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,3
* Department of Human Nutrition, The Ohio State University, Columbus, OH and
Linus Pauling Institute, Oregon State University, Corvallis, OR
3To whom correspondence and reprint requests should be addressed. E-mail: tammy.bray{at}oregonstate.edu.
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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B activation, reduced expression of proinflammatory cytokines [interleukin (IL)-1ß and tumor necrosis factor (TNF)-
], and a decrease in neutrophil infiltration during the early stage of cutaneous wound healing. We used a cutaneous, full-thickness excisional wound model in CD-1 mice to examine the rate of wound closure as well as mRNA levels of inhibitory (I)B, IL-1ß, and TNF- and infiltration of neutrophils at the wound site of mice fed a diet containing <1 (deficient), 50 (control), 500, or 1000 µg zinc/g diet. Zinc deficiency reduced the rate of wound closure and mRNA levels of IL-1ß and TNF- and attenuated infiltration of neutrophils at the wound site compared with controls. Interestingly, zinc supplementation at 1000 µg/g delayed the rate of wound closure and decreased mRNA levels of TNF- and infiltration of neutrophils compared with mice fed the control diet. These findings demonstrate that zinc deficiency and high-dose zinc supplementation delay wound healing as a result of altered inflammatory responses and suggest that adequate zinc supplementation may have beneficial effects on the inflammatory responses to enhance cutaneous wound healing.
KEY WORDS: • wound healing • zinc • inflammation • proinflammatory cytokines
Zinc plays an essential role in growth, immune function, antioxidant defense, and wound healing (1–3). Zinc deficiency was shown to increase the time for wound closure and to decrease wound strength (4,5). In addition, zinc has been used as a topical agent to treat diaper rash and as a nutritional supplement in patients with bedsores, ulcers, and incisional wounds (6–8). Although the role of zinc in wound healing has been investigated since the 1950s (9), the mechanisms by which zinc affects healing processes are not clear.
Wound healing is a complicated network that involves the interaction and coordination of various cell types, structural proteins, cytokines, and reactive oxygen species (ROS).4 In general, there are three major stages of wound healing, i.e., inflammation, proliferation, and remodeling (10,11). Although it is a highly dynamic process, inflammation is considered to be a critical stage for establishing an environment that facilitates the subsequent stages of the healing process (12). The initial event during the inflammatory stage is the infiltration of neutrophils into the wound site to prevent infection through phagocytic processes and to induce vascular endothelial growth factor (VEGF) production in macrophages by ROS production (13–15). However, excessive ROS production can cause tissue damage and may impair wound healing (16,17).
The recruitment and function of neutrophils were shown to be regulated by several chemotactic factors, which are stimulated by interleukin-1ß (IL-1ß) and tumor necrosis factor- (TNF-) (18,19). Both mRNA levels of IL-1ß and TNF- are regulated by nuclear factor B (NFB), a critical transcription factor that regulates the expression of inflammatory mediators such as cytokines, chemokines, and cell adhesion molecules (20,21). Hence, NFB plays a pivotal role during the inflammatory stage, and NFB activation is thus proposed to be a critical event of early wound healing.
Despite the considerable importance of inflammation, little research has focused on the effect of zinc with regard to pathways that control the early inflammatory responses during wound healing. Recently, zinc deficiency was shown to reduce NFB nuclear binding activity in vitro and in vivo (22,23). However, there is limited evidence that zinc may alter NFB binding activity and subsequently regulate early wound healing processes.
We hypothesized that the mechanism by which zinc deficiency delays wound healing involves an alteration in NFB activation during the inflammatory stage of cutaneous wound healing. Moreover, we proposed that dietary zinc supplementation with high levels of zinc may enhance and accelerate inflammatory responses and promote wound healing. In this study, we used a cutaneous, full-thickness, excisional wound model to examine the effects of dietary zinc deficiency and zinc supplementation on the rate of wound closure, and the temporal kinetics of IL-1ß, TNF-, and inhibitory (I)B mRNA expression and neutrophil infiltration during the early inflammatory stage of cutaneous wound healing.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Female CD-1 mice (3 wk old, 15–18 g) were obtained from Harlan and housed in individual metal wire cages in a temperature- and humidity-controlled room (12-h light:dark cycle) with free access to tap water and food. Food intake and body weights were measured every other day. Mice were acclimated for 2 d before initiation of dietary treatments. They were fed a modified AIN-93G rodent powder diet (24) containing <1, 50, 500, or 1000 µg zinc/g diet for 2 wk (Dyets). All mice were used in accordance with animal protocols approved by the Ohio State University Institutional Laboratory Animal Care and Use Committee.
