Effects of electroacupuncture at <italic style="font-style: italic">changbing fang</italic> on autophagy of colonic cells and gut microbiota in ulcerative colitis of rats

XU Huichao; WU Tian; HAO Jianheng; WU Ronglin; YAN Bingbei; WANG Haijun; JI Laixi

doi:10.13703/j.0255-2930.20240912-k0003

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Effects of electroacupuncture at changbing fang on autophagy of colonic cells and gut microbiota in ulcerative colitis of rats

Study on Mechanism | 更新时间：2025-05-15

- Effects of electroacupuncture at changbing fang on autophagy of colonic cells and gut microbiota in ulcerative colitis of rats
- Chinese Acupuncture & Moxibustion Vol. 45, Issue 5, Pages: 657-669(2025)
- 作者机构：
  
  山西中医药大学第二临床学院，晋中　030619
- 作者简介：
- 基金信息：
- DOI：10.13703/j.0255-2930.20240912-k0003
  CLC：
- Received：12 September 2024，
  
  Online First：05 February 2025，
  
  Published：12 May 2025
- 稿件说明：
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XU Huichao, WU Tian, HAO Jianheng, et al. Effects of electroacupuncture at changbing fang on autophagy of colonic cells and gut microbiota in ulcerative colitis of rats[J]. Chinese Acupuncture & Moxibustion, 2025, 45(5): 657-669. DOI: 10.13703/j.0255-2930.20240912-k0003.
DOI：

XU Huichao, WU Tian, HAO Jianheng, et al. Effects of electroacupuncture at changbing fang on autophagy of colonic cells and gut microbiota in ulcerative colitis of rats[J]. Chinese Acupuncture & Moxibustion, 2025, 45(5): 657-669. DOI: 10.13703/j.0255-2930.20240912-k0003. DOI：

摘要

目的：

观察电针“肠病方”对溃疡性结肠炎（UC）大鼠结肠细胞自噬、肠道菌群的影响，探讨电针治疗UC的作用机制。

方法：

将32只雄性SD大鼠随机分为对照组、模型组、电针（EA）组、假电针（SEA）组，每组8只。除对照组外，其余组大鼠自由饮用5%葡聚糖硫酸钠（DSS）溶液7 d建立UC大鼠模型。EA组大鼠予电针“肠病方”，穴取“中脘”和双侧“天枢”“上巨虚”，同侧“天枢”与“上巨虚”连接电针，选择疏密波，频率10 Hz/ 50 Hz，每次20 min；SEA组在EA组穴位外侧5 mm处进行浅透皮穿刺，电针参数与电针组相同，均每日1次，连续干预3 d。每日测量大鼠体质量，干预前后评估大鼠疾病活动指数（DAI）评分。干预后，测量大鼠结肠长度；采用HE法观察大鼠结肠组织形态，并评定结肠组织病理学评分；ELISA法检测大鼠血清肿瘤坏死因子-α（TNF-α）、白细胞介素（IL）-1β、IL-2、IL-10含量；透射电镜观察大鼠结肠组织超微结构；Western blot法检测大鼠结肠组织微管相关蛋白1轻链3（LC3）Ⅱ、LC3Ⅰ、自噬相关基因（ATG）5、ATG12、顺序相关泛素结合蛋白1（p62）、磷脂酰肌醇3-激酶（PI3K）、磷酸化蛋白激酶B（p-AKT）、蛋白激酶B（AKT）、磷酸化哺乳动物雷帕霉素靶蛋白（p-mTOR）、哺乳动物雷帕霉素靶蛋白（mTOR）蛋白表达；实时荧光定量PCR法检测大鼠结肠组织PI3K、AKT、mTOR mRNA表达；16S rRNA基因测序分析大鼠粪便肠道菌群结构。

