the increase in CAT activity (Fig. 2C). We next investigated the protective effect of crocetin in small intestine tissue. As shown in Figure 3A, the level of MDA was markedly increased after burns. Different doses of crocetin significantly decreased the content of MDA. In addition, burns significantly decreased the activities of SOD, CAT, and GPx, and different doses of crocetin significantly increased the enzymatic activities (Fig. 3BeD).
3.3. Crocetin administration reduced the levels of proinflammatory response
Fig. 4 e Crocetin administration reduced burn-induced inflammatory response in serum. Inflammatory cytokines IL-6 (A) and TNF-a (B) in serum were measured using an ELISA standardized procedure. Data are expressed as the mean ± standard deviation, *P < 0.05 compared with the basal, **P < 0.05 compared with the control. ELISA [ enzyme-linked immunosorbent assay; IL-6 [ interleukin 6; TNF-a [ tumor necrosis factor a.
3.
Results
To evaluate the inhibitory effect of crocetin on inflammatory reactions, the levels of two proinflammatory cytokines, IL-6 and TNF-a, were assessed. As shown in Figure 4, the serum levels of IL-6 and TNF-a were increased after burn injury. Treatment with crocetin significantly decreased the IL-6 and TNF-a levels. We next measured the levels of IL-6 and TNF-a in intestinal tissue, and found that they were significantly increased after burns. Additionally, a significant accumulation of neutrophils in intestinal tissues, as assessed by MPO activity, was also detected. Treatment with crocetin significantly reduced the tissue levels of IL-6 and TNF-a, and attenuated the neutrophil infiltration (Fig. 5AeC). To investigate the mechanism of crocetin on the inflammatory response, we further evaluated the activity of the transcription factor NF-kB. As shown in Figure 5D, nuclear NF-kB p65 phosphorylation was increased after burn injury; however, it was attenuated by crocetin administration.
3.1. Antioxidant activity levels of different doses of crocetin
3.4. Protective effect of crocetin on burn-induced intestinal injury
To evaluate the free radical scavenging activities of crocetin, several free radicalegenerating models were used. As shown in Figure 1A, crocetin remarkably inhibited DPPH free radical, hydroxyl radical, and superoxide anion radical. These data indicate that crocetin might be an effective free radical scavenger. Lipid peroxidation was induced rapidly by incubation of rat liver microsomes with vitamin C/Fe2þ, CHP, or CCl4/ NADPH. As shown in Figure 1B, different doses of crocetin remarkably inhibited lipid peroxidation in all three models. These data indicate that crocetin might be an effective lipid peroxidation inhibitor.
We first investigated the effect of crocetin on burn-induced intestinal permeability. As shown in Figure 6, the intestinal permeability to intraluminally injected FITCedextran was markedly increased after burn injury, whereas the systemic treatment with crocetin significantly attenuated the burninduced intestinal permeability. We next investigated burn-mediated histologic changes in the intestinal tissue. Compared with the sham group (Fig. 7A), the burn group (Fig. 7B) clearly showed mucosal ulceration and focal necrosis, and these changes were attenuated by treatment with different doses of crocetin (Fig. 7C and D). This result indicated that crocetin could significantly decrease the intestinal injury in burn injury model.
3.2.
Crocetin administration reduced oxidative stress
To evaluate the protective effect of crocetin against oxidative stress, several parameters in serum were detected, including MDA content, SOD, CAT, and GPx activity. As shown in Figure 2A, burns significantly increased the content of MDA,
4.
Discussion
The present study investigated the antioxidant activities of crocetin systematically and evaluated its role in burn-induced
104
j o u r n a l o f s u r g i c a l r e s e a r c h 1 9 8 ( 2 0 1 5 ) 9 9 e1 0 7
Fig. 5 e Crocetin administration reduced burn-induced inflammatory response in small intestine. IL-6 (A), TNF-a (B), MPO activities (C), and NF-kB activation (D) in intestinal tissues were measured. Data are expressed as the mean ± standard deviation, *P < 0.05 compared with the basal, **P < 0.05 compared with the control. IL-6 [ interleukin 6; MPO [ myeloperoxidase; NF-kB [ nuclear factor kB; TNF-a [ tumor necrosis factor a.
intestinal injury. Our findings suggest that crocetin is an effective free radical scavenger and lipid peroxidation inhibitor. Importantly, systemic treatment with crocetin effectively improved antioxidant biomarkers, attenuated inflammatory reaction, decreased intestinal permeability, and histologic changes in rats after burn injury. Some studies have demonstrated that crocetin has certain antioxidant ability [17e19]. However, studies on the mechanism of its antioxidant effects are currently limited.
