Objective

2

To explore the effect mechanism of electroacupuncture (EA) for ameliorating diabetic peripheral neuropathy (DPN) based on the analysis of gut microbiota and metabolomics.

Methods

2

Thirty SPF-grade male SD rats were randomly divided into a normal group

a model group

and an EA group

with 10 rats in each one. Except in the normal group

the intraperitoneally injection with streptozotocin was used to induce diabetes mellitus model in the rest groups. In the EA group

acupuncture was delivered at bilateral "Zusanli" (ST36)

"Sanyinjiao" (SP6)

"Pishu" (BL20) and "Shenshu" (BL23)

and electric stimulation was attached to "Zusanli" (ST36)-"Sanyinjiao" (SP6) and "Pishu" (BL20)-"Shenshu" (BL23)

on the same side

with continuous wave and a frequency of 2 Hz

for 10 min in each intervention. The intervention measure of each group was delivered once every 2 days

3 times a week

for 8 consecutive weeks. Body weight

random blood glucose (RBG)

thermal withdrawal latency (TWL)

and mechanical withdrawal threshold (MWT) before intervention

and in 4 and 8 weeks of intervention

separately

as well as sensory nerve conduction velocity (SCV) and motor nerve conduction velocity (MCV) of the sciatic nerve after intervention were measured. Metagenomic sequencing (MS) was used to analyze gut microbiota and screen for differential species. Liquid chromatography-mass spectrometry (LC-MS) was employed to detect the differential metabolites in plasma

and the metabolic pathway enrichment analysis was performed on the differential metabolites. Spearman correlation analysis was adopted to assess the relationship between gut microbiota and metabolomics.

Results

2

After 4 and 8 weeks of intervention

when compared with the model group

the EA group showed the increase in body weight

TWL

MWT (

P

<

0.01)

and the decrease in RBG (

P

<

0.01). Compared with the normal group

SCV and MCV

as well as Chao1 index were dropped in the model group (

P

<

0.01)

and those were elevated in the EA group when compared with those in the model group (

P

<

0.01). The dominant bacterial phyla of each group were

Firmicutes

(F) and

Bacteroidota

(B)

the ratio of them (F/B) in the model group was lower than that of the normal group (

P

<

0.05)

and F/B in the EA group was higher when compared with that in the model group (

P

<

0.05). In comparison with the normal group

the relative abundance increased in

Prevotella

Segatella

Prevotella-hominis

and

Segatella-copri

(

P

<

0.05); and it decreased in

Ligilactobacillus

Eubacterium

Pseudoflavonifractor

Ligilactobacillus-murinus

(

P

<

0.05) in the model group. Compared with the model group

the relevant abundance of the above mentioned gut bacteria was all ameliorated in the EA group (

P

<

0.05

P

<

0.01). Among the three groups

120 differential metabolites were identified and enriched in

28 key metabolic pathways

such as glycerophospholipid and linoleic acid

of which

glycerophospholipid was the most significantly affected pathway in EA intervention. Spearman correlation analysis showed that 6 phosphatidylcholine metabolites were significantly positively correlated with

Pseudoflavonifractor

and were negatively with

Prevotella

Segatella

Prevotella-hominis

Segatella-copri

; 5 phosphatidylethanolamine metabolites were significantly negatively correlated with

Pseudoflavonifractor

and positively correlated with

Prevotella

Segatella

Prevotella-hominis

Segatella-copri

.

Conclusion

2

EA may regulate metabolic pathways such as glycerophospholipid

modulate specific gut microbiota such as

Pseudoflavonifractor

Prevotella

and

Segatella

and the co-expressed differential metabolites like phosphatidylcholine and phosphatidylethanolamine

thereby reducing blood glucose and protecting nerve function

so as to relieve the symptoms of DPN of rats.