蓬莪术醋制前后改善斑马鱼急性肝损伤的作用机制研究

Mechanism of raw and vinegar-processed Curcuma phaeocaulis on improving acute liver injury in zebrafish

目的基于核因子E2相关因子2(nuclear factor-erythroid 2-related factor 2,Nrf2)/血红素加氧酶-1(heme oxygenase-1,HO-1)和转化生长因子-β(transforming growth factor-β,TGF-β)/Smad3信号通路探讨醋制前后蓬莪术改善硫代乙酰胺(thioacetamide,TAA)诱导斑马鱼急性肝损伤的作用机制。方法以醋制前后的蓬莪术为研究对象,建立TAA诱导斑马鱼急性肝损伤模型,将醋制前后蓬莪术水煎液分别设置高、中、低剂量(8、4、2 mg/mL)组,以异甘草酸镁为阳性对照药物组,斑马鱼培养水为对照组,给药72 h后观察斑马鱼幼鱼肝脏形态,通过苏木素-伊红(HE)染色观察肝组织病理变化;采用试剂盒检测斑马鱼幼鱼中丙氨酸氨基转移酶(alanineaminotransferase,ALT)、天冬氨酸氨基转移酶(aspartate aminotransferase,AST)活性及谷胱甘肽(glutathione,GSH)、丙二醛(malondialdehyde,MDA)水平;利用qRT-PCR检测斑马鱼幼鱼Nrf2、白细胞介素-10(interleukin-10,IL-10)、Smad3和Smad7基因表达;Western blotting测定斑马鱼幼鱼Nrf2/HO-1和TGF-β/Smad3信号通路相关蛋白表达。结果醋制前后蓬莪术对TAA导致的肝组织细胞形态损伤具有保护作用。生化结果表明,与模型组比较,醋制前后蓬莪术水煎液组ALT、AST活性及GSH水平均显著升高(P<0.05、0.01、0.001),MDA水平均显著降低(P<0.001)。qRT-PCR结果表明,与模型组比较,醋制前后蓬莪术水煎液组Nrf2、IL-10、Smad7 m RNA表达水平显著升高(P<0.05、0.01、0.001),醋蓬莪术水煎液高剂量组Smad3 m RNA表达水平显著降低(P<0.001);其中,与等剂量蓬莪术组相比,醋蓬莪术高剂量组显著抑制Nrf2、Smad3的m RNA表达(P<0.05、0.001),且醋蓬莪术具有促进IL-10和Smad7 m RNA表达的趋势,但没有显著性差异。Western blotting结果表明,与模型组比较,蓬莪术水煎液高、中剂量组和醋蓬莪术水煎液各剂量组Nrf2蛋白表达水平显著升高(P<0.001),蓬莪术高剂量组和醋蓬莪术各剂量组Smad3蛋白表达水平显著降低(P<0.05、0.01、0.001),蓬莪术水煎液高剂量组和醋蓬莪术水煎液高、中剂量组TGF-β蛋白表达水平显著降低(P<0.05、0.01、0.001);其中,与等剂量蓬莪术组相比,醋蓬莪术各剂量组Nrf2蛋白表达水平显著升高(P<0.001),且醋蓬莪术具有抑制Smad3和TGF-β蛋白表达的趋势,但没有显著性差异。结论醋制前后的蓬莪术可以不同程度地改善TAA诱发的急性肝损伤,其作用机制可能与调控Nrf2/HO-1和TGF-β/Smad3信号通路有关。

Objective To investigate the mechanism of raw and vinegar-processed Curcuma phaeocaulis on improving thioacetamide(TAA)-induced acute liver injury in zebrafish based on nuclear factor-erythroid 2-related factor 2(Nrf2)/heme oxygenase-1(HO-1)and transforming growth factor-β(TGF-β)/Smad3 signaling pathway.Methods The raw and vinegar-processed C.phaeocaulis were studied and the TAA-induced acute liver injury model of zebrafish was established.High-,medium-and low-doses(8,4,2 mg/mL)of raw and vinegar-processed C.phaeocaulis water decoction were set.Magnesium isoglycyrrhizinate was used as positive control group,and zebrafish culture water was used as normal control group.The liver morphology of zebrafish larvae was observed 72 h after administration,and the pathological changes of liver tissue were observed by hematoxylin-eosin(HE)staining.Alanine aminotransferase(ALT),aspartate aminotransferase(AST)activities and glutathione(GSH),malondialdehyde(MDA)levels in zebrafish larvae were detected by kit;qRT-PCR was used to detect Nrf2,interleukin-10(IL-10),Smad3 and Smad7 gene expressions in zebrafish larvae;Western blotting was used to determine the expressions of Nrf2/HO-1 and TGF-β/Smad3 signaling pathway related proteins in zebrafish larvae.Results Raw and vinegar-processed C.phaeocaulis water decoction had protective effect on the morphological injury of liver cells caused by TAA.Biochemical results showed that compared with model group,ALT,AST activities and GSH level in raw and vinegar-processed C.phaeocaulis water decoction group were significantly increased(P<0.05,0.01,0.001),MDA level was significantly decreased(P<0.001).The results of qRT-PCR showed that compared with model group,the mRNA expression levels of Nrf2,IL-10 and Smad7 in raw and vinegar-processed C.phaeocaulis group water decoction were significantly increased(P<0.05,0.01,0.001),while the mRNA expression levels of Smad3 in vinegar-processed C.phaeocaulis group were significantly decreased(P<0.001).Compared with the same dose of C.phaeocaulis group,vinegar-processed C.phaeocaulis highdose group significantly inhibited the mRNA expression of Nrf2 and Smad3(P<0.05,0.001),and the vinegar-processed C.phaeocaulis showed a tendency to promote IL-10 and Smad7 mRNA expression,but there was no significant difference.Western blotting results showed that compared with model group,the expression level of Nrf2 protein in the high-dose and medium-dose groups of C.phaeocaulis and the dosage groups of vinegar-processed C.phaeocaulis were significantly increased(P<0.001).The expression level of Smad3 protein in high-dose C.phaeocaulis and the dosage groups of vinegar-processed C.phaeocaulis was significantly decreased(P<0.05,0.01,0.001).The expression level of TGF-βprotein in high-dose C.phaeocaulis and high-,medium-dose groups of vinegarprocessed C.phaeocaulis was significantly decreased(P<0.05,0.01,0.001).Among them,the Nrf2 protein level of the dosage groups of vinegar-processed C.phaeocaulis was significantly higher than that of the same dose of C.phaeocaulis group(P<0.001),and vinegar-processed C.phaeocaulis showed a tendency to inhibit the expression of Smad3 and TGF-β,but there was no significant difference.Conclusion The raw and vinegar-processed C.phaeocaulis can improve TAA-induced acute liver injury to varying degrees,and the mechanism of action may be related to the regulation of Nrf2/HO-1 and TGF-β/Smad3 signaling pathways.