基于Nrf2信号通路探讨茵陈蒿汤对阻塞性黄疸大鼠肾氧化应激损伤的影响及其作用机制

刘军舰; 陈帅; 袁红霞; 许艳; 张西波; 李忠廉

doi:10.3969/j.issn.1001-5256.2023.05.019

基于Nrf2信号通路探讨茵陈蒿汤对阻塞性黄疸大鼠肾氧化应激损伤的影响及其作用机制

DOI: 10.3969/j.issn.1001-5256.2023.05.019

刘军舰¹,
陈帅²,
袁红霞³,
许艳⁴,
张西波¹,
李忠廉^1, , ,

1.
天津市中西医结合医院肝胆胰外科二，天津 300100
2.
徐州市中医院胸外科，江苏徐州 221003
3.
天津中医药大学管理学院，天津 301617
4.
天津医科大学研究生院，天津 300070

基金项目:

国家自然科学基金面上项目 (81273952);

天津市应用基础研究多元投入基金 (21JCZDJC01150);

天津市卫生健康委员会中医药重点领域科研项目 (2019003);

天津市卫生健康委员会中医中西医结合科研课题 (2021042);

2022中央财政转移支付地方项目

伦理学声明：本研究方案于2021年3月经由天津市中西医结合医院(天津市南开医院)审批，批号：NKYY-DWLL-2021-102，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：刘军舰负责课题设计，资料分析，撰写论文；陈帅、许艳参与实验操作及数据分析；袁红霞、张西波参与修改论文；李忠廉负责拟定写作思路，指导撰写文章并最后定稿。

详细信息

通信作者:
李忠廉，nkyylzl@163.com (ORCID: 0000-0001-5211-7612)

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出版历程
- 收稿日期: 2022-10-05
- 录用日期: 2022-11-10
- 出版日期: 2023-05-20

Effect of Yinchenhao decoction on renal oxidative stress injury in rats with obstructive jaundice and its mechanism of action based on the nuclear factor erythroid 2-related factor 2 signaling pathway

1.
Second Department of Hepatopancreatobiliary Surgery, Tianjin Hospital of Integrated Traditional Chinese and Western Medicine, Tianjin 300100, China
2.
Department of Thoracic Surgery, Xuzhou Hospital of Traditional Chinese Medicine, Xuzhou, Jiangsu 221003, China
3.
School of Management, Tianjin University of Traditional Chinese Medicine, Tianjin 301617, China
4.
Graduate School of Tianjin Medical University, Tianjin 300070, China

Research funding:

General Project National Natural Science Foundation of China (81273952);

Tianjin Applied Basic Research Diversified Investment Fund (21JCZDJC01150);

Scientific and Technological Project in Key Areas of Traditional Chinese Medicine of Tianjin Municipal Health and Health Committee (2019003);

Integrated Traditional Chinese and Western Medicine Project of Tianjin Municipal Health and Health Committee (2021042);

2022 Central Finance Transfer Payment Local Project

More Information

Corresponding author: LI Zhonglian, nkyylzl@163.com (ORCID: 0000-0001-5211-7612)

