绿原酸通过抑制NF-κB/NLRP3通路介导的细胞焦亡改善慢性应激致大鼠肠道损伤

doi:10.11843/j.issn.0366-6964.2025.12.050

摘要/Abstract

摘要：

旨在探究绿原酸(CGA)对慢性应激致大鼠肠道损伤的保护作用及其对核因子-κB/NOD样受体蛋白3(NF-κB/NLRP3)通路介导的细胞焦亡的调控作用，为防治畜牧业应激性肠道疾病提供新策略。本试验选用32只初始体重为(200±20)g的SD大鼠，随机分为对照组(CON组)、绿原酸组(CGA组)、慢性束缚应激组(CRS组)和绿原酸组干预组(CRS+CGA组)。CON组未作干预。CGA组按100 mg·kg^-1剂量灌胃CGA。CRS组每天束缚6 h，连续束缚21 d。CRS+CGA组先灌胃同等剂量CGA后实施与CRS组相同的束缚操作。第22天进行旷场试验以评估模型构建是否成功，第23天处死大鼠并采集血液和回肠样本。测定血清皮质酮(CORT)水平，苏木精-伊红(HE)染色观察回肠病理变化，酶联免疫吸附法(ELISA)测定回肠炎症因子水平，免疫组织化学法(IHC)和实时荧光定量PCR(RT-qPCR)检测紧密连接(TJs)相关蛋白及基因表达水平，蛋白免疫印迹法(Western blot)检测细胞焦亡相关蛋白表达情况。结果显示，慢性应激模型构建成功。CGA干预显著促进慢性应激大鼠的日均体重增长(P < 0.01)，减轻回肠结构损伤，降低炎症因子水平(P < 0.01)，上调TJs相关蛋白及基因咬合蛋白(Occludin)、闭合蛋白3(Claudin3)和闭锁小带蛋白-1(ZO-1)的表达(P < 0.01)，抑制细胞焦亡相关蛋白核因子-κB p65(NF-κB p65)、NLRP3、胱天蛋白酶-1 p20(Caspase-1 p20)和N-消皮素D(N-GSDMD)的表达(P < 0.01)。综上表明，CGA缓解慢性应激诱导的大鼠回肠损伤，其机制可能与抑制NF-κB/NLRP3通路，减轻细胞焦亡，降低炎症因子水平，进而增强肠道屏障功能有关。

关键词: 绿原酸, 慢性应激, 回肠损伤, 肠道屏障功能, 细胞焦亡

Abstract:

The aim of this study was to investigate the protective effect of chlorogenic acid (CGA) against chronic stress-induced intestinal injury in rats and its regulatory role in the nuclear factor-kappa B/NOD-like receptor protein 3 (NF-κB/NLRP3) pathway-mediated pyroptosis, in order to provide new strategies for preventing stress-related intestinal diseases in livestock. Thirty-two Sprague-Dawley (SD) rats with an initial body weight of 200±20 g were randomly divided into four groups: control group (CON group), chlorogenic acid group (CGA group), chronic restraint stress group (CRS group), and chlorogenic acid intervention group (CRS+CGA group). The CON group received no intervention. The with CGA group was administered CGA via gavage at a dose of 100 mg·kg^-1. The CRS group was subjected to 6 h of daily restraint for 21 d consecutively. The CRS+CGA group was gavaged with an equivalent dose of CGA and followed by the same restraint procedures as the CRS group. Open-field test was conducted on day 22 to evaluate the model establishment. Rats were euthanized on day 23 for blood and ileum collection. Serum corticosterone (CORT) levels were measured, ileum histopathology was observed by hematoxylin-eosin (HE) staining, inflammatory cytokine levels in the ileum were detected by enzyme-linked immunosorbent assay (ELISA), tight junction (TJs) proteins and gene expression were analyzed by immunohistochemistry (IHC) and real-time quantitative PCR (RT-qPCR), and pyroptosis-related proteins were assayed by Western blot. The results showed that the chronic stress model was successfully established. CGA intervention significantly promoted average daily body weight gain of rats under chronic stress (P < 0.01), alleviated ileum structural damage, reduced inflammatory cytokine levels (P < 0.01), upregulated TJs-related proteins and gene Occludin, Claudin3, Zonula occludens-1 (ZO-1) expression (P < 0.01), and inhibited pyroptosis-related proteins nuclear factor-kappa B p65 (NF-κB p65), NLRP3, cysteinyl aspartate specific proteinase-1 p20 (Caspase-1 p20), and N-gasdermin D (N-GSDMD) expression (P < 0.01). Collectively, CGA alleviates chronic stress-induced ileum injury in rats, possibly by inhibiting the NF-κB/NLRP3 pathway, attenuating pyroptosis, reducing inflammatory cytokine levels, and thereby enhancing intestinal barrier function.

