基于Nrf2/ARE抗氧化应激途径探究乌梅丸对溃疡性结肠炎小鼠的作用机制

doi:10.11958/20230533

天津医药 ›› 2024, Vol. 52 ›› Issue (3): 278-254.doi: 10.11958/20230533

基于Nrf2/ARE抗氧化应激途径探究乌梅丸对溃疡性结肠炎小鼠的作用机制

陈静¹(), 魏运姣²^,^△(), 罗超¹, 黄利华³, 陈橙¹, 段莎莎¹

1.武汉市第四医院儿科（邮编430034），2.药学部，3.病理科

收稿日期:2023-04-18 修回日期:2023-05-18 出版日期:2024-03-15 发布日期:2024-03-13
通讯作者: ^△E-mail：grqxafrou5@163.com
作者简介:陈静（1978），女，主治医师，主要从事经方现代应用、中药药理及临床研究。E-mail：chenjing7851@126.com
基金资助:
武汉市中医药科研项目(WZ22Z15)

The mechanism of Wumei pill on ulcerative colitis in mice based on Nrf2/ARE antioxidant stress pathway

CHEN Jing¹(), WEI Yunjiao²^,^△(), LUO Chao¹, HUANG Lihua³, CHEN Cheng¹, DUAN Shasha¹

1. Department of Pediatrics, 2. Department of Pharmacy, 3. Department of Pathology, the Fourth Hospital of Wuhan, Wuhan 430034, China

Received:2023-04-18 Revised:2023-05-18 Published:2024-03-15 Online:2024-03-13
Contact: ^△E-mail: grqxafrou5@163.com

陈静, 魏运姣, 罗超, 黄利华, 陈橙, 段莎莎. 基于Nrf2/ARE抗氧化应激途径探究乌梅丸对溃疡性结肠炎小鼠的作用机制[J]. 天津医药, 2024, 52(3): 278-254.

CHEN Jing, WEI Yunjiao, LUO Chao, HUANG Lihua, CHEN Cheng, DUAN Shasha. The mechanism of Wumei pill on ulcerative colitis in mice based on Nrf2/ARE antioxidant stress pathway[J]. Tianjin Medical Journal, 2024, 52(3): 278-254.

摘要/Abstract

摘要：

目的探究乌梅丸基于核因子E2相关因子2（Nrf2）/抗氧化反应元件（ARE）抗氧化应激途径对溃疡性结肠炎（UC）小鼠的作用机制。方法将70只SPF级雄性C57BL/6小鼠随机分为对照组、UC组、美沙拉嗪组（MES组，0.82 g/kg MES）、乌梅丸低剂量组（WMW-L组，按生药5 g/kg）、乌梅丸中剂量组（WMW-M组，按生药10 g/kg）、乌梅丸高剂量组（WMW-H组，按生药20 g/kg）和乌梅丸高剂量+Nrf2抑制剂ML-385组（WMW-H+ML-385组，乌梅丸按生药20 g/kg+20 mg/kg ML-385），每组10只。末次给药后，对小鼠进行疾病活动指数（DAI）评分及结肠黏膜损伤评分。HE染色观察小鼠结肠黏膜组织的病理学变化。酶联免疫吸附试验（ELISA）测定小鼠血清和结肠组织白细胞介素（IL）-1β、肿瘤坏死因子（TNF）-α、IL-6水平。硫代巴比妥酸比色法（TBA）测定小鼠血清和结肠组织丙二醛（MDA）含量。黄嘌呤氧化酶法测定小鼠血清和结肠组织超氧化物歧化酶（SOD）活性。二硫代二硝基苯甲酸（DTNB）直接法测定小鼠血清和结肠组织谷胱甘肽过氧化物酶（GSH-px）活力。免疫组织化学染色法观察小鼠结肠组织中Nrf2的阳性表达。Western blot检测小鼠结肠组织血红素加氧酶1（HO-1）和NAD（P）H：醌氧化还原酶1（NQO1）蛋白的表达情况。结果与对照组相比，UC组小鼠DAI评分、结肠黏膜损伤评分、结肠组织病理学评分升高，血清和结肠组织中IL-1β、TNF-α、IL-6、MDA水平升高，结肠组织Nrf2、HO-1、NQO1蛋白表达升高，血清和结肠组织中SOD、GSH-px水平降低（P<0.05），结肠黏膜损伤严重。与UC组相比，MES组、WMW-M组、WMW-H组小鼠相应指标变化与上述相反，而结肠组织Nrf2、HO-1、NQO1蛋白表达升高（P<0.05），结肠黏膜损伤减轻。WMW-L组、WMW-M组、WMW-H组各指标变化呈剂量依赖性。WMW-H组与MES组差异无统计学意义；ML-385减弱了高剂量乌梅丸对UC小鼠结肠黏膜损伤的改善作用。结论乌梅丸可能通过激活Nrf2/ARE抗氧化应激途径减轻UC小鼠的结肠黏膜损伤。

