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中华危重症医学杂志(电子版)

中华危重症医学杂志(电子版) ›› 2016, Vol. 09 ›› Issue (05) : 315 -320. doi: 10.3877/cma.j.issn.1674-6880.2016.05.006

所属专题： 文献；

论著

血必净对脓毒性急性肾损伤大鼠肾小管细胞凋亡和相关蛋白表达的影响
孙雪东1,†(), 严一核1, 刘芳2, 褚韦韦1, 张亦婷1, 应利君1   
  1. 1. 312000　浙江绍兴，绍兴市人民医院重症医学科
    2. 312000　浙江绍兴，绍兴市人民医院病理科
  • 收稿日期:2016-06-05 出版日期:2016-10-01
  • 通信作者: 孙雪东
  • 基金资助:
    浙江省绍兴市科技计划项目(2014B70066)

Effect of xuebijing injection on tubular apoptosis and related proteins expression in rat model of sepsis-induced acute kidney injury

Xuedong Sun1,†(), Yihe Yan1, Fang Liu2, Weiwei Chu1, Yiting Zhang1, Lijun Ying1   

  1. 1. Department of Intensive Medicine, Shaoxing People's Hospital, Shaoxing 312000, China
    2. Department of Pathology, Shaoxing People's Hospital, Shaoxing 312000, China
  • Received:2016-06-05 Published:2016-10-01
  • Corresponding author: Xuedong Sun
  • About author:
    Corresponding author: Sun Xuedong, Email:
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引用本文：

孙雪东, 严一核, 刘芳, 褚韦韦, 张亦婷, 应利君. 血必净对脓毒性急性肾损伤大鼠肾小管细胞凋亡和相关蛋白表达的影响[J]. 中华危重症医学杂志(电子版), 2016, 09(05): 315-320.

Xuedong Sun, Yihe Yan, Fang Liu, Weiwei Chu, Yiting Zhang, Lijun Ying. Effect of xuebijing injection on tubular apoptosis and related proteins expression in rat model of sepsis-induced acute kidney injury[J]. Chinese Journal of Critical Care Medicine(Electronic Edition), 2016, 09(05): 315-320.

目的

探讨早期应用血必净对脓毒性急性肾损伤大鼠肾小管细胞凋亡，及相关蛋白B细胞淋巴瘤2（Bcl-2）和Bcl-2相关X蛋白（Bax）表达的影响。

方法

54只雄性大鼠分为假手术组（Sham组）、脓毒症组（CLP组）、血必净组（CLP + XBJ组）三组，每组18只；以术后12、24和48 h作为时间观察点，将各组再分为3个亚组，每组6只。在各时间点检测大鼠的肌酐清除率（CrCl）、肾脏血流灌注量，并留取肾脏组织观察病理学变化，计算肾小管损伤评分，采用原位末端标记（TUNEL）法并通过计算积分光密度值（IOD）测定肾小管凋亡细胞，采用Western-blotting法测定肾脏髓质Bcl-2、Bax的变化。

结果

三组大鼠的CrCl和肾脏血流灌注量在术后各时间点差异均有统计学意义（F = 6.405、18.821，P < 0.05）；且CLP + XBJ组在术后48 h时CrCl和肾脏血流灌注量明显高于CLP组[（2.1 ± 0.5）ml/min/100 g vs.（1.1 ± 0.3）ml/min/100 g，（159 ± 38）BPU vs.（79 ± 32）BPU；P均< 0.05]。三组大鼠肾小管损伤评分比较，不同时间点差异存在统计学意义（F = 5.461，P < 0.05）；且CLP + XBJ组与CLP组比较，24、48 h时评分均有显著下降[（1.6 ± 0.5）vs.（2.8 ± 0.8），（1.8 ± 0.8）vs.（3.6 ± 0.6）；P均< 0.05]。三组大鼠各时间点凋亡细胞IOD值，差异存在统计学意义（F = 7.259，P < 0.05）；CLP + XBJ组各时间点的IOD值均明显小于CLP组[（26.6 ± 6.9）vs.（34.4 ± 5.0），（38.2 ± 5.3）vs.（48.0 ± 5.8），（37.6 ± 2.2）vs.（53.8 ± 6.7）；P均< 0.05]。同时，CLP + XBJ组能升高大鼠肾脏髓质Bcl-2的表达，抑制Bax的表达，维持Bcl-2/Bax的平衡，24、48 h时其Bcl-2[（1.30 ± 0.09）vs.（0.76 ± 0.09），（2.12 ± 0.38）vs.（0.51 ± 0.07）；P均< 0.05]和Bax[（1.19 ± 0.37）vs.（1.95 ± 0.90），（1.48 ± 0.15）vs.（2.69 ± 0.39）；P均< 0.05]的表达水平与脓毒症组相比差异均有统计学意义。

