Experimental Research on Stimulation of Wound Healing by n-3 Fatty Acids

Index: WOUNDS. 2013;25(7):186-192.

  Abstract: Objective. The objective of this study was to investigate the wound healing effects of n-3 fatty acids and to identify factors that stimulate wound healing. Materials and Methods. Four-week-old male Wistar rats were subjected to full-thickness skin wounds and assigned to 3 experimental diet groups (an n-3 fatty acid-fortified diet, a diet with a 1:3 ratio of n-3 to n-6 fatty acids, and an n-6 fatty acid-fortified diet). Intergroup comparisons were conducted for the changes in wound areas, the number of days to wound healing, and blood cytokines, blood hydroxyproline, and blood chemistry test values on the day before and after wound healing. Results. The number of days to wound healing in the n-3/n-6 fatty acid group (18.4 ± 1.8 days) was significantly shorter than in the n-3 fatty acid-fortified diet (21.6 ± 1.6 days) and n-6 fatty acid-fortified diet groups (21.9 ± 1.8 days). This suggests that the n-3/n-6 fatty acid diet stimulates wound healing (P < 0.05). Changes in wound area, however, were not significantly different. The n-3 fatty acid-fortified diet was found to have potent immunopotentiating and anti-inflammatory effects in the group receiving this diet, as evidenced by total blood lymphocyte count and plasma levels of interleukin-1 beta (IL-1β) and sialic acid on day 1 after wounding. The plasma hydroxyproline concentrations noted in the groups with a diet containing n-3 fatty acids indicate that this fatty acid type stimulates wound healing. Conclusions. Findings suggest that n-3 fatty acids have anti-inflammatory and immunopotentiating effects, and are beneficial in the wound healing process, particularly during early inflammation.

Introduction

  Persisting pressure, rubbing, and underlying disease morbidity are the major causes of pressure ulcers, but malnutrition is another important factor. Research comparing the body measurements of patients with pressure ulcers, and the finding that 46% of these patients have serum albumin levels < 3.5 g/dL, highlight the importance of better nutrition for the prevention and healing of pressure ulcers.^1-3   Among nutrients, fatty acids are precursors of bioactive substances. The types and ratios consumed are therefore important considerations. The n-6 fatty acid linoleic acid, for instance, produces eicosanoids derived from arachidonic acid within the body and is a key contributor to the initiation of the inflammatory response among other functions associated with the body’s defenses.⁴ In contrast, the n-3 fatty acids competitively inhibit the conversion of arachidonic acid into bioactive substances, thereby suppressing inflammatory cytokine production. Resolvins, metabolites of n-3 fatty acids, have recently been shown to stimulate the resolution of inflammation as anti-inflammatory mediators.^5-6 Moreover, the n-3 fatty acids are immunonutrients that activate intestinal immunity, protect the cardiovascular system, suppress weight loss in cancer patients, and play other important roles in maintaining and improving immunity.⁶ Research is now characterizing the anti-inflammatory and immunopotentiating effects of n-3 fatty acids, but it is the authors’ understanding that the role of these substances in the wound healing process remains quite limited. The effects of n-3 fatty acids on inflammatory cytokines in the wound healing process, and the ability of n-3 fatty acids to stimulate wound healing in wounded rats fed a diet fortified with n-3 fatty acids, were investigated.

