荔枝核总黄酮对CCl4诱导的大鼠肝纤维化的影响及作用机制和潜在Q-marker的预测

Effect and mechanism of total flavonoids of Lichi Semen on CCl4-induced liver fibrosis in rats,and prediction of Q-marker

考察荔枝核总黄酮(total flavonoids of Lichi Semen,TFL)对四氯化碳(CCl4)诱导的大鼠肝纤维化的作用,并分析和预测TFL的作用机制和潜在质量标志物(quality marker,Q-marker)。雄性SD大鼠皮下注射40%CCl4植物油溶液,每周2次,连续8周,建立肝纤维化大鼠模型。将肝纤维化大鼠随机分成模型组、水飞蓟宾组(43.19 mg·kg^-1)、扶正化瘀胶囊组(462.75 mg·kg^-1)、TFL不同剂量组(100,25 mg·kg^-1),以正常大鼠作为空白组,每组10只。除空白组外,其余各组大鼠均皮下注射维持剂量的40%CCl4植物油溶液,每周1次。同时,各治疗组灌胃给予相应药物,空白组和模型组大鼠均灌胃给予等体积生理盐水,每天1次,连续4周。实验末,腹主动脉取血,收集肝脏。采用自动生化检测仪检测血清中总胆红素(total bilirubin,TBiL)、直接胆红素(direct bilirubin,DBiL)、间接胆红素(indirect bilirubin,IBiL)、谷丙转氨酶(alanine aminotransferase,ALT)、谷草转氨酶(aspartate aminotransferase,AST)水平,通过Masson染色观察大鼠肝组织病理学变化。然后,收集TFL的化学成分,通过SWISS数据库、反向分子对接服务器(DRAR-CPI)预测化学成分作用靶点。通过GeneCards数据库筛选肝纤维化的疾病靶点,STRING数据库分析蛋白-蛋白的相互作用并进行GO(gene ontology)及KEGG(Kyoto encyclopedia of genes and genomes)分析。接着,iTRAQ蛋白质组学技术测定TFL组、模型组、空白组大鼠肝脏组织蛋白表达,验证靶点。最后,通过Cytoscape软件建立和可视化化学成分、靶点、通路网络并分析TFL的潜在Q-marker。结果显示,与空白组比较,模型组大鼠血清中TBiL,DBiL,IBiL,ALT,AST水平明显升高(P<0.05);与模型组比较,各给药组大鼠血清中TBiL,DBiL,IBiL,ALT,AST水平呈降低趋势,但没有显著性。Masson染色显示模型组大鼠肝损伤和纤维化程度均严重,各给药组大鼠的肝损伤程度和纤维化程度均呈现不同程度减轻。该文筛选得到74个化学成分,其可作用于EGFR,SRC等865个靶点,参与调控癌症途径、PI3K-Akt信号通路、HIF-1信号通路等多条与肝纤维化密切相关的信号通路。乔松素、槲皮素、表儿茶素、原花青素A2、柚皮素、川皮苷、根皮苷、芦丁与肝纤维化相关的靶点、通路关联度最高。蛋白质组学结果显示,在网络药理学预测的45个蛋白中,共有18个蛋白被鉴定。其中,与空白组相比,模型组有6个蛋白差异表达,其中上调蛋白5个、下调蛋白1个。TFL干预后,ALB,PLG,HSP90AA1,EGFR和MAP2K1显著回调。以上可见,TFL通过干预ALB,PLG,HSP90AA1,EGFR和MAP2K1表达发挥抗肝纤维化作用和潜在保肝作用,可能与调控PI3K-Akt通路等多条与肝纤维化相关的信号通路有关。乔松素、槲皮素、表儿茶素、原花青素A2、柚皮素、川皮苷、根皮苷、芦丁可作为TFL的潜在Q-marker用于制剂的质量控制。

This paper was to investigate the effect of total flavonoids of Lichi Semen(TFL)on carbon tetrachloride(CCl4)-induced liver fibrosis in rats,analyze and predict its mechanism of action and potential quality markers(Q-marker).Firstly,male SD rats were taken and injected subcutaneously with a 40% CCl4-vegetable oil solution twice a week for 8 consecutive weeks to establish a rat model of liver fibrosis.The rats with liver fibrosis were randomly divided into model group,silybin group(43.19 mg·kg^-1),Fuzheng Huayu Capsules group(462.75 mg·kg^-1),and TFL groups(100 mg·kg^-1 and 25 mg·kg^-1),with normal rats as a blank group,10 rats in each group.Except for the blank group,the rats in the other groups were subcutaneously injected with 40%CCl4-vegetable oil solution of a maintenance dose,once a week.The rats in various treatment groups received corresponding doses of drugs,while the rats in the blank group and model group received the same volume of normal saline once a day for 4 weeks.At the end of the experiment,blood was collected from the abdominal aorta and the liver tissues were collected.The levels of total bilirubin(TBiL),direct bilirubin(DBiL),indirect bilirubin(IBiL),alanine aminotransferase(ALT),and aspartate aminotransferase(AST)in serum were detected by using an automatic biochemical detector.Masson staining was used to observe the histopathological changes of rat liver.Then,the chemical compositions of TFL were collected,and the action targets of these chemical compositions were predicted through SWISS database and reverse molecular docking server(DRAR-CPI).After screening of disease targets of liver fibrosis by Gene Cards database,the protein-protein interaction was analyzed with use of STRING database,and GO(gene ontology)analysis and KEGG(Kyoto encyclopedia of genes and genomes)enrich analysis were also carried out.Moreover,an iTRAQ proteomics technology was used to determine protein expression in liver tissues of rats in TFL,model and blank groups to verify the targets.Furthermore,Cytoscape software was used to establish and visualize the network of chemical components,targets and pathways,and predict the potential Q-marker of TFL.The results showed that the levels of TBiL,DBiL,IBiL,ALT,and AST in the model group were significantly higher than those in the blank normal group(P<0.05),and the above levels in the treatment groups were lower than those in the model group,but with no significant differences.Masson staining showed that the liver damage and the degree of fibrosis were severe in the model group,and were relieved to different degrees in the treatment groups.Then,74 chemical components were screened,which could act on 865 targets such as EGFR and SRC,participating in the regulation of cancer pathways,PI3 K-Akt signaling pathway,HIF-1 signaling pathway and other signaling pathways closely related to liver fibrosis.Pinocembrin,quercetin,epicatechin,procyanidin A2,naringenin,nobiletin,phlorizin and rutin showed the highest correlation with liver fibrosis-related targets and pathways.Proteomics results showed that a total of 18 proteins among the 45 proteins predicted by internet pharmacology were identified,among which 6 proteins were significantly expressed,including 5 up-regulated proteins and 1 down-regulated protein.The protein expression of ALB,PLG,HSP90 AA1,EGFR and MAP2 K1 was significantly returned to a normal state in the TFL treatment groups.In conclusion,TFL may demonstrate the anti-hepatic fibrosis and potential hepatoprotective effects by regulating the expression of ALB,PLG,HSP90 AA1,EGFR and MAP2 K1,which may be associated with the regulation of multiple signaling pathways related to liver fibrosis such as PI3 K-Akt pathway.Pinocembrin,quercetin,epicatechin,procyanidin A2,naringenin,nobiletin,phlorizin and rutin could be regarded as potential Q-markers of TFL for quality control.