Background

Triptolide is naturally isolated from Tripterygium wilfordii Hook F., possessing multiple biological activities. Hepatotoxicity is one of the main side effects of triptolide. However, the effect of triptolide on nonalcoholic fatty liver disease remains unknown (NAFLD).

Purpose

This study aimed to observe the amelioration of triptolide against NAFLD and investigate the engaged mechanism.

Methods

Two typical animal models of NAFLD, obese db/db mice and methionine/choline-deficient (MCD) diet-fed mice, were used. Hepatic steatosis, inflammation, and fibrosis were evaluated by H&E and Masson staining. Oil red O staining and lipid extraction analysis were used to detect fat content in mice livers. Expression of lipid metabolism, inflammatory and fibrogenic genes was also detected by Real-time PCR and Western blotting, respectively. Phosphoproteomics, molecular docking, and TR-FRET assay were performed to provide further insight into how triptolide improved NAFLD.

Results

Intraperitoneal injection of triptolide at a daily dose of 50 μg/kg significantly alleviated MCD diet-induced nonalcoholic steatohepatitis (NASH), but 100 μg/kg triptolide caused severe hepatotoxicity. Pathological staining confirmed low-dose triptolide treatment reducing hepatic lipid deposition, inflammation, and fibrosis in NASH. Serum biochemical analysis revealed a reduction in the level of liver enzymes and bilirubin. MCD also induced rising expression of typical genes and proteins related to fibrosis (fibronectin, α-SMA, collagens, TGF-β) and inflammation (ILs, TNF-α, MCP-1), which was suppressed by low-dose triptolide. Data from the proteomics/phosphoproteomics and TR-FRET assay indicated triptolide was a potential allosteric AMPK agonist to increase the phosphorylation on Thr172 residue, with the EC₅₀ of 277.78 μM and 231.02 μM for AMPKα1 and AMPKα2, respectively. Moreover, triptolide exhibited an ability to activate AMPK and further led to increasing ACC1 phosphorylation in the liver. The positive results that triptolide ameliorated hepatic lipogenesis, fatty acid oxidation, and fibrosis of NAFLD via activating AMPK were further confirmed in db/db mice with 10-week intervention (50 μg/kg, i.v., twice a week).

Conclusion

This study demonstrates that dose-related triptolide as an allosteric AMPK agonist has the potential to alleviate NAFLD without hepatotoxicity.

中文翻译：

---

雷公藤内酯激活 AMPK 通过改善肝脏脂质代谢、炎症和纤维化减轻非酒精性脂肪肝

背景

雷公藤内酯是从雷公藤 Hook F. 中天然分离出来的，具有多种生物活性。肝毒性是雷公藤内酯的主要副作用之一。然而，雷公藤内酯对非酒精性脂肪性肝病（NAFLD）的影响仍然未知。

目的

本研究旨在观察雷公藤内酯对 NAFLD 的改善作用并探讨其作用机制。

方法

使用了两种典型的 NAFLD 动物模型，即肥胖 db/db 小鼠和蛋氨酸/胆碱缺乏 (MCD) 饮食喂养的小鼠。通过 H&E 和 Masson 染色评估肝脏脂肪变性、炎症和纤维化。油红O染色和脂质提取分析用于检测小鼠肝脏中的脂肪含量。脂质代谢、炎症和纤维化基因的表达也分别通过实时 PCR 和蛋白质印迹检测。进行了磷酸化蛋白质组学、分子对接和 TR-FRET 测定，以进一步了解雷公藤内酯如何改善 NAFLD。

结果

雷公藤内酯以 50 μg/kg 日剂量腹腔注射显着减轻 MCD 饮食诱导的非酒精性脂肪性肝炎 (NASH)，但 100 μg/kg 雷公藤内酯引起严重的肝毒性。病理染色证实低剂量雷公藤内酯治疗可减少 NASH 中的肝脏脂质沉积、炎症和纤维化。血清生化分析显示肝酶和胆红素水平降低。MCD 还诱导与纤维化（纤连蛋白、α-SMA、胶原蛋白、TGF-β）和炎症（ILs、TNF-α、MCP-1）相关的典型基因和蛋白质的表达升高，而低剂量雷公藤内酯可抑制这种表达。来自蛋白质组学/磷酸蛋白质组学和 TR-FRET 测定的数据表明雷公藤内酯是一种潜在的变构 AMPK 激动剂，可增加 Thr172 残基的磷酸化，EC ₅₀AMPKα1 和 AMPKα2 分别为 277.78 μM 和 231.02 μM。此外，雷公藤内酯表现出激活 AMPK 的能力，并进一步导致肝脏中 ACC1 磷酸化的增加。雷公藤内酯通过激活 AMPK 改善肝脏脂肪生成、脂肪酸氧化和 NAFLD 纤维化的阳性结果在 db/db 小鼠中得到进一步证实，干预 10 周（50 μg/kg，静脉注射，每周两次）。

结论

这项研究表明，剂量相关的雷公藤内酯作为一种变构 AMPK 激动剂有可能减轻 NAFLD 而没有肝毒性。