| Objective: In recent years, the incidence caused by over-training-induced acute kidney injury increased year by year, as a common diseasecaused by exercise has been more and more attention of scholars at home andabroad, but the pathogenesis of OTIAKI has not yet fully understood, and haslacked effective prevention and control measures. Our past studies also provedthat the cell apoptosis was the main pathogenesis of OTIAKI, but thesestudies about the signal transduction pathways of apoptosis caused byover-training-induced acute kidney injury were less. This animal model ofover-training-induced acute kidney injury was established by exhaustiveswimming, and we observe the expression of cytochrome C and Caspase-3inrenal tissue and the influence of cytochrome C and Caspase-3expression byanisodamine,to explore the mitochondria pathway of the renal cell apoptosisand the renal protection mechanism of anisodamine. The study providedOTIAKI with new prevention targets.Methods:(1) Select48healthy SD rats, weighing180-220g. The ratswere randomly divided into three groups: control group (CN, n=8),exhaustive swimming group (ES, n=24) and Anisodamine group (AD, n=16). The rat of ES swimed to exhaustive state and sacrificed atimmediately(ESI),6h (ES6h) and24h (ES24h). The rats of AD receivedintraperitoneal injection of Anisodamine at the dose of10mg/kg body weightat20minutes before swimming and then swimed to exhaustive condition. Therats of AD were sacrificed at6h (AD6h) and24h (AD24h) after exhaustiveswimming. The OTIAKI animal model was established using rats with onceexhaustive swimming with no burden.(2)The two kidneys were sampled afteranaesthesia using intraperitoneal injection of5%Chloral Hydrate (3ml/kg). The left kidney was left in an EP tube with liquid-nitrogen inside and then thetube with sample was store at-80℃for detection of cytochrome C andCaspase-3expressed using Western blot. The right kidney was mainly used forfixing tissue and paraffin section and placed in4%paraform of4℃forstorage.(3) The renal histopathology change were observed with lightmicroscope in HE staining.(4)The expression of cytochrome C and Caspase-3were detected by immunohistochemistry. And the results were analyzed withthe pathological image analysis system.(5)The expression of cytochrome Cand Caspase-3were detected by western blot analysis.(6) Statistical analysis:All statistical analysis of the experimental data were used SPSS13.0statisticalsoftware package, the results were expressed as mean±standarddeviation(X±S). Differences are compared using single factor analysis ofvariance (one-way ANOVA), between groups comparison using SNK method,the P<0.05was considered statistically significant.Results:(1)Rats after once exhaustive swimming with no burdenexhibited extreme fatigue with dim eyes, slouch, obtuse reaction to the outerstimulation, lying flat on the belly, standing instability, difficulty in activitiesand even bleeding in the bulbar conjunctiva in some severe ones. Two hoursafter exhaustive exercise, the rats in exhaustive group could move freely butthey usually exhibited poor state in eating and drinking. The rats inanisodamine group, however, could move freely and take normal diet only20min after exhaustive swimming.(2) No obvious changes in renal tissuestructure were detected in CN group and some light changes, such asdilatation of glomerular capsule and renal tubules, edema of renal proximaltubular epithelial cells and vacuolar degeneration, were found in ESI groupand ES6h group. However, rats with significantly changed renal tissuestructure were found in ES24h group. These changes including numerousdarkly stained cells and cell pyknosis in medulla and the juncture of cortexand medulla, infiltrating cells in interstitium and the irregular or evendeciduous brush border observed in renal tubular epithelial cells. The rats inAD group showed significantly slight in structure changes when compared to the corresponding sub-group of exhaustive group.(3) Immunohistochemicalresults：①t he expression of cytochrome C:most positive cells stainsed brownwere renal tubular epithelial cells and no positive ones were found inglomerulus and renal interstitial. Normal control group had a few expressionof cytochrome C, the expression in ESI, ES6h, ES24h was graduallyincreased, and reached the higher level in ES24h after exhaustion (p<0.05),the expression of ES were more than CN (p<0.05).The expression ofcytochrome C in AD group significantly decreased compared with the sameperiod exhaustive group (p<0.05).②the expression of Caspase-3: Normalcontrol group had weekly expression, but the expression of Caspase-3in ESI,ES6h, ES24h was gradually increased, and reached the higher level in ES24hafter exhaustive swimming (p<0.05), the expression of ES Caspase-3weremore than CN (p<0.05). The expression of Caspase-3in AD groupsignificantly decreased compared with the same period exhaustive group(p<0.05).(4) The results of Western Blotting:①the expression of cytochromeC: The expression of cytochrome C in CN (2.055±0.375) was the weakestthan that in ESI(2.665±0.329),ES6h(3.316±0.343)and ES24h (3.808±0.517)(P<0.05). The expression of cytochrome C in AD6h (2.475±0.336),AD24h(3.195±0.268) were significantly decreased compared with the same periodexhaustive group (p<0.05).②the expression of Caspase-3: The expression ofCaspase-3in CN (0.140±0.022) was the weakest than that in ESI (0.295±0.019), ES6h (0.636±0.041) and ES24h(1.150±0.017)(p<0.05).The expressionof Caspase-3in AD6h(0.384±0.022)， AD24h(0.905±0.027) significantlydecreased compared with the same period exhaustive group (p<0.05).Conclusions:(1)Cytochrome C and Caspase-3together involve in renalcell apoptosis, suggesting that the mitochondria apoptosis pathways is theimportant signaling pathways;(2)Anisodamine can reduce the expression ofcytochrome C and Caspase-3. It prompt that anisodamine can inhibit kidneytissues cell apoptosis by suppressing the mitochondria pathways to play aprotective role in renal tissue. |
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