收稿日期: 2024-05-31
修回日期: 2024-09-05
网络出版日期: 2025-03-26
基金资助
兵团南疆重点项目(S2021AB3280);国家卫生健康委中亚高发病防治重点实验室开放基金(KF202105);国家（兵团）科技计划(S2022EB5188)
Echinococcus granulosus cyst fluid promotes activation and migration of hepatic stellate cells
Received date: 2024-05-31
Revised date: 2024-09-05
Online published: 2025-03-26
Supported by
XPCC Southern Xinjiang Key Project(S2021AB3280);National Health Commission Key Laboratory for Prevention and Treatment of High Disease Diseases Open Fund(KF202105);National Science and Technology Plan Project(S2022EB5188)
目的 探究人细粒棘球蚴囊液（CF）对肝星状细胞（HSC）激活和迁移的影响及其作用机制。方法 收集细粒棘球蚴病患者的肝病灶组织，使用苏木精-伊红（HE）染色和Masson染色观察肝组织的纤维化情况。分别用不含CF和含有10%、20%、40% CF的培养基体外培养HSC 48 h和72 h，蛋白质免疫印迹（Western blotting）检测α平滑肌肌动蛋白（α-SMA）、Ⅰ型胶原α1（Col1a1）、磷脂酰肌醇3激酶（PI3K）、磷酸化PI3K（p-PI3K）、蛋白激酶B（Akt）、磷酸化Akt（p-Akt）等的相对表达水平，实时荧光定量PCR（qPCR）检测α-SMA、Col1a1和Ⅲ型胶原α1（Col3a1）的mRNA相对转录水平，划痕实验和Transwell实验检测CF对HSC迁移的影响。结果 HE染色结果显示，细粒棘球蚴病患者肝病灶组织中的细粒棘球蚴外囊呈透明均一的纤维条索样，Masson染色结果显示细粒棘球蚴外囊壁处有大量的胶原纤维沉积。Western blotting结果显示，干预72 h后，对照组、10% CF组、20% CF组和40% CF组的α-SMA蛋白相对表达水平分别为0.359 ± 0.043、0.641 ± 0.088、0.900 ± 0.084、1.111 ± 0.027，10% CF组、20% CF组、40% CF组均高于对照组（t = 5.236、10.050、13.980，均P < 0.05）；各组Col1a1的相对表达水平分别为0.392 ± 0.057、0.546 ± 0.022、0.854 ± 0.034、1.127 ± 0.057，10% CF组、20% CF组、40% CF组均高于对照组（t = 4.228、12.630、20.090，均P < 0.05）。qPCR结果显示，干预72 h后，对照组、10% CF组、20% CF组和40% CF组α-SMA的mRNA相对转录水平分别为1.000 ± 0.093、1.437 ± 0.106、1.453 ± 0.105、1.697 ± 0.154，10% CF组、20% CF组、40% CF组均高于对照组（t = 4.604、4.769、7.339，均P < 0.05）；各组Col1a1的mRNA相对转录水平分别为0.856 ± 0.042、1.067 ± 0.049、1.283 ± 0.128、1.582 ± 0.046，20% CF组、40% CF组均高于对照组（t = 6.932、11.790，均P < 0.05）；各组Col1a3的mRNA相对转录水平分别为0.611 ± 0.054、0.908 ± 0.041、1.000 ± 0.045、1.239 ± 0.101，10% CF组、20% CF组、40% CF组均高于对照组（t = 5.599、7.346、11.850，均P < 0.05）。Western blotting结果显示，对照组、10% CF组、20% CF组和40% CF组的p-PI3K/PI3K蛋白相对表达水平分别为0.346 ± 0.050、0.716 ± 0.054、0.941 ± 0.114、1.276 ± 0.004，10% CF组、20% CF组、40% CF组均高于对照组（t = 6.669、10.740、16.780，均P < 0.01）；各组p-Akt/Akt蛋白相对表达水平分别为0.524 ± 0.111、0.815 ± 0.019、1.043 ± 0.052、1.333 ± 0.054，10% CF组、20% CF组、40% CF组均高于对照组（t = 5.268、9.413、14.670，均P < 0.01）。划痕实验结果显示，干预24 h后，10% CF组、20% CF组和40% CF组的细胞迁移率分别为（73.6 ± 2.1）%、（88.2 ± 2.1）%和（96.5 ± 1.2）%，均高于对照组的（55.1 ± 2.9）%（t = 10.510、18.820、23.540，均P < 0.05）。Transwell实验结果显示，干预24 h后，对照组、10% CF组、20% CF组和40% CF组的平均每视野下细胞数分别为（62.333 ± 8.145）、（82.333 ± 8.505）、（132.333 ± 17.620）、（164.333 ± 11.060）个，20% CF组、40% CF组均高于照组（t = 7.173、10.450，均P < 0.01）。结论 细粒棘球蚴CF可以通过PI3K/Akt通路促进HSC激活，并提高HSC的迁移能力。
崔杰 , 伍希雅 , 林芷伊 , 蔡克丰 , 吴正展 , 熊梓彤 , 黄延鑫 , 房福众 , 辛子睿 , 张宏伟 . 细粒棘球蚴囊液促进肝星状细胞激活和迁移[J]. 中国寄生虫学与寄生虫病杂志, 2025 , 43(1) : 69 -75 . DOI: 10.12140/j.issn.1000-7423.2025.01.011
Objective To explore the effects and mechanisms of Echinococcus granulosus cyst fluid (CF) on the activation and migration of hepatic stellate cells (HSC). Methods Liver lesion tissues of cystic echinococcosis (CE) patients were obtained, and the fibrosis of liver tissues was observed using hematoxylin and eosin (HE) staining and Masson staining. HSCs were cultured in vitro for 48 and 72 hours in culture medium without CF and with 10%, 20% and 40% CF, respectively. Western blotting was used to detect the relative protein expression levels of α-mooth muscle actin (α-SMA), collagen type I alpha 1 (Col1a1), phosphatidylinositol 3 kinase (PI3K), phosphorylated PI3K (p-PI3K), protein kinase B (Akt) and phosphorylated Akt (p-Akt). Real-time quantitative PCR (qPCR) was used to detect the relative mRNA transcription levels of α-SMA, Col1a1 and Col3a1. Scratch assay and