Zinc concentrations in serum and skin.
Serum was separated by centrifugation at 2000 x g for 10 min. Zinc concentration in serum or skin was assessed according to the method of Luterotti et al. (25). Serum or skin tissues were digested in 2 mol/L hydrochloric acid for 24 h at room temperature. Samples were then centrifuged at 7000 x g for 25 min, and the supernatant was used for direct measurement of metal concentrations using an atomic absorption spectrometer (Model AA-5, Varian Techtron).
Wound biopsy.
Mice were anesthetized with isoflurane and the back of the mouse was shaved and sterilized using an alcohol swab. The wound biopsy model used in this experiment was described previously (26). The shaven skin was then pinched and folded, and a sterile biopsy punch (3.5-mm diameter, Miltex Instrument Company) was used to punch through the full-thickness of the folded skin. This yielded two circular wounds, identical in size, on the dorsum, below the shoulder blades, of each mouse. A wound placed in this area cannot be reached by the mice, thereby eliminating self-licking.
Measurement of wound closure.
Wounds from individual mice were photographed digitally every day, beginning on the day of wounding (d 0) with a standard dot equivalent to the initial wound area placed beside the wound. Wound closure was quantified using Canvas 7SE software (Deneba). The rate of wound closure was expressed as the ratio of wound area (each day after wounding) compared with the initial wound area. A smaller wound ratio indicates faster wound closure.
Harvesting and histologic analysis.
Mice were killed with an overdose of isoflurane to collect tissue samples at the wound site for examination of neutrophil infiltration into the wound area. Wounds at 0, 6, 12, and 24 h after wounding were removed by cutting a square area that covered the entire wound site from 4 mice from each treatment group. Harvested tissues were immediately stored in 4% formaldehyde solution in PBS (pH 7.4) and were then washed in PBS, dehydrated in a series of alcohols, and embedded in paraffin. Microtome sections (5-µm thick) were cut vertically across the wound site, adhered to slides, and stained with hematoxylin and eosin. Photographs of the wound site were obtained; the images were digitized using Adobe Photoshop (Adobe Systems), and the density of neutrophil infiltration quantified using ImageJ (NIH). Neutrophil infiltration into the wound site was quantified at 3 spots on the wound edge and within the wound bed in each wound by densitometric analysis.
In situ hybridization (immunofluorescence).
The in situ hybridization protocols were performed as described previously for ribonucleotide (cDNA) probes (26,27). Antisense probes were transcribed using the Riboprobe System (Promega Biotech) with T7 RNA polymerase. Mouse cDNAs of IB and TNF- were generously provided by Dr. Rebecca Taub (University of Pennsylvania, Philadelphia, PA) and Dr. Karl Decker (Albert-Ludwigs-Universitat, Freiburg, Germany) respectively. A 672-bp DNA sequence of murine IL-1ß mRNA was prepared from commercially available insert #963357 in vector pT7T3D-pac cloned in host Escherichia coli (American Type Culture Collection). Immunofluorescence kits were obtained from Dako Company. In situ hybridization was carried out with digoxigenin-labeled probes for IB, IL-1ß, or TNF- mRNA on slide sections as previously described (26). Diluted antidigoxigenin antibody was added onto the slides, followed by anti-mouse antibody, primary streptavidin, biotinyl tyramide solution, and finally CY2-streptavidin. The slides were then viewed with a microscope through a fluorescence filter. The mRNA level was quantified by measuring the intensity of cells (neutrophils) expressing mRNA of IB, IL-1ß, and TNF- using Adobe Photoshop and ImageJ software. mRNA quantified spots were located at the wound edges and within the wound bed (3 spots per slide). One slide per wound from each of 4 mice/group was examined.
Statistical analysis.
All values are expressed as means ± SEM. Data were analyzed by 1-way ANOVA at each time point, and then differences among means were analyzed using Duncan’s test. The paired t test was used to compare differences in zinc concentration between unwounded and wounded skin in each group. For all tests, differences were considered significant at P < 0.05.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Weight change and food intake did not differ among the groups throughout the 2-wk dietary treatment (data not shown).
Zinc concentrations in serum and skin.
Serum zinc concentration was reduced in mice fed the zinc-deficient diet and was increased in mice fed zinc-supplemented diets (500 or 1000 µg/g) compared with mice fed the control diet (P < 0.05) (Fig. 1A). Zinc concentrations of unwounded and wounded skin in mice fed the zinc-deficient diet were significantly lower than those of mice fed the control diet and mice fed zinc-supplemented diets (Fig. 1B). However, zinc concentration in wounded skin did not differ from unwounded skin in the same mouse from all groups. Notably, serum and skin zinc concentrations did not differ between mice fed the 500 and the 1000 µg/g zinc diet.