结论

早期应用血必净可以减少肾小管细胞在脓毒症进程中的凋亡，改善脓毒性急性肾损伤大鼠肾脏的病理学改变，维持Bcl-2/Bax的平衡，减轻脓毒症导致的肾功能损害。

关键词: 脓毒症, 细胞凋亡, 血必净, 急性肾损伤
Objective

To investigate the effect of early application of xuebijing in rat model of sepsis-induced acute kidney injury by observing tubular apoptosis, B cell lymphoma 2 (Bcl-2) and Bcl-2 associated X protein (Bax) expressions.

Methods

Fifty-four male SD rats were randomly divided into three groups: Sham group (n = 18), cecal ligation and puncture (CLP) group (n = 18), and xuebijing (CLP + XBJ) group (n = 18). After rat model establishment, 12, 24, 48 h were regarded as observation time points, and each group was accordingly divided into 3 subgroups (6 rats per subgroup). Creatine clearance (CrCl), renal blood perfusion unit, renal pathological changes and renal tubular injury score were measured; renal tubular cell apoptosis was determined by terminal-deoxynucleoitidyl transferase mediated nick end labeling (TUNEL) method and integrated optical density (IOD) was calculated. The expressions of Bcl-2 and Bax were detected by Western-blotting.

Results

The CrCl and renal blood perfusion unit in all three groups had significant differences at each time point (F = 6.405, 18.821; P < 0.05). The CrCl and renal blood perfusion unit in the CLP + XBJ group were significantly higher than that in the CLP group [(2.1 ± 0.5) ml/min/100 g vs. (1.1 ± 0.3) ml/min/100 g, (159 ± 38) BPU vs. (79 ± 32) BPU; all P < 0.05]. The renal tubular damage scores in all three groups had significant differences at each time point (F = 5.461, P < 0.05). Tubular damage scores decreased significantly in the CLP + XBJ group compared with the CLP group at 24, 48 h [(1.6 ± 0.5) vs. (2.8 ± 0.8), (1.8 ± 0.8) vs. (3.6 ± 0.6); all P < 0.05]. The IOD had a significant difference in all three groups at each time point (F = 7.259, P < 0.05). In addition, the IOD in the CLP + XBJ group was much lower than that in the CLP group at each time point [(26.6 ± 6.9) vs. (34.4 ± 5.0), (38.2 ± 5.3) vs. (48.0 ± 5.8), (37.6 ± 2.2) vs. (53.8 ± 6.7); all P < 0.05]. Meanwhile, xuebijing injection could increase Bcl-2 expression, inhibit Bax expression, and keep balance of Bcl-2/Bax ratio. There were significant differences of Bcl-2 [(1.30 ± 0.09) vs. (0.76 ± 0.09), (2.12 ± 0.38) vs. (0.51 ± 0.07); all P < 0.05] and Bax [(1.19 ± 0.37) vs. (1.95 ± 0.90), (1.48 ± 0.15) vs. (2.69 ± 0.39); all P < 0.05] in the CLP + XBJ group compared with the CLP group at 24 and 48 h.

Conclusion

Early application of xuebijing might improve renal dysfunction and the pathological changes, reduce the tubular cell apoptosis, keep balance of Bcl-2/Bax ratio and relieve renal impairment in rat model of sepsis-induced acute kidney injury.

