黄芪对大黄诱导大鼠腹泻的治疗作用及其机制

doi:10.13481/j.1671-587X.20220508

摘要/Abstract

摘要：

目的探讨黄芪对于大黄诱导腹泻大鼠肠道炎症、肠道屏障和肠道菌群的治疗作用，并阐明其可能的作用机制。方法 50只大鼠随机分为对照组、模型组、阳性对照组（2.468 g·kg^－1参苓白术散）、低剂量（1.35 g·kg^－1）黄芪组和高剂量（2.70 g·kg^－1）黄芪组，每组10只。采用大黄灌胃结合饮食不节复制大鼠腹泻模型，检测各组大鼠体质量、粪便含水量和腹泻评分；HE染色观察各组大鼠结肠组织病理形态表现，Western blotting法检测各组大鼠结肠组织中蛋白酶激活受体2（PAR-2）、磷酸化p38（p-p38）、肌球蛋白轻链激酶（MLCK）、磷酸化肌球蛋白轻链（p-MLC）、闭锁蛋白1（ZO-1）、闭合蛋白（Occludin）、Toll样受体4（TLR4）、肿瘤坏死因子受体相关因子6（TRAF6）、髓样分化因子88（MyD88）和核因子κB（NF-κB）蛋白表达水平；采用酶联免疫吸附测定（ELISA）法检测各组大鼠血清中胃动素（MTL）、胃泌素（GAS）、肿瘤坏死因子α（TNF-α）、白细胞介素1β（IL-1β）、白细胞介素6（IL-6）、白细胞介素10（IL-10）、丙二醛（MDA）和分泌型免疫球蛋白A（SIgA）水平及超氧化物歧化酶（SOD）活性；16S r DNA 基因测序分析各组大鼠肠道菌群情况。结果与对照组比较，模型组大鼠体质量明显降低（P<0.01），粪便含水量和腹泻评分明显升高（P<0.01），血清中MTL和GAS水平明显升高（P<0.01）；与模型组比较，阳性对照组、低剂量黄芪组和高剂量黄芪组大鼠体质量明显升高（P<0.01），粪便含水量和腹泻评分明显降低（P<0.01），血清中MTL和GAS水平明显降低（P<0.01）。HE染色，与对照组比较，模型组大鼠结肠组织上皮黏膜损伤，有大量炎症细胞浸润，腺体排列紊乱；与模型组比较，阳性对照组和高剂量黄芪组大鼠结肠组织上皮黏膜结构较规则，炎症细胞浸润较少，未见明显腺体紊乱，低剂量黄芪组大鼠结肠组织上皮黏膜结构可见轻微损伤，有部分炎症细胞浸润，腺体排列较整齐。Western blotting法检测，与对照组比较，模型组大鼠结肠组织中PAR-2、MLCK、TLR4、TRAF6、MyD88、NF-κB、p-p38和p-MLC蛋白表达水平明显升高（P<0.01），ZO-1和Occludin蛋白表达水平明显降低（P<0.01）；与模型组比较，阳性对照组、低剂量黄芪组和高剂量黄芪组大鼠结肠组织中PAR-2、MLCK、TLR4、TRAF6、MyD88、NF-κB、p-p38和p-MLC蛋白表达水平明显降低（P<0.05或P<0.01），ZO-1和Occludin蛋白表达水平明显升高（P<0.01）。与对照组比较，模型组大鼠血清中TNF-α、IL-1β、IL-6和MDA水平明显升高（P<0.01），IL-10和SIgA水平及SOD活性明显降低（P<0.01）；与模型组比较，阳性对照组、低剂量黄芪组和高剂量黄芪组大鼠血清中TNF-α、IL-1β、IL-6和MDA水平明显降低（P<0.01），IL-10和SIg A水平及SOD活性明显升高（P<0.01）。肠道菌群检测，在门水平上，与对照组比较，模型组大鼠肠道菌群中变形菌门和疣微菌门丰度明显升高（P<0.01）；与模型组比较，低和高剂量黄芪组大鼠肠道菌群中变形菌门和疣微菌门丰度明显降低（P<0.01），高剂量黄芪组大鼠肠道菌群中ε-变形菌门丰度明显升高（P<0.01）。结论黄芪对大黄诱导的腹泻模型大鼠有治疗作用，其机制可能与通过TLR4/TRAF6/NF-κB信号通路抑制肠道炎症，进而修复肠道屏障，恢复肠道菌群组成有关。

