Study On The Change Of Riboflavin Status Influenced By Supplementing Different Doses Of Riboflavin In Severely Burned Rats

ObjectiveTo contrast the effects on severely burned rats with different dosages of (50mg/kg,100mg/kg and200mg/kg) riboflavin and the associated changes in erythrocyte glutathione reductase activity coefficient (EGRAC) and serum total antioxidant capacity (T-AOC), and their impacts on the pathology of wound collagen deposition. To investigate the appropriate supplementary dosage and to deepen the research understanding of proper riboflavin supplement dosage in burn patients by providing a scientific basis.Methods(1) Establishment of Ⅲ°burn model in Wistar rats:64adult male Wistar rats,6-8weeks of age, with weight ranging150-180g, underwent intraperitoneal injection of the5%chloral hydrate (250mg/kg) anesthetics to the back using the4%sodium sulfide hair-removal. The anesthetics was reintroduced on the second day; then the back areas with hair loss is submerged in100℃water for15seconds, thereby inducing a10%TBSAⅢ°scalding (confirmed by biopsy, hereinafter referred to as a burn). After the burn, isotonic saline (4ml/100g) was injected to counteract shock symptoms, the wounded area was covered with cold gauze. Each rat was then returned to a single cage, allowing free feeding.(2) Grouping of experimental animals:The64burned Wistar rats were randomly divided as such:16for treatment group Ⅰ,16for treatment group Ⅱ, and16for treatment group Ⅲ, with riboflavin regimen initiated2hours after the burn. The rats in each treatment group were given different dosages of riboflavin:group Ⅰ with50mg/kg, group Ⅱ with100mg/kg, group Ⅲ with200mg/kg. The riboflavin is then administered once a day for2weeks. The control group rats were not given riboflavin injections.(3) OUTCOME MEASURES1) EGRAC and serum T-AOC:The rat tail blood was acquired from rats in experimental group and controls groups1、3、7and14days after the burn using BECKMANLX20automatic biochemical analyzer to determine the EGRAC, and721spectrophotometer to determine the T-AOC in serum.2) Histological examination:7days after injury, half of the rats were randomly chosen from each group to be sacrificed; the wounded tissues were cut and isolated, embedded in paraffin, HE stained, and finally observed under light microscope. For each slide series,5samples were randomly selected for high field (10x40) microscopy, with observation on the number of fibroblasts per high-power field, the degree of inflammatory response (in number of inflammatory cells and necrotic exudate), epidermal cell growth, and collagen fibers.14days after the injury, the remaining rats were sacrificed, undergoing the same procedure.3) Hydroxyproline(HOP) content detection on burn wounds:Upon obtaining the aforementioned wound tissue, Chemical colorimetry was employed to measure the change of the wound HOP content, and to reveal the changes of collagen content during the wound healing process.4) Statistics:SPSS13.0statistical software was used for dataanalysis. All data measurements are expressed as mean±standard deviation (x±s). The results within each group were compared at different times using analysis ofvariance. Different groups at the same time point were compared using analysis ofvariance; pairwise comparisons of the different groups at the same time was doneusing the q-test; count data was compared using the chi-square test. P<0.05isconsidered statistically significant.Result1.The determination of EGRAC and serum T-AOC:The treatment group was given riboflavin, the number of cases of riboflavin deficiency on identical time is significantly lower than that of the control group, and the serum T-AOC significantly increased comparing with the control group; the differences were statistically significant (P<0.05).7to14days after the injury, rats in treatment group Ⅱ and Ⅲ had better improvements on riboflavin deficiency and serum T-AOC comparing with treatment group Ⅰ, with differences statistically significant (P<0.05); Comparing rats from treatment group Ⅱ and Ⅲ, the difference in improving riboflavin deficiency and serum T-AOC is unremarkable, with the difference not statistically significant (P>0.05).2. Histological examination:7and14days after the injury, all groups exhibited acute and chronic inflammatory reactions on wounded areas.3treatment groups have significantly fewer fibroblasts and neonatal epidermal layers than that of the control group; the difference is statistically significant (P<0.05); Treatment group Ⅲ is significantly greater than group Ⅰ or group Ⅱ, there is no significant difference between treatment group Ⅰ and Ⅱ. Wounded tissues with HE staining for treatment group Ⅲ shows milder inflammatory response after injury compared with treatment group Ⅰ, treatment group Ⅱ, and the control group.3.Determination of wound HOP:HOP quantity of both the treatment and control groups gradually increased over time with wound healing. The average content of HOP for the wounded areas of each treatment group has a lower degree of growth comparing to that of the control group; for identical time, all treatment groups’ wound HOP content was lower than that of the control group, a statistically significant difference (P<0.05). There is no significant difference among the treatment groups (P>0.05).Conclusion1. A deficiency of riboflavin incurs in rats with severe burn, but proper daily supplements could compensate this deficiency, improve the overall antioxidant capacity, and significantly enhance the riboflavin nutritional status of rats.2. The wound HE staining showed:on the PBD7and PBD14after the injury, each treatment group displays fewer fibroblasts and neonatal epidermal layers than that of the control group. indicating the inhibitory effects of riboflavin on fibroblasts; treatment group Ⅲ exhibits less inflammatory response, illustrating an achievement of anti-inflammatory effects when riboflavin supplement doses exceed200mg/kg.3. The Hydroxyproline test results demonstrated:supplementing riboflavin reduces severe-burn wound collagen deposition in rats and promotes the process of wound healing.