基于CAR/PXR交叉谈话与TLRs/NF-κB信号通路茵陈甘草组方对胆汁淤积肝损伤的保护作用与机制研究

Persistent intrahepatic cholestasis will eventually lead to liver failure, and there is still lack of effective drugs. The recent study indicated that the toxic bile compounds such as bile acid and bilirubin accumulated in hepatocytes can trigger an inflammatory response and aggravate liver injury through activation of TLRs/NF-kappa B pathway. Our previous findings suggested that Yinchenhao Decoction and Gancao Decoction could up-regulate gene expression of drug metabolic enzymes and transporters via activation of nuclear receptor CAR and PXR. The induction of the drug metabolic enzymes and transporters can reduce the levels of toxic bile compounds in hepatocytes. We found that Yin Gan mixture has obvious protective effect on liver injury via reducing recruitment the inflammatory cells to attacking injured hepatocytes. As such,we proposed the scientific hypothesis that Yinchen Gancao formula has a protective effects on cholestatic liver injury because Yinchen Gancao formula not only regulate with network metabolism and transport of toxic bile components to reduce their toxicity via crosstalk between CAR and PXR , but also has an anti-inflammatory effect through inhibiting the TLRs/NF-kappa B pathway to reduce the synthesis and release of cytokines and chemokines. Modern medical research technology will be used to explore the protective mechanism of Yinchen Gancao formula on cholestatic liver injury in knockout mouse model and gene silencing cells. The protective mechanism of Yinchen Gancao formula on cholestatic liver injury could attribute to the anti-inflammatort activaties,which are associated with the inhibition of the TLRs/NF-kappa B pathway and reducing the synthesis and release of cytokines and chemokines, and the detoxification activaties,which are associated with the up-regulation of expression of drug metabolic enzymes and transporters through crosstalk between CAR and PXR. It will provide scientific support for Yinchen Gancao formula being used for treating cholestatic liver injury in clinical practice.

持续肝内胆汁淤积最终会导致肝衰竭，尚缺乏有效药物。最新研究表明毒性胆汁成分积聚引发肝脏损伤，激活TLRs/NF-κB通路促使炎性细胞募集和炎性反应而加重肝损伤。前期研究发现:茵陈蒿和甘草单煎液可激活CAR和PXR增加药物代谢酶及转运体表达而减少胆汁成分淤积；合煎液可减少模型鼠肝窦炎性细胞募集与保护肝损伤。由此提出“茵陈甘草组方通过CAR与PXR交叉谈话网络调控胆汁酸和胆红素代谢与转运的解毒作用，抑制TLRs/NF-κB通路减少细胞因子和趋化因子产生的抗炎作用，治疗胆汁淤积性肝损伤”的科学假说。采用现代医学研究方法在基因敲除小鼠和基因沉默细胞水平开展茵陈甘草组方治疗胆汁淤积性肝损伤的保护机制研究，阐明通过CAR/PXR交叉谈话网络调控药物代谢酶及转运体作用的解毒作用和抑制TLRs/NF-κB通路减少细胞因子和趋化因子产生的抗炎作用，明确其治疗胆汁淤积肝损伤的保护机制，为临床使用提供科学依据。

肝内胆汁淤积症（IC）是多种原因导致胆汁酸在肝细胞内积聚诱发的肝脏病变。由于肝脏高浓度胆汁酸可诱发炎性细胞募集及炎性反应而加重肝脏损伤，因此，其治疗策略应从利胆和抗炎两方面入手。熊去氧胆酸和奥贝胆酸是FDA批准的治疗原发性IC的药物，因部分病人应答不完全或严重瘙痒等副作用而限制临床广泛使用。我们利用本项目研究发现茵陈甘草组方对于治疗胆汁淤积症具有良好疗效。主要研究内容和结果：1. 在ANIT诱导IC模型小鼠中发现甘草及体内活性物质甘草次酸抑制TLR2/NF-κB通路，减轻肝脏炎症反应和炎症细胞浸润；经流式细胞技术发现甘草次酸抑制TLR2/NF-κB通路减少CCL2，抑制CCR2+炎性细胞肝脏浸润，对ANIT模型小鼠肝损伤具有保护作用。并通过体外TLR2基因沉默技术证实甘草次酸的作用靶点为TLR2。2. 灌服甘草水煎液及甘草酸对PXR敲除小鼠制作的ANIT诱导IC模型小鼠肝损伤仍有保护作用，对胆汁酸代谢蛋白UGT1A1、CYP3A11表达无明显影响，仍抑制p-NF-κB p65和NLRP3的表达。提示甘草及甘草酸对ANIT诱导IC模型小鼠肝损伤的保护作用不是通过激动PXR，与抑制肝脏炎症反应有关。3. 甘草次酸对石胆酸诱导的IC模型肝脏TLR2、TLR4和p-NF-κB-p65蛋白及细胞因子（IL-1β、IL-6、TNF-α、CCL2和CXCL2）表达具有抑制作用，提示甘草次酸通过抑制TLRs/NF-κB通路的肝脏保护作用。5. 茵陈水煎液明显升高1%CA诱导IC模型小鼠FXR及下游靶基因BSEP、MRP2、OATP1A1、MRP3、MRP4等蛋白表达；抑制NTCP及CYP7A1表达；促进肠道FXR、FGF15及肝脏FGFR4蛋白表达而减少肝内胆汁酸蓄积，对1%CA诱导胆汁淤积肝损伤具有明显保护作用。相比较，茵陈对FXR敲除小鼠制作的1%CA诱导IC模型小鼠肝损伤无保护作用。6. 茵陈甘草（5:1）和（3:1）组明显增加肝脏BSEP表达，减轻肝脏胆汁酸潴留，抑制NLRP3及炎症反应，对1%CA诱导的肝诱导IC肝损伤的保护作用优于茵陈和甘草单剂量组。本项目利用基因敲除小鼠和基因沉默技术明确甘草及活性物质甘草次酸抑制TLR2/NF-κB通路的抗炎作用，茵陈激活FXR诱导BSEP促进胆汁酸排泌作用，明确茵陈甘草组方从利胆和抗炎两方面协同治疗胆汁淤积症，为临床使用提供依据。