Key words: chlorogenic acid, chronic stress, ileum injury, intestinal barrier function, pyroptosis

中图分类号:

S856.4

张鹤馨, 曲悠扬, 陈若瑄, 何欢, 唐琦超, 尹柏双, 王奔, 冯秀晶. 绿原酸通过抑制NF-κB/NLRP3通路介导的细胞焦亡改善慢性应激致大鼠肠道损伤[J]. 畜牧兽医学报, 2025, 56(12): 6502-6512.

ZHANG Hexin, QU Youyang, CHEN Ruoxuan, HE Huan, TANG Qichao, YIN Baishuang, WANG Ben, FENG Xiujing. Chlorogenic Acid Ameliorates Chronic Stress-Induced Intestinal Injury in Rats by Inhibiting NF-κB/NLRP3 Pathway-Mediated Pyroptosis[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(12): 6502-6512.

图/表 8

表 1

图 1

图 2

图 3

图 4

图 5

图 6

图 7

参考文献 29

1	ZEESHAN M H , VAKKALAGADDA N P , SREE G S , et al.Irritable bowel syndrome in adults: prevalence and risk factors[J]. Ann Med Surg (Lond),2022,81,104408.
2	郭昶彤. 异戊酸通过NF-κB途径减缓束缚应激引发小鼠肠道炎症的机制探究[D]. 武汉: 华中农业大学, 2024.
	GUO C T. Mechanistic investigation of isovaleric acid alleviating intestinal inflammation induced by restraint stress in mice via the NF-κB pathway[D]. Wuhan: Huazhong Agricultural University, 2024. (in Chinese)
3	蔡志远, 张飞宇, 刘永杰."微生物-肠-脑"轴减轻舍饲期间动物应激的研究进展[J].草原与草坪,2023,43(6):162-176.
	CAI Z Y , ZHANG F Y , LIU Y J .Research progress on the "microbiota-gut-brain" axis in alleviating animal stress during feeding[J].Grassland and Turf,2023,43(6):162-176.
4	WANG Q , YE W , TAO Y , et al.Transport stress induces oxidative stress and immune response in juvenile largemouth bass (micropterus salmoides): analysis of oxidative and immunological parameters and the gut microbiome[J]. Antioxidants (Basel),2023,12(1):157. doi: 10.3390/antiox12010157
5	WANG L , LI W , XIN S , et al.Soybean glycinin and β-conglycinin damage the intestinal barrier by triggering oxidative stress and inflammatory response in weaned piglets[J].Eur J Nutr,2023,62(7):2841-2854. doi: 10.1007/s00394-023-03188-8
6	WU K , WANG Q , ZHANG Z , et al.Honokiol ameliorates pyroptosis in intestinal ischemia-reperfusion injury by regulating the SIRT3-mediated NLRP3 inflammasome[J].Int J Mol Med,2025,55(6):96.
7	SHAO Y , JIANG Y , WANG J , et al.Inhibition of circulating exosomes release with GW4869 mitigates severe acute pancreatitis-stimulated intestinal barrier damage through suppressing NLRP3 inflammasome-mediated pyroptosis[J].Int Immunopharmacol,2024,126,111301. doi: 10.1016/j.intimp.2023.111301
8	梁慧, 赵静, 王妍雅, 等.饲粮中添加绿原酸对热应激母兔繁殖性能及其仔兔生长的影响[J].畜牧兽医学报,2025,56(2):755-764. doi: 10.11843/j.issn.0366-6964.2025.02.025
