| Objective:To evaluate the effects of vitamin D on inflammatory response in severe burned mice and provide theoretical basis of clinical application of vitamin D on alleviating post-burn inflammatory response.Methods:1. Vitamin D-depleted mice modelyoung (3-week-old) and healthy KM mice (20 male and female mice respectively) were kept in ultraviolet (UV) light-free surroundings with a vitamin D-depleted diet containing 2% calcium and 1.25% phosphorus.12 breeding pairs were obtained through detection of concentration of 25-hydroxyvitamin D3 (25(OH)D3), which dropped below detection limit in mice (<2.5 ng/ml) by after 8 weeks ages. One cage included one breeding pair. Their offspring as second generation were kept with the same UV-free surroundings. After 3 weeks, they were weaned and received the same diet as their parents. The deep secondary burn model was made at the time of 6 weeks of age.2. Vitamin D-depleted mice burn model80 mice(6-week-old) chosen from second generation undertook anesthesia by intra-peritoneal injection of 10% chloral hydrate (40mg/kg). Then, remove the back hair with 4% sodium sulfide. The back of mice awake were placed in 98℃water for 13s next day, causing 20% TBSAⅡwound (2.5×2.5cm2) (confirmed by biopsy). The mice were intra-peritoneal injected with 0.6ml lactated ringer's solution immediately for anti-shock. The 20% SD-Ag cream was put on the wound surface bid for anti-infection. Then, the mice received single cage feeding.3. Groups of the mice80 burn mice were randomly divided into treatment group and control group, which included 40 mice respectively. The sixteen 6-week-old mice, which came from the same breeding institution were treated as blank group. Treatment group were immediately intra-peritoneal injected with same amount of vitamin D 2200IU/Kg every 24 hours, except for the control group. The blank group was not burned.4. Outcome measures 1) Serum TNF-α,IL-1β,IL-4, and IL-10concentration levels The 8 mice of treatment and control groups were sacrificed in order to collect blood at every time point of 6 h,12h,24h,48h and 96h after burn respectively. The serum level of TNF-α, IL-1β, IL-4 and IL-10 were measured by ELISA.8 mice randomly selected in the blank group undergone the same procedure of treatment and control groups.2) Histological observation of the intestine and amount of intestinal bacterial translocationHomogenate was made from sterile separation of the liver, spleen, mesenteric lymph nodes 48 hours after burn. Then, the homogenate was diluted and inoculated in LB agar, which was placed into CO2 incubator under 37℃to do aerobic bacterial culture; the colony number of bacteria per gram of tissue colony forming units (CEU) and its standard deviation were calculated 48 hours later. The HE stained conventional Paraffin sections which were made from jejunum were used to compare pathological changes of intestinal mucosa in the optical microscopy of two groups.8 mice left in the blank group undergone the same course.5. StatisticsAll data was depicted as mean±standard deviation (x±s) and analyzed using SPSS 11.7 software. The intra-group and intergroup inflammatory factors of treatment group and control group undergone one-way ANOVA and Student-Newman-Keuls text. The intergroup differences of inflammatory factors and germiculture results between blank group and treatment, control group were analyzed using two independent sample t-test.Results:1. Mental status and dietThe mental status and diet of mice in treatment group and control group are obviously listlessness, poor feeding, slow response and inactive, compared with the blank group.2. Serum level of TNF-αand IL-1β1) The serum concentration level of TNF-αand IL-1βin the treatment and control group were obviously elevated at every time point after injury compared to blank group (P<0.01).2) The serum concentration level of TNF-αand IL-1βin the treatment group were obviously lower at the time point of 24h,48h,96h after injury compared to control group (P<0.01).3) Peak value of TNF-a and IL-1βconcentration:the serumTNF-a and IL-1βconcentration level in the treatment and control group reached its peak value 3.53±0.63ng/ml,0.48±0.12ng/ml and 5.42±0.45 ng/ml,0.33±0.07 ng/ml both at the time point of 24h respectively.3. Serum IL-4 and IL-10 concentration levels1) The serum concentration level of IL-4 in the treatment and control group were obviously higher at the time point of 24h,48h and 96h after injury compared to blank group (P<0.01). And the serum concentration level of IL-10 in the treatment and control group were obviously higher at the time point of 12h,24h,48h and 96h after injury compared to blank group (P <0.01).2) The scrum concentration level of IL-4 in the treatment group were obviously higher at the time point of 24h,48h,96h after injury compared to control group (P<0.01). And the serum concentration level of IL-10 in the treatment group were obviously higher at the time point of 12h,24h,48h, 96h after injury compared to control group (P<0.01)3) The serum IL-4/IL-10 concentration level in the treatment and control group elevated over time.4. Amount of intestinal bacterial translocationBacterial colony was formed through visceral tissue culturing in control group andtreatment group, except for the blank group. The amount of bacterial translocation in the treatment group(mesenteric lymph nodes 780±28.7, liver147±15.3, spleen 254±18.7) was significantly lower than the control group (mesenteric lymph nodes 850±32.8,liver180±17.5. spleen 295±28.2)(P<0.01).5. Histological observation of the intestineSmall intestine gland in the treatment group expended mildly and, the epithelial cells were unbroken and aligned, While the intestinal mucosa in the control group were edema obviously, which contained expansion of small intestine gland, loose and fell of intestinal villi epithelial, some inflammatory cell were infiltrated in the intestinal submucosa. The intestinalvilli epithelial of blank group were normal.Conclusions: 1) The remarkable elevating of serum TNF-αand IL-1βconcentration level in the treatment group and control group while the lower serum TNF-αand IL-1βconcentration level in the treatment group compared to control group at the time point of 24h,48h and 96h after burn demonstrated that vitamin D reduced the synthesis of pro-inflammatory cytokines (TNF-αand IL-1β) in mice after severe burn.2) The obvious increasing of serum IL-4 concentration level in the treatment group and control group from start point of 24h after wound, while higher serum IL-4 concentration level at the same time point in treatment group compared to control group and the obvious elevating of serum IL-10 concentration level in the treatment group and control group from the start point of 12h and 24h respectively after wound while higher serum IL-10 concentration level at the same time point in treatment group compared to control group demonstrated that vitamin D promoted the synthesis of anti-inflammatory cytokines (IL-4 and IL-10) in mice after severe burn.3) The amount of bacterial translocation in the treatment group was lower than the control group significantly, and the intestinal mucosa in the control group were edema obviously than the treatment group. It indicated that vitamin D play the protective role for the intestine mucosa after sever burn, by preventing the bowel bacteria immigration and reducing intestine mucosa edema. |
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