硫化氢通过P物质介导促进小鼠肺组织的神经源性炎性反应

摘要

目的：探讨硫化氢对小鼠肺组织神经源性炎性反应的影响及其作用机制。方法：饲养8周龄的雄性Balb／c小鼠，分别予以CP‐96345（2．5 mg／kg ，腹腔注射）、辣椒素（50 mg／kg ，皮下注射）或capsazepine（15 mg／kg ，皮下注射）预处理，然后随机腹腔注射0．9％氯化钠溶液或硫氢化钠（NaHS ；10 mg／kg），即分为对照组、NaHS组、CP‐96345＋NaHS组、辣椒素＋NaHS组和capsazepine＋NaHS组。用药1 h后处死小鼠，采用酶联免疫吸附试验（ELISA）法检测血浆P物质以及肺组织匀浆中肿瘤坏死因子‐α（tumor necrosis factor‐alpha ，TNF‐α）和白细胞介素‐1β（interleukine‐1 beta ，IL‐1β）水平，并用四甲基联苯胺反应液测定肺组织匀浆中髓过氧化物酶（myeloperoxidase ，MPO）活性，通过HE染色法观察肺组织的病理学变化。另选8周龄、雄性、前速激肽原A（pre‐pro‐totchykinin A ，PPT‐A）基因敲除小鼠（PPT‐A‐／‐）和相同遗传背景的同龄、野生型Balb／c小鼠（PPT‐A＋／＋），分别随机腹腔注射0．9％氯化钠液或NaHS（2．5 mg／kg），1 h后处死小鼠，检测肺组织匀浆中 MPO活性、TNF‐α和IL‐1β水平，并观察肺组织的病理学变化。结果：NaHS可显著升高小鼠血浆 P物质浓度（ P＜0．05），提高肺组织匀浆中的MPO活性以及TNF‐α和IL‐1β水平（均 P＜0．05），导致肺组织炎性损伤。敲除小鼠编码P物质的PPT‐A基因或者予以CP‐96345、辣椒素或capsazepine预处理阻断P物质信号通路后，NaHS不能显著升高肺组织中MPO活性以及 TNF‐α和IL‐1β水平，与单纯NaHS处理组相比差异有统计学意义（均 P＜0．05）；而且NaHS所致的肺组织损伤也明显减轻。结论：硫化氢可能通过刺激神经末梢纤维释放P物质，促进肺组织发生神经源性炎性反应，最终导致肺组织损伤。%Objective:To investigate the effect of hydrogen sulfide on the neurogenic inflammation in mouse lung tissues ,and its mechanism .Methods:Eight‐week old ,male Balb/c mice were randomly divided into the following groups:control group , NaHS group ,CP‐96345 + NaHS group、capsaicin + NaHS group and capsazepine + NaHS group .The mice were pretreated with vehicle ,CP‐96345 (2 .5 mg/kg ,i .p .) ,capsaicin (50 mg/kg ,s .c .) or capsazepine (15 mg/kg ,s .c .) and then received NaHS (10 mg/kg ,i .p .) or 0 .9% NaCl solution .One hour after intervention ,the mice were sacrificed ,then the concentration of substance P in plasma and the levels of tumor necrosis factor‐alpha (TNF‐α) and interleukine‐1 beta (IL‐1β) in lung homoge‐nate were measured by ELISA assay .Myeloperoxidase (MPO) activity in lung tissues was detected by tetramethyl benzidine (TMB) .The pathological changes of lung tissues were stained with hematoxylin and eosin .On the other hand ,the prepro‐tachykinin‐A (PPT‐A) gene knockout mice (eight‐week old ,female ,PPT‐A‐/‐) and their corresponding wild‐type Balb/c mice (eight‐week old ,female ,PPT‐A+ /+ ) were randomly given 0 .9% NaCl solution or NaHS (10 mg/kg ,i .p .) .One hour after intervention ,the mice were sacrificed ,then MPO activity ,TNF‐α and IL‐1β levels in lung tissues as well as the pathological changes of lung tissues were examined .Results:Hydrogen sulfide donor drug NaHS elevated significantly plasma substance P concentration (P<0 .05) ,lung MPO activity as well as lung TNF‐αand IL‐1βlevels (all P<0 .05) ,resulting in lung inflam‐matory injury .The knockout of PPT‐A gene ,or pretreatment with CP‐96345 ,capsaicin or capsazepine abolished significantly the elevation of lung MPO activity ,lung TNF‐αand IL‐1βlevels ,as well as the lung injury induced by NaHS (all P<0 .05) . Conclusions :Hydrogen sulfide may induce lung inflammatory injury through stimulating the release of substance P in nerve end‐ing s .