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The rate of wound closure in mice fed the zinc-deficient diet was significantly slower than that of mice fed the control diet during the early inflammatory stage (d 1–4) of wound healing (Fig. 2). The rate of wound closure in mice fed the 500 µg/g zinc diet was faster than that in mice fed the zinc-deficient diet. Interestingly, the rate of wound closure of mice fed the 1000 µg/g zinc diet was significantly slower than that of mice fed the control diet or the 500 µg/g zinc diet from d 1 to 4.
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Levels of IB mRNA tended to be greater (P = 0.055–0.085) in mice fed the 500 µg/g zinc diet at each time point compared with mice fed the control diet (Fig. 3). Moreover, the IB mRNA level in mice fed the 500 µg/g zinc diet was significantly higher at 6, 12, and 24 h than in mice fed the 1000 µg/g zinc diet, and at 6 and 24 h compared with mice fed the zinc-deficient diet (P < 0.05).
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(Fig. 4B) mRNAs were detected in the dermis along the wound edge and clot. The IL-1ß mRNA level in mice fed the zinc-deficient diet was lower than that of control mice at 6 and 12 h after wounding (P < 0.05) (Fig. 4A). On the other hand, TNF- mRNA was differently expressed in mice fed the various levels of dietary zinc. The TNF- mRNA level in mice fed the zinc-deficient diet was lower (43–64%) than in control mice at 6 and 12 h (P < 0.05) (Fig. 4B). The TNF- mRNA level in mice fed the 1000 µg/g zinc diet was lower (47–58%) than in control mice at 6 and 24 h after wounding (P < 0.05).
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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, and neutrophil infiltration during the early inflammatory stage of wound healing. Moreover, these experiments demonstrated that although zinc supplementation at 500 µg/g diet had beneficial effects on the inflammatory response and wound closure rate, high-dose zinc supplementation (1000 µg/g) negatively affected the inflammatory response of wound healing. As expected, wound and serum zinc levels were significantly lower, and wound size was significantly greater, in mice fed the zinc-deficient diet (<1 µg/g zinc) compared with mice fed the control diet (50 µg/g zinc) or the 500 µg/g zinc- supplemented diet (Figs. 1, 2). However, neither skin nor circulating zinc level was an unambiguous predictor of the rate of wound closure. In particular, the rate of wound closure was significantly slower in mice fed the high zinc diet (1000 µg/g) compared with mice fed the control diet or the 500 µg/g zinc-supplemented diet, despite the fact that there was no significant difference in skin and serum zinc levels between the two zinc-supplemented groups. Thus, it appears that the deleterious effects of dietary zinc supplementation at 1000 µg/g zinc may be mediated by a mechanism that is independent of either serum or wound tissue zinc level. Previous work showed that very high zinc intakes may decrease copper absorption, leading to copper deficiency and anemia and may play an important role in the observed immunodepression of patients supplemented with high levels of zinc (28–30). Hence, both zinc deficiency and high-dose zinc supplementation may be deleterious for cutaneous wound healing.
To investigate the molecular mechanisms through which dietary zinc alters the rate of wound closure, we examined NFB activation through the expression of IB, a downstream gene that is regulated by and mirrors the activity of NFB (31,32). NFB is a redox-sensitive transcription factor that regulates inflammatory mediators such as cytokines, chemokines, cell adhesion molecules, and extracellular matrix molecules during inflammation, and is normally maintained in an inactive form in the cytoplasm bound to the inhibitory protein, IB (33). During activation by various stimuli such as UV light, ROS, proinflammatory cytokines, and virus infection, IB is phosphorylated and degraded, facilitating the translocation of NFB to the nucleus where it regulates the expression of immune and inflammatory genes (33). Our data demonstrated that IB mRNA levels generally were lower in both the zinc-deficient and the 1000 µg/g zinc-supplemented groups than in mice fed the control or 500 µg/g zinc diet (Fig. 3). Furthermore, the delayed rate of wound closure in zinc-deficient or 1000 µg/g zinc-supplemented mice paralleled the decrease in IB mRNA level. Zinc plays a role as an antioxidant in protecting sulfhydryl groups, essential in protein stability and activation, from oxidation and prevents superoxide and hydroxyl radical production by prooxidant metals, copper, and iron (2,34,35). Therefore, zinc deficiency may increase oxidative stress–induced tissue damage by decreasing antioxidant functions. Previous studies showed that zinc deficiency reduces NFB binding activity after oxidative stress in vitro and in vivo and impairs NFB translocation in vivo (22,23,36). Therefore, our data suggest that zinc deficiency may delay wound healing as a result of impaired NFB binding activity secondary to increased oxidative stress.