Fig. 6 e Protective effect of crocetin on burn-induced intestinal permeability. Different doses of crocetin were administered immediately after burn injury. FITCedextran assay was used to detect the intestinal permeability. Data are expressed as the mean ± standard deviation, *P < 0.05 compared with the basal, **P < 0.05 compared with the control.
Therefore, in the first part of this study, the antioxidant activity of crocetin was evaluated systematically in vitro. Antioxidants function through several mechanisms, one of which is the scavenging of free radicals. Hydroxyl radical and superoxide anion radical are two kinds of the most important free radicals in organisms. Hydroxyl radical production plays a significant role in the initiation of lipid peroxidation [28]. The superoxide anion radical is a relatively low-energy radical, but it is responsible for the production of highly reactive and damaging hydroxyl radical [29]. DPPH is a stable free radical and used to evaluate the radical scavenging activity of natural substances [22,29]. Several free radicalegenerating models were used to evaluate the free radical scavenging activity of crocetin. Our results found that different doses of crocetin effectively inhibited the generation of DPPH, hydroxyl, and superoxide anion radicals. Consistent with our results, Yoshino et al. [18] have reported that crocetin significantly inhibited hydroxyl radical generation using in vitro X-band electron spin resonance and spin trapping. Taken together, these results indicate that crocetin could be an effective free radical scavenger. Membrane lipids are particularly susceptible to oxidation because of their high concentration of polyunsaturated fatty acids and their association with the enzymatic and nonenzymatic systems in the cell membrane, which are able to generate free radical species [30]. Crocetin was also tested for its antioxidant activity by measuring its ability to inhibit lipid peroxidation induced by vitamin C/Fe2þ, CHP, or CCl4/NADPH in rat liver microsomes [22,25]. The results of lipid peroxidation in rat liver microsomes suggested that crocetin exhibited antioxidant potential and may act as a lipid peroxidation inhibitor.
j o u r n a l o f s u r g i c a l r e s e a r c h 1 9 8 ( 2 0 1 5 ) 9 9 e1 0 7
105
Fig. 7 e Effects of crocetin on burn-induced intestinal injury. The intestinal microscopic appearances of four groups are shown (AeD). Each image was taken at 3100 magnification. Data shown are the typical results. The burn group (B) clearly shows mucosal ulceration and focal necrosis, and these changes were attenuated by treatment with different doses of crocetin (C and D). (Color version of figure is available online.)
Previously mentioned results are very significant findings from the point of view of clarifying the detailed antioxidant mechanism of crocetin. In the following experiments, we detected the antioxidant ability of crocetin in vivo. As we know, there is an intricate balance between the production and destruction of ROS in living organism. Excess ROS react with many biomolecules such as DNA [31], lipids [32], or proteins [33]. The body has developed an antioxidant defense system against harmful effects of ROS. There are several enzymes that play critical roles in the removal of excess ROS in living organism. Among them, SOD, CAT, and GPx are the three most crucial enzymes in the cellular antioxidant system [22,29]. Modulation of these antioxidant enzymes may be able to protect against oxidative stress. Therefore, we detected the effect of crocetin on these enzymes after burns. Results show that burns significantly decreased the activities of SOD, CAT, and GPx in intestine tissue, and different doses of crocetin increased the enzymatic activities (Fig. 3BeD). This could be responsible for the increased resistance to oxidative stress. Consistent with our results, Shen and Qian [34] and Shen et al. [35] have reported that crocetin markedly increased GPx and SOD activities in norepinephrine-induced cardiac injury. A similar trend was observed in the serum enzymatic activities of SOD and GPx, but not of CAT. As shown in Figure 2C, serum CAT activity was significantly increased after burns. It may be attributed to an instinct protective effect in response. Under normal conditions, there is no CAT in extracellular fluid of
human body, but in certain conditions, such as infection or stress, CAT would appear in serum. The higher activity of CAT could make cells capable of tolerating higher H2O2 concentrations. Tissue MDA content, the final product of lipid breakdown caused by oxidative stress, is considered to be a good indicator for radical-induced lipid peroxidation [26,27]. In our present study, MDA level was increased markedly in animals subjected to burns, and crocetin administration significantly decreased the MDA concentration in a dose-dependent manner. It indicated that crocetin may inhibit the lipid peroxidation induced by burns. Take together, previously mentioned results indicated that crocetin, which possesses both radical scavenging and antioxidant activities, could improve the antioxidant defense system and inhibit the oxidative damage of small intestine. In addition to radical scavenging and antioxidant activities, crocetin also exhibits anti-inflammation property [20,21]. There is increasing evidence showing that the inflammatory response plays a key role in burn-induced intestinal injury and suppression of inflammatory signaling can protect the injured intestinal tract [10e12]. In the present study, we found that crocetin treatment limited PMN accumulation in small intestine and decreased the levels of TNF-a and IL-6. Also, burn injury increases the level of NF-kB p65, whereas treatment with crocetin reduced burn-induced NF-kB activation. These results suggested that crocetin
106
j o u r n a l o f s u r g i c a l r e s e a r c h 1 9 8 ( 2 0 1 5 ) 9 9 e1 0 7
significantly inhibited the inflammatory response in rats after burn. On the basis of the antioxidative and anti-inflammatory properties of crocetin, we then investigated if crocetin could attenuate burn-induced intestinal injury. Consistent with others, the intestinal permeability to FITCedextran was strongly increased after burns [9]. Our treatment with different doses of crocetin attenuated intestinal permeability in a dose-dependent manner. We additionally observed changes in the intestinal tissue after burns microscopically. Damage to the crypt and shortening of the villi in the intestine are typical histologic changes after burns described previously [9,11]. Treatment with crocetin attenuated both of these in intestinal injury. Previously mentioned results indicated that crocetin could inhibit oxidative tissue damage and inflammatory response, resulting in the inhibition of burn-induced intestinal injury.
