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丹参酮Ⅰ在肝缺血再灌注损伤小鼠模型中的保护作用
DOI: 10.3969/j.issn.1001-5256.2021.01.021
利益冲突声明：本研究不存在研究者、伦理委员会成员、受试者监护人以及与公开研究成果有关的利益冲突，特此声明。
作者贡献声明：易小康、杜毅超、钱保林、付文广等负责课题设计，资料分析，撰写论文; 易小康、黄治伟、温剑等参与收集数据，修改论文; 李秋、付文广、温剑等负责拟定写作思路、指导撰写文章并最后定稿。
Protective effect of tanshinone I in a mouse model of hepatic ischemia-reperfusion injury
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摘要:
目的 探讨丹参酮Ⅰ(T-Ⅰ)在小鼠肝缺血再灌注损伤(HIRI)模型中的保护作用。 方法 C57BL/6J小鼠36只随机分为假手术(sham)组(n=6)、缺血再灌注(IR)组(n=6)、IR+T-Ⅰ(5 mg/kg)组(n=6)、IR+T-Ⅰ(10 mg/kg)组(n=6)、IR+T-Ⅰ(20 mg/kg)组(n=6)和IR+T-Ⅰ(40 mg/kg)组(n=6)，各组均腹腔注射给药，sham组与IR组注射等量溶剂橄榄油，IR+T-Ⅰ组每日给药1次，连续给药7 d, 末次给药2 h后建立70%的HIRI模型，再灌注6 h后收集血清及肝脏标本; 试剂盒检测血清ALT、AST水平，检测肝组织内超氧化物歧化酶(SOD)、丙二醛(MDA)、Caspase-3及还原型谷胱甘肽(GSH)指标; HE染色观察肝组织病理情况，TUNEL法检测肝细胞凋亡水平，免疫组化检测Caspase-3、血红素加氧酶-1(HO-1)蛋白表达水平。计量资料多组间比较采用单因素方差分析，进一步两两比较采用LSD-t检验。 结果 IR+T-Ⅰ(20 mg/kg)组的血清ALT[(192.48±23.67)U/L]、AST[(123.19±9.16)U/L]较IR组[ALT:(336.90±41.52)U/L, AST:(206.90±18.81)U/L]均显著下降(P值均＜0.01)，确定了20 mg/kg为最佳浓度; 与IR组[MDA：(3.48±0.95)μmol/mg; Caspase-3:(1.04±0.35)μmol/mg; SOD:(160.29±27.37)U/mg; GSH:(1.03±0.42)μmol/mg]比较，IR+T-Ⅰ(20 mg/kg)组的MDA[(1.34±0.21)μmol/mg]、Caspase-3[(0.69±0.97)μmol/mg]均显著降低(P值均＜0.05)，而SOD[(274.47±30.53)U/mg]及GSH[(2.12±0.27)μmol/mg]均明显升高(P值均＜0.05);HE染色显示，IR组肝小叶结构紊乱，肝细胞灶性或大面积变性坏死; 与IR组相比，IR+T-Ⅰ(20 mg/kg)组肝细胞坏死面积减小，肝组织结构基本完整; 免疫组化结果显示，与IR组比较，IR+T-Ⅰ(20 mg/kg)组的小鼠肝细胞凋亡数目明显减少，Caspase-3蛋白表达明显减少，HO-1蛋白表达明显增加。 结论 T-Ⅰ通过抑制肝脏氧化应激反应和肝细胞凋亡对小鼠HIRI中起到保护作用。 Abstract:Objective To investigate the protective effect of tanshinone I (T-I) on hepatic ischemia-reperfusion injury (HIRI) in mice. Methods A total of 36 C57BL/6J mice were randomly divided into sham-operation group, ischemia-reperfusion (IR) group, IR+T-I (5 mg/kg) group, IR+T-I (10 mg/kg) group, IR+T-I (20 mg/kg) group, and IR+T-I (40 mg/kg) group, with 6 mice in each group. Each group was given intraperitoneal injection. The mice in the sham-operation group and the IR group were injected with an equal volume of the solvent olive oil; the mice in the IR+T-I groups were administered once a day for 7 consecutive days, a model of 70% HIRI was established at 2 hours after the last administration, and serum and liver samples were collected after 6 hours of reperfusion. Related kits were used to measure the serum level of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) and the content of superoxide dismutase (SOD), malondialdehyde (MDA), caspase-3, and reduced glutathione (GSH) in liver tissue; HE staining was used to observe liver histopathology; the TUNEL method was used to measure the level of hepatocyte