关键词: 结肠炎, 溃疡性, NF-E2相关因子2, 抗氧化反应元件, 氧化性应激, 乌梅丸

Abstract:

Objective To explore the mechanism of Wumei pill on ulcerative colitis (UC) in mice based on the anti oxidative stress pathway of nuclear factor erythroid 2-related factor 2 (Nrf2)/antioxidant response element (ARE). Methods Seventy SPF male C57BL/6 mice were randomly divided into the control group, the UC group, the mesalazine group (MES group, 0.82 g/kg MES), the low dose Wumei pill group (WMW-L group, 5 g/kg crude drug), the middle dose Wumei pill group (WMW-M group, 10 g/kg crude drug), the high dose Wumei pill group (WMW-H group, 20 g/kg crude drug) and the high dose Wumei pills+Nrf2 inhibitor ML-385 group (WMW-H+ML-385 group, Wumei pills crude drug 20 g/kg+20 mg/kg ML-385), with 10 rats in each group. The disease activity index (DAI) score and colonic mucosa injury score were performed in mice after the last administration. Pathological changes of colonic mucosa in mice were observed by HE staining. The levels of interleukin (IL) -1β, tumor necrosis factor-α (TNF-α) and IL-6 in serum and colon tissue of mice were measured by enzyme-linked immunosorbent assay (ELISA). The content of malondialdehyde (MDA) in serum and colon tissue of mice was determined by thiobarbituric acid colorimetry (TBA). The activity of superoxide dismutase (SOD) in serum and colon tissue of mice was measured by xanthine oxidase method. The activity of glutathione peroxidase (GSH-px) in serum and colon tissue of mice was determined by direct method with dithiodinitrobenzoic acid (DTNB). The positive expression of Nrf2 in colon tissue of mice was observed by immunohistochemistry. The expression of heme oxygenase-1 (HO-1) and NAD(P)H:quinone oxidoreductase-1 (NQO1) proteins in colon tissue of mice were detected by Western blot assay. Results Compared with the control group, the DAI score, colonic mucosa injury score, colonic histopathology score, levels of IL-1β, TNF-α, IL-6 and MDA in serum and colonic tissue, and expression levels of Nrf2, HO-1 and NQO1 protein in colonic tissue of mice were increased in the UC group, levels of SOD and GSH-px in serum and colon tissue decreased (P<0.05), the colon mucosa of mice was seriously damaged. Compared with the UC group, changes of corresponding indexes were contrary to the above in the MES group, the WMW-M group and the WMW-H group. However, the expression levels of Nrf2, HO-1 and NQO1 proteins in colon tissue were increased (P<0.05), and the damage of colon mucosa in mice was alleviated. Changes of the above indexes were dose-dependent in the WMW-L group, the WMW-M group and the WMW-H group. There were no significant differences in the above indexes between the WMW-H group and the MES group. ML-385 attenuated the improvement effect of high dose Wumei pill on colon mucosa injury. Conclusion Wumei pill may alleviate the colon mucosal damage of UC mice by activating Nrf2/ARE antioxidant stress pathway.

Key words: colitis, ulcerative, NF-E2-related factor 2, antioxidant response elements, oxidative stress, Wumei pill

中图分类号:

R285.5

图/表 8

参考文献 25

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组别	DAI（n=10）	结肠黏膜损伤评分（n=10）	组织病理学评分（n=5）
对照组	0	0	0
UC组	3.86±0.43^a	4.47±0.49^a	3.76±0.21^a
MES组	1.04±0.07^b	1.59±0.13^b	1.15±0.10^b
WMW-L组	3.80±0.41	4.39±0.46	3.71±0.32
WMW-M组	2.13±0.15^bc	2.73±0.22^bc	2.25±0.17^bc
WMW-H组	0.96±0.08^bcd	1.54±0.14^bcd	1.08±0.11^bcd
WMW-H+ML-385组	2.97±0.22^e	3.75±0.31^e	3.14±0.28^e
F	363.996^＊	312.249^＊	270.269^＊

组别	DAI（n=10）	结肠黏膜损伤评分（n=10）	组织病理学评分（n=5）
对照组	0	0	0
UC组	3.86±0.43^a	4.47±0.49^a	3.76±0.21^a
MES组	1.04±0.07^b	1.59±0.13^b	1.15±0.10^b
WMW-L组	3.80±0.41	4.39±0.46	3.71±0.32
WMW-M组	2.13±0.15^bc	2.73±0.22^bc	2.25±0.17^bc
WMW-H组	0.96±0.08^bcd	1.54±0.14^bcd	1.08±0.11^bcd
WMW-H+ML-385组	2.97±0.22^e	3.75±0.31^e	3.14±0.28^e
F	363.996^＊	312.249^＊	270.269^＊

