Objective: To investigate the effects of persistent Echinococcus multilocularis infections on hepatic fibrosis in mice, so as to provide insights into the understanding of liver fibrogenesis induced by E. multilocularis infections and the treatment of alveolar echinococcosis.
Methods: Hepatic stellate HSC-T6 and LX-2 cells were exposed to the sera (25, 50 and 100 μL) from Meriones unguiculatus infected with E. multilocularis, and E. multilocularis, germinal layer cells (GCs) and protoscoleces (PSCs) for 48 hours, respectively. The cell proliferation was measured using a CCK-8 assay, and the levels of collagen 1 (Col1) and α-smooth muscle actin (α-SMA) were measured in the culture supernatant of HSC-T6 cells using ELISA. In addition, the serum and liver samples were collected 1, 2, 4, 6, 8 months post-infection with E. multilocularis, respectively. The serum Col1 and α-SMA concentrations were measured using enzyme-linked immunosorbent assay (ELISA), and the deposition of collagen fibers was examined in mice livers using Sirius red staining.
Results: The sera of E. multilocularis-infected gerbils promoted the proliferation of HSC-T6 and LX-2 cells in vitro, and there were significant differences seen in the proliferative rate of HSC-T6 (FHSC-T6 = 126.50, P < 0.05) and LX-2 cells (FLX-2 = 201.50, P < 0.05) among different serum groups, with the highest proliferative rate of HSC-T6 (573.36% ± 206.34%) and LX-2 cells (940.38% ± 61.65%) found following exposure to 100 μL mouse sera. Exposure to serum from E. multilocularis-infected gerbils resulted in an increase in the Col1 and α-SMA levels in the culture supernatant of HSC-T6 cells, with the greatest Col1 (20.99 ng/mL ± 2.01 ng/mL) and α-SMA levels (305.52 pg/mL ± 16.67 pg/mL) measured following exposure to 100 μL sera. The metacestodes (142.65% ± 9.17% and 189.99% ± 7.75%), GCs (118.55% ± 8.96% and 122.54% ± 0.21%) and PSCs of E. multilocularis (156.34% ± 17.45% and 160.59% ± 31.41%) all promoted the proliferation of HSC-T6 and LX-2 cells in vitro, and there were significant differences in the proliferative rates of HSC-T6 (FHSC-T6 = 11.24, P < 0.05) and LX-2 cells among groups (FLX-2 = 47.72, P < 0.05). Exposure to E. multilocularis resulted in an increase in Col1 and α-SMA levels in the culture supernatant of HSC-T6 cells, and the highest Col1 (4.43 ng/mL ± 2.23 ng/mL) and α-SMA levels (285.20 pg/mL ± 90.67 pg/mL) were detected following treatment with E. multilocularis metacestodes. In addition, a persistent increase was seen in the deposition of collagen fibers in mice livers 1 to 8 months post-infection with E. multilocularis, with the greatest Col1 level (280.26 ng/mL ± 23.04 ng/mL) seen 6 months post-infection and the highest α-SMA level (33.68 ng/mL ± 4.45 ng/mL) detected 8 months post-infection, respectively.
Conclusions: Persistent E. multilocularis infections promote hepatic stellate cell proliferation, induce an increase in mouse serum Col1 and α-SMA levels, and cause elevated deposition of collagen fibers in mice livers. The infective stage of E. multilocularis is a critical period for inducing hepatic fibrosis of alveolar echinococcosis.
［摘要］ 目的 评价泡球蚴持续感染对小鼠肝脏纤维化的影响, 为研究泡型棘球蚴病肝纤维化进展及其治疗方法提供 参考。方法 以泡球蚴感染长爪沙鼠血清 (25、50、100 μL) 和泡球蚴及其生发层细胞、原头节分别对肝星状HSC-T6和 LX-2细胞进行体外刺激 48 h, 采用CCK-8法检测细胞增殖, 应用酶联免疫吸附试验 (enzyme-linked immunosorbent assay, ELISA) 检测HSC-T6细胞培养上清中Ⅰ型胶原蛋白 (collagen 1, Col1) 和α-平滑肌动蛋白 (α-smooth muscle actin, α-SMA) 表达量。收集泡球蚴感染 1、2、4、6、8 个月小鼠血清和肝脏, 分别采用ELISA法检测血清中Col1和α-SMA含量, 采用天狼 猩红染色法动态观察肝脏胶原纤维沉积情况。结果 泡球蚴感染沙鼠血清体外可诱导HSC-T6和LX-2细胞增殖, 不同 血清剂量组细胞增殖率差异均有统计学意义 (FHSC-T6 = 126.50、FLX-2 = 201.50, P 均< 0.05); 其中100 μL血清对HSC-T6和 LX-2细胞促增殖率最高, HSC-T6和LX-2细胞增殖率分别为 (573.36 ± 206.34) % 和 (940.38 ± 61.65) %。泡球蚴感染沙鼠血清体外刺激后, HSC-T6细胞培养上清中Col1和α-SMA蛋白含量均上升; 且以 100 μL血清刺激后Col1和α-SMA含量最 高, 分别为 (20.99 ± 2.01) ng/mL和 (305.52 ± 16.67) pg/mL。泡球蚴及其生发层细胞、原头节均可体外诱导 HSC-T6 和 LX-2 细胞增殖, 增殖率分别为 (142.65 ± 9.17) % 和 (189.99 ± 7.75) %、 (118.55 ± 8.96) %和 (122.54 ± 0.21) %、 (156.34 ± 17.45) % 和 (160.59 ± 31.41) %, 不同刺激组细胞增殖率差异均有统计学意义 (FHSC-T6 = 11.24、FLX-2 = 47.72, P 均< 0.05); 泡球蚴及其生发层细胞、原头节刺激后, HSC-T6细胞培养上清中Col1和α-SMA含量均增高; 且以泡球蚴作用最为明显, Col1 和α-SMA含量分别为 (4.43 ± 2.23) ng/mL和 (285.20 ± 90.67) pg/mL。泡球蚴感染后 1 ~ 8 个月, 小鼠肝脏中胶原纤维 沉积持续增加; 小鼠血清中Col1水平在感染后 6 个月达最高, 为 (280.26 ± 23.04) ng/mL; α-SMA 水平在感染后 8 个月达最 高, 为 (33.68 ± 4.45) ng/mL。结论 泡球蚴持续感染可促进肝星状细胞体外增殖及小鼠血清中Col1和α-SMA蛋白含量升高, 引起小鼠肝脏中胶原纤维沉积增多。泡球蚴感染阶段是引起泡型棘球蚴病肝纤维化的关键期。.
Keywords: Collagen fiber; Echinococcus multilocularis; Germinal layer cell; Hepatic stellate cell; Protoscolcece.