分子標靶治療藥物在肝纖維化進展之療效

Abstract

慢性B型、C型肝炎以及後續產生的肝纖維化、肝硬化、肝癌及死亡一直是重要的健康議題。肝臟細胞長期發炎及壞死，是導致肝纖維化進展及肝臟癌變的主要原因。目前對於慢性B型、C型肝炎或是肝癌已經有抗病毒藥物或抗癌的標靶藥物可以使用，然而對於肝纖維化及肝硬化，目前並沒有任何有效的治療，因此仍是臨床上重要且未解決的問題。 肝臟星狀細胞（hepatic stellate cells）的增生及活化是引起肝臟纖維化的主要原因。若能了解星狀細胞活化，表現膠原蛋白基因的分子生物機轉，可以協助我們發展新的抗纖維化藥物。轉化生長因子（transforming growth factor-β）及血小板衍生性生長因子(platelet-derived growth factor)均可以促進肝臟星狀細胞的增生及活化。信號轉導與轉錄激活因子3（signal transducer and activator of transcription 3）是一個跟肝臟損傷、發炎及再生有關的轉錄因子。SHP-1去磷酸酶調控信號轉導與轉錄激活因子3，並可以直接影響血小板衍生性生長因子受體，這些因子均可能作為纖維化治療的重要標的。 Sorafenib目前是肝癌治療的標靶藥物，可以抑制包括Raf激酶（kinase）、酪氨酸激酶（tyrosine kinase）、血管內皮生長因子受體（vascular endothelial growth factor receptor）及血小板衍生性生長因子受體等。其藉由抑制肝臟星狀細胞的增殖及促其細胞凋亡而具有抗纖維化的能力，且可以增加基質金屬蛋白酶（matrix metalloproteinases）並減少基質金屬蛋白酶組織抑制劑（tissue inhibitor of matrix metalloproteinases），因而減少肝臟星狀細胞中膠原蛋白的合成。最近的研究發現sorafenib及其缺乏Raf激酶活性的結構衍生物（SC-1）均可以抑制信號轉導與轉錄激活因子3傳導路徑。而另一個sorafenib結構衍生物（SC-43）為SHP-1去磷酸酶促進劑亦可以抑制信號轉導與轉錄激活因子3傳導路徑。本研究的目的是探討SHP-1/信號轉導與轉錄激活因子3訊息傳導路徑在肝纖維化進展的角色，且研究sorafenib及其衍生物SC-1及SC-43之抗肝纖維化進展的能力及探討可能的分子機轉，並進一步探討SHP-1/信號轉導與轉錄激活因子3訊息傳導路徑在慢性B型肝炎伴隨肝纖維化患者的臨床運用。 我們使用硫代乙醯胺（thioacetamide）、四氯化碳（carbon tetrachloride）及總膽管結紮誘發肝纖維化的小鼠模式，以評估sorafenib、SC-1及SC-43之抗纖維化的能力。另外，我們利用大鼠的肝臟星狀細胞株（HSC-T6）、人類肝臟星狀細胞株（LX2）及小鼠的初代肝臟星狀細胞(primary hepatic stellate cells)探討sorafenib、SC-1及SC-43抗纖維化的能力：包含藥物對於細胞增生、細胞凋亡評估，利用西方墨點法檢測與信號轉導與轉錄激活因子3相關的蛋白質表現。另外，我們藉由細胞轉染實驗活化信號轉導與轉錄激活因子3及轉染SHP-1去磷酸酶及其結構突變型以活化SHP-1去磷酸酶活性，來探討這兩個因子對於肝臟星狀細胞的增生及sorafenib、SC-1及SC-43等藥物作用的機轉。最後利用在台大醫院接受肝切片的慢性B型肝炎患者中，我們隨機挑選了40位肝纖維化程度由低到高的患者，檢測他們肝組織中磷酸化-信號轉導與轉錄激活因子3、平滑肌肌動蛋白-α及SHP-1的表現，並進行分析這些因子與肝纖維化的相關性。 在第一部分的研究，我們發現經過硫代乙醯胺或總膽管結紮誘導產生肝纖維化後，給予小鼠sorafenib及SC-1治療後顯著減少肝纖維化。在細胞研究的結果指出sorafenib及SC-1可以降低星狀細胞的增生及增加細胞凋亡，且隨著劑量及用藥時間增加效果更顯著。Sorafenib及SC-1都可以抑制轉信號轉導與轉錄激活因子3訊息傳導路徑，過度表達信號轉導與轉錄激活因子3後，可以減少sorafenib及SC-1誘導的細胞凋亡。SHP-1去磷酸酶會下調磷酸化-信號轉導與轉錄激活因子3。使用SHP-1去磷酸酶抑制劑增加磷酸化-信號轉導與轉錄激活因子3，且逆轉sorafenib或SC-1誘導的肝臟星狀細胞的細胞凋亡。我們也發現sorafenib及SC-1會增強SHP-1去磷酸酶活性且抑制信號轉導與轉錄激活因子3。我們進一步發現慢性B型肝炎的患者中，肝纖維化越嚴重的患者肝組織中表達顯著較高的磷酸化-信號轉導與轉錄激活因子3蛋白。介白素-6（interleukin-6）是信號轉導與轉錄激活因子3的主要刺激信號，我們證明了介白素-6的刺激活化了肝臟星狀細胞中的信號轉導與轉錄激活因子3路徑，且發現慢性B型肝炎患者血漿介白素-6濃度與肝纖維化程度及肝臟中磷酸化-信號轉導與轉錄激活因子3蛋白的表現量有高度正相關。 在第二部分的研究中，我們進一步探討SHP-1去磷酸酶在肝纖維化進展的角色以及利用SC-43研究SHP-1去磷酸酶促進劑治療肝纖維化的療效。在細胞研究的結果中，我們發現SC-43可以促進肝臟星狀細胞的凋亡，且抑制轉化生長因子受體及血小板衍生性生長因子受體訊息傳導路徑。另外，SC-43可以透過與血小板衍生性生長因子受體無關的機轉抑制信號轉導與轉錄激活因子3。活化SHP-1去磷酸酶顯著抑制肝臟星狀細胞的增生，而抑制SHP-1活性則可以逆轉SC-43導致的細胞凋亡。細胞轉染SHP-1去磷酸酶結構突變型(N-SH2結構區)後，我們發現SC-43無法再度活化SHP-1去磷酸酶及抑制下游的信號轉導與轉錄激活因子3且降低SC-43導致的細胞凋亡，代表SC-43作用在SHP-1結構中的N-SH2結構區以提升其SHP-1去磷酸酶活性。我們在小鼠的四氯化碳或總膽管結紮誘導產生肝纖維化模型中，發現SC-43可以有效的預防及治療肝纖維化的進展，甚至可以增加小鼠的存活。最後，我們發現纖維化的肝臟組織中，SHP-1蛋白表現隨著肝纖維化的程度而增加，且主要分佈在纖維化的區域。這些研究結果證實了SHP-1促進劑的確可以治療肝纖維化，且特定作用在纖維化的肝組織中。 從我們的研究中，確認SHP-1/信號轉導與轉錄激活因子3是肝纖維化進展的重要訊息傳導途徑，SC-1及SC-43均可以有效藉由調控SHP-1/信號轉導與轉錄激活因子3而抑制肝纖維化。信號轉導與轉錄激活因子3可做為慢性B型肝炎患者纖維化的生物標記，而SHP-1去磷酸酶促進劑相關的抗纖維化療法可做為治療肝纖維化的新策略。

