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目的观察旋毛虫(Trichinella spiralis)成虫排泄-分泌蛋白(adult-worm excretory-secretory protein,AES)对小鼠脓毒症急性肝损伤的保护作用。方法 24只雄性BALB/c小鼠采用抽签法随机分为假手术组(Sham组)、盲肠结扎穿孔组(CLP组)、AES治疗组(AES+CLP组)和AES对照组(AES+Sham组),每组6只。CLP组和AES+CLP组采用盲肠结扎穿孔术制备脓毒症急性肝损伤动物模型,Sham组和AES+Sham组不结扎、不穿孔盲肠,其余操作与CLP组相同。术后30 min,Sham组和CLP组腹腔注射PBS,AES+CLP组和AES+Sham组腹腔注射AES(25μg/只)。术后12 h,检测小鼠血清中丙氨酸转氨酶(ALT)和天门冬氨酸转氨酶(AST)水平,观察小鼠肝脏组织病理改变,蛋白质印迹(Western blotting)测定肝脏组织中Toll样受体4(TLR4)、髓样分化因子88(My D88)的表达水平,免疫组化法测定肝细胞核因子-κB(NF-κB)p65的表达水平并分析NF-κB p65核阳性率,ELISA检测小鼠肝脏组织中α肿瘤坏死因子(TNF-α)、白细胞介素-6(IL-6)和IL-1β的水平。结果术后12 h,CLP组小鼠血清中ALT和AST水平分别为(269.83±77.60)和(712.00±186.52)IU/L,明显高于Sham组的(45.00±15.01)和(132.50±36.95)IU/L,及AES+CLP组的(136.67±30.27)和(419.00±60.86)IU/L(P<0.05);AES+Sham组分别为(50.83±13.24)和(134.67±35.78)IU/L,与Sham组比较差异无统计学意义(P>0.05)。HE染色显示:CLP组肝索紊乱,肝细胞水肿,有炎性细胞浸润;AES+CLP组肝脏结构损伤明显减轻。Western blotting检测显示:CLP组小鼠术后肝脏组织中TLR4/β-actin和My D88/β-actin相对表达量分别为0.66±0.12和0.69±0.13,明显高于Sham组的0.31±0.06和0.22±0.04(P<0.05);AES+CLP组TLR4/β-actin相对表达量为0.56±0.09,与CLP组的比较差异无统计学意义(P>0.05),而My D88/β-actin相对表达量为0.47±0.06,较CLP组降低(P<0.05)。免疫组化分析结果显示:CLP组肝细胞中NF-κB p65核阳性率为(18.93±2.91)%,明显高于Sham组的(0.50±0.21)%及AES+CLP组的(9.60±1.59)%(P<0.05)。ELISA检测结果显示:CLP组小鼠肝脏组织中TNF-α、IL-6和IL-1β水平分别是(26.21±2.96)、(80.80±9.94)和(28.99±3.70)pg/mg,明显高于Sham组的(15.42±1.21)、(47.70±4.77)和(16.86±1.68)pg/mg,及AES+CLP组的(18.93±2.04)、(61.82±7.24)和(23.18±2.21)pg/mg(P<0.05)。结论AES可通过降低肝脏组织中My D88表达和NF-κB p65核阳性率,从而降低肝脏组织中TNF-α、IL-6和IL-1β水平,减轻脓毒症对肝脏的损伤。
Objective To observe the protective effect of adult-worm excretory-secretory protein (Trichinella spiralis) on acute liver injury induced by sepsis in mice. Methods Twenty-four male BALB / c mice were randomly divided into three groups: Sham group, CLP group, AES + CLP group and AES + Sham group ), 6 in each group. CLP group and AES + CLP group were prepared cecal ligation and puncture of animal models of sepsis-induced acute liver injury, sham group and AES + Sham group did not ligation, cecum perforated cecum, the rest of the operation and CLP group the same. Thirty minutes after operation, Sham group and CLP group were intraperitoneally injected with PBS (PBS), AES + CLP group and AES + Sham group (25 μg / mouse). At 12 h after operation, the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in the serum of mice were detected, and pathological changes in liver tissues of mice were observed. Toll-like receptors in liver tissues were detected by Western blotting 4 (TLR4) and myeloid differentiation factor 88 (My D88) were detected by immunohistochemistry. The expression of nuclear factor-kappaB (NF-κB) p65 in hepatocytes was detected by immunohistochemistry and the positive rate of NF- Levels of TNF-α, IL-6 and IL-1β in rat liver tissue. Results The levels of ALT and AST in serum of CLP group were (269.83 ± 77.60) and (712.00 ± 186.52) IU / L at 12 h after operation, which were significantly higher than those in Sham group (45.00 ± 15.01) and (132.50 ± 36.95) (136.67 ± 30.27) and (419.00 ± 60.86) IU / L in the AES + CLP group and (50.83 ± 13.24) and (134.67 ± 35.78) IU / L in the AES + Sham group , No significant difference compared with Sham group (P> 0.05). Hematoxylin-eosin staining showed that hepatic cord disturbance, hepatocellular edema and inflammatory cell infiltration were found in CLP group. Liver structural damage in AES + CLP group was significantly reduced. Western blotting showed that the relative expression of TLR4 / β-actin and My D88 / β-actin in CLP group were 0.66 ± 0.12 and 0.69 ± 0.13, respectively, which were significantly higher than those in Sham group (0.31 ± 0.06 and 0.22 ± 0.04 (P <0.05). The relative expression of TLR4 / β-actin in AES + CLP group was 0.56 ± 0.09, which was not significantly different from CLP group (P> 0.05) The amount was 0.47 ± 0.06, which was lower than CLP group (P <0.05). The results of immunohistochemistry showed that the positive rate of nuclear NF-κB p65 in CLP group was (18.93 ± 2.91)%, significantly higher than that in Sham group (0.50 ± 0.21)% and (9.60 ± 1.59) in AES + CLP group % (P <0.05). The results of ELISA showed that the levels of TNF-α, IL-6 and IL-1β in CLP group were (26.21 ± 2.96), (80.80 ± 9.94) and (28.99 ± 3.70) pg / mg respectively (15.42 ± 1.21), (47.70 ± 4.77) and (16.86 ± 1.68) pg / mg in sham group and (18.93 ± 2.04), (61.82 ± 7.24) and (23.18 ± 2.21) pg / mg in AES + (P <0.05). Conclusions AES can reduce the level of TNF-α, IL-6 and IL-1β in liver tissue and reduce the damage of liver caused by sepsis by decreasing the expression of My D88 and the positive rate of NF-κB p65 in liver tissue.