Characteristics of renal oxidative stress injuries in rats with high-voltage electric burns and the intervention effects of breviscapine
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摘要:
目的 探讨高压电烧伤后大鼠肾脏氧化应激损伤的特点及灯盏花素的干预作用。 方法 该研究为实验研究。将160只8~10周龄雄性SD大鼠按随机数字表法分为假伤组、电烧伤组、盐水组、低灯盏花素组、中灯盏花素组、高灯盏花素组,其中假伤组、电烧伤组各60只大鼠,余4组各10只大鼠。将假伤组和电烧伤组大鼠按伤后0 h（即刻）、8 h、24 h、48 h、72 h和1周,分为每个时间点10只。将假伤组大鼠不通电致假伤,余5组大鼠致高压电烧伤。伤后对假伤组和电烧伤组大鼠不行干预,对盐水组、低灯盏花素组、中灯盏花素组、高灯盏花素组大鼠分别另经腹腔按5 mL/kg剂量注射生理盐水及0.4、1.6、4.0 g/L灯盏花素,每24小时1次,至伤后72 h。模型制作成功后14只大鼠死亡,包括伤后24 h、48 h、72 h、1周电烧伤组各1、2、2、1只,伤后72 h盐水组、低灯盏花素组、中灯盏花素组、高灯盏花素组各4、1、2、1只。取6组大鼠的肾脏组织,称重后计算肾/体比。取假伤组,伤后8 h、24 h、48 h、72 h和1周的电烧伤组,伤后72 h的盐水组、低灯盏花素组、中灯盏花素组、高灯盏花素组各4只大鼠的左肾上极组织,行苏木精-伊红染色后采用半定量病理评分系统行肾小管与肾间质损伤评分。取6组大鼠下腔静脉血,采用肌氨酸氧化酶法检测血清肌酐水平,采用脲酶法检测血清尿素氮水平；取6组大鼠右肾皮质,采用钼酸铵法检测肾组织上清液中过氧化氢酶（CAT）活力,采用酶联免疫吸附测定法检测肾组织上清液中晚期氧化蛋白产物（AOPP）水平和Klotho蛋白水平。 结果 伤后8 h、48 h、72 h、1周,电烧伤组大鼠肾/体比均明显高于假伤组（t值分别为-0.52、-3.75、-4.05、-2.25,P<0.05）。伤后72 h,与电烧伤组、盐水组、低灯盏花素组、中灯盏花素组比较,高灯盏花素组大鼠肾 / 体比明显降低（P值均<0.05）。与假伤组比较,电烧伤组大鼠伤后48 h、72 h、1周肾小管与肾间质损伤评分均明显升高（P<0.05）；与电烧伤组伤后8、24 h比较,电烧伤组大鼠伤后1周肾小管与肾间质损伤评分明显升高（P值均<0.05）。伤后72 h,5组电烧伤大鼠肾小管与肾间质损伤评分相近（P>0.05）。伤后8 h、24 h、48 h、72 h、1周,电烧伤组大鼠血清肌酐和尿素氮水平均明显高于假伤组（Z值分别为-2.00、-2.37、-2.62、-2.67、-3.67和-2.34、-3.11、-3.43、-3.11、-3.51,P<0.05）。与电烧伤组伤后0 h比较,电烧伤组大鼠伤后72 h、1周血清肌酐水平均明显升高（P<0.05）；与电烧伤组伤后8 h比较,电烧伤组大鼠伤后72 h、1周血清肌酐水平均明显升高（P<0.05）；与电烧伤组伤后24 h比较,电烧伤组大鼠伤后1周血清肌酐水平明显升高（P<0.05）。伤后72 h,5组电烧伤大鼠血清肌酐水平相近（P>0.05）；与电烧伤组比较,低灯盏花素组、中灯盏花素组、高灯盏花素组大鼠血清尿素氮水平均明显降低（P<0.05）；与盐水组比较,中灯盏花素组、高灯盏花素组大鼠血清尿素氮水平均明显降低（P<0.05）。伤后48 h、72 h、1周,电烧伤组大鼠肾组织上清液中CAT活力均明显低于假伤组（Z值分别为-2.22、-2.13、-3.51,P<0.05）；伤后8 h、24 h、48 h、72 h、1周,电烧伤组大鼠肾组织上清液中AOPP水平均明显高于假伤组（Z值分别为-2.00、-3.15、-2.71、-2.04、-2.33,P<0.05）；伤后0 h~1周,假伤组和电烧伤组大鼠肾组织上清液中Klotho蛋白水平均相近（P>0.05）。与电烧伤组伤后0 h比较,电烧伤组大鼠伤后72 h和1周肾组织上清液中CAT活力及伤后48 h、72 h、1周肾组织上清液中Klotho蛋白水平均明显降低（P<0.05）；与电烧伤组伤后8 h比较,电烧伤组大鼠伤后72 h和1周肾组织上清液中CAT活力及伤后48 h、72 h、1周肾组织上清液中Klotho蛋白水平均明显降低（P<0.05）；与电烧伤组伤后24 h比较,电烧伤组大鼠伤后72 h和1周肾组织上清液中CAT活力均明显降低（P<0.05）；与电烧伤组伤后48 h比较,电烧伤组大鼠伤后1周肾组织上清液中CAT活力明显降低（P<0.05）。伤后72 h,5组电烧伤大鼠肾组织上清液中Klotho蛋白水平相近（P>0.05）；与电烧伤组[14.6（12.6,23.6）U/mgprot]比较,低灯盏花素组、中灯盏花素组、高灯盏花素组大鼠肾组织上清液中CAT活力[20.5（18.0,39.8）、24.9（14.7,28.9）、28.0（21.9,39.1）U/mgprot]均明显升高（P<0.05）；与盐水组[15.7（13.7,25.6）U/mgprot]比较,中灯盏花素组、高灯盏花素组大鼠肾组织上清液中CAT活力均明显升高（P<0.05）；与低灯盏花素组比较,高灯盏花素组大鼠肾组织上清液中CAT活力明显升高（P<0.05）；与电烧伤组及盐水组比较,中灯盏花素组、高灯盏花素组大鼠肾组织上清液中AOPP水平均明显降低（P<0.05）。 结论 高压电烧伤后大鼠肾脏发生氧化应激损伤,且随时间的延长呈进行性加重,应用灯盏花素可减轻高压电烧伤大鼠肾脏氧化应激损伤。 Abstract:Objective To explore the characteristics of renal oxidative stress injuries in rats with high-voltage electric burns and the intervention effects of breviscapine. Methods This study was an experimental study. One hundred and sixty 8-10-week-old male Sprague Dawley rats were divided into sham injury group, electric burn group, saline group, low breviscapine group, middle breviscapine group, and high breviscapine group, with 