针刺研究2012，Vol.37Issue(6)：440-446,7.
电针耳甲区对糖尿病大鼠脑微血管内皮细胞肿瘤坏死因子-α mRNA、细胞黏附因子-1和血管间黏附分子-1蛋白表达的影响
Effects of Serum Derived from Rats Undergone Auricular Acupuncture Intervention on Expression of TNF-α mRNA, Cell Adhesion Factor-1 and Vascular Intercellular Adhesion Molecule-1 Proteins of Incubated Cerebral Microvascular Endotheliocytes with Diabetic I
摘要
Abstract
Objective To observe the protective effect o1 serum derived from rats undergone auricular electroacupunc-ture (EA) stimulation on the incubated cerebral microvascular endotheliocytes with diabetic Injury so as to investigate the underlying mechanism of cholinergic anti-inflammatory action. Methods SD rats were randomized into normal group (n = 10), diabetic 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 oc-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 % strep-tozotocin 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), nexamethonium (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-oc) mRNA expression of the cultured microvascular endotheliocytes was assayed using RT-PCR, and the soluble cell adhesion factor-1 (slCAM-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 a-bungarotoxin groups. TNF-oc mRNA expression and slCAM-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 a-bungarotoxin groups in the levels of TNF-a mRNA expression and slCAM-1 and sVCAM-1 concentrations (P>0. 05). Conclusion Auricular EA intervention rat serum can lighten diabe-tic cellular injury, suppress TNF-a mRNA expression and reduce slCAM-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.关键词
耳针/糖尿病损伤/脑微血管内皮细胞/胆碱能抗炎通路Key words
Auricular acupuncture/Diabetic injury/Cerebral microvascular endotheliocytes/Cholinergic anti-inflammatory pathway分类
医药卫生引用本文复制引用
梅志刚,曾永保,王明智,刘晓洁,肖长义,黎家华..电针耳甲区对糖尿病大鼠脑微血管内皮细胞肿瘤坏死因子-α mRNA、细胞黏附因子-1和血管间黏附分子-1蛋白表达的影响[J].针刺研究,2012,37(6):440-446,7.基金项目
国家自然科学基金项目(30801486)、三峡大学博士科研启动项目(0620070106). （30801486）
