抗菌肽Cathelicidin-PY治疗急性肝衰竭小鼠的初步研究  

Preliminary study of antimicrobial peptide cathelicidin-PY therapy in mice with acute liver failure

作　　者：王艳[1] 黄小平[1] 甘建和[1] Wang Yan;Huang Xiaoping;Gan Jianhe(Department of Infectious Diseases,First Hospital Affiliated to Soochow University,Suzhou 215006,China)

机构地区：[1]苏州大学附属第一医院感染病科,215006

出　　处：《中华肝脏病杂志》2020年第3期254-258,共5页Chinese Journal of Hepatology

基　　金："十二五"国家科技部"艾滋病和病毒性肝炎等重大传染病防治"科技重大专项子课题:免疫调节结合人工肝支持技术治疗重型乙型肝炎(肝衰竭)的应用研究(2012ZX10002004-008)。

摘　　要：目的通过小鼠动物模型探讨阳离子抗菌肽Cathelicidin-PY(PY)治疗急性肝衰竭的可行性。方法内毒素鲎试验(LAL assay)检测不同浓度抗菌肽PY体外中和内毒素/脂多糖(LPS)能力;CCK-8法检测不同浓度抗菌肽PY对小鼠单核巨噬细胞(RAW264.7)的毒性作用;体外溶血实验评价抗菌肽PY对健康人红细胞的溶血活性;构建D-氨基半乳糖联合LPS诱导的小鼠急性肝衰竭模型,观察抗菌肽PY对模型小鼠存活率的影响;通过HE染色观察肝脏病理学变化,并通过免疫组织化学、蛋白质印迹法检测凋亡相关蛋白Caspase-3的表达。组间比较采用t检验、方差分析,率的比较采用χ^2检验。结果体外实验显示极低剂量(0.01μmol/L)可达到较高的内毒素中和率,中等剂量(10~40μmol/L)中和率可超过70%,且不同浓度组间比较,差异有统计学意义(F=569.22,P<0.05)。中等剂量抗菌肽PY有较强的中和内毒素作用,较低的细胞毒性及溶血活性。进一步体内实验显示中等剂量抗菌肽PY可改善模型小鼠的肝脏损伤程度并显著提高存活率,且免疫组织化学检测结果显示中等剂量抗菌肽组较肝衰竭组肝组织内Caspase-3表达明显减少,与蛋白质印迹法检测结果一致。结论抗菌肽PY具有较强中和内毒素的能力且毒副作用小,特定剂量抗菌肽PY可以减轻肝细胞凋亡,并显著提高模型动物存活率,为肝衰竭治疗的研究提供了新思路。Objective To investigate the feasibility of cationic antimicrobial peptide cathelicidin-PY(PY)therapy through a mouse model of acute liver failure.Methods The ability of different concentrations of antimicrobial peptide PY to neutralize endotoxin/lipopolysaccharide(LPS)in vitro was detected by Limulus Amebocyte Lysate(LAL)assay.Cell counting kit-8(CCK-8)was used to detect the toxic effect of different concentrations of antimicrobial peptide PY on mouse monocyte macrophages(RAW264.7).An in vitro hemolysis experiment was used to evaluate the activity of antimicrobial peptide PY on healthy human erythrocytes.D-galactosamine combined with LPS-induced mouse model of acute liver failure was constructed.The antimicrobial peptide PY effect on survival rate of mouse model was observed.HE staining was used to observe the pathological changes of liver tissue.Immunohistochemistry and Western blotting were used to detect the expression of apoptosis-associated protein caspase-3.Intra-group comparisons were performed using t-test and analysis of variance.χ^2 test was used for the comparison of rates.Results An in vitro experiment showed that the endotoxin neutralization rate was higher at very low dose(0.01μmol/L),and exceeded 70%at medium-dose(10-40μmol/L),and the difference between groups with different concentration was statistically significant(F=569.22,P<0.05).Medium-dose antimicrobial peptide PY had strong endotoxin neutralizing effect,low cytotoxicity and hemolytic activity.Moreover,in vivo experiments showed that the degree of liver injury and survival rate of mouse model was significantly improved with the medium-dose of antimicrobial peptide PY.Immunohistochemistry results showed that the expression of caspase-3 in the liver tissue was significantly depleted in the medium-dose group than that of the liver failure group,and the results were consistent with protein immunoblotting testing.Conclusion Antimicrobial peptide PY possesses a strong ability to neutralize endotoxin and few toxic side effects.A specific dose o

关 键 词：急性肝衰竭 内毒素/脂多糖 抗菌肽 阳离子抗菌肽 Cathelicidin-PY 

分 类 号：R575[医药卫生—消化系统] R51[医药卫生—内科学]