| ObjectiveTo explore the effect of1,25-dihydroxyvitamin D3on insulin resistance and inflammatory response in mice with severe burns under stress.Methods1. Set up an established mouse model of30%of the total body surface area (TBSA) Ⅲ°burnsChoosing130healthy SPF KM mice (Chinese Academy of Military Medical Experimental Animal Center), an equal split of male and female, weighing28～32g, which aged6to8weeks. The back hair of mice which is30%TBSA is dehairinged with6%Na2S.4%chloral hydrate (40mg·kg-1) intraperitoneal injection is the way of anesthesia. After anesthesia, the mice Hair removal area is placed100℃hot water to12s, made burn area of30%TBSA Ⅲ°burn model (biopsy confirmed, the normal group does not burn), immediately infecting2mL·kg-11%TBSA-1Lactated Ringer’s Solution after the injury by intraperitoneal injection, giving half the amount on the2day. Wound is smeared with2%SD-Ag1times a day to anti-infect.2. The grouping of experimental animalsThe mice were randomly divided into four groups, the health groups (not scalding)10, the experimental group Ⅰ (treatment after burn), the experimental group Ⅱ (treatment after burn) and the control group (no treatment after burn) each40. Feed each group separately with the conventional animal feed. After modeled successfully, the every other morning at8:00, giving the experimental group Ⅰ mice1,25-(OH)2VitD31μg·kg-1+0.6mL peanut oil gavage and the mice given the experimental group Ⅱ1,25-(OH)2VitD34μg·kg-1+0.6mL peanut oil gavage. At the same time,the control mice given0.6mL peanut oil gavage. Gavage lasts two weeks. 3. Outcome measures(1) The fasting blood glucose (FBG)The laboratory mice were fasted overnight, collecting blood after beheading death at8o’clock in the morning. Healthy mice were killed once. The experimental group I,the experimental group II and the control group were killed10mice respectively on the1,3,7and14day after burned. Use blood glucose test strips and glucometer test instant FBG when you are collecting blood after beheading death.(2) Fasting insulin (FIns)When10mice of experimental group Ⅰ, experimental group Ⅱ and the control group were killed at1,3,7,14d respectively (the10mice of healthy group were killed once),2mL blood samples were collected. After3000r/min,30min,4℃centrifugation,0.8mL of serum is collected and stored at-80℃for measuring fasting insulin. End of the experiment,0.5mL of the prepared samples were placed in serum cup (remaining0.3mL of serum for determination of serum TNF-a), placed on automated chemiluminescence immunoassay analyzer (using chemiluminescent immunoassay measure FIns content), started testing after adjusting the detection parameters, read light quantum number, calculated the FIns content according to the standard curve.(3) calculate the value of IRCalculate HOMA-IR using homeostasis model equation method (HOMA) as: HOMA-IR=FBG (mmol/L)×FINS (mU/L)/22.5.(4) Measurement of serum levels of TNF-aRemoving serum samples from-80℃to room temperature, after half an hour, using mouse TNF-α ELISA test kit to detect serum TNF-α concentrations.(5) Measure wound tissue NF-κB-positive rate by immunohistochemistryWound tissue specimens were embedded in paraffin sections, staining by immunohistochemical kit. Brownish yellow particles mean that NF-κB (mainly expressed in the cytoplasm and/or nucleus) is positive. Read5different horizons randomly at high magnification (x400times), counting100cells and the number of positive cells. The wound tissue NF-KB-positive rate (%)=(number of positive cells /total cells)×100%.4. Statistical MethodsStatistics and data were processed using the SPSS18.0software. Measurement data are showed by x±s, using single-factor analysis of variance (ANOVA), multiple comparisons between groups using LSD-t test. P<0.05was considered statistically significant.Results1. The determination of fasting blood glucose (FBG) at each group of miceUse blood glucose test strips and glucometer test instant FBG when mice were killed. The results show that mice’s FBG at different times after injury of experimental group Ⅰ、experimental group Hand the control group were higher than the healthy group.(the14th day of experimental group Ⅱ mice compared with healthy group, P>0.05, the remnant compared with healthy group, P<0.05). Comparison at the same time between the different groups, experimental group Ⅰ and experimental group Ⅱ FBG were lower than the control group. And experimental group Hare lower than experimental group Ⅰ. At the same time point in each experimental group, FBG differences are statistically significant (P<0.05).2. mice fasting serum insulin (FIns) determination in each groupUsing chemiluminescent immunoassay measure FIns content. The results show that mice’s FIns content at different times after injury of experimental group Ⅰ、 experimental group Ⅱand the control group were higher than the healthy group,(the14th day of experimental group Ⅱ mice compared with healthy group, P>0.05, the remnant compared with healthy group, P<0.05). Comparison at the same time between the different groups, experimental group Ⅰ and experimental group Ⅱ FIns content were lower than the control group. And experimental group Ⅱ are lower than experimental group Ⅰ. At the same time point in each experimental group, FIns content differences are statistically significant (P<0.05). 