Key words: Sepsis, Apoptosis, Xuebijing, Acute kidney injury
表1 三组大鼠各时间点CrCl和肾脏血流灌注量的比较（±s）
组别 只数 CrCl（ml·min-1·100 g-1） 血流灌注量(BPU)
12 h 24 h 48 h 12 h 24 h 48 h
Sham组 6 1.8 ± 0.3 1.9 ± 0.8 1.8 ± 0.6 185 ± 22 195 ± 20 189 ± 19
CLP组 6 1.8 ± 0.1 1.4 ± 0.4ab 1.1 ± 0.3abc 295 ± 52a 155 ± 30ab 79 ± 32abc
CLP + XBJ组 6 1.7 ± 0.4 2.0 ± 0.3bd 2.1 ± 0.5bd 249 ± 32a 175 ± 30bd 159 ± 38bcd
F值 ? 6.405 18.821
P值 ? < 0.05 < 0.05
图1 各组大鼠肾脏组织不同时间点病理染色图。注：a~c图为Sham组；d~f图为CLP组，24、48 h可见明显的肾小管退化、空泡变和坏死，以及管型或透明管型细胞并伴有大量单核细胞浸润；g~i图为CLP + XBJ组，24、48 h可见肾小管空泡变性减少，单核细胞浸润未见明显增加；Sham组：假手术组；CLP组：脓毒症组；CLP + XBJ组：血必净组（HE染色× 400）
表2 三组大鼠各时间点肾小管损伤评分（分，±s）
组别 只数 12 h 24 h 48 h
Sham组 6 0.5 ± 0.4 0.6 ± 0.4 0.6 ± 0.6
CLP组 6 1.4 ± 0.6a 2.8 ± 0.8ab 3.6 ± 0.6abc
CLP + XBJ组 6 1.2 ± 0.4a 1.6 ± 0.5abd 1.8 ± 0.8abcd
F值 ? ? 5.461 ?
P值 ? ? < 0.05 ?
图2 各组大鼠肾脏免疫荧光染色图。注：a~c图为Sham组；d~f图为CLP组，24 h后可见凋亡细胞逐步增加，48 h后显示大量簇状、成堆凋亡细胞；g~i图为CLP + XBJ组，24、48 h凋亡细胞稍增加；Sham组：假手术组；CLP组：脓毒症组；CLP + XBJ组：血必净组（TUNEL染色× 400）
表3 三组大鼠各时间点肾小管凋亡细胞的IOD比较（±s）
组别 只数 12 h 24 h 48 h
Sham组 6 10.4 ± 1.5 10.6 ± 2.1 10.4 ± 2.6
CLP组 6 34.4 ± 5.0a 48.0 ± 5.8ab 53.8 ± 6.7abc
CLP + XBJ组 6 26.6 ± 6.9ad 38.2 ± 5.3abd 37.6 ± 2.2abd
F值 ? ? 7.259 ?
P值 ? ? < 0.05 ?
表4 三组大鼠肾脏髓质各时间点Bcl-2的相对表达水平（±s）
组别 只数 12 h 24 h 48 h
Sham组 6 1.50 ± 0.07 1.47 ± 0.06 1.50 ± 0.10
CLP组 6 1.18 ± 0.22a 0.76 ± 0.09ab 0.51 ± 0.07abc
CLP + XBJ组 6 1.12 ± 0.03a 1.30 ± 0.09abd 2.12 ± 0.38abcd
F值 ? ? 42.702 ?
P值 ? ? < 0.05 ?
表5 三组大鼠肾脏髓质各时间点Bax的相对表达水平（±s）
组别 只数 12 h 24 h 48 h
Sham组 6 0.50 ± 0.12 0.56 ± 0.18 0.52 ± 0.21
CLP组 6 1.85 ± 0.92a 1.95 ± 0.90ab 2.69 ± 0.39abc
CLP + XBJ组 6 1.12 ± 0.07ad 1.19 ± 0.37abd 1.48 ± 0.15abcd
F值 ? ? 52.748 ?
P值 ? ? < 0.05 ?
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