Protective effect of ferulic acid regulating AMPK pathway on H2O2-induced intestinal epithelial barrier injury

doi:10.3877/cma.j.issn.1674-0793.2021.05.011

Abstract

Abstract:

Objective

To observe the protective effect of ferulic acid (FA) on hydrogen peroxide (H₂O₂)-induced intestinal epithelial barrier injury and explore its mechanism.

Methods

Caco-2 cells were cultured in vitro to establish intestinal epithelial barrier model. They were randomly divided into control group, H₂O₂ group, 10 μmol/L FA group, 20 μmol/L FA group, 40 μmol/L FA group, AMPK inhibitor group and FA+AMPK inhibitor group. Methyl thiazolyl tetrazolium (MTT) assay and lactate dehydrogenase (LDH) method were used to detect the survival and injury of Caco-2 cells. The changes of intestinal epithelial barrier function were evaluated by transepithelial electrical resistance (TEER), and malondialdehyde (MDA) and superoxide dismutase (SOD) kits were used to detect the changes of intestinal epithelial barrier oxidative stress. Western blotting analysis was used to detect the protein expressions of Occludin, ZO-1 and p-AMPK in intestinal epithelial barrier; the protein distribution and changes of Occludin and ZO-1 in intestinal epithelial barrier were observed by immunofluorescence.

Results

Compared with those in the control group, the survival rate of Caco-2 cells, TEER value, SOD activity, Occludin, ZO-1 and p-AMPK protein expressions in H₂O₂ group were significantly lower (P<0.05), the LDH activity and MDA expression were significantly higher (P<0.05). Compared with H₂O₂ group, the survival rate of Caco-2 cells, TEER value, SOD activity, Occludin, ZO-1 and p-AMPK protein expressions in 10 μmol/L FA group, 20 μmol/L FA group and 40 μmol/L FA group increased in turn (P<0.05), and the LDH activity and MDA expression decreased in turn (P<0.05), but the changes of the above indexes in AMPK inhibitor group showed opposite trend. Confocal microscopy showed that the proteins were evenly distributed along the cell membrane in the control group, in a continuous network structure. In H₂O₂ group, the distribution of ZO-1 and Occludin protein was disorderly; in AMPK inhibitor group, the protein distribution was further disordered and the structure was seriously damaged; in 40 μmol/L FA group, the protein distribution tended to be uniform; in FA+AMPK inhibitor group, the continuity of ZO-1 and Occludin protein distribution was slightly restored.

Conclusion

FA may protect intestinal epithelial barrier function by activating AMPK signaling pathway.

Key words: Ferulic acid, Intestinal epithelial barrier, Adenosine monophosphate activated protein kinase, Tight junction zonula occludens 1, Occludin

Juncheng Wu, Liguo Zhao. Protective effect of ferulic acid regulating AMPK pathway on H2O2-induced intestinal epithelial barrier injury[J]. Chinese Archives of General Surgery(Electronic Edition), 2021, 15(05): 364-369.

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Figures/Tables 5

References 20

[1]	Dokladny K, Zuhl MN, Moseley PL. Intestinal epithelial barrier function and tight junction proteins with heat and exercise[J]. J Appl Physiol (1985), 2016, 120(6): 692-701.
[2]	Odenwald MA, Turner JR. The intestinal epithelial barrier: A therapeutic target?[J]. Nat Rev Gastroenterol Hepatol, 2017, 14(1): 9-21.
[3]	Olivier S, Leclerc J, Grenier A, et al. AMPK activation promotes tight junction assembly in intestinal epithelial Caco-2 cells[J]. Int J Mol Sci, 2019, 20(20): 5171-5183.
[4]	Sun X, Yang Q, Rogers CJ, et al. AMPK improves gut epithelial differentiation and barrier function via regulating Cdx2 expression[J]. Cell Death Differ, 2017, 24(5): 819-831.
[5]	Zduńska K, Dana A, Kolodziejczak A, et al. Antioxidant properties of ferulic acid and its possible application[J]. Skin Pharmacol Physiol, 2018, 31(6): 332-336.
[6]	Cao YJ, Zhang YM, Qi JP, et al. Ferulic acid inhibits H₂O₂-induced oxidative stress and inflammation in rat vascular smooth muscle cells via inhibition of the NADPH oxidase and NF-κB pathway[J]. Int Immunopharmacol, 2015, 28(2): 1018-1025.
[7]	杨涛, 谢克亮, 陈红光, 等. 富氢液对脂多糖刺激离体肠上皮屏障功能障碍的影响[J]. 中华危重病急救医学, 2016, 28(3): 230-234.
[8]	王明星, 张艳秋, 肖旭朗. 基于H₂O₂诱导Caco-2细胞模型的猴头菌多糖抗溃疡性结肠炎活性研究[J]. 时珍国医国药, 2017, 28(10): 2355-2357.
[9]	Ugwu FN, Ho J. Preclinical evidence of sphingosine kinase 1 inhibition in alleviation of intestinal epithelial injury in polymicrobial sepsis[J]. Inflamm Res, 2019, 68(9): 723-726.
[10]	Wong M, Ganapathy AS, Suchanec E, et al. Intestinal epithelial tight junction barrier regulation by autophagy-related protein ATG6/beclin 1[J]. Am J Physiol Cell Physiol, 2019, 316(5): C753-C765.
[11]	Thiagarajah JR, Chang J, Goettel JA, et al. Aquaporin-3 mediates hydrogen peroxide-dependent responses to environmental stress in colonic epithelia[J]. Proc Natl Acad Sci U S A, 2017, 114(3): 568-573.
[12]	Zhong H, Liu M, Ji Y, et al. Genipin reverses HFD-induced liver damage and inhibits UCP2-mediated pyroptosis in mice[J]. Cell Physiol Biochem, 2018, 49(5): 1885-1897.
[13]	Li Y, Tian X, Li S, et al. Total polysaccharides of adlay bran (Coix lachryma-jobi L.) improve TNF-α induced epithelial barrier dysfunction in Caco-2 cells via inhibition of the inflammatory response[J]. Food Funct, 2019, 10(5): 2906-2913.
[14]	Sangsefidi ZS, Yaghoubi F, Hajiahmadi S, et al. The effect of coenzyme Q10 supplementation on oxidative stress: A systematic review and Meta-analysis of randomized controlled clinical trials[J]. Food Sci Nutr, 2020, 8(4): 1766-1776.
[15]	Yang J, Wang Y, Liu H, et al. C2-ceramide influences alveolar epithelial barrier function by downregulating Zo-1, occludin and claudin-4 expression[J]. Toxicol Mech Methods, 2017, 27(4): 293-297.
[16]	Han X, Lee A, Huang S, et al. Lactobacillus rhamnosus GG prevents epithelial barrier dysfunction induced by interferon-gamma and fecal supernatants from irritable bowel syndrome patients in human intestinal enteroids and colonoids[J]. Gut Microbes, 2019, 10(1): 59-76.
[17]	Salin Raj P, Swapna SUS, Raghu KG. High glucose induced calcium overload via impairment of SERCA/PLN pathway and mitochondrial dysfunction leads to oxidative stress in H9c2 cells and amelioration with ferulic acid[J]. Fundam Clin Pharmacol, 2019, 33(4): 412-425.
[18]	Bumrungpert A, Lilitchan S, Tuntipopipat S, et al. Ferulic acid supplementation improves lipid profiles, oxidative stress, and inflammatory status in hyperlipidemic subjects: A randomized, double-blind, placebo-controlled clinical trial[J]. Nutrients, 2018, 10(6): 713-720.
[19]	Xia Z, Huang L, Yin P, et al. L-Arginine alleviates heat stress-induced intestinal epithelial barrier damage by promoting expression of tight junction proteins via the AMPK pathway[J]. Mol Biol Rep, 2019, 46(6): 6435-6451.
[20]	Chen X, Guo Y, Jia G, et al. Ferulic acid regulates muscle fiber type formation through the Sirt1/AMPK signaling pathway[J]. Food Funct, 2019, 10(1): 259-265.

