Objective

2

To investigate the effects of moxibustion at "Tianshu" (ST25) on intestinal inflammation in mice with Crohn's disease (CD)

and to explore the mechanism of p53 regulating the SLC7A11/GSH/GPX4 axis on intestinal ferroptosis lipid peroxidation in CD mice.

Methods

2

Fifty SPF-grade C57BL/6 wild-type mice were randomly divided into a blank group (10 mice) and a modeling group (40 mice). The modeling group was given 2% dextran sulfate sodium (DSS) solution prepared with distilled water to establish a CD mouse model. One mouse was randomly selected from the blank group and four mice were randomly selected from the modeling group for model comparison and evaluation. After confirming successful modeling

the modeling group was divided into a model group

a moxibustion group

an activator group

and an inhibition group

with 9 mice in each group

and 9 mice in the blank group. In the moxibustion group

mice were treated with thin moxa stick at bilateral "Tianshu" (ST25) for 15 min each time

once a day

for 14 consecutive days. The activator group received the same moxibustion intervention as the moxibustion group

and at the same time

RITA (10 mg/kg)

an agonist

was injected subcutaneously around "Tianshu" (ST25) once a day for 14 consecutive days. The inhibition group was injected subcutaneously with the inhibitor Pifithrin-α (2.2 mg/kg) around "Tianshu" (ST25) once a day for 14 consecutive days. Before and after intervention

the general condition

body weight

and disease activity index (DAI) of each group were observed. After intervention

HE staining was used to observe the colonic tissue morphology of mice. ELISA was used to detect the serum contents of tumor necrosis factor-α (TNF-α)

interleukin (IL)-1β

IL-17

and IL-6

as well as the contents of 4-hydroxynonenal (4-HNE)

malondialdehyde (MDA)

GSH

superoxide dismutase (SOD)

and ferrous ion (Fe

2+

) in colonic tissue. Western blot was used to detect the protein expressions of p53

SLC7A11

and GPX4 in colonic tissue.

Results

2

Compared with the blank group

the mice in the model group showed dry and dull fur

huddled posture

sluggish responses

reduced food intake

mucus and purulent blood around the anus and in the cages

loose and mushy stools

and positive fecal occult blood tests; the body weight was decreased (

P

<

0.05)

and the DAI scores were increased (

P

<

0.05); the structural integrity of the colonic tissue was destroyed

with swollen and disorganized glands

a large number of infiltrating inflammatory cells

and epithelial rupture and loss; the serum levels of TNF-α

IL-1β

IL-17

and IL-6 were elevated (

P

<

0.05); the contents of 4-HNE

MDA

and Fe

2+

and the protein expression of p53 in colonic tissue were increased (

P

<

0.05)

while

the contents of SOD and GSH

and the protein expressions of SLC7A11 and GPX4 were decreased (

P

<

0.05). Compared with the model group

the mice in the moxibustion group

inhibitor group

and activator group showed alleviated symptoms; the body weight was increased (

P

<

0.05)

and DAI scores were decreased (

P

<

0.05). In the moxibustion and inhibitor groups

the colonic epithelial continuity was restored

with mildly swollen mucosa

orderly arranged glands

and only a few scattered inflammatory cells. In the activator group

the swelling of the colonic mucosa was milder than that in the model group

but the improvement was less than that observed in the moxibustion and inhibitor groups

and epithelial continuity was not restored. The serum levels of TNF-α

IL-1β

IL-17

and IL-6 in the moxibustion group and inhibitor group were decreased (

P

<

0.05); the contents of 4-HNE

MDA

Fe

2+

and protein expression of p53 in colonic tissue were decreased (

P

<

0.05); the levels of GSH

SOD

and the protein expressions of SLC7A11 and GPX4 were increased (

P

<

0.05). In the activator group

the serum levels of TNF-α

IL-1β

and IL-17 were decreased (

P

<

0.05); the contents of MDA

Fe

2+

and protein expressions of p53 in the colonic tissue were decreased (

P

<

0.05); and the content of GSH and protein expressions of SLC7A11 and GPX4 were increased (

P

<

0.05). Compared with the moxibustion group

the serum levels of TNF-α

IL-1β

IL-17

and IL-6

as well as the contents of 4-HNE and protein expression of p53

were increased in the activator and inhibitor groups (

P

<

0.05); the body weight

the contents of SOD and GSH

and the protein expressions of SLC7A11 and GPX4 in colonic tissue were decreased (

P

<

0.05). The contents of MDA and Fe

2+

in colonic tissue in the activator group were increased (

P

<

0.05). Compared with the inhibitor group

the serum level of IL-6

the contents of 4-HNE and MDA

and protein expression of p53 in colonic tissue in the activator group were elevated (

P

<

0.05)

while the body weight

the contents of SOD and GSH

and protein expression of GPX4 in colonic tissue were reduced (

P

<

0.05).

Conclusion

2

Moxibustion at "Tianshu" (ST25) could alleviate intestinal inflammation in CD mice

possibly by downregulating the protein expression of p53

enhancing the activity of the SLC7A11/GSH/GPX4 pathway

reducing the production of lipid peroxides

maintaining intestinal iron homeostasis

and reducing colonic ferroptosis.