Transwell assays were conducted to assess the effects of CF on HSC migration. Results HE staining showed that the outer capsule of E. granulosus in the liver lesion tissues of CE patients was transparent and homogeneous with fibrous strips, and Masson staining showed a large amount of collagen fiber deposition in the outer capsule. Western blotting results showed that after 72 hours of intervention, the relative protein expression levels of α-SMA in the control group, 10% CF group, 20% CF group and 40% CF group were 0.359 ± 0.043, 0.641 ± 0.088, 0.900 ± 0.084 and 1.111 ± 0.027, respectively. The 10% CF group, 20% CF group, and 40% CF group had higher levels than the control group (t = 5.236, 10.050, 13.980; all P < 0.05). The relative expression levels of Col1a1 in each group were 0.392 ± 0.057, 0.546 ± 0.022, 0.854 ± 0.034 and 1.127 ± 0.057, respectively. The 10% CF group, 20% CF group and 40% CF group had higher levels than the control group (t = 4.228, 12.630, 20.090; all P < 0.05). qPCR results showed that after 72 hours of intervention, the relative mRNA transcription levels of α-SMA in the control group, 10% CF group, 20% CF group and 40% CF group were 1.000 ± 0.093, 1.437 ± 0.106, 1.453 ± 0.105 and 1.697 ± 0.154, respectively. The 10% CF group, 20% CF group and 40% CF group had higher levels than the control group (t = 4.604, 4.769, 7.339; all P < 0.05). The relative mRNA transcription levels of Col1a1 in each group were 0.856 ± 0.042, 1.067 ± 0.049, 1.283 ± 0.128 and 1.582 ± 0.046, respectively. The 20% CF group and 40% CF group had higher levels than the control group (t = 6.932, 11.790; both P < 0.05). The relative mRNA transcription levels of Col3a1 in each group were 0.611 ± 0.054, 0.908 ± 0.041, 1.000 ± 0.045 and 1.239 ± 0.101, respectively. The 10% CF group, 20% CF group and 40% CF group had higher levels than the control group (t = 5.599, 7.346, 11.850; all P < 0.05). Western blotting results showed that the relative protein expression levels of p-PI3K/PI3K in the control group, 10% CF group, 20% CF group and 40% CF group were 0.346 ± 0.050, 0.716 ± 0.054, 0.941 ± 0.114 and 1.276 ± 0.004, respectively. The 10% CF group, 20% CF group, and 40% CF group had higher levels than the control group (t = 6.669, 10.740, 16.780; all P < 0.01). The relative protein expression levels of p-Akt/Akt in each group were 0.524 ± 0.111, 0.815 ± 0.019, 1.043 ± 0.052 and 1.333 ± 0.054, respectively. The 10% CF group, 20% CF group and 40% CF group had higher levels than the control group (t = 5.268, 9.413, 14.670; all P < 0.01). Scratch assay results showed that after 24 hours of intervention, the cell migration rates in the 10% CF group, 20% CF group and 40% CF group were (73.6 ± 2.1)%, (88.2 ± 2.1)% and (96.5 ± 1.2)%, respectively, all higher than (55.1 ± 2.9)% in the control group (t = 10.510, 18.820, 23.540; all P < 0.05). Transwell assay results showed that after 24 hours of intervention, the average number of cells per field in the control group, 10% CF group, 20% CF group and 40% CF group were (62.333 ± 8.145), (82.333 ± 8.505), (132.333 ± 17.620) and (164.333 ± 11.060), respectively. The 20% CF group and 40% CF group had higher numbers than the control group (t = 7.173, 10.450; both P < 0.01). Conclusion E. granulosus CF could promote the activation of HSC and enhance their migration ability through the PI3K/Akt pathway.
Key words： Echinococcus granulosus; Hepatic stellate cells; Cyst fluid; Fibrosis
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