Investigation of pyrrolizidine alkaloid-induced liver fibrosis and therapeutic intervention

Ph.D.Pyrrolizidine alkaloids (PAs) are widely presented in plants and about half of them have been reported to be toxic. Hepatic sinusoidal obstruction syndrome (HSOS) is the major acute toxicity induced by PAs. Despite well-studied pathological changes of PA-induced acute HSOS, the progression of chronic liver disease caused by PA intoxication is largely unexplored. Liver fibrosis is a complex pathophysiological process of chronic liver diseases. The inevitable and consequence of sustainable liver fibrosis is cirrhosis or cancer, and therefore, preventing liver fibrosis is the primary measure to avoid further deterioration of liver diseases.Thus, the aims of the present study are to:a) Develop a rat model for investigating the disease progression from PA-induced HSOS to liver fibrosis and underlying mechanisms;b) Using the established rat model, to evaluate the potential activity of the selected anti-fibrotic agents on PA-induced liver fibrosis.Using retrorsine (RTS), a representative toxic PA, we investigated the progression of RTS-induced HSOS to liver fibrosis in male SD rats via two dosage regimens, including a single oral dose of RTS at 40 mg/kg and twice oral doses of RTS at 40 and 20 mg/kg on day 0 and day 7. RTS caused typical HSOS at the early stage, which further progressed to liver fibrosis in the two treatment groups. Depending upon the dosage regimens, RTS-induced liver fibrosis was either regressed if there was no further PA exposure, or progressed with a second RTS treatment. The mechanistic study revealed that RTS caused liver fibrosis through the following three major pathways: 1) the increased expression of transforming growth factor-β1 (TGF-β1); 2) the activation of hepatic stellate cells (HSCs); 3) the overproduction of extracellular matrix (ECM) regulated by the decreased expression of matrix metalloproteinase-9 (MMP-9) and increased expression of tissue inhibitor of metalloproteinase-1 (TIMP- 1).Using the established fibrosis model with twice doses of RTS, the antifibrotic effects of Fuzheng Huayu capsule (FZHY), a commonly used Traditional Chinese Medicine (TCM) proprietary product for treating fibrosis, salvianolic acid B (SalB) and baicalein (BAI), the TCM-derived bioactive compounds, were investigated. FZHY (202.5 mg/kg/day), SalB (20 mg/kg/day or twice a day) or BAI (40 mg/kg/day or twice a day) attenuated RTS-induced distortion of liver architecture and inhibited the fibrosis progression. All the three agents could decrease the expression of plasminogen activator inhibitor-1 (PAI-1) to accelarate the regression from RTS-induced liver fibrosis, and inhibit the production of ECM via reducing the expression of TIMP-1. Also, these three agents alleviated RTS-induced liver fibrosis via inhibiting the expression of TGF-β1 or ⍺-smooth muscle actin (α-SMA), a marker of activated HSCs. Metabolomics study demonstrated that FZHY treatment rectified the RTS-induced abnormal glycerophospholipid metabolism.All these results demonstrated the success of model development and also revealed the disease progression from RTS-induced acute liver injury to liver fibrosis and the underlying mechanisms. In addition, FZHY, SalB and BAI all exerted promising antifibrotic activities on RTS-induced liver fibrosis and deserved further investigations for their therapeutic values for PA-induced liver fibrosis in clinic.吡咯里西啶類生物鹼（Pyrrolizidine alkaloids, PA）廣泛存在於植物中，超過半數的PA 具有毒性。肝小靜脈閉塞症（Hepatic sinusoidal obstructionsyndrome, HSOS）是PA 急性中毒的主要症狀。PA 導致的HSOS 病理變化已得到深入研究，但PA 導致的慢性肝臟疾病則鮮有研究。肝纖維化是一種慢性複雜的病理過程。持續的肝纖維化將發展為肝硬化或肝癌，治療肝纖維化是避免慢性肝臟疾病進一步惡化的首要措施。因此，本研究旨在：a) 建立PA 誘導的大鼠肝纖維化模型，用以研究疾病進展和相關機制；b) 基於建立的肝纖維化模型，評估擬選藥物的治療作用。本研究使用倒千里光碱（Retrorsine, RTS）作為毒性PA 代表，研究了兩種給藥方案下，RTS 誘導的肝纖維化在雄性SD 大鼠體內的進展。給藥方案一是單次灌胃給予40 mg/kg RTS，方案二是第一及第七天分別灌胃給予40 mg/kg 和20 mg/kg RTS。两组大鼠均顯現了HSOS 症狀，隨後進一步發展為肝纖維化。若再次暴露于PA 下，肝纖維化將繼續發展；反之，可自行恢复。機制研究表明，RTS 誘導肝纖維化主要通過以下途徑：1）促進轉化生長因子β1（Transforming growth factor-β1，TGF-β1）的表達；2）促進肝星狀細胞（Hepatic stellate cells, HSCs）的激活；3）降低基質金屬蛋白脢-9（Matrix metalloproteinase, MMP-9）和增加基質金屬蛋白酶抑制劑-1（Tissue inhibitor of metalloproteinase-1, TIMP-1）的表達，導致細胞外基質（Extracellular matrix, ECM）過度產生。採用方案二建立大鼠肝纖維化模型，第15 天開始連續灌胃兩週給予治療藥物。結果表明，扶正化淤膠囊（202.5 mg/kg/day），黃芩素（40 mg/kg/day ortwice a day）和丹酚酸B（20 mg/kg/day or twice a day）均有治療效果。三者通過抑制TGF-β1 或α-SMA 表達發揮作用，亦可通過降低纖溶酶原激活物抑制物-1（Plasminogen activator inhibitor-1, PAI-1）和TIMP-1 的表達發揮治療作用。代謝組學研究還表明，扶正化淤膠囊還能夠逆轉RTS 導致的甘油磷脂代謝異常。總之，本研究建立了RTS 誘導的肝纖維化模型，並闡明了從急性肝損傷到肝纖維化的疾病進展和機制。扶正化淤膠囊，黃芩素和丹酚酸B 均對RTS 誘導的肝纖維化有治療作用，其對PA 誘導的肝纖維化的治療價值值得進一步研究。Chen, Xinmeng.Thesis Ph.D. Chinese University of Hong Kong 2020.Includes bibliographical references (leaves 107-121).Abstracts also in Chinese.Title from PDF title page (viewed on February 11, 2022)