Chronic hepatitis B and C and their subsequent liver fibrosis progression, cirrhosis, hepatocellular carcinoma (HCC) and death are important health challenges. Repeated liver inflammation and hepatocyte damage are major pathogenesis for liver fibrosis progression and hepatocellular carcinogenesis. Currently, there are several antiviral agents against viral hepatitis B and C, and there is also one molecular target agent against HCC. However, effective antifibrotic therapy is unavailable, which remains an important unmet clinical need. The proliferation and activation of hepatic stellate cells (HSC) is the major contributor for hepatic fibrogenesis. A comprehensive understanding of the molecular mechanisms involved in HSC activation, perpetuation and collagen gene expression, will provide insight to develop antifibrotic therapy. The transforming growth factor-β (TGF-β) and platelet-derived growth factor (PDGF) are key mitogens for HSC proliferation and activation. The signal transducer and activator of transcription 3 (STAT3) is a transcription factor associated with liver injury, inflammation and regeneration. Src-homology protein tyrosine phosphatase-1 (SHP-1) regulates STAT3 and can directly interact with the PDGF receptor. These molecules are potential targets for antifibrotic therapy. Sorafenib is the only FDA-approved molecular target agent against advanced HCC by inhibition of the Raf kinase, the receptor tyrosine kinase, vascular endothelial growth factor receptor, and PDGF receptor. Sorafenib is found to have antifibrotic effect by reducing HSC proliferation and inducing apoptosis. In addition, sorafenib increases the ratio of matrix metalloproteinases to tissue inhibitor of matrix metalloproteinases and reduces collagen synthesis in HSC. Recent studies found sorafenib, and its derivative (SC-1), which lacks Raf kinase activity, all inhibit the STAT3 pathway. Another sorafenib derivative, SC-43, a SHP-1 agonist, can also inhibit the STAT3 pathway. The aim of this study is to investigate the role of SHP-1/STAT3 signaling pathway in fibrogenesis. The antifibrotic activity of sorafenib, SC-1 and SC-43 are examined and the molecular mechanisms are explored. Furthermore, the clinical implications of SHP-1/STAT3 in chronic hepatitis B (CHB) patients with liver fibrosis are studied. In this study, the antifibrotic activities of sorafenib, SC-1, and SC-43 are investigated in experimental murine liver fibrosis models by the induction of thioacetamide or carbon tetrachloride, or by bile duct ligation. We use the rat (HSC-T6) and human (LX2) HSC cell lines and mouse primary HSC for mechanistical investigation, including cell proliferation and apoptosis assays. The expression of STAT3 pathway related proteins are evaluated by western blot. The STAT3, or SHP-1 with its N-SH2 domain mutants are transfected to investigate their role for HSC proliferation