血必净通过调控自噬减轻脓毒症炎症反应和肺损伤的实验研究

Experimental study of Xuebijing on reducing sepsis inflammatory reaction and lung injury by regulating autophagy

目的分析血必净对脓毒症小鼠的炎症反应、肺损伤及肺组织细胞的自噬情况。方法选择40只雄性SPF级ICR小鼠,按照随机数字表法平均分为假手术组(Sham组,在无菌条件下只进行开腹操作)、脓毒症模型组〔采用盲肠结扎穿孔术(CLP)建立脓毒症模型〕、脓毒症+自噬抑制剂3-甲基腺嘌呤(3-MA)组(CLP+3-MA组)、脓毒症+血必净组(CLP+血必净组)和CLP+血必净+3-MA组。CLP+3-MA组CLP前1 h腹腔注射3-MA 15 mg/kg;CLP+血必净组CLP前2 h腹腔注射血必净注射液6 mL/kg;CLP+血必净+3-MA组在CLP+血必净组基础上于术前1 h腹腔注射3-MA 15 mg/kg。制膜后24 h取小鼠静脉血及肺脏,采用酶联免疫吸附试验(ELISA)分别检测各组小鼠静脉血中白细胞介素(IL-6、IL-1β)、肿瘤坏死因子-α(TNF-α)水平,采用蛋白质免疫印迹试验(Western blotting)测定各组小鼠肺组织中自噬标志物微管相关蛋白1轻链3(LC3-Ⅱ、LC3-Ⅰ)的相对蛋白表达,采用苏木素-伊红(HE)染色观察各组小鼠肺脏组织病理学改变。结果与Sham组比较,CLP组IL-6、IL-1β、TNF-α水平均明显升高,肺组织LC3-Ⅰ、LC3-Ⅱ蛋白表达及LC3-Ⅱ/LC3-Ⅰ比值明显增加,表明制模成功。与CLP组比较,CLP+3-MA组IL-6、IL-1β、TNF-α水平均明显升高〔IL-6(ng/L):1441.41±88.64比847.69±182.03,IL-1β(ng/L):73.97±65.17比11.66±4.82,TNF-α(ng/L):499.10±621.42比49.19±29.61,均P<0.05〕,而CLP+血必净组IL-6、IL-1β、TNF-α水平均明显下降〔IL-6(ng/L):380.33±168.49比847.69±182.03,IL-1β(ng/L):4.65±1.85比11.66±4.82,TNF-α(ng/L):8.20±6.21比49.19±29.61,均P<0.05〕;CLP+3-MA组LC3-Ⅰ、LC3-Ⅱ蛋白表达及LC3-Ⅱ/LC3-Ⅰ比值均显著降低〔LC3-Ⅰ(mg/L):37.60比41.80,LC3-Ⅱ(mg/L):11.60比25.99,LC3-Ⅱ/LC3-Ⅰ比值:0.31比0.62,均P<0.05〕;而CLP+血必净组LC3-Ⅰ、LC3-Ⅱ蛋白表达均显著降低,但LC3-Ⅱ/LC3-Ⅰ比值显著增加(LC3-Ⅱ/LC3-Ⅰ比值:0.66比0.62,均P<0.05);CLP+血必净+3-MA组上述指标均无明显变化。HE染色结果显示,CLP组与CLP+血必净+3-MA组肺组织肺泡腔出现大量红细胞、炎症细胞和血浆样物质渗出,肺泡间隔和肺泡壁明显增厚不均,部分肺泡腔塌陷;CLP+3-MA组严重程度更甚于CLP组;CLP+血必净组肺组织呈一定程度水肿和炎症浸润,但严重程度不及CLP组。结论血必净注射液可以改善脓毒症小鼠的肺损伤和炎症反应、抑制自噬。

Objective To analyze the inflammatory response,lung injury and autophagy of lung tissue cells of Xuebijing on septic mice.Methods Forty male SPF ICR mice were randomly divided into sham operation group(sham group,only open operation under sterile conditions),sepsis model group(sepsis model was established by cecal ligation and perforation(CLP),sepsis+autophagy inhibitor 3-methyladenine(3-MA)group(CLP+3-MA group),sepsis+Xuebijing group(CLP+Xuebijing group)and CLP+Xuebijing+3-MA group.CLP+3-MA group received intraperitoneal injection of 3-MA 15 mg/kg 1 hour before CLP,CLP+Xuebijing group received intraperitoneal injection of Xuebijing injection 6 mL/kg 2 hours before CLP,CLP+Xuebijing+3-MA group was injected intraperitoneally with 3-MA 15 mg/kg 1 hour before operation on the basis of CLP+Xuebijing group.24 hours after the CLP,the venous blood and lungs of mice were taken,and the interleukins(IL-6,IL-1β),tumor necrosis factor-α(TNF-α)in the venous blood of mice in each group was detected by enzyme-linked immunosorbent assay(ELISA).The relative protein expression of autophagy marker microtubule associated protein 1 light chain 3(LC3-Ⅱ,LC3-Ⅰ)in the lung tissue of mice in each group was measured by Western blotting,and the pathological changes of lung tissue of mice in each group were observed by hematoxylineosin(HE)staining.Results Compared with sham group,the levels of IL-6,IL-1βand TNF-αin CLP group were significantly increased,and the expression of LC3-Ⅰ,LC3-Ⅱprotein and the ratio of LC3-Ⅱ/LC3-Ⅰin lung tissue were significantly increased,indicating the success of the CLP model.Compared with CLP group,the levels of IL-6,IL-1βand TNF-αin CLP+3-MA group were significantly higher[IL-6(ng/L):1441.41±88.64 vs.847.69±182.03,IL-1β(ng/L):73.97±65.17 vs.11.66±4.82,TNF-α(ng/L):499.10±621.42 vs.49.19±29.61,all P<0.05].The levels of IL-6,IL-1βand TNF-αin CLP+Xuebijing group decreased significantly[IL-6(ng/L):380.33±168.49 vs.847.69±182.03,IL-1(ng/L):4.65±1.85 vs.11.66±4.82,TNF-α(ng/L):8.20±6.21 vs.49.19±29.61,all P<0.05].The protein expression of LC3-Ⅰand LC3-Ⅱand the ratio of LC3-Ⅱ/LC3-Ⅰin CLP+3-MA group were significantly decrease[LC3-Ⅰ(mg/L):37.60 vs.41.80,LC3-Ⅱ(mg/L):11.60 vs.25.99,LC3-Ⅱ/LC3-Ⅰratio:0.31 vs.0.62,all P<0.05].In CLP+Xuebijing group,the expression of LC3-Ⅰand LC3-Ⅱprotein decreased significantly,but the ratio of LC3-Ⅱ/LC3-Ⅰincreased significantly(LC3-Ⅱ/LC3-Ⅰratio:0.66 vs.0.62,all P<0.05).There was no significant change in the above indicators in CLP+Xuebijing+3-MA group.The results of HE staining showed that in CLP group and CLP+Xuebijing+3-MA group,a large number of red blood cells,inflammatory cells and plasma like substances appeared in the alveolar cavity of lung tissue,the alveolar septum and alveolar wall were significantly thickened unevenly,and some alveolar cavities collapsed.The severity of CLP+3-MA group was more serious than that of CLP group.The lung tissue of CLP+Xuebijing group showed edema and inflammatory infiltration to a certain extent,but the severity was less than that of CLP group.Conclusion Xuebijing injection can improve the lung injury and inflammatory reaction of sepsis mice and inhibit autophagy.