Abstract: Objective　To investigate the development, role, and regulatory mechanism of macrophage efferocytosis in the liver of hosts infected with Schistosoma japonicum. Methods　The expression of efferocytosis⁃related gene like efferocytosis receptors, efferocytosis⁃related bridging molecules,"eat me" signal and "don't eat me" signal was detected in the livers of patients and mice infected with S. japonicum in the Gene Expression Omnibus (GEO) database. Ten wild⁃type (WT) male mice (6 ~ 8 weeks old, weighing 20 ~ 25 g) were randomly divided into a Schistosoma japonicum infection (SJ) group and a normal control (NC) group, with 5 mice in each group. The efferocytosis of neutrophils and T cells by liver macrophages was detected in mice from SJ group and NC group using flow cytometry and immunofluorescence assay, respectively. The expression of efferocytosis⁃related Mer receptor tyrosine kinase (MerTK) and Axl receptor tyrosine kinase (Axl) proteins was determined in mouse liver tissues using Western blotting assay, and the proportion of MerTK＋ macrophages and the average fluorescence intensity of macrophage MerTK were detected in mouse livers using flow cytometry. Changes in liver granulomas and fibrosis were observed in mice infected with S. japonicum following injection of efferocytosis inhibitors, and S. japonicum⁃infected mice without injection of efferocytosis inhibitors served as controls. Bone marrow⁃derived macrophages (BMDMs) were isolated from macrophage scavenger receptor class A (SR⁃A) conditional knockout (CKO) and wild⁃type (WT) mice, and changes in apoptotic neutrophils were detected in SR⁃A CKO mouse macrophages in vitro using flow cytometry. Then, BMDMs was divided into the WT mono⁃culture group, the WT and apoptotic neutrophils co⁃culture group, the SR⁃A CKO mono⁃culture group, and the SR⁃A CKO and neutrophils co⁃culture group, and the expression of MerTK and Axl was quantified in vitro using Western blotting and real⁃time quantitative PCR (RT⁃qPCR) assays during neutrophil efferocytosis. The efferocytosis of neutrophils and T cells by mouse liver macrophages was detected in the SR⁃A CKO SJ group and the WT SJ group using flow cytometry and immunofluorescence assay, and the expression of MerTK and Axl proteins was determined in mouse liver tissues in both groups using Western blotting. In addition, the proportion of MerTK＋ macrophages and the average fluorescence intensity of macrophage MerTK were detected in mouse livers in both groups using flow cytometry. Results　Data from the GEO database showed that the expression of some efferocytosis receptors and efferocytosis⁃related bridging molecules, and "eat me" and "don't eat me" signals all appeared a tendency towards a rise in