60 rats in each of the sham injury group and electric burn group, 10 rats in each of the other 4 groups, respectively. The rats in sham injury group and electric burn group were divided into 10 rats at each time point, including post injury hour (PIH) 0 (immediately), 8, 24, 48, and 72, and post injury week (PIW) 1. The rats in sham injury group were not conducted with electrical current to cause sham injury. The rats in the other 5 groups were caused high-voltage electric burns. The rats in sham injury group and electric burn group were not treated after injury. The rats in saline group, low breviscapine group, middle breviscapine group, and high breviscapine group were intraperitoneally injected with 5 mL/kg normal saline or 0.4, 1.6, and 4.0 g/L breviscapine, repeated every 24 h until PIH 72. After the model was successfully made, 14 rats died, including 1, 2, 2, and 1 rat (s) at PIH 24, 48, and 72 and PIW 1 in electric burn group, 4, 1, 2, and 1 rat (s) at PIH 72 in saline group, low breviscapine group, middle breviscapine group, and high breviscapine group, respectively. The kidney tissue collected from rats in the 6 groups was weighed and the kidney/body weight ratio was calculated. The left upper pole tissue of kidney was collected from each 4 rats in sham injury group, and in electric burn group at PIH 8, 24, 48, and 72 and PIW 1, and in saline group, low breviscapine group, middle breviscapine group, and high breviscapine group at PIH 72. The renal tubular and renal interstitial injury was evaluated by a semi-quantitative histological scoring system after hematoxylin-eosin staining. The inferior vena cava blood samples were collected from rats in the 6 groups to measure the serum creatinine levels via sarcosine oxidase method, and serum urea nitrogen levels via urease method. The right renal cortices were collected from rats in the 6 groups to measure the catalase (CAT) activity in the supernatant of renal tissue via molybdic acid method, and the levels of advanced oxidation protein product (AOPP) and Klotho protein in the supernatant of renal tissue via enzyme-linked immunosorbent assay. Results At PIH 8, 48, and 72 and PIW 1, the kidney/body weight ratios of rats in electric burn group were significantly higher than those in sham injury group (with t values of -0.52, -3.75, -4.05, and -2.25, respectively, P<0.05). At PIH 72, compared with those in electric burn group, saline group, low breviscapine group, and middle breviscapine group, the kidney/body weight ratio of rats in high breviscapine group was significantly decreased (with P values all <0.05). Compared with those in sham injury group, the renal tubular and renal interstitial injury scores of rats in electric burn group at PIH 48 and 72 and PIW 1 were significantly increased (P<0.05). Compared with those in electric burn group at PIH 8 and 24, the renal