阿魏酸调控AMPK通路对过氧化氢诱导的肠上皮屏障损伤的保护作用研究

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

目的

观察阿魏酸（FA）对过氧化氢（H₂O₂）诱导的肠上皮屏障损伤保护作用，并初步探讨其作用机制。

方法

体外培养Caco-2细胞，建立肠上皮屏障模型，随机分为对照组、H₂O₂组、10 μmol/L FA组、20 μmol/L FA组、40 μmol/L FA组、单磷酸腺苷活化蛋白激酶（AMPK）抑制剂组、FA+AMPK抑制剂组。噻唑蓝（MTT）实验和乳酸脱氢酶（LDH）法检测Caco-2细胞存活和损伤情况；跨上皮电阻（TEER）测定各组评估肠上皮屏障功能变化；丙二醛（MDA）和超氧化物歧化酶（SOD）试剂盒检测各组肠上皮屏障氧化应激状态变化；Western blotting法检测各组肠上皮屏障闭塞蛋白（Occludin）、紧密连接小带闭塞蛋白1（ZO-1）和p-AMPK蛋白表达情况；免疫荧光法观察各组肠上皮屏障中Occludin和ZO-1蛋白分布及变化情况。

结果

与对照组相比，H₂O₂组Caco-2细胞存活率、TEER值、SOD活性、Occludin、ZO-1和p-AMPK蛋白表达显著降低（P<0.05），LDH活力、MDA表达显著升高（P<0.05）；与H₂O₂组相比，10 μmol/L FA组、20 μmol/L FA组和40 μmol/L FA组Caco-2细胞存活率、TEER值、SOD活性、Occludin、ZO-1和p-AMPK蛋白表达依次升高（P<0.05），LDH活力、MDA表达依次降低（P<0.05），AMPK抑制剂组上述指标变化呈完全相反趋势。共聚焦显微镜观察显示，对照组ZO-1和Occludin蛋白沿细胞膜均匀分布，呈连续网状结构；H₂O₂组ZO-1和Occludin蛋白分布杂乱；AMPK抑制剂组蛋白分布进一步杂乱，结构破坏严重；40 μmol/L FA组ZO-1和Occludin蛋白分布趋于均匀；FA+AMPK抑制剂组ZO-1和Occludin蛋白分布连续性稍有恢复。

结论

FA可能通过激活AMPK信号通路，发挥对肠上皮屏障功能的保护作用。

关键词: 阿魏酸, 肠上皮屏障, AMP激活蛋白激酶, 紧密连接小带闭塞蛋白1, 闭塞蛋白

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

表1 各组Caco-2细胞存活率和LDH活力变化情况（±s，n=6）

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

表1 各组Caco-2细胞存活率和LDH活力变化情况（±s，n=6）

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

表2 FA作用后H₂O₂诱导的肠上皮屏障损伤MDA和SOD表达变化（±s，n=6）

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

表2 FA作用后H₂O₂诱导的肠上皮屏障损伤MDA和SOD表达变化（±s，n=6）

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

图1 各组肠上皮屏障损伤模型Occludin、ZO-1和AMPK蛋白表达电泳图　A为对照组；B为H₂O₂组；C为10 μmol/L FA组；D为20 μmol/L FA组；E为40 μmol/L FA组；F为AMPK抑制剂组；G为FA+AMPK抑制剂组

表3 各组肠上皮屏障损伤模型Occludin、ZO-1和AMPK蛋白表达（±s，n=6）

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

表3 各组肠上皮屏障损伤模型Occludin、ZO-1和AMPK蛋白表达（±s，n=6）

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

图2 免疫荧光法观察肠上皮屏障损伤模型ZO-1和Occludin蛋白表达和分布情况（×400） A为对照组；B为H₂O₂组；C为40 μmol/L FA组；D为AMPK抑制剂组；E为FA+AMPK抑制剂组

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