探討hydroxychloroquine如何緩解脂肪性肝炎

Abstract

NAFLD（非酒精性脂肪肝病）由於肥胖、糖尿病、胰島素抵抗、基因遺傳及肝臟脂肪代謝紊亂等因素發展而來，導致肝臟脂肪積累。這種脂肪積累會引起肝損傷、炎症和纖維化，進而可能發展成NASH（非酒精性脂肪性肝炎）、肝硬化甚至肝癌。在一項回顧性研究中，我們測試了hydroxychloroquine (HCQ)對脂肪性肝炎患者的治療潛力。結果顯示，接受HCQ治療的患者在一個月內serum alanine transaminase（ALT）水平顯著降低（治療前94.1 ± 44.9 U/L vs. 治療後一個月46.7 ± 24.0 U/L，N=26，p < 0.001）。因此，HCQ在緩解脂肪性肝炎方面非常有效。

為了理解HCQ如何緩解脂肪性肝炎的機制，我們將HCQ應用於包括hepatocytes、stellate cells（fibroblast）和Kupffer cells （monocytes/macrophages）在內的肝臟常駐活細胞。結果顯示，治療濃度下（1.45-2.90 μM）的HCQ不影響HepG2細胞的活力。此外，當使用palmitic acid（PA）刺激肝臟free fatty acid（FFA）積累時，HCQ對肝臟內的脂肪積累沒有顯著影響。有趣的是，我們發現HCQ可以抑制飢餓誘導下的肝細胞油滴生成，HCQ不僅減少了細胞內的油滴數量，還抑制了新油滴的形成。

然而，在細胞活力和活性方面，我們觀察到肝臟星狀細胞（HSCs）對HCQ比HepG2細胞更為敏感。HCQ通過自噬抑制減少了活化HSC中的F-actin信號，但對α-SMA和Collagen I的mRNA和蛋白表達水平沒有影響。

在免疫微環境方面，我們利用ELISA測量了涉及肝臟發炎的關鍵inflammatory cytokines，包括IL-1β、IL-6和TNF-α。結果顯示，治療濃度下的HCQ並不抑制THP-1免疫細胞和HSCs的cytokine secretion。

總結來說，我們發現HCQ不會改變營養過剩誘導的油滴形成，但會抑制飢餓誘導的油滴生成。然而，它可能通過靶向肝臟星狀細胞的活力來減少纖維化。通過建立這些平台，我們對HCQ緩解NAFLD的機制有了更好的理解。因此，HCQ未來可能成為治療NAFLD的新型潛力藥物。

NAFLD (Non-Alcoholic Fatty Liver Disease) arises from factors such as obesity, diabetes, insulin resistance, genetics, and disruptions in liver fat metabolism, which result in the accumulation of fat in the liver. This accumulation can cause liver damage, inflammation, and fibrosis, potentially progressing to NASH (Non-Alcoholic Steatohepatitis), cirrhosis, or even liver cancer. In a previous retrospective study, we tested the therapeutic potential of hydroxychloroquine (HCQ) in patients with steatohepatitis. The results showed that the patients receiving HCQ had significantly reduced serum alanine transaminase (ALT) levels within one month (94.1 ± 44.9 U/L before HCQ vs. 46.7 ± 24.0 U/L one month after HCQ, N=26, p < 0.001). Therefore, HCQ is very effective in alleviating steatohepatitis.

To understand the mechanism of how HCQ alleviated steatohepatitis, we applied HCQ to resident live cells including hepatocytes, stellate cells (fibroblast), and Kupffer cells (monocytes/macrophages). HCQ at therapeutic concentrations (1.45-2.90 μM) did not impact the viability of HepG2 cells. Additionally, when free fatty acid (FFA) accumulation in the liver was stimulated using palmitic acid (PA), HCQ did not significantly influence lipid accumulation within the liver. Interestingly, we discovered that HCQ can inhibit starvation-induced lipid droplet biogenesis in hepatocytes. HCQ not only decreased the number of lipid droplets within the cells but also prevented the formation of new lipid droplets.

However, regarding cell viability and activity, we observed that hepatic stellate cells (HSCs) were more sensitive to HCQ than HepG2 cells. HCQ reduced F-actin signals in activated HSCs by inhibiting autophagy, but it did not impact the mRNA and protein expression levels of α-SMA and Collagen I.

In terms of the immune microenvironment, we utilized ELISA to measure key inflammatory cytokines involved in liver inflammation, including IL-1β, IL-6, and TNF-α. Our results indicated that HCQ, at therapeutic concentrations, did not inhibit cytokine secretion from THP-1 immune cells and HSCs.

In conclusion, we found that HCQ did not alter nutrient surplus-induced lipid droplet formation but inhibited starvation-induced lipid droplet formation. However, it may target the viability of hepatic stellate cells to reduce fibrosis. Through the establishment of these platforms, we have gained a better understanding of how HCQ alleviates NAFLD. Therefore, HCQ has the potential to become a novel therapeutic drug for NAFLD in the future.