[探讨青蒿琥酯与扶正化瘀方对血吸虫病肝纤维化治疗中线粒体的影响]

[Exploring the effects of artesunate and fuzheng huayu decoction on mitochondria in the treatment of schistosomiasis liver fibrosis].

作者信息

Luo J T, Qian S N, Wu K Y, Zhu S C, Huang Y L, Ye J P, Shen S

机构信息

Shanghai Jiao Tong University Affiliated Sixth People's Hospital, Shanghai 200233, China.

Shanghai University of Medicine & Health Sciences, Shanghai 201318, China.

出版信息

Zhonghua Gan Zang Bing Za Zhi. 2022 Jan 20;30(1):45-51. doi: 10.3760/cma.j.cn501113-20201024-00577.

Abstract

To compare the effects of artesunate (Art) and fuzheng huayu decoction on mitochondrial autophagy in the treatment of schistosomiasis liver fibrosis. Eighty C57BL/6 female mice were randomly divided into healthy control group, infection group, Art treatment group and Fuzheng Huayu Decoction treatment group, with 20 mice in each group. Mice in the infection group and treatment group were infected with 16 Schistosoma japonicum cercariae. After 6 weeks, praziquantel (300 mg/kg) was used for 2 days to kill the worms. The Art treatment group was treated with intraperitoneal injection of 100 mg/kg/day, while the Fuzheng Huayu Decoction treatment group was fed 16g of fuzheng huayu decoction per 1kg per day. After 6 weeks, fresh liver tissues of the four groups were collected. Masson staining and Western blot were used to observe the succinate dehydrogenase subunit A (SDHA) and malate dehydrogenase (MDH2), citrate synthase (CS), ketoglutarate dehydrogenase (OGDH), and target of rapamycin 1 (mTORC1) pathway involved in mitochondrial tricarboxylic acid cycle in liver tissues. The relative expression levels of adenylate activated protein kinase (AMPK) and mitochondrial autophagy pathway kinase (PINK1) were detected. Liver tissue samples were extracted from each group to detect the mitochondrial oxygen consumption rate. Two-way ANOVA was used to compare the significance and difference between two sets of samples. Masson staining showed that the infection group mice had significantly higher liver fibrosis area than the healthy control group, while the Art treatment group and Fuzheng Huayu Decoction treatment group mice had lower liver fibrosis area than the infection group. Western blot analysis showed that the infection group (0.82 ± 0.05) had significantly lower relative expression of SDHA protein than the healthy control group (1.00 ± 0.05) ( = 11.23, = 0.0035), while the Art treatment group (0.73 ± 0.05) had significantly higher relative expression of SDHA protein than the infection group ( = 10.79, = 0.0073). However, there was no significant change in Fuzheng Huayu Decoction treatment group (0.98±0.05) ( = 1.925, = 0.1266). The relative expression of p-AMPK protein was significantly higher in the infection group (1.15 ±0.05) than in the healthy control group (0.98 ± 0.07, = 12.18, = 0.0029), and the expression of p-AMPK in the Art treatment group (0.50 ± 0.05) was significantly lower than the infection group ( = 11.78, = 0.0032). The relative protein expression of AMPK was significantly lower in the infection group (0.80 ± 0.05) than in the healthy control group (1.00 ± 0.05, = 10.53, = 0.0046). The expression of AMPK was significantly lower in the Art treatment group (0.54 ± 0.05) than in the infection group ( = 13.98, = 0.0036). The relative expression of p-mTORC1 protein (0.93 ± 0.08) was not significantly different in the infection group than in the healthy control group ( = 2.28, = 0.065), while the Art treatment group (0.63 ± 0.05) had significantly lower relative expression of p-mTORC1 protein than the infection group ( = 10.58, = 0.029). The expression of p-mTORC1/ m-TORC1 was not significantly different in the infection group (0.98 ± 0.03) than in the healthy control group (0.97 ± 0.03, = 0.98, P = 0.085), while the Art treatment group (0.63 ± 0.05) had significantly lower relative expression of p-mTORC1/ m-TORC1 than the infection group ( = 14.58, = 0. 009). The relative protein expression of PINK1 was significantly lower in the infection group (0.55 ± 0.05) than in the healthy control group (1.00 ± 0.03, = 13.49, = 0.0011), while the Art treatment group (1.21 ± 0.05, = 9.98, = 0.0046) and Fuzheng Huayu Decoction treatment group (1.31 ±0.35, = 6.98, = 0.027) had significantly higher relative protein expression of PINK1 than the infection group. Mitochondrial function tests showed that after adding substrate complex II, the oxygen consumption of the infection group was lower than the healthy control group, while the Art treatment group and the Fuzheng Huayu Decoction treatment group had higher oxygen consumption than the infection group. The oxygen consumption was significantly lower after adding the substrate complex III in the infection group than the healthy control group, while the Art treatment group and Fuzheng Huayu Decoction treatment group had higher oxygen consumption than the infection group. Art can alleviate schistosomiasis liver fibrosis by inhibiting AMPK/mTORC1 signaling pathway activity and enhancing mitochondrial oxygen consumption, autophagy and SDHA expression.