tubular and renal interstitial injury score of rats in electric burn group at PIW 1 was significantly increased (with P values all <0.05). At PIH 72, the renal tubular and renal interstitial injury scores of rats in the 5 groups of rats with electric burns were similar (P>0.05). At PIH 8, 24, 48, and 72 and PIW 1, the levels of serum creatinine and serum urea nitrogen of rats in electric burn group were significantly higher than those in sham injury group (with Z values of -2.00, -2.37, -2.62, -2.67, -3.67, -2.34, -3.11, -3.43, -3.11, and -3.51, respectively, P<0.05). Compared with that in electric burn group at PIH 0, the levels of serum creatinine of rats in electric burn group at PIH 72 and PIW 1 were significantly increased (P<0.05). Compared with that in electric burn group at PIH 8, the levels of serum creatinine of rats in electric burn group at PIH 72 and PIW 1 were significantly increased (P<0.05). Compared with that in electric burn group at PIH 24, the level of serum creatinine of rats in electric burn group at PIW 1 was significantly increased (P<0.05). At PIH 72, the levels of serum creatinine of rats in the 5 groups of rats with electric burns were similar (P>0.05). Compared with that in electric burn group, the levels of serum urea nitrogen of rats in low breviscapine group, middle breviscapine group, and high breviscapine group were significantly decreased (P<0.05). Compared with that in saline group, the levels of serum urea nitrogen in middle breviscapine group and high breviscapine group were significantly decreased (P<0.05). At PIH 48 and 72 and PIW 1, the CAT activities in the supernatant of renal tissue of rats in electric burn group were significantly lower than those in sham injury group (with Z values of -2.22, -2.13, and -3.51, respectively, P<0.05). At PIH 8, 24, 48, and 72 and PIW 1, the levels of AOPP in the supernatant of renal tissue of rats in electric burn group were significantly higher than those in sham injury group (with Z values of -2.00, -3.15, -2.71, -2.04, and -2.33, respectively, P<0.05). At PIH 0-PIW 1, the levels of Klotho protein in the supernatant of renal tissue of rats in sham injury group and electric burn group were all similar (P>0.05). Compared with that in electric burn group at PIH 0, the CAT activities in the supernatant of renal tissue of rats in electric burn group at PIH 72 and PIW 1 and the levels of Klotho protein in the supernatant of renal tissue of rats in electric burn group at PIH 48 and 72 and PIW 1 were significantly decreased (P<0.05). Compared with that in electric burn group at PIH 8, the CAT activities in the supernatant of renal tissue of rats in electric burn group at PIH 72 and PIW 1 and the levels of Klotho protein in the supernatant of renal tissue of rats in electric burn group at PIH 48 and 72 and PIW 1 were significantly decreased (P<0.05). Compared