Objective To observe the protective effect of serum derived from rats undergone auricular electroacupuncture(EA) stimulation on the incubated cerebral microvascular endotheliocytes with diabetic injury so as to investigate the underl-ying mechanism of cholinergic anti-inflammatory action.Methods SD rats were randomized into normal group(n=10)

diabe-tic model group(n=6)

auricular EA group(n=8)

vagotomy+EA group(n=7

received ipsilateral vagotomy before auricular EA stimulation)

atropine+EA group(n=8)

hexamethonium+EA group(n=7) and α-bungarotoxin+EA group(n=7).Diabetic mellitus model was established by feeding the rats with high sugar

high fat forage and intraperitoneal injection of 1% streptozotocin injection(STZ

35 mg/kg).EA was applied to ipsilateral "Yi-Dan"-point and "Er-Shenmen" for 30 min

once daily for 10 days.Atropine(0.1 mg/kg

an anticholinergic drug)

hexamethonium(10 mg/kg

an antagonist of the nicotinic acetylcholine receptors located in sympathetic and parasympathetic ganglia) and α-bungarotoxin(1.0 μg/kg

a type of α-neurotoxin that is known to bind irreversibly and competitively to the nicotinic acetylcholine receptors) were given to the rats by tail venous injection

respectively

before ipsilateral auricular EA intervention

once daily for 10 days.Blood samples from rats of each group were then collected.Normally cultured rat brain microvascular endotheliocytes were randomly divided into the same 7 groups.The diabetic-like damage model of cerebral microvascular endotheliocytes was established in the 6 groups except the normal group by adding the fluid containing glucose(20 mmol/L)

insulin(100 mU/L) and oxidized low density lipoprotein(200 mg/L) to the culture medium.After 48 hours' incubation

the conditional culture solutions were collected for filtration and degerming.Morphological changes and cellular ultra-microstructure were examined using light microscope and transmission electron microscope

respectively.Tumor necrosis factor-alpha(TNF-α) mRNA expression of the cultured microvascular endotheliocytes was assayed using RT-PCR

and the soluble cell adhesion factor-1(sICAM-1) and soluble vascular intercellular adhesion molecule-1(sVCAM-1) concentrations in 1 mL culture fluid were measured using ELISA.Results Compared to the normal control group

the cultured cerebral microvascular endotheliocytes in the model group displayed a cluster-like or floating state

enlargement of the space

and increase of refractivity under light microscope

and showed swelling of the mitochondria with broken cristae and expansion of the space and even with membrane fusion or disappearance under electron microscope.This situation was relatively lighter in both auricular EA and atropine groups

and severe in the vagotomy

hexamethonium and α-bungarotoxin groups.TNF-α mRNA expression and sICAM-1 and sVCAM-1 concentrations were significantly higher in the model group than in the normal group

but significantly lower in both auricular EA group and atropine group than in the model group(P<0.01

P<0.05).No remarkable diffe-rences were found among the model

vagotomy

hexamethonium and α-bungarotoxin groups in the levels of TNF-α mRNA expression and sICAM-1 and sVCAM-1 concentrations(P>0.05).Conclusion Auricular EA intervention rat serum can lighten diabe-tic cellular injury

suppress TNF-α mRNA expression and reduce sICAM-1 and sVCAM-1 concentrations of rat cerebral microvascular endotheliocytes

which is closely associated with the intact vagus nerve and normal nicotinic acetylcholine receptors in rats.