RESUMEN
Puerarin, a major isoflavonoid derived from the Chinese medical herb radix puerariae (Gegen), has been reported to inhibit neuronal apoptosis and play an anti-inflammatory role in focal cerebral ischemia model rats. Recent findings regarding stroke pathophysiology have recognized that anti-inflammation is an important target for the treatment of ischemic stroke. The cholinergic anti-inflammatory pathway is a highly robust neural-immune mechanism for inflammation control. This study was to investigate whether activating the cholinergic anti-inflammatory pathway can be involved in the mechanism of inhibiting the inflammatory response during puerarin-induced cerebral ischemia/reperfusion in rats. Results showed that puerarin pretreatment (intravenous injection) reduced the ischemic infarct volume, improved neurological deficit after cerebral ischemia/reperfusion and decreased the levels of interleukin-1ß, interleukin-6 and tumor necrosis factor-α in brain tissue. Pretreatment with puerarin (intravenous injection) attenuated the inflammatory response in rats, which was accompanied by janus-activated kinase 2 (JAK2) and signal transducers and activators of transcription 3 (STAT3) activation and nuclear factor kappa B (NF-κB) inhibition. These observations were inhibited by the alpha7 nicotinic acetylcholine receptor (α7nAchR) antagonist α-bungarotoxin (α-BGT). In addition, puerarin pretreatment increased the expression of α7nAchR mRNA in ischemic cerebral tissue. These data demonstrate that puerarin pretreatment strongly protects the brain against cerebral ischemia/reperfusion injury and inhibits the inflammatory response. Our results also indicated that the anti-inflammatory effect of puerarin may partly be mediated through the activation of the cholinergic anti-inflammatory pathway.
RESUMEN
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 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 alpha-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 alpha-bungarotoxin (1.0 microg/kg, a type of alpha-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-alpha) 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 alpha-bungarotoxin groups. TNF-alpha 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 alpha-bungarotoxin groups in the levels of TNF-alpha mRNA expression and sICAM-1 and sVCAM-1 concentrations (P > 0 05). CONCLUSION: Auricular EA intervention rat serum can lighten diabetic cellular injury, suppress TNFalpha mRNA expression and reduce ICAM-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.