with that in electric burn group at PIH 24, the CAT activities in the supernatant of renal tissue of rats in electric burn group at PIH 72 and PIW 1 were significantly decreased (P<0.05). Compared with that in electric burn group at PIH 48, the CAT activity in the supernatant of renal tissue of rats in electric burn group at PIW 1 was significantly decreased (P<0.05). At PIH 72, the levels of Klotho protein in the supernatant of renal tissue of rats in the 5 groups of rats with electric burns were similar (P<0.05). Compared with 14.6 (12.6, 23.6) U/mgprot in electric burn group, the CAT activities in the supernatant of renal tissue of rats in low breviscapine group (20.5 (18.0, 39.8) U/mgprot), middle breviscapine group (24.9 (14.7, 28.9) U/mgprot), and high breviscapine group (28.0 (21.9, 39.1) U/mgprot) were significantly increased (P<0.05). Compared with 15.7 (13.7, 25.6) U/mgprot in saline group, the CAT activities in the supernatant of renal tissue of rats in middle breviscapine group and high breviscapine group were significantly increased (P<0.05). Compared with that in low breviscapine group, the CAT activity in the supernatant of renal tissue of rats in high breviscapine group was significantly increased (P<0.05). Compared with those in electric burn group and saline group, the levels of AOPP in the supernatant of renal tissue of rats in middle breviscapine group and high breviscapine group were significantly decreased (P<0.05). Conclusions After high-voltage electric burns, oxidative stress injury occur in the kidneys of rats, which is aggravated with time extension. Breviscapine can alleviate oxidative stress injuries in the kidneys of rats with high-voltage electric burns. -
Key words:
- Burns, electric /
- Oxidative stress /
- Kidney /
- Advanced oxidation protein products /
- Catalase /
- Breviscapine
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李聪颖：酝酿、设计和实施实验,采集和整理数据,统计学分析,撰写和修改论文；赵学刚、郝嘉文：酝酿、设计和实施实验,统计学分析及修改论文；盖晨阳、鲁梦远、张晶：实施实验,采集数据及修改论文；张庆富：指导研究,修改论文,经费支持；冯建科、屠丽红：指导研究所有作者均声明不存在利益冲突
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图 1 6组大鼠伤后各时间点肾脏组织病理学变化 苏木精-伊红×200。1A.假伤组大鼠肾小管上皮细胞排列整齐、大小一致、染色均匀、界限清楚,未见水肿征象,肾间质中未见炎症细胞浸润；1B、1C、1D、1E、1F.分别为电烧伤组伤后8 h、24 h、48 h、72 h、1周情况,相较于图1A,图1B~1F出现不同程度肾小球系膜细胞增生和基质增多、肾小管上皮细胞坏死脱落、肾间质炎症细胞浸润；1G、1H、1I、1J.分别为盐水组、低灯盏花素组、中灯盏花素组、高灯盏花素组伤后72 h情况,相较于图1E、1G,图1H~1J肾小球系膜细胞增生和基质增多、肾小管上皮细胞坏死脱落、肾间质炎症细胞浸润程度减轻
注：假伤组大鼠仅不通电致假伤,电烧伤组、盐水组、低灯盏花素组、中灯盏花素组、高灯盏花素组大鼠均致高压电烧伤,其中电烧伤组大鼠不进行药物干预,盐水组、低灯盏花素组、中灯盏花素组、高灯盏花素组大鼠分别经腹腔注射生理盐水或低、中、高浓度灯盏花素
Table 1. 2组大鼠伤后各时间点肾/体比比较（%,
组别 0 h 8 h 24 h 48 h 72 h 1周 假伤组 0.79±0.07 0.76±0.03 0.79±0.04 0.78±0.04 0.81±0.04 0.82±0.10 电烧伤组 0.84±0.08 0.87±0.06a 0.83±0.06 0.87±0.05a 0.93±0.08a 0.95±0.14a 注：假伤组大鼠不通电致假伤,各时间点样本数均为10；电烧伤组大鼠致高压电烧伤,伤后0 h（即刻）、8 h、24 h、48 h、72 h和1周样本数分别为10、10、9、8、8、9；处理因素主效应,F=43.43,P<0.001；时间因素主效应,F=3.88,P=0.003；两者交互作用,F=1.09,P=0.370；与假伤组比较,aP<0.05 
