S100A12在小鼠脓毒症继发急性肺损伤的表达及功能研究

Expression and functional study of S100A12 in sepsis-induced acute lung injury in mice

目的探究S100钙结合蛋白A12(S100A12)在小鼠脓毒症继发急性肺损伤的表达及功能,寻找潜在的急性肺损伤标志物及可能的干预靶点。方法共48只雄性C57BL/6小鼠,采用随机分组法分为两大组:盲肠结扎穿孔(CLP)组、假手术(SHAM)组。两组动物按实验后4、8、12、24 h分为4个亚组,每亚组各6只。CLP小鼠结扎盲肠全长,无菌针头穿孔3个;SHAM组仅做剖腹术。各组在相应时间点对各组小鼠进行麻醉,麻醉方式为腹腔注射戊巴比妥钠。采集小鼠心脏血液0.5 ml左右,制备血清,并采集小鼠右肺组织。酶联免疫吸附测定(ELISA)法检测小鼠血清中S100A12蛋白含量;逆转录聚合酶链反应(RT-PCR)测定小鼠右肺中的S100A12、晚期糖基化终末产物受体(RAGE)、核因子κB(NF-κB)、肿瘤坏死因子α(TNF-α)等mRNA表达水平;苏木精-伊红(HE)染色法对小鼠右肺组织进行病理学观察、病理学评分。结果CLP组小鼠肺部有淤血、肿胀表征,随着实验时间的加长,情况越发严重。小鼠肺部颜色偏向暗红,肺部表面有明显的出血点。SHAM组小鼠肺部组织完整,表面无明显出血点,肺部色泽偏向红润,质地相对柔软。两组小鼠标本有明显的不同。CLP组小鼠血清中的S100A12水平、肺组织病理学评分均在实验后4 h时明显升高,12 h到达峰值,与SHAM组小鼠存在明显差异,差异有统计学意义(P<0.05);CLP组小鼠RAGEmRNA、NF-κBp65 mRNA、TNF-αmRNA相对表达量大于SHAM组,其中RAGE mRNA在实验后8 h开始升高,至12 h到达最高值,NF-κBp65mRNA、TNF-αmRNA相对表达量在实验后4h即明显升高,差异有统计学意义(P<0.05)。结论随着脓毒症的加重,小鼠肺组织受到损坏,肺部出现明显损伤,S100A12、RAGE、NF-κB、TNF-α表达水平上升,S100A12水平与促氧因子、肺部损伤存在正相关,提示S100A12可能与脓毒症后急性肺损伤存在一定的关系。

Objective To investigate the expression and function of S100 calcium bin ding protein A12(S100A12)in sepsis-induced acute lung injury in mice,and to identify potential markers and intervention targets for the treatment of acute lung injury.Methods A total of 48 male C57BL/6 mice were randomly divided into two groups:the cecal ligation and puncture(CLP)group and the sham group(SHAM)group.Two groups of animals were further divided into four subgroups at 4 h,8 h,12 h,and 24 h after the experiment,with 6 animals in each subgroup.The entire length of the cecum in CLP group were ligated,with 3 sterile needle punctures.The SHAM group only underwent laparotomy.Each group of mice was anesthetized at the corresponding time point through intraperitoneal injection of pentobarbital sodium.About 0.5 ml of blood were collected from the mouse heart for preparation of serum.And right lung tissues were collected from the mice.The content of S100A12 protein in mouse serum was detected using the enzyme-linked immunosorbent assay(ELISA)method.The expression level of such mRNA as S100A12,receptor for advanced glycation end products(RAGE),nuclear factor kappa-B(NF-κB)and tumor necrosis factorα(TNF-α)in the right lung of mice was determined using the reverse transcription polymerase chain reaction(RT-PCR)method,And hematoxylin-eosin(HE)staining method was used to observe and obtain histopathologic score of the right lung tissues in mice.Results The mice in CLP group showed signs of congestion and swelling in the lungs,which worsened as the experimental time increased.The lung color tended to be dark red,and there were obvious bleeding spots on the surface of lungs.The lung tissues of the mice in SHAM group were intact,with no obvious bleeding spots on the surface of lungs.The lung color tended to be reddish,with relatively soft tissue texture.There were significant differences between the two groups of mouse specimens.The S100A12 level in the m ice serum and lung his topathology score in the CL P group significantly increased at 4 h and reached a peak at 12 h,which were significantly different from the condition in the mice serum in the SHAM group,and the difference was statistically significant(P<0.05).The relative expression level of RAGEmRNA,NF-κB p65 mRNA and TNF-α mRNA in the CLP group was higher than that in the SHAM group,with RAGEmRNA increasing after 8 h of the experiment and reaching its peak value at 12 h.The relative expression level of NF-κB p65 mRNA and TNF-α mRNA significantly increased after 4 h of the experiment,and the difference was statistically significant(P<0.05).Conclusion As sepsis worsens,the lung tissue of mice is damaged,causing significant lung damages.The expression level of S100A12,RAGE,NF-κB and TNF-αwill increase,and S100A12 level is positively correlated with inflammatory factors and lung injury,indicating that S100A12 may have a certain relationship with se psis-induced acute lung injury.