p38丝裂原活化蛋白激酶抑制剂对妊娠晚期合并急性胰腺炎相关胎鼠肺脏损伤的作用研究

Effects of p38MAPK inhibitor on fetal lung injury in a rat model of acute pancreatitis in late pregnancy

目的探讨p38丝裂原活化蛋白激酶(p38MAPK)抑制剂SB203580对妊娠晚期合并急性胰腺炎(acute pancreatitis in late pregnancy,APILP)相关胎鼠肺脏损伤中的作用及可能机制。方法24只SPF级妊娠晚期SD大鼠随机(随机数字法)分为假手术(Sham-operation,SO)组、APILP组和p38MAPK抑制剂SB203580处理(SB)组。采用5%牛磺胆酸钠(sodium taurocholate,STC)逆行胰胆管注射法建立APILP模型,SB组大鼠在建立APILP模型前0.5 h给予SB203580腹腔注射(10 mg/kg)。SO组大鼠仅在开腹后翻动暴露胰腺。SO和APILP组大鼠在开腹前0.5 h给予对应体积的SB203580溶剂,各组大鼠均在术后12 h剖杀取材。测定大鼠AMY和TNF-α水平,光镜下观察大鼠胰腺、胎鼠肺脏的病理学改变,并进行评分。免疫组织化学法检测胎鼠肺脏中NF-κB的表达及定位,免疫荧光法检测胎鼠肺脏中MPO的表达,免疫印迹法检测胎鼠肺脏中p-p38MAPK、p38MAPK、TNF-α、细胞间黏附分子-1(ICAM-1)以及高迁移率族蛋白1 (high mobility group box-1 protein, HMGB1)的表达水平。单因素方差分析进行统计学处理,组间比较采用Tukey事后多重比较,以P<0.05为差异具有统计学意义。结果与SO组相比,APILP组妊娠大鼠血清中AMY、TNF-α的水平显著升高[(7 871.3±623.5) vs (1 915.3±452.3),(193.8±25.4) vs (107.0±13.3),(P<0.05)],APILP组大鼠胰腺、胎鼠肺脏的病理评分显著增高[(12.44±1.08) vs (1.56±0.56),(2.50±0.53) vs (0.88±0.64),(P<0.05)],胎鼠肺脏中NF-κB、MPO阳性细胞计数显著多于SO组[(150.63±34.58) vs (29.50±8.80),(53.38±8.30) vs (11.75±3.33),(P<0.05)],并且NF-κB的表达量和核转位更为明显;ANPIP组胎鼠肺脏中p-p38MAPK[(0.6367±0.0386) vs (0.2282±0.0220)]、TNF-α[(0.6313±0.0395) vs (0.0725±0.0076)]、ICAM-1[(0.8958±0.0776) vs (0.1372±0.0388)]和HMGB1[(0.6478±0.0209) vs (0.2825±0.0533)]的表达水平显著升高(P<0.05)。与APILP组比较,SB组妊娠大鼠血清中AMY(4,162.1±642.1)、TNF-α(139.6±21.1)水平显著下降(P<0.05),妊娠大鼠胰腺(9.38±1.58)和胎鼠肺脏(1.63±0.52)的病理评分显著降低(P<0.05),胎鼠肺脏中NF-κB(93.00±18.88)和MPO(27.38±4.75)阳性细胞计数显著下降(P<0.05),并且NF-κB表达量和核转位水平明显减少,胎鼠肺脏中p-p38MAPK(0.2578±0.0170)、TNF-α(0.3240±0.0326)、ICAM-1(0.4177±0.0823)和HMGB1(0.4923±0.0457)的表达水平显著下降(P<0.05)。结论p38MAPK及其下游炎症信号通路参与了APILP相关胎鼠肺脏损伤的过程;SB203580干预能够显著改善APILP相关胎鼠肺脏损伤,其机制可能与其抑制p38MAPK的磷酸化水平,阻断其下游信号通路激活引起的炎症级联反应有关。

Objective To investigate the effect and underlying mechanisms of p38 mitogen-activated protein kinase inhibitor SB203580 on fetal lung injury in a rat model of acute pancreatitis in late pregnancy. Methods Twenty-four pregnant Sprague-Dawley rats in last gestation were randomly(random number) divided into the SO group, APILP group, and SB203580 treatment (SB) group. APILP model was induced by retrograde injection of 5% sodium taurocholate into the biliary-pancreatic duct. SB203580 administration (10 mg/kg body weight, intraperitoneal injection) was performed 0.5 h before surgery. All the rats in the SO and APILP groups received intraperitoneal injection of equivoluminal solvent at the same time point. Animals were sacrificed at 12 h after the induction of APILP, then the blood and tissue samples were harvested. Serum levels of AMY and TNF-α were analyzed. Histopathological changes of maternal pancreas and fetal lung were observed and evaluated. The expression and location of NF-κB in fetal lungs were detected by immunohistochemistry and MPO expression in fetal lungs was examined by immunofluorescence. The expression of p-p38MAPK, p38MAPK, TNF-α and ICAM-1 was determined by Western blot. One-way ANOVA and Tukey's multiple comparison tests were used for statistical analysis. Results The levels of AMY and TNF-α in maternal serum were markedly increased after APILP [(7 871.3±623.5) vs (1 915.3±452.3),(193.8±25.4) vs (107.0±13.3),(P<0.05)]. Obvious pathological changes presented in maternal pancreas and fetal lung after the attack of APILP, and their pathological scores were significantly higher than those of the SO group [(12.44±1.08) vs (1.56±0.56),(2.50±0.53) vs (0.88±0.64),(P<0.05)]. The number of NF-κB and MPO positive cells in fetal lungs were significantly higher than those in the SO group [(150.63±34.58) vs(29.50±8.80),(53.38±8.30) vs (11.75±3.33);P<0.05)]. In addition, the expression and nuclear translocation were pervasive in fetal lungs in the APILP group. Furthermore, the levels of p-p38MAPK [(0.6367±0.0386) vs (0.2282±0.0220)], TNF-α[(0.6313±0.0395) vs (0.0725±0.0076)], ICAM-1 [(0.8958±0.0776) vs (0.1372±0.0388)] and HMGB1 [(0.6478±0.0209) vs (0.2825±0.0533)] expression in fetal lungs were significantly increased after the establishment of APILP model (P<0.05). However, with the pre-administration of SB203580, the pathological scores of maternal pancreases (9.38±1.58) and fetal lungs (1.63±0.52) were decreased significantly (P<0.05), as well as the levels of AMY (4162.1±642.1) and TNF-α(139.6±21.1) in maternal serum (P<0.05). The number of NF-κB (93.00±18.88) and MPO (27.38±4.75) positive cells in fetal lungs were dramatically reduced (P<0.05) and fewer nuclear translocation was observed in the SB group. Interestingly, the expression levels of p-p38MAPK (0.2578±0.0170), TNF-α(0.3240±0.0326), ICAM-1 (0.4177±0.0823) and HMGB1 (0.4923±0.0457) in fetal lungs were markedly decreased with the treatment of SB203580 (P<0.05). Conclusions P38MAPK and its downstream inflammatory signaling pathway were involved in the process of APILP-related fetal lung injury;SB203580 administration could significantly attenuate fetal lung injury induced by APILP, which may be closely related to the inhibition of p38MAPK phosphorylation and inflammatory cascade caused by the activation of downstream signal pathways.