livers of patients and mice in the SJ group relative to the NC group, suggesting that S. japonicum infection⁃induced liver diseases may be associated with efferocytosis. Flow cytometry detected higher proportions of Ly6G＋cells [(13.13 ± 0.45)% vs. (6.48 ± 0.25)%; t = 22.30, P < 0.05] and CD3＋ cells [(7.60 ± 0.33)% vs. (3.30 ± 0.42)%; t = 13.98, P < 0.05] in mouse liver macrophages in the SJ group than in the NJ group, and the expression of MerTK protein [(2.30 ± 0.14) vs. (1.14 ± 0.46); t = 4.19, P < 0.05], the mean fluorescence intensity of macrophages [(160.67 ± 15.28) vs. (94.50 ± 19.61); t = 4.81, P < 0.05], and the proportion of MerTK＋ macrophages [(20.78 ± 4.17)% vs. (6.85 ± 0.39)%; t = 6.57, P < 0.05] were significantly higher in mouse liver tissues in the SJ group than in the NC group. The Axl expression was lower in mouse liver tissues in the SJ group than in the NC group [(1.25 ± 0.08) vs. (1.93 ± 0.37); t = 2.79, P < 0.05], and the area of granulomas around single eggs [(9.18 ± 1.81) × 104 μm2  vs.  (5.24 ± 1.35) × 104 μm2; t = 3.03, P < 0.05] and proportion of collagen fibers [(25.27 ± 3.99)% vs. (15.14 ± 4.02)%; t = 3.10, P < 0.05] were significantly greater in livers of S. japonicum⁃infected mice with injection of efferocytosis inhibitors than in mice without injection of efferocytosis inhibitors. The in vitro efferocytosis efficiency of BMDMs [(32.83 ± 3.17)% vs. (45.43 ± 2.34)%; t = 5.54, P < 0.05], and the proportions of Ly6G＋ [(9.37 ± 0.48)% vs. (13.13 ± 0.72)%; t = 7.50, P < 0.05] and CD3＋ cells [(4.95 ± 0.17)% vs. (7.64 ± 0.50)%; t = 8.87, P < 0.05] in liver macrophages post⁃infection with S. japonicum were significantly lower in SR⁃A CKO mice than in WT mice, and the expression of MerTK protein [(0.65 ± 0.25) vs. (1.96 ± 0.69); t = 3.10, P < 0.05], the average fluorescence intensity of macrophages [(138.33 ± 8.39) vs. (160.67 ± 15.28); t = 3.03, P < 0.05] and the proportion of MerTK＋ macrophages [(13.17 ± 5.01)% vs. (22.63 ± 2.06)%; t = 3.56, P < 0.05] were significantly lower in mouse liver tissues in the SR⁃A CKO SJ group than in the WT group. Western blotting detected no significant difference in the Axl protein expression in mouse liver tissues between the SR⁃A CKO SJ group and the WT SJ group [(0.48 ± 0.07) vs. (0.68 ± 0.30); t = 1.09, P > 0.05]. There were significant differences in the relative MerTK mRNA and protein expression during efferocytosis of BMDMs among the WT mono⁃culture group, the WT and apoptotic neutrophils co⁃culture group, the SR⁃A CKO mono⁃culture group, and the SR⁃A CKO and neutrophils co⁃culture group (F = 9.41 and 40.68, both P values < 0.05). In addition, there was a significant difference in the relative Axl mRNA expression during efferocytosis of BMDMs among the four groups (F = 13.62, P < 0.05); however, no significant difference was seen in the relative Axl protein expression (F = 1.27, P > 0.05). Conclusions　Efferocytosis of liver macrophages is seen in mice infected with S. japonicum and inhibits liver fibrosis. SR⁃A may up⁃regulate the efficiency of macrophage efferocytosis through regulating the expression of efferocytosis receptors.