5.
Conclusions
The present study, for the first time, investigated the antioxidant activity of crocetin systematically by using a variety of in vitro models and examined its protective effect against burn-induced intestinal injury. We found that crocetin, which showed both radical scavenging and antioxidant activities, could inhibit oxidative tissue damage, improve the antioxidant defense system, decrease the inflammatory response, and exhibit a protective effect on small intestine insulted by burn injury. These results suggested that crocetin would be a promising candidate against intestine injury after burn. However, the potential mechanisms of crocetin on signal transduction pathways, cellular homeostasis, and biologic effects as a potential clinical agent still remain to be clarified.
Acknowledgment This work was in part supported by grants from the National Natural Science Foundation of China (No. 81201463) and the Shanxi Province Natural Science Foundation of China (No. 2014JQ4153). We thank all other members of our laboratory for their insight and technical support. All authors have contributed significantly. Chunxiang Zhou and Wei Bai performed the experiment and collected the data; Qiaohua Chen and Zhigang Xu performed data analysis; Xiongxiang Zhu did critical revision; Aidong Wen contributed toward conception and design; Xuekang Yang contributed toward analysis, interpretation, funding, and writing. Each author certifies that they have made a direct and substantial contribution to the work reported in the manuscript by participating in each of the following three areas: (1) conceiving and designing the study, collecting the data, or analyzing and interpreting the data; (2) writing the manuscript or providing critical revisions that are important for the intellectual content; and (3) approving the final version of the manuscript.
Disclosure The authors declare that they have no conflicts of interest.
references
[1] Zhou B, Zhou X, Ouyang LZ, et al. An epidemiological analysis of paediatric burns in urban and rural areas in south central China. Burns 2014;40:150. [2] Zhu L, Zhang Y, Liu L, et al. Hospitalized pediatric burns in North China: a 10-year epidemiologic review. Burns 2013;39: 1004. [3] Wang C, Zhao R, Du WL, Ning FG, Zhang GA. Firework injuries at a major trauma and burn center: a five-year prospective study. Burns 2014;40:305. [4] Zhang LJ, Cao J, Feng P, et al. Influencing factors of the quality of life in Chinese burn patients: investigation with adapted Chinese version of the BSHS-B. Burns 2014;40:731. [5] Xu JH, Qiu J, Zhou JH, et al. Pediatric burns in military hospitals of China from 2001 to 2007: a retrospective study. Burns 2014;40:1780. [6] Zhu L, Zhang H, Shi F, Yi D, Zhu G. Epidemiology and outcome analysis of scalds in children caused by “guo lian kang”: an 11-year review in a burn center in China. Burns 2015;41:289. [7] Magnotti LJ, Deitch EA. Burns, bacterial translocation, gut barrier function, and failure. J Burn Care Rehabil 2005;26:383. [8] Gosain A, Gamelli RL. Role of the gastrointestinal tract in burn sepsis. J Burn Care Rehabil 2005;26:85. [9] Costantini TW, Peterson CY, Kroll L, et al. Role of p38 MAPK in burn-induced intestinal barrier breakdown. J Surg Res 2009;156:64. [10] Liu DM, Sun BW, Sun ZW, Jin Q, Sun Y, Chen X. Suppression of inflammatory cytokine production and oxidative stress by CO-releasing molecules-liberated CO in the small intestine of thermally-injured mice. Acta Pharmacol Sin 2008;29:838. [11] Yang XK, Chen J, Bai H, et al. Inhibition of Naþ/Hþ exchanger 1 by cariporide reduces burn-induced intestinal barrier breakdown. Burns 2013;39:1557. [12] Schwacha MG. Macrophages and post-burn immune dysfunction. Burn 2003;29:1. [13] Ha H, Park J, Kim Y, Endou H. Oxidative stress and chronic allograft nephropathy. Yonsei Med J 2004;45:1049. [14] Flessas II, Papalois AE, Toutouzas K, Zagouri F, Zografos GC. Effects of lazaroids on intestinal ischemia and reperfusion injury in experimental models. J Surg Res 2011; 166:265. [15] Hernandez LA, Grisham MB, Twohig B, Arfors KE, Harlan JM, Granger DN. Role of neutrophils in ischemia reperfusion-induced microvascular injury. Am J Phys 1987; 253:H699. [16] Katada K, Bihari A, Mizuguchi S, et al. Carbon monoxide liberated from CO-releasing molecule (CORM-2) attenuates ischemia/reperfusion (I/R)-induced inflammation in the small intestine. Inflammation 2010;33:92. [17] Selim K, Tsimidou M, Biliaderis CG. Kinetic studies of degradation of saffron carotenoids encapsulated in amorphous polymer matrices. Food Chem 2000;71:199. [18] Yoshino F, Yoshida A, Umigai N, Kubo K, Lee MC. Crocetin reduces the oxidative stress induced reactive oxygen species in the stroke-prone spontaneously hypertensive rats (SHRSPs) brain. J Clin Biochem Nutr 2011;49:182.
