| ObjectiveTo investigate and explore the effect of hydrogen-rich saline on acute kidney injury in severely burned rats and its detailed regulation on burn-induced renal inflammation.Methods56 SD rats were randomly divided into 7 groups according to the random number table method,as the sham group(n=8),the burn group(Burn,n=8 at each time point,total of 24),the hydrogen-rich saline group(8 at each time point,total of 24).The rats in sham group was only treated by 20 ℃ water bath for 15 s,and the rats in bum group and hydrogen-rich saline received 100℃ water bath for 15 s to build a rat model of severe burn(about 30%of TBSA),and the sodium lactate solution(LRS)was given by intraperitoneal injection of 4 ml/kg/TBSA at 6 hours after the injury.After injury,saline at the dose of 10 ml/kg were intraperitoneally injected to the rats in the sham group and the burn group immediately after injury for only one time.The rats in the hydrogen-rich saline group were given a one-time injection of hydrogen rich water(10 ml/kg)immediately after the injury.1)the rats in the sham group were sacrificed at 72 h after injury,and those in the burn and hydrogen rich water group were sacrificed at 6,24 and 72 h after the injury.After the anesthesia,the blood from abdominal aorta was collected respectively.The serum creatinine and blood urea nitrogen were detected by the clinical blood biochemical analyzer.After the blood samples were taken,the rats were sacrificed and the bilateral kidney was placed in the 4%polyformaldehyde or-80 ℃ refrigerator respectively for subsequent histology or molecular biology.2)the renal tissue specimens of each group were stained with hematoxylin and eosin(HE),and 4 tissue sections were randomly selected to observe histopathological changes and renal tubular injury scores to evaluate renal tissue injury.The changes of renal tissue inflammatory indexes were evaluated by immunohistochemical staining,RT-PCR,and western blot respectively.Kruskal-Wallis test was used for the count data followed by a Dunn ’s post-hoc test,and the measurement data were analyzed by one-way ANOVA followed by a Bonferroni post-hoc test.Results(1)The renal tubular structure was complete in renal tissue of the sham group,showing no inflammatory cell infiltration,no cell degeneration and necrosis,no disintegrating necrotic cell fragments in the lumen,and the renal tubule injury score was lower(0.10±0.31).In the burn group,the changes in the formation,deformation and aggregation of broken protein in the tubular structure were observed in all the phase points(6,24,72 h)after injury.The renal tubule injury scores were significantly higher than those in the sham group(2.19±0.70,2.06±0.81,2.44±0.97,P value were less than 0.05).The renal tubular structure injury in the renal tissue of the rats in the hydrogen-rich saline group was significantly lower than that in the burn group,and the renal tubular injury scores of each time point(6,24,72 h)after the injury were significantly lower than those in the burn group(1.35±0.84,1.25±0.70,1.58±0.94,P value were less than 0.05).(2)the levels of blood creatinine(33.75±5.05 μmol/L,37.504.35 μmol/L,26.81±5.31 μmol/L)and blood urea nitrogen(10.00±2.11 mmol/L,13.48±2.69 mmol/L,9.78±2.71 mmol/L)at 6,24 and 72 h after burn injury were significantly higher than those of the sham group(creatinine 18.56±4.46 μmol/L,urea nitrogen 6.15±0.82 mmol/L,P value were less than 0.05).The blood creatinine(22.0±05.80 μmol/L,25.75±3.96 μmol/L,20.50±3.92 μmol/L)at 6,24 and 72 h after insult in the hydrogen-rich saline group was significantly lower than that of the burn group(P value were less than 0.05),and the blood urea nitrogen at 24 and 72 h after injury(9.07±1.14 mmol/L,7.72±1.13 mmol/L)was less than those of the burn group(P was less than 0.05),while the changes of blood urea nitrogen at 6 h in hydrogen-rich saline group was not obvious compared to the sham group(7.60±1.45 mmol/L,P value is 0.05).(3)the renal mRNA levels of inflammatory factors:TNF-alpha(4.67±1.26,10.73±2.12,13.30±0.73)and IL-1 beta(12.19±1.37,19.18±2.91,8.53±1.40),IL-6(15.46±2.73,12.87±1.97,14.00±0.99)at 6,24 and 72 h after injury in the burn group were significantly higher than those of the sham group(1.00±0.38,1.00±0.19,1.00±0.10)(P value were less than 0.05).The expression level of TNF-a mRNA in hydrogen-rich saline group was not significantly changed at 6 h after injury(5.25±0.92,P value was greater than 0.05),and the mRNA levels of TNF-α at 24 and 72 h in the hydrogen-rich saline groups were significantly lower than that of the sham group(4.93±1.18,2.75±0.48,P value were less than 0.05).The mRNA level of IL-1β and IL-6 at 6,24,72 h in the hydrogen-rich saline group was significantly lower than those in the corresponding phase of the burn group(IL-1β:1.78±0.19,1.59±0.48,2.14±0.20,IL-6:10.07±1.62,1.42±0.43,0.84±0.05,P values were less than 0.05).(4)The expression of high mobility group protein 1(HMGB1)of renal tissue in 6,24,72 h burns group was significantly increased(1.19±0.07,1.00±0.06,0.80±0.05,P value were less than 0.05),compared with the sham group(0.39±0.02).Besides,the expression of HMGB1 at 6,24 and 72 h in the hydrogen-rich saline group was significantly lower than that of the burn group(0.35±0.08,0.47±0.06,0.42±0.06,P value were less than 0.05),but there was no significant difference between these time points and the sham group(P value were greater than 0.05).ConclusionsHydrogen-rich saline showed significant benefit to the progression of AKI in severely burned rats;the protective effect is mediated by the regulation of HMGB1 on release of inflammatory cytokines in renal tissues. |
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