Methods and Materials

  Three study diets were prepared: an n-3 fatty acid-fortified diet was prepared by adding perilla oil in place of soybean oil in the basic diet (AIN-76A); a diet with a 1:3 ratio of n-3 to n-6 fatty acids was prepared by adding perilla oil and corn oil at a 1:3 ratio in place of soybean oil in the basic diet; and an n-6 fatty acid-fortified diet was prepared by adding corn oil in place of soybean oil in the basic diet. The compositions of the diets are shown in (Table 1).   The study was conducted using 4-week-old male Wistar rats (Japan SLC, Hamamatsu, Japan). Animals were assigned to 3 groups of 25 animals each (n = 75); group A was fed the n-3 fatty acid-fortified diet, group B was fed the diet with a 1:3 n-3 to n-6 fatty acid ratio, and group C was fed the n-6 fatty acid-fortified diet. Animals were allowed to consume up to 80 kcal per day, and were given water ad libitum. Full-thickness defects were introduced after 1 week. Specifically, the backs of the rats, anesthetized with pentobarbital Nembutal, were shaved, and full-thickness defects were introduced using a hole-punch with a head 15 mm in diameter. Before wounding, blood samples were collected by cardiac puncture from 6 ether-anesthetized animals from each group to investigate the effects of the diets on blood fatty acid fraction. These animals were subsequently sacrificed. Feeding of the study diets under the same conditions continued in each group after wounding. Blood samples were collected by cardiac puncture on day 1 and day 3 after wounding, from 6 animals in each group, to monitor changes over time to inflammatory and wound markers. Animals were subsequently sacrificed. The remaining 7 animals in each group were fed the study diets until the day of complete epithelial restitution of the wound surface. Blood samples were collected as described above, and animals were sacrificed. Changes in body weight, calories consumed, and cumulative calories consumed in each group were measured daily throughout the study. No wound treatment to stimulate wound healing was administered. The study was conducted with the approval of the Institutional Animal Care and Use Committee of Fujita Health University.   The number of days to wound healing was calculated as the number of days, beginning on the day of wounding, required for complete gross resolution of scabbing and erythema. The size of the wound surface to epithelial restitution was adjusted for body weight (1 cm² = 6.75 mg) to calculate wound area. Blood fatty acid fraction, plasma sialic acid levels, IL-1β, hydroxyproline, serum total protein, albumin, total cholesterol, triglycerides, alkaline phosphatase, retinol binding protein levels, and total lymphocyte count were measured in blood tests conducted the day before wounding, day 1 and day 3 after wounding, and the day of wound healing. The procedures and reagents used were as follows: serum sialic acid levels were measured using the enzyme method with Runpia SIAL reagent (Kyokuto Pharmaceutical Industrial Co Ltd, Tokyo, Japan) with an autoanalyzer; IL-1β was measured using the Rat IL-1β Immunoassay Kit (Invitrogen, Carlsbad, CA); and plasma hydroxyproline levels were measured with the rat hydroxyproline enzyme-linked immunosorbent assay (ELISA) Kit (Cusabio, Wuhan, China). ELISA for plasma hydroxyproline and IL-1β was performed according to the manufacturer’s protocol based on the solid phase sandwich principle. Finally, blood fatty acid fractions were measured by analyzing blood samples by gas chromatography after methyl esterification with a derivatization reagent.

Statistical Analysis

  Data was expressed as mean ± standard deviation (SD) and statistical analysis was carried out employing one way analysis of variance (ANOVA) followed by Tukey-Kramer multiple comparisons test at P < 0.05 significance level using InStat version 3.00 (GraphPad software, San Diego, CA).   The mean differences among the 3 groups were compared using Student’s t test.

Results

  Blood fatty acid concentrations on the day before wounding reflected the fatty acid compositions present in the respective diets (Table 2). Cumulative calorie consumption did not differ significantly among the groups at 438.8 ± 10.5 kcal in group A, 416.3 ± 15.8 kcal in group B, and 438.4 ± 14.7 kcal in group C. Similarly, changes in calorie consumption and body weight did not differ significantly among the 3 groups.   The number of days from wounding to complete gross resolution of scabbing and erythema was significantly shorter in group B at 18.4 ± 1.8 days than in groups A and C (group A vs group B: P < 0.05; group B vs group C: P < 0.05) (Figure 1). The changes in wound area decreased over time in each of the 3 groups, and changes in area did not differ significantly among the groups. Levels of IL-1β at the time of acute inflammation, or day 1 of wounding, were significantly lower in group A at 2.19 ± 0.52 pg/mL than in group C at 6.44 ± 1.31 pg/mL.   Similarly, plasma sialic acid levels were significantly lower in group A at 54.0 ± 1.7 mg/dL than in group C at 62.3 ± 2.4 mg/dL (P < 0.05) (Figure 2). Plasma hydroxyproline concentrations before wounding, which were 1034.4 ± 67.1 ng/mL in group A, 962.4 ± 78.9 ng/mL in group B, and 945.8 ± 68.5 ng/mL in group C, did not differ significantly among the 3 groups, but hydroxyproline concentrations increased in proportion to the percentage of n-3 fatty acids in the diet.   Changes in concentration after wounding decreased significantly in all groups, particularly in group C (Figure 3). Total cholesterol was significantly lower in group A than group C on the day before wounding, on days 1 and 3 after wounding, and on the day of wound healing; and was significantly lower than in group B on days 1 and 3 after wounding, and on the day of wound healing. Triglycerides were significantly lower in group A than group C on day 1 after wounding and on the day of wound healing; and were significantly lower in group A than group B on the day of wound healing. Levels of albumin, alkaline phosphatase, total protein, and retinol-binding protein did not differ significantly among the 3 groups. However, total lymphocyte count on the day before wounding was significantly higher in group A at 48.2 ± 6.1 µg/L than in group C at 40.7 ± 7.6 µg/L (P < 0.05) (Table 3).