结果：

第1~7天，与对照组比较，模型组、EA组、SEA组体质量降低（

0.05，

0.01）；第9~10天，与模型组和SEA组比较，EA组体质量增加（

0.05，

0.01）。干预前，与对照组比较，模型组、EA组、SEA组DAI评分升高（

0.01）；干预后，与模型组和SEA组比较，EA组DAI评分降低（

0.01）。与对照组比较，模型组结肠长度缩短（

0.01），结肠组织出现畸变隐窝，黏膜层变薄，杯状细胞减少，有炎性细胞浸润，组织整体结构混乱，结肠组织病理学评分升高（

0.01）；血清TNF-α和IL-1β含量升高（

0.01），IL-2和IL-10含量降低（

0.01）；结肠上皮细胞结构紊乱，纤毛脱落，细胞质内有大量空泡，线粒体肿胀、结构不清、部分嵴消失，少见自噬体存在；结肠组织LC3Ⅱ/LC3Ⅰ值及ATG5、ATG12蛋白表达降低（

0.01），p62、PI3K蛋白表达及p-AK

T/AKT、p-mTOR/mTOR值升高（

0.01），PI3K、AKT、mTOR mRNA表达升高（

0.01）；Chao1、Ace、Shannon指数降低（

0.01），门水平上，厚壁菌门（

Firmicutes

）相对丰度降低（

0.05），拟杆菌门（

Bacteroidetes

）和变形菌门（

Proteobacteria

）相对丰度升高（

0.05，

0.01），属水平上，乳杆菌属（

Lactobacillus

）相对丰度降低（

0.05），拉克诺斯皮拉科NK4A136 组（

Lachnospiraceae

_NK4A136_group）和考拉杆菌属（

Phascolarctobacterium

）相对丰度升高（

0.01，

0.05）。与模型组和SEA组比较，EA组结肠长度增加（

0.01），结肠组织炎性细胞浸润减少，肠上皮细胞排列较整齐，有少量脱落，结肠病理组织学评分降低（

0.01）；血清TNF-α和IL-1β含量降低（

0.01），IL-2和IL-10含量升高（

0.01）；结肠上皮细胞排列较整齐，细胞器形态基本正常，可见明显的自噬体；结肠组织LC3Ⅱ/LC3Ⅰ值及ATG5、ATG12蛋白表达升高（

0.01，

0.05），p62、PI3K蛋白表达及p-AKT/AKT、p-mTOR/mTOR值降低（

0.01），PI3K、AKT、mTOR mRNA表达降低（

0.01）；Chao1、Ace、Shannon指数升高（

0.01），门水平上，

Firmicutes

相对丰度升高（

0.01），

Bacteroidetes

相对丰度降低（

0.01），属水平上，

Lactobacillus

相对丰度升高（

0.05），

Lachnospiraceae

_NK4A136_group相对丰度降低（

0.01）。与模型组比较，EA组门水平

Proteobacteria

相对丰度降低（

0.05），属水平

Phascolarctobacterium

相对丰度降低（

0.05）。

结论：

电针“肠病方”可能通过抑制PI3K/AKT/mTOR信号通路调控结肠细胞自噬，并改善肠道菌群，从而缓解UC症状。

Abstract

Objective

To observe the effects of electroacupuncture (EA) at

changbing fang

(prescription for intestinal disease) on autophagy of colonic cells and gut microbiota in rats with ulcerative colitis (UC)

and to explore the mechanism of EA in the treatment of UC.

Methods

Thirty-two SD male rats were randomly divided into a control group

a model group

an EA group and a sham-EA group

with 8 rats in each group. Except the control group

the UC rat model was established by free drinking of 5% dextran sulfate sodium solution for 7 days in the other groups. In the EA group

changbing fang

was adopted

in which

EA was applied at "Tianshu" (ST25) and "Shangjuxu" (ST37)

at disperse-dense wave and frequency of 10 Hz/50 Hz

for 20 min in each intervention. In the sham-EA group

shallow transcutaneous puncture was performed at the sites

5 mm away from the points as the EA group

with the same parameters as the EA group. The intervention was delivered once daily for 3 consecutive days. The body weight was measured daily and the disease activity index (DAI) score was calculated before and after intervention. After intervention completion

the colon length was measured. Using HE staining

the colon morphology was observed and the score of colonic pathology was assessed. With ELISA adopted

the contents of tumor necrosis factor (TNF-α)

interleukin (IL)-1β

IL-2 and IL-10 in the serum of the rats were detected. The ultrastructure of the colon tissue was observed under electron microscopy. Using Western blotting

the protein expression was detected for microtubule-associated protein 1 light chain 3 (LC3)Ⅱ

LC3Ⅰ

autophagy-related genes (ATG) 5

ATG12

sequestosome 1 (p62)

phosphatidylinositol 3-kinase (PI3K)

phosphorylated protein kinase B (p-AKT)

protein kinase B (AKT)

and phosphorylated mammalian target of rapamycin (p-mTOR)

mammalian target of rapamycin (mTOR) in the colon tissue. The mRNA expression of PI3K

AKT and m-TOR in the colon tissue was detected by real-time fluorescence quantitative PCR. The 16S rRNA gene sequencing was used to analyze the structure of gut flora in the feces of rats.

