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探讨青蒿琥酯和扶正化瘀方治疗血吸虫病肝纤维化对线粒体的影响

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

摘要:

目的:比较青蒿琥酯(Art)和扶正化瘀方治疗血吸虫病肝纤维化对线粒体自噬的影响。方法:将80只C57BL/6雌性小鼠随机分为健康组、感染组、Art治疗组和扶正化瘀方治疗组,每组20只。感染组及治疗组小鼠感染日本血吸虫尾蚴16条。6周后,吡喹酮(300 mg/kg)2日疗法杀虫。Art治疗组采用每天100 mg/kg腹腔注射的方式进行治疗,扶正化瘀方治疗组采用每天饲料给药,每1 kg饲料含16 g扶正化瘀方,6周后,取四组小鼠新鲜肝组织。Masson染色观察、蛋白质印迹分析肝组织中参与线粒体三羧酸循环反应的琥珀酸脱氢酶亚单位A(SDHA)、苹果酸脱氢酶(MDH2)、柠檬酸合酶(CS)、酮戊二酸脱氢酶(OGDH);雷帕霉素靶蛋白1(mTORC1)通路;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。提取各组肝脏组织样品的线粒体检测线粒体的耗氧率。双因素方差分析比较各组间蛋白表达的差异。结果:Masson染色显示,感染组小鼠肝脏纤维化面积显著高于健康组并且Art治疗组和扶正化瘀方治疗组小鼠肝脏纤维化面积均低于感染组。蛋白质印迹法分析结果显示,SDHA蛋白相对表达量感染组(0.82±0.05)与健康组(1.00±0.05)相比显著下降( t = 11.23, P = 0.004),并且Art治疗组(0.73±0.05)显著高于感染组( t = 10.79, P = 0.007),但是扶正化瘀方组(0.98±0.05)与健康组比较,差异无统计学意义( t = 1.925, P= 0.127);感染组p-AMPK的蛋白质相对表达量(1.15±0.05)显著高于健康组(0.98±0.07, t = 12.18, P = 0.003),Art治疗组(0.50±0.05)相比较于感染组p-AMPK的表达显著降低( t= 11.78, P= 0.003)。感染组AMPK的蛋白质相对表达量(0.80±0.05)显著低于健康组(1.00±0.05, t = 10.53, P = 0.005),Art治疗组(0.54±0.05)相比较于感染组AMPK的表达显著降低( t= 13.98, P= 0.004);感染组p-mTORC1的蛋白质相对表达量(0.93±0.08)与健康组相比差异不显著( t = 2.28, P= 0.065),而Art治疗组的表达量(0.63±0.05)显著低于感染组( t= 10.58, P = 0.029);感染组p-mTORC1/m-TORC1(0.98±0.03)相对于健康对照组(0.97±0.03, t = 0.98, P= 0.085)差异无统计学意义,Art治疗组(0.48±0.05)相对于感染组显著下降( t= 14.58, P = 0.009)。感染组PINK1的蛋白质相对表达量(0.55±0.05)显著低于健康组(1.00±0.03, t= 13.49, P= 0.001),Art治疗组(1.21±0.05, t= 9.98, P= 0.005)和扶正化瘀方治疗组(1.31±0.35, t= 6.98, P= 0.027)均显著高于感染组。线粒体功能检测显示,加入复合物Ⅱ底物后感染组耗氧量低于健康组而Art治疗组和扶正化瘀方治疗组均高于感染组,加入复合物Ⅲ底物后感染组耗氧量低于健康组而Art治疗组和扶正化瘀方治疗组显著高于感染组。 结论:Art通过抑制AMPK/mTORC1信号通路活性并增强线粒体耗氧量和自噬及SDHA表达而缓解血吸虫病肝纤维化。

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abstracts:

Objective:To compare the effects of artesunate (Art) and fuzheng huayu decoction on mitochondrial autophagy in the treatment of schistosomiasis liver fibrosis.Methods: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.Results: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) ( t = 11.23, P = 0.0035), while the Art treatment group (0.73±0.05) had significantly higher relative expression of SDHA protein than the infection group ( t = 10.79, P = 0.0073). However, there was no significant change in Fuzheng Huayu Decoction treatment group (0.98±0.05) ( t = 1.925, P= 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, t= 12.18, P = 0.0029), and the expression of p-AMPK in the Art treatment group (0.50±0.05) was significantly lower than the infection group ( t = 11.78, P= 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, t= 10.53, P= 0.0046). The expression of AMPK was significantly lower in the Art treatment group (0.54±0.05) than in the infection group (T = 13.98, P = 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 ( t = 2.28, P = 0.065), while the Art treatment group (0.63±0.05) had significantly lower relative expression of p-mTORC1 protein than the infection group ( t = 10.58, P = 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, t = 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 ( t = 14.58, P = 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, t = 13.49, P = 0.0011), while the Art treatment group (1.21±0.05, t = 9.98, P = 0.0046) and Fuzheng Huayu Decoction treatment group (1.31 ±0.35, t = 6.98, P = 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. Conclusion:Art can alleviate schistosomiasis liver fibrosis by inhibiting AMPK/mTORC1 signaling pathway activity and enhancing mitochondrial oxygen consumption, autophagy and SDHA expression.

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作者: 罗军涛 朱思成 黄依磊 叶建平 沈双
期刊: 《中华肝脏病杂志》2021年29卷7期 148-154页 MEDLINEISTICPKUCSCDCA
栏目名称: 肝纤维化·肝硬化
DOI: 10.3760/cma.j.cn501113-20201024-00577
发布时间: 2021-04-06
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