Pathological Mechanism of HSOS in Rats Induced by Different Doses of Monocrotaline

Si-qi GAO; Zhun XIAO; Ya-dong FU; Yong-hong HU; Gao-feng CHEN; Xiao-ning WANG; Wei LIU; Jia-mei CHEN; Ping LIU

doi:10.13422/j.cnki.syfjx.20220823

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Pathological Mechanism of HSOS in Rats Induced by Different Doses of Monocrotaline

更新时间：2022-05-18

- Pathological Mechanism of HSOS in Rats Induced by Different Doses of Monocrotaline
- Chinese Journal of Experimental Traditional Medical Formulae Vol. 28, Issue 12, Pages: 48-60(2022)
- 作者机构：
  
  1.上海中医药大学附属曙光医院，上海市中医药研究院肝病研究所，上海 201203
  2.上海中医药大学交叉科学研究院，上海 201203
  3.肝肾疾病病证教育部重点实验室，上海市中医临床重点实验室，上海 201203
- 作者简介：
- 基金信息：
- DOI：10.13422/j.cnki.syfjx.20220823
  CLC： R22;R242;R2-031;R285.5
- Received：07 December 2021，
  
  Published Online：15 February 2022，
  
  Published：20 June 2022
- 稿件说明：
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高思琦,肖准,付亚东等.野百合碱诱导大鼠肝窦阻塞综合征模型的病理机制[J].中国实验方剂学杂志,2022,28(12):48-60.

GAO Si-qi,XIAO Zhun,FU Ya-dong,et al.Pathological Mechanism of HSOS in Rats Induced by Different Doses of Monocrotaline[J].Chinese Journal of Experimental Traditional Medical Formulae,2022,28(12):48-60.
高思琦,肖准,付亚东等.野百合碱诱导大鼠肝窦阻塞综合征模型的病理机制[J].中国实验方剂学杂志,2022,28(12):48-60. DOI： 10.13422/j.cnki.syfjx.20220823.

GAO Si-qi,XIAO Zhun,FU Ya-dong,et al.Pathological Mechanism of HSOS in Rats Induced by Different Doses of Monocrotaline[J].Chinese Journal of Experimental Traditional Medical Formulae,2022,28(12):48-60. DOI： 10.13422/j.cnki.syfjx.20220823.

摘要

目的

观察不同剂量野百合碱（MCT）诱导大鼠肝窦阻塞综合征（HSOS）的病理变化，探讨造模剂量-时间致肝损伤的病理特征，并解析其部分作用机制。

方法

将72只雄性SD大鼠随机分为正常组（

12），MCT低、中、高剂量组（每组

=20）。MCT低、中、高剂量组分别采用MCT 80、120、160 mg·kg

-1

灌胃一次制备模型，各剂量组大鼠于造模48、120 h处死取材。观测各组大鼠的存活率，检测大鼠体质量、肝质量与血清肝功能变化，采用扫描电镜、苏木素-伊红（HE）染色及天狼星红（SR）染色观察肝组织病理变化，微板法检测肝组织匀浆谷胱甘肽S转移酶（GST）、总超氧化物歧化酶（T-SOD）活性、丙二醛（MDA）含量，实时荧光定量聚合酶链式反应（Real-time PCR）、蛋白免疫印迹法（Western blot）和免疫组化检测肝组织相关指标的表达。

结果

与正常组比较，MCT各组血清丙氨酸氨基转移酶/天冬氨酸氨基转移酶（ALT/AST）活性随造模剂量增加而升高（

0.05，

0.01）；随造模时间增加，MCT低剂量组血清ALT和AST活性均下降（

0.01），MCT中、高剂量组ALT和AST活性均显著升高（

0.01）。HE染色显示MCT低、中、高剂量组肝组织肝细胞坏死、炎性细胞浸润及红细胞淤积，电镜下可见肝窦内皮窗孔扩大，“筛状”结构消失，其损伤程度随剂量提高而加重。与正常组比较，MCT低、中、高剂量组肝窦内皮细胞标志物CD44表达减少（

0.05，

0.01）。SR染色显示造模48 h MCT各组未见阳性染色，120 h MCT各组可见汇管区及肝窦胶原沉积。与正常组比较，MCT各剂量组肝组织MDA含量及GST活性增加，T-SOD活性降低，以MCT中、高剂量组变化显著（