apoptosis; immunohistochemistry was used to measure the protein expression of caspase-3 and heme oxygenase-1 (HO-1). A one-way analysis of variance was used for comparison of continuous data between multiple groups, and the least significant difference t-test was used for further comparison between two groups. Results Compared with the IR group, the IR+T-I (20mg/kg) group had significant reductions in the serum levels of ALT (192.48±23.67 U/L vs 336.90±41.52 U/L, P < 0.01) and AST (123.19±9.16 U/L vs 206.90±18.81 U/L, P < 0.01), and thus 20 mg/kg was determined as the optimal concentration. Compared with the IR group, the IR+T-I (20 mg/kg) group had significant reductions in MDA (1.34±0.21 μmol/mg vs 3.48±0.95 μmol/mg, P < 0.05) and caspase-3 (0.69±0.97 μmol/mg vs 1.04±0.35 μmol/mg, P < 0.05) and significant increases in SOD (274.47±30.53 U/mg vs 160.29±27.37 U/mg, P < 0.05) and GSH (2.12±0.27 μmol/mg vs 1.03±0.42 μmol/mg, P < 0.05). HE staining showed that the IR group had disordered structure of hepatic lobules and focal or extensive degeneration and necrosis of hepatocytes; compared with the IR group, the IR+T-I (20 mg/kg) group had a reduction in the area of hepatocyte necrosis and a basically complete structure of the liver. Immunohistochemistry showed that compared with the IR group, the IR+T-I (20 mg/kg) group had significant reductions in the number of apoptotic hepatocytes and the protein expression of caspase-3 and a significant increase in the protein expression of HO-1. Conclusion T-I exerts a protective effect against HIRI in mice by inhibiting liver oxidative stress response and hepatocyte apoptosis. -
Key words:
- Reperfusion Injury /
- Oxidative Stress /
- TANSHINONE
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表 1 5组间ALT、AST水平的比较
指标 Sham组
(n=6)IR组
(n=6)IR+T-Ⅰ
(5 mg/kg)
(n=6)IR+T-Ⅰ
(10 mg/kg)
(n=6)IR+T-Ⅰ
(20 mg/kg)
(n=6)IR+T-Ⅰ
(40 mg/kg)
(n=6)F值 P值 ALT(U/L) 18.64±3.72 336.90±41.521) 287.09±18.47 208.83±29.112) 192.48±23.672) 220.47±19.802) 87.06 ＜0.01 AST(U/L) 34.59±12.71 206.90±18.811) 174.25±16.072) 145.78±8.392) 123.19±9.162) 137.09±22.072) 69.14 ＜0.01 注：1)与Sham组比较，P＜0.01; 2)与IR组比较，P＜0.05。 
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表 2 3组间MDA、SOD、GSH、Caspase-3水平的比较
指标 Sham组
(n=6)IR组
(n=6)IR+T-Ⅰ(20 mg/kg)组
(n=6)F值 P值 SOD(U/mg) 345.08±43.81 160.29±27.371) 274.47±30.532) 27.76 ＜0.01 MDA(μmol/mg) 0.86±0.17 3.48±0.951) 1.34±0.212) 31.15 ＜0.01 GSH(μmol/mg) 2.76±0.09 1.03±0.421) 2.12±0.272) 36.62 ＜0.01 Caspase-3(μmol/mg) 0.47±0.43 1.04±0.351) 0.69±0.972) 85.99 ＜0.01 注：1)与Sham组比较，P＜0.01; 2)与IR组比较，P＜0.05。
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