To confirm the effect of dietary zinc on NFB activation and subsequent events during the early inflammatory stage, mRNA levels of the proinflammatory cytokines IL-1ß and TNF-, target genes of NFB activation, were investigated at different time points. Because of their biological roles in the activation of neutrophils, stimulation of fibroblast and keratinocyte growth, synthesis and breakdown of extracellular matrix proteins, and regulation of immune responses (37), it is possible that decreased expression of these cytokines may delay the wound healing process by impairing tissue regeneration, thus delaying restoration of normal tissue structure and function. Although the mRNA level of IL-1ß was reduced only by zinc deficiency (Fig. 4A), TNF- mRNA levels were reduced by zinc deficiency as well as high-dose zinc supplementation (1000 µg/g) (Fig. 4B). Interestingly, mRNA levels of IL-1ß and TNF- returned to control levels 24 h after wounding in zinc-deficient mice, whereas high-dose zinc supplementation significantly depressed TNF- levels throughout the 24-h period. Therefore, these results suggest that zinc deficiency or high-dose zinc supplementation may exert their deleterious effects through alterations in the expression of proinflammatory cytokines.
In addition to their putative role in stimulating the expression of growth factor genes that are important in cutaneous wound healing (38), IL-1ß and TNF- modulate the expression of chemokines and adhesion molecules necessary for the recruitment of inflammatory cells to the site of injury (39). Neutrophils are the predominant cell type at the wound site within 24 h of wounding where they function not only as phagocytic cells, but also as cells that synthesize and release various cytokines and chemokines that are essential to promoting subsequent steps in the wound healing process (40). Similar to our other results, consumption of either the zinc-deficient (<1 µg/g) or the high-zinc (1000 µg/g) diet may impair the wound healing process through their effects on neutrophil infiltration at the wound site (Fig. 5). However, neutrophil infiltration at the wound site in mice fed the 500 µg/g zinc diet was comparable to that of control mice. Other studies have shown that not only the recruitment of neutrophils but also their chemotactic activity is disturbed by zinc deficiency or supplementation in vitro (41–43). As noted previously, high-dose zinc supplementation may induce copper deficiency, which can cause both neutropenia and functional impairment of neutrophils including the chemotactic response and phagocytic activity (28–30). Indeed, it was demonstrated in human studies that a zinc intake 20-fold greater (300 mg/d) than the current RDA (15 mg/d) decreased several indices of immune function, including the chemotactic response and phagocytic activity of neutrophils (44). Hence, these findings suggest that both zinc deficiency and high-dose zinc supplementation may impair wound healing by adversely affecting the recruitment and phagocytic activity of neutrophils.
Taken together, the results of this study support the hypothesis that zinc deficiency delays the early inflammatory response resulting in delayed wound healing. Furthermore, these data clearly demonstrate that although moderate zinc supplementation may be beneficial, excessive zinc supplementation is unequivocally deleterious in terms of its influence on cutaneous wound healing. Because of the central role of NFB, which is required for the induction of proinflammatory cytokines such as IL-1ß and TNF- during inflammation, decreased activation of NFB may lead to a delayed inflammatory cascade, culminating in immunosuppression and delayed wound healing in zinc deficiency and high-dose zinc supplementation.
In summary, the current study demonstrated that zinc deficiency exerts negative effects on the early inflammatory response of cutaneous wound healing. Understanding the molecular mechanisms that regulate cutaneous wound healing is critical not only in zinc deficiency, but also in numerous other disorders associated with abnormal wound repair. Hence, the results of this work may provide critical insight into future nutritional intervention strategies designed to enhance immune function not only in patients suffering from zinc deficiency, but also in patients suffering from malnutrition-related disorders.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Supported by National Institutes of Health Grant DK55847 and NS38315 to T.M.B.
4 Abbreviations used: IB, inhibitory B; IL-1ß, interleukin-1ß; NFB, nuclear factor B; ROS, reactive oxygen species; TNF-, tumor necrosis factor-; VEGF, vascular endothelial growth factor.
Manuscript received 2 September 2003. Initial review completed 26 September 2003. Revision accepted 27 January 2004.
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