Objective

2

To investigate the mechanism of electroacupuncture (EA) at "Zusanli" (ST36) and "Taichong" (LR3) for attenuating duodenal injury in rats with functional dyspepsia (FD) based on ferroptosis mediated by Kelch-like epichlorohydrin-associated protein 1 (Keap1)/nuclear factor E2-related factor 2 (Nrf2)/glutathione peroxidase 4 (GPX4) pathway.

Methods

2

A total of 21 SPF-grade SD male rats were randomly divided into a normal group

a model group and an EA group

7 rats in each one. In the model and EA groups

FD (liver stagnation and spleen deficiency) models were prepared by dual-factor combination method

using mild tail clamping

stimulation and intermittent diet. After model preparation

in the EA group

EA was operated at bilateral "Zusanli" (ST36) and "Taichong" (LR3)

with a disperse-dense wave

a frequency of 2 Hz/100 Hz and for 20 min each session

once a day

and the duration of intervention was composed of 14 days. The general condition of the rats was observed. The gastric emptying rate and intestinal propulsion rate were measured. The pathomorphologic changes of duodenal tissue were observed by HE staining method; the positive expression of Keap1

Nrf2 and GPX4 in duodenal tissue was detected using immunohistochemistry and the positive expression of reactive oxygen species (ROS) in duodenal tissue was detected using immunofluorescence. The levels of ferrous ions (Fe

2+

) in duodenal tissue

and malondialdehyde (MDA)

reduced glutathione (GSH) and superoxide dismutase (SOD) in serum were detected using biochemical methods

ATP level in serum was detected using ELISA

and the mRNA expression of Keap1

Nrf2

and GPX4 in duodenal tissue was detected by real-time fluorescence quantitative PCR (RT-qPCR); and the protein expression of Keap1

Nrf2

GPX4

acyl-CoA synthase long-chain family 4 (ACSL4) and solute carrier family 7 member 11 (SLC7A11) was detected using Western blot.

Results

2

Compared with the normal group

the rats in the model group exhibited the decrease in food intake

body weight

gastric emptying rate and intestinal propulsion rate (

P

<

0.05). In the duodenal tissue

the intestinal villi were disordered

and the inflammatory cells infiltrated. The positive expression of ROS

and the contents of Fe

2+

and MDA were elevated (

P

<

0.05)

the levels of GSH

SOD and ATP decreased (

P

<

0.05)

the mRNA expression of Nrf2 and GPX4 and the protein expression of Nrf2

GPX4 and SLC7A11 were reduced (

P

<

0.05)

while the mRNA expression of Keap1 and the protein expression of Keap1 and ACSL4 were elevated (

P

<

0.05). When compared with the model group

the rats in the EA group showed the increase in food intake

body weight

gastric emptying rate and intestinal propulsion rate (

P

<

0.05). In the duodenal tissue

the structure of the intestinal mucosa was intact basically

without inflammatory cell infiltration in the lamina propria. The positive expression of ROS

and the contents of Fe

2+

and MDA were dropped (

P

<

0.05)

the contents of GSH

SOD and ATP rose (

P

<

0.05)

the mRNA expression of Nrf2 and GPX4 and the protein expression of Nrf2

GPX4 and SLC7A11 were elevated (

P

<

0.05)

while the mRNA expression of Keap1 and the protein expression of Keap1 and ACSL4 were reduced (

P

<

0.05).

Conclusion

2

Electroacupuncture at "Zusanli" (ST36) and "Taichong" (LR3) alleviates lipid peroxidation and oxidative stress injury in the duodenum of FD rats

maintains intracellular iron homeostasis

and inhibits ferroptosis probably by regulating Keap1/Nrf2/GPX4 pathway

so as to attenuate FD.