Objective

2

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

2

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

2

From day 1 to day 7

compared with the control group

the body weight decreased in the model group

EA group

and SEA group (

P

<

0.05

P

<

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 (

P

<

0.05

P

<

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 (

P

<

0.01). After intervention

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

P

<

0.01). Compared with the control group

in the model group

the colon length of rats was shorter (

P

<

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 (

P

<

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

P

<

0.01)

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

P

<

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 (

P

<

0.01)

the protein expression of p62 and PI3K

and the values of p-AKT/AKT

and p-mTOR/mTOR increased (

P

<

0.01)

and mRNA expression of PI3K

AKT and mTOR was elevated (

P

<

0.01). The indexes of Chao1

Ace and Shannon decreased (

P

<

0.01). At the phylum level

the relative abundance of

Firmicutes

decreased (

P

<

0.05)

that of

Bacteroidetes

and

Proteobacteria

increased (

P

<

0.05

P

<

0.01). At the genus level

the relevant abundance of

Lactobacillus

decreased

(

P

<

0.05)

while that of

Lachnospiraceae

_NK4A136_group and

Phascolarctobacterium

increased (

P

<

0.01

P

<

0.05 ). Compared with the model group and SEA group

in the EA group

the colon length increased (

P

<

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 (

P

<

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

P

<

0.01)

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

P

<

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 (

P

<

0.01

P

<

0.05)

the protein expression of p62 and PI3K

and the values of p-AKT/AKT

and p-mTOR/mTOR decreased (

P

<

0.01); and mRNA expression of PI3K

AKT

m-TOR was reduced (

P

<

0.01). The indexes of Chao1

Ace and Shannon increased (

P

<

0.01). At the phylum level

the relative abundance of

Firmicutes

increased (

P

<

0.01)

while that of

Bacteroidetes

decreased (

P

<

0.01). At the genus level

the relative abundance of

Lactobacillus

increased (

P

<

0.05)

whereas that of

Lachnospiraceae

_NK4A136_group decreased (

P

<

0.01). When compared with the model group

the relative abundance of

Proteobacteria

decreased (

P

<

0.05)

and that of

Phascolarctobacterium

was reduced (

P

<

0.05) in the EA group.

Conclusion

2

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.