摘要

摘要: 目的探讨茵陈蒿汤对阻塞性黄疸大鼠肾氧化应激损伤的作用与调节核因子E2相关因子2(Nrf2)表达及核异位的关系。方法 32只雄性SD大鼠，随机分为假手术组(S组)、模型组(O组)、低剂量茵陈蒿汤组(LY组)和高剂量茵陈蒿汤组(HY组)，每组8只，S组大鼠仅游离上段胆总管但不予结扎，剩余各组大鼠胆总管中上1/3行双重结扎建立阻塞性黄疸模型，7天后LY组、HY组分别予茵陈蒿汤6.3 mL/kg和18.9 mL/kg灌胃，S组、O组每日给予等体积蒸馏水灌胃，连续7天，于第14天处理大鼠。采用ELISA法测定大鼠血清总胆红素(TBil)、直接胆红素(DBil)、丙氨酸转氨酶(ALT)、谷氨酰转移酶(GGT)、尿素氮(BUN)和血肌酐(Cr)水平；分光光度法检测大鼠肾组织氧化应激因子超氧化物歧化酶(SOD)和丙二醛(MDA)活性；实时荧光定量PCR和蛋白免疫印迹法分别检测肾组织中Nrf2、Kelch样环氧氯丙烷相关蛋白1(Keap1)、醌氧化还原酶1(NQO1)mRNA和蛋白表达水平；免疫组化检测肾组织中Nrf2蛋白的核异位情况。计量资料多组间比较采用单因素方差分析，组内进一步两两比较采用LSD-t检验。结果与S组比较，O组大鼠TBil、DBil、ALT、GGT、BUN、Cr水平升高，SOD活性减弱，MDA水平升高，差异均有统计学意义(P值均＜0.05)；与O组比较，LY组和HY组肝、肾功能指标水平均下降，SOD活性升高，MDA水平降低，差异均有统计学意义(P值均＜0.05)。与S组比较，O组大鼠肾组织中Nrf2、NQO1 mRNA和蛋白表达水平均明显下降(P值均＜0.05)；与O组比较，LY组和HY组Nrf2、NQO1 mRNA和蛋白表达水平均明显升高(P值均＜0.05)；各组大鼠肾组织中Keap1蛋白表达水平无明显差异(P＞0.05)。与S组比较，O组大鼠肾组织中Nrf2细胞核内阳性率显著降低(P＜0.05)；与O组比较，LY组和HY组Nrf2细胞核内阳性率均显著升高(P值均＜0.05)。结论茵陈蒿汤可以有效减轻阻塞性黄疸引起的肾损伤，其作用机制可能是通过上调大鼠肾组织中Nrf2蛋白的表达，并调控Nrf2蛋白核异位，从而介导下游NQO1蛋白的表达，调节阻塞性黄疸引起的氧化应激反应，进而减轻大鼠肾损伤。
- 黄疸, 阻塞性 /
- 肾损伤 /
- 茵陈蒿汤 /
- 氧化性应激 /
- 大鼠, Sprague-Dawley
Abstract: Objective To investigate the effect of Yinchenhao decoction on renal oxidative stress injury in rats with obstructive jaundice and its association with the regulation of the expression of nuclear factor erythroid 2-related factor 2 (Nrf2) and nuclear translocation. Methods A total of 32 male Sprague-Dawley rats were randomly divided into sham-operation group (S group), model group (O group), low-dose Yinchenhao decoction group (LY group), and high-dose Yinchenhao decoction group (HY group), with 8 rats in each group. For the rats in the S group, the upper common bile duct was isolated without ligation, and for those in the other groups, double ligation of the middle and upper 1/3 of the common bile duct was performed to establish a model of obstructive jaundice. After 7 days, the rats in the LY group and the HY group were given Yinchenhao decoction by gavage at a dose of 6.3 and 18.9 mL/kg, respectively, while those in the S and O groups were given an equal volume of distilled water by gavage every day for 7 consecutive days, and the rats were treated on day 14. ELISA was used to measure the serum levels of total bilirubin (TBil), direct bilirubin (DBil), alanine aminotransferase (ALT), gamma-glutamyl transpeptidase (GGT), blood urea nitrogen (BUN), and creatinine (Cr); spectrophotometry was used to measure the activity of the oxidative stress factors superoxide dismutase (SOD) and malondialdehyde (MDA) in renal tissue; quantitative real- time PCR and Western blotting were used to measure the mRNA and protein expression levels of Nrf2, Kelch-like ECH-associated protein 1 (Keap1), and NAD(P)H quinone dehydrogenase 1 (NQO1) in renal tissue; immunohistochemistry was used to measure observe the nuclear translocation of Nrf2 protein in renal tissue. 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 pairwise comparison within groups. Results Compared with the S group, the O group had significant increases in the levels of TBil, DBil, ALT、GGT, BUN, and Cr, a significant reduction in the activity of SOD, and a significant increase in the level of MDA (all P < 0.05). Compared with the O group, the LY group and the HY group had significant reductions in liver and renal function parameters, a significant increase in the activity of SOD, and a significant reduction in the level of MDA (all P < 0.05). Compared with the S group, the O group had significant reductions in the mRNA and protein expression levels of Nrf2 and NQO1 in renal tissue (all P < 0.05), and compared with the O group, the LY group and the HY group had significant increases in the mRNA and protein expression levels of Nrf2 and NQO1 (all P < 0.05), while there was no significant difference in the protein expression level of Keap1 between groups (P > 0.05). Compared with the S group, the O group had a significant reduction in the positive rate of Nrf2 in cell nucleus in renal tissue (P < 0.05), and compared with the O group, the LY group and the HY group had a significant increase in the positive rate of Nrf2 in cell nucleus (P < 0.05). Conclusion Yinchenhao decoction can effectively alleviate renal injury caused by obstructive jaundice, possibly by upregulating the protein expression of Nrf2 in renal tissue and regulating the nuclear translocation of Nrf2 protein, so as to mediate the protein expression of downstream NQO1, regulate oxidative stress response caused by obstructive jaundice, and thereby alleviate renal injury in rats.
- Jaundice, Obstructive /
- Renal Injury /
- Yinchenhao Decoction /
- Oxidative Stress /
- Rats, Sprague-Dawley