关键词: 黄芪, 肠道屏障, 肠道炎症, 肠道菌群, 革兰阴性菌

Abstract:

Objective To investigate the therapeutic effects of Astragali Radix on the intestinal inflammation， intestinal barrier and intestinal flora in the rats with rhubarb-induced diarrhea，and to elucidate the possible mechanisms. Methods Fifty rats were randomly divided into control group， model group， positive control group （2.468 g·kg^－1 Shenlingbaizhu Powder）， low dose（1.35 g·kg^－1） of Astragali Radix group， and high dose（2.70 g·kg^－1） of Astragali Radix group，with 10 rats in each group. The rat diarrhea models were replicated by rhubarb gavage in combination with diet indiscipline.The body weights， fecal water contents and diarrhea scores of the rats in various groups were detected； HE staining was used to observe the pathomorphology of colon tissue of the rats in various groups， and Western blotting method was used to detect the expression levels of protease activated receptor-2（PAR-2），phosphorylated p38 （p-p38）， myosin light chain kinase （MLCK）， phosphorylated myosin light chain （p-MLC）， zonula occludens-1（ZO-1），Occludin，Toll-like receptor 4 （TLR4），tumor necrosis factor receptor-associated factor 6（TRAF6）， myeloid differentiation factor 88 （MyD88）， and nuclear factor-κB （NF-κB） proteins in colon tissue of the rats in various groups；the levels of motilin （MTL）， gastrin （GAS）， tumor necrosis factor-α （TNF-α）， interleukin-1β （IL-1β）， interleukin-6 （IL-6），interleukin-10 （IL-10），malondialdehyde （MDA），and secretory immunoglobulin A （SIgA） and the activities of superoxide dismutase （SOD） in serum of the rats in various groups were measured by enzyme linked immunosorbent assay（ELISA） method；16S r DNA gene sequencing was performed to analyze the intestinal flora of the rats in various groups. Results Compared with control group，the body weight of the rats in model group was significantly decreased（P<0.01）， the fecal water content and diarrhea score were significantly increased （P<0.01），and the levels of MTL and GAS in serum were significantly increased （P<0.01）； compared with model group，the body weights of the rats in positive control and low and high doses of Astragali Radix groups were significantly increased（P<0.01）， the fecal water contents and diarrhea scores were significantly decreased （P<0.01），and the levels of MTL and GAS in serum of the rats in were significantly decreased （P<0.01）. The HE staining results showed that compared with control group， the colonic epithelial mucosa of the rats in model group was damaged， with a large number of infiltrated inflammatory cells and disordered glands. Compared with model group， the mucosal structures of the epithelium in positive control and high dose of Astragali Radix groups were more regular， with less inflammatory cell infiltration and without obvious glandular disorder； the mucosal structure of the colonic epithelium in low dose of Astragali Radix group had slight mucosal damage， with partial inflammatory cell infiltration and more neatly arranged glands.The Western blotting results showed that compared with control group，the expression levels of PAR-2，MLCK，TLR4，TRAF6，MyD88，NF-κB，p-p38 and p-MLC proteins in colon tissue of the rats in model group were significantly increased （P<0.01），and the expression levels of ZO-1 and Occludin proteins in colon tissue of the rats in model group were significantly decreased （P<0.01）； compared with model group，the expression levels of PAR-2， MLCK， TLR4， TRAF6， MyD88， NF-κB，p-p38 and p-MLC proteins in colon tissue of the rats in positive control group，low dose of Astragali Radix group and high dose of Astragali Radix group were significantly decreased （P<0.05 or P<0.01）， and the expression levels of ZO-1 and Occludin proteins in colon tissue of the rats in positive control group， low dose of Astragali Radix group and high dose of Astragali Radix group were significantly increased （P<0.05 or P<0.01）.Compared with control group，the levels of TNF-α， IL-1β， IL-6 and MDA in serum of the rats in model group were significantly increased （P<0.01），the levels of IL-10， and SIgA in serum and the SOD activity of the rats were significantly decreased（P<0.01）； compared with model group， the levels of TNF-α， IL-1β， IL-6 and MDA in serum of the rats in positive control group， low dose of Astragali Radix group and high dose of Astragali Radix group were significantly decreased （P<0.01）， and the levels of IL-10 and SIgA in serum and the SOD activities were significantly increased （P<0.01）.At the level of phylum，the intestinal flora detection results showed that compared with control group， the abundance of Proteobacteria and Verrucomicrobia in model group was significantly increased （P<0.01）； compared with model group， the abundance of Proteobacteria and Verrucomicrobia in low and high doses of Astragali Radix groups was significantly decreased （P<0.01）， and the abundance of Epsilonbacteraeota in intestinal flora in high dose of Astragali Radix group was significantly increased （P<0.01）. Conclusion Astragali Radix has a therapeutic effect in the rhubarb-induced diarrhea model rats， and its mechanism may be related to inhibiting intestinal inflammation through TLR4/TRAF6/NF-κB signaling pathway， thereby repairing the intestinal barrier and restoring the composition of intestinal flora.