	LIANG H , ZHAO J , WANG Y Y , et al.Effects of dietary chlorogenic acid on reproductive performance of female rabbits and growth of suckling rabbits under heat stress conditions[J].Acta Veterinaria et Zootechnica Sinica,2025,56(2):755-764. doi: 10.11843/j.issn.0366-6964.2025.02.025
9	ZHAO Y , WANG C , YANG T , et al.Chlorogenic acid alleviates chronic stress-induced intestinal damage by inhibiting the P38MAPK/NF-κB pathway[J].J Agric Food Chem,2023,71(24):9381-9390. doi: 10.1021/acs.jafc.3c00953
10	CHEN J , LUO Y , LI Y , et al.Chlorogenic acid attenuates oxidative stress-induced intestinal epithelium injury by co-regulating the PI3K/Akt and IκBα/NF-κB signaling[J].Antioxidants (Basel),2021,10(12):1915. doi: 10.3390/antiox10121915
11	房尚萍, 孙任珂, 苏慧, 等.绿原酸减轻脓毒症诱导的小鼠急性肾损伤: 基于抑制caspase-1经典细胞焦亡信号通路[J].南方医科大学学报,2024,44(2):317-323.
	FANG S P , SUN R K , SU H , et al.Chlorogenic acid alleviates sepsis-induced acute kidney injury in mice: based on inhibition of the caspase-1 classical pyroptosis signaling pathway[J].Journal of Southern Medical University,2024,44(2):317-323.
12	ZHANG S , HU J , LIU G , et al.Chronic clomipramine treatment increases hippocampal volume in rats exposed to chronic unpredictable mild stress[J].Transl Psychiatry,2022,12(1):245. doi: 10.1038/s41398-022-02006-9
13	COWEN P J .Not fade away: the HPA axis and depression[J].Psychol Med,2010,40,1-4.
14	ZHAO Y , WANG C , YANG T , et al.Chlorogenic acid alleviates chronic stress-induced duodenal ferroptosis via the inhibition of the IL-6/JAK2/STAT3 signaling pathway in rats[J].J Agric Food Chem,2022,70(14):4353-4361. doi: 10.1021/acs.jafc.2c01196
15	CHEN Y , FENG X , HU X , et al.Dexmedetomidine ameliorates acute stress-induced kidney injury by attenuating oxidative stress and apoptosis through inhibition of the ROS/JNK signaling pathway[J].Oxid Med Cell Longevity,2018,2018,4035310. doi: 10.1155/2018/4035310
16	JIN X , MA Y , LIU D , et al.Role of pyroptosis in the pathogenesis and treatment of diseases[J].Med Comm,2023,4(3):e249.
17	AL MAMUN A , WU Y , JIA C , et al.Role of pyroptosis in liver diseases[J].Int Immunopharmacol,2020,84,106489. doi: 10.1016/j.intimp.2020.106489
18	RAO Z , ZHU Y , YANG P , et al.Pyroptosis in inflammatory diseases and cancer[J].Theranostics,2022,12(9):4310-4329. doi: 10.7150/thno.71086
