亚低温对脓毒症小鼠肺组织高迁移率族蛋白B1表达的影响

Effects of mild hypothermia on the expression of high mobility group protein B1 in lung tissues of septic mice

目的探讨自发性亚低温和干预性亚低温对脓毒症小鼠肺组织高迁移率族蛋白B1(high mobility group box 1,HMGB1)表达的影响。方法120只BALB/C小鼠(SPF级),随机编号,将号码为10的整数倍的小鼠共12只作为对照(normal control,NC)组,其余108只小鼠作为脓毒症组,以腹腔注射脂多糖(lipopolysaccharide,LPS)10 mg/kg的方法建立脓毒症小鼠模型,NC组给予同等剂量的生理盐水。脓毒症组造模成功1 h后测量肛温,按T≤36℃和>36℃脓毒症小鼠分为自发性亚低温组和非亚低温组;在自发性亚低温组中,T<34℃的小鼠予以剔除,将剩余的脓毒症小鼠随机分成自发性亚低温观察(naturally occurring mild hypothermia,NOMH)组和保持正常体温(keep normothermia,KN)组,其中NOMH组不给予预热干预,而KN组放在保温箱保持肛门温度在36.0~37.5℃之间;非亚低温组脓毒症小鼠随机分成非亚低温观察(nonhypothermia,NH)组和人工获得性亚低温(artificial mild hypothermia,ATMH)组,NH组不给予降温治疗,而ATMH组给予物理降温法降温,使小鼠肛温维持在34~36℃。各组中分别在建模后6、12 h随机选取4只小鼠,采用酶联免疫吸附试验(enzyme-linked immunosorbent assay,ELISA)检测小鼠血清肿瘤坏死因子α(tumor necrosis factor-α,TNF-α)、白细胞介素6(interleukin-6,IL-6)、HMGB1浓度。在12 h时,观察各组小鼠的生存率;选取4只小鼠处死后取肺组织,常规苏木素-伊红(hematoxylin-eosin,HE)染色观察肺组织病理变化,免疫组化染色观察HMGB1在肺组织中的表达;实时荧光定量PCR和蛋白质免疫印迹法法分别从mRNA和蛋白水平检测HMGB1的相对表达变化。结果(1)建模后12 h,NOMH组、ATMH组、KN组及NH组生存只数分别为36(40)、6(11)、27(40)、4(11),4组间比较,差异有统计学意义(χ^2=32.286,P=0.002),与另外3组脓毒症小鼠相比,NOMH组的生存率最高(与ATMH组:χ^2=5.222,P=0.022;与KN组:χ^2=6.050,P=0.013;与NH组:χ^2=11.672,P=0.001),但其余各两组之间比较,差异均无统计学意义(P均>0.05);(2)与NC组相比,各组脓毒症小鼠在6 h、12 h的血清TNF-α、IL-6及HMGB1浓度均明显升高(P均<0.05);与NOMH组相比,ATMH组、KN组、NH组小鼠在6 h、12 h的TNF-α、IL-6及HMGB1浓度均明显升高(P均<0.05);NH组在各时间点的TNF-α、IL-6、HMGB1浓度均为最高(P均<0.05);各组脓毒症小鼠组内不同时间点比较,12 h TNF-α浓度较6 h时下降(P均<0.05),而IL-6、HMGB1浓度在12 h时较6 h时上升(P均<0.05);(3)HE染色显示NOMH组肺组织损伤程度最轻,其次为ATMH组;(4)免疫组化染色显示HMGB1蛋白表达由少至多依次为NOMH、ATMH组、KN组和NH组;(5)NOMH组、ATMH组、KN组、NH组脓毒症小鼠肺组织HMGB1蛋白含量分别为0.280±0.013、0.320±0.016、0.340±0.018、0.380±0.014,HMGB1 mRNA相对表达量分别为4.86±0.22、6.02±0.18、6.26±0.20、7.98±0.28,分别较NC组(HMGB1蛋白含量:0.240±0.013,HMGB1 mRNA相对表达量:2.21±0.12)明显升高(P均<0.05);与NOMH组相比较,ATMH组、KN组、NH组肺组织中HMGB1蛋白、HMGB1 mRNA相对表达量均明显升高(P均<0.05),而NH组表达水平为最高(P均<0.05)。结论亚低温可能通过下调脓毒症小鼠肺组织HMGB1的表达,减轻肺组织损伤,自发性亚低温的改善更为显著。

