重组旋毛虫53000抗原蛋白通过激活M2型巨噬细胞减轻脂多糖对肝脏的损害

Recombinant trichinella spiralis-53000 protein alleviates liver damage due to lipopolysaccharides via M2 macrophage activation

目的 研究重组旋毛虫53 000抗原蛋白（rTsP53）是否通过激活M2巨噬细胞减轻脂多糖（LPS）所致肝损害.方法 60只雄性BALB/c小鼠,按随机数字表法分为LPS组、LPS＋磷酸盐缓冲液（PBS）组及rTsP53干预组,每组20只.3组动物禁食8h后腹腔注射15 μg/kg LPS;LPS＋ PBS组在注射LPS后1h注射等量PBS;rTsP53干预组在注射LPS后1h注射5 mg/kg rTsP53蛋白.干预后48 h处死小鼠,提取腹腔巨噬细胞,用流式细胞仪检测巨噬细胞标志物CCR7（M1型）及CD206（M2型）表达变化;取肝脏组织制作切片,苏木素-伊红（HE）染色观察病理改变,双染色免疫荧光检测F4/80（＋）HLA-DR（＋）及F4/80（＋）CD163（＋）表达情况;取外周血,检测血清天冬氨酸转氨酶（AST）及丙氨酸转氨酶（ALT）水平.结果 与LPS组、LPS＋ PBS组比较,rTsP53干预组小鼠生存率明显提高（90％比25％、30％,均P＜0.01）;肝脏病理损害减轻,组织结构明显改善;血清ALT、AST水平明显降低[ALT （U/L）：97.7±8.5比181.7±19.5、173.7±17.2,AST（U/L）：142.7±12.1比235.7±9.9、213.7±6.7,均P＜0.05];腹腔巨噬细胞FITC-CD206（＋）比例明显升高[（17.75±0.30）％比（1.38±0.13）％、（1.36±0.05）％,均P＜0.05],腹腔巨噬细胞PE-CCR7（＋）比例明显下降[（6.89±0.11）％比（15.30±0.64）％、（14.96±0.93）％,均P＜ 0.05];肝组织切片内F4/80（＋）CD163（＋）细胞表达荧光强度明显增强（0.36±0.01比0.29±0.02、0.31±0.01,均P＜0.05）,而F4/80（＋）HLA-DR（＋）荧光强度则无明显差异（0.30±0.01比0.30±0.02、0.31±0.01,均P＞0.05）.LPS组与LPS＋ PBS组各指标比较差异均无统计学意义（均P＞0.05）.结论 rTsP53蛋白可以通过促进体内M2型巨噬细胞活化,减轻LPS所致肝组织损害,提高动物生存率.

Objective To evaluate if recombinant trichinella wpiralis-53 000 protein （rTsP53） could alleviate liver damage caused by lipopolysaccharides （LPS） via M2 macrophage activation.Methods Sixty male BALB/c mice were randomly divided into LPS group,LPS ＋ phosphate buffer saline （PBS） group and rTsP53 intervention group by random number table,with 20 mice in each group.Intraperitoneal injection of 15 μg/kg LPS was performed for all the mice in the three groups after 8 hours of fasting.The mice in LPS ＋ PBS group were injected with PBS after 1 hour of LPS injection.The mice in the rTsP53 intervention group were injected with rTsP53 （5 mg/kg） after 1 hour of LPS injection.After 48 hours all the mice were sacrificed.Peritoneal macrophages were harvested and flow cytometry （FCM） was used to detect markers CCR7 （M1） and CD206 （M2） of macrophages.Hepatic tissue was harvested for pathological study after hematoxylin-eosin （HE） staining,and double staining immunofluorescence was used to detect F4/80（＋） HLA-DR（＋） and F4/80 （＋） CD163（＋）.Peripheral blood serum was harvested to detect the levels of aspartate transaminase （AST） and alanine transaminase （ALT）.Results Compared with LPS and LPS ＋ PBS groups,survival rate of mice of rTsP53 intervention group was significantly elevated （90% vs.25%,30%,both P〈 0.01）,and the pathological injury of the liver was significantly ameliorated,and the hepatic structure was better preserved.The transaminase in rTsP53 intervention group was significantly lower than that of LPS and LPS ＋ PBS groups [ALT （U/L）：97.7 ± 8.5 vs.181.7 ± 19.5,173.7 ± 17.2; AST （U/L）：142.7 ± 12.1 vs.235.7 ± 9.9,213.7 ± 6.7,all P〈 0.05],FITC-CD206 （＋） proportion of peritoneal macrophage was significantly higher [（17.75 ± 0.30）% vs.（1.38 ± 0.13）%,（1.36±0.05）%,both P〈0.05] while PE-CCR7 （＋） proportion [（6.89 ± 0.11）% vs.（15.30±0.64）%,（14.96 ± 0.93） %,both P 〈 0.05] was significantly lower.Fluorescence intensity of macrophages with F4/80 （＋） CD163（＋） double staining for liver sections was significantly increased （0.36 ± 0.01 vs.0.29 ± 0.02,0.31 ± 0.01,both P〈0.05）,while there was no significant difference in the fluorescence intensity of macrophages with F4/80（＋）HLA-DR （＋） double staining （0.30 ± 0.01 vs.0.30 ± 0.02,0.31 ± 0.01,both P〉0.05）.There was no significant difference of above results between LPS group and LPS ＋ PBS group （all P〉0.05）.Conclusion rTsP53 could ameliorate liver damage caused by LPS and improve animal＇s survival via the activation of M2 macrophage.