j o u r n a l o f s u r g i c a l r e s e a r c h 1 9 8 ( 2 0 1 5 ) 9 9 e1 0 7
[19] Venkatraman M, Konga D, Peramaiyan R, Ganapathy E, Dhanapal S. Reduction of mitochondrial oxidative damage and improved mitochondrial efficiency by administration of crocetin against benzo[a]pyrene induced experimental animals. Biol Pharm Bull 2008;31:1639. [20] Yang R, Yang L, Shen X, et al. Suppression of NF-kB pathway by crocetin contributes to attenuation of lipopolysaccharide-induced acute lung injury in mice. Eur J Pharmacol 2012;674:391. [21] Nam KN, Park YM, Jung HJ, et al. Anti-inflammatory effects of crocin and crocetin in rat brain microglial cells. Eur J Pharmacol 2010;648:110. [22] Wang X, Ye XL, Liu R, et al. Antioxidant activities of oleanolic acid in vitro: possible role of Nrf2 and MAP kinases. Chem Biol Interact 2010;184:328. [23] Satav JG, Katyare SS. Effect of experimental thyrotoxicosis on oxidative phosphorylation in rat liver, kidney and brain mitochondria. Mol Cell Endocrinol 1982;28:173. [24] Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem 1951;193:265. [25] Xi MM, Hai CX, Tang H, Chen M, Fang K, Liang X. Antioxidant and antiglycation properties of total saponins extracted from traditional Chinese medicine used to treat diabetes mellitus. Phytother Res 2008;22:228. [26] Wang X, Hai CX, Liang X, Yu SX, Zhang W, Li YL. The protective effects of Acanthopanax senticosus harms aqueous extracts against oxidative stress: role of Nrf2
[27]
[28]
[29]
[30]
[31] [32] [33] [34]
[35]
107
and antioxidant enzymes. J Ethnopharmacol 2010;127: 424. Sato Y, Itagaki S, Oikawa S, et al. Protective effect of soy isoflavone genistein on ischemia-reperfusion in the rat small intestine. Biol Pharm Bull 2011;34: 1448. Mallick IH, Yang W, Winslet MC, Seifalian AM. Ischemiarepefusion injury of the intestine and protective strategies against injury. Dig Dis Sci 2004;49:1359. Yang XK, Bai H, Cai WX, et al. Lycium barbarum polysaccharides reduce intestinal ischemia/reperfusion injuries in rats. Chem Biol Interact 2013;204:166. Aruoma OI. Free radicals, oxidative stress, and antioxidants in human health and disease. J Am Oil Chem Soc 1998;75:199. Marnett LJ. Oxyradicals and DNA damage. Carcinogenesis 2000;21:361. Yla-Herttuala S. Oxidized LDL and atherogenesis. Ann N Y Acad Sci 1999;874:134. Stadtman ER, Levine RL. Protein oxidation. Ann N Y Acad Sci 2000;899:191. Shen XC, Qian ZY. Effects of crocetin on antioxidant enzymatic activities in cardiac hypertrophy induced by norepinephrine in rats. Pharmazie 2006;61:348. Shen XC, Qian ZY, Wang YJ, Duan JA. Crocetin attenuates norepinephrine-induced cytotoxicity in primary cultured rat cardiac myocytes by antioxidant in vitro. J Asian Nat Prod Res 2009;11:417.