3insulin resistance (IR) of mice of each groupHealthy mice’s mean HOMA-IR was4.75±0.48. The results show that mice’s mean HOMA-IR at different times after injury of experimental group Ⅰ experimental group Ⅱ and the control group were higher than the healthy group.(the14th day of experimental group Ⅱmice compared with healthy group, P>0.05, the remnant compared with healthy group, P<0.05). Comparison at the same time between the different groups, experimental groupⅠand experimental group Ⅱ mean HOMA-IR were lower than the control group. And experimental group Ⅱ are lower than experimental group Ⅰ. At the same time point in each experimental group, mean HOMA-IR differences are statistically significant (P<0.05). Comparison at different time points in the same group shows that scalded mice’s mean HOMA-IR on the3day reached the peak, then gradually reduced; within the same group at different time points,the HOMA-IR differences were statistically significant (P<0.05).4. Comparison of different points of serum TNF-a levels in each group of miceSerum TNF-α level in healthy mice is (364.10±46.13) ng/L. The results show that mice’s mean serum TNF-a level at different times after injury of experimental group Ⅰ、experimental group Ⅱ and the control group were higher than the healthy group. On the14day after injury, the experimental group Ⅱ mice’s serum TNF-α level were close to the level of the healthy group (the14th day of experimental group Ⅱ mice compared with healthy group, P>0.05, the remnant compared with healthy group, P<0.05). Comparison at the same time between the different groups, experimental group Ⅰand experimental group Ⅱ mice’s serum TNF-α level were lower than the control group. And experimental group Ⅱ are lower than experimental group Ⅰ. At the same time point in each experimental group, mean serum TNF-α level differences are statistically significant (P<0.05). Comparison at different time points in the same group shows that scalded mice’s mean serum TNF-α level on the1day reached the peak, then gradually reduced; within the same group at different time points,the serum TNF-α level differences were statistically significant (P<0.05).5. wound tissue NF-κB immunohistochemical staining results in each group mice The NF-κB expression positive rate of healthy mice skin tissue is (13.67±6.28)%. The results show that mice’s mean positive rate of wound tissue expression of NF-κB at different times after injury of experimental group Ⅰ、experimental group Ⅱ and the control group were higher than the healthy group. On the7day after injury, the experimental group Ⅱ mice’s positive rate of wound tissue expression of NF-κB were close to the level of the healthy group (the7th day of experimental group II mice compared with healthy group, P>0.05, the remnant compared with healthy group,P<0.05). Comparison at the same time between the different groups, experimental group Ⅰ and experimental group Ⅱ mice’s positive rate of wound tissue expression of NF-κB were lower than the control group. And experimental group Ⅱ are lower than experimental group Ⅰ. At the same time point in each experimental group, mean positive rate of wound tissue expression of NF-κB differences are statistically significant (P<0.05). Comparison at different time points in the same group shows that scalded mice’s mean positive rate of wound tissue expression of NF-κB on the1day reached the peak, then gradually reduced; within the same group at different time points,the positive rate of wound tissue expression of NF-κB differences were statistically significant (P<0.05).Conclusion1. Scalded mice’s FBG and FIns content of experimental group Ⅰ、experimental group Ⅱ and the control group were higher than the healthy group. It provide that mouse model of30%of the total body surface area (TBSA) Ⅲ°burns have been modeled successfully (biopsy confirmed), producing significant insulin resistance (IR).2. Scalded mice’s FBG and Fins content of experimental group Ⅰ、experimental group Ⅱ and the control group compare, differences were statistically significant (P <0.05). It provide that1,25-(OH)2VitD3can effectively control IR.3. Mice’s mean serum TNF-a levels at different times after injury of experimental group Ⅰ、experimental group Ⅱ and the control group compare in pares and different time points in the same group compare in pares, differences were statistically significant (P<0.05). Mice’s mean positive rate of wound tissue expression of NF-κB at different times after injury of experimental group I experimental group Hand the control group compare in pares and different time points in the same group compare in pares, differences were statistically significant (P<0.05).1,25-(OH)2VitD3can suppress mice’s mean of serum TNF-α levels and positive rate of wound tissue expression of NF-κB actively, thereby inhibit IR, improve and promote wound healing. |
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