黄芪提取物调节IL-6/STAT3信号通路治疗马兜铃酸Ⅰ诱导肝肾损伤小鼠模型的效果观察

朱哿瑞; 皮亚妮; 王静; 黄恺; 彭渊; 陈高峰; 刘成海; 陶艳艳

doi:10.3969/j.issn.1001-5256.2023.08.020

黄芪提取物调节IL-6/STAT3信号通路治疗马兜铃酸Ⅰ诱导肝肾损伤小鼠模型的效果观察

DOI: 10.3969/j.issn.1001-5256.2023.08.020

朱哿瑞^{1, 2},
皮亚妮¹,
王静¹,
黄恺³,
彭渊¹,
陈高峰¹,
刘成海^{1, 3, 4},
陶艳艳^1, , ,

1.
上海中医药大学附属曙光医院·肝病研究所，上海 201203
2.
上海中医药大学中医药国际标准化研究所，上海 201203
3.
上海市中医临床重点实验室，上海 201203
4.
肝肾疾病病证教育部重点实验室(上海中医药大学)，上海 201203

基金项目:

上海市科委科技支撑项目 (19401901500);

上海市中医药三年行动计划 (ZY-〔2018-2020〕-CCCX- 5001)

伦理学声明：本研究方案于2021年8月经由上海中医药大学实验动物伦理委员会审批，批号：PZSHUTCM-210926011，符合实验室动物管理与使用准则。

利益冲突声明：本文不存在任何利益冲突。

作者贡献声明：朱哿瑞负责动物实验，资料分析，撰写论文；皮亚妮协助动物实验和数据整理；王静、黄恺、彭渊、陈高峰负责修改论文；刘成海指导课题设计，修改论文；陶艳艳负责拟定课题设计和写作思路，指导撰写文章并最后定稿。

详细信息

通信作者:
陶艳艳，taoyanyan1023@126.com (ORCID：0000-0002-8962-3137)

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出版历程
- 收稿日期: 2022-11-23
- 录用日期: 2023-01-18
- 出版日期: 2023-08-20

Efficacy of Astragali Radix extract in treatment of a mouse model of aristolochic acid Ⅰ-induced liver and renal injury by regulating the IL-6/STAT3 signaling pathway

Gerui ZHU^{1, 2},
Yani PI¹,
Jing WANG¹,
Kai HUANG³,
Yuan PENG¹,
Gaofeng CHEN¹,
Chenghai LIU^{1, 3, 4},
Yanyan TAO^{1
, ,
,}

1.
Institute of Liver Diseases, Shuguang Hospital Affiliated to Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China
2.
Institute of TCM International Standardization, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China
3.
Shanghai Key Laboratory of Traditional Chinese Clinical Medicine, Shanghai 201203, China
4.
Key Laboratory of Liver and Kidney Diseases, Ministry of Education, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China

Research funding:

Science and Technology Innovation Action Plan of the Shanghai Municipal Science and Technology Commission (19401901500);

Three-Year Action Plan of for the Development of TCM in Shanghai (ZY-〔2018-2020〕-CCCX- 5001)

More Information

Corresponding author: TAO Yanyan, taoyanyan1023@126.com (ORCID: 0000-0002-8962-3137)