19	HAN Y L , KANG Z X , JIN S W , et al.Electroacupuncture improves low-grade duodenal inflammation in FD rats by reshaping intestinal flora through the NF-κB p65/NLRP3 pyroptosis pathway[J].Heliyon,2024,10(10):e31197. doi: 10.1016/j.heliyon.2024.e31197
20	XUE Y , LI F , LI R , et al.Chlorogenic acid alleviates IPEC-J2 pyroptosis induced by deoxynivalenol by inhibiting activation of the NF-κB/NLRP3/caspase-1 pathway[J].J Anim Sci Biotechnol,2024,15(1):159. doi: 10.1186/s40104-024-01119-z
21	LIU H , MENG H , DU M , et al.Chlorogenic acid ameliorates intestinal inflammation by inhibiting NF-κB and endoplasmic reticulum stress in lipopolysaccharide-challenged broilers[J].Poult Sci,2024,103(5):103586. doi: 10.1016/j.psj.2024.103586
22	CHIKINA AS , NADALIN F , MAURIN M , et al.Macrophages maintain epithelium integrity by limiting fungal product absorption[J].Cell,2020,183(2):411-428. doi: 10.1016/j.cell.2020.08.048
23	OSHIMA T , MIWA H .Gastrointestinal mucosal barrier function and diseases[J]. J Gastroenterol,2016,51(8):768-778. doi: 10.1007/s00535-016-1207-z
24	LANDY J , RONDE E , ENGLISH N , et al.Tight junctions in inflammatory bowel diseases and inflammatory bowel disease associated colorectal cancer[J].World J Gastroenterol,2016,22(11):3117-3126. doi: 10.3748/wjg.v22.i11.3117
25	LI Y , LIU J , PONGKORPSAKOL P , et al.Relief effects of icariin on inflammation-induced decrease of tight junctions in intestinal epithelial cells[J].Front Pharmacol,2022,13,903762. doi: 10.3389/fphar.2022.903762
26	HAN H , YOU Y , CHA S , et al.Multi-species probiotic strain mixture enhances intestinal barrier function by regulating inflammation and tight junctions in lipopolysaccharides stimulated Caco-2 cells[J].Microorganisms,2023,11(3):656. doi: 10.3390/microorganisms11030656
27	FU Y , LI E , CASEY T M , et al.Impact of maternal live yeast supplementation to sows on intestinal inflammatory cytokine expression and tight junction proteins in suckling and weanling piglets[J].J Anim Sci,2024,102,1-10.
28	TAN H , ZHEN W , BAI D , et al.Effects of dietary chlorogenic acid on intestinal barrier function and the inflammatory response in broilers during lipopolysaccharide-induced immune stress[J].Poult Sci,2023,102(5):102623. doi: 10.1016/j.psj.2023.102623
29	LI Y Q , ZHANG Y , BAI D Y , et al.Effects of dietary chlorogenic acid on ileal intestinal morphology, barrier function, immune factors and gut microbiota of broilers under high stocking density stress[J].Front Physiol,2023,14,1169375. doi: 10.3389/fphys.2023.1169375