摘要

比较青蒿琥酯(Art)与扶正化瘀方对血吸虫病肝纤维化线粒体自噬的影响。将80只C57BL/6雌性小鼠随机分为健康对照组、感染组、Art治疗组和扶正化瘀方治疗组,每组20只。感染组和治疗组小鼠经16只日本血吸虫尾蚴感染。6周后,用吡喹酮(300mg/kg)连续治疗2天以杀灭虫体。Art治疗组腹腔注射100mg/kg/天,扶正化瘀方治疗组每天每1kg喂饲16g扶正化瘀方。6周后,收集四组的新鲜肝组织。采用Masson染色和蛋白质免疫印迹法观察肝组织中参与线粒体三羧酸循环的琥珀酸脱氢酶亚基A(SDHA)、苹果酸脱氢酶(MDH2)、柠檬酸合酶(CS)、酮戊二酸脱氢酶(OGDH)以及雷帕霉素靶蛋白1(mTORC1)信号通路。检测腺苷酸活化蛋白激酶(AMPK)和线粒体自噬通路激酶(PINK1)的相对表达水平。从每组中提取肝组织样本检测线粒体氧耗率。采用双向方差分析比较两组样本间的显著性和差异。Masson染色显示,感染组小鼠肝纤维化面积显著高于健康对照组,而Art治疗组和扶正化瘀方治疗组小鼠肝纤维化面积低于感染组。蛋白质免疫印迹分析显示,感染组(0.82±0.05)SDHA蛋白相对表达显著低于健康对照组(1.00±0.05)(F=11.23,P=0.0035),而Art治疗组(0.73±0.05)SDHA蛋白相对表达显著高于感染组(F=10.79,P=0.0073)。然而,扶正化瘀方治疗组(0.98±0.05)无显著变化(F=1.925,P=0.1266)。感染组p-AMPK蛋白相对表达(1.15±0.05)显著高于健康对照组(0.98±0.07,F=12.18,P=0.0029),Art治疗组(0.50±0.05)p-AMPK表达显著低于感染组(F=11.78,P=0.0032)。感染组AMPK相对蛋白表达(0.80±0.05)显著低于健康对照组(1.00±0.05,F=10.53,P=0.0046)。Art治疗组AMPK表达(0.54±0.05)显著低于感染组(F=13.98,P=0.0036)。感染组p-mTORC1蛋白相对表达(0.93±0.08)与健康对照组无显著差异(F=2.28,P=0.065),而Art治疗组(0.63±0.05)p-mTORC1蛋白相对表达显著低于感染组(F=10.58,P=0.029)。感染组p-mTORC1/m-TORC1表达(0.98±0.03)与健康对照组无显著差异(0.97±0.03,F=0.98,P=0.085),而Art治疗组(0.63±0.05)p-mTORC1/m-TORC1相对表达显著低于感染组(F=14.58,P=0.009)。感染组PINK1相对蛋白表达(0.55±0.05)显著低于健康对照组(1.00±0.03,F=13.49,P=0.0011),而Art治疗组(1.21±0.05,F=9.98,P=0.0046)和扶正化瘀方治疗组(1.31±0.35,F=6.98,P=0.027)PINK1相对蛋白表达显著高于感染组。线粒体功能检测显示,加入底物复合物II后,感染组氧耗低于健康对照组,而Art治疗组和扶正化瘀方治疗组氧耗高于感染组。加入底物复合物III后,感染组氧耗显著低于健康对照组,而Art治疗组和扶正化瘀方治疗组氧耗高于感染组。青蒿琥酯可通过抑制AMPK/mTORC1信号通路活性,增强线粒体氧耗、自噬及SDHA表达,减轻血吸虫病肝纤维化。

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