Key words: Astragali Radix, Intestinal barrier, Intestinal inflammation, Intestinal flora, Gram-negative bacillus

中图分类号:

R285.5

庄雪峰,律广富,林贺,黄晓巍,周佳,李禹墨,赵嘉睿,林喆,王雨辰. 黄芪对大黄诱导大鼠腹泻的治疗作用及其机制[J]. 吉林大学学报(医学版), 2022, 48(5): 1156-1166.

Xuefeng ZHUANG,Guangfu LYU,He LIN,Xiaowei HUANG,Jia ZHOU,Yumo LI,Jiarui ZHAO,Zhe LIN,Yuchen WANG. Therapeutic effect and its mechanism of Astragali Radix on rhubarb-induced diarrhea in rats[J]. Journal of Jilin University(Medicine Edition), 2022, 48(5): 1156-1166.

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Group	MTL	GAS
Control	64.11±5.29	63.79±2.29
Model	83.03±3.36^*	49.20±1.51^*
Positive control	73.84±6.64^△	59.67±2.01^△
High dose of Astragali Radix	75.06±4.89^△	56.30±1.97^△
Low dose of Astragali Radix	75.23±3.02^△	55.40±2.32^△

Group	Firmicutes	Bacteroidetes	Proteobacteria	Verrucomicrobia	Thaumarchaeota	Epsilonbacteraeota
Control	46.98±13.53	50.14±13.78	1.18±0.49	0.04±0.02	0.02±0.01	0.08±0.05
Model	47.81±7.42	29.35±6.73	8.35±3.91^*	11.54±8.33^*	0.01±0.01	0.03±0.02
Low dose of Astragali Radix	48.08±8.08	38.84±15.14	7.61±13.86^△	0.69±1.74^△	0.88±0.85	0.12±0.09^△
High dose of Astragali Radix	52.46±15.09	38.44±16.06	1.87±0.90^△	0.04±0.03^△	0.46±0.44	0.03±0.03

Group	TNF-α[ρ_B/（ng·L^－1）]	IL-1β[ρ_B/（μg·L^－1）]	IL-6[ρ_B/(ng·L^－1)]	IL-10[ρ_B/(ng·L^－1)]
Control	78.58±5.15	26.12±2.91	75.20±19.95	10.54±0.88
Model	90.88±3.26^*	34.08±3.20^*	240.10±15.37^*	8.20±0.52^*
Positive control	79.95±5.85^△	27.73±2.21^△	93.24±18.04^△	10.13±0.91^△
Low dose of Astragali Radix	80.86±6.70^△	27.47±1.15^△	143.81±11.19^△	9.80±0.58^*△
High dose of Astragali Radix	81.54±4.54^△	27.28±1.98^△	118.76±15.88^△	10.10±0.74^△

Group	MDA[ρ_B /(g·L^－1)]	SIgA［ρ_B /（mg·L^－1）］	SOD[λ_B /(U·mL^－1)]
Control	9.45±1.56	6.38±0.08	6.17±0.59
Model	11.94±0.85^*	4.47±0.02^*	3.97±0.76^*
Positive control	9.63±1.77^△	6.28±0.05^△	5.59±1.12^△
Low dose of Astragali Radix	10.81±1.32^△	5.77±0.04^△	5.08±0.67^△
High dose of Astragali Radix	9.98±1.13^△	5.90±0.05^△	5.29±0.85^△

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