基因名称 Gene name	引物序列(5′→3′) Primer sequence(5′→3′)	产物长度/bp Product length
β-actin	F：CCAGCCTTCCTTCTTGGGTAT	276
β-actin	R：TGCTGGAAGGTGGACAGTGAG	276
Occludin	F：GTCTTGGGAGCCTTGACATCTTG	174
Occludin	R：GCATTGGTCGAACGTGCATC	174
Claudin3	F：GACAAAGACACCTCGCCCTT	91
Claudin3	R：TGCCCACTATGAGCCTTCTG	91
ZO-1	F：CCATCTTTGGACCGATTGCTG	123
ZO-1	R：TAATGCCCGAGCTCCGATG	123

[1]	王志浩, 郭龙, 王培莉, 李建基, 王亨. 细胞焦亡及其在角膜炎疾病中的作用机制研究进展[J]. 畜牧兽医学报, 2025, 56(3): 1089-1099.
[2]	梁慧, 赵静, 王妍雅, 龙润泽, 刘旭阳, 吴英杰, 刘宁, 秦应和. 饲粮中添加绿原酸对热应激母兔繁殖性能及其仔兔生长的影响[J]. 畜牧兽医学报, 2025, 56(2): 755-764.
[3]	扆妍妍, 孙盼盼, 孙娜, 李宏全. 基于蛋白质组学探讨绿原酸抗玉米赤霉烯酮致卵泡颗粒细胞死亡的作用机制[J]. 畜牧兽医学报, 2025, 56(12): 6477-6486.
[4]	王峥钰, 付春宇, 黄子轩, 高滢, 于泳, 鲁文赓. 脂多糖诱导奶牛子宫内膜上皮细胞焦亡的分析[J]. 畜牧兽医学报, 2025, 56(10): 5232-5238.
[5]	王靖, 关淑文, 赵小博, 王琳玮, 郭刚, 蒋林树. 竹叶黄酮对H₂O₂诱导奶牛乳腺上皮细胞焦亡的保护作用[J]. 畜牧兽医学报, 2025, 56(1): 281-294.
[6]	李美艳, 邢晓莹, 李娜, 张椰莉, 乔宏萍, 武晓英. 甘草多糖对LPS诱导小鼠睾丸炎的调节作用[J]. 畜牧兽医学报, 2024, 55(6): 2741-2750.
[7]	苏依曼, 叶嘉莉, 邱文粤, 章心婷, 庞晓玥, 王荣梅, 唐兆新, 苏荣胜. 积雪草酸通过抑制HMGB1/TLR4/NF-κB通路减轻脂多糖诱导肉鸡肾细胞焦亡[J]. 畜牧兽医学报, 2024, 55(4): 1777-1786.
[8]	何婷, 刘思颖, 全锦雯, 苏伟谦, 黄江利, 李玙萌, 刘忠华, 余文兰. 高硼暴露通过诱导细胞焦亡加剧鸡肝细胞损伤[J]. 畜牧兽医学报, 2024, 55(10): 4747-4759.
[9]	马亚军, 焦智慧, 刘笑凝, 陆翔羽, 刘涛, 王月, 朴晨曦, 王洪斌. 脂肪间充质干细胞对小型猪肝缺血再灌注合并肝切除组织细胞焦亡的影响[J]. 畜牧兽医学报, 2024, 55(1): 355-364.
[10]	陈喜宏, 路桂聪, 王浩磊, 苟少校, 玉永雄, 林涛, 蒋曹德. 利用牛乳腺细胞和小鼠乳腺组织分析异绿原酸C通过NF-κB信号通路对乳腺炎症反应的抑制效应[J]. 畜牧兽医学报, 2023, 54(9): 3931-3940.
[11]	杨梦然, 杨成林, 吴悠, 王思琪, 孔祥祎, 宁灿, 肖文广, 范慧, 邬静, 袁志航. 桔青霉素诱导小鼠肠道屏障功能障碍的机制研究[J]. 畜牧兽医学报, 2023, 54(11): 4805-4816.
[12]	常伟辰, 李帅奇, 李琰, 闫微, 张红英, 王彦彬, 杨明凡, 张昂克. 白头翁散煎剂发酵物对感染猪流行性腹泻病毒仔猪肠道屏障功能的影响[J]. 畜牧兽医学报, 2023, 54(10): 4403-4410.
[13]	梁文清, 刘忠华, 常晓月, 何婷, 陈嘉, 马晓莉, 唐兆新, 余文兰. 长期高铜暴露通过影响线粒体自噬和细胞焦亡诱导大鼠肝组织损伤[J]. 畜牧兽医学报, 2022, 53(12): 4490-4500.
[14]	石玉华, 傅心雨, 郑梦洁, 吕倩, 师福山. 非经典细胞焦亡体外模型的构建[J]. 畜牧兽医学报, 2021, 52(8): 2284-2290.
[15]	卓春柳, 王方方, 钟翠红, 王斌, 石玉祥, 徐美利, 武亚南, 张永英. 甜叶菊绿原酸增强大肠杆菌感染蛋雏鸡免疫力研究[J]. 畜牧兽医学报, 2021, 52(8): 2200-2212.