组别	血清（n=10）			结肠组织（n=5）
组别	IL-1β	TNF-α	IL-6	IL-1β	TNF-α	IL-6
对照组	38.26±6.87	165.22±18.65	11.76±2.43	47.80±9.33	178.95±19.45	25.63±3.29
UC组	114.35±12.56^a	396.87±32.15^a	158.49±19.36^a	136.92±15.14^a	415.57±34.68^a	178.48±12.30^a
MES组	52.17±7.20^b	228.41±20.52^b	62.30±5.38^b	64.48±6.24^b	237.73±21.43^b	75.26±7.15^b
WMW-L组	109.54±8.82	385.47±30.26	152.79±16.22	130.68±13.34	409.50±30.47	174.34±11.92
WMW-M组	73.42±5.94^bc	297.15±23.08^bc	93.51±8.42^bc	90.27±7.49^bc	356.53±24.26^bc	118.92±10.16^bc
WMW-H组	48.79±4.85^bcd	219.84±18.57^bcd	58.37±5.06^bcd	59.82±5.56^bcd	231.46±20.36^bcd	69.44±6.39^bcd
WMW-H+ML-385组	82.06±7.69^e	324.25±25.36^e	117.54±9.30^e	104.52±8.33^e	388.24±26.84^e	142.50±11.08^e
F	137.824^＊	126.668^＊	234.576^＊	62.662^＊	70.542^＊	186.631^＊

组别	血清（n=10）			结肠组织（n=5）
组别	IL-1β	TNF-α	IL-6	IL-1β	TNF-α	IL-6
对照组	38.26±6.87	165.22±18.65	11.76±2.43	47.80±9.33	178.95±19.45	25.63±3.29
UC组	114.35±12.56^a	396.87±32.15^a	158.49±19.36^a	136.92±15.14^a	415.57±34.68^a	178.48±12.30^a
MES组	52.17±7.20^b	228.41±20.52^b	62.30±5.38^b	64.48±6.24^b	237.73±21.43^b	75.26±7.15^b
WMW-L组	109.54±8.82	385.47±30.26	152.79±16.22	130.68±13.34	409.50±30.47	174.34±11.92
WMW-M组	73.42±5.94^bc	297.15±23.08^bc	93.51±8.42^bc	90.27±7.49^bc	356.53±24.26^bc	118.92±10.16^bc
WMW-H组	48.79±4.85^bcd	219.84±18.57^bcd	58.37±5.06^bcd	59.82±5.56^bcd	231.46±20.36^bcd	69.44±6.39^bcd
WMW-H+ML-385组	82.06±7.69^e	324.25±25.36^e	117.54±9.30^e	104.52±8.33^e	388.24±26.84^e	142.50±11.08^e
F	137.824^＊	126.668^＊	234.576^＊	62.662^＊	70.542^＊	186.631^＊

组别	血清（n=10）			结肠组织（n=5）
组别	SOD/（U/mg）	MDA/（μmol/L）	GSH-px/（U/mg）	SOD/（U/mg）	MDA/（μmol/L）	GSH-px/（U/mg）
对照组	114.26±3.20	1.85±0.14	203.50±12.46	127.49±8.62	2.24±0.17	234.26±17.34
UC组	23.53±1.89^a	8.67±0.45^a	96.21±5.29^a	33.27±2.38^a	10.38±0.86^a	117.16±6.85^a
MES组	89.08±5.21^b	3.04±0.20^b	167.34±10.18^b	89.46±6.43^b	4.25±0.25^b	210.34±16.47^b
WMW-L组	24.68±2.14	8.25±0.63	98.07±8.69	34.29±3.18	10.16±0.72	119.72±8.86
WMW-M组	60.35±5.36^bc	5.74±0.30^bc	143.25±10.26^bc	66.82±5.33^bc	6.79±0.44^bc	165.83±12.45^bc
WMW-H组	96.48±8.93^bcd	2.58±0.16^bcd	173.34±14.30^bcd	92.78±7.45^bcd	3.88±0.28^bcd	217.33±16.09^bcd
WMW-H+ML-385组	43.57±5.84^e	6.25±0.44^e	126.34±10.09^e	56.28±6.18^e	7.49±0.65^e	143.15±12.27^e
F	485.133^＊	548.147^＊	145.492^＊	161.288^＊	170.028^＊	64.769^＊

基于Nrf2/ARE抗氧化应激途径探究乌梅丸对溃疡性结肠炎小鼠的作用机制

The mechanism of Wumei pill on ulcerative colitis in mice based on Nrf2/ARE antioxidant stress pathway

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