摘要

摘要: 目的探讨黄芪提取物(AR)改善马兜铃酸Ⅰ(AA Ⅰ)致小鼠急性肝、肾损伤效果及其调控IL-6/STAT3信号通路的作用机制。方法健康雄性C57BL/6小鼠38只，采用简单随机分组法分为正常组(n=8)、模型组(n=10)、AR组(n=10)和N-乙酰半胱氨酸(NAC)组(n=10)。模型组小鼠以20 mg/kg AAⅠ腹腔注射，1次/d，持续5 d。正常组小鼠腹腔注射相同容积羧甲基纤维素钠。AR组、NAC组20 mg/kg AAⅠ腹腔注射，1次/d，持续3 d；第4天分别按AR 75 mg/kg、NAC 150 mg/kg小鼠体质量剂量灌胃，1次/d，持续8 d。NAC为阳性对照药。给药造模结束后，处死小鼠并收集血清及肝、肾组织。试剂盒检测血清ALT、AST、肌酐(SCr)、尿素氮(BUN)水平；HE染色观察肝、肾组织病理；荧光PCR及免疫组化分析肝、肾组织中p-STAT3表达量；酶联免疫吸附实验检测肝、肾组织IL-6、IL-1β及TNF-α表达水平。计量资料多组间比较采用单因素方差分析，进一步两组间比较采用SNK-q检验。结果与正常组小鼠相比，模型组小鼠肾体比上升(P < 0.05)；与模型组相比，AR组ALT、AST、SCr和BUN水平显著降低(F值分别为49.29、31.31、58.89、85.88，P值均 < 0.01)；HE染色结果表明，AR可有效减轻AAⅠ导致的肝、肾组织结构破坏和炎性细胞浸润；荧光PCR及免疫组化染色结果表明，AR可减少肝、肾组织p-STAT3表达；酶联免疫吸附检测发现，AR可下调IL-6、IL-1β及TNF-α表达。NAC与AR效应相似，两者间无明显差异。结论 AR对AAⅠ所致急性肝、肾损伤有保护作用，其部分作用机制可能与抑制IL-6/STAT3信号通路激活，减轻炎症反应有关。
- 马兜铃酸 /
- 化学性与药物性肝损伤 /
- 急性肾损伤 /
- STAT3转录因子 /
- 黄芪
Abstract: Objective To investigate the mechanism of action of Astragali Radix (AR) extract in improving aristolochic acid Ⅰ (AA Ⅰ)-induced acute liver and renal injury in mice by regulating the IL-6/STAT3 signaling pathway. Methods A total of 38 healthy male C57BL/6 mice were randomly divided into normal group with 8 mice, model group with 10 mice, AR group with 10 mice, and N-Acetyl-L-cysteine (NAC) group with 10 mice. The model group mice were intraperitoneally injected with 20 mg/kg AAⅠ once a day for 5 days. Normal mice were intraperitoneally injected with the same volume of Carboxymethyl cellulose sodium. AR group and NAC group received intraperitoneal injection of 20 mg/kg AAⅠ once a day for 3 days; On the 4th day, mice were gavaged with AR 75 mg/kg and NAC 150 mg/kg body mass doses, once a day, for 8 days. NAC was used as a positive control drug. After the end of administration and modeling, the mice were sacrificed to collect serum samples and liver and renal tissue samples. The kit was used to measure the serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), serum creatinine (SCr), and blood urea nitrogen (BUN); HE staining was used to observe liver and renal histopathology; quantitative real-time PCR and immunohistochemistry were used to measure the expression level of p-STAT3 in the liver and renal tissue; ELISA was used to measure the expression levels of interleukin-6 (IL-6), interleukin-1β (IL-1β), and tumor necrosis factor-α (TNF-α) in the liver and renal tissue. A one-way analysis of variance was used for comparison of continuous data between multiple groups, and the SNK-q test was used for further comparison between two groups. Results Compared with the normal group, the model group had a significant increase in kidney-to-body ratio (P < 0.05). Compared with the model group, the AR group had significant reductions in the levels of ALT, AST, SCr, and BUN (F=49.29, 31.31, 58.89, and 85.88, all P < 0.01). HE staining showed that AR could effectively alleviate AAⅠ -induced structural damage and inflammatory cell infiltration in the liver and renal tissue; quantitative real-time PCR and immunohistochemistry showed that AR could reduce the expression of p-STAT3 in the liver and renal tissues; ELISA showed that AR could downregulate the expression of IL-6, IL-1β, and TNF-α. NAC and AR had a similar effect with no significant differences. Conclusion AR exerts a protective effect against AAⅠ-induced acute liver and renal injury, possibly by inhibiting the activation of the IL-6/STAT3 signaling pathway and alleviating inflammatory response.
- Aristolochic Acid /
- Chemical and Drug Induced Liver Injury /
- Acute Kidney Injury /
- STAT3 Transcription Factor /
- Astmgali Radix

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图 1 各组小鼠体质量变化

Figure 1. Changes in body weight of mice in each group

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图 2 各组小鼠肝、肾外观改变

Figure 2. Appearance changes of liver and kidney in each group

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图 3 各组小鼠肝、肾组织HE染色(×200)

Figure 3. Liver and kidney tissues of mice in each group HE staining (×200)

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图 4 各组小鼠肝、肾组织中p-STAT3免疫组化结果(×100)

Figure 4. Immunohistochemical results of p-STAT3 in liver and kidney tissues of mice in each group(×100)

下载: 全尺寸图片幻灯片

表 1 荧光定量PCR基因引物序列

Table 1. qRT-PCR primer sequences of each gene

基因	引物	序列	产物长度(bp)
STAT3	上游	5′-TGTCAGATCACATGGGCTAAAT-3′	88
	下游	5′-GGTCGATGATATTGTCTAGCCA-3′
β-actin	上游	5′-TGACGAGGCCCAGAGCAAGA-3′	330
	下游	5′-ATGGGCACAGTGTGGGTGAC-3′