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Table 2. 2组大鼠伤后各时间点血清肌酐和尿素氮水平比较[M（min,max）]
组别与指标 0 h 8 h 24 h 48 h 72 h 1周 假伤组 肌酐（μmol/L） 46（35,64） 45（33,63） 45（31,63） 51（31,66） 46（30,64） 47（30,59） 尿素氮（mmol/L） 9.0（6.4,11.4） 8.6（5.0,10.5） 7.9（5.2,11.4） 8.7（7.0,11.4） 8.1（6.4,12.6） 8.2（4.1,12.6） 电烧伤组 肌酐（μmol/L） 55（37,79） 55（45,80）a 59（54,88）a 66（57,89）a 78（7,95）abc 74（62,102）abcd 尿素氮（mmol/L） 10.1（6.7,30.5） 13.7（6.8,20.4）a 12.6（9.3,44.4）a 16.3（10.9,30.9）a 13.9（8.9,45.6）a 18.8（11.2,37.4）a 注：假伤组大鼠不通电致假伤,各时间点样本数均为10；电烧伤组大鼠致高压电烧伤,伤后0 h（即刻）、8 h、24 h、48 h、72 h和1周样本数分别为10、10、9、8、8、9；与假伤组比较,aP<0.05；与电烧伤组伤后0 h比较,bP<0.05；与电烧伤组伤后8 h比较,cP<0.05；与电烧伤组伤后24 h比较,dP<0.05
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Table 3. 5组电烧伤大鼠伤后72 h血清肌酐和尿素氮水平比较[M（min,max）]
组别 样本数 肌酐（μmol/L） 尿素氮（mmol/L） 电烧伤组 8 78（7,95） 13.9（8.9,45.6） 盐水组 6 80（18,91） 12.5（8.9,30.5） 低灯盏花素组 9 74（6,92） 10.5（7.0,11.9）a 中灯盏花素组 8 61（50,95） 8.6（7.3,16.9）ab 高灯盏花素组 9 57（43,89） 8.7（7.5,13.0）ab H值 8.97 15.37 P值 0.062 0.004 注：5组大鼠均致高压电烧伤,其中电烧伤组大鼠不进行药物干预,盐水组、低灯盏花素组、中灯盏花素组、高灯盏花素组大鼠分别经腹腔注射生理盐水或低、中、高浓度灯盏花素；H值、P值为组间各指标总体比较所得；与电烧伤组比较,aP<0.05；与盐水组比较,bP<0.05
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Table 4. 2组大鼠伤后各时间点肾组织上清液中CAT活力、AOPP水平及Klotho蛋白水平比较[M（min,max）]
组别与指标 0 h 8 h 24 h 48 h 72 h 1周 假伤组 CAT活力（U/mgprot） 36.2（17.9,109.1） 40.0（13.7,94.6） 37.5（3.6,124.4） 44.6（15.1,61.5） 38.5（3.6,140.2） 43.0（14.7,120.9） AOPP水平（μmol/L） 43.8（7.2,117.4） 44.4（16.3,92.4） 50.5（25.7,60.1） 47.2（38.3,74.4） 48.6（36.5,96.0） 46.3（8.9,92.4） Klotho蛋白水平（ng/mL） 1.8（0.3,3.5） 2.2（0.3,3.6） 1.4（0.1,5.5） 1.8（0.2,4.1） 1.8（0.3,5.4） 1.6（0,7.8） 电烧伤组 CAT活力（U/mgprot） 45.9（9.7,84.9） 47.2（17.3,89.6） 31.8（18.0,43.6） 27.3（11.7,35.0）a 14.6（12.6,23.6）abcd 9.0（3.9,21.9）abcde AOPP水平（μmol/L） 53.1（42.4,88.7） 72.7（38.5,127.3）a 64.9（52.7,116.3）a 71.3（46.8,97.6）a 76.0（32.6,88.7）a 75.2（55.9,92.5）a Klotho蛋白水平（ng/mL） 1.8（1.1,3.5） 2.3（1.5,2.8） 1.8（1.2,2.2） 1.2（0.6,1.8）bc 0.7（0.5,2.0）bc 0.7（0.2,2.7）bc 注：假伤组大鼠不通电致假伤,各时间点样本数均为10；电烧伤组大鼠致高压电烧伤,伤后0 h（即刻）、8 h、24 h、48 h、72 h和1周样本数分别为10、10、9、8、8、9；CAT为过氧化氢酶,AOPP为晚期氧化蛋白产物；与假伤组比较,aP<0.05；与电烧伤组伤后0 h比较,bP<0.05；与电烧伤组伤后8 h比较,cP<0.05；与电烧伤组伤后24 h比较,dP<0.05；与电烧伤组伤后48 h比较,eP<0.05
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Table 5. 5组电烧伤大鼠伤后72 h肾组织上清液中CAT活力、AOPP水平及Klotho蛋白水平比较[M（min,max）]
组别 样本数 CAT活力（U/mgprot） AOPP水平（μmol/L） Klotho蛋白水平（ng/mL） 电烧伤组 8 14.6（12.6,23.6） 76.0（32.6,88.7） 0.7（0.5,2.0） 盐水组 6 15.7（13.7,25.6） 75.7（47.0,82.9） 0.8（0.7,1.2） 低灯盏花素组 9 20.5（18.0,39.8）a 59.0（46.9,82.9） 0.7（0.5,1.2） 中灯盏花素组 8 24.9（14.7,28.9）ab 54.1（48.8,76.6）ab 0.7（0.2,0.9） 高灯盏花素组 9 28.0（21.9,39.1）abc 52.7（46.8,71.6）ab 0.7（0.2,0.9） H值 23.90 13.00 4.31 P值 <0.001 0.011 0.366 注：5组大鼠均致高压电烧伤,其中电烧伤组大鼠不进行药物干预,盐水组、低灯盏花素组、中灯盏花素组、高灯盏花素组大鼠分别经腹腔注射生理盐水或低、中、高浓度灯盏花素；CAT为过氧化氢酶,AOPP为晚期氧化蛋白产物；H值、P值为组间各指标总体比较所得；与电烧伤组比较,aP<0.05；与盐水组比较,bP<0.05；与低灯盏花素组比较,cP<0.05
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