辛伐他汀纳米粒通过调节诱导型一氧化氮合酶/内皮型一氧化氮合酶系统对小鼠脓毒症相关急性肺损伤的影响

Effects of simvastatin nanoparticles on septic-associated acute lung injury in mice by regulating inducible nitric oxide synthase/endothelial nitric oxide synthase system

目的探讨辛伐他汀纳米粒对脓毒症相关急性肺损伤小鼠肺组织中诱导型一氧化氮合酶(i NOS)/内皮型一氧化氮合酶(eNOS)平衡的调节以及对脓毒症小鼠预后的影响。方法将90只C57/BL6小鼠分为假手术组、脓毒症组、灌胃组、静脉制剂组及纳米粒组,每组各18只。采用盲肠结扎穿孔术(CLP)建立脓毒症小鼠模型;灌胃组小鼠通过灌胃针,给予辛伐他汀口服制剂灌胃治疗后进行CLP术;静脉制剂组及纳米粒制剂组小鼠CLP术后,立即分别通过尾静脉注射预配置好的辛伐他汀静脉制剂和辛伐他汀纳米粒制剂。其中每组12只小鼠用于7 d生存评估,另外6只用于24 h时间点标本采集。每24小时观察小鼠的生存情况,然后记算各组小鼠每日生存情况。采用苏木素-伊红(HE)染色观察5组小鼠病理变化并计算肺损伤病理评分,免疫组织化学法检测5组小鼠肺组织i NOS、eNOS表达水平。结果 Kaplan-Meier生存曲线结果显示,5组小鼠7 d生存情况比较,差异有统计学意义(χ2=3.780,P <0.001)。进一步两两比较发现,脓毒症组及灌胃组小鼠的7 d生存情况均较假手术组明显下降(P均<0.001),而纳米粒组小鼠的7 d生存情况显著优于脓毒症组(P=0.001)。HE染色结果显示,假手术组小鼠肺组织未见明显病理征象;脓毒症组小鼠肺组织弥漫性中性粒细胞浸润、肺泡腔变小、肺泡间中隔增厚、肺间质弥漫性水肿、细胞排列紊乱、部分肺组织完整性遭破坏;灌胃组小鼠病理所示与脓毒症组相似;静脉制剂组及纳米粒组小鼠中性粒细胞渗出均较脓毒症组损伤减少、肺泡完整性较好、损伤程度较轻。5组小鼠肺损伤病理评分、i NOS及eNOS表达水平比较,差异均有统计学意义(F=889.200、9.633、6.918,P均<0.05)。进一步两两比较发现,脓毒症组小鼠的肺损伤病理评分、i NOS及eNOS表达水平与假手术组比较,差异均有统计学意义(P均<0.05);静脉制剂组及纳米粒组小鼠病理评分、i NOS及eNOS表达水平与脓毒症组比较,差异均有统计学意义(P均<0.05),且纳米粒组小鼠的肺损伤病理评分较静脉制剂组显著降低(P <0.05)。结论不同的辛伐他汀制剂具有不同的效应,其中纳米粒制剂对于脓毒症相关的肺损伤最具保护价值,建立eNOS与i NOS之间的平衡,可以成为具有保护效应的重要处理位点。

Objective To investigate effects of simvastatin nanoparticles on the balance of inducible nitric oxide synthase(iNOS)/endothelial nitric oxide synthase(eNOS)and the prognosis of septic mice with acute lung injury.Methods A total of 90 C57/BL6 mice were divided into the sham operation group,sepsis group,gavage group,intravenous preparation group and nanoparticle group,with 18 mice in each group.The sepsis mouse model was established by cecal ligation and puncture(CLP).Mice in the gavage group received CLP operation after oral administration of simvastatin,and mice in intravenous preparation and nanoparticle groups were immediately injected preconfigured simvastatin intravenous preparations and simvastatin nanoparticles via tail vein respectively after CLP operation.Twelve mice in each group were used for 7-day survival assessment and the other 6 were collected specimen at 24 h time point.The survival of mice was observed every 24 hours,and the daily survival of each group was counted.In the meantime,the pathological changes were observed by hematoxylin-eosin(HE)staining,the pathological score of lung injury was calculated,and the expressions of iNOS and eNOS in lung tissues of 5 groups were detected by the immunohistochemical method.Results The Kaplan-Meier survival curve showed that the survival of mice in 5 groups on 7 days was significantly different(χ^2=3.780,P<0.001).Further comparison showed that the survival rate was significantly lower in sepsis and gavage groups than in the sham operation group(both P<0.001),while it was significantly better in the nanoparticle group than in sepsis group(P=0.001).HE staining results showed that no obvious pathological signs were found in lung tissues of mice in the sham operation group.Diffuse neutrophil infiltration,small alveolar space,thickened interalveolar septum,diffuse pulmonary interstitial edema,disordered cell arrangement and damaged integrity of some lung tissues were found in the sepsis group.The pathological findings in the gavage group were similar to those in sepsis group.Mice in intravenous preparation and nanoparticle groups revealed less neutrophil exudation,better alveolar integrity and lighter degree of injury than those in the sepsis group.The pathological scores of lung injury and expressions of iNOS and eNOS in the 5 groups were significantly different(F=889.200,9.633,6.918;all P<0.05).Further comparison showed that there were significant differences at pathological scores of lung injury and expressions of iNOS and eNOS between sepsis and sham operation groups(all P<0.05).Compared with the sepsis group,pathological scores of lung injury and expressions of iNOS and eNOS in intravenous preparation and nanoparticles groups were significantly different(all P<0.05),and the pathological score of lung injury in nanoparticle group was significantly lower than that in intravenous preparation group(P<0.05).Conclusions Different simvastatin formulations have different effects,among which nanoparticle preparation has the most protective value for septic-related lung injury.Establishing the balance between eNOS and iNOS can be an important treatment site with protective effects.