0.01）；且造模120 h MCT各组变化呈现剂量依赖性（

0.01）。免疫组化及Western blot结果显示，与正常组比较，促炎巨噬细胞标志物CD68蛋白表达随剂量升高而增加（

0.01），抗炎巨噬细胞CD163蛋白及mRNA表达随剂量升高而减少（

0.01）。与正常组比较，造模后MCT中、高剂量组磷酸化核转录因子-

B/核转录因子-

B（p-NF-

B/NF-

B）、磷酸化蛋白激酶B/蛋白激酶B（p-Akt/Akt）明显升高（

0.05，

0.01）。MCT造模后肝组织

-平滑肌肌动蛋白（

-SMA）蛋白表达随时间延长、剂量升高增加，

-SMA、Ⅰ型胶原蛋白基因

1（Col 1a1）和Ⅳ型胶原蛋白基因

1（Col 4a1）mRNA表达也随时间延长、剂量升高而明显增加（

0.05，

0.01）。TUNEL染色结果显示造模后凋亡细胞数量随造模剂量升高而增加，B细胞淋巴瘤-2（Bcl-2）蛋白与Bcl-2相关X蛋白（Bax）比值显著降低（

0.01）。

结论

不同剂量MCT灌胃诱导的大鼠肝窦阻塞综合征，病变随剂量增加而加重。80 mg·kg

-1

MCT导致的肝脏损伤可自愈，超过120 mg·kg

-1

的MCT导致的肝脏损伤会随着时间的延长而加重，甚至出现纤维化、死亡。MCT导致大鼠HSOS的病理机制可能是MCT引发肝组织内强烈的氧化应激，激活促炎巨噬细胞大量分泌炎症因子，进而激活NF-

B/Akt信号通路，导致严重的细胞损伤和死亡。

Abstract

Objective

To observe the pathological changes of hepatic sinusoidal obstruction syndrome （HSOS） induced by different doses of monocrotaline （MCT） in rats， investigate the dose and duration of modeling， and elucidate the mechanism.

Method

A total of 72 male SD rats were randomized into normal group （

=12）， and low-， medium-， and high-dose MCT groups （

=20 per group， 80，120，160 mg·kg

-1

， respecctively）. In the model groups， different doses of MCT were intragastrically administered to induce the HSOS in rats. After 48 h and 120 h separately， rats in each group were sacrificed and sampling was performed. The survival rate of rats in each group was calculated， and the body weight， liver weight， and and serum liver function indexes of the rats were examined. The histopathological changes of the liver were observed based on scanning electron microscopy， hematoxylin and eosin （HE） staining， and Sirius red （SR） staining. Glutathione S-transferase （GST） activity， total superoxide dismutase （T-SOD） activity， and malondialdehyde （MDA） content of liver tissue homogenate were measured with microplate method. The expression of liver tissue-related indexes was detected by real-time polymerase chain reaction （PCR）， Western blot， and immunohistochemistry.

Result

The activity of serum alanine aminotransferase （ALT） and aspartate aminotransferase （AST） in MCT groups rose with the increase in MCT dose （

0.05，

0.01） compared with that in the normal group. With the extension of modeling time， the activity of serum ALT and AST in the low-dose group decreased （

0.01）， while the activity of them in the medium-dose and high-dose groups increased （

0.01）. HE staining showed that hepatocyte necrosis， inflammatory cell infiltration， and erythrocyte accumulation in MCT groups. Electron microscopy demonstrated that fenestrae of liver sinusoidal endothelial cells widened and the sieve plates disappeared. Morever， the injury was worsened with the increase in MCT dose. In addition， the expression of CD44 in MCT groups was significantly reduced compared with that in the normal group （

0.05，

0.01）. SR staining showed that no positive staining was found in model groups after 48 h， while collagen deposition in portal areas and liver sinusoids could be seen in model groups after 120 h. MCT groups showed increase in MDA content and GST activity and decrease in T-SOD activity compared with the normal group， particularly the medium-dose and high-dose groups （

0.01）， and the changes were dose-dependent after 120 h （

0.01）. The protein expression of CD68 （pro-inflammatory macrophage marker） was raised with the increase in dosage， which was consistent with the results of immunohistochemistry （

0.01）， while CD163 （anti-inflammatory macrophage marker） protein and mRNA expression was significantly decreased with the increase in dosage （

0.01）. Western blot results showed that the expression of phosphorylated nuclear factor-

B/nuclear factor-

B （p-NF-

B/NF-

B） and phosphorylated protein kinase B/protein kinase B （p-Akt/t-Akt） was significantly increased in medium-dose and high-dose MCT groups （

0.05，

0.01）. The protein expression of

-smooth muscle actin （

-SMA） in liver tissues in MCT groups was significantly increased over time and with the increase in dose， and the mRNA expression of

-SMA， collagen type I

1 （Col1a1）， and collagen type Ⅳ

1 （Col4a1） showed the same trend （

0.05，

0.01）. The results of TUNEL staining showed that apoptotic cells were increased with the rise of MCT dose， while B-cell lymphoma-2（Bcl-2） /Bcl-2 associated X protein （Bax） was remarkably decreased （

0.01）.

Conclusion

HSOS in rats induced by intragastric administration of different doses of MCT was aggravated with the increase of dosage. In the low-dose （80 mg·kg

-1

） MCT group， the liver healed spontaneously over time. However， liver damage caused by MCT of 120 mg·kg

-1

and 160 mg·kg

-1

aggravated over time， and even fibrosis and death occurred. The pathological mechanism of MCT-induced HSOS in rats may be that MCT triggered intense oxidative stress in liver tissue， thus activated pro-inflammatory macrophages to secrete large amounts of inflammatory factors， and further activated the NF-

B/Akt signalling pathway， leading to severe cell damage and death.

关键词

Keywords

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