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表 2 AR对AAⅠ小鼠模型脏器指数的影响

Table 2. Effect of AR on organ index of AAⅠ model mice

组别	动物数(只)	肝体比(%)	肾体比(‰)
正常组	8	5.36±0.59	7.56±0.66
模型组	8	5.09±0.27	10.76±1.03¹⁾
AR组	10	5.26±0.32	9.98±0.99¹⁾²⁾
NAC组	9	5.24±0.32	10.68±0.45¹⁾
F值		0.38	22.63
P值		0.70	< 0.01
注：与正常组相比，1)P < 0.01；与模型组相比，2)P < 0.01。

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表 3 AR对AAⅠ模型小鼠肝、肾功能的影响

Table 3. Effects of AR on liver and kidney function of AAⅠ model mice

组别	动物数(只)	肝功能		肾功能
组别	动物数(只)	ALT(U/L)	AST(U/L)	SCr (mmol/L)	BUN (mmol/L)
正常组	8	36.50±17.52	79.75±35.63	13.50±1.07	8.78±2.44
模型组	8	124.40±15.62¹⁾	453.90±99.91¹⁾	288.40±2.39¹⁾	109.70±13.52¹⁾
AR组	10	95.80±16.10²⁾	263.20±92.20²⁾	195.70±48.56²⁾	80.82±19.18²⁾
NAC组	9	112.20±13.83	357.80±77.76³⁾	241.30±67.76	79.49±9.54²⁾
F值		49.29	31.31	58.89	85.88
P值		< 0.01	< 0.01	< 0.01	< 0.01
注：与正常组相比，1)P < 0.01；与模型组相比，2)P < 0.01，3)P < 0.05。

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表 4 各组小鼠肝、肾组织STAT3 mRNA变化

Table 4. Changes of STAT3 mRNA in liver and kidney of mice in each group

组别	动物数(只)	肝STAT3 mRNA	肾STAT3 mRNA
正常组	8	0.63±0.20	0.71±0.09
模型组	8	1.53±0.16¹⁾	1.61±0.15¹⁾
AR组	10	0.97±0.20²⁾	1.15±0.15²⁾
NAC组	9	1.02±0.23²⁾	1.18±0.10²⁾
F值		29.88	72.40
P值		< 0.01	< 0.01
注：与正常组相比，1)P < 0.01；与模型组相比，2)P < 0.01。

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表 5 各组小鼠肝、肾组织中p-STAT3免疫组化结果半定量分析

Table 5. Semi-quantitative analysis of p-STAT3 immunohistochemical results in liver and kidney tissues of mice in each group

组别	动物数(只)	肝脏p-STAT3阳性染色面积比(%)	肾脏p-STAT3阳性染色面积比(%)
正常组	8	0.36±0.08	0.21±0.03
模型组	8	8.62±0.24¹⁾	12.70±10.13¹⁾
AR组	10	4.78±0.13²⁾	6.20±0.16²⁾
NAC组	9	6.78±0.12²⁾	9.29±0.10²⁾
F值		491.97	2 101.97
P值		< 0.01	< 0.01
注：与正常组相比，1)P < 0.01；与模型组相比，2)P < 0.01。

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表 6 AR对AAⅠ模型肝、肾组织中IL-6、IL-1β和TNF-α水平的影响

Table 6. Effects of AR on Levels of IL-6, IL-1β and TNF-α in liver and kidney tissues

组别	动物数(只)	肝组织(pg/mg)			肾组织(pg/mg)
组别	动物数(只)	IL-6	IL-1β	TNF-α	IL-6	IL-1β	TNF-α
正常组	8	24.52±2.92	57.06±8.91	147.40±17.18	31.00±6.11	42.11±7.78	99.18±32.36
模型组	8	55.70±10.73¹⁾	141.50±30.27¹⁾	355.90±15.48¹⁾	158.60±12.53¹⁾	329.80±45.23¹⁾	1 051.00±12.53¹⁾
AR组	10	29.05±3.03²⁾	70.67±13.70²⁾	185.20±19.82²⁾	36.51±23.65²⁾	67.54±28.79²⁾	105.30±31.71²⁾
NAC组	9	45.89±13.89³⁾	75.98±9.53²⁾	192.70±9.33²⁾	45.56±9.08²⁾	76.08±17.54²⁾	171.80±31.24²⁾
F值		22.05	38.03	270.37	123.21	186.31	2 098.69
P值		< 0.01	< 0.01	< 0.01	< 0.01	< 0.01	< 0.01
注：与正常组相比，1)P < 0.01；与模型组相比，2)P < 0.01，3)P < 0.05。

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