红花清肝十三味丸通过激活HNF4α改善肝功能的保肝机制研究

Improving Liver Function by Activating HNF4αIs a Mechanism Underlying the Hepatoprotective Effects of Honghua Qinggan 13 Flavor Pill,a Traditional Mongolian Medicine

[目的]探讨红花清肝十三味丸(HHQG)通过激活肝细胞核因子4α(hepatocyte nuclear factor 4α,HNF4α)改善肝功能的保肝作用机理。[方法]将45只C57BL/6J小鼠随机分为对照组、CCl_(4)损伤组、HHQG组,每组15只;HHQG组小鼠灌胃剂量为0.6165 g/(kg·BW)的HHQG,每天1次,持续7 d,对照组和CCl_(4)损伤组灌胃等体积的无菌蒸馏水;末次灌胃4 h后,对照组小鼠腹腔注射剂量为5 mL/(kg·BW)的橄榄油,CCl_(4)损伤组和HHQG组小鼠腹腔注射相同剂量的CCl_(4)与橄榄油混合液(CCl_(4)∶橄榄油=1∶4,V/V)进行肝损伤造模;分别在造模后第2、5、7天采集各组小鼠血液及肝脏组织样本;记录各组小鼠在试验期间的体重;测定血清谷草转氨酶(AST)、谷丙转氨酶(ALT)及总超氧化物歧化酶(SOD)活力;采用HE染色法及Masson染色法观察肝脏病理组织学变化;应用转录组测序(RNA-seq)技术分析差异基因富集通路;利用免疫组织化学技术检测HNF4α蛋白的表达情况。[结果]与对照组相比,在造模后第1~4天以及第6~7天,CCl_(4)损伤组小鼠体重显著(P<0.05)或极显著(P<0.01或P<0.001)降低;与CCl_(4)损伤组相比,在造模后第4天以及第6~7天,HHQG组小鼠体重显著(P<0.05)或极显著(P<0.01)增加。与对照组相比,在造模后第2天,CCl_(4)损伤组小鼠血清ALT和AST活力极显著(P<0.001)升高;与CCl_(4)损伤组相比,在造模后第2天,HHQG组小鼠血清ALT和AST活力显著(P<0.05)降低。HE染色及Masson染色观察结果显示,在造模后第2、5、7天,与CCl_(4)损伤组相比,HHQG组小鼠肝脏组织损伤程度明显改善。KEGG信号通路富集分析表明,与CCl_(4)损伤组相比,HHQG组小鼠肝脏组织中过氧化物酶体增殖物激活受体、脂肪酸降解、脂肪酸代谢等信号通路上调,核糖体、甘油磷脂代谢、甲状腺癌症等信号通路下调。与CCl_(4)损伤组相比,HHQG组小鼠肝脏组织中与肝脏分化相关的基因群(HNF4α、ATF5、Cebpb等)显著上调。免疫组织化学分析显示,与CCl_(4)损伤组相比,在造模后第2、5天,HHQG组小鼠肝脏组织中HNF4α表达量明显增加。[结论]HHQG通过激活HNF4α,抑制肝脏氧化损伤、促进肝脏组织修复以及维持肝脏功能,改善小鼠肝损伤,从而发挥保肝效应。

[Objective]This study was conducted to reveal the molecular mechanism underlying the hepatoprotective effects of Honghua Qinggan 13 Flavor Pill(HHQG),a traditional Mongolian medicine,by improving liver function via activating hepatocyte nuclear factor 4α(HNF4α).[Method]A total of 45 C57BL/6J mice were randomly assigned into control group,CCl_(4) injury group and HHQG group,with 15 mice in each group.The mice in HHQG group were given HHQG at a dose level of 0.6165 g/(kg·BW)by gavage once daily for 7 consecutive days,and those in control group and CCl_(4) injury group were given an equal volume of sterile distilled water by gavage.Four hours after the last gavage,the mice in control group were intraperitoneally injected with 5 mL/(kg·BW)of olive oil,and those in CCl_(4) injury group and HHQG group were intraperitoneally injected with the same dose of a CCl_(4) and olive oil mixture(CCl_(4)∶olive oil=1∶4,V/V)for liver injury modeling.Blood and liver tissue samples from mice in each group were collected on the 2nd,5th and 7th days after modeling.The body weights of the mice were monitored during the experiment period.The serum activities of aspartate aminotransferase(AST),alanine aminotransferase(ALT)and total superoxide dismutase(SOD)were measured.HE staining and Masson staining were used for observing liver histopathological changes.Differential gene enrichment pathways were analyzed by transcriptome sequencing(RNA-seq).The expression of HNF4αprotein in liver tissue was detected using immunohistochemical assay.[Result]Compared with control group,the body weight of CCl_(4) injury group significant(P<0.05)or extremely significant(P<0.01 or P<0.001)dropped on the 1st to 4th and 6th to 7th days after modeling,respectively.In comparison to CCl_(4) injury group,the body weight of HHQG group significant(P<0.05)or extremely significant(P<0.01)rose on the 4th and 6th to 7th days after modeling,respectively.On the 2nd day after modeling,CCl_(4) injury group had extremely significantly(P<0.001)increased serum activities of ALT and AST than control group,while significantly(P<0.05)decreased serum activities of ALT and AST were observed in HHQG group compared with CCl_(4) injury group.HE staining and Masson staining demonstrated that the liver damages in HHQG group were obviously ameliorated compared with CCl_(4) injury group on the 2nd,5th and 7th days after modeling.KEGG signaling pathway enrichment analysis showed that the signaling pathways such as peroxisome proliferator-activated receptor,fatty acid degradation and fatty acid metabolism in the liver tissue of HHQG group mice were upregulated,while ribosome,glycerophospholipid metabolism,thyroid cancer and some others were downregulated compared with CCl_(4) injury group.In comparison to CCl_(4) injury group,the gene groups associated with liver differentiation(such as HNF4α,ATF5 and Cebpb)in the liver tissue of HHQG group were significantly upregulated.Immunohistochemical analysis showed that HHQG group had obviously elevated HNF4αexpression level in liver tissue than CCl_(4) injury group on the 2nd and 5th days after modeling.[Conclusion]As a mechanism underlying the hepatoprotective effects,HHQG ameliorated liver injury in mice by inhibiting liver oxidative damage,promoting liver tissue repair and maintaining liver function via activating HNF4α.