Objective To investigate the effects of naturally occurring mild hypothermia and artificial mild hypothermia on the expression of high mobility group box 1(HMGB1)in lung tissues of septic mice.Methods One hundred and twenty BALB/C mice(SPF level)were randomly numbered.Twelve mice with integer multiples of 10 were used as the normal control(NC)group,and the remaining 108 mice were chosen as the septic group.The septic mouse model was established by intra abdominal injection of lipopolysaccharide(LPS)10 mg/kg.The NC group was given the same dose of normal saline.Anal temperature of the septic mice were measured 1 hour after the model was established successfully,and then were divided into naturally occurring mild hypothermia group and non-mild hypothermia group according to T≤36℃and T>36℃.In the naturally occurring mild hypothermia group,the mice with T<34℃were eliminated,and the remaining septic mice were randomly divided into the naturally occurring mild hypothermia(NOMH)observation group and the keep normothermia(KN)group.NOMH group was not given preheating intervention,while KN group was placed in an incubator to maintain the anal temperature between 36.0℃and 37.5℃.Septic mice in the non-mild hypothermia group were randomly divided into the nonhypothermia(NH)observation group and the artificial mild hypothermia(ATMH)group.The NH group was not treated with hypothermia,while the ATMH group was treated with physical hypothermia,so that the anal temperature of the mice were maintained at 34℃-36℃.Four mice in each group were randomly selected at 6 and 12 hours after modeling,and the concentrations of tumor necrosis factor-α(TNF-α),interleukin-6(IL-6)and HMGB1 in serum were detected by enzyme-linked immunosorbent assay(ELISA).At 12 hours,the survival rate of each group of mice was observed.Then 4 mice of each group were sacrificed and lung tissues were taken.The pathological changes of lung tissues were observed by hematoxylin-eosin(HE)staining,and the expression of HMGB1 in lung tissues was observed by immunohistochemical staining.Real time fluorescence quantitative PCR and Western blot were used to detect the relative expression of HMGB1 at mRNA and protein levels.Results(1)Twelve hours after modeling,the survival number of NOMH group,ATMH group,KN group and NH group were 36(40),6(11),27(40),4(11),respectively,and there were differences between the four groups(χ^2=32.286,P=0.002).Compared with the other three groups of septic mice,the survival rate was highest in the NOMH group(compared with ATMH group:χ^2=5.222,P=0.022;compared with the KN group:χ^2=6.050,P=0.013;and the NH group:χ^2=11.672,P=0.001),but the differences between the other two groups were not statistically significant(all P>0.05).(2)Compared with the NC group,the concentrations of serum TNF-α,IL-6 and HMGB1 of septic mice in each group were significantly increased at 6 h and 12 h(all P<0.05).Compared with NOMH group,the concentrations of TNF-α,IL-6 and HMGB1 in ATMH group,KN group and NH group were significantly increased at 6 h and 12 h(all P<0.05),and the concentrations of TNF-α,IL-6 and HMGB1 in NH group were the highest at all time points(all P<0.05).The concentrations of TNF-αat 12 h decreased compared with 6 h(all P<0.05),while the concentrations of IL-6 and HMGB1 at 12 h increased compared with 6 h(all P<0.05).(3)HE staining showed that the lung tissue damage were minimal in NOMH group,followed by ATMH group.(4)Immunohistochemical staining showed that the expression of HMGB1 protein was in order of NOMH group,ATMH group,KN group and NH group;(5)The relative expressions of HMGB1 protein in lung tissues of septic mice in NOMH group,ATMH group,KN group,and NH group was 0.280±0.013,0.320±0.016,0.340±0.018,and 0.380±0.014,respectively,and the relative expression level of HMGB1 mRNA was 4.86±0.22,6.02±0.18,6.26±0.20,and 7.98±0.28,respectively,compared with NC group(HMGB1 protein content was 0.240±0.013,and the relative expression level of HMGB1 mRNA was 2.21±0.12)significantly increased(all P<0.05).Cmpared with NOMH group,the relative expression levels of HMGB1 protein and HMGB1 mRNA in the lung tissues of the ATMH group,KN group and NH group were significantly increased(all P<0.05),with the highest expression level in the NH group(all P<0.05).Conclusion Mild hypothermia may reduce lung tissue damage by down-regulating the expression of HMGB1 in lung tissues of septic mice,and the improvement of spontaneous mild hypothermia was more significant.