组别	Caco-2细胞存活率（%）	LDH活力（U/10⁴个细胞）
对照组	100.00±0.00	15.24±1.29
H₂O₂组	53.19±2.74^a	63.47±2.06^a
10 μmol/L FA组	65.37±2.22^b	50.64±2.44^b
20 μmol/L FA组	75.19±2.68^bc	33.81±2.63^bc
40 μmol/L FA组	87.37±3.06^bcd	21.04±2.28^bcd
AMPK抑制剂组	34.68±3.05^b	82.47±2.42^b
FA+AMPK抑制剂组	56.87±2.93^e	59.82±2.68^e

组别	Caco-2细胞存活率（%）	LDH活力（U/10⁴个细胞）
对照组	100.00±0.00	15.24±1.29
H₂O₂组	53.19±2.74^a	63.47±2.06^a
10 μmol/L FA组	65.37±2.22^b	50.64±2.44^b
20 μmol/L FA组	75.19±2.68^bc	33.81±2.63^bc
40 μmol/L FA组	87.37±3.06^bcd	21.04±2.28^bcd
AMPK抑制剂组	34.68±3.05^b	82.47±2.42^b
FA+AMPK抑制剂组	56.87±2.93^e	59.82±2.68^e

组别	MDA（nmol/L）	SOD（U/ml）
对照组	1.83±0.16	12.56±0.52
H₂O₂组	4.67±0.22^a	4.08±0.26^a
10 μmol/L FA组	3.81±0.18^b	5.47±0.17^b
20 μmol/L FA组	3.02±0.21^bc	7.33±0.36^bc
40 μmol/L FA组	2.22±0.24^bcd	10.58±0.40^bcd
AMPK抑制剂组	7.26±0.15^b	2.32±0.13^b
FA+AMPK抑制剂组	4.49±0.25^e	3.98±0.22^e

组别	MDA（nmol/L）	SOD（U/ml）
对照组	1.83±0.16	12.56±0.52
H₂O₂组	4.67±0.22^a	4.08±0.26^a
10 μmol/L FA组	3.81±0.18^b	5.47±0.17^b
20 μmol/L FA组	3.02±0.21^bc	7.33±0.36^bc
40 μmol/L FA组	2.22±0.24^bcd	10.58±0.40^bcd
AMPK抑制剂组	7.26±0.15^b	2.32±0.13^b
FA+AMPK抑制剂组	4.49±0.25^e	3.98±0.22^e

组别	p-AMPK/AMPK	Occludin/GAPDH	ZO-1/GAPDH
对照组	0.92±0.09	1.39±0.08	1.22±0.09*
H₂O₂组	0.17±0.01a	0.21±0.04^a	0.18±0.03^a
10 μmol/L FA组	0.46±0.06^b	0.50±0.03^b	0.37±0.02^b
20 μmol/L FA组	0.81±0.05^bc	0.95±0.05^bc	0.83±0.03^bc
40 μmol/L FA组	0.98±0.07^bcd	1.19±0.04^bcd	1.02±0.05^bcd
AMPK抑制剂组	0.06±0.01^b	0.05±0.01^b	0.07±0.01^b
FA+AMPK抑制剂组	0.20±0.04^e	0.22±0.02^e	0.21±0.02^e

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