Discussion

  Numerous animal studies using rat models of skin wounds have recently been conducted.^7-11 In certain tissue disorders, and with the presence of inflammation, IL-1β TNF-α, and eicosanoids are released from macrophages migrating to wound sites.¹² The essential n-6 and n-3 polyunsaturated fatty acids are vital in wound healing.^9-10 By activating macrophages, n-3 fatty acids stimulate the proliferation of fibroblasts, which in turn increases collagen synthesis and promotes granulation.¹³ In addition, n-3 fatty acids stabilize the phospholipid composition in the cell membrane and suppress inflammation by suppressing cyclooxygenase 2, the substrate of which is arachidonic acid released from monocytes and neutrophils that have migrated to the wound site, thereby inhibiting the production of prostaglandin E2.¹⁴ The n-6 fatty acids and inflammatory mediators derived from these substances, in contrast, enhance inflammation by triggering inflammatory cytokine production and the amplification cascade, even promoting cellular proliferation by activating transcription factors.^15-16 However, diets with a large proportion of n-6 fatty acids have been shown to actually promote inflammation and delay wound healing.^15,17-18 The significantly lower levels of IL-1β and serum sialic acid in group A than in group C on day 1 after wounding in this study appear to be a result of the anti inflammatory effects of n-3 fatty acids. The significantly higher total lymphocyte count on the day before wounding in group A in comparison to group C also indicates that n-3 fatty acids are closely associated with immunity and may regulate inflammatory cytokine production.   Collagen serves as the cellular substrate in the wound healing process. The growth and polymerization of collagen fibers confer tensile strength to the wound and profoundly affect cellular proliferation.^19-22 The authors used hydroxyproline, an amino acid component of collagen, to assess cellular proliferation during wound healing.^23-24 Before wounding, plasma hydroxyproline concentrations increased in proportion to the n-3 fatty acid content of the diet. After wounding, the decrease in plasma hydroxyproline concentrations was more gradual in rats in groups A and B, which were fed a diet containing n-3 fatty acids, than those in group C, which were not. The high hydroxyproline concentration in group A on day 3 after wounding suggests that this amino acid also operates in the repair mechanism in cellular proliferation.   The dietary administration of eicosapetaenoic acid and docosahexaenoic acid in an animal study on n-3 fatty acids lowered levels of cholesterol and triglycerides.²⁵ Similarly, levels of total cholesterol and triglycerides were significantly lower in group A than group C in this study, which suggests that the α-linoleic acid in perilla oil more strongly reduces total cholesterol and triglycerides than linoleic acid.   The relative ratios of fatty acids consumed in the human diet have been investigated.²⁶ A ratio of n-3 to n-6 fatty acids in the range of 1:1 to 1:5 is recommended. A noteworthy finding of this study is the significantly smaller number of days to wound healing in the rats in group B, whose diet was formulated at a 1:3 ratio, in comparison to those in groups A and C. The larger reduction in wound area on day 6 and day 10 after wounding in group B indicates that certain fatty acids may have specific actions in each of the inflammation and proliferation stages of the wound healing process, and this issue merits further investigation. At the inflammation stage, n-6 fatty acids are necessary to induce inflammatory cytokines, but n-3 fatty acids are also necessary to inhibit excess inflammation. Maintaining n-3 fatty acids in the diet until cytokine-activated macrophages migrate to the site of inflammation to affect collagen synthesis appears to facilitate the wound healing process. In other words, a diet fortified with n-3 fatty acids is apparently more effective at suppressing inflammation, and both n-3 and n-6 fatty acids are essential for wound healing. Studies in patients with pressure ulcers indicate the use of semi-digested enteral nutrients formulated with a 1:3 ratio of n-3 to n-6 fatty acids helps heal pressure ulcers by improving protein synthesis and lipid metabolism.^17,27-28

Conclusion

  These findings suggest that n-3 fatty acids have anti-inflammatory and immunopotentiating effects, and are beneficial in the wound healing process, particularly during early inflammation. The fact that a diet with a 1:3 ratio of n-3 to n-6 fatty acids stimulated wound healing indicates that the ratio of n-3 to n-6 fatty acids must be adjusted according to wound healing stage.

References

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Fish oil supplementation alters levels of lipid mediators of inflammation in microenvironment of acute human wounds. Wound Repair Regen. 2011;19(2):189-200. Akihiko Futamura, PhD; Takashi Higashiguchi, MD, PhD; and Akihiro Ito, MD, PhD are from the Department of Surgery and Palliative Medicine, Fujita Health University School of Medicine, Toyoake, Aichi, Japan. Yoshiyuki Kodama, MD is from Digestive Disease Center, Tokeidai-Memorial Hospital, Sapporo, Japan. Takeshi Chihara, PhD; Takaaki Kaneko; Akiko Tomatsu; and Kan Shimpo, PhD are from Fujita Memorial Nanakuri Institute, Fujita Health University, Toyoake, Aichi, Japan. Address correspondence to: Akihiko Futamura, PhD Department of Surgery and Palliative Medicine Fujita Health University School of Medicine 424-1, Ohtori-cho Tsu-City, Mie, Japan, 514-1295 myk@zc.ztv.ne.jp Disclosure: The authors disclose no financial or other conflicts of interest.