Results

From day 1 to day 7

compared with the control group

the body weight decreased in the model group

EA group

and SEA group (

0.05

0.01). From day 9 to day 10

the EA group showed an increase in body weight compared with the model group and SEA group (

0.05

0.01). Before intervention

the DAI score in the model group

EA group

and SEA group was higher than the score of the control group

respectively (

0.01). After intervention

the DAI score in the EA group was reduced compared with the model group and SEA group (

0.01). Compared with the control group

in the model group

the colon length of rats was shorter (

0.01); it showed the distorted crypts

thinner mucosal layer

reduced goblet cells

inflammatory cell infiltration and the disarranged histological structure; and the pathological score of the colon tissue increased (

0.01); the serum contents of TNF-α and IL-1β increased (

0.01)

and those of IL-2 and IL-10 decreased (

0.01). The structure of colon epithelial cells was disarranged

with cilia pelt off

and a large number of vacuoles in the cytoplasm; the mitochondria were swollen

with unclear structure and cristae partially disappeared; and few autophagosomes were observed. The value of LC3Ⅱ/LC3Ⅰand the protein expression of ATG5 and ATG12 in the colon tissues were reduced (

0.01)

the protein expression of p62 and PI3K

and the values of p-AKT/AKT

and p-mTOR/mTOR increased (

0.01)

and mRNA expression of PI3K

AKT and mTOR was elevated (

0.01). The indexes of Chao1

Ace and Shannon decreased (

0.01). At the phylum level

the relative abundance of

Firmicutes

decreased (

0.05)

that of

Bacteroidetes

and

Proteobacteria

increased (

0.05

0.01). At the genus level

the relevant abundance of

Lactobacillus

decreased

(

0.05)

while that of

Lachnospiraceae

_NK4A136_group and

Phascolarctobacterium

increased (

0.01

0.05 ). Compared with the model group and SEA group

in the EA group

the colon length increased (

0.01)

the infiltration of inflammatory cells was reduced

the arrangement of intestinal epithelial cells was arranged regularly

with a small amount of shedding

and the pathological score of the colon tissue decreased (

0.01). The serum contents of TNF-α and IL-1β decreased (

0.01)

and those of IL-2 and IL-10 increased (

0.01). The colonic epithelial cells were arranged relatively

the morphology of organelles was basically normal

and autophagosomes were visible. The value of LC3Ⅱ/LC3Ⅰand the protein expression of ATG5 and ATG12 in colon tissue increased (

0.01

0.05)

the protein expression of p62 and PI3K

and the values of p-AKT/AKT

and p-mTOR/mTOR decreased (

0.01); and mRNA expression of PI3K

AKT

m-TOR was reduced (

0.01). The indexes of Chao1

Ace and Shannon increased (

0.01). At the phylum level

the relative abundance of

Firmicutes

increased (

0.01)

while that of

Bacteroidetes

decreased (

0.01). At the genus level

the relative abundance of

Lactobacillus

increased (

0.05)

whereas that of

Lachnospiraceae

_NK4A136_group decreased (

0.01). When compared with the model group

the relative abundance of

Proteobacteria

decreased (

0.05)

and that of

Phascolarctobacterium

was reduced (

0.05) in the EA group.

Conclusion

EA at

changbingfang

alleviates UC sympt

oms probably through inhibiting the PI3K/AKT/mTOR signaling pathway to regulate colonic autophagy and improve the intestinal flora.

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Effects of electroacupuncture at changbing fang on autophagy of colonic cells and gut microbiota in ulcerative colitis of rats

DOI：10.13703/j.0255-2930.20240912-k0003

摘要

Abstract

关键词

Keywords

references