Key words: Schistosomiasis japonica, Hepatic damage, Macrophage, Efferocytosis, Scavenger receptor class A 

摘要： 目的　观察日本血吸虫感染宿主肝脏中巨噬细胞胞葬现象及其作用和调控机制。方法　基于基因表达综合数据库（Gene Expression Omnibus，GEO）中的数据集，探究日本血吸虫感染患者及小鼠肝脏内胞葬受体、胞葬相关桥接分子、“吃我”信号、“不要吃我”信号等胞葬相关基因的表达情况。将10只WT小鼠（雄性，6 ~ 8周龄，体质量20 ~ 25 g）随机分为日本血吸虫感染组（SJ组）和正常对照组（NC组），每组5只。通过流式细胞术与免疫荧光技术检测，观察SJ组、NC组小鼠肝脏巨噬细胞对中性粒细胞、T细胞的胞葬作用；采用蛋白质免疫印迹（Western Blot，WB）实验检测两组小鼠肝脏组织胞葬相关Mer受体酪氨酸激酶（MerTK）和Axl受体酪氨酸激酶（Axl）的蛋白表达情况，并采用流式细胞术检测两组小鼠肝脏中MerTK＋巨噬细胞比例和巨噬细胞MerTK的平均荧光强度。以未注射胞葬抑制剂的日本血吸虫感染小鼠为对照组，观察注射胞葬抑制剂后感染小鼠肝脏肉芽肿和纤维化改变。提取A类清道夫受体（scavenger receptor class A，SR⁃A）巨噬细胞条件性基因敲除（conditional knockout，CKO）小鼠及野生型（wild type，WT）小鼠骨髓来源巨噬细胞（bone marrow⁃derived macrophages，BMDM），采用流式细胞术检测SR⁃A CKO小鼠巨噬细胞体外胞葬凋亡中性粒细胞的变化情况，随后将BMDM分为WT单独培养组、WT＋凋亡中性粒细胞共培养组、SR⁃A CKO单独培养组、SR⁃A CKO＋中性粒细胞共培养组，采用WB和RT⁃qPCR检测体外胞葬中性粒细胞过程中MerTK及Axl的表达情况。使用流式细胞术及免疫荧光术检测SR⁃A CKO SJ组及WT SJ组小鼠肝脏巨噬细胞对中性粒细胞和T细胞的胞葬作用，采用WB检测两组小鼠肝脏组织MerTK及Axl的蛋白表达情况，采用流式细胞术检测两组小鼠肝脏中MerTK＋巨噬细胞比例和巨噬细胞MerTK的平均荧光强度。结果　GEO数据库分析结果显示，与NC组相比，日本血吸虫感染患者及小鼠肝脏内部分胞葬受体、胞葬相关桥接分子、“吃我”信号、“不要吃我”信号均呈上调趋势，提示日本血吸虫性肝病可能与胞葬现象有关。SJ组小鼠肝脏巨噬细胞中Ly6G＋细胞占比［（13.13 ± 0.45）%］和CD3＋细胞占比［（7.60 ± 0.33）%］均显著高于NC组［（6.48 ± 0.25）%、（3.30 ± 0.42）%］（t = 22.30、13.98，P均< 0.05）；胞葬受体MerTK在SJ组小鼠肝脏组织中的蛋白表达水平、巨噬细胞平均荧光强度和MerTK＋巨噬细胞比例［2.30 ± 0.14、160.67 ± 15.28、（20.78 ± 4.17）%］均显著高于NC组［1.14 ± 0.46、94.50 ± 19.61、（6.85 ± 0.39）%］（t = 4.19、4.81、6.57，P均< 0.05）。Axl在SJ组小鼠肝脏组织中的蛋白表达水平（1.25 ± 0.08）显著低于NC组（1.93 ± 0.37）（t = 2.79，P < 0.05）。SJ＋注射胞葬抑制剂组小鼠肝脏单个虫卵肉芽肿面积和胶原纤维面积比例［（9.18 ± 1.81） × 104 μm2、（25.27 ± 3.99）%］均高于未注射胞葬抑制剂SJ组［（5.24 ± 1.35） × 104 μm2、（15.14 ± 4.02）%］（t = 3.03、3.10，P均< 0.05）。SR⁃A CKO小鼠BMDM体外胞葬效率、日本血吸虫感染后肝脏内巨噬细胞中Ly6G＋细胞占比和CD3＋细胞占比［（32.83 ± 3.17）%、（9.37 ± 0.48）%、（4.95 ± 0.17）%］均显著低于WT小鼠［（45.43 ± 2.34）%、（13.13 ± 0.72）%、（7.64 ± 0.50）%］（t = 5.54、7.50、8.87，P均< 0.05）。胞葬受体MerTK在SR⁃A CKO SJ组小鼠肝脏组织中的蛋白表达水平、巨噬细胞平均荧光强度和MerTK＋巨噬细胞占比［0.65 ± 0.25、138.33 ± 8.39、（13.17 ± 5.01）%］均显著低于WT小鼠［1.96 ± 0.69、160.67 ± 15.28、（22.63 ± 2.06）%］（t = 3.10、3.03、3.56，P均< 0.05）。Axl在SR⁃A CKO SJ组小鼠肝脏组织中的蛋白表达水平（0.48 ± 0.07）与WT SJ组（0.68 ± 0.30）差异无统计学意义（t = 1.09，P > 0.05）。体外实验结果表明，胞葬过程中4组BMDM MerTK的mRNA和蛋白相对表达量差异均有统计学意义（F = 9.41、40.68，P均< 0.05）；4组BMDM Axl的mRNA相对表达量差异亦有统计学意义（F = 13.62，P < 0.05），而蛋白相对表达量差异无统计学意义（F = 1.27，P > 0.05）。结论　日本血吸虫感染小鼠肝脏巨噬细胞呈显著胞葬现象，该现象具有抑制肝纤维化的作用，SR⁃A则可通过调节胞葬受体的表达上调巨噬细胞胞葬效率。

关键词: 日本血吸虫病, 肝脏损伤, 巨噬细胞, 胞葬, A类清道夫受体