and the mechanisms of sorafenib, SC-1 or SC-43. Among CHB patients who received liver biopsies at National Taiwan University Hospital, we randomly select 40 patients with various stage of liver fibrosis. Their hepatic phospho-STAT3 (p-STAT3), α-SMA, and SHP-1 expressions are correlated with the severity of fibrosis. In the first part of this study, after thioacetamide induction or bile duct ligation, sorafenib or SC-1 treatment significantly reduce hepatic fibrosis. In vitro studies show that both sorafenib and SC-1 have dose and time-dependent effects against cell viability and promote the apoptosis of HSCs. Both sorafenib and SC-1 downregulate p-STAT3 signaling pathway. The sorafenib- and SC-1-induced apoptosis are rescued in STAT3-overexpressing HSC cells. SHP-1 phosphatase is involved in downregulation of p-STAT3, and SHP-1 phosphatase inhibition can upregulate p-STAT3 and reverse sorafenib- or SC-1-induced HSC apoptosis. Both sorafenib and SC-1 may increase SHP-1 activity. CHB patients with advanced fibrosis have overexpressed p-STAT3. Finally, interleukin-6 (IL-6) is the main activating signal of STAT3 and we demonstrate IL-6 stimulation activates the STAT3 pathway in HSCs. We further find plasma IL-6 concentration significantly correlates with the severity of liver fibrosis and hepatic p-STAT3 level. In the second part of this study, we further investigate the role of SHP-1 phosphatase in fibrogenesis and the antifibrotic activity of SC-43, a SHP-1 agonist. In vitro studies show that SC-43 may promote the apoptosis of HSC and inhibit the TGF-β and PDGF receptor pathways. In addition, the inhibition of STAT3 is independent to the PDGF receptor inhibition. The enhanced SHP-1 activity significantly inhibits the proliferation of HSC; while knockdown SHP-1 may rescue the SC-43-induced apoptosis. After transfection of the N-SH2 domain mutants of SHP-1, the SC-43 cannot further increase SHP-1 activity or downregulate p-STAT3, and the pro-apoptotic effect of SC-43 is significantly abolished, indicating that SC-43 interacts with the N-SH2 domain of SHP-1 to enhance its SHP-1 phosphatase activity. In the carbon tetrachloride or the bile duct ligation fibrotic murine models, SC-43 effectively prevents and regresses the fibrogenesis, and promotes the mouse survival. Finally, the SHP-1 is overexpressed in the area of significant fibrosis, indicating that SHP-1 agonist may ameliorate fibrosis with tissue specificity. According to our study, we confirm that SHP-1/STAT3 is an important signaling pathway in fibrogenesis. Both SC-1 and SC-43 can regulate the SHP-1/STAT3 pathway to inhibit liver fibrogenesis effectively. STAT3 may be a fibrotic biomarker in CHB patients with active fibrogenesis, and SHP-1 phosphatase-directed antifibrotic therapy may represent a novel strategy for antifibrotic drug discovery.