黄芩汤通过调控内质网应激减轻小鼠溃疡性结肠炎

doi:10.12122/j.issn.1673-4254.2024.11.14

南方医科大学学报 ›› 2024, Vol. 44 ›› Issue (11): 2172-2183.doi: 10.12122/j.issn.1673-4254.2024.11.14

• • 上一篇

黄芩汤通过调控内质网应激减轻小鼠溃疡性结肠炎

邱建国¹^,²(), 邱一桐¹, 李国荣¹, 张林生¹, 郑雪¹^,³, 姚泳江¹, 王熙丹², 黄海阳², 张凤敏², 苏冀彦⁴, 郑学宝¹^,³(), 黄晓其¹^,³()

^1.广州中医药大学中药学院，广东广州 510006
^2.广州中医药大学东莞医院，广东东莞 523000
^3.东莞广州中医药大学研究院，广东东莞 523000
^4.佛山市妇幼保健院，广东佛山 528000

收稿日期:2024-07-07 出版日期:2024-11-20 发布日期:2024-11-29
通讯作者: 郑学宝,黄晓其 E-mail:1194883143@qq.com;xuebaozheng@ gzucm.edu.cn;huangxiaoqi@gzucm.edu.cn
作者简介:邱建国，硕士，E-mail: 1194883143@qq.com
基金资助:
国家自然科学基金(82274227);广东省基础与应用基础研究基金(2022A1515140011)

Huangqin Decoction alleviates ulcerative colitis in mice by reducing endoplasmic reticulum stress

Jianguo QIU¹^,²(), Yitong QIU¹, Guorong LI¹, Linsheng ZHANG¹, Xue ZHENG¹^,³, Yongjiang YAO¹, Xidan WANG², Haiyang HUANG², Fengmin ZHANG², Jiyan SU⁴, Xuebao ZHENG¹^,³(), Xiaoqi HUANG¹^,³()

^1.School of Pharmaceutical Sciences, Guangzhou University of Chinese Medicine, Guangzhou 510006, China
^2.Dongguan Hospital of Guangzhou University of Chinese Medicine, Dongguan 523000, China
^3.Dongguan Institute of Guangzhou University of Chinese Medicine, Dongguan 523000, China
^4.Foshan Maternal and Child Health Hospital, Foshan 528000, China

Received:2024-07-07 Online:2024-11-20 Published:2024-11-29
Contact: Xuebao ZHENG, Xiaoqi HUANG E-mail:1194883143@qq.com;xuebaozheng@ gzucm.edu.cn;huangxiaoqi@gzucm.edu.cn
Supported by:
National Natural Science Foundation of China(82274227)

摘要/Abstract

摘要：

目的观察黄芩汤对小鼠溃疡性结肠炎（UC）细胞凋亡的影响并探讨其作用机制。方法将雄性Balb/c小鼠随机分为：正常组、模型组、美沙拉嗪组（5-ASA，200 mg/kg）、黄芩汤低、中、高剂量组（HQDL，2.275 g/kg；HQDM，4.55 g/kg；HQDH，9.1 g/kg），8只/组。各组自由饮食，除正常组自由饮用无菌水外，其余各组小鼠自由饮用3% DSS溶液，持续7d以建立UC模型。取结肠组织采用HE、AB-PAS和TUNEL染色分别观察结肠损伤和细胞凋亡情况，采用ELISA法检测炎症因子表达变化；采用Western blotting、免疫组化和qRT-PCR法分别检测肠道化学屏障、机械屏障、内质网应激等相关指标的蛋白或基因表达变化。结果与模型组相比，黄芩汤干预下的UC小鼠，DAI评分和宏观评分下降（P<0.01），TUNEL染色荧光强度下降（P<0.01）。促炎因子IL-6、TNF-α、IL-1β、IL-8表达减少（P<0.01），MUC2和TFF3的基因表达升高（P<0.05），Claudin-1、Occludin和E-cadherin的蛋白表达升高（P<0.05），GRP78、CHOP和Caspase-12的基因和蛋白表达下降（P<0.01）、PERK、eIF2α和IRE1α的磷酸化表达降低（P<0.05），Bcl-2/Bax蛋白表达比例升高（P<0.01）和Caspase-3的蛋白表达降低（P<0.01）。结论黄芩汤能够抑制UC小鼠的细胞凋亡反应并改善肠道屏障功能，其机制可能与PERK和IRE1α信号通路介导的内质网应激有关。

关键词: 溃疡性结肠炎, 黄芩汤, 内质网应激, 细胞凋亡

Abstract:

Objective To evaluate the therapeutic effect of Huangqin Decoction (HQD) on ulcerative colitis (UC) in mice and explore its mechanism. Methods Male Balb/c mice were randomly divided into normal control group, model group, mesalazine group (5-ASA, 200 mg/kg), and low-, medium- and high-dose HQD groups (2.275, 4.55 and 9.1 g/kg, respectively). With the exception of those in the normal control group, all the mice were exposed to 3% DSS solution in drinking water for 7 days to establish UC models. After treatment with the indicated drugs, the mice were assessed for colon injury and apoptosis using HE, AB-PAS and TUNEL staining, and the expression levels of inflammatory factors were detected with ELISA. Western blotting, immunohistochemistry and qRT-PCR were used to detect the changes in protein expressions associated with the intestinal chemical barrier, mechanical barrier and endoplasmic reticulum stress (ERS). Results HQD treatment significantly reduced DAI score and macro score of UC mice, decreased colonic epithelial cell apoptosis, lowered expressions of IL-6, TNF-α, IL-1β and IL-8, and enhanced the expressions of MUC2 and TFF3. HQD treatment also upregulated the protein expressions of claudin-1, occludin and E-cadherin, reduced the expressions of GRP78, CHOP, caspase-12 and caspase-3, decreased the phosphorylation levels of PERK, eIF2α and IRE1α, and increased the Bcl-2/Bax ratio in the colon tissues of UC mice. Conclusion HQD inhibits colonic epithelial cell apoptosis and improves intestinal barrier function in UC mice possibly by reducing ERS mediated by the PERK and IRE1α signaling pathways.

Key words: ulcerative colitis, Huangqin Decoction, endoplasmic reticulum stress, apoptosis

邱建国, 邱一桐, 李国荣, 张林生, 郑雪, 姚泳江, 王熙丹, 黄海阳, 张凤敏, 苏冀彦, 郑学宝, 黄晓其. 黄芩汤通过调控内质网应激减轻小鼠溃疡性结肠炎[J]. 南方医科大学学报, 2024, 44(11): 2172-2183.

Jianguo QIU, Yitong QIU, Guorong LI, Linsheng ZHANG, Xue ZHENG, Yongjiang YAO, Xidan WANG, Haiyang HUANG, Fengmin ZHANG, Jiyan SU, Xuebao ZHENG, Xiaoqi HUANG. Huangqin Decoction alleviates ulcerative colitis in mice by reducing endoplasmic reticulum stress[J]. Journal of Southern Medical University, 2024, 44(11): 2172-2183.

图/表 12

表 1 DAI评分量化表

Tab.1 Quantitative table of DAI score

Weight loss (%)	Feces consistency	Hemafecia	Score
0	Normal	N/A	0
1-5	Mild soft	Slight bleeding	1
5-10	Soft and wet	Moderate bleeding	2
10-20	Half loose stool	Gross bleeding	3
>20	loose stool	Blood clot around anus	4

表 2 结肠组织病理学评分

Tab.2 Histopathological score of the colon

Inflammation degree	Inflammation range	Colonic crypt damage	Score
N/A	N/A	Normal	0
Mild inflammation	Inflammation of the mucosa	One-third of crypt damage	1
Moderate inflammation	Inflammation of the mucosa and submucosa	Two thirds of crypt damage	2
Severe inflammation	Inflammation of the mucosa and intestinal wall	Crypts disappeared but epithelium remains	3
Acute severe inflammation	Transmural inflammation	Both crypts and epithelium disappeared	4

表 3 引物序列

Tab.3 Primer sequence

Gene	Orientation	Sequence (5′ to 3′)
MUC2	Forward	AGGGCTCGGAACTCCAGAAA
MUC2	Reverse	CCAGGGAATCGGTAGACATCG
TFF3	Forward	TTGCTGGGTCCTCTGGGATAG
TFF3	Reverse	TACACTGCTCCGATGTGACAG
AGR2	Forward	ACAACTGACAAGCACCTTTCTC
AGR2	Reverse	GTTTGAGTATCGTCCAGTGATGT
GRP78	Forward	ACTTGGGGACCACCTATTCCT
GRP78	Reverse	GTTGCCCTGATCGTTGGCTA
CHOP	Forward	AAGCCTGGTATGAGGATCTGC
CHOP	Reverse	TTCCTGGGGATGAGATATAGGTG
Caspase-12	Forward	TTGGAAGGTAGGCAAGACTGGTTC
Caspase-12	Reverse	TCAGTTCACCTGGGACCTCAAATG
ATF4	Forward	AACCTATAAAGGCTTGCGGC
ATF4	Reverse	GATTTCGTGAAGAGCGCCAT
XBP1s	Forward	AAGAACACGCTTGGGAATGG
XBP1s	Reverse	CTGCACCTGCTGCGGAC

图 1 各组小鼠造模期间摄水、摄食及体质量、DAI评分动态变化

Fig.1 Changes of water intake (A), food intake (B), body weight (C) and DAI score (D) of the mice during modeling (Mean±SD, n=8). ##P<0.01 vs CON group; **P<0.01 vs DSS group.

图 2 黄芩汤对UC小鼠结肠长度及病理形态的影响

Fig. 2 Effect of Huangqin Decoction on colon length and pathology in UC mice. A: Colon length of the mice in each group. B: Statistics of colon length in each group. C: Histopathological score of the colon in each group (Mean±SD, n=8). D: HE staining of the colon tissues in each group (Original magnification: ×100 or 200). ##P<0.01 vs CON group; *P<0.05, **P<0.01 vs DSS group.

图 3 黄芩汤对UC小鼠结肠上皮细胞凋亡的影响

Fig.3 Effect of Huangqin decoction on apoptosis of colonic epithelial cells in UC mice. A: TUNEL staining of the colon tissue in each group (×400 or 200). B: Mean fluorescence intensity in each group (Mean±SD, n=3) . ##P<0.01 vs CON group;**P<0.01 vs DSS group.

图 5 黄芩汤对UC小鼠黏蛋白和杯状细胞分泌的影响

Fig.5 Effect of Huangqin Decoction on mucin and goblet cell secretion in UC mice (Mean±SD, n=3). A: AB-PAS staining of mice in each group (×100, ×200). B:Expression of MUC2 MRNA. C: Expression of TFF3 MRNA. D: Expression of AGR2 mRNA. #P<0.005, ##P<0.01 vs CON group; *P<0.05, **P<0.01 vs DSS group.

图 6 黄芩汤对UC小鼠结肠上皮机械屏障蛋白表达的影响

Fig 6 Effect of Huangqin Decoction on mechanical barrier function of the colonic epithelium in UC mice (Mean±SD, n=3). ##P<0.01 vs CON group; *P<0.05, **P<0.01 vs DSS group. A: E-cadherin. B: Occludin. C: Claudin-1.

图 7 黄芩汤对UC小鼠GRP78基因和蛋白表达的影响

Fig.7 Effect of Huangqin Decoction on expression of GRP78 mRNA and protein in UC mice (Mean±SD, n=6). A: Immunohistochemistry staining of GRP78 protein of mice in each group (×200, ×400). B: Area fraction of GRP78 protein of mice in each group. C: Expression of GRP78 mRNA; ##P<0.01 vs CON group; *P<0.05, **P<0.01 vs DSS group.

图 8 黄芩汤对UC小鼠内质网应激通路相关蛋白和基因的影响

Fig.8 Effect of Huangqin decoction on protein and mRNA expressions related to endoplasmic reticulum stress pathway in UC mice (Mean±SD, n=3). ##P<0.01 vs CON group; *P<0.05, **P<0.01 vs DSS group. A: p-PERK/PERK. B: p-IRElα/IRElα. C: p-eIF2α/eIF2α. D: ATF4 mRNA level (fold change). E: XBPls mRNA level (fold change).

图 9 黄芩汤对UC小鼠ERS中CHOP和Caspase-12的影响

Fig.9 Effect of Huangqin Decoction on CHOP and Caspase-12 expressions in UC mice (Mean±SD, n=3). A: Immunohistochemistry staining of CHOP and Caspase-12 protein of mice in each group (×200, ×400). B-C: Area fraction of immunohistochemistry staining of CHOP and Caspase-12 protein of mice in each group. D-E: Expression of CHOP and Caspase-12 mRNA; ##P<0.01 vs CON group; **P<0.01 vs DSS group.

图 10 黄芩汤对UC小鼠Bcl-2、Bax、Cleaved-Caspase-3的影响

Fig 10 Effect of Huangqin Decoction on Bcl-2, Bax and Caspase-3 expressions in UC mice (Mean±SD, n=3). ##P<0.01 vs CON group; **P<0.01 vs DSS group. A: Bcl-2/Bax. B: Cleaved-Caspase-3/β-tubulin.

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黄芩汤通过调控内质网应激减轻小鼠溃疡性结肠炎

Huangqin Decoction alleviates ulcerative colitis in mice by reducing endoplasmic reticulum stress

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