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

doi:10.12122/j.issn.1673-4254.2024.11.14

Abstract

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

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.

Figures/Tables 12

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

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

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

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.

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.

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.

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.

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.

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.

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).

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.

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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