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图 1 大鼠肾组织中Nrf2、Keap1、NQO1 mRNA表达水平比较

Figure 1. Comparison of Nrf2, Keap1 and NQO1 mRNA expression in renal tissue of rats

下载: 全尺寸图片幻灯片

图 2 大鼠肾组织中Nrf2、Keap1、NQO1蛋白表达

Figure 2. Expression of Nrf2, Keap1 and NQO1 proteins in rat kidney

下载: 全尺寸图片幻灯片

图 3 大鼠肾组织中Nrf2、Keap1、NQO1蛋白表达水平比较

Figure 3. Comparison of Nrf2, Keap1 and NQO1 protein expression in renal tissue of rats

下载: 全尺寸图片幻灯片

图 4 大鼠肾组织中Nrf2蛋白核异位情况(免疫组化染色)

Figure 4. Nuclear heterotopia of Nrf2 protein in rat kidney (IHC)

下载: 全尺寸图片幻灯片

图 5 大鼠肾组织中Nrf2蛋白细胞核阳性率比较

Figure 5. Comparison of nuclear positive rate of Nrf2 protein in renal tissues of rats in each group

下载: 全尺寸图片幻灯片

表 1 引物序列

Table 1. Primer sequence

基因	引物序列	扩增片段长度(bp)
Nrf2	上游5′-CTTTAGTCAGCGACAGAAGGAC-3′	227
	下游5′-AGGCATCTTGTTTGGGAATGTG-3′
Keap1	上游5′- CGGGGACGCAGTGATGTATG-3′	85
	下游5′-TGTGTAGCTGAAGGTTCGGTTA-3′
NQO1	上游5′- AGGATGGGAGGTACTCGAATC-3′	127
	下游5′-TGCTAGAGATGACTCGGAAGG-3′
β-actin	上游5′- GTGACGTTGACATCCGTAAAGA-3′	245
	下游5′-GCCGGACTCATCGTACTCC-3′

下载: 导出CSV

表 2 大鼠血清中TBil、DBil、ALT、GGT、BUN、Cr水平比较

Table 2. Comparison of serum TBil, DBil, ALT, GGT, BUN and Cr levels in rats

组别	动物数(只)	TBil (μmol/L)	DBil (μmol/L)	ALT (U/L)	GGT (U/L)	BUN (mmol/L)	Cr (μmol/L)
S组	8	1.83±0.33¹⁾	0.87±0.44¹⁾	63.17±10.29¹⁾	1.34±0.65¹⁾	3.90±0.60¹⁾	28.05±1.75¹⁾
O组	8	209.80±17.47	146.99±13.74	415.23±55.93	47.57±10.97	7.28±1.01	33.41±1.77
LY组	8	151.51±13.47¹⁾	114.11±14.48¹⁾	291.67±30.68¹⁾	30.53±6.69¹⁾	5.27±1.16¹⁾	30.45±1.95¹⁾
HY组	8	122.30±7.26¹⁾²⁾	85.70±13.26¹⁾²⁾	189.41±47.32¹⁾²⁾	20.83±4.61¹⁾²⁾	4.01±0.26¹⁾²⁾	28.38±1.76¹⁾²⁾
F值		455.164	229.455	111.746	63.779	30.085	18.335
P值		＜0.001	＜0.001	＜0.001	＜0.001	＜0.001	＜0.001
注：与O组比较，1)P＜0.05；与LY组比较，2)P＜0.05。

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表 3 大鼠肾组织SOD、MDA水平比较

Table 3. Comparison of SOD and MDA levels in renal tissues of rats

组别	动物数(只)	SOD(U/mg)	MDA(nmol/mg)
S组	8	67.57±6.50¹⁾	2.66±0.68¹⁾
O组	8	45.38±4.98	4.01±0.43
LY组	8	71.75±10.89¹⁾	3.23±0.38¹⁾
HY组	8	86.00±6.70¹⁾²⁾	2.53±0.37¹⁾²⁾
F值		39.336	15.919
P值		＜0.001	＜0.001
注：与O组比较，1)P＜0.05；与LY组比较，2)P＜0.05。

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