Stimulation of Wound Healing by n-3 Fatty Acids
Stimulation of Wound Healing by n-3 